Novel approaches to

management of congestive heart failure

in dogs and cats

Mariko Yata

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Sydney School of Veterinary Science, Faculty of Science

The University of Sydney

2018

i

Statement of authentication

This thesis is submitted to The University of Sydney in fulfilment of the requirements for the

degree of Doctor of Philosophy.

The work presented in this thesis is, to the best of my knowledge and belief, original except

as acknowledged in the text. I certify that the intellectual content of this thesis is the product

of my own work and that all assistance received in preparing this thesis has been

acknowledged. I hereby declare that I have not submitted this material, either in full or in part,

for a degree at this or any other institution.

Mariko Yata

August, 2018

ii

Summary of thesis

Congestive heart failure (CHF) is the most commonly diagnosed and treated condition in

veterinary cardiology as it is the sequela of a multitude of cardiovascular diseases.

Development of this syndrome marks the beginning of the end, with many losing the battle

against the collapse in circulatory function and excessive fluid retention within a year and a

half of diagnosis. Medical therapy plays the vital role of providing relief of symptoms, a good

quality of life, and time. In this thesis, current practices in CHF therapeutics within veterinary

medicine are reviewed, and novel approaches to medical therapy of CHF are explored.

The work begins by examining the existing literature surrounding the topic in

Chapter 1. Common aetiologies and pathophysiology of CHF in dogs and cats are discussed.

Common practices in veterinary medicine are scrutinised in light of existing scientific

evidence, and areas requiring further research support are highlighted. Throughout the

chapter, references to human literature are made to underline inter-species differences and

contemplate alternative treatment methods. Finally, the aims of this thesis are presented.

The following three chapters (Chapters 2, 3, and 4) extend upon the knowledge basis

supporting current CHF therapeutics in veterinary patients and present insights for

improvements to treatment regimen. Chapters 2 and 3 examine the pharmacokinetics and

pharmacodynamics of pimobendan in dogs and cats, respectively. Major findings include the

discovery of greater metabolism of pimobendan and cardiovascular effects in dogs

compared to cats; in addition, echocardiography is proven to be an effective method of

monitoring treatment effects in the former but not the latter. Thus, the importance of further

investigations on use of pimobendan in cats is stressed. The focus moves on from

pimobendan to the realm of multi-drug therapy of CHF in dogs with myxomatous mitral valve

disease (MMVD) in Chapter 4. This is a descriptive chapter detailing the disease course in

eleven dogs from the time of first diagnosis of CHF till death or the conclusion of the study.

The use of pimobendan, frusemide, benazepril, and spironolactone resulted in a median

survival time of 419 days, with recurrence of CHF being the most common cause of death or

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euthanasia in this cohort of dogs. In the absence of published studies evaluating the benefit

of such combination therapy, this work serves to present objective survival data in a small,

yet relevant, group of individuals for veterinary practitioners.

In the final two chapters of the body (Chapters 5 and 6), the natriuretic peptide

system is evaluated as a novel therapeutic target in dogs with CHF. Due to existing

concerns regarding cross-reactivity between active and inactive components of the system

when immunoassays are employed, a specific method of detecting active brain natriuretic

peptide (BNP1-32) in canine plasma utilising mass spectrometry is investigated in Chapter 5.

Concurrently, a method of stimulating endogenous brain natriuretic peptide production

through administration of high-volume intravenous fluids is tested, with the aim of using the

aforementioned assay to characterise the response of this system in healthy dogs.

Subsequently, the effect of augmenting this system in dogs with CHF due to MMVD through

administration of synthetic canine BNP1-32 is examined in Chapter 6. The results suggest a

peripheral natriuretic peptide resistance and raise interesting questions regarding the

pathophysiology of, and potential future therapeutics of, CHF in these dogs.

The ensuing discussion brings together the findings of these five studies and

highlights their significance within the context of current and future veterinary practices. The

limitations of each study are considered, and suggestions for future endeavours to elucidate

answers to remaining questions are made. Ultimately, the conclusions drawn from this work

are hoped to contribute small pieces in the knowledge of CHF therapeutics that will help

guide ongoing investigations into its pathophysiology and optimal treatments.

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Acknowledgements

A PhD is a journey of all sorts. At its surface, this thesis presents a journey of scientific

discovery; yet below it lies that of personal and interpersonal developments. No journeys

exist without characters, a setting, plot twists, and a record of these crucial elements. What

follows is but a mere fragment of the credit roll for this particular journey.

To start, I would like to thank my supervisory team – Niek Beijerink, Andrew

McLachlan, and Ben Crossett. Your wonderful brains helped me overcome many challenges

over the past few years. Niek, I think of you as my “research dad”. You have spent countless

hours on meetings, discussions, data collection, and reviewing my studies and manuscripts

over the past few years. Andrew and Ben, your guidance in the fields of pharmacology and

mass spectrometry helped me grow as a scientist beyond clinical veterinary practice.

Without all of you, the work presented in this thesis would not have existed in its current form

– so thank you, thank you, thank you.

None of this would have started without the generous support and sponsorship from

Stephen Page and Luoda Pharma. Stephen, your advice on various aspects of my project

were always much appreciated. Thank you also for the opportunity to dip my toes into the

world of pharmaceutical research beyond the scope of this project alone.

I would also like to thank the many other scholars whose knowledge and expertise

were invaluable in the successful completion of my studies. Evelyn Hall, Peter Thompson,

and Navneet Dhand – thank you for your advice on all things “stats-related”. To my

collaborator, David Foster – your expertise facilitated a greater understanding of

pharmacometrics – a world I knew nothing about prior to this project. Thank you to Brett

Hambley and Yaxin Lu for holding my hand as I learned about ELISAs.

This journey could not have taken place in the lack of a setting. Thank you to Bob

Pigott, Maurice Webster, and the staff at Invetus (formerly Wongaburra Research Centre) for

accommodating and guiding us as we embarked on the journey into pharmacological

research. Thank you to the kind, patient staff at the Mass Spectrometry Core Facility

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(Charles Perkins Centre) for answering my many questions and for helping me with the

misbehaving, angry, and seemingly suicidal hardware and software, time and time again, so

I could make some progress with my research. Thank you to the staff at the University

Veterinary Teaching Hospital, Sydney for welcoming me as an honorary staff member and

supporting all my research endeavours at this site.

Thank you to my fellow office mates: Kendy Teng, Aaron Izes, Amanda Shapiro, Alan

Marcus, Holly Cope, Emily Sellens, Gemma Ma, Soraya Gharibi, Laura Schmertmann, Kate

Worthing, Caroline Marschner, Mark Westman, Judy Sung, Iona Maher, and many others

who have come and gone. I thoroughly enjoyed our collegial discussions and light-hearted

banter.

Last but not the least. Thank you to my family – mom, dad, Yoshi, Hiro, and my

partner, Alan Marcus, for your support, your belief in me, your patience, and your

understanding. Thank you to all of my friends who helped me stay sane. Thank you to the

invisible forces that shaped this journey and to those who provided me with opportunities to

engage in so much outside of this project. You were the glue that held the pieces together

and the breath that helped breathe life into this story.

vi

Table of Contents

Statement of authentication………………………………………………………………………..i

Summary of thesis………………………………………………………………………………….ii

Acknowledgements………………………………………………………………………………..iv

Table of contents…………………………………………………………………………………..vi

List of publications and presentations……………………………………………………….vii

List of abbreviations ……………………………………………………………………………..viii

Chapter 1: Introduction, literature review, and aims of this thesis………………………...1

Chapter 2: Pharmacokinetics and cardiovascular effects following a single oral

administration of a nonaqueous pimobendan solution in healthy dogs….40

Chapter 3: Single-dose pharmacokinetics and cardiovascular effects of oral

pimobendan in healthy cats…………………………………………………………..51

Chapter 4: Evaluation of the natural course of disease in eleven dogs with congestive

heart failure due to myxomatous mitral valve disease treated with combination

medical therapy…………………………………….…………………………………….69

Chapter 5: Brain natriuretic peptide assay development and evaluation in dogs

undergoing acute intravascular volume expansion………………………93

Chapter 6: Cardiorenal and endocrine effects of synthetic canine BNP1-32 in dogs

with compensated congestive heart failure due to myxomatous mitral valve

disease: a pilot study..........................................................…………………….….133

Chapter 7: Discussion and conclusions……………………………………………………..156

References………………………………………………………………………………………...177

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List of publications and presentations arising from candidature

Journal articles:

Yata M, McLachlan AJ, Foster DJR, Page SW, Beijerink NJ. (2016) Pharmacokinetics and

cardiovascular effects following a single oral administration of a nonaqueous pimobendan

solution in healthy dogs. J Vet Pharmacol Ther. 39:45-53. DOI: 10.1111/jvp.12243.

Yata M, McLachlan AJ, Foster DJR, Hanzlicek AS, Beijerink NJ. (2016) Single-dose

pharmacokinetics and cardiovascular effects of oral pimobendan in healthy cats. J Vet

Cardiol. 18:310-325. DOI: 10.1016/j.jvc.2016.07.001.

Conference presentations:

Yata M, McLachlan AJ, Foster DJR, Hanzlicek AS, Beijerink NJ. Evaluation of pimobendan

in healthy cats: An echocardiographic study of acute cardiovascular effects. 25th European

College of Veterinary Internal Medicine – Companion Animals Congress, September 10-12th,

2015. Lisbon, Portugal.

Yata M, Kooistra HS, Beijerink N. Effects of BNP1-32 in dogs with congestive heart failure

due to myxomatous mitral valve disease. 2018 American College of Veterinary Internal

Medicine Forum, June 14-16th, 2018. Seattle, USA.

viii

List of abbreviations

ACC/AHA American College of Cardiology/American Heart Association

ACE Angiotensin converting enzyme

ACE-I Angiotensin converting enzyme inhibitor

ACVIM American College of Veterinary Internal Medicine

ANP Atrial natriuretic peptide

Ao Aortic root diameter

Ao Vel Maximal aortic velocity

ARNI Angiotensin receptor neprilysin inhibitor

ARVC Arrhythmogenic right ventricular cardiomyopathy

ATE Aortic thromboembolism

AUC0-∞ Area under the plasma concentration-time curve to infinity

BNP Brain natriuretic peptide

AUC0-t Area under the plasma concentration-time curve to final concentration

BP Blood pressure

cANP, ANP1-28 c-terminal ANP (active fragment)

cBNP, BNP1-32 c-terminal BNP (active fragment)

cGMP Cyclic guanosine monophosphate

CHF Congestive heart failure

CL/F Apparent clearance

Cmax Maximal plasma concentration

CO Cardiac output

CV Coefficient of variation

CVP Central venous pressure

DBP Diastolic blood pressure

DCM Dilated cardiomyopathy

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EDV End diastolic volume

EF Ejection fraction

ESI Electrospray ionisation

ESV End systolic volume

FEK Fractional excretion of potassium

FENa Fractional excretion of sodium

FS Fractional shortening

HCM Hypertrophic cardiomyopathy

HFmrEF Heart failure with mid-range ejection fraction

HFpEF Heart failure with preserved ejection fraction

HFrEF Heart failure with reduced ejection fraction

HOCM Hypertrophic obstructive cardiomyopathy

HR Heart rate

ISACHC International Small Animal Cardiac Health Council

IVSd Thickness of interventricular septum in diastole

IVSs Thickness of interventricular septum in systole

Kel Elimination rate constant

LA Left atrial diameter

LA:Ao Left atrium to aorta ratio

LLOQ Lower limit of quantification

LVET Left ventricular ejection time

LVIDd Left ventricular internal diameter in diastole

LVIDs Left ventricular internal diameter in systole

LVmax dP/dt Rate of rise in maximal left ventricular pressure

LVOT Left ventricular outflow tract

LVPWd Thickness of left ventricular posterior wall in diastole

LVPWs Thickness of left ventricular posterior wall in systole

x

MALDI Matrix-assisted laser desorption/ionisation

MBP Mean blood pressure

MMC Matrix matched calibration control

MMVD Myxomatous mitral valve disease

MRA Mineralocorticoid receptor antagonists

MS Mass spectrometer

MS/MS Tandem mass spectrometry

MST Median survival time

NP Natriuretic peptide

NPR-A Natriuretic peptide receptor-A

NPR-C Natriuretic peptide receptor-C

NPS Natriuretic peptide system

NT-proANP N-terminal ProANP (inactive fragment)

NT-proBNP N-terminal proBNP (inactive fragment)

NYHA New York Health Association

ODMP O-demethylated metabolite of pimobendan

PcGMP Plasma cGMP concentrations

PEP Pre-ejection period

RAAS Renin-angiotensin-aldosterone system

RCM Restrictive cardiomyopathy

RRR Resting respiratory rate

SAM Systolic anterior motion

SBP Systolic blood pressure

SNS Sympathetic nervous system

SRR Sleeping respiratory rate

SV Stroke volume

SVR Systemic vascular resistance

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t1/2 Elimination half-life

Tmax Time to reach maximum plasma concentration

TOF Time of flight mass spectrometer

UAldo:C Urinary aldosterone to creatinine ratio

UcGMP Urinary cGMP concentration

UCM Unclassified cardiomyopathy

UD-CG 212 CL O-demethylated metabolite of pimobendan

UHPLC Ultra-high performance liquid chromatography

UOP Urine output

UEcGMP Urinary excretion of cGMP

V/F, Vz/F Apparent volume of distribution

VHS Vertebral heart score

wnLVIDd Weight-normalised left ventricular internal diameter in diastole

wnLVIDs Weight-normalised left ventricular internal diameter in systole

Chapter 1:

Introduction, literature review, and aims of this thesis

1

1.1) Introduction to canine and feline congestive heart failure

Early descriptions of the cardiovascular system date back to as far as 300 – 400 years BC

(Buchanan, 2013). Yet the foundation of modern day cardiology is most accepted to have

been laid in the 1600's, when William Harvey conducted a series of experiments to explain

basic cardiovascular physiology (Buchanan, 2013). Since then, large advancements in

medical technology have led to increasing knowledge regarding various cardiovascular

diseases affecting both humans and animals. Although descriptions of affected individuals

existed prior to the 1900's, it was not until the early 1900's that heart disease in dogs was

first characterised (Detweiler & Patterson, 1965). Publications describing feline

cardiovascular diseases also emerged in the mid 1900’s (Buchanan, 2013). Fast-forward

some 60-odd years to today, and numerous congenital and acquired heart diseases, as well

as their natural course of pathology and pathophysiology, have been discovered and

described in our veterinary patients.

Despite the myriad of shades that colour the spectrum of cardiovascular diseases in

dogs and cats, many share a common sequela known as congestive heart failure (CHF).

CHF is a syndrome that is characterised by the presence of cardiovascular disease, fluid

retention, and ensuing oedema. This manifests most commonly in cats and dogs as

pulmonary oedema, pleural or peritoneal effusions, or both. The effect of CHF on the

lifespan of affected populations has been characterised by the wealth of literature supporting

estimated median survival times (MST) of around a year and a half or less (De Madron et al.,

2011; Ettinger et al., 1998; Ferasin et al., 2003; Finn et al., 2010; Fox et al., 2014; Fox et al.,

2018; Häggström et al., 2008; Hambrook & Bennett, 2012; Harvey et al., 2005; Jung et al.,

2016; Petric et al., 2002; Rush et al., 2002; Slupe et al., 2008; S. A. Smith et al., 2003; The

BENCH Study Group, 1999; Tidholm et al., 1997; Vollmar, 2000); recurrent hospitalisation,

the need for constant monitoring, and daily medical therapy pose a burden, not only for the

patients, but also their owners, who must be “fearfully vigilant, at the mercy of the disease

and its treatments, while worrying about that which remained unspoken” (Bursch, 2012).

Such is the context in which numerous research efforts into the pathophysiology and

2

treatment of CHF were born, and to which this thesis aims to contribute towards as a small

cog in the ever-revolving wheel of medical advancements.

In this literature review, an overview of canine and feline CHF and existing

therapeutics will be provided, and key knowledge gaps will be identified. It will start with a

description of the leading aetiologies of CHF in dogs, cats, and people, followed by a

discussion of the pathophysiology of CHF in individuals with specific heart disease. The

existing therapeutic approaches in both veterinary and human cardiology will be analysed in

light of these processes. Specific areas warranting further investigation in veterinary CHF

therapeutics will be identified, and the aims of this thesis will be outlined.

1.2) Aetiology of CHF

It is well accepted in both the veterinary and medical professions that cardiovascular

diseases leading to CHF most commonly affect older individuals. A variety of pathological

processes can occur to affect virtually all parts of the cardiovascular anatomy, but primary

valvulopathies and cardiomyopathies predominate in canine and feline acquired heart

diseases. In humans, cardiomyopathies secondary to diabetes, systemic hypertension, and

coronary artery disease also form an important class of cardiovascular disease.

1.2.1) Aetiology of canine CHF

Myxomatous mitral valve disease (MMVD) is the leading acquired cause of CHF in dogs. Its

high prevalence was noted as early as 1965, when an epidemiological study of 4831 dogs

identified chronic valvular disease in around 75% of cases diagnosed with an acquired

cardiovascular disease (Detweiler & Patterson, 1965). There is a known association with age,

with higher prevalence in older animals (Detweiler & Patterson, 1965; Olsen et al., 1999;

Thrusfield et al., 1985). Breeds most commonly affected by MMVD include small to medium

breed dogs, with a particular predisposition towards the disease in the Miniature Poodle,

Cocker Spaniel, Chihuahua, Cavalier King Charles Spaniel, Dachshund, and Boston Terrier,

amongst others (Beardow & Buchanan, 1993; Häggström et al., 1992; O'Leary & Wilkie,

3

2009; Olsen et al., 1999; D. Pedersen et al., 1999; Thrusfield et al., 1985). In the Cavalier

King Charles Spaniel, Dachshund, Whippet, and Bull Terrier, this disease is suspected to be

heritable, with a polygenic mode of inheritance (Madsen et al., 2011; Meurs et al., 2018;

O'Leary & Wilkie, 2009; Olsen et al., 1999; Stern et al., 2015; Swenson et al., 1996).

Several characteristic gross and histological abnormalities occur in the cardiac

structures of dogs affected by MMVD. The affected valves, which most commonly include

the mitral and tricuspid valves, grossly show elongation, thickening, haemorrhage, distortion,

and grey-white nodular changes (Fox, 2012; Kittleson & Kienle, 1998; Whitney, 1974). At a

cellular level, the disease involves alterations to the valvular cell population that resemble

inflammatory and ageing changes. Valvular interstitial cells become activated, the net

number of valvular interstitial and endothelial cells reduce, and macrophages and mast cells

infiltrate the tissue; this, in turn, causes an increase in glycosaminoglycans and

proteoglycans in the extracellular matrix (Aupperle et al., 2009; Connell et al., 2012; Han et

al., 2010; Han et al., 2008; Han et al., 2013). The increase in proteoglycans and net loss of

collagen and elastin results in obliteration of the definition between the spongiosa and

fibrosa layers of the valve (Aupperle et al., 2009; Fox, 2012; Hadian et al., 2007; Han et al.,

2010; Kittleson & Kienle, 1998). Valvular integrity is lost, leading to elongation or rupture of

chordae tendinae. Valvular insufficiency also contributes towards development of atrial jet

lesions, endomyocardial splitting, atrial enlargement, eccentric ventricular hypertrophy, and

annular dilatation (Fox, 2012; Kittleson & Kienle, 1998; Ware, 2011).

Other causes of CHF in dogs include pericardial diseases such as idiopathic

pericarditis; primary cardiomyopathies such as dilated cardiomyopathy (DCM); conduction

disorders leading to severe tachyarrhythmias or bradyarrhythmias; cardiac neoplasia;

myocarditis; valvular endocarditis; and congenital diseases including patent ductus

arteriosus, aortic and pulmonic stenosis, septal defects, and anomalous vessels (Sisson,

2010).

4

1.2.2) Aetiology of feline CHF

Cats, unlike dogs, suffer predominantly from cardiomyopathies (Fox et al., 2018). Of 408

cats presenting over a seven-year period at one referral clinic, 287 were diagnosed with a

primary cardiomyopathy (Riesen et al., 2007). Hypertrophic cardiomyopathy (HCM) or

hypertrophic obstructive cardiomyopathy (HOCM) occurs most frequently, comprising

around 58 – 68% of cardiomyopathies in cats (Ferasin et al., 2003; Riesen et al., 2007).

Males appear to be affected more commonly than females, and a breed predilection exists

for the domestic shorthair, Persian, Maine Coon, British Blue, Ragdoll, Birman, Sphinx,

Scottish Fold, Siamese, and American and British shorthairs, amongst others (Ferasin et al.,

2003; Granström et al., 2011; Riesen et al., 2007). Two different single point mutations in the

gene encoding myosin binding protein C have been identified in the Maine Coon and

Ragdoll (Meurs et al., 2007; Meurs et al., 2005), and heritability of the disease has also been

documented in these and the Norwegian Forest, Sphinx, and a family of mixed-breed cats

(Borgeat, Casamian-Sorrosal, et al., 2014; Kittleson et al., 1999; Kraus et al., 1999; März et

al., 2015; Silverman et al., 2012).

Grossly, HCM is characterised by variable degrees of left ventricular concentric and

papillary muscle hypertrophy (Fox, 2003a). Histologically, the normally parallel distribution of

myofibrils becomes disrupted, resulting in a disorganised array of fibres displaying

perpendicular and oblique orientations (Fox, 2003a). Additional features include myocyte

hypertrophy, extracellular fibrosis, arteriosclerosis, and abnormal proliferation of intramural

coronary arteries (Fox, 2003a). These contribute towards myocardial ischemia and

functional abnormalities predominantly consisting of impaired ventricular relaxation and

compliance (Abbott, 2010). In those with HOCM, a dynamic left ventricular outflow tract

(LVOT) obstruction caused by systolic anterior motion (SAM) of the mitral valve apparatus

accompanies and compounds these hypertrophic changes. Factors that are thought to

contribute towards the development of this dynamic obstruction include the development of

an intraventricular pressure gradient due to the Venturi effect, the presence of anatomical

variants such as anterior displacement of the papillary muscle, and the effect of drag forces

5

which draw the anterior mitral leaflet into the LVOT; more recently, the Venturi effect theory

has fallen somewhat out of favour based on observations of SAM at lower LVOT velocities in

people with HOCM (Abbott, 2010; Sherrid, 2006; Sherrid et al., 2000).

In addition to primary HCM and HOCM, reversible ventricular hypertrophy secondary

to systemic hypertension and hyperthyroidism can occur in cats (Fox, 2003a; Riesen et al.,

2007). Other cardiomyopathies which may result in CHF include restrictive cardiomyopathy

(RCM), DCM, unclassified cardiomyopathy (UCM), and arrhythmogenic right ventricular

cardiomyopathy (ARVC); more recently, transient myocardial thickening has been identified,

particularly in younger cats (Novo Matos et al., 2018). Myocarditis, valvular endocarditis,

pericardial diseases, and congenital diseases such as septal defects and valvular dysplasia

are other causes of CHF in cats (Riesen et al., 2007).

1.2.3) Aetiology of CHF in humans

There are several primary valvular and myocardial diseases in people that mimic those

which occur in veterinary patients. Idiopathic DCM, for example, shares similar histological

characteristics, functional abnormalities, and familiar inheritance as in dogs (Dec & Fuster

1994). HCM and HOCM also share similarities with the diseases seen in cats; however, the

genetic basis of these have been better characterised in people, and sudden death due to

arrhythmias comprises a major concern with HCM and HOCM in people (Maron, 2002). In

the class of valvulopathies, mitral valve prolapse is one of the most commonly encountered

diseases, with myxomatous degeneration being the leading cause of significant mitral

regurgitation (Guy & Hill, 2012; Hayek et al., 2005). Histological and gross characteristics

are akin to those observed in dogs with MMVD (Delling & Vasan, 2014).

Yet by far, the most common conditions associated with CHF in people include

systemic hypertension and coronary artery disease. These are present in up to 85-90% of

people with CHF (Ho et al., 1993). Both are primary vascular diseases, but they induce

gross and functional cardiac pathology that can mimic primary cardiomyopathies and lead to

development of CHF. For example, the high afterload created by systemic hypertension

6

leads to concentric myocardial hypertrophy and diastolic dysfunction as seen with HCM

(Levy et al., 1996; Viau et al., 2015); the increase in myocardial stress can also contribute

towards ischemia in latter stages of this type of secondary cardiomyopathy. In contrast, a

DCM-like phenotype can result with myocardial ischemia caused by coronary artery disease

(Gheorghiade & Bonow, 1998). These conditions are associated closely with the metabolic

syndrome, which arises from a combination of hypertension, obesity, dyslipidaemia, insulin

resistance, and hyperglycaemia (Alberti et al., 2005). Despite genetic predisposition towards

this syndrome in some individuals, environmental factors consisting of diet and lifestyle

choices are thought to play a major role in its development (Kaur, 2014). These preventable,

secondary cardiomyopathies are not prevalent in veterinary patients but form important

causes of CHF in people.

1.3) Introduction to the pathophysiology of CHF

Irrespective of the underlying heart disease, there are common mechanisms involved in the

development of chronic CHF. The process almost always begins with a slight reduction in

cardiac output (CO) and blood pressure (BP) caused by the primary abnormality (Sisson,

2010). This may be the result of valvular regurgitation; intrinsic or relative myocardial systolic

dysfunction; reduced ventricular filling due to diastolic dysfunction or an inflow obstruction;

the presence of an outflow tract obstruction; or inappropriate shunting (Sisson, 2010). In

response, the renin-angiotensin-aldosterone system (RAAS), sympathetic nervous system

(SNS), and a variety of local vascular and tissue processes are activated to cause sodium

and water retention, arteriolar and venoconstriction, and positive inotropic, lusitropic, and

chronotropic effects (Sisson, 2010). The resultant increase in preload, afterload, venous

pressures, and cardiac contractility causes normalisation of BP and CO. Early in the course

of disease, these processes are appropriately antagonised by feedback mechanisms and

counter-regulatory systems once normality is established. However, chronic activation and

pathological anomalies to these systems, together with progression of the underlying

structural disease, result in excessive preload, afterload, and inefficient myocardial

7

remodelling (Sisson, 2010). The ability to maintain cardiovascular homeostasis becomes

compromised, ultimately leading to CHF.

An important characteristic of CHF is the presence of congestion caused by

excessive sodium and water retention and exacerbated by excessive vasoconstriction. The

RAAS and its counter-regulatory system, the natriuretic peptide system (NPS), are of

particular importance here. Excessive RAAS activation is thought to occur due to the chronic

reduction in forward output caused by the structural or functional heart disease. Indeed,

enhanced RAAS activity, reflected by elevation in plasma renin, angiotensin II, and

aldosterone concentrations, has been noted in CHF in many experimental and clinical

studies (H. H. Chen & Burnett Jr, 2000; Francis et al., 1990; Häggström et al., 2014;

Knowlen et al., 1983; Watkins Jr et al., 1976; Weber, 2001; Weber & Villarreal, 1993).

More recently, evidence to support the activation of local or tissue RAAS, even in the lack of

circulatory RAAS activation, has emerged (Carey & Siragy, 2003; Dzau et al., 2002; Fujii et

al., 2007; Paul et al., 2006). Angiotensin II and aldosterone are the final products of both

circulating and tissue RAAS. These bind to angiotensin type 1 receptor and

mineralocorticoid receptors, respectively, resulting in vasoconstriction, renal sodium and

water reabsorption, platelet activation and aggregation, stimulation of the SNS, and

promotion of inflammatory and reparative processes through cytokine production,

chemotaxis, macrophage activation, and stimulation of pro-fibrotic mechanisms – all of which

propagate the syndrome of CHF (Reid, 1992; Weber, 2001).

The effects of the RAAS are countered by the NPS, which is triggered in response to

RAAS activation to promote diuresis, natriuresis, vasodilation, and RAAS antagonism (Potter

et al., 2009). In the context of CHF, the NPS can thus be considered a “protective”

mechanism. Despite high circulating concentrations of immunoreactive natriuretic peptides

(NP), a decline in NP responsiveness, resulting in vasoconstriction and reduced natriuresis,

has been demonstrated in those with CHF (Cody et al., 1986; Eiskjær et al., 1991; Jensen et

al., 1999; Moesgaard et al., 2011; Nakamura et al., 1998; Schmitt et al., 2004). Although

there is some evidence to support suppression of NPS due to excess RAAS activity (Abassi

8

et al., 1990; H. H. Chen & Burnett Jr, 2000; Margulies et al., 1991; Villarreal & Freeman,

1991; Weber & Villarreal, 1993), a relative state of NP resistance or altered NP metabolism

may also contribute towards the development of CHF.

Indeed, this concept of a “NP paradox” contributing towards development of CHF has

received much attention in human cardiology over the past 10 – 15 years (Baerts et al.,

2012). Several mechanisms have been proposed to explain this paradox, including

alterations to the synthesis, activity, and degradation of NP as well as limitations to available

methods of NP detection.

Of the natriuretic peptides, atrial and brain NP (ANP, BNP), play a significant role in

cardiovascular physiology. Both originate as inactive pro-forms, with proANP1-126 being

stored in atrial granules while proBNP1-108 is produced mainly by ventricular myocytes

upon demand (Potter et al., 2009). Activation of ANP and BNP is triggered through detection

of hypervolemia via atrial and ventricular stretch and involves enzymatic cleavage of the pro-

forms mediated by transmembrane serine proteases, corin and furin (Ichiki et al., 2013). The

products of this cleavage consist of a biologically inactive N-terminal portion

(NT-proANP and NT-proBNP) and a biologically active c-terminal portion (ANP1-28 and

BNP1-32, or cANP and cBNP), both of which are released into circulation. These active

molecules bind to the natriuretic peptide receptor-A (NPR-A) and produce a cascade of

intracellular signalling effects mediated by cyclic guanosine monophosphate (cGMP) to

produce biological effects (Potter et al., 2009). Breakdown of the active molecules involves

either internalisation and degradation following binding to NPR-A; degradation via the

clearance receptor, natriuretic peptide receptor-C (NPR-C); or extracellular fragmentation to

less active or biologically inactive fragments via circulating enzymes such as neprilysin,

meprin, dipeptidyl peptidase IV, and insulin degrading enzyme (Ichiki et al., 2013; Potter et

al., 2009). The circulating half-life of ANP is shorter than that of BNP, measuring around two

minutes and twenty minutes, respectively (Potter et al., 2009). Though ANP is normally

present in higher concentrations in circulation, a shift towards greater BNP production occurs

9

with progressive CHF, and as such, the latter has received more attention in this context

(Langenickel et al., 2000; Luchner et al., 1998).

In states of CHF, there is some evidence to support alterations to NP synthesis,

degradation, and activity. Plasma and tissue concentrations of corin were reduced in animals

(Tripathi et al., 2016) and humans (Barth et al., 2006; Dong et al., 2010; Ibebuogu et al.,

2011; Shrestha et al., 2010) with CHF. However, the relationship between concentrations of

corin and its activity in CHF appears to be ill-defined, with some studies observing the lack of

a clear relationship between measured corin and NP concentrations (S. Chen et al., 2010;

Miller et al., 2011; Shrestha et al., 2010; Zhou et al., 2016). This may have been due, at

least in part, to the complex nature of corin activation, which relies on a particular

conformation and sequence of the extracellular component of the molecule (J. Jiang et al.,

2011; Knappe et al., 2004; W. Wang et al., 2008; Yue Zhang et al., 2014), glycosylation (X.

Liao et al., 2007; H. Wang et al., 2015), and presence of activating substances (S. Chen et

al., 2015; J. Jiang et al., 2011). There has also been evidence to support enhanced

degradation of NPs, with high neprilysin activity being suggested in an experimental model

of CHF (Margulies et al., 1995) and increased density of NPR-C being reported in platelets

and myocardial samples from people with CHF (Andreassi et al., 2001; Kuhn et al., 2004).

Furthermore, high circulating concentrations of neprilysin were detected in people with CHF,

and this was associated with increased CHF-related mortality and morbidity (Bayés-Genís et

al., 2015). Enhanced degradation of active peptides could result in diminished effects of NPs,

but reduced numbers of, or productive interactions with, NPR-A could also attenuate their

activity (Schiffrin, 1988; Tsunoda et al., 1988; Tsutamoto et al., 1993).

The theory of enhanced degradation and stunted production of ANP1-28 and

BNP1-32 in the face of CHF are also supported through advancements in assay techniques

to measure circulating concentrations of these peptides. Immunoassays have traditionally

been used for the detection of these peptides; yet because these rely on antibody-antigen

interaction between specific fragments of these proteins, an inherent limitation of this

technique is the inability to differentiate between active peptides and inactive proforms or

10

fragments. Indeed, a handful of studies have demonstrated cross-activity between

ProBNP1-108 and BNP1-32 (Liang et al., 2007; Nishikimi et al., 2010), ProBNP1-108 and

NT-proBNP (Heublein et al., 2007; Liang et al., 2007), as well as BNP1-32 and its less-active

fragments (Heublein et al., 2007; Rawlins et al., 2005) in commercially available

immunoassays. The predominance of high-molecular weight BNP (consistent with

ProBNP1-108) over the low-molecular form (consistent with BNP1-32) in the plasma of

people with CHF has been identified through high-performance liquid chromatography and

western blotting techniques (Giuliani et al., 2006; Liang et al., 2007; Yandle et al., 1993).

When mass spectrometry was utilised to differentiate BNP1-32 from its less active fragments,

a significantly lower concentration of specific BNP1-32 compared to the immunoassay-

derived “BNP-32” has been demonstrated in people with varying degrees of CHF

(Hawkridge et al., 2005; Miller et al., 2011; Niederkofler et al., 2008). Thus, it appears that

people with CHF may, in fact, have a lower concentration of biologically active NP despite

the detection of high concentrations of NP using commercial immunoassays. Although these

studies have as limitations their small size and lack of a control group for comparison in

some cases, they raise the possibility of altered NP metabolism in states of CHF which may

be masked by the inherent limitations of existing commercial immunoassays.

In addition to the RAAS and aberrant NPS, vasopressin, or anti-diuretic hormone, is

another contributor towards the fluid retentive and vasoconstrictive states observed in CHF.

Vasopressin causes free water reabsorption at the level of the collecting ducts in addition to

arteriolar vasoconstriction. Increased plasma concentrations of vasopressin and

hyponatremia have been observed in some, but not all, patients with CHF, and these are

recognised negative prognostic indicators linked to reduced survival and increased

frequency of hospitalisation (De Luca et al., 2005; Lanfear et al., 2013; Sica, 2005). Although

the overall reduction in forward blood flow and increased angiotensin-II activity are thought

to play a role, the exact mechanisms contributing towards inappropriate vasopressin

production in some individuals with CHF remains unclear (Gassanov et al., 2011; Imamura

et al., 2014).

11

Another arm of the pathophysiology of CHF involves stimulation of the SNS, which

contributes towards vasoconstriction, increased heart rate, and reduced cardiac contractility.

SNS stimulation may occur directly via baroreceptor-mediated pathways or through

circulating and tissue RAAS-mediated pathways (Cox et al., 1999; Lal et al., 2004; Lewis &

Reit, 1965; Luoh & Chan, 1998; Ma et al., 1999; Reid, 1992). In human and veterinary

patients with CHF, heightened SNS activity is evidenced by high circulating concentrations

of its major neurotransmitter and hormone, noradrenaline (Francis et al., 1990; O'Sullivan et

al., 2007; Santos et al., 2006; Triposkiadis et al., 2009; Ware et al., 1990). Chronic SNS

activation results in desensitisation and malfunction of β1 adrenoreceptors, which are its

major effect-producing receptors in the heart (Böhm et al., 1995; Triposkiadis et al., 2009).

High levels of catecholamines are directly cardiotoxic, causing interstitial fibrosis, myocyte

apoptosis, and oxidative damage (Behonick et al., 2001; Bindoli et al., 1992; Goldspink et al.,

2004; Liaudet et al., 2014; Singal et al., 1982; Triposkiadis et al., 2009). In humans, several

mutations to genes encoding ɑ- and β- receptors have also been associated with heart

failure (Triposkiadis et al., 2009). Finally, diminished baroreceptor sensitivity and

suppression of the parasympathetic pathways in CHF further amplify these detrimental

effects of SNS activation (Eckberg et al., 1971; Grassi et al., 1995; Higgins et al., 1972;

Sabbah, 2012; Triposkiadis et al., 2009).

At the vascular level, disruptions in function and production of its most potent locally-

derived vasoconstrictor, endothelin-1, and vasodilator, nitric oxide, have also been

documented in CHF. In naturally occurring and experimentally induced chronic CHF,

endothelin-1 concentrations are increased, seemingly due to enhanced production and

reduced clearance (Giaid et al., 1993; Picard et al., 1998; Sakai et al., 1996; Zolk et al.,

1999). Impaired nitrous oxide responsiveness occurs in humans with CHF, despite some

evidence to support increased levels of nitric oxide (Bauersachs et al., 1999; Kubo et al.,

1991; Winlaw et al., 1994). In veterinary medicine, high levels of circulating endothelin-1 has

been described in dogs (O'Sullivan et al., 2007; Prošek et al., 2004b; Tessier-Vetzel et al.,

2006) and cats with CHF (Prošek et al., 2004a). Endothelial dysfunction, as measured

12

through impairment of flow-mediated vasodilation and reactive hyperaemia, has also been

demonstrated in Cavalier King Charles Spaniels with MMVD (Moesgaard et al., 2012) and

dogs with CHF (S. M. Cunningham et al., 2012). Aside from contributing towards an increase

in afterload, this elevation in endothelin-1, reduced nitric oxide synthase, and overall

endothelial dysfunction can cause development of complications in the form of

disproportionate pulmonary hypertension secondary to left-sided CHF (Cooper et al., 1998;

Giaid et al., 1993; Porter et al., 1993; Sakai et al., 1996). Thus, such imbalances at the

vascular level not only propagate the syndrome of CHF but also trigger the development of

further comorbidities.

There is also evidence to support pathological alterations to the mechanisms

involved in the contractile function of the myocardium in states of CHF due to

cardiomyopathies. Abnormal cardiomyocyte calcium cycling occurs in the sarcoplasmic

reticulum due to altered functions of its components such as the L-type calcium channels,

ryanodine receptors, SERCA, and Phospholamban (Hasenfuss & Pieske, 2002; Luo &

Anderson, 2013). The net effects are a reduction in available calcium to facilitate actin-

myosin cross-bridge formation, contributing towards systolic dysfunction; reduced

sarcoplasmic reticulum sequestration of calcium, resulting in diastolic dysfunction; and

prolongation of the cardiac action potential, which increases the arrhythmogenic potential

(Luo & Anderson, 2013). Abnormal expression of, and structural anomalies to, myosin, actin,

and associated proteins including troponins and tropomyosin are also thought to contribute

towards altered cardiac function, leading to CHF (Yin et al., 2015).

Other cellular mechanisms which appear to be involved in development and

propagation of CHF include alterations to cardiovascular and renal functions due to reduced

intracellular signal transduction via enhanced expression and function of

phosphodiesterases (Ding et al., 2005; Pokreisz et al., 2009; Takimoto et al., 2005) and

enhanced cardiac and valvular remodelling mediated by transforming growth factor beta and

various matrix metalloproteinases (Aupperle & Disatian, 2012; Dobaczewski et al., 2011;

Khan & Sheppard, 2006; Moesgaard et al., 2014; Orton et al., 2012). Finally, enhancement

13

of inflammatory pathways has also been identified as a contributor in some studies (Anker &

von Haehling, 2004; Cesari et al., 2003; Mann & Young, 1994; Reimann et al., 2016; Zois et

al., 2012).

1.4) Comparative pathophysiology of CHF

Despite the many shared mechanisms between various aetiologies that lead to CHF, there

are also differences in pathophysiology between these aetiologies which are particularly

important to consider in the context of therapeutics. For example, systolic dysfunction is an

important contributor towards CHF in certain aetiologies such as DCM; in contrast, diastolic

dysfunction predominates in others such as HCM and RCM. CHF resulting primarily from

systolic dysfunction is characterised by reduced myocardial contractility. Governed by the

Frank-Starling mechanism, the ventricles undergo eccentric hypertrophy to accommodate for

the increase in volume required to maintain CO. Maximising CO in this context relies on

reducing afterload, improving myocardial contractility, and preventing excessively high

preload (Zile & Brutsaert, 2002). On the other hand, CHF resulting primarily from diastolic

dysfunction is characterised by impaired myocardial relaxation and an increase in filling

pressure. Due to the reduced capacity for passive ventricular filling, increasing atrial function

and reducing heart rate to allow for prolonged filling time become more important in

maintaining CO in this context (Zile & Brutsaert, 2002). Although both systolic and diastolic

dysfunction can be present in many conditions that result in CHF, identifying the

predominant form of myocardial dysfunction can therefore guide optimal therapeutic

approaches.

There are also a number of associated abnormalities which are specific to certain

aetiologies or species that can accelerate the onset of CHF or contribute towards refractory

disease. Atrial fibrillation, supraventricular tachyarrhythmias, and ventricular

tachyarrhythmias have been described in dogs with DCM (M. W. S. Martin et al., 2009;

Monnet et al., 1995; Wess et al., 2010), ARVC (Meurs et al., 2014; Palermo et al., 2011),

and MMVD (Crosara et al., 2010). Acute chordal rupture occurs in around 16% of dogs with

14

MMVD (Serres, Chetboul, Tissier, et al., 2007), and tearing of the atrial septum or caudal

wall may also develop with this disease (Buchanan & Kelly, 1964; Peddle & Buchanan,

2010; Reineke et al., 2008); however, chordal rupture and atrial tears have not been

reported in the literature for common canine or feline cardiomyopathies. Disproportionate

pulmonary hypertension is also a relatively common complication of MMVD that is seen in

up to 40% of affected dogs, and it is thought to occur as a result of increased pulmonary

venous pressures, combined with various neuroendocrine pathways and endothelial

dysfunction as previously described (Borgarelli et al., 2015; Kellihan & Stepien, 2012).

Cardiomyopathies or myocarditis are less commonly associated with secondary pulmonary

hypertension in dogs (Johnson et al., 1999; Serres, Chetboul, Gouni, et al., 2007). Although

some clinicians have speculated upon the possibility of severe left atrial dysfunction and

subsequent pulmonary hypertension contributing towards pleural effusion in cats with CHF

(Johns et al., 2012), the significance and prevalence of pulmonary hypertension in feline

cardiomyopathies have not yet been characterised. Finally, thromboembolic disease is a

unique feature of feline, but not canine, cardiomyopathies, with caudal aortic

thromboembolism (ATE) being the most common location and a well-recognised component

of HCM (Fox et al., 2018; Rush et al., 2002; Trehiou-Sechi et al., 2012) and other

cardiomyopathies (S. A. Smith et al., 2003). These comorbidities cause an increase in

preload, increase in afterload, reduced myocardial contractility, or a combination of these,

and thus, negatively impact both individuals with CHF and those at risk of developing it.

1.5) Classifications of CHF

There are various methods of defining and describing severity of CHF, with some differences

existing between aetiologies and species. These definitions and methods of categorising

severity of symptoms play an important role in guiding diagnosis and treatment

recommendations.

In humans, several established methods of CHF classifications exist. The most

commonly utilised classification scheme to describe various functional stages of CHF in

15

people is the New York Heart Association (NYHA) classification scheme (Table 1.5a). More

recently, the American College of Cardiology/American Heart Association (ACC/AHA)

created guidelines for staging disease severity which also include presence or absence of

structural abnormalities (Table 1.5b). Treatment guidelines for CHF are also governed by

the presence or absence of a reduction in ejection fraction (EF). Those with CHF with an EF

of ≥ 50% are said to have heart failure with preserved ejection fraction (HFpEF), whereas

those with an EF of < 40% are said to have heart failure with reduced ejection fraction

(HFrEF); those with an EF between 40 – 49% fall in between these categories and are said

to have heart failure with mid-range ejection fraction (HFmrEF) (Ponikowski et al., 2016). It is

important to note that EF is used as a measure of ventricular function and does not refer to

specific aetiologies or types of myocardial dysfunction. However, in general, HFpEF tends to

accompany disorders resulting in either a normal or concentric hypertrophy of the left

ventricle, with or without left atrial enlargement; therefore, it may occur more commonly in

diseases resulting in primary diastolic dysfunction (Ponikowski et al., 2016). In contrast,

HFrEF often reflects the predominance of systolic dysfunction.

16

Table 1.5a: New York Heart Association (NYHA) classification of heart failure based

on severity of symptoms in people [adapted from Web Table 3.2, (Ponikowski et al.,

2016)].

NYHA Class Description

Class I No limitation of physical activity. Ordinary physical activity does

not cause undue breathlessness, fatigue, or palpitations.

Class II Slight limitation of physical activity. Comfortable at rest, but

ordinary physical activity results in undue breathlessness,

fatigue, or palpitations.

Class III Marked limitation of physical activity. Comfortable at rest, but

less than ordinary physical activity results in undue

breathlessness, fatigue, or palpitations.

Class IV Unable to carry on any physical activity without discomfort.

Symptoms at rest can be present. If any physical activity is

undertaken, discomfort is increased.

Table 1.5b: American College of Cardiology/American Heart Association (ACC/AHA)

classification of heart failure based on symptoms and structural diseases [adapted

from Web Table 3.3, (Ponikowski et al., 2016)].

ACC/AHA Stage Description

A At high risk for heart failure but without structural heart disease or

symptoms of heart failure.

B Structural heart disease but without signs or symptoms of heart

failure.

C Structural disease with prior or current symptoms of heart failure.

D Refractory heart failure requiring specialised interventions.

17

In the world of veterinary cardiology, CHF classifications and guidelines for diagnosis

are somewhat less structured and varies based on the aetiology and species. In dogs, the

two largest classification schemes utilised include the American College of Veterinary

Internal Medicine (ACVIM) classification scheme for MMVD (Table 1.5c) and the

International Small Animal Cardiac Health Council (ISACHC) scheme (Table 1.5d).

Importantly, the definition of presence or absence of CHF differs between the two, with the

ACVIM scheme necessitating radiographic evidence of pulmonary oedema in order to

differentiate between CHF and primary respiratory causes of such symptoms (Atkins et al.,

2009; International Small Animal Cardiac Health Council (ISACHC), 1999). As such, it is

considered a more stringent classification scheme than the ISACHC scheme. These

classification systems are not commonly utilised in dogs with diseases other than MMVD or

for cats. Dogs with DCM, for example, are typically classified into occult or symptomatic

DCM based on the presence or absence of symptoms and a point-based system of

diagnostic testing (Dukes-McEwan et al., 2003). In cats with HCM, there are no formal

definitions of disease severity; mild, moderate, and severe disease is defined based on an

individual clinician’s preference regarding left ventricular thickness and left atrial size

(MacDonald, 2010). CHF is defined as the point at which pulmonary oedema or body cavity

effusions become appreciable in the presence of left ventricular and atrial enlargements.

Contrary to the situation in humans, where EF is used for treatment and prognostic purposes,

EF is uncommonly used in veterinary cardiology.

18

Table 1.5c: The American College of Veterinary Internal Medicine (ACVIM)

classification scheme for congestive heart failure (CHF) due to myxomatous mitral

valve disease (MMVD) in dogs [adapted from (Atkins et al., 2009)).

ACVIM Stage Description

A Patients at risk for developing heart disease with no current

structural abnormalities.

B Patients with structural heart disease but without clinical signs of

CHF.

Substage B1 Asymptomatic patients without radiographic or echocardiographic

evidence of cardiac remodelling in response to MMVD.

Substage B2 Asymptomatic patients with radiographic or echocardiographic

evidence of left heart enlargement (atrial and ventricular) in

response to MMVD.

C Patients with past or current clinical signs of CHF associated with

structural heart disease. Radiographic evidence of CHF.

D Patients with end-stage disease with clinical signs of CHF due to

MMVD that are refractory to standard therapy and that require

advanced or specialised treatments to maintain comfort.

19

Table 1.5d: The International Small Animal Cardiac Health Council (ISACHC)

classification scheme for congestive heart failure (CHF) in dogs [adapted from

(International Small Animal Cardiac Health Council (ISACHC), 1999)].

ISACHC Class Description

IA Patients with heart disease without clinical signs or cardiac

remodelling.

IB Patients with heart disease without clinical signs but with

radiographic or echocardiographic evidence of cardiac

remodelling (e.g. left ventricular or atrial enlargement)

II Patients with mild to moderate CHF. Clinical signs (such as

exercise intolerance, cough, tachypnoea, mild respiratory

distress, and ascites) present at rest or mild exercise but without

significant effect on the quality of life.

No evidence of hypo-perfusion at rest.

IIIA Patients with advanced CHF in which home treatment is

possible. Clinical signs (such as dyspnoea, marked ascites,

profound exercise intolerance, hypo-perfusion at rest,

cardiogenic shock) are obvious. Death or severe debilitation is

likely without treatment.

IIIB Patients with advanced CHF which require hospitalisation for

treatment. Clinical signs (such as dyspnoea, marked ascites,

profound exercise intolerance, hypo-perfusion at rest,

cardiogenic shock) are obvious. Death or severe debilitation is

likely without treatment.

20

These differences in definition and grading schemes of CHF across aetiologies and

between species make it difficult to extrapolate and compare findings of various studies. For

example, a human study evaluating treatment effects in people with HFpEF may include a

large number of people with systemic hypertension; thus, the assumption that these study

results could be applied to dogs with MMVD or cats with primary HCM should not be made.

Treatment recommendations in people with mitral valve prolapse may not align with those

recommended in dogs with MMVD due to the different cut-off points used in people (e.g. use

of NYHA class, EF) compared to dogs (e.g. use of ACVIM staging). Careful inspection of the

inclusion criteria, including disease aetiology and description of disease severity, is essential

when attempting such extrapolations.

1.6) Treatment of CHF

The underlying disease process and various pathways involved in the development of CHF

provide potential therapeutic targets for this syndrome. Some of these have been

investigated more rigorously than others, but the evidence provided by existing research

efforts, as well as cross-species extrapolation of these concepts, have led to a multi-modal

approach to treatment of CHF in veterinary patients. With MMVD and HCM/HOCM being the

most common forms of heart disease leading to CHF in dogs and cats, respectively, the

proceeding discussion will focus on treatment of CHF resulting from these. For comparative

purposes, and to present avenues for future investigation, treatment approaches in human

medicine have also been reviewed.

1.6.1) Medical therapy of canine CHF due to MMVD

The therapeutic approach to CHF in canine patients with MMVD has been reasonably well-

described, with a moderate to large supply of evidence to support clinical practices. These

have formed the basis of guidelines for the treatment of this condition, which were published

by the ACVIM (Atkins et al., 2009). Essential components of treatment include the use of

loop diuretics to reduce preload, RAAS modulation through angiotensin converting enzyme

21

(ACE) inhibition with or without a mineralocorticoid receptor antagonist (MRA), and an

inodilator to provide positive inotropic and vasodilatory support. Yet certain knowledge gaps

still exist, as highlighted below.

1.6.1.1) Loop diuretics

Loop diuretics form the cornerstone of CHF therapeutics, and in human medicine, a meta-

analysis of a few small, randomised, controlled trials supported the role of loop and thiazide

diuretics in reducing CHF-related morbidity and mortality (Faris et al., 2002). In contrast,

controlled clinical studies in veterinary patients with CHF do not exist. This is an important

consideration as the use of loop diuretics is not entirely benign. Frusemide is the most widely

used diuretic within this class, though increasing use of torsemide has emerged in recent

years (Chetboul et al., 2017). Frusemide acts through inhibition of sodium reabsorption via

the sodium-potassium-chloride cotransporter in the ascending limb of the loop of Henle, and

as such, its use can result in side effects including hypokalaemia and hypochloraemic

metabolic alkalosis (Plumb, 2015a). It can also unmask or exacerbate renal disease in those

with suboptimal kidney function. In humans with chronic CHF, initiation of frusemide caused

detrimental short-term hemodynamic effects such as a rise in ventricular filling pressure,

increase in SVR, and increase in heart rate accompanied by activation of both RAAS and

SNS (Francis et al., 1985). High doses of frusemide were also associated with increased

mortality in several clinical trials (Hasselblad et al., 2007; Peacock et al., 2009). This last

statement is confounded by the fact that greater disease severity may have necessitated use

of higher doses of frusemide; nevertheless, the range of deleterious side effects associated

with the use of loop diuretics should not be ignored.

One method of reducing the negative impact of diuretics may be through therapeutic

monitoring and goal-oriented approaches to diuretic therapy. A clinical measure that may be

useful in guiding treatment with diuretics in dogs is the sleeping or resting respiratory rate.

Indeed, there is data identifying the upper limit of normal respiratory rates in dogs with

subclinical and medically controlled CHF (Ohad et al., 2013; Porciello et al., 2016; Schober

22

et al., 2011); however, the benefits of using respiratory rates to guide the amount of long-

term diuretic use in these patients has not yet been formally evaluated.

1.6.1.2) RAAS antagonism

The use of ACE inhibitors (ACE-I) in chronic management of CHF due to MMVD has been

established through several prospective, controlled clinical studies in dogs with MMVD and

DCM which observed haemodynamic, clinical, and survival benefits of treatment (Ettinger et

al., 1998; Sisson, 1995; The BENCH Study Group, 1999; Woodfield, 1995). This evidence,

together with the wealth of information supporting its benefits in the treatment of CHF in

humans (Ponikowski et al., 2016; Yancy et al., 2017) and its theoretical role in

neuroendocrine modulation led to its inclusion in the routine therapy of CHF due to MMVD in

dogs. Yet it is interesting to note that there are no published studies in the veterinary

literature which have reported successful attenuation of angiotensin II, aldosterone, or their

effects in dogs with naturally occurring CHF treated with ACE-I. In fact, a recent study in the

target population observed no effects of short-term benazepril therapy on these endocrine or

cardiovascular variables (Häggström, Lord, et al., 2013). Other studies in normal dogs

demonstrated that ACE-I therapy did not attenuate diuretic-induced activation of RAAS (M. K.

Ames et al., 2015; Lantis, Ames, Werre, et al., 2015), thereby raising concerns about their

effectiveness for this purpose in dogs with CHF. These findings are in contrast to the clinical

benefits observed in the aforementioned larger trials examining the use of ACE-I in dogs

with CHF and bring to question the true role of ACE-I therapy.

This is an interesting point to contemplate, as there are reasons why ACE-I may not

provide adequate RAAS suppression in dogs with CHF. ACE-independent pathways for

angiotensin conversion exist, with tissue chymase-driven conversion being the dominant

alternate pathway in several species including the dog (Dell'Italia et al., 1995; Paul et al.,

2006). In addition, loss of ACE-I activity through the aldosterone escape mechanism has

been observed with chronic ACE-I therapy in humans (Struthers, 2004). In normal dogs

undergoing frusemide or amlodipine-induced RAAS activation, a more immediate form of

23

aldosterone breakthrough has been reported (M. K. Ames et al., 2016; Lantis, Ames, Atkins,

et al., 2015), and aldosterone breakthrough has been demonstrated in around 30% of dogs

with and without CHF being treated with an ACE-I over a longer period (M. K. Ames et al.,

2017). The small numbers of dogs included in the study; the variable length, doses, and

types of ACE-I therapy; and lack of strict definitions for aldosterone breakthrough in

veterinary medicine are some of the limitations associated with this latter study, but these

findings nevertheless raise questions regarding the efficacy of ACE-I in dogs with CHF.

These concerns regarding ACE-I led to the investigation of MRA as an additional

method of overcoming RAAS effects in people with CHF. In humans with HFrEF, several

large, double-blinded, randomised controlled trials have been conducted in which the

addition of MRA to standard therapy resulted in prolonged survival and reduced morbidity

(Pitt et al., 2003; Pitt et al., 1999; Zannad et al., 2011). The reason for these benefits

appears to be related to its anti-fibrotic effects (Deswal et al., 2011; Zannad et al., 2000).

However, similar survival benefits have yet to be proven in dogs with CHF due to MMVD. In

one double-blinded, randomised controlled trial of dogs with MMVD and varying degrees of

CHF, ~ 55% reduction in risk to combined cardiac death, euthanasia, or severe progression

of disease was observed in the spironolactone treated group compared to placebo (Bernay

et al., 2010). Unfortunately, only 18% of dogs in this study reached this endpoint. In another

smaller study of dogs with MMVD and DCM, addition of spironolactone to standard therapy

reduced the risk of progression of disease but did not result in improved survival, circulating

endocrine markers, or hemodynamic measures over the six month duration of treatment

(Schuller et al., 2011). However, this study was limited by the short follow-up duration and

very small numbers of dogs. Currently, there is also a lack of information to support

successful attenuation of cardiac remodelling in dogs with CHF due to MMVD treated with

MRA. Thus, despite the growing popularity of MRA as a potassium-sparing adjunctive

diuretic and RAAS-modulating drug, whether they offer a true benefit in canine CHF remains

ambiguous.

24

The presence of conflicting evidence surrounding ACE-I and MRA in dogs with CHF

due to MMVD raises questions regarding the role of the RAAS in the development and

propagation of CHF in these dogs. Indeed, RAAS activation here is relatively ill-defined

compared to the situation in dogs and people with cardiomyopathies resulting in significant

systolic dysfunction (Dzau et al., 1981; Koch et al., 1995; Tidholm et al., 2001). In

asymptomatic dogs with MMVD, the RAAS may (H. D. Pedersen et al., 1995) or may not

(Häggström et al., 1997) be activated, and valvular insufficiency may cause differential

activation of circulating and tissue RAAS (Fujii et al., 2007). Additionally, RAAS activation

may be pulsatile, increasing only around times of rapid ventricular remodelling (Hezzell,

Boswood, Chang, Moonarmart, & Elliott, 2012). In contrast to findings of one retrospective

study in Doberman Pinschers with DCM (O'Grady et al., 2009), where benefits of ACE-I in

delaying the onset of CHF were suggested, ACE-I have not been proven to attenuate the

progression to CHF in neither humans with valvular diseases nor in dogs with MMVD, and

are therefore not routinely recommended for use in preclinical disease (Atkins et al., 2007;

Baumgartner et al., 2017; Kvart et al., 2002). One retrospective study observed longer

survival in dogs treated with benazepril in the pre-clinical stage of MMVD compared with

those who were not, but this difference was less likely to be related to cardiac benefits of

ACE-I as the causes of death were mainly non-cardiac in nature, and there was no

difference in time to CHF or cardiac-related deaths between the groups (Pouchelon et al.,

2008). Interestingly, there is also the possibility that some of the aforementioned studies

presenting haemodynamic and survival benefits of ACE-I in dogs with CHF may have been

influenced significantly by the inclusion of a large portion of dogs with DCM (Sisson, 1995;

Woodfield, 1995). Compared to dogs with MMVD, those with DCM treated with ACE-I were

observed to have greater improvements in overall clinical evaluation (Sisson, 1995) and

improvements in a larger number of haemodynamic and clinical variables evaluated

(Woodfield, 1995). Despite this, fewer dogs with MMVD experienced progressive disease on

benazepril treatment in one large, multi-centre, double-blinded, randomised controlled study

(The BENCH Study Group, 1999), and this finding was supported by the prolonged time to

25

treatment failure in MMVD dogs on enlaparil in another long-term study (Ettinger et al., 1998)

Ultimately, these existing studies evaluating the role of RAAS in the development and

propagation of CHF due to MMVD are few in number and limited by small sample sizes in

some cases; as such, it is impossible to reach compelling conclusions regarding this matter.

As can be appreciated, the history behind ACE-I and MRA in the treatment of CHF

due to MMVD is rather convoluted. The role of the RAAS, as well as ACE-I therapy and any

alternate or additional methods targeting the RAAS, in the development and treatment of

CHF due to MMVD, is an area requiring further investigation.

1.6.1.3) Inodilators

Much like with diuretics and ACE-I, inodilators were initially used in people prior to their rise

in veterinary medicine. Yet in contrast to the success of ACE-I, inodilators experienced a

spectacular course of demise in human medicine.

Pimobendan is a benzimidazole-pyridazinone derivative that produces both positive

inotropic and vasodilatory effects via selective phosphodiesterase III inhibition and calcium

sensitisation (Brunkhorst et al., 1989; C. H. Chen et al., 1997; Fujimoto, 1994; Fujimoto &

Matsuda, 1990; Fujino et al., 1988b; Rüegg et al., 1984; Solaro et al., 1989). Following

administration, it is metabolized to an active metabolite (UD-CG 212 CL) via oxidative

demethylation (O-demethylation) in the liver (Hagemeijer, 1993; Pahernik et al., 1995). In

humans with HFrEF, pimobendan caused an acute increase in EF, cardiac index and output,

stroke volume (SV) and SV index, and rate of rise in maximal left ventricular pressure

(LVmax dP/dt); in addition, a decrease in right atrial and pulmonary pressures, systemic

vascular resistance (SVR), peripheral vascular resistance, systemic blood pressure (BP),

and end systolic and diastolic volumes were also observed (Baumann et al., 1989;

Hagemeijer, Brand, & Roth, 1989; Hagemeijer, Brand, & Van Mechelen, 1989; Hasenfuss et

al., 1989; Katz et al., 1992; Permanetter et al., 1989; Pouleur et al., 1989; Remme et al.,

1989; Renard et al., 1988; Walter et al., 1988). Despite this, it failed to demonstrate any

significant longer-term benefits in haemodynamic, quality of life, or survival measures across

26

three major randomised, double-blinded, and placebo-controlled and clinical trials (Kubo et

al., 1992; Lubsen et al., 1996; The EPOCH Study Group, 2002). This, along with the

unfortunate discovery of adverse effects associated with other positive inotropes which were

being investigated at the time, led to it disappearing from all but a small Asian market of

cardiovascular pharmaceuticals across the world.

Yet in the early 2000’s, when pimobendan was introduced to the world of veterinary

cardiology, convincing evidence in favour of its use emerged. The largest and most

compelling study examining its use in dogs with CHF due to MMVD was the “QUEST” study

– a multi-centre, single-blinded, randomised study in 260 dogs (Häggström et al., 2008). In

contrast to the long-term outcome in humans, a definitive benefit of pimobendan over

benazepril was highlighted in the significantly longer time to reach the combined endpoint of

cardiovascular death/euthanasia or treatment failure in pimobendan-treated dogs (median

267 days for pimobendan vs 140 days for benazepril). Two smaller studies examining long-

term effects of pimobendan compared with an ACE-I in dogs with CHF due to MMVD also

reported similar benefits. Conclusions from both studies favoured treatment with

pimobendan, with one study showing a 25% reduction in likelihood of an adverse outcome

due to CHF in the pimobendan group (P. J. Smith et al., 2005) while the other observed a

significantly longer MST (430 days, compared with 228 days in those treated with an ACE-I)

in dogs treated with pimobendan (Lombard et al., 2006).

The positive outcomes observed in clinical trials were supported by findings of

beneficial haemodynamic changes induced by pimobendan in dogs with experimental or

naturally occurring mitral valve insufficiency. Kanno et al., (2007) identified significant

reductions in left ventricular internal diameter in systole (LVIDs), left ventricular internal

diameter in diastole (LVIDd), left ventricular end systolic volume (ESV), left ventricular end

diastolic volume (EDV), and regurgitant stroke volume in four dogs with experimentally

induced mitral valve insufficiency over a four-week period of time. Using another

experimental model of mitral regurgitation, Suzuki et al. (2011) also observed a reduction in

left atrial pressure and mitral regurgitation indices after seven days of pimobendan treatment.

27

An increase in fractional shortening (FS) was also found after the same period of treatment

in another study on 20 dogs with MMVD (Caro et al., 2009). In dogs with MMVD and

pulmonary hypertension, a chronic reduction in LVIDs and increase in left ventricular

shortening area were also documented (Atkinson et al., 2009). These studies confirmed a

lack of significant changes to heart rate (HR) and BP in dogs treated with pimobendan.

Together, these experimental and clinical studies highlighted the benefits of

pimobendan in dogs with CHF due to MMVD. Moreover, the larger clinical studies

specifically demonstrated the superiority of pimobendan over use of an ACE-I. To add to this

pool of evidence, a recent multi-centre, placebo-controlled, double-blinded, randomised

clinical trial of 360 dogs with advanced pre-clinical MMVD demonstrated a significant delay

in the onset of either CHF or cardiac-related death/euthanasia when treated with

pimobendan (Boswood et al., 2016). This study, known as the “EPIC” study, therefore raised

the possibility that pimobendan may modulate the pathophysiology leading to CHF in dogs

with MMVD. This is in contrast to the situation with ACE-I, where a clear benefit of treatment

in the preclinical phase could not be demonstrated. It is interesting to note that despite this

pool of evidence to support the advantages of pimobendan over ACE-I, both drugs are

recommended in the gold standard of treatment in dogs with MMVD. Although a large,

single-blinded, randomised study investigating the benefits of adding an ACE-I to

pimobendan and frusemide was commenced in 2005, results have not yet been published

(Wess, 2017); as such, it is not yet known whether such combination therapy would offer

additional advantages.

Pimobendan has also been used in clinical practice at a higher frequency (three

times daily at 0.2 – 0.3 mg/kg) than the labelled twice daily dosing regimen. This practice is

typically reserved for dogs with refractory CHF (Atkins et al., 2009) but may also be utilised

in cases of concurrent renal insufficiency (to optimise haemodynamics in the face of diuretic

intolerance) or severe airway disease (to reduce compression of mainstem bronchi). The

reported pharmacokinetic properties of this drug include rapid absorption and elimination,

with achievement of maximal plasma concentrations at two and three hours following oral

28

dosing, and elimination half-life of 0.5 and two hours, for pimobendan and its active

metabolite, respectively (Plumb, 2015b). Its duration of action also appears to be long, with

LVmax dP/dt remaining elevated for over eight hours following oral dosing at 0.5 mg/kg (Van

Meel & Diederen, 1989). Based on this information, it would appear that the off-label dosage

regimen may provide effective, or potentially better, inodilation. However, information

regarding effectiveness of this regimen is lacking.

1.6.2) Medical therapy of feline CHF

In contrast to the situation in canine medicine, where a body of evidence exists to support

practices undertaken in the therapy of CHF, there is a distinct lack of research into feline

CHF therapeutics. This is likely due to their small body size and blood volume, tendency to

display resistance towards training and unsolicited handling, and the historically small size of

the feline market (Downes et al., 2009; Slater et al., 2008; Toribio et al., 2009). Nevertheless,

current practices in feline medicine have been guided by the few published literature, expert

opinions, personal experience, and theoretical benefits (Rishniw & Pion, 2011). Key

components here include the use of loop diuretics to reduce volume overload, RAAS

modulation through ACE inhibition, and an antiplatelet agent to reduce the risk of

thromboembolic disease. Most recently, the use of inodilators in this population has been

debated.

1.6.2.1) Loop diuretics

Much like the situation in dogs, diuretics form an integral component of CHF therapeutics in

cats despite the lack of randomised, controlled trials to support survival and quality of life

benefits. In cats, there is, perhaps, an even greater need for more robust guidelines on

frusemide use as there is a widespread belief amongst clinicians that cats have a tendency

to be more “sensitive” to the diuretic effects of frusemide. This is partially, but not entirely,

supported by an experimental study in which a difference in diuretic and natriuretic effects of

frusemide between dogs and cats was suggested (Klatt et al., 1975); in addition, cats suffer

29

from concomitant chronic kidney disease more commonly than dogs, and they appear to be

more particular in their feeding and drinking habits – thus making them prone to dehydration.

As such, lower doses of diuretics are recommended for use in this species (Barrett, 2010;

Plumb, 2015a); however, a goal-oriented approach would, of course, be ideal. Sleeping or

resting respiratory rates may be a useful tool, but further evaluation of its use in this setting

is required (Ljungvall et al., 2014; Porciello et al., 2016).

1.6.2.2) ACE inhibitors

The lack of prospective clinical trials to investigate the use of frusemide in cats with CHF is

not surprising. What is surprising, however, is the similar lack of prospective clinical trials

investigating the use of ACE-I in this context. One retrospective study described small

reductions in left ventricular wall thickness and left atrial sizes when enalapril was used with

or without other medications in 19 cats with clinical and subclinical HCM; however, the effect

of treatment on survival or long-term outcome measures could not be identified due to the

variable treatment regimen utilised in each case and the lack of a control group (Rush et al.,

1998). Three small prospective studies evaluated the use of ACE-I in cats with HCM with

and without CHF, with no obvious benefit of ACE-I therapy compared to an active control or

placebo (MacDonald et al., 2006; Taillefer & Di Fruscia, 2006) except for a small reduction in

left ventricular free wall thickness in one study (Amberger et al., 1999). Preliminary results of

one study evaluating long-term outcomes of ACE-I in cats with CHF due to HCM, HOCM,

UCM, or RCM suggested a shorter period of time to hospitalisation for recurrent CHF in cats

treated with ACE-I and frusemide when compared with frusemide alone, and addition of an

ACE-I did not affect overall mortality rates (Fox, 2003b). Unfortunately, this study was never

published, and as such, effects of ACE-I on survival or long-term outcomes remain ill-defined.

Despite this, its use in cats with CHF has been advocated by experts based on

extrapolations from human and canine literature and as an attempt to attenuate RAAS

activation secondary to frusemide therapy (Gordon & Côté, 2015).

30

1.6.2.3) Antithrombotic agents

Depending on the population studied, roughly 4 – 17% of cats with HCM may present with

ATE (Fox et al., 2018; Rush et al., 2002; Trehiou-Sechi et al., 2012). This is thought to occur

in part, but not entirely, due to the presence of hypercoagulability (Stokol et al., 2008).

Although ATE can occur in the absence of CHF, it is a significant cause of death or

euthanasia, with reported MST in those surviving the acute period of hospitalisation ranging

from 94 to around 345 days (Borgeat, Wright, et al., 2014; Hogan et al., 2015; Laste &

Harpster, 1995; Schoeman, 1999; S. A. Smith et al., 2003). Recurrent thrombosis is

common in those surviving the initial insult, and a handful of studies have evaluated the

effects of aspirin and clopidogrel as a means of reducing recurrence or prolonging the time

to recurrence of ATE (Hogan et al., 2015; Schoeman, 1999; S. A. Smith et al., 2003). To

date, the most compelling evidence exists for clopidogrel, which was shown to reduce the

recurrence of ATE and double the time to recurrence of ATE in a randomised, multi-centre,

double-blinded clinical trial utilising aspirin as an active control (Hogan et al., 2015). Yet

there is no solid evidence to guide the criteria for initiation of antithrombotic medications prior

to the development of a thromboembolic episode. Due to the high morbidity and mortality

associated with this disease, many clinicians advocate the use of antithrombotic medications

in the presence of severe left atrial enlargement, spontaneous echocardiographic contrast,

or an intra-atrial thrombus (Ferasin, 2009; Hogan, 2017).

1.6.2.4) Inodilators

The strong evidence to support pimobendan use in dogs with CHF led to investigations into

its off-label use in cats with CHF. To date, there have been no prospective studies

evaluating the use of pimobendan in this population; however, a number of retrospective

studies have been conducted (Gordon et al., 2012; Hambrook & Bennett, 2012; MacGregor

et al., 2011; Reina-Doreste et al., 2014). Pimobendan appears to be well-tolerated, with only

a single report of significant adverse effects being reported in a cat with mitral valve

dysplasia, systolic anterior motion, and a ventral septal defect (Gordon et al., 2012). The

31

majority of these studies examined its use in cats with systolic dysfunction, with one case-

control study reporting around a four-fold increase in MST in cats with CHF due to DCM

treated with pimobendan compared to a group of controls receiving either digoxin or no

positive inotropes (49 days with pimobendan versus 12 days in the controls) (Hambrook &

Bennett, 2012). This difference just reached significance (p = 0.048), but pimobendan

treatment was not identified as a significant prognostic indicator. Another recent case-control

study also observed a longer MST in cats with CHF due to HCM and HOCM without systolic

dysfunction when pimobendan was used in addition to standard therapy (626 days with

pimobendan versus 103 days in the controls, p = 0.024) (Reina-Doreste et al., 2014).

Despite suggestions that pimobendan may offer survival benefits in cats with CHF, these

findings were clouded by the relatively small sample size, changes in medical practices

during the ~ 10 year period from which the data were collected, and the lack of control over

various aspects of medical management due to the retrospective design.

Thus, prospective studies are still required to elucidate the role of pimobendan in

feline CHF and to identify the population that truly benefits from treatment. For example, its

effect in cats with HOCM remains controversial. In theory, presence of an outflow tract

obstruction is a contraindication for treatment with inodilators; although some studies have

commented on successful use of pimobendan in cats with HOCM (MacGregor et al., 2011;

Reina-Doreste et al., 2014), negative effects have been observed in cats with outflow tract

obstructions (Gordon et al., 2012), and the small numbers of cats in the existing studies

prohibits any conclusions to be drawn. In addition, the two aforementioned case-control

studies observed a relatively high drop-out rate of ~ 30 – 40% in both cases and controls in

the first part of the survival curve (equivalent to ~ 25 days in the study by Hambrook and

Bennett (2012)). Thus, any benefit of pimobendan treatment may only be in individuals who

survive this initial period of disease. There may also be a portion of “non-responders” who

experience an early demise. In the Hambrook and Bennett (2012) study, more cats treated

with pimobendan died of sudden death (n = 7) compared to controls (n = 4). The incidence

of sudden death was not clarified in the study by Reina-Doreste et al. (2014). With some

32

evidence to suggest inter-species differences in pharmacokinetics of the drug between dogs

and cats (Hanzlicek et al., 2012), further evaluation of optimal dosing regimen and potential

differences in cardiovascular effects between species is warranted.

1.7) Medical therapy of CHF in humans

Human medicine has influenced aspects of veterinary medicine throughout history, and the

treatment of CHF has not been an exception. As such, a review of current medical practices

is essential to provide additional evidence for or against current practices in veterinary

medicine. In addition, these may provide inspiration for prospective novel therapeutics in

veterinary patients.

The prevalence of CHF amongst people world-wide is estimated to be around

1 – 2% (Ponikowski et al., 2016). The often poor prognosis associated with it is well-

recognised amongst medical professionals, with a one year mortality rate for those requiring

hospitalisation reaching around 17% (Ponikowski et al., 2016). Those suffering from HFrEF

due to dilated and ischemic cardiomyopathies have a worse prognosis than those with

HFpEF, and this has driven numerous investigations into novel approaches to the medical

management of CHF arising from these aetiologies. Thus, it is interesting to note that even

in humans, there is little evidence to support medical therapy of CHF in those with diseases

encountered in our veterinary patients such as mitral valve disease and HCM; in fact, even

the use of treatments such as ACE-I and diuretics for CHF secondary to these diseases is

not well-supported, with the level of evidence comprising primarily of expert opinions and

extrapolations from studies in people with dilated and ischemic cardiomyopathies

(Baumgartner et al., 2017; Elliott et al., 2014). Due to the superiority of surgery in most

situations, medical therapy consisting of ACE-I with or without beta-blockers and MRA is

only recommended in people with symptomatic mitral valve disease with an EF of < 30%,

and there are no recommendations on the use of positive inotropes (Baumgartner et al.,

2017). The diastolic dysfunction and the arrhythmogenic tendencies associated with HCM

and HOCM in people have led to beta blockers, calcium channel blockers, and anti-

33

arrhythmics forming the cornerstone of therapy (Elliott et al., 2014; Gersh et al., 2011).

ACE-I are only recommended in people with symptomatic HCM if the ejection fraction is

< 50%; interestingly, the use of diuretics and ACE-I in people with HOCM appears

controversial, with recommendations to avoid these or to use with caution due to potential

exacerbation of the obstructive pathology (Elliott et al., 2014; Gersh et al., 2011). Here, the

use of positive inotropes is absolutely contraindicated, even in the presence of hypotension

and pulmonary oedema (Elliott et al., 2014; Gersh et al., 2011).

The cornerstone of medical therapy in people with symptomatic HFrEF involves the

use of an ACE-I, beta-blocker, and a MRA (Ponikowski et al., 2016; Yancy et al., 2017).

A significant body of evidence in the form of large randomised, controlled, clinical trials exist

to support their survival and quality of life benefits in people with HFrEF. Some of these also

advocate the use of combination therapy. For example, a reduced risk of sudden death and

death due to CHF was observed when beta-blockers and MRAs were used with ACE-I

(Packer et al., 2001; Pitt et al., 1999; The MERIT-HF Study Group, 1999; Zannad et al.,

2011). Other treatments that are recommended for use in certain people include diuretics in

those who require this to control symptoms, along with angiotensin receptor type-I

antagonists in those suffering from side effects of ACE-I therapy.

Most recently, the combined angiotensin receptor neprilysin inhibitor (ARNI) has

been added to these guidelines as a replacement for ACE-I (Ponikowski et al., 2016; Yancy

et al., 2017). This class of medication combines an angiotensin receptor type-I antagonist

with a neprilysin inhibitor. Neprilysin is a metalloproteinase that is involved in the breakdown

of numerous peptides in the body, including those involved in cardiovascular homeostasis

such as natriuretic peptides and adrenomedullin (McMurray et al., 2014). This combination

of drugs therefore results in RAAS antagonism and enhancement of the NPS. In a double-

blinded, randomised, controlled clinical trial of 8442 people with HFrEF, treatment with ARNI

resulted in ~20% reduction in risk of reaching the combined primary outcome of

cardiovascular death and rehospitalisation due to CHF compared to ACE-I therapy

(McMurray et al., 2014). ARNI was also associated with a lower rate of all-cause mortality

34

and increased functional capacity. The benefits of neprilysin inhibitors on cardiovascular and

renal systems have also been documented in pacing-induced models of CHF in dogs, with

the effect of combined neprilysin and RAAS inhibition providing superior increase in CO and

SV, reduction in mean BP (MBP) and SVR, and renal function (H. H. Chen et al., 1999;

Margulies et al., 1991; F. L. Martin et al., 2005; Seymour et al., 1993). In addition, ARNI

reduced myocardial cell hypertrophy and fibrosis in rats with myocardial infarction (von

Lueder et al., 2014). This approach of combined NPS augmentation and RAAS inhibition

therefore appears to provide superior treatment of CHF than with RAAS inhibition alone and

highlights the significance of the NPS in the pathophysiology of CHF in those with systolic

dysfunction.

The success of ARNI in people with HFrEF supports the theory of relative NP

deficiency in states of CHF, which was previously discussed in section 1.3. Yet interestingly,

NPS augmentation via administration of exogenous NP was not effective in people with CHF.

Much like the situation with pimobendan, exogenous NP resulted in acute benefits such as a

reduction in pulmonary capillary wedge pressure, right atrial pressure, systemic vascular

resistance, systolic and pulmonary blood pressures, left ventricular volume indices, and

symptoms such as dyspnoea and fatigue, in people with symptomatic CHF participating in

double-blinded, randomised, controlled trials (H. H. Chen et al., 2012; Colucci et al., 2000;

Publication Committee for the VMAC Investigators, 2002). This resulted in the registration of

Nesiritide, a recombinant BNP1-32 product, for use in people with acute CHF. However, a

subsequent trial examining the effect of this product on 30-day rehospitalisation and

mortality rates showed no clear benefits over standard therapy; furthermore, Nesiritide

resulted in greater incidences of hypotension (O'Connor et al., 2011). This led to Nesiritide

falling out of favour in human cardiology. This contrast between Nesiritide and ARNI may

suggest a greater role of enhanced NP degradation in CHF; alternatively, neprilysin inhibition

could be producing beneficial effects outside of the NPS.

35

1.8) Knowledge gaps and aims of the thesis

As can be appreciated, many knowledge gaps exist in the pathophysiology and treatment of

CHF in both veterinary and human medicine. Considering the timeline, physical capacity,

and financial capacity in the context of this project, the following areas were selected for

further investigation.

Despite the established use of pimobendan in dogs with CHF due to MMVD, there

has been little evidence to support off-label practices such as the higher frequency of dosing

in dogs with latter stages of disease (Atkins et al., 2009). Of particular importance here is the

lack of published pharmacodynamic studies investigating the effects of pimobendan

following single, oral administration of labelled doses. This makes it difficult to predict the

effect of these treatment regimens. In addition, therapeutic monitoring is not employed; as

such, benefits of altering treatment regimen at an individual level are currently unknown.

There are also concerns related to treatment compliance. It is well-known that

treatment compliance reduces in people with increased frequency and number of dosing

(Claxton et al., 2001; Eisen et al., 1990). In veterinary medicine, an additional concern is the

willingness of pets to receive medications, with solid dose forms proving troublesome for

many. Pimobendan is particularly challenging to work with as its dosing recommendations

call for administration without food. Finally, the life-long nature of treatment adds up to a

considerable expense – thus necessitating evidence of benefit in order to maintain owner

compliance.

The second chapter of this thesis will aim to address these concerns through

investigation of pharmacokinetic and pharmacodynamic properties of a non-aqueous, liquid

formulation of pimobendan in healthy dogs. In addition to assessing the suitability of this

formulation as an alternative to existing solid dose forms, this study will explore the utility of

echocardiography as a non-invasive method of monitoring drug effects and provide a

rationale for alternative treatment regimen.

The debate surrounding the use of pimobendan in cats exists due, at least in part, to

the questions regarding its pharmacokinetics and pharmacodynamics which have yet to be

36

answered. The in-vivo disposition of pimobendan appears to differ in cats compared to dogs,

with one published study raising a hypothesis that it may not undergo significant metabolism

in this species (Hanzlicek et al., 2012); however, this has not been proven, and the

relationship between the parent drug and its active metabolite remains unknown. This is

important to clarify as there is data to suggest that pimobendan produces greater positive

inotropic and vasodilatory effects than its metabolite at physiological concentrations (Böhm

et al., 1991; Endoh et al., 1991; Fujimoto, 1994; Verdouw et al., 1987). There are two

existing studies that have investigated the acute effects of pimobendan in cats, but these did

not comment on the magnitude, duration, or range of its effects (Kent, 2011; Von Meel,

1985). Thus, further pharmacological studies are required to guide optimal treatment

strategies and further investigations into the benefit, if any, of pimobendan in cats with CHF.

In the third chapter of this thesis, the dose-effect relationship of pimobendan will be

investigated in healthy cats through evaluating its pharmacokinetics and pharmacodynamics.

The ability to detect treatment effects using echocardiography will also be tested, and by

employing a similar study design to that of the preceding chapter, the suitability of this

method to monitor treatment effects across species will be evaluated. Inter-species

differences in drug disposition, particularly regarding its metabolism, will be considered.

The conflicting information regarding the role of the RAAS in CHF in dogs with

MMVD, together with the proven benefits of pimobendan over ACE-I in the treatment of this

condition, raise the need for further evaluations regarding optimal medical therapy for this

condition. This is an important consideration in veterinary medicine, where cost and

compliance form major determinants of viable treatment options. Although a large

prospective study investigating potential benefits of combination therapy compared to

treatment with pimobendan and frusemide alone has been performed, results have not yet

been published (Wess, 2017). With some evidence to suggest the presence of aldosterone

escape and ACE-independent pathways in dogs, another unanswered question relates to

whether concomitant use of MRBs and ACE-I to provide a more comprehensive approach to

RAAS antagonism might positively alter the course of this disease.

37

Interestingly, even the natural course of disease on these combination therapies

utilising pimobendan and RAAS inhibition is minimally documented in the existing literature.

Aside from the major studies evaluating survival outcomes in dogs with MMVD being treated

with an ACE-I or pimobendan (Ettinger et al., 1998; Häggström et al., 2008; Lombard et al.,

2006; The BENCH Study Group, 1999), several other longitudinal studies evaluating survival

and risk factors for negative outcomes in dogs with CHF due to MMVD exist (Beaumier et al.,

2018; Borgarelli et al., 2008; De Madron et al., 2011; Häggström, Boswood, et al., 2013;

Hezzell, Boswood, Chang, Moonarmart, Souttar, et al., 2012; Hezzell, Boswood,

Moonarmart, et al., 2012; M. Mizuno et al., 2017; Serres, Chetboul, Tissier, et al., 2007;

Serres et al., 2008; Slupe et al., 2008; Wolf et al., 2012). However, the treatments used were

highly variable, and pimobendan and RAAS inhibition were often not administered

concurrently. In addition, some studies focused more on prognostic clinical variables without

evaluating the role of varying treatment regimen. Finally, many of these studies failed to

enrol dogs at the first onset of CHF. As such, the expected course of disease on

combination therapy utilising pimobendan and RAAS inhibition remains ill-defined.

The fourth chapter of this thesis will consist of a prospective study evaluating the

natural course of disease in dogs with newly diagnosed CHF due to MMVD undergoing

treatment with frusemide, pimobendan, benazepril, and spironolactone. This study will aim to

add to existing survival data in dogs with CHF due to MMVD by describing the evolution of

disease when pimobendan and comprehensive RAAS inhibition are included in the

treatment strategy.

In humans, aspects of the NPS have been investigated for their role in the

pathophysiology of CHF, and this has resulted in the development of ARNI as a novel

therapeutic agent. Although a pilot study examining the short-term use of ARNI in dogs with

subclinical MMVD has very recently been published (Newhard et al., 2018), the various

components of the NPS have not been thoroughly investigated in the context of MMVD in

dogs. Thus, it remains a mystery as to whether reduced production of, accelerated

degradation of, or peripheral resistance to, the active BNP1-32 and ANP1-28 may be

38

present in those that develop CHF. Aside from the aforementioned pilot study and one other,

in which synthetic BNP1-32 was administered to healthy dogs and those with subclinical

MMVD (Oyama et al., 2017), the NPS has not been examined as a therapeutic target in

veterinary medicine.

The fifth chapter of this thesis will examine methods of evaluating the BNP system in

healthy dogs as a starting point to its investigation in dogs with MMVD. A primary aim will be

to develop an assay utilising mass spectrometry to identify active BNP1-32 in canine plasma.

A secondary aim will be to examine the BNP system in healthy dogs undergoing an increase

in preload induced through high-volume fluid therapy.

Finally, the effect of augmenting the BNP system in dogs with CHF due to MMVD will

be examined in the sixth chapter. This study will document the efficacy of synthetic BNP1-32

administered subcutaneously to dogs with compensated CHF due to MMVD and aim to

provide support for further investigations into its use in this population.

39

Chapter 2:

Pharmacokinetics and cardiovascular effects

following a single oral administration

of a nonaqueous pimobendan solution in healthy dogs

40

Author contribution statement

This chapter was published in the Journal of Veterinary Pharmacology and Therapeutics as:

Yata M, McLachlan AJ, Foster DJR, Page SW, Beijerink NJ. (2016) Pharmacokinetics and

cardiovascular effects following a single oral administration of a nonaqueous pimobendan

solution in healthy dogs. J Vet Pharmacol Ther. 39:45-53. DOI: 10.1111/jvp.12243.

Mariko Yata was the primary investigator and author of the work reported in this manuscript

and was involved in all aspects of study design, data collection, data analysis, and reporting.

Niek Beijerink provided guidance on the study design, assisted with data collection, and

critically reviewed this manuscript.

Andrew McLachlan and David Foster contributed towards the study design, provided

guidance on pharmacokinetic analysis, and critically reviewed this manuscript.

Stephen Page contributed towards the study design and critically reviewed this manuscript.

Mariko Yata Date__ 14/8/2018____

Niek Beijerink Date___19/08/2018___

Andrew McLachlan Date___14/8/2018____

David Foster Date___14/8/2018____

Stephen Page Date___14/8/2018___

41

Pharmacokinetics and cardiovascular effects following a single oral

administration of a nonaqueous pimobendan solution in healthy dogs

M. YATA*

A. J. MCLACHLAN†

D. J. R. FOSTER‡

S. W. PAGE§ &

N. J. BEIJERINK*

*University Veterinary Teaching Hospital,

Faculty of Veterinary Science, University of

Sydney, Sydney, NSW, Australia; †Faculty

of Pharmacy, University of Sydney, Sydney,

NSW, Australia; ‡School of Pharmacy and

Medical Sciences, University of South Aus-

tralia, Adelaide, South Australia, Australia;§Luoda Pharma, Caringbah, NSW, Australia

Yata, M., McLachlan, A. J., Foster, D. J. R., Page, S. W., Beijerink N. J.

Pharmacokinetics and cardiovascular effects following a single oral

administration of a nonaqueous pimobendan solution in healthy dogs. J. vet.

Pharmacol. Therap. 39, 45–53.

Pimobendan is an inodilator used in the treatment of canine congestive heart fail-

ure (CHF). The aim of this study was to investigate the pharmacokinetics and car-

diovascular effects of a nonaqueous oral solution of pimobendan using a single-

dose, operator-blinded, parallel-dose study design. Eight healthy dogs were divided

into two treatment groups consisting of water (negative control) and pimobendan

solution. Plasma samples and noninvasive measures of cardiovascular function

were obtained over a 24-h period following dosing. Pimobendan and its active

metabolite were quantified using an ultra-high-performance liquid chromatogra-

phy–mass spectrometer (UHPLC-MS) assay. The oral pimobendan solution was

rapidly absorbed [time taken to reach maximum concentration (Tmax) 1.1 h] and

readily converted to the active metabolite (metabolite Tmax 1.3 h). The elimination

half-life was short for both pimobendan and its active metabolite (0.9 and 1.6 h,

respectively). Maximal cardiovascular effects occurred at 2–4 h after a single oral

dose, with measurable effects occurring primarily in echocardiographic indices of

systolic function. Significant effects persisted for <8 h. The pimobendan nonaqu-

eous oral solution was well tolerated by study dogs.

(Paper received 18 February 2015; accepted for publication 30 April 2015)

Niek Jozef Beijerink, University Veterinary Teaching Hospital, Faculty of

Veterinary Science, University of Sydney, NSW 2006, Australia.

E-mail: niek.beijerink@sydney.edu.au

INTRODUCTION

Pimobendan is a benzimidazole-pyridazinone derivative that is

widely used in veterinary medicine for treatment of canine

congestive heart failure (CHF). Its mechanism of action is via

calcium sensitization and inhibition of phosphodiesterase III;

vasodilation and an increase in myocardial contractility with-

out a disproportionate increase in myocardial oxygen con-

sumption result (Boyle & Leech, 2012). In the latest published

guidelines for treatment of canine CHF, the American College

of Veterinary Internal Medicine (ACVIM) recommends pimob-

endan as one of three essential drugs for the treatment of

symptomatic CHF (ACVIM heart disease stage C and D) (Atkins

et al., 2009). This recommendation is supported by a number

of prospective, randomized, controlled trials that have reported

improvements in survival and time to treatment failure in dogs

with CHF due to myxomatous mitral valve disease (MMVD)

treated with pimobendan when compared to those treated with

an angiotensin converting enzyme inhibitor (ACE-I) (Smith

et al., 2005; Lombard et al., 2006; H€aggstr€om et al., 2008).

Currently, several registered oral formulations of pimoben-

dan are commercially available. These formulations consist of

solid dosage forms (flavoured and chewable tablets and cap-

sules). Some limitations of these pimobendan formulations

include difficulties in accurately dosing small patients, making

dose adjustments and administering the substance to non-

tractable patients. Successful medical treatment of veterinary

patients relies on acceptable owner and patient compliance.

The availability of a range of effective pharmaceutical prepa-

rations of a medication will aid in achieving this aim and will

be particularly beneficial when long-term therapy is required

as in the case of CHF. This study was designed to evaluate

the pharmacokinetics and cardiovascular effects of pimoben-

dan and its active metabolite after administration of a novel

nonaqueous oral pimobendan solution which may serve as an

alternative to currently existing registered formulations of this

drug.

MATERIALS AND METHODS

Animals

Eight healthy, adult dogs (Beagles; four male, four female;

weight 13.6–23.8 kg; age 4–6 years old) were recruited for

© 2015 John Wiley & Sons Ltd 45

J. vet. Pharmacol. Therap. 39, 45--53. doi: 10.1111/jvp.12243.

42

the study. Ethics approval for the study was obtained from the

Wongaburra Research Centre Animal Care and Ethics Commit-

tee and University of Sydney Animal Ethics Committee prior to

the study.

Study design

A randomized, operator-blinded, single-dose, parallel-dose study

design was used. Dogs were block-randomized into the two

treatment groups (four dogs per group). Randomization was

also stratified by dog weight and sex to give an even male to

female ratio and distribution of body weights between groups.

Investigators involved in pharmacokinetic analysis and mea-

surement of pharmacodynamic parameters were blinded to the

treatment allocation until plasma concentrations of the drug

and metabolite were quantified and all cardiovascular measure-

ments were assessed.

Schedule of events

The study was conducted over 2 weeks in February 2014. All

animals underwent a full physical examination on the first day

of the study and were deemed to be fit and healthy. Recorded

body weight from this day was used to calculate treatment

dose given to each dog.

On the day prior to dosing, all dogs underwent a second

physical examination. Blood pressure measurements and echo-

cardiography (details below) were performed twice to familiar-

ize dogs with the procedures and to ensure normal cardiac

anatomy and function. Following these procedures, dogs were

sedated lightly with butorphanol (Zoetis Australia, Rhodes,

NSW, Australia). A jugular catheter (B. Braun Certofix Duo or

B. Braun Cavafix Certo with Splittocan; B. Braun Australia Pty

Ltd, Bella Vista, NSW, Australia) was placed aseptically in the

right jugular vein under local anaesthesia (lignocaine; Troy

Laboratories Australia Pty Ltd., Glendenning, NSW, Australia).

Dogs were fed in the evening to provide a minimum fasting

time of 10 h prior to dosing. Water was available ad libitum

overnight.

On the day of dosing, dogs were treated with a single oral

dose of treatment according to their allocated treatment

groups. Negative control dogs received 10 mL water, and the

pimobendan solution dogs received a nonaqueous oral pimob-

endan solution (3.5 mg/mL pimobendan supplied by Luoda

Pharma, Caringbah NSW, Australia) at 0.27 mg/kg (range:

0.26–0.28 mg/kg). Each dose was followed by 5–10 mL of

water administered orally to ensure the dose was swallowed.

Echocardiography, blood pressure and heart rate (HR) monitor-

ing were performed 30 min prior to dosing, immediately prior

to dosing, then at 15, 30 min, 1, 1.5, 2, 3, 4, 8, 12 and 24 h

postdosing. Five millilitres of blood were acquired via the

jugular catheter prior to dosing and at 5, 10, 15, 30, 45 min,

1, 1.5, 2, 3, 4, 8, 12, and 24 h following oral dose

administration.

Throughout the sampling period, water was available ad

libitum. Dogs were fed after obtaining the 4 h sample.

Blood sample processing and storage

Blood samples were collected in lithium heparin-coated blood

tubes and centrifuged for 5 min within 2 h of collection to

harvest plasma. Plasma samples were stored frozen at �30 °Cuntil the time of analysis.

Analytical method

The analytical method was developed and validated at a com-

mercial laboratory (PIA PHARMA Pty Ltd., Gladesville, NSW,

Australia). Identification of plasma concentrations of pimoben-

dan and its O-demethylated metabolite (ODMP), i.e. UD CG

212 Cl, was performed using ultra high-performance liquid

chromatography (UHPLC) (Eksigent ekspert ultra LC100; AB

Sciex, Redwood City, CA, USA) coupled with a mass spectrom-

eter (MS) (AB Sciex API3200, AB Sciex, Framingham, MA,

USA). Quantification, analysis and generation of the calibration

curve were performed with AB Sciex Multiquant software

(version 2.1; Foster City, CA, USA). Analytical grade racemic

pimobendan (Toronto Research Chemicals, Toronto, Ontario,

Canada) and ODMP (BDG Synthesis Ltd., Wellington, New

Zealand) were utilized as reference standards for the analysis.

Analytical grade fenbendazole (Sigma-Aldrich Pty Ltd., Castle

Hill, NSW, Australia) was utilized as the internal standard for

all analysis. All reagents utilized throughout analysis were of

analytical grade.

Procedure. Each plasma sample was thawed at room

temperature on the day of analysis. Fenbendazole internal

standard (50 lL containing 2.5 ng Fenbendazole) was added

to plasma (250 lL) and mixed gently. Deproteinization was

performed by adding acetonitrile (450 lL). The mixture was

vortex mixed and centrifuged prior to addition of water

(250 lL) to improve peak shape. The sample was centrifuged

again, and the supernatant was removed and passed through

a 0.45-lm Hydraflon filter into a collection plate for analysis.

Quantification method. For identification of pimobendan and

ODMP, 10 lL of the extract was injected into the ultra-

high-performance liquid chromatography–mass spectrometer

(UHPLC-MS) system. Reverse-phase chromatography utilizing a

Supelco Discovery column (C18, 50 9 2.1 mm, 5 lm; Sigma-

Aldrich Pty Ltd.) maintained at 40 °C was performed. A mobile

phase consisting of 1–99% water and acetonitrile was used for

gradient elution at a flow rate of 0.8 mL/min.

Both compounds were detected using mass spectrometry

with a triple quadrupole mass analyser. Ionization was per-

formed with positive electrospray ionization at 5500 V. The

desolvation temperature was set to 500 °C. Multiple reaction

monitoring (MRM) was utilized to obtain transitions for identi-

fication of each compound. The transitions for ODMP, pimob-

endan and fenbendazole were 321.3 ? 305.2, 335.2 ?319.1 and 300.2 ? 159.2, respectively. The retention times

for ODMP, pimobendan and fenbendazole were 1.55, 1.66 and

1.89 min, respectively.

© 2015 John Wiley & Sons Ltd

46 M. Yata et al.

43

Quantification of pimobendan and ODMP was performed

using a calibration curve derived from a series of matrix-

matched calibration controls (MMC) created on each day of

analysis. Each MMC was prepared using blank plasma and

contained pimobendan and ODMP added at incremental con-

centrations ranging between 0.5 and 20 ng/mL. Each curve

was generated from area ratios between the analyte and fen-

bendazole utilizing a minimum of six MMCs. Linear regression

analysis using a 1/x weighting system to maximize accuracy

and reduce the coefficient of variation (CV) at the lower end of

the curve was performed. The coefficient of determination (R2)

was >0.99 for all calibration curves.

Analytical validation and stability. Validation of the assay

followed standard operating procedures for the laboratory and

consisted of analyte identification, selectivity, determination of

carryover, acceptable identification of the limits of

quantification, accuracy and precision.

Analyte identification was assessed through visual observa-

tion of chromatograms. Adequate selectivity of the assay and

lack of carry-over between analyses were assessed through

sequential testing of samples containing the analyte. The per-

centage area ratio was <20% of the lower limit of quantifica-

tion (LLOQ) for pimobendan and ODMP and <5% of the peak

for fenbendazole in all samples.

Intraday accuracy and precision were determined using six

replicates of fortified samples at concentrations ranging

between 0.22 and 13.23 ng/mL for pimobendan and

0.32–19.13 ng/mL for ODMP and were represented as the

ratio between the measured and nominal concentrations. The

mean percentage of accuracy ranged between 97.2–108.6%and 89.8–103.8% for pimobendan and ODMP, respectively.

Precision was represented as %CV, and ranged between 2.7–9.7% for pimobendan and 4.0–13.6% for ODMP. At fortified

concentrations of 0.55 ng/mL (pimobendan) and 0.32 ng/mL

(ODMP), the mean percentage of accuracy was 96.4% (pimob-

endan) and 97.2% (ODMP), and the %CV was 9.7% (pimoben-

dan) and 7.9% (ODMP). The LLOQ for pimobendan and ODMP

was identified at 0.5 ng/mL.

Interday assay performance was assessed using quality con-

trol samples for accuracy and precision. A total of 10% of the

samples run on each day of analysis consisted of fortified

quality control samples (minimum of four concentrations,

assayed in duplicate between 0.54–16.28 ng/mL for pimoben-

dan and 0.64–19.13 ng/mL for ODMP) prepared on the day

of the analysis. The mean percentage of accuracy

(n = 3 days) was 94.8–102.6% for pimobendan at 0.54 ng/mL

and 90.1–119.9% at all other concentrations. The mean

percentage of accuracy (n = 3 days) was 94.7–99.8% for

ODMP at 0.64 ng/mL and 92.3–119.4% at all other concen-

trations.

Stability testing on fortified samples was also performed to

determine whether storage and freeze–thaw cycles contributed

toward degeneration of the analyte. Analysis of samples stored

frozen for 2 months (n = 6) showed >90% recovery. Following

three freeze–thaw cycles (n = 6), recovery was >85%.

Pharmacokinetic analysis

The area under the plasma concentration–time curve to the

final measured concentration value (AUC0-t) was determined

using the trapezoidal rule. The elimination rate constant (kel)

was calculated using the log-linear slope of the terminal por-

tion of the concentration–time profile.

The kel was used to calculate the area under the plasma con-

centration–time curve extrapolated to infinity (AUC0-∞). The

median dose administered to the pimobendan solution group

was 5 mg. The AUC0-∞ for pimobendan was adjusted to this

dose using the following equation:

Adjusted AUC0�1 ¼ AUC0�1 � Dose=5

The elimination half-life (t1/2) was calculated as ln2/kel.

For pimobendan, the apparent clearance (CL/F) and appar-

ent volume of distribution (V/F) were also determined using

the equations:

CL/F ¼ Dose=AUC0�1

V/F ¼ ðCL/FÞ=kel

Results of pharmacokinetic analyses were reported as the med-

ian and range.

Echocardiography and blood pressure measurements

Dogs were trained to lie in lateral recumbency for the proce-

dures prior to the study, and no chemical restraint was utilized

at any point during the sampling period. Echocardiography

was performed by a single investigator (NB) throughout the

course of the study. A commercially available high-frame-rate

ultrasound system equipped with a 3.5–8.0 MHz sector trans-

ducer and simultaneous electrocardiography (Vivid I; General

Electric Healthcare, Silverwater, NSW, Australia) was used. All

images were recorded and analysed at a later time.

The aortic root diameter (Ao), left atrial diameter (LA),

systolic and diastolic measurements of interventricular septal

thickness (IVSs, IVSd), left ventricular internal diameter

(LVIDs, LVIDd) and left ventricular posterior wall thickness

(LVPWs, LVPWd) were measured using 2D and M-mode func-

tions from standard right parasternal short-axis views as previ-

ously described (Chetboul et al., 2004). The fractional

shortening (FS) was calculated using the following equation:

FS = (LVIDd � LVIDs)/LVIDd.

The maximal aortic velocity (Ao vel), pre-ejection period

(PEP) and left ventricular ejection time (LVET) were obtained

via ECG-gated pulsed-wave Doppler measurements of aortic

outflow derived from the subcostal view (Dukes-McEwan et al.,

2002; Abbott & Maclean, 2003; Rajabioun et al., 2008). Addi-

tionally, the HR was taken from recorded ECG-gated M-mode

images obtained during echocardiography. For each parameter

measured at each time point, three separate cardiac cycles

were measured and averaged.

© 2015 John Wiley & Sons Ltd

PK and PD of pimobendan solution in dogs 47

44

Blood pressure was measured indirectly using an oscillomet-

ric device (VetHDO monitor; Memo Diagnostic, Darmstadt,

Germany) coupled with an inflatable cuff, measuring 36–50%of the tail diameter, placed at the tail base (Egner, 2006; Meyer

et al., 2010; Mitchell et al., 2010). The systolic (SBP), diastolic

(DBP) and mean (MBP) blood pressures were also measured

three times at each time point and averaged.

Observer variability in echocardiography

The CV for each of the echocardiographic parameters for the

investigator performing echocardiography (NB) was established

prior to the study. Echocardiography was performed on a total of

six healthy adult dogs (three male, three female) over the course

of 6 h in 1 day (11 am to 5 pm). Dogs were examined five times

in succession in the same order throughout the examination per-

iod. Images were recorded and analysed at a later time. For each

parameter measured in each time period, six cardiac cycles were

measured. The overall CV (mean � standard deviation) was

<10% for Ao (5.8% � 0.6%), LA (6.9% � 2.5%), Ao vel

(8.7% � 2.4%), IVSs (8.7% � 2.0%), LVIDd (4.4% � 1.4%),

LVIDs (5.3% � 0.9%), LVPWd (8.6% � 3.1%), LVPWs

(6.8% � 2.6%) and LVET (6.1% � 2.2%). This value was

slightly higher for IVSd (10.2% � 2.6%) and PEP

(12.8% � 3.0%).

The potential effect of time on the CV (e.g. increase in varia-

tion due to operator fatigue) was also examined using a simple

linear regression model. Non-normally distributed data were

log-transformed prior to analysis. Other assumptions were eval-

uated post hoc via inspection of residual plots. The results

showed no significant effect of time (P > 0.05) on CV for any

of the measured parameters.

Pharmacodynamic and statistical analysis

Descriptive analysis of the cardiovascular effects was performed

through visual observation of graphs representing the effect

plotted against time for each parameter. Because the size of

dog has been previously identified as an influencing factor for

IVSs, IVSd, LVIDs, LVIDd, LVPWs, LVPWd, LA and Ao, these

were normalized to body weight (Cornell et al., 2004).

The effects of treatment and time on the measured pharma-

codynamic parameters were assessed via linear mixed model-

ling with restricted maximal likelihood estimation using a

commercially available software program (Genstat 16th Edi-

tion; VSN International Ltd., Hemel Hempstead, UK). The time

point following dosing (time) and treatment group (treatment)

were specified as fixed effects, with individual dogs comprising

the random effects in this model. The interaction between time

and treatment was included in the model to enable assessment

of differences arising between treatment groups at each time

point, as well as differences from the baseline value within a

treatment group. Significance was set at an alpha of 0.05.

Parameters that were not normally distributed were log or

power transformed to resemble a normal distribution prior to

analysis. Fulfilment of assumptions for the model was checked

via visual inspection of residual plots.

Results of pharmacodynamic analysis are reported as the

mean and standard deviation.

RESULTS

Drug dosing

Dosing was tolerated well in all dogs receiving the pimobendan

solution. Potential adverse effects of treatment manifested as

minor transient hypersalivation in one dog in the treated

group.

Pharmacokinetics

Individual plasma concentration–time profiles of pimobendan

and ODMP following treatment with the oral liquid pimoben-

dan formulation are presented in Fig. 1. Plasma concentrations

were below LLOQ from 8 h onwards for pimobendan and from

12 h onwards for ODMP.

The pharmacokinetic parameters for pimobendan and ODMP

for dogs receiving pimobendan are summarized in Table 1. The

pimobendan nonaqueous oral solution was rapidly absorbed

(median time taken to reach maximum concentration (Tmax)

1.1 h, range: 0.5–2.0 h). There was a large range in V/F

(median 15.5 L/kg, range: 5.9–22.3 L/kg) and CL/F (median

176.5 mL/min/kg, range: 105.0–301.0 mL/min/kg).

The concentration–time profile of ODMP lagged slightly

behind that of pimobendan. Conversion of pimobendan to

ODMP appeared to occur rapidly, with only a minor delay in

Tmax for ODMP (median 1.3 h, range: 0.8–2.0 h). The AUC for

ODMP tended to be higher than for pimobendan (median

AUC0-∞ ODMP to AUC0-∞ pimobendan ratio 1.6, range:

1.2–1.7).Both pimobendan and ODMP displayed first-order elimina-

tion at the dose administered (Fig. 1), and the t1/2 of both

compounds following a single dose of the nonaqueous solution

was short (median 0.9 h, range: 0.7–1.1 h for pimobendan

and median 1.6 h, range: 1.3–1.9 h for ODMP).

Pharmacodynamics

Baseline cardiovascular values were not significantly different

between treatment groups (Table 2).

Significant differences (P < 0.05) from the baseline value

over time were observed in dogs treated with pimobendan for

the following echocardiographic parameters: Ao Vel (1–4 h),

FS (2–4 h), LVIDd (2–4 h), LVIDs (1.5–4 h), LVPWs (1.5–4 h)

and PEP (1, 2, 3 h) (Fig. 2). Ao Vel, FS and LVPWs increased

from baseline, whereas LVIDd, LVIDs and PEP decreased. Maxi-

mal effects were observed between 2 and 4 h following treat-

ment, and significant effects were lost within 8 h of dosing. Of

these, a significant difference (P < 0.05) was also observed

between pimobendan treated and control groups from 1.5 to

© 2015 John Wiley & Sons Ltd

48 M. Yata et al.

45

4 h for Ao Vel, at 3 h for FS and LVPWs, from 3 to 4 h for

LVIDs and from 2 to 3 h for PEP.

No significant effect over time was observed in dogs in the

treated group for Ao, LA, HR, IVSs, IVSd, LVET, LVPWd, DBP,

MBP and SBP. Values from dogs in this group were not signifi-

cantly different to those of control dogs for these parameters.

DISCUSSION

In this study, the pharmacokinetics and acute cardiovascular

effects of an oral, nonaqueous solution of pimobendan were

investigated in order to evaluate its potential as an alternative

to the currently existing solid preparations of this medication.

These evaluations play an important role in the establishment

of a drug’s efficacy and safety profile.

The oral nonaqueous pimobendan solution was absorbed

quickly, with maximal average plasma concentrations detected

at around 1 h following dosing. Pimobendan solution showed

a large V/F and this, in addition to the known lipophilic nature

of the drug, suggests that it is readily distributed into tissues.

Pimobendan was rapidly converted to ODMP, with maximal

average plasma concentrations occurring at 1.3 h following

dosing. The systemic exposure was larger for ODMP than for

pimobendan. Inspection of the graph and comparison of t1/2and kel for pimobendan and ODMP in treated individuals sug-

gest an elimination rate-limited disposition of the metabolite.

0 1 2 3 4 50

10

20

30Pimobendan

Time (h)

Con

c (n

g/m

L)

Con

c (n

g/m

L)C

onc

(ng/

mL)

Con

c (n

g/m

L)

0 1 2 3 4 50.1

1

10

100Pimobendan

Time (h)

0 2 4 6 8 100

5

10

15

20

25ODMP

Time (h)0 2 4 6 8 10

0.1

1

10

100ODMP

Time (h)

Fig. 1. Individual concentration–time plots of total pimobendan and O-demethylated metabolite (ODMP) for dogs (n = 4) treated with a single dose

of nonaqueous oral pimobendan solution. Conc, concentration. Concentrations fell below the lower limit of quantification (LLOQ) by 8 and 12 h for

pimobendan and ODMP, respectively.

Table 1. Summary of pimobendan and O-demethylated metabolite

(ODMP) pharmacokinetic parameters (median, range) for dogs treated

with a single dose of nonaqueous pimobendan oral solution

Parameter (units)

Pimobendan oral solution (n = 4)

Pimobendan ODMP

Tmax (h) 1.1 (0.5–2.0) 1.3 (0.8–2.0)Cmax (ng/mL) 18.6 (6.1–25.3) 16.2 (6.0–22.3)AUC0-∞ (ng/mL�h) 27.1 (15.2–44.2) 42.1 (25.1–52.7)AUC0-∞ (ng/mL�h)* 31.9 (12.7–42.1) –t1/2 (h) 0.9 (0.7–1.1) 1.6 (1.3–1.9)CL/F (mL/min/kg) 176.5 (105.0–301.0) –V/F (L/kg) 15.5 (5.9–22.3) –

Tmax, time taken to reach maximum concentration; Cmax, maximum

concentration; AUC0-∞, area under the plasma concentration–time

curve extrapolated to infinity; t1/2, elimination half-life; CL/F, apparent

clearance; V/F, apparent volume of distribution.

*AUC0-∞ normalised to a dose of 5 mg pimobendan

© 2015 John Wiley & Sons Ltd

PK and PD of pimobendan solution in dogs 49

46

Therefore, the greater AUC0-∞ for ODMP appears largely attrib-

utable to prolonged duration of time spent in the body by this

compound as a result of slower elimination rather than as a

result of slower formation.

In terms of tolerability, the pimobendan solution was well

tolerated by the study dogs, with no major adverse effects

observed during the study.

In this study, noninvasive methods of monitoring cardiovas-

cular changes were employed. This is the first study to evalu-

ate the acute effects of pimobendan in this fashion. Both

echocardiography and oscillometric blood pressure monitoring

are established modes of monitoring hemodynamic changes

and utilized commonly in veterinary practice. Aside from posi-

tive animal welfare implications of using a noninvasive mode

of evaluation, an additional benefit to utilizing these methods

include the potential for identifying practical methods of evalu-

ating therapeutic effects which may be used in routine clinical

practice. They do not require general anaesthesia or sedation,

and as such, may also be a more accurate method of detecting

true drug effects.

Pimobendan solution caused a significant rise in Ao Vel, FS,

and LVPWs and a reduction in LVIDs and PEP from around

1–2 h after dosing. Maximal effects, which were significantly

different to values from dogs in the control group, were

observed between 3 and 4 h following treatment. These

parameters relate to enhanced left ventricular systolic function

and are consistent with findings of prior studies in dogs in

which invasive monitoring methods were used (Von Meel,

1985; Pouleur et al., 1988, 1989; Van Meel & Diederen,

1989; Ohte et al., 1997; FDA-CVM, 2007).

The maximal effects observed in this study occurred slightly

later (3–4 h) than in prior invasive studies evaluating systolic

function after oral doses (1.5–2 h) (Von Meel, 1985; Van Meel

& Diederen, 1989). This is most likely attributable to the use of

higher dosages (0.5–1 mg/kg) in prior studies as a shorter time

to maximal effect for higher dosages has been previously

reported in dogs (FDA-CVM, 2007) and in humans (Walter

et al., 1988). The duration of significant effects observed in this

study (<8 h) was also shorter than what has been previously

reported (Von Meel, 1985; Van Meel & Diederen, 1989; FDA-

CVM, 2007). This may be attributable to the fact that the sig-

nificance of the effect in relation to the baseline or an

untreated animal was not evaluated in prior studies or that

the small number of dogs in our study resulted in failure to

detect significant differences at later time points.

Pimobendan has been reported to reduce blood pressure in

prior studies conducted on anesthetized dogs (Kitzen et al., 1988;

Lynch et al., 1989; Van Meel & Diederen, 1989; Pagel et al.,

1996). In the present study, a significant effect on blood pressure

was not observed. This may be due to the use of an indirect

method of measurement. In addition, >10% variability in the

triplicate blood pressure readings was present at times and may

have contributed toward reduced accuracy of blood pressure

assessment. It is also possible that the effect on blood pressure is

not readily measurable in conscious dogs, and findings of one

study on conscious instrumented dogs (Von Meel, 1985) and a

handful of others evaluating the effect of chronic pimobendan

treatment (Chetboul et al., 2007; Ouellet et al., 2009; Suzuki

et al., 2011) suggest that this is a plausible theory.

The findings from this study would suggest that the plasma

concentration of pimobendan and ODMP is unlikely to accu-

mulate at the dosage regimen currently recommended for this

drug (0.4–0.6 mg/kg/day divided twice daily). Although the

cardiovascular response to pimobendan may be different in

dogs with heart disease, it appears that to achieve a narrower

range of daily effects, an increase in dosing frequency would

be required. Interestingly, there are currently no data available

regarding the optimal magnitude and duration of effects associ-

ated with outcome variables such as survival, morbidity, and

development of adverse effects. It is known that the current

dosage regimen leads to a favourable increase in longevity of

dogs with CHF (Smith et al., 2005; Lombard et al., 2006;

H€aggstr€om et al., 2008). It is not known, however, if it is

advantageous or disadvantageous to increase the daily pimob-

endan dose. Thus, it could very well be possible that enhancing

the systolic function continuously could be detrimental to the

animal in the long term. Further evaluation of pimobendan

pharmacokinetic–pharmacodynamic relationship and charac-

terization of the long-term effects in dogs with heart disease is

required to clarify the role of pimobendan in managing canine

Table 2. Table of baseline cardiovascular measurements (prior to dos-

ing, i.e. time zero) in dogs dosed with water only (Control; n = 4) or a

nonaqueous oral pimobendan solution (pimobendan; n = 4)

Parameter (units) Control Pimobendan

Ao Vel (m/s) 1.5 (0.2) 1.7 (0.2)

Ao* (cm) 0.9 (0.1) 0.9 (0.1)

FS (%) 34.3 (8.2) 33.3 (6.9)

HR (bpm) 117.0 (18.2) 108.3 (21.6)

IVSd* (cm) 0.5 (0.1) 0.5 (0.1)

IVSs* (cm) 0.7 (0.1) 0.6 (0.1)

LA* (cm) 1.2 (0.1) 1.2 (0.1)

LVET (ms) 185.8 (20.9) 182.3 (25.1)

LVIDd* (cm) 1.5 (0.1) 1.6 (0.1)

LVIDs* (cm) 0.9 (0.1) 1.0 (0.1)

LVPWd* (cm) 0.5 (0.0) 0.5 (0.1)

LVPWs* (cm) 0.7 (0.1) 0.7 (0.1)

PEP (ms) 49.3 (5.5) 50.1 (6.9)

MBP (mmHg) 102.8 (8.2) 99.3 (9.4)

DBP (mmHg) 71.0 (6.5) 66.1 (5.5)

SBP (mmHg) 162.0 (23.8) 161.6 (20.8)

Ao Vel, aortic velocity; Ao, aortic diameter; FS, fractional shortening;

HR, heart rate; IVSd, interventricular septum (diastole); IVSs, interven-

tricular septum (systole); LA, left atrium; LVET, left ventricular ejection

time; LVIDd, left ventricular internal diameter (diastole); LVIDs, left

ventricular internal diameter (systole); LVPWd, left ventricular poster-

ior wall (diastole); LVPWs, left ventricular posterior wall (systole); PEP,

pre-ejection period; MBP, mean blood pressure; DBP, diastolic blood

pressure; SBP, systolic blood pressure.

No values were significantly different between treatment groups

(P > 0.05).

Values expressed as mean (standard deviation).

*Indexed to body weight (Cornell et al., 2004).

© 2015 John Wiley & Sons Ltd

50 M. Yata et al.

47

heart disease. Such information could also be utilized to indi-

vidualize treatments to achieve better outcomes.

A number of limitations were present in this study. At the

time of planning this study, a registered intravenous formula-

tion of pimobendan was not available. As such, absolute bio-

availability could not be elucidated. The health status of the

dogs included in this study was determined with physical

examination, evaluation of history and cardiovascular testing.

Because clinical pathology was not performed to ascertain spe-

cific organ function, this may be considered a weakness in the

inclusion criteria. In addition, the study utilized a small

number of dogs. This is not an uncommon occurrence when

0 50.0

0.5

1.0

1.5

2.0

2.5

10 15 20 25

Aortic velocity

Time (h)

Max

Ao

Vel

(m/s

ec)

***

**

† † † † †

Tx P 0.016Time P 0.006Tx*Time P <0.001

0 51.2

1.4

1.6

1.8

10 15 20 25

Left ventricular internal diameter (diastole)

Time (h)

LVID

d (c

m, w

t nor

m)

**

*Tx P 0.838Time P 0.001

Tx*Time P 0.004

0 50

20

40

60

10 15 20 25

Fractional shortening

Time (h)

FS (%

)

*

**

†

Tx P 0.308Time P 0.047Tx*Time

TxTime

Tx*Time

TxTimeTx*Time

P <0.001

0 50.0

0.5

1.0

1.5

10 15 20 25

Left ventricular internal diameter (systole)

Time (h)

LVID

s (c

m, w

t nor

m)

****

† †

Tx P 0.488Time P <0.001Tx*Time P <0.001

05

0.5

0.6

0.7

0.8

0.9

1.0

1.1

10 15 20 25

Left ventricular posterior wall (systole)

Time (h)

LVP

Ws

(cm

, wt n

orm

) ****

†

P 0.276P 0.017

P <0.001

0 50

20

40

60

80

10 15 20 25

Pre-ejection period

Time (h)

PE

P (m

s)

***

††

P 0.074P 0.021P 0.014

Fig. 2. Graphical representation of change in select cardiovascular effects over time for dogs treated with pimobendan oral solution (pimobendan group;

n = 4) and those treated with water (control group; n = 4). Solid line represents control group; dashed line represents pimobendan group. Ao Vel, aortic

velocity; FS, fractional shortening, LVIDs, left ventricular internal diameter (systole); PEP, pre-ejection period; LVIDd, left ventricular internal diameter

(diastole); LVPWs, left ventricular posterior wall (systole); cm, wt norm, weight-normalized centimeters (indexed to body weight, as per Cornell et al.,

2004). *Significant difference (P < 0.05) from baseline (time 0) within the pimobendan group at indicated time. There were no significant differences

from baseline in the control group. †Significant difference (P < 0.05) between control and pimobendan groups at indicated time. Group mean and

standard deviation are represented by point and bars, respectively. P-values associated with fixed (Tx = treatment; Time = time) and interaction

(Tx*Time = treatment*time) terms in the model are displayed on each graph.

© 2015 John Wiley & Sons Ltd

PK and PD of pimobendan solution in dogs 51

48

companion animal models are utilized in pharmacokinetic

studies; however, this raises the possibility of both type I and II

errors in our findings, with the latter being more likely.

Although the observed haemodynamic trends in systolic func-

tion are convincing, we were unable to clarify the exact dura-

tion of effect due to reduced sampling points at later time

points. The inclusion of repeated baseline measurements on

the day of sampling to further characterize the resting cardio-

vascular status would also have been ideal. Finally, the cardio-

vascular parameters measured during this study were not

adjusted for the preceding R-R interval. It is well known that a

longer R-R interval would result in higher end-diastolic vol-

ume. The Frank-Starling mechanism dictates that this would

increase the contractile energy and left ventricular systolic

function. Therefore, the possibility exists that part of the total

variability observed in our data set may have been attributable

to the effect of HR, and adjusting for this effect may result in

better assessment of systolic function.

CONCLUSION

The nonaqueous oral solution of pimobendan was absorbed,

metabolized and excreted rapidly. Following a single oral dose,

pimobendan treatment produced positive inotropic effects

which were able to be measured utilizing echocardiography.

Maximal aortic velocity and left ventricular internal diameter

in systole were particularly useful for this evaluation. Depend-

ing on the measured variable, maximal effects appeared to

occur around 2–4 h following a dose and were maintained at

a significant level for <8 h. This may be useful in detecting an

individual’s response to dosing.

ACKNOWLEDGMENTS

This study received financial support from Luoda Pharma

(Caringbah, NSW). We would like to thank Joe Pippia (PIA

PHARMA Pty Ltd, Gladesville, NSW) for development, valida-

tion and performance of the UHPLC-MS assay; Bob Pigott,

Maurice Webster and Vanessa Andrews (Wongaburra

Research Centre operated by Vetx Research) for provision of

facilities and their support in planning and conducting this

study; and Dr Evelyn Hall and Alan Marcus for advice on sta-

tistical analysis.

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© 2015 John Wiley & Sons Ltd

PK and PD of pimobendan solution in dogs 53

50

Chapter 3:

Single-dose pharmacokinetics and cardiovascular effects

of oral pimobendan in healthy cats

51

Author contribution statement

This chapter was published in the Journal of Veterinary Cardiology as:

Yata M, McLachlan AJ, Foster DJR, Hanzlicek AS, Beijerink NJ. (2016) Single-dose

pharmacokinetics and cardiovascular effects of oral pimobendan in healthy cats. J Vet

Cardiol. 18:310-325. DOI: 10.1016/j.jvc.2016.07.001

Mariko Yata was the primary investigator and author of the work reported in this manuscript

and was involved in all aspects of study design, data collection, data analysis, and reporting.

Niek Beijerink provided guidance on the study design, assisted with data collection, and

critically reviewed this manuscript.

Andrew McLachlan and David Foster contributed towards the study design, provided

guidance on pharmacokinetic analysis, and critically reviewed this manuscript.

Andrew Hanzlicek contributed towards the study design and critically reviewed this

manuscript.

Mariko Yata Date__ 14/8/2018____

Niek Beijerink Date__19-08-2018___

Andrew McLachlan Date___14/8/2018____

David Foster Date___14/8/2018____

Andrew Hanzlicek Date___14/8/2018____

52

* CorrespondE-mail addr

http://dx.doi.o1760-2734/ª 2

Journal of Veterinary Cardiology (2016) 18, 310e325

www.elsevier.com/locate/jvc

Single-dose pharmacokinetics andcardiovascular effects of oral pimobendanin healthy cats

M. Yata, BVSc (Hons) a, A.J. McLachlan, BPharm (Hons), PhDb, D.J.R. Foster, BSc (Hons), PhD c,d, A.S. Hanzlicek, MS, DVMe, N.J. Beijerink, DVM, PhD a,*

aThe University of Sydney, Faculty of Veterinary Science, University VeterinaryTeaching Hospital Sydney, 65 Parramatta Road, Camperdown, NSW 2050, AustraliabThe University of Sydney, Faculty of Pharmacy, A15 Pharmacy and Bank Building,University of Sydney, Sydney, NSW 2006, AustraliacAustralian Centre for Pharmacometrics, School of Pharmacy and Medical Sciences,University of South Australia, Division of Health Sciences, GPO Box 2471, Adelaide, SA5001, Australiad Sansom Institute for Health Research, School of Pharmacy and Medical Sciences,University of South Australia, Division of Health Sciences, GPO Box 2471, Adelaide, SA5001, AustraliaeDepartment of Veterinary Clinical Sciences, Oklahoma State University, Stillwater,OK 74078, USA

Received 18 December 2015; received in revised form 13 July 2016; accepted 16 July 2016

KEYWORDSMetabolite pharmaco-kinetics;UD-CG 212;Inodilator;Feline;Pharmacodynamics

ing author.ess: niek.beijerink@syd

rg/10.1016/j.jvc.2016016 Elsevier B.V. All rig

Abstract Introduction: To investigate the pharmacokinetics and pharmacody-namics of oral pimobendan in conscious, healthy cats.Animals: Eight healthy adult cats.Materials and methods: A randomised, single-blinded, crossover design was used.Two oral doses of pimobendan (0.625-mg [LD], 1.25-mg [HD]) and a control sub-stance (3-mL water) were administered to each cat. Blood collection, echocardio-graphy, and oscillometric blood pressure measurements were performedrepeatedly for 12 h following each dose. Plasma concentrations of pimobendanand the active metabolite, O-desmethylpimobendan (ODMP), were quantified using

ney.edu.au (N.J. Beijerink).

.07.001hts reserved.

53

Pimobendan PK/PD in cats 311

Abbreviations

Ao Vel peak aortic velocAUC0eN area under the pCL/F apparent clearanCmax maximal plasmaDBP diastolic blood pFS fractional shorteHD high-dose (1.25 mHR heart rateIVSd thickness of theIVSs thickness of theLD low-dose (0.625LVIDd left ventricular iLVIDs left ventricular iLVPWd thickness of theLVPWs thickness of theMBP mean blood presODMP O-desmethylpimoPO per oralSBP systolic blood prt1/2 elimination half-Tmax time of maximalUHPLC-MS ultra-high-perforVz/F apparent volume

ultra-high-performance liquid chromatography tandem mass spectrometry. Cardio-vascular parameters were evaluated for between- and within-treatment effectsover time using linear mixed modelling.Results: Pimobendan was rapidly absorbed and converted to ODMP with the pimo-bendan AUC0eN greater than ODMP AUC0eN (ODMP:pimobendan AUC0eN ratio 0.6[LD] and 0.5 [HD]) despite a longer elimination half-life of ODMP (pimobendan t1/2 0.8 h vs. ODMP t1/2 1.6 h [LD]; pimobendan t1/2 0.7 h vs. ODMP t1/2 1.3 h [HD]).Averaged across all time points, pimobendan increased several measures of systolicfunction; however, its effect could not be further characterised. Although treat-ment was well-tolerated, two cats vomited following HD and another had a ventri-cular premature beat recorded following LD.Conclusions: The lower ODMP:pimobendan AUC0eN ratio compared to that ob-served previously in dogs suggests reduced metabolism in cats. Treatment effectswere observed in measures of systolic function; however, the duration of actionand differences in effects between the two pimobendan doses could not be charac-terised. Further studies are required to evaluate pimobendan in feline cardiovascu-lar medicine.ª 2016 Elsevier B.V. All rights reserved.

itylasma concentration curve extrapolated to infinityceconcentrationressureningg) pimobendan treatment

interventricular septum in diastoleinterventricular septum in systolemg) pimobendan treatmentnternal diameter in diastolenternal diameter in systoleleft ventricular posterior wall in diastoleleft ventricular posterior wall in systolesurebendan

essurelifeplasma concentrationmance liquid chromatography tandem mass spectrometryof distribution

Introduction

Pimobendan is utilised in the treatment of caninecongestive heart failure. It primarily producespositive inotropic and vasodilatory effects viaphosphodiesterase III inhibition and calcium

54

sensitisation [1e6]. Additional effects such aspositive lusitropy, improvements in myocardialcompliance, regional increases in coronary bloodflow, and positive effects on myocardial oxygenconsumption have also been reported [6e13].Following administration, it is metabolised to an

f Vetmedin 1.25-mg chewable tablets, Boehringer Ingelheim,North Ryde, NSW, Australia.

312 M. Yata et al.

active metabolite, O-desmethylpimobendan(ODMP, or UD-CG 212 CL), which induces similarinodilatory effects, primarily through phospho-diesterase III inhibition [14e16].

In the past decade, the off-label use of pimo-bendan in cats with congestive heart failure hasreceived increasing attention. A number of retro-spective studies evaluating pimobendan use in catswith heart failure due to a variety of aetiologiessuggested that the drug was tolerated well and didnot negatively impact morbidity and mortality[17e20]. Despite this, published literature on thecardiovascular effects of treatment in this speciesremains sparse.

To date, only one study has reported the directhaemodynamic effect of pimobendan in cats [21].This study utilised anaesthetised cats and cumu-lative dosing methods. Pimobendan produced adose-dependent increase in ventricular con-tractility and heart rate and reduction in diastolicblood pressure. The interpretation of these find-ings, however, is complicated by the influences ofanaesthesia, and the duration of drug effectswere not evaluated. More recently, the effect ofmultiple doses of pimobendan on left atrial func-tion and size in healthy cats was evaluated indi-rectly via echocardiography [22]. Althoughpimobendan treatment reduced maximal leftatrial diameter and volume, the left atrial func-tion did not change significantly. The cats utilisedin this study were sedated for echocardiography,and the duration of effect was not evaluated. Twoof the aforementioned retrospective studies alsoevaluated haemodynamic effects after a period oftreatment with multimodal therapy which inclu-ded pimobendan [17,18]. Limitations to the datafrom these studies include a wide range of time tofollow-up evaluation, lack of information regard-ing the temporal relationship of measured effectsto time of dosing, inability to differentiate theeffect of pimobendan from that of other medi-cations which were administered concurrently,and low numbers of animals for which follow-updata were available. Thus, the dose-responserelationship of pimobendan in cats remains poorlyunderstood.

In 2012, Hanzlicek et al. prospectively eval-uated the pharmacokinetics of oral pimobendanadministered to healthy cats at a dose similar tothat used in dogs (0.25e0.30 mg/kg administeredorally twice daily) [23]. A major finding from thisstudy was that the peak plasma concentration ofpimobendan was approximately ten times higherthan that previously described in dogs given similardoses [24], and the elimination half-life of pimo-bendan in cats was also longer. Unfortunately, the

55

active metabolite was not measured; as such, thetotal body exposure to all of the active moietiesrelated to pimobendan could not be clarified. It ispossible that the presence of higher pimobendanconcentrations may, for example, be due to arelative lack of conversion of this compound toODMP. This pimobendan metabolite is more potentbut less effective, than pimobendan in vitro[15,16]. Thus, inter-species differences in drugdisposition may contribute towards a difference incardiovascular response. In particular, reducedmetabolism of pimobendan may result in greatercardiovascular effects in cats. The importance ofclarifying the response to drug administration(pharmacodynamics) and need for further evalua-tion of pharmacokinetics in this species washighlighted.

The aim of the present study was to address thisknowledge gap by investigating the pharmacoki-netics and cardiovascular effects of two singledoses of pimobendan administered orally tohealthy cats. We hypothesized that conversion ofpimobendan to the active metabolite may be lowin cats and that treatment with pimobendan wouldcause measurable effects in echocardiographicparameters representing systolic function.

Animals, materials and methods

Animals

Eight healthy adult cats (two entire males, fiveneutered males, and one spayed female; weight3.7e4.8 kg; age 1e4 years) that were amenable tohandling were utilised in this study. The cats weresourced from a contract research facility. Each catunderwent a full physical examination, echo-cardiography, electrocardiography, and bloodpressure evaluation to ensure that no significantphysical or cardiovascular abnormalities werepresent. The methods utilised in this study wereapproved by the University of Sydney Animal EthicsCommittee (Project 678).

Study design

A randomised, single-blinded, cross-over designwas used to investigate the pharmacokinetics andcardiovascular effects of two single doses ofpimobendanf in these cats. Three treatment con-ditions consisting of low-dose pimobendan (LD e

Pimobendan PK/PD in cats 313

0.625-mg pimobendan PO; equivalent to a mediandose of 0.14 mg/kg with range 0.13e0.17 mg/kg),high-dose pimobendan (HD e 1.25-mg pimobendanPO; equivalent to a median dose of 0.28 mg/kgwith range 0.27e0.32 mg/kg), and water as acontrol (3-mL water PO) were tested in each cat.Before the study, each cat was assigned to receiveone of three treatment sequences (water, LD, HD;HD, water, LD; and LD, HD, water) via a randomdraw by an assistant. All investigators involved insampling procedures remained blinded to theorder of treatments until measurements werecompleted.

g Eksigent ekspert ultra LC100; AB Sciex, Redwood City, CA,USA.h AB Sciex API3200, AB Sciex, Framingham, MA, USA.i

Schedule of events

The study was conducted over a two-month period(JanuaryeMarch, 2015) at the University Veteri-nary Teaching Hospital, Sydney. Following arrivalat the clinic, cats were acclimatised for a mini-mum of three days before receiving the first doseof treatment. During this time, cats were alsofamiliarised with echocardiography and bloodpressure techniques. Cats were fasted overnightfor a minimum of 10 h before administration ofeach dose.

On the first day of dosing, cats were treatedwith the first assigned treatment by an assistantwho did not perform any of the sampling proce-dures or recording of measurements. The inves-tigators were not present in the room at the timeof dosing. Oral tablet dosing was followed byadministration of water (3 mL) to encourageoesophageal transit. Blood samples (3 mL) werecollected via jugular or cephalic venipuncture at20 min, 1, 2, 4, and 6 h following dosing. Echo-cardiography, heart rate (HR), and blood pressuremeasurements were obtained before dosing, then20, 40 min, 1, 2, 4, 6, 8, 10, and 12 h followingdosing. Cats were fed 2 h following dosing. Theseprocedures were repeated for each subsequentdose, which was administered 1 week apart to givea seven-day wash-out period between doses. Thiswas deemed to be adequate for recovery fromstudy procedures and was greater than five timesthe elimination half-life of pimobendan (w1.3 h)as described in a previous study [23].

Deviations from this protocol occurred on sev-eral occasions. Cat 7 received a dose of treatment(HD) 2 h before the scheduled time on the first dayof treatment. The cat was fed immediatelythereafter in an attempt to reduce absorption ofthe drug, and no cardiovascular measurementswere taken on this day. This cat was allocated adifferent treatment order, and the cat received

56

the newly allocated treatment (water) 48 h later.Because cardiovascular measurements were nottaken on this day, and based on the previouslyreported elimination half-life of pimobendan incats [23], the 48 h wash-out period was deemed tobe adequate.

On another occasion, one dose of treatment (HD)thought to have been given to cat 8 was found intactin the cage 3.5 h following dosing. It was suspectedthat this tablet was most likely regurgitated shortlyfollowing administration. In order to maintainblinding, the investigator performing echocardiog-raphy (N.J. Beijerink)was not advised of this finding.The data collected on this day was categorised asbeing under the water treatment, and the cat wastreated the subsequent week with HD pimobendaninstead of the scheduled water treatment. Bloodwas not collected from one cat (cat 8) that did nottolerate venipuncture, andone other cat (cat 1) onlyhad blood collected twice after treatment with LDdue to development of strong aversion towards thisprocedure at this occasion.

Analytical method

Blood samples were collected in plastic tubescoated with lithium heparin, placed on ice, andcentrifuged at 15,800 RPM for 150 s within 20 minof collection. Plasma was separated and placed onice for up to 1 h before freezing. Plasma wasstored at or below �20 �C for four to six monthsbefore analysis. Greater than 90% recovery ofpimobendan from frozen canine plasma samplesstored for two months has been reported [25], butthe stability of pimobendan in frozen feline plasmais unknown.

Plasma concentrations of pimobendan andODMP were determined by ultra-high-performanceliquid chromatographyg coupled with a massspectrometerh (UHPLC-MS) at a commercial ana-lytical laboratoryi.

A previously validated assay with minor adjust-ments was employed [25]. Briefly, 40 mL ofpimobendan-d3 (internal standard) and 350 mL ofacetonitrile were added to 200 mL of plasma afterthawing at room temperature. The mixture wasvortexed and centrifuged. Subsequently, 200 mL ofwater was added to improve the chromatographyand centrifuged again. The supernatant waspassed through a 0.45-mm Hydraflon� filter, and5 mL of this solution were injected into the system.

PIA PHARMA Pty Ltd, Gladesville, NSW, Australia.

314 M. Yata et al.

Reverse-phase chromatography with a DionexAcclaim Polar Advantage II column (2.1 � 150 mm,3 mm)j maintained at 40 �C was performed. Themobile phase consisted of formic acid (0.1% inwater) and acetonitrile in gradient mode at0.5 mL/min, and the total run time was 3 min foreach sample. Tandem mass spectrometry was uti-lised for identification of the compounds, andcalibration curves (R2 > 0.99) spanning 0.2e60 ng/mL (pimobendan) and 0.4e25 ng/mL (ODMP) gen-erated from matrix matched calibration standardswere utilised for quantitation of each compound.The lower limit of quantitation was 0.2 ng/mL forpimobendan and 0.4 ng/mL for ODMP. All sampleswere analysed on a single day. For concentrationsabove the lower limit of quantitation, the meanaccuracy and precision (% CV) for pimobendanranged between 99.9e116% and 0.9e7.4% respec-tively and the accuracy and precision (% CV) forODMP ranged between 105.7e116.9% and1.8e8.0% respectively.

Echocardiography and blood pressuremeasurements

Cats were manually restrained in right and leftlateral recumbency without sedation. Echo-cardiography was performed by a veterinary car-diologist certified by the European College ofVeterinary Internal Medicine (N.J. Beijerink) usinga commercially available ultrasound systemequipped with a 3.5e8 MHz sector transducer andsimultaneous electrocardiographyk. 2D evaluationin the right parasternal short axis view was utilisedto obtain the thickness of the interventricularseptum in systole and diastole (IVSs, IVSd), leftventricular internal diameter in systole and dia-stole (LVIDs, LVIDd), and thickness of the leftventricular posterior wall in systole and diastole(LVPWs, LVPWd). This method of evaluation of theleft ventricular indices was selected as end-systolic cavity obliteration, which produced inter-ference caused by papillary muscles entering thesampling beam on M-mode, was present at timesduring the study. The fractional shortening (FS)was calculated as (LVIDd e LVIDs)/LVIDd * 100. Theleft apical five chamber view was utilised tomeasure the peak aortic velocity (Ao Vel). The HRwas calculated using the ReR interval betweenfour to five consecutive beats captured during AoVel interrogation.

j Thermo Fisher Scientific, Scoresby, VIC, Australia.k Vivid I; General Electric Healthcare, Silverwater, NSW,

Australia.l VetHDO monitor; Memo Diagnostic, Darmstadt, Germany.

57

An oscillometric blood pressure devicel coupledwith an appropriately-sized inflatable cuff(40e50% of the circumference of the limb) placedon the tail base or the right foreleg was utilised toobtain the systolic (SBP), mean (MBP), and dia-stolic (DBP) blood pressures. The cats were placedin sternal recumbency for tail measurements orrestrained in a sitting position with the limb heldup at the level of the chest for forelimb meas-urements. Measurements were taken repeatedlyuntil three consecutive or near-consecutive read-ings with good-quality pressure curves and littlevariability were obtained.

For each cat at each time point, echocardio-gram, HR, and blood pressure measurements wereobtained in triplicates and averaged. Consecutivebeats were utilised for the majority ofmeasurements.

Observer variation

On the day after arrival at the clinic, six of theeight cats underwent echocardiography at 9 AM, 11AM, 1 PM, 3 PM, and 5 PM. Echocardiography wasperformed by a single investigator (N.J. Beijerink),and six cardiac cycles from each time point wereutilised to obtain the coefficient of variation forthe investigator. The overall coefficient of varia-tion (mean � standard deviation) was 7 � 3% forIVSd; 10 � 5% for IVSs; 4 � 2% for LVIDd; 8 � 5% forLVIDs; 9 � 3% for LVPWd; 10 � 4% for LVPWs; and3 � 2% for Ao Vel. The effect of time on thecoefficient of variation for each parameter wasassessed using linear mixed modellingm. The timewas set as a fixed effect, and the cat ID was set asa random effect. There were no significant effectsof time on the coefficient of variation (p>0.05).Inter-day and inter-cat variations were notassessed.

Data analysis

Pharmacokinetic analysis was performed usingnon-compartmental analysis on the plasmaconcentration-time data using a Microsoft Excelplug-in software program (PK Solver) [26]. Thelinear trapezoidal method was utilised for calcu-lation of the area under the curve to the lastobserved concentration. The slope of the terminalportion of the concentration-time plot (kel) wasutilised to extrapolate this to infinity (AUC0eN).The elimination half-life was calculated as ln2/kel.The apparent clearance (CL/F ¼ dose/AUC0eN)

m GenStat 16th Edition, VSN International Ltd, Hemel Hemp-stead, UK.

Pimobendan PK/PD in cats 315

and the apparent volume of distribution (CL/F/kel)were also calculated for pimobendan. The phar-macokinetic data were reported as median andrange unless otherwise specified.

The effect of each treatment over time wasdepicted graphically for descriptive analysis.Statistical analysis was performed using GenStatm.Evaluation of the QeQ plot for normality wasperformed on the haemodynamic data collected.Where the data were not normally distributed, log(Ao Vel, IVSd, LVIDs, LVPWs) or power (FS) trans-formations were applied to approximate normal-ity. A linear mixed model with restricted maximallikelihood estimation was performed to identifythe effect of treatment and time on the car-diovascular parameters. The assumptions of thismodel were assessed through visual inspection ofresidual plots. The model used to evaluate theeffect of treatment and time on each of the car-diovascular parameters was as follows:

bYðcardiovascular parameterÞ ¼ Treatment

þ Timeþ Treatment � Time

þCat IDðrandom effectÞ þ ε

Where the Treatment*Time interaction was notsignificant (p>0.05), the term was dropped fromthe model. For all significant effects (p<0.05),subsequent evaluation of pairwise comparisonswas performed using least square differencesof the means with a nominated significance levelof 5%.

Results

Drug dosing and adverse effects

None of the cats voluntarily ate the treatmentsoffered; however, treatment was tolerated well inall cats during the study.

Possible minor adverse effects were observedduring the study. One cat (cat 6) developed self-limiting vomiting within 10 min of treatment withHD. Another cat (cat 5) developed self-limitingvomiting and diarrhoea around 24 h followingtreatment with HD. A third cat (cat 3) had a singleventricular premature beat recorded on the ECGduring echocardiography 1 h following treatmentwith LD. No other arrhythmias were observed inany of the cats (including this cat at subsequenttreatments) during the remainder of the studyperiod. No serious adverse effects were encoun-tered at any point in time during or after the study.

58

Pharmacokinetics

Blood samples were collected from seven catsfollowing HD treatment (cats 1e7), six cats fol-lowing LD treatment (cats 2e7), and seven catsfollowing water treatment (cats 1e7). One cat (cat6) vomited a small amount of liquid within 10 minof the HD treatment. Although the tablet could notbe identified in the vomitus, the measured plasmaconcentrations were not higher than thoseobtained following LD treatment in this cat. Inanother cat (cat 7), the measured plasma con-centrations were much lower than the concen-trations observed in other cats at both the HD andLD treatments. There was no discernible reason(e.g. vomiting, diarrhoea, hypersalivation, incom-plete dosing, physical exam abnormalities, orother abnormalities in assessment of clinical well-being) throughout the course of the study for this.The data for these two cats (cats 6 and 7) werereported separately.

Pimobendan treatment resulted in detectableconcentrations of pimobendan and ODMP from thetime of first sampling in the majority of cats(Fig. 1). As expected, HD tended to produce higherplasma concentrations of both compounds com-pared to LD. In the majority of cats, the maximalplasma concentration of pimobendan occurred atthe first sampling point (20 min); as such, theascending phase of plasma drug concentrations forpimobendan could not be captured for these cats.However, the elimination phase was adequatelycaptured (percentage extrapolation for AUC0eN

was <7% for pimobendan and <14% for ODMP forall cats, including cats 6 and 7).

A summary of pharmacokinetic parameters ispresented in Table 1. Pimobendan and ODMPpharmacokinetics in dogs treated with0.26e0.28 mg/kg of oral pimobendan solution [25]has also been included in this table for comparison.

Both the HD and LD treatments resulted in rapidabsorption of pimobendan and conversion intoODMP (median pimobendan Tmax 40 min [LD] and20 min [HD] and ODMP Tmax 1 h [LD and HD]). Forboth doses, the AUC0eN for pimobendan appearedto be higher than for ODMP. Inspection of the ratiosof AUC0eN of ODMP to pimobendan showed amedian AUC0eN ODMP:pimobendan of 0.6 (range0.5e0.8) for LD treatment and 0.5 (range 0.4e0.7)for HD treatment.

A summary of pharmacokinetic parameters forcats 6 and 7 are presented in Table 2.

No active compounds were detected in samplesfrom all seven cats in which plasma samples wereobtained following water treatment.

Figure 1 Individual concentration-time plots for plasma concentrations of pimobendan (A) and O-desmethylpi-mobendan (ODMP) (B) following single oral administration of low-dose (0.625 mg; n ¼ 4) and high-dose (1.25 mg;n ¼ 5) pimobendan. Concentration-time plots of pimobendan and ODMP in cats 6 (C and D) and 7 (E and F) arepresented separately. Dashed line ¼ low-dose pimobendan; solid line ¼ high-dose pimobendan.

316 M. Yata et al.

59

Table

1Pharm

aco

kineticparameters

(median,range

)followingnon-compartmentalanalysisofplasm

aco

nce

ntrationsofpim

obendan(Pim

o)anditsmetabolite

(ODMP)in

cats

dosedwithsingleoraltreatm

ents

oflow-dose

(0.625

mg;

n¼

4)andhigh-dose

(1.25mg;

n¼

5)pim

obendan.

Pim

o0.62

5mga

ODMP

0.62

5mg

Pim

o1.25

mgb

ODMP

1.25

mg

Pim

o(Dog,

w5mgc)[25]

ODMP(Dog,

w5mg)

[25]

Cmax(ng/

ml)

29.9

(24.7e34

.7)

9.7(8.1e14

.2)

45.4

(41.4e70

.9)

16.5

(11.3e21

.8)

18.6

(6.1e25

.3)

16.2

(6.0e22

.3)

Tmax(h)

0.7(0.3e1.0)

1.0(1.0e1.0)

0.3(0.3e1.0)

1.0(0.3e2.0)

1.1(0.5e2.0)

1.3(0.8e2.0)

AUC0eN

(ng/

ml*h)

53.5

(50.1e65

.3)

36.8

(23.1e

38.8)

89.9

(66.3e16

0.5)

61.1

(29.9e71

.0)

27.1

(15.2e

44.2)

42.1

(25.1e52

.7)

Vz/F(L/k

g)2.9(2.8e3.0)

e3.1(2.6e3.9)

e15

.5(5.9e22

.3)

eCL/

F(m

l/min/k

g)42

.9(33.7e55

.9)

e51

.1(27.4e84

.9)

e17

6.5(105

.0e30

1.0)

et 1

/2(h)

0.8(0.6e1.0)

1.6(1.3e1.8)

0.7(0.5e1.1)

1.3(1.1e1.9)

0.9(0.7e1.1)

1.6(1.3e1.9)

AUC0eN

ODMP:Pim

o0.6(0.5e0.8)

0.5(0.4e0.7)

1.6(1.2e1.7)

Forreference

,pharm

acokineticparameters

(median,range

)previouslyreportedforpim

obendanandODMPin

dogs

(n¼

4)from

anotherstudyin

whichasimilarassaywasutilised[25]

hasbeenincludedin

thelast

twoco

lumns.

Cmax¼

maximalplasm

aco

nce

ntration;Tmax¼

timeofmaximalplasm

aco

nce

ntration;AUC0eN

¼areaundertheco

nce

ntration-tim

eplotextrapolatedto

infinity;

Vz/F

¼apparent

volumeofdistribution;CL/

F¼

apparentclearance

;t 1

/2¼

eliminationhalf-life.

aEquatesto

amedianof0.14

mg/

kgwithrange

0.13

e0.17

mg/

kg.

bEquatesto

amedianof0.28

mg/

kgwithrange

0.26

e0.34

mg/

kg.

cEquatesto

amedianof0.27

mg/

kgwithrange

0.26

e0.28

mg/

kg.

Pimobendan PK/PD in cats 317

60

Cardiovascular effects

Data from 6 cats (cats 1e5 and cat 8) wereincluded in the cardiovascular analysis. Thedata from cats 6 and 7 were excluded foraforementioned reasons pertaining to thepharmacokinetics. Although cat 8 did not haveblood samples taken (and therefore, did notundergo all of the same procedures as theother seven cats in the study), this was con-sistent across all three treatments. The datafrom cat 1, who did not complete the fullextent of blood collection after LD treatment,were also included as this cat largely under-went the same conditions for all threetreatments.

The baseline haemodynamic values weresimilar for all treatments (Table 3). However,the MBP and DBP at baseline for HD treatmentwere significantly lower than for water treat-ment (p<0.05). The MBP for HD was also sig-nificantly lower than for LD treatment(p<0.05).

The effect of each treatment over time isdemonstrated in Fig. 2. On visual inspection ofthe graphs, pimobendan treatment appearedto increase Ao Vel, FS, IVSs, IVSd, and LVPWdwithin the first few hours of treatment. LVIDsand LVIDd appeared to decrease. However,similar trends were observed following watertreatment for many of these parameters. Inaddition, a high degree of variability wasobserved for all of these parameters.

Statistical analysis revealed that therewere no significant differences betweenwater and pimobendan treatments at specifictime points following dosing. This was evi-denced by the non-significant interactionterm for all cardiovascular parameters otherthan DBP. Within this parameter, thesignificant between- and within-treatmentinteractions occurred sporadically and withno discernible pattern to indicate a true bio-logical effect of treatment (Fig. 2). Thus, forall parameters other than DBP, the model wasreduced to:

bYðcardiovascular parameterÞ ¼ Treatment

þ TimeþCat IDðrandom effectÞ þ ε

Using this model, the effect of treatmentaveraged across the entire sampling period(0e12 h) was evaluated. The relevant groupmeans are presented in Table 4. Averagedacross all time points, both HD and LD treat-ments were associated with a significantly

Table 2 Pharmacokinetic parameters following non-compartmental analysis of plasma concentrations of pimo-bendan (Pimo) and its metabolite (ODMP) in cats 6 and 7 dosed with single oral treatments of low-dose (0.625 mg)and high-dose (1.25 mg) pimobendan.

Pimo0.625 mga

ODMP0.625 mg

Pimo1.25 mgb

ODMP1.25 mg

Cat 6 Cmax (ng/ml) 23.9 8.9 24.0 8.2Tmax (h) 0.3 1.0 0.3 0.3AUC0eN (ng/ml*h) 32.3 20.4 30.0 22.5Vz/F (L/kg) 3.6 e 7.0 eCL/F (ml/min/kg) 74.1 e 159.1 et1/2 (h) 0.6 1.2 0.5 1.4

AUC0eN ODMP:Pimo 0.6 0.8Cat 7 Cmax (ng/ml) 12.4 5.5 8.3 5.4

Tmax (h) 0.3 0.3 2.0 2.0AUC0eN (ng/ml*h) 16.4 13.9 20.4 16.1Vz/F (L/kg) 9.8 e 17.1 eCL/F (ml/min/kg) 169.1 e 272.3 et1/2 (h) 0.7 1.2 0.7 1.3

AUC0eN ODMP:Pimo 0.8 0.8

Cmax ¼ maximal plasma concentration; Tmax ¼ time of maximal plasma concentration; AUC0eN ¼ area under the concentration-time plot extrapolated to infinity; Vz/F ¼ apparent volume of distribution; CL/F ¼ apparent clearance; t1/2 ¼ elimination half-life.a Equates to 0.14 mg/kg (cat 6) and 0.17 mg/kg (cat 7).b Equates to 0.29 mg/kg (cat 6) and 0.33 mg/kg (cat 7).

Table 3 Baseline values (mean, SD) of car-diovascular parameters for water (3-mL water), low-dose pimobendan (0.625 mg), and high-dose pimo-bendan (1.25 mg) treatments in six cats.

Parameter(units)

Water Low dose High dose

Ao Vel (m/s) 1.0 (0.1) 1.0 (0.1) 1.1 (0.1)HR (bpm) 178.4 (32.1) 176.7 (28.2) 172.1 (17.5)IVSd (mm) 3.9 (0.2) 3.8 (0.4) 3.8 (0.4)IVSs (mm) 6.5 (0.8) 6.6 (0.9) 6.4 (0.7)LVIDd (mm) 16.1 (1.8) 16.2 (1.4) 16.2 (1.7)LVIDs (mm) 9.0 (1.8) 8.5 (1.9) 9.0 (2.1)LVPWd (mm) 4.4 (0.8) 4.5 (0.4) 4.5 (0.7)LVPWs (mm) 6.4 (0.8) 6.4 (0.7) 6.7 (0.8)FS (%) 44.3 (10.0) 46.9 (13.1) 44.8 (11.1)DBP (mmHg) 82.6 (4.0) 77.7 (7.0) 72.1 (5.4)a

MBP (mmHg) 104.4 (6.7) 102.4 (9.6) 93.6 (10.5)a,b

SBP (mmHg) 144.2 (16.9) 147.7 (20.8) 132.7 (22.2)

Key: Ao Vel ¼ aortic velocity; HR ¼ heart rate; IVSd/s ¼ interventricular septum thickness in diastole/systole;LVIDd/s ¼ left ventricular internal diameter in diastole/systole; LVPWd/s ¼ left ventricular posterior wall thicknessin diastole/systole; FS ¼ fractional shortening;DBP ¼ diastolic blood pressure; MBP ¼ mean blood pressure;SBP ¼ systolic blood pressure; SD, standard deviation.Model: Y (cardiovascular parameter) ¼ Treatment þ Cat ID(random effect) þ Ɛa Denotes significant differences (p<0.05) from water.b Denotes significant differences (p<0.05) from low dose.

318 M. Yata et al.

61

higher Ao Vel, IVSs, IVSd, and significantly lowerLVIDs, than water treatment (p<0.05). HD treat-ment was also associated with a significantlyhigher FS than water treatment (p<0.05). The SBPwas higher with LD treatment than with HD orwater (p<0.05). The HR was lower with HD treat-ment than with LD or water (p<0.05). The LVPWswas also higher for HD treatment than for LDtreatment (p<0.05).

Discussion

In this study, the pharmacokinetics and pharma-codynamics of two single doses of pimobendanwere evaluated in healthy, conscious cats. Thepharmacokinetic relationship between pimo-bendan and ODMP was identified and the haemo-dynamic responses following pimobendantreatment were described.

The pimobendan pharmacokinetics from thisstudy were generally in agreement with thosepreviously reported in the cat [23]. There weresome differences in the magnitude of parametersthat may be attributable to differences in samplingschedule, analytical method, and method ofpharmacokinetic evaluation. In both studies,pimobendan was absorbed rapidly after oral

Figure 2 Graphical representation of cardiovascular parameters over time following water (3-mL water), low-dose(0.625-mg pimobendan), and high-dose (1.25-mg pimobendan) treatments in six cats. The mean and standard devi-ation have been plotted. Abbreviations: Ao Vel ¼ aortic velocity; DBP ¼ diastolic blood pressure; FS ¼ fractionalshortening; HR ¼ heart rate; IVSd/s ¼ interventricular septum thickness in diastole/systole; LVIDd/s ¼ left ventricularinternal diameter in diastole/systole; LVPWd/s ¼ left ventricular posterior wall thickness in diastole/systole;MBP ¼ mean blood pressure; SBP ¼ systolic blood pressure. Solid lines represent water (3 mL water) treatment.Dotted lines represent low-dose (0.625-mg pimobendan) treatment. Dashed lines represent high-dose (1.25-mgpimobendan) treatment. a and b denote significant deviations from time zero (p<0.05) at the specified time for thehigh-dose (1.25-mg pimobendan) and water (3-ml water) treatments respectively. X and Y indicate significant dif-ferences (p<0.05) between the high-dose and water treatments and low-dose and water treatments respectively atthe specified time.

Pimobendan PK/PD in cats 319

62

Fig. 2 (continued)

320 M. Yata et al.

administration, displayed a high apparent volumeof distribution, and was eliminated readily fromthe plasma. High plasma concentrations of pimo-bendan were observed, with the Cmax and AUC0eN

being higher than previously reported values fromstudies evaluating pimobendan pharmacokinetics

63

in dogs given similar mg/kg doses [24,25]. Poten-tial reasons for these findings include a higherbioavailability or a lower clearance(i.e. conversion to ODMP or elimination via faecal/urinary excretion) in the cat.

Table 4 Treatment means averaged across time(0e12 h post treatment) for select cardiovascularparameters following water (3-mL water), low-dose(0.625-mg pimobendan), and high-dose (1.25-mgpimobendan) treatments in six cats.

Parameter (units) Water Low dose High dose

Ao Vel (m/s) 1.18 1.23a 1.27a

HR (bpm) 203.5 202.6 195.9a,b

IVSd (mm) 3.80 4.07a 4.01a

IVSs (mm) 6.78 7.16a 7.03a

LVIDs (mm) 7.83 7.38a 7.16a

LVPWs (mm) 6.46 6.34 6.62b

FS (%) 52.09 53.44 54.78a

SBP (mmHg) 143.1 148.7a 142.9b

Key: Ao Vel ¼ aortic velocity; HR ¼ heart rate; IVSd/s ¼ interventricular septum thickness in diastole/systolerespectively; LVIDs ¼ left ventricular internal diameter insystole; LVPWs ¼ left ventricular posterior wall thickness insystole; FS ¼ fractional shortening; SBP ¼ systolic bloodpressure.Model: Y (cardiovascular parameter) ¼ Treatment þ Time þCat ID (random effect) þ Ɛa Denotes significant differences (p<0.05) from water.b Denotes significant differences (p<0.05) from low dose.

Pimobendan PK/PD in cats 321

Intravenous administration of pimobendan andODMP was not performed in our study. As such, theabsolute bioavailability could not be determined.Clearance and volume of distribution reportedhere are also estimated values corrected for anunknown bioavailability. This is a limitation of thestudy that prohibits between-species evaluationsof these parameters. However, an important fac-tor that can be explored through our data is thepotential for inter-species differences in themetabolism of this drug.

Comparisons of our data with published data indogs show that the elimination half-life of pimo-bendan and ODMP were similar between cats anddogs (Table 1). Interestingly, the AUC0eN ofpimobendan appeared higher than the AUC0eN ofODMP in cats, which is in contrast to what hasbeen reported in dogs (Table 1). This resulted inthe ratio of AUC0eN ODMP:pimobendan beinglower in cats than in dogs (median 0.5, range0.4e0.7 in cats for HD vs. median 1.6, range1.2e1.7 in dogs). This reduced AUC0eN ratio inthe face of similar elimination profiles in dogs andcats suggests a relatively reduced conversion ofpimobendan to ODMP in cats. Pimobendan con-version to ODMP involves specific cytochrome P-450 isoenzymes in humans [27]. In addition, bothpimobendan and ODMP are ultimately glucur-onidated and excreted [28]. Because differencesin the activity of cytochrome P-450 isoforms and

64

ability to glucuronidate compounds have beenidentified between dogs and cats [29e34], thesepathways could be contributing towards the dif-ference in metabolism of pimobendan in thesetwo species.

A difference in pharmacokinetics between spe-cies may result in inter-species variation inresponse to treatment. Thus, two doses of pimo-bendan were utilised in our study to investigatethe potential effect of dose. A non-invasive studydesign which we have previously employed for thedetection of pimobendan effects in dogs [25] waschosen to avoid the impacts of general anaesthesiaand instrumentation whilst providing the addi-tional benefit of being clinically applicable.

Averaged across the sampling period, HD and LDtreatments were associated with significant dif-ferences from water in some echocardiographicparameters representing systolic function(increased Ao Vel and FS and decreased LVIDs forHD; increased Ao Vel and decreased LVIDs for LD).These changes reflect the positive inotropiceffects of pimobendan. In general, HD was sig-nificantly different to water in a greater number ofparameters than LD was to water.

We were unable to characterise the duration ofeffects, timing of maximal effects, and any dif-ference in effects between the two doses ofpimobendan. Major complicating factors includedthe high degree of variability in the data, the smallnumber of cats in our study, and the presence ofsimilar haemodynamic trends which were observedfollowing the water treatment. These contributedtowards obscuring both the within- and between-treatment effects over time as evidenced throughthe non-significant interaction term in the model.It is possible that the intensive handling, ven-ipuncture, and greater environmental activity inthe hospital during the first few hours of samplingmay have contributed towards increased anxiety inthe cats regardless of the treatment administered.With so many similarities in the cardiovascularchanges expected with anxiety and with pimo-bendan treatment, and because pimobendantreatment did not result in haemodynamic changesthat extended beyond reported reference ranges[35] on the majority of occasions, it would beimpossible to fully separate the relative con-tributions of anxiety and treatment in consciousanimals. Retrospectively, more extensive trainingof the cats for these procedures may have beenhelpful to reduce some of the anxiety experiencedduring the study.

It is also possible that non-invasive methods ofmonitoring may not have been sensitive enough to

322 M. Yata et al.

detect consistent treatment effects in cats. Onseveral occasions with both HD and LD treatment,end-systolic cavity obliteration of the left ven-tricle was observed. This may have contributedtowards measurement errors and prohibited dif-ferentiation of the maximal positive inotropiceffects between the two doses of pimobendan.With the size of the heart being small in cats, smallinconsistencies in measurements between indi-vidual cats and across time points could contributetowards enough variability in the data to obscuretreatment effects. Variable correlations betweendirect and oscillometric techniques of blood pres-sure monitoring have also been observed pre-viously in this species [36e39]. It is possible thatthis contributed towards the wide range of bloodpressures observed.

In our study, there was a focus on measuringventricular systolic indices as they were commonlymeasured parameters which reflect the primaryeffect of pimobendan. However, diastolic dysfunc-tion plays a large role in feline heart disease due tothe propensity of this species to develop hyper-trophic and restrictive cardiomyopathies. In theseconditions, atrial systolic function, which was notevaluated in the present study, becomes vital inmaintaining ventricular filling. Therefore, possibleeffects of pimobendan on ventricular diastolic andatrial systolic functions need further evaluation.

Treatment with a single oral dose of pimo-bendan did not result in any major adverse eventsin the study cats. Vomiting occurred in cat 6shortly after HD treatment. In cat 5, vomitingoccurred around 24 h following HD treatment. Thetemporal relationship between dosing and symp-toms, as well as the nature of the symptomsobserved (productive vomiting associated withnausea), make an adverse drug reaction possiblefor cat 6 but questionable in cat 5. Minor gastro-intestinal signs have been observed in 0e11% ofpimobendan-treated cats in prior studies[17,19,20,23], and none of the cats in this studydeveloped gastrointestinal signs with LD or watertreatment. Thus, it is possible that any potentialminor gastrointestinal adverse effects may bedose-dependent or not necessarily repeatable.

One cat (cat 3) had a single ventricular pre-mature complex recorded around 1 h followingtreatment with LD. No further arrhythmias werenoted in this cat on this occasion, and arrhythmiaswere not noted following HD or water treatment inthis cat. Although further testing to evaluate othercauses of this arrhythmia were not performed, it ispossible that this cat may have had a pre-existingarrhythmia which was not otherwise captured

65

during brief electrocardiography preceding andaccompanying the screening echocardiogram.There are contrasting reports regarding the effectsof pimobendan on cardiac rhythm in other species.Some studies suggested pro-arrhythmic effects[40,41] while others did not document this[42e52]. To date, no pro-arrhythmic effects ofpimobendan have been identified in cats [18,20];however, large-scale evaluations for this effecthave not been conducted.

One cat (cat 7) had lower plasma concentrationsof pimobendan and ODMP following both LD and HDwhen compared to other cats and was thereforeexcluded from group analysis. However, theAUC0eN ODMP:pimobendan ratio was similar tothat of other cats, thus suggesting a similarity inthe extent of metabolism of pimobendan to ODMP.Although no abnormalities were identified in theinitial health assessment or during the study, it ispossible that this cat had undetected gastro-intestinal, hepatic, or plasma protein abnormal-ities that contributed towards lower bioavailabilityor altered distribution of the drug. The inclusion ofserum biochemistry in the screening examinationor retrospective diagnostic testing of gastro-intestinal function may have provided furtherinsight. Alternatively, it is possible that the datafrom this cat may be representative of one end ofthe spectrum of drug concentrations in the generalpopulation of cats. A population-based pharma-cokinetic study in a larger number of individualsmay aid in further evaluation of this anomaly.

There were a number of other limitations in thisstudy that may have influenced our findings. Thisstudy utilised a reduced sampling protocol forblood collection which resulted in depletion ofaround 6% of the total blood volume each week inthe smallest cat. This protocol did not allow foridentification of the ascending phase of plasmaconcentrations in the majority of cats. Thus, thetrue Cmax and Tmax may not have been captured.Although a higher number of plasma samples mayhave facilitated a more accurate pharmacokineticanalysis, this had to be balanced with the effect ofbleeding on the cardiovascular system. Ideally, thebetween-day coefficient of variation for the car-diologist should also have been assessed, and thegender of cats and order of treatment should havebeen fully balanced. The small number of animalsalso precluded adjustments for these imbalancesin the statistical model.

Had the treatment effects been more readilyappreciable, the data could have also been ana-lysed using simultaneous pharmacokinetic-pharmacodynamic modelling to provide further

Pimobendan PK/PD in cats 323

insight on the dose-response relationship. Based onour findings, however, it would seem that evaluat-ing a wider range of cardiovascular parameters tomonitor treatment effects and employing furthermeasures to minimise anxiety in the study catswould be advisable to obtain more reliable data forthis purpose. Pimobendan is marketed as a racemicmixture; however, stereoselective pharmacoki-netics and pharmacodynamics have been observedin prior studies [6,53e56]. Thus, evaluation ofenantiomer kinetics and effects may also aid inrefining such a model to describe the dos-eeresponse relationship in this species.

The data from this study were collected fromhealthy cats; however, the effect of drug treat-ment may be different in cats with cardiovasculardisease. In addition, this drug is typically admin-istered chronically, thus necessitating furtherevaluations in the form of multidose studies.Ultimately, further pharmacological inves-tigations and rigorous prospective clinical trials incats with cardiovascular diseases are required toclarify the optimal dosing regimen, identifywhether cardiovascular effects are detectable inthe target animals for this drug, and whetherthese effects translate to positive clinicaloutcomes.

Conclusions

Pimobendan treatment was well-tolerated infasted cats. Minor potential adverse effects con-sisting of vomiting and documentation of a singleventricular premature contraction were observedin our study. The results from this study suggest areduced metabolism of pimobendan into theactive metabolite in cats compared to dogs.Treatment effects with pimobendan at both1.25 mg (HD) and 0.625 mg (LD) were observed inmeasures of systolic function; however, theduration of action and differences in effectsbetween the two pimobendan doses could not becharacterised. These findings can be used toguide further investigations into the safety andefficacy of pimobendan treatment in felinepatients.

Conflicts of Interest

M. Yata received financial support from LuodaPharma, the Eric Horatio Maclean Scholarship, andGoldia and Susie Lesue Scholarship whilst under-taking this project.

66

Luoda Pharma sponsored additional studiesevaluating pimobendan in dogs which were con-ducted by M. Yata, A. McLachlan, D. Foster, and N.Beijerink. Luoda Pharma also contributed towardsUHPLC-MS analysis of plasma samples; however,Luoda Pharma did not provide the pimobendanproduct used in this study.

Interim results of this study were presented atthe 2015 ECVIM congress.

Acknowledgements

This study was funded by the Thea Jean Schna-kenberg Bequest, J.M & J.R. Stewart Legacy, andMargot Roslyn Flood Bequest. Luoda Pharma pro-vided funds for UHPLC-MS analysis ofplasma samples. The authors would like to thankJoe Pippia (PIA PHARMA Pty Ltd, Gladesville,NSW) for development and performance of theUHPLC-MS assay; the University VeterinaryTeaching Hospital, Sydney for provision of facili-ties to conduct this study; Kelly Amaro and Eli-sabeth Newfield for their assistance with handlingof the cats; Dr Navneet Dhand, Dr Peter Thomson,and Dr Alan Marcus for advice on statisticalanalysis.

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Available online at www.sciencedirect.com

ScienceDirect

Chapter 4:

Evaluation of the natural course of disease in eleven dogs

with congestive heart failure due to myxomatous mitral valve

disease treated with combination medical therapy

69

Author contribution statement

Mariko Yata was the primary investigator and author of the work reported in this chapter and

was involved in all aspects of study design, data collection, data analysis, and reporting.

Niek Beijerink provided guidance on the study design, assisted with data collection, and

critically reviewed this chapter.

Stephen Page provided guidance on the study design and critically reviewed this chapter.

Mariko Yata Date__ 14/8/2018____

Niek Beijerink Date___19/08/2018___

Stephen Page Date___14/8/2018____

70

Introduction

The most commonly acquired cardiovascular disease in dogs is myxomatous mitral valve

disease (MMVD), which is characterised by progressive degeneration of affected valves.

The accompanying effects on cardiovascular haemodynamics and activation of various

neuroendocrine systems, though initially beneficial, become maladaptive and deleterious

with chronicity. The natural course of disease is variable; yet typically, several years of

asymptomatic disease precedes development of congestive heart failure (CHF) (Borgarelli &

Buchanan, 2012). Progression to CHF depends on many factors including the age of the

individual and the severity of disease. In a recent study, around 33 – 43% of dogs with

evidence of cardiomegaly developed CHF within 3.5 years (Boswood et al., 2016). Once in

CHF, both morbidity and mortality are negatively impacted.

Medical therapy is the primary treatment of CHF in dogs with MMVD; yet this is not

curative. Rather, the aims of therapy are to palliate the clinical signs and to achieve the best

quality of life for as long as possible. To this end, the benefit of a variety of medications has

been evaluated in large-scale clinical studies over the past three decades (Amberger et al.,

2004; Bernay et al., 2010; Chetboul et al., 2017; Ettinger et al., 1998; Häggström et al.,

2008; The BENCH Study Group, 1999; Woodfield, 1995).

Based on these findings, the American College of Veterinary Internal Medicine

(ACVIM) produced guidelines for the treatment of dogs with CHF due to MMVD in 2009

(Atkins et al., 2009). According to these guidelines, the gold standard therapy of chronic

CHF due to MMVD, supported by all panel members, would consist of combination therapy

including frusemide (a diuretic), pimobendan (an inodilator), and an angiotensin converting

enzyme inhibitor (ACE-I). The majority of panellists also recommended the concurrent use of

spironolactone as an aldosterone antagonist in order to provide more robust RAAS

antagonism. These medications act through mediating the neuroendocrine pathways

contributing towards CHF (ACE-I and aldosterone antagonists) or by overcoming their

effects (diuretics and inodilators). Thus, combination therapy would theoretically result in

71

successful obliteration of multiple pathways contributing towards CHF, and therefore, offer

the best outcome.

It is interesting to note, however, that there is a relative lack of literature evaluating

the true effects of such combination therapy. Prior to the commencement of this study, the

long-term effects of the aforementioned combination therapy had only been evaluated in a

single retrospective study which described the outcome of dogs treated with frusemide,

pimobendan, an ACE-I, spironolactone, and amlodipine (De Madron et al., 2011). Dogs

included in this study had a median survival time of 430 days from the time of diagnosis of

CHF, with the majority of deaths being cardiac-related. These findings appear similar to, or

possibly better than, survival outcomes reported by existing studies evaluating effects of

various components of this combination therapy (160 days till treatment failure in the LIVE

study (Ettinger et al., 1998); mean survival time of 428 days in the BENCH study (The

BENCH Study Group, 1999); median time to combined cardiac death, euthanasia, and

treatment failure of 267 days with pimobendan or 140 days with benazepril in the QUEST

study (Häggström et al., 2008); median survival time of 430 days with pimobendan or 228

days with benazepril in a study by Lombard et al., (2006); however, such comparisons

between studies are speculative at best, due to the vast differences in study design, cohort

of dogs enrolled, and classification systems for CHF. In addition, the retrospective nature of

the study by De Madron et al., (2011) made it more susceptible to selection bias and

confounders.

The aim of this study was therefore to evaluate, prospectively, the natural course of

disease in dogs with newly diagnosed CHF due to MMVD when treated with a combination

therapy consisting of frusemide, pimobendan, an ACE-I, and spironolactone.

Materials and Methods

Animals

Eleven client-owned dogs with ACVIM class C (chronic) CHF due to MMVD were recruited

from first opinion or referral caseload presenting to the University Veterinary Teaching

72

Hospital, Sydney (Sydney, NSW, Australia) between February, 2015 and March, 2016.

Ethics approval was obtained from the University of Sydney Animal Ethics Committee prior

to commencement of the study (project approval numbers 2013/532 and 2014/727).

Study design

This study was a prospective observational study examining the natural course of disease in

dogs with CHF due to MMVD on oral treatment with frusemide tablets or solution (APO

Frusemide; Apotex Pty Ltd, Macquarie Park, NSW, Australia or Flusapex; Apex laboratories

Pty Ltd, Somersby, NSW, Australia), pimobendan solution (Vetmedin 3.5 mg/ml oral

solution; Boehringer Ingelheim Pty Ltd, North Ryde, NSW, Australia), benazepril tablets

(Fortekor; Elanco Australasia Pty Ltd, West Ryde, NSW, Australia), and spironolactone

tablets (Aldactone; Pfizer Australia Pty Ltd, West Ryde, NSW, Australia) from the time of first

onset of CHF to death or the conclusion of the study period. The pimobendan was a liquid

formulation in the process of gaining registration by the Australian Pesticides and Veterinary

Medicines Authority at the time this study was commenced. Registration was obtained

during the course of the study (15 September, 2016; APVMA approval no 81552/103342).

It was a single-centre study conducted at the University Veterinary Teaching Hospital,

Sydney between February, 2015 and June, 2017.

Enrolment Criteria

Prior to enrolment, each dog had a screening examination which consisted of a review of the

history, physical examination, oscillometric blood pressure measurements (VetHDO monitor;

Memo Diagnostic, Darmstadt, Germany), electrocardiogram, echocardiogram, and thoracic

radiography. Select blood and urine tests were also performed in-house at the clinic using

the IDEXX Catalyst One® Chemistry Analyser (IDEXX Laboratories Inc., Rydalmere, NSW,

Australia), the Multistix 10 SG Reagent Strips (Siemens Healthcare Pty Ltd., Bayswater, VIC,

Australia), and a refractometer. The PCV, total protein, electrolytes, urea, creatinine,

73

phosphate, calcium, alanine aminotransferase, alkaline phosphatase, urine dipstick, and

urine specific gravity were evaluated.

The inclusion criteria consisted of older (greater than 5 years of age), small breed

dogs (weight less than 20 kg) diagnosed with first onset of left-sided CHF due to MMVD

(ACVIM Stage C) within 14 days of the screening exam. This diagnosis was confirmed

during the screening consultation and necessitated documentation of characteristic

radiographic changes (left atrial enlargement, pulmonary oedema, and venous congestion)

and symptoms (such as dyspnoea, orthopnoea, tachypnoea, changes to lung sounds on

auscultation, cyanosis/reduced oxygen saturation, cough, loud systolic left apical murmur,

and tachycardia) of CHF at the time of original diagnosis, as well as echocardiographic

findings consistent with severe MMVD at the screening exam (mitral valve thickening with

valve prolapse or flail valve, presence of mitral regurgitation, and evidence of cardiomegaly

based on left atrium to aorta ratio > 1.6 with increased left ventricular internal diameter in

diastole (Gonçalves et al., 2002). The signs of CHF needed to be adequately controlled

(normal respiratory rate and effort, no side effects of initial medical therapy, and resolution of

pulmonary oedema on thoracic radiographs at screening) for a minimum of two days and

maximum of 14 days prior to enrolment.

The exclusion criteria consisted of CHF due to aetiologies other than MMVD (e.g.

cardiomyopathies, arrhythmias, endocarditis, congenital heart disease); primary right-sided

CHF at the time of screening; haemodynamically significant arrhythmias refractory to

treatment at the time of screening; treatment with frusemide or pimobendan prior to onset of

initial CHF; presence of significant systemic diseases which may interfere with treatment of

CHF (e.g. chronic gastrointestinal, hepatic, or renal diseases) at the time of screening; and

chronic treatment with medications which may interfere with treatment of CHF (e.g.

sucralfate, antacids).

Pre-set exit criteria for discharge from the study included severe adverse reactions to

drugs used for treatment of CHF (frusemide, pimobendan, benazepril, spironolactone);

significant lack of owner compliance to adhere to treatment regimen or prescribed follow-up

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examinations; and development of significant unrelated diseases which may affect

cardiovascular function or require treatment with cardiotoxic agents. The development of

symptomatic pulmonary hypertension, right-sided CHF, arrhythmias, and other complications

of MMVD were not criteria for discharge from the study.

Schedule of visits

Upon successful enrolment into the study, each dog was required to commence quadruple

therapy with frusemide, benazepril, spironolactone, and pimobendan. The doses of

medications were adjusted as required throughout the course of the study, with acceptable

standard dosing defined as 3 – 12 mg/kg/day divided for frusemide; 0.25 – 0.5 mg/kg/day for

benazepril; 2 – 6 mg/kg/day divided for spironolactone; and 0.4 – 0.6 mg/kg/day divided for

pimobendan. Routine evaluations consisting of history, physical examination, blood pressure

measurements, electrocardiogram, standard echocardiography, thoracic radiographs, and

basic clinical pathology (as per the screening exam) were performed at one week, one

month, then every three months following enrolment until death or the conclusion of the

study period.

In addition to these routine scheduled visits, dogs were also seen for non-routine

visits by the investigators or other clinicians if required. These visits were not controlled and

consisted of any diagnostic and medical interventions which were deemed necessary by the

investigators or the attending clinician at the time.

Variables evaluated

Clinical variables evaluated in the study included body weight (kg); total dose of medications

per day; identifications of any arrhythmias present; identification of any concurrent diseases

or medications; adherence to treatment regimen; identification of any gastrointestinal or

behavioural abnormalities; sleeping or resting respiratory rates (SRR, RRR); murmur grade,

heart rate (HR); body condition score (BCS); and systolic, mean, and diastolic blood

pressure (SBP, MBP, DBP). In addition, a 32-point clinical scoring system adapted from

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Häggström et al., (2008) consisting of exercise tolerance, demeanour, appetite, respiratory

effort, coughing, nocturnal comfort levels, pulmonary oedema score, and ACVIM heart

failure class was also evaluated based on history and exam findings (Table 1).

The aforementioned blood and urine tests were also performed in-house at each visit

where possible.

Standard three-view thoracic radiographs consisting of right and left lateral and

dorsoventral views were performed. The vertebral heart score (VHS) was calculated by

measuring the long and short axis of the cardiac silhouette on a lateral projection of the

thorax and adding the number of vertebral bodies equivalent to these distances starting from

the fourth thoracic vertebral body (Buchanan & Bücheler, 1995).

Echocardiography was performed by a board-certified specialist in cardiology (NB), a

cardiology resident, or a veterinarian (MY) under the guidance of the cardiologist or

cardiology resident. An ultrasound system (Vivid I; General Electric Healthcare, Silverwater,

NSW Australia) coupled with a 3.5 – 8 mHz sector probe and simultaneous

electrocardiography was used. Two dimensional right parasternal short axis views were

used to measure the left atrium (LA) and aorta (Ao), and left ventricular indices were

obtained using M-mode at the level of the chordae tendinae. The left ventricular internal

diameter in systole and diastole were normalised for body weight (wnLVIDs, wnLVIDd)

(Cornell et al., 2004). Fractional shortening (FS) was calculated as (LVIDd – LVIDs) / LVIDd.

The aortic velocity was measured from the left apical five chamber view. Moderate to severe

pulmonary hypertension was detected in animals with a tricuspid regurgitation if the velocity

of the regurgitant jet exceeded 3.5 m/s (Borgarelli et al., 2015; Kellihan & Stepien, 2012).

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Table 1: Clinical scoring system for evaluation (maximum 32 points) of general well-

being in dogs with congestive heart failure (CHF) due to myxomatous mitral valve

disease (MMVD). Adapted from Häggström et al. (2008).

Exercise tolerance 1 Very good: Dog moved around with ease, was able to fully exercise 2 Good: Dog moved around with ease, was not able to fully exercise; ability to run was reduced 3 Moderate: Dog was less active than normal, moved around a few times per day, avoided long walks 4 Poor: Dog was inactive and would only get up to eat, drink, or urinate Demeanour 1 Alert, responsive 2 Mildly lethargic 3 Moderately lethargic 4 Minimally responsive Appetite 1 Increased 2 Normal 3 Decreased (2/3 normal) 4 Markedly decreased (< 2/3 normal) Respiratory effort 1 Normal 2 Mildly increased rate or effort 3 Moderately laboured 4 Severe respiratory distress Coughing 1 None 2 Occasional (a few times a week) 3 Frequent (a few times a day) 4 Persistent (frequently during the day) Nocturnal discomfort 1 None 2 Mild: Restlessness experienced <2 nights a week or need to toilet; no dyspnoea or coughing 3 Moderate: Restlessness experienced 3-5 nights a week or frequent need to toilet; mild dyspnoea or coughing 4 Severe: Restless on a nightly basis, frequent need to toilet, and presence of moderate dyspnoea Pulmonary oedema 1 None 2 Mild 3 Moderate 4 Severe ACVIM heart failure classification 1 Class C (chronic) 2 Class D (chronic) 3 Class C (acute) 4 Class D (acute)

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Endpoints

The primary endpoint of this study was death. This included both cardiovascular and non-

cardiovascular causes, as well as euthanasia. An alternate endpoint was reached at the

conclusion of the study period (30 June, 2017) if an animal was still alive at this time.

Statistical analysis

Analysis consisted of summary statistics and visual observation of graphs depicting

longitudinal data. The median and ranges were reported unless otherwise specified.

A Kaplan-Meier curve was created to obtain an estimation of median survival time to all-

cause death and cardiovascular death. All animals alive at the end of the study period were

censored in the former analysis, while animals suspected of non-cardiac death were also

censored in the latter analysis.

Results

A total of eleven dogs were enrolled between February, 2015 and March, 2016. The median

age was 12 years (range 6 – 16), median body weight was 5.9 kg (range 2.7 – 8.6), and

there was even distribution of the sexes (1 entire female and 5 spayed females; 1 entire

male and 4 neutered males). Breeds represented included the Japanese Chin (n = 1),

Chihuahua (n = 1) or Chihuahua cross (n = 2), Shih Tzu cross (n = 1), Miniature Fox Terrier

(n = 2), Jack Russell Terrier cross (n = 1), Maltese cross (n = 2), and Cavalier King Charles

Spaniel (n = 1). At the time of enrolment, all dogs were receiving treatment with frusemide

(median dose 3.7 mg/kg/day; range 2.9 – 6.3) and pimobendan (median dose

0.5 mg/kg/day; range 0.3 – 0.8). One dog was also receiving spironolactone (2.1 mg/kg/day),

and seven dogs were also receiving benazepril (median dose 0.2 mg/kg/day; range 0.2 –

0.6). Dogs had been on treatment for a median of seven days (range 6 – 14 days) prior to

screening.

Relevant baseline clinical information at enrolment has been listed in Table 2. Two of

the eleven dogs were suspected to have suffered acute chordal rupture which caused them

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to develop CHF; they had less cardiomegaly compared to other dogs in the study. Left atrial

rupture was suspected in one dog at the time of initial diagnosis with CHF and in another

dog around two months prior to presentation with initial onset of CHF. One dog had both

right and left-sided CHF at the time of initial onset of CHF. Five dogs had evidence of

moderate to severe pulmonary hypertension, two of which displayed clinical signs

associated with this (syncope and right-sided CHF).

Concurrent diseases at the time of enrolment included a history of seizures in two

dogs; dynamic airway disease or suspected chronic bronchitis in four dogs; skin allergies in

three dogs; and testicular neoplasia in one dog. One dog had mild systemic hypertension at

the time of screening and was subsequently diagnosed with, and successfully treated for,

hyperadrenocorticism. One dog developed evidence of kidney disease within one month of

the screening exam. This was thought to be attributable to exacerbation of pre-existing

subclinical kidney disease as a result of treatment with frusemide and benazepril.

Throughout the course of the study, the five dogs with moderate to severe pulmonary

hypertension all developed clinical signs (syncope, n = 2; right-sided CHF, n = 2; cyanosis,

n = 1) for which treatment with sildenafil was commenced at 0.7 – 2.2 mg/kg twice daily. One

dog developed supraventricular tachycardia which was treated with digoxin (1.7 mg/kg two

to three times daily) and diltiazem (3.6 μg/kg twice daily); another developed atrial fibrillation

which was treated with diltiazem at 2.3 mg/kg twice daily (18% for combined

tachyarrhythmias). Six dogs (55%) developed at least one episode of decompensated left

(n = 5) or right (n = 1) sided CHF prior to reaching the endpoint of death. Decompensation

first occurred between 2 – 6.5 months prior to death.

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Table 2: Clinical variables at enrolment in eleven dogs with congestive heart failure

(CHF) due to myxomatous mitral valve disease (MMVD). The median and range were

reported. Abbreviations: BCS – body condition score; SBP, MBP, DBP – systolic,

mean, and diastolic blood pressure; LA:Ao – left atrium to aorta ratio; wnLVIDs/d –

weight normalised left ventricular internal diameter in systole/diastole; FS – fractional

shortening; VHS – vertebral heart score; Crea – creatinine; ALT – alanine

aminotransferase.

Variable Median Range

Heart rate (bpm) 150 80 - 180

Murmur grade (/6) 4 4 - 5

BCS (/5) 3 1.5 - 3.5

SBP (mmHg) 146 125 - 169

MBP (mmHg) 97 80 - 120

DBP (mmHg) 71 51 - 83

LA:Ao 2.0 1.6 - 3.4

wnLVIDs (cm) 1.0 0.8 - 1.7

wnLVIDd (cm) 2.2 1.6 - 3.0

FS (%) 49.4 37 - 62.2

Aortic velocity (m/s) 1.1 0.7 - 1.5

VHS 11 10.0 - 14.0

Clinical Score (/32) 10 9 - 13

Crea (μmol/L) 98 59 - 128

Urea (mmol/L) 13.1 7.6 - 20.8

ALT (U/L) 102 42 - 164

Nine out of the eleven enrolled dogs (82%) reached the endpoint of death. The most

common cause of death was death or euthanasia due to recurrence of decompensated CHF

(n = 6; 55%). Other causes of death included euthanasia for suspected CHF (n = 1; 9%),

sudden death following a seizure-like episode (n = 1; 9%), and death of unknown cause

following development of lethargy, inappetence, and diarrhoea (n = 1; 9%).

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The proportion of dogs alive from the day of diagnosis of CHF till the end of the study

period is represented in Figure 1. The median survival time was 419 days to all-cause death

(95% confidence interval 184 – 616). The two dogs that originally developed CHF following a

suspected acute chordal rupture had lived for 481 and 773 days and were alive at the end of

the study period. When the one dog that possibly died of extra-cardiac causes (following

lethargy, inappetence, and diarrhoea in the other) was also censored to give the median

survival time to cardiovascular causes of death and euthanasia, the figure did not change

(Figure 2).

The trends in select clinical variables measured have been depicted in

Figures 3. The two dogs alive at the end of the study period (represented by dotted lines)

tended to have smaller measurements of heart size (LA:Ao, wnLVIDd, wnLVIDs, VHS) and

lower aortic velocity and FS (Figure 3). In contrast, dogs that reached the primary endpoint

earlier tended to have larger LA:Ao, wnLVIDs, and wnLVIDd. The LA:Ao appeared to

increase before the primary endpoint was reached.

The clinical exam findings recorded in this study remained largely static. The SRR

remained below 30 breaths per minute on the whole. The murmur grade was static except in

one dog, in which this reduced noticeably over the course of the study. This dog was alive at

the end of the study period and was one of the dogs that had chordal rupture with minimal

cardiac remodelling.

There were no discernible trends in each of the eight measures included in the

clinical scoring system (exercise tolerance, demeanour, appetite, respiratory effort, coughing,

nocturnal discomfort, pulmonary oedema score, and ACVIM heart failure class), body

condition score, urea, creatinine, or ALT. The urea and creatinine were abnormally high in

one dog that developed kidney disease. The ALT increased in two dogs that developed

supraventricular tachycardia and atrial fibrillation, which necessitated addition of

antiarrhythmic therapy (diltiazem and digoxin). Of these dogs, it is unclear as to why one

tended to have a high ALT throughout the course of the study.

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Overall, the treatment compliance was high throughout the study, with all owners

reporting adherence to treatment regimen all or most of the time. In the latter case, the most

common deviation to the treatment regimen related to the timing of administering

pimobendan in relation to feeding. All owners were instructed to dose this medication on an

empty stomach, one to two hours before food. Where deviations to treatment occurred, this

was due to the need to either administer pimobendan with food or to feed pets shortly

following treatment. In addition, the timing of medications was not always consistent, and

minor difficulties administering solid dose forms were reported by some of the owners. This

was usually overcome through administering medications with various food items considered

to be treats.

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Figure 1: Kaplan-Meier curve depicting percentage of dogs alive in relation to time,

starting from the day of diagnosis of congestive heart failure (CHF). Data from eleven

dogs with myxomatous mitral valve disease (MMVD) have been included. Deaths were

represented by vertical lines and included both cardiac and non-cardiac causes; the

two dogs alive at the end of the study period were censored and are represented by

the ticks. The median survival time was 419 days.

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Figure 2: Kaplan-Meier curve depicting percentage of dogs alive in relation to time,

starting from the day of diagnosis of congestive heart failure (CHF). Data from eleven

dogs with myxomatous mitral valve disease (MMVD) have been included. Deaths were

represented by vertical lines and represent only cardiac-related causes; the two dogs

alive at the end of the study period and the one dog that possibly died of extra-cardiac

causes were censored and are represented by the ticks. The median survival time was

419 days.

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Figure 3: Individual trends in echocardiographic and radiograph findings over time in

eleven dogs with congestive heart failure (CHF) due to myxomatous mitral valve

disease (MMVD). The two dogs that were alive at the conclusion of the study are

represented by dotted lines. Abbreviations: LA:Ao – left atrium to aorta ratio;

wnLVIDs/d – weight normalised left ventricular internal diameter in systole/diastole;

FS – fractional shortening; VHS – vertebral heart score.

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Discussion

The primary aim of this study was to describe the natural course of disease in dogs with

CHF due to MMVD receiving treatment with a quadruple combination therapy consisting of

frusemide, pimobendan, ACE-I, and spironolactone. This aim was achieved through analysis

of survival data, identification of comorbidities, and characterisation of repeated exam

findings in the study population.

The median survival time of 419 days (95% confidence interval 184 – 616) in dogs

with MMVD following initial diagnosis of CHF is comparable to findings of prior retrospective

studies evaluating survival in dogs treated with this combination of medications. De Madron

et al. (2011) reported a median survival time of 430 days (75% survival 99 days; 25%

survival N/A) from time of onset of CHF in 21 dogs with MMVD treated with quadruple

therapy and amlodipine. More recently, another retrospective study found that the median

time to development of advanced CHF was 163 days (range 10 – 743 days) in dogs with

newly diagnosed CHF due to MMVD, with a median survival time of 281 days (range 3 – 885

days) in advanced CHF (Beaumier et al., 2018). The quadruple combination therapy utilised

in the present study was employed in 36/54 dogs in the study by Beaumier et al. (2018).

Although the small number of enrolled dogs is a limitation to all three studies, this agreement

in central tendency of the data across three studies utilising similar inclusion and exclusion

criteria strengthens these findings. It can be deduced, then, that combination therapy

utilising early initiation of pimobendan, comprehensive RAAS inhibition, and frusemide

results in a median survival time of around 14 – 15 months in these dogs.

Interestingly, this figure is ~ 100 days longer than the reported median survival time

from a recently published retrospective study evaluating the effects of treatment utilising

frusemide, pimobendan, and ACE-I (M. Mizuno et al., 2017). As the inclusion criteria for this

study were also similar to that of the aforementioned studies, it is possible that the use of

comprehensive RAAS inhibition (i.e. spironolactone + ACE-I) may provide survival benefits.

Unfortunately, these findings are somewhat clouded by the use of spironolactone in ~ 25%

of the dogs in the study by Mizuno et al. (2017). The results from another prospective study

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evaluating the benefits of treatment with pimobendan, ACE-I, and frusemide over that of

pimobendan and frusemide alone are pending (Wess, 2017) but may facilitate further

comparative assessments of survival. To date, only one study has evaluated long-term

benefits of adding spironolactone to conventional therapy with ACE-I +/- frusemide +/-

digoxin in dogs with CHF (Bernay et al., 2010); although therapy including spironolactone

was associated with ~ 55% reduction in risk of cardiac death, euthanasia, or worsening

disease, the event rate for the primary endpoint was low (~ 18%), and this study did not

include pimobendan in the treatment regimen. Despite these suggestions that quadruple

therapy may provide enhanced survival benefits, further studies are required to confirm this

suspicion.

It is also important to note that the studies by Beaumier et al. (2018), De Madron et al.

(2011), and the present study represent the only studies to date which have examined

survival outcomes utilising early institution of quadruple therapy in dogs diagnosed with CHF

based on the ACVIM heart failure classification scheme. A key aspect which differentiates

this classification scheme from others (such as the ISACHC and modified NYHA) is the

requirement of pulmonary oedema demonstrated on thoracic radiography in order to

diagnose CHF; thus, it may be considered a more stringent system for providing a diagnosis,

with associated implications on survival outcomes. With this being the predominant

classification scheme utilised to guide diagnosis and treatment of CHF in Australia, reports

of survival outcomes using this scheme are most relevant to the population of dogs treated

in this country. Perhaps the single largest survival study in dogs with CHF diagnosed utilising

this classification scheme is the QUEST study, in which treatment with frusemide +

benazepril or frusemide + pimobendan was evaluated (Häggström et al., 2008). However,

“double therapy” is seldom used today; in addition, the primary endpoint of this study was a

composite of cardiac death, euthanasia, and treatment failure, and dogs were not enrolled

within a specified period following diagnosis of CHF. Thus, it would appear that despite the

small numbers, the findings of the present study and those by De Madron et al. (2011) and

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Beaumier et al. (2018) represent important survival information in dogs with CHF due to

MMVD when treated with such combination therapy.

Recurrent CHF was, by far, the largest contributor towards increased morbidity and

mortality and accounted for 78% of deaths observed in this study. This is in line with findings

of existing survival studies, in which cardiac causes comprised the majority (54 – 86%) of

deaths and euthanasias in dogs with CHF due to MMVD (Beaumier et al., 2018; De Madron

et al., 2011; Häggström et al., 2008; The BENCH Study Group, 1999). Thus, across a variety

of treatment methods, it appears death due to CHF or other cardiac causes remains the

most likely outcome in dogs with CHF due to MMVD. In addition, a majority of dogs (six out

of nine dogs that died) in this study experienced at least one episode of recurrent CHF prior

to death or euthanasia, starting from 2 – 6.5 months prior to death. This may be a negative

prognostic indicator but requires further studies to evaluate its significance.

Pulmonary hypertension and arrhythmias are important comorbidities which may

develop in cases of severe MMVD. Both have been associated with increased morbidity and

risk of death (Borgarelli et al., 2015; Borgarelli et al., 2008). The estimated prevalence of

pulmonary hypertension is 14 – 53% across all stages of MMVD, with higher prevalence in

advanced stages of the disease (Kellihan & Stepien, 2012). In line with this, five dogs (or

around 45% of dogs) had moderate to severe pulmonary hypertension at enrolment in this

study; in all of these dogs, clinical symptoms warranting treatment emerged. Ultimately, four

of these five dogs died or were euthanized because of confirmed or suspected CHF. Of

known arrhythmias, atrial fibrillation and paroxysmal supraventricular tachycardia were most

common in dogs with MMVD in one report (Borgarelli et al., 2008), and indeed, these were

the two significant arrhythmias observed in our study population. Both dogs were euthanized

due to recurrent CHF. These findings reinforced reports of previous studies regarding

pulmonary hypertension and arrhythmias in dogs with CHF due to MMVD.

Throughout the course of the study, only a few clinical variables showed discernible

trends in relation to survival. Dogs with larger LA:Ao, wnLVIDs, and wnLVIDd tended to die

earlier, and the LA:Ao appeared to increase prior to onset of death (Figure 3). This is in line

88

with previous reports which identified these factors as being negative prognostic indicators in

those treated with other combinations of medications (Borgarelli et al., 2008; Häggström et

al., 2008; Hezzell, Boswood, Moonarmart, et al., 2012). Body condition, VHS, and the clinical

score have also been identified as potential risk factors in previous studies, but this was not

obvious in our study cohort (Borgarelli et al., 2008; Häggström et al., 2008; Slupe et al.,

2008). This is likely owing to the small number of enrolled dogs and vastly heterogeneous

nature of the disease. Additionally, bias towards monitoring variables relating to the

cardiorespiratory systems (e.g. respiratory rate, murmur grade) with few items representative

of systemic well-being (e.g. body condition score) and inability to document clinical changes

leading up to and at the time of death in every case are also thought to have contributed.

Despite this, the emergence of trends in LA:Ao, wnLVIDs, and wnLVIDd in the present study

would suggest that these may be more robust prognostic variables with greater utility in

clinical practice.

Interestingly, the two dogs that were alive at the end of the study period were both

suspected to have had an acute chordal rupture which led to manifestation of CHF. They

had stable heart disease and even displayed a reducing trend in LA:Ao, wnLVIDd, and VHS

(Figure 3). One of these dogs subsequently developed syncope and was diagnosed with

pulmonary hypertension and decompensated CHF 287 days following completion of the

study. A large increase in LA:Ao and wnLVIDd was also noted at this time. Despite

escalation of therapy and addition of further medications, this dog died suddenly 37 days

later, 805 days from the time of first onset of CHF. The other dog was alive without

significant cardiac complications 333 days following completion of the study and 1106 days

from the time of first onset of CHF (as at 25 May 2018). The reported prognosis of dogs

suffering rupture of chordae tendinae are variable, with some reporting generally poor

outcomes (Borgarelli & Buchanan, 2012) and others observing better outcomes (median

survival time of 394 days in dogs with symptomatic CHF) (Serres, Chetboul, Tissier, et al.,

2007). However, to date, there are no studies that have evaluated the effect of combined

pimobendan and RAAS antagonism on the prognosis of dogs with CHF due to chordal

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rupture. Both dogs in the present study survived for an extremely long time and

demonstrated beneficial cardiac remodelling following initial onset of CHF. Whilst it is

possible that this particular combination of treatments facilitated positive cardiac remodelling

following chordal rupture and prolonged survival, additional studies are required to

investigate this theory.

There are several ways in which this study could have been modified to strengthen

the findings and contribute further data. As previously mentioned, greater numbers of

enrolled animals would have strengthened the observations made in the study. Though the

initial goal was to enrol up to 50 dogs over the course of the first year, this goal was not

achieved due to the stringent inclusion criteria, single-centre study design, and small number

of personnel available for case recruitment and data collection (three veterinarians

consisting of a specialist cardiologist, cardiology resident, and PhD candidate). Unfortunately,

extension of the recruitment period was deemed unfeasible due to the longitudinal nature of

the study and the time constraints to complete data collection within the duration of this PhD

candidature. A larger number of dogs may have facilitated more accurate extrapolations of

these findings to the population level or identification of specific risk factors for the primary

endpoint of death.

This study was designed as an observational study of dogs receiving quadruple

therapy, and therefore, did not include a control group to compare findings. Including a

positive control group could have provided additional information on the efficacy of

quadruple therapy in comparison to double or triple therapy. This is an important

consideration as increasing the number of medications can result in greater financial and

practical burdens on owners and reduced treatment compliance. However, such a study

would require very large numbers of dogs to be recruited, and the resources of this research

team did not allow for this type of study design.

Another limitation of this study was the lack of assessment of biomarkers. Natriuretic

peptides, and in particular, plasma concentrations of NT-proBNP, increase with disease

severity, and concentrations before and following therapy for CHF have been identified as

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significant predictors of mortality (Oyama et al., 2008; Serres et al., 2009; Wolf et al., 2012).

Other biomarkers which may provide information on disease severity and prognosis include

cardiac troponins, endothelin-1, and various markers of inflammation and endothelial

function (Boswood, 2009); however, many of these are not specific to the disease process,

and their clinical utility remains ill-defined at large. The combination of NT-proBNP and

serum high-sensitivity cardiac troponin I has been suggested to provide superior prognostic

information than either one alone (Hezzell, Boswood, Chang, Moonarmart, Souttar, et al.,

2012), and such a method of using multiple biomarkers has been advocated by some

experts in veterinary cardiology (Boswood, 2009; Oyama, 2014). Unfortunately, biomarker

assessment was limited through the lack of commercially available validated assays and

concerns over sample stability, given the long duration of storage throughout this study.

The method of data collection was yet another limitation. Some of the measured

items were subjective (e.g. degree of nocturnal discomfort, pulmonary oedema score, etc.);

where objective data was collected, some (such as sleeping respiratory rate) relied on owner

observations whereas others (such as echocardiographic measurements) were performed

by operators with varying levels of experience. The resultant inter-observer variability was

not quantified or monitored, thus making it possible that some inaccuracies existed in our

dataset. Nevertheless, it is interesting to note that some trends such as those in LA:Ao,

wnLVIDs, and wnLVIDd were still apparent. This appears to provide affirmation of the

robustness of these variables and provides support for their use by those less experienced

in the skill of echocardiography, such as general practitioners.

Conclusions

Eleven dogs with CHF due to MMVD were treated with combination medical therapy

consisting of frusemide, pimobendan, benazepril, and frusemide, and the natural course of

disease was prospectively evaluated. Nine dogs reached the endpoint of death, with death

or euthanasia due to confirmed or suspected cardiac causes comprising the majority of

these deaths. The median survival time from the time of CHF diagnosis was 419 days.

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Clinically significant pulmonary hypertension and recurrent decompensated CHF were

commonly encountered in this study cohort. Larger LA:Ao, wnLVIDs, and wnLVIDd

appeared to be associated with shorter survival times. These findings are in line with

previous studies evaluating survival outcomes in dogs with MMVD receiving treatment with a

variety of medications but suggest the potential for prolonged survival using this combination

of medications; ultimately, however, the benefit of quadruple therapy over other

combinations remains unproven. Further studies are required to shed light on this matter.

Conflicts of interest

This study was supported financially by the manufacturers of the liquid pimobendan product

and was also a field study for long-term safety assessment of this product. The cost of

medications (pimobendan, frusemide, benazepril, and spironolactone) and testing performed

at routine follow-up evaluations were covered under this sponsorship. No significant adverse

effects of treatment were encountered throughout the study, and registration for this product

was obtained during the course of the study.

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Chapter 5:

Brain natriuretic peptide assay development and evaluation

in dogs undergoing acute intravascular volume expansion

93

Author contribution statement

Mariko Yata was the primary investigator and author of the work reported in this chapter and

was involved in all aspects of study design, data collection, assay development, analysis,

and reporting.

Niek Beijerink provided guidance on the study design, assisted with data collection, and

critically reviewed this chapter.

Ben Crossett provided guidance with assay development and critically reviewed this chapter.

Mariko Yata Date__ 14/8/2018___

Niek Beijerink Date___19/08/2018__

Ben Crossett Date__ 15/8/2018____

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Introduction

Brain natriuretic peptide (BNP) is a peptide in the natriuretic peptide system (NPS) which

has been used as a biomarker for congestive heart failure (CHF) in humans and veterinary

patients (Oyama et al., 2013; Ponikowski et al., 2016). The predominant effects of BNP

include natriuresis, diuresis, vasodilation, and inhibition of the renin-angiotensin-aldosterone

system (RAAS); yet CHF occurs in the face of high circulating concentrations of BNP

(DeFrancesco et al., 2007; Häggström et al., 2000). This paradox has prompted further

research into the role of BNP and the NPS in CHF in humans (Colucci et al., 2000;

McMurray, 2015; Publication Committee for the VMAC Investigators, 2002; Vanderheyden et

al., 2010).

One of the reasons for this BNP paradox could be attributed to the many forms of this

peptide and the relatively non-specific nature of immunoassays, which are the primary

diagnostic tools used to detect circulating concentrations of BNP. BNP is primarily stored in

an inactive proform (proBNP1-108), which, upon activation, undergoes enzymatic cleavage

into a biologically active c-terminal component (BNP1-32) and a biologically inactive

N-terminal component (NT-proBNP) (Vanderheyden et al., 2010). BNP1-32 is further broken

down into less active or inactive fragments by a variety of circulating proteases

(Vanderheyden et al., 2010). Immunoassays designed for quantification of plasma BNP1-32

and NT-proBNP in humans display cross-reactivity with the inactive proBNP1-108 and less

active or inactive fragments of BNP1-32 (Heublein et al., 2007; Luckenbill et al., 2008;

Yandle et al., 1993). The problems associated with this lack of specificity prompted research

efforts into other techniques for detection and quantification of BNP1-32, the primary active

molecule, in humans (Vanderheyden et al., 2010).

A candidate for such a specific technique of peptide detection is mass spectrometry.

Mass spectrometers (MS) operate by ionising molecules and measuring the resultant mass-

to-charge ratios – a unique physicochemical footprint that can be used to identify the

substance of interest. There are numerous methods of ionising molecules, of which

electrospray (ESI) and matrix-assisted laser desorption/ionisation (MALDI) are most

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commonly used for peptides and proteins (D. Liebler, 2001). This is coupled with a mass

analyser and detector to sort and identify the various charged particles. One of the simpler

MS is a time-of-flight MS, which, when coupled with a MALDI source, uses a laser to ionise

the analyte and measures the time taken for each ion to reach the detector to determine the

mass. This method generally yields low numbers of fragmentation ions and can be used to

measure the mass of a broad range of peptides and proteins (D. Liebler, 2001).

In certain circumstances, the intact peptide mass alone can be diagnostic of a

particular peptide sequence; more commonly, however, peptide fragments are monitored.

With many MS, it is possible to isolate a particular ion (the parent ion) within the instrument,

fragment it, and analyse the fragment (or product) ions that are produced (D. Liebler, 2001).

This process is known as tandem mass spectrometry (MS/MS). Triple quadrupole MS are

particularly adept at monitoring certain parent ion – product ion combinations (or

“transitions”), which are unique to the analyte of interest due to its specific physicochemical

properties (D. Liebler, 2001). This makes mass spectrometry an extremely specific method

of detection and overcomes the issue of cross-reactivity encountered in immunoassays (D.

Liebler, 2001; D. C. Liebler & Zimmerman, 2013).

Though the real forte of mass spectrometry is in peptide detection, it can also be

used as a tool for peptide quantification, and therefore, as a bioanalytical tool. Aside from the

specificity, this technique also offers the ability to detect peptide modifications and quantify

multiple peptides simultaneously (Hoofnagle et al., 2012). Advancements in instrument

technology, analytical software, and development of standardised techniques have also

aided in overcoming some of its limitations, which have traditionally included a relatively low

sensitivity compared to immunoassays, the need for more extensive sample preparation

prior to analysis, and dependence on operator skills (Addona et al., 2009; Kingsmore, 2006;

D. C. Liebler & Zimmerman, 2013).

The successful application of mass spectrometry to quantifying levels of BNP1-32 in

human plasma has been demonstrated in a small number of published studies (Hawkridge

et al., 2005; Miller et al., 2011; Niederkofler et al., 2008). When the results of mass

96

spectrometry and immunoassays were compared, the former showed far lower

concentrations of circulating BNP1-32 in people with heart failure (Hawkridge et al., 2005;

Miller et al., 2011; Niederkofler et al., 2008). This discovery reinforced the possibility of a

relative natriuretic peptide deficiency in individuals with CHF and highlighted the importance

of immunoreactivity between active and inactive forms of natriuretic peptides.

In veterinary medicine, immunoassays are offered by commercial diagnostic

laboratories to detect plasma concentrations of immunoreactive BNP-32 (Antech® Cardio-

BNP test; Antech Diagnostics) and NT-proBNP (Cardiopet® proBNP and SNAP Feline

Cardiopet proBNP tests; IDEXX Laboratories Inc.). A handful of species-specific commercial

immunoassay kits are also available for research purposes (Phoenix Pharmaceuticals, Inc.,

Peninsula Laboratories International, Inc., Cloud-Clone Corporation, and other internet-

based distributors); yet the vast majority of these have not been validated. Development of a

mass spectrometry assay for veterinary patients may therefore enable further clarification of

the role of the BNP system in the pathophysiology of CHF in dogs and cats. Moreover, it

may contribute towards identification of a new therapeutic target for this disease.

In order to evaluate the clinical utility of such an assay, testing it on clinical samples

containing a wide range of concentrations of BNP1-32 and evaluating the results against

corresponding measures of cardiorenal function would be ideal. To facilitate this, a study

was designed in which the NPS in healthy dogs was experimentally challenged. Though

numerous experimental methods of NPS stimulation are available, some of these (such as

submersion in water or use of a hyperbaric chamber (Anderson et al., 1986; Rico et al.,

1989), strenuous exercise (Ohba et al., 2001), or balloon inflation of the atrium (Dietz, 2005)

were deemed to be impractical, while others (such as induction of heart failure or

ischemic/hypoxic damage (Dietz, 2005)) were deemed to be overly invasive. An alternative

method was the rapid administration of high volumes of intravenous fluids. This technique

causes a relatively non-invasive, acute, and reversible physiological challenge to the NPS

through preload augmentation (Borgeson et al., 1998; Hori et al., 2010; Kamp-Jensen et al.,

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1990; Lobo et al., 2010; Watenpaugh et al., 1992; Weil et al., 1985) and appeared to be an

ideal method for the purposes of this study.

The aims of this chapter, therefore, were to:

1. Develop a mass spectrometry method for identification and quantification of endogenous

concentrations of active BNP1-32 in canine plasma.

2. Evaluate the BNP system and its effects in normal dogs experiencing an acute expansion

of intravascular volume and cardiac preload via fluid therapy.

Part 1: Development of a mass spectrometry method for identification of canine BNP1-32

Materials and Methods

Canine synthetic BNP1-32 and canine BNP1-32 IgG antibody (Catalogue no 011-22 and

G-011-22; Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) were used as a standard

for assay development and immunoprecipitation. All solvents and protease inhibitors were

obtained from Sigma Aldrich (Castle Hill, NSW, Australia) unless otherwise specified. The

following abbreviations were employed: formic acid (FA), trifluoroacetic acid (TFA), sodium

bicarbonate (NaHCO3), trichloroacetic acid (TCA), phosphate-buffered saline (PBS),

Acetonitrile (ACN), Ethylenediaminetetraacetic acid (EDTA), 4-(2-aminoethyl)

benzenesulfonyl fluoride hydrochloride (AEBSF). ACN was obtained from Life Technologies

Australia Pty Ltd. (Scoresby, VIC Australia).

Canine serum and plasma anticoagulated with EDTA, with or without protease

inhibitors (final concentration 5 mM AEBSF + 10 mM Benzamidine), were used during

development of sample preparation methods. Blood samples were obtained from client-

owned, healthy, adult dogs presenting to the University Veterinary Teaching Hospital,

Sydney during routine examinations (University of Sydney Animal Ethics Committee

approved protocol number 2014/727).

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The assay development involved three stages: 1. Identification of BNP1-32 standard

using matrix-associated laser desorption/ionization time-of-flight spectrometry (MALDI-TOF);

2. Identification of BNP1-32 standard by online liquid chromatography coupled with

electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) using multiple reaction

monitoring (MRM); and 3. Sample preparation.

1. Identification of BNP1-32 standard using MALDI-TOF:

Serial dilutions of BNP1-32 standard in 0.1% (v/v) FA and 1 x PBS were made (final

concentrations ranging from 0.001 – 2 pM/μl). Various concentrations of MALDI matrix

(700 μl ACN; 40 μl 2.5% (v/v) TFA; 260 μl MilliQ water; and either 2, 5, or 10 mg of α-cyano-

4-hydroxycinnamic acid per ml of matrix solution) were made. 1 μl of each dilution was

mixed with 1 μl of the MALDI matrix on a MALDI plate and air-dried. MALDI-TOF analysis

was performed using positive ionisation mode with a laser power of 50% with pulse rate of

20 Hz as per standard operating procedures within the laboratory. The QSTAR Elite

spectrometer and the Analyst QS software 2.0 (Applied Biosystems/MDS Sciex Instruments)

were used for MS and data analysis.

2. Identification of BNP1-32 standard by LC-ESI-MS/MS (MRM):

Prior to MRM method development, 5+ and 6+ precursor ions were identified as being most

abundant in unmodified BNP1-32 using information dependent acquisition (IDA) on a Triple

TOF 6600 spectrometer (AB Sciex).

Skyline v3.5 (MacCross Lab, Department of Genome Sciences, University of

Washington) was used for MRM method development and optimisation, as well as for data

analysis. Peptide settings were initially set to include peptide lengths of 2-35 peptides with

no digestion or modifications. The transition settings were set to monitor the monoisotopic

mass of both precursor and product ions. 5+ and 6+ precursor ions and product ions in the

Y- and B- ion series with charge states of 1+, 2+, and 3+ were monitored. The collision

energy and declustering potential were automatically selected to start.

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The Sciex Eksigent ekspert nano LC 415 system (AB Sciex) was coupled with the

Sciex QTRAP 6500 mass spectrometer (AB Sciex). Serial dilutions of BNP1-32 standard in

0.1% (v/v) FA were made (ranging from 0.0001 – 1 pM/μl). 5 μl were injected into a nano-

flow reverse-phase liquid chromatography column containing 5 μm diameter C18 matrix (Dr

Maisch, Ammerbuch-Entringen, Germany) which was packed in-house. The mobile phase

consisted of 0.1% (v/v) FA (Buffer A) and 80% ACN plus 0.1% (v/v) FA (Buffer B), and a

30-minute gradient elution method (from 2 – 95% Buffer B) was used with a flow rate of

300 nl/min as per standard operating procedures in the laboratory. Nanospray ionisation was

performed using the negative ion mode with ion spray voltage 2700 V, Curtain gas 25 units,

collision gas set to high, and interface heater temperature set to 200°C. The entrance

potential was set to 10, with collision cell exit potential set to 24.

Method optimisation (D. C. Liebler & Zimmerman, 2013) was performed through the

selection of the top three transitions from each precursor-product charge states to monitor,

followed by collision energy optimisation. Scheduling was not performed as only one peptide

was being monitored. Further optimisation was performed through inclusion of methionine

oxidation and deamination modifications.

3. Sample preparation

A variety of sample preparation methods were explored for the identification of BNP1-32 in

plasma:

a) Bulk purification using C4, C8, C18, or HLB sorbent material

b) Reduction, alkylation, and digestion with Endoproteinase Asp-N (Product P3303; Sigma

Aldrich, Saint Louis, MO, USA) followed by bulk purification using C18 sorbent material.

c) Solvent precipitation using TCA, Acetone (L. Jiang et al., 2004), ACN, or ACN/TFA

(Chertov et al., 2004; Merrell et al., 2004) followed by bulk purification using C8 or C18

sorbent material.

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d) Immunoprecipitation using protein A beads (MSIA D.A.R.T.’S ™ Tips, Thermo Fisher;

Dynabeads ™ Protein A, Thermo Fisher Scientific) cross-linked to canine BNP1-32 antibody,

with or without final purification using C8 material.

Method optimisation for immunoprecipitation involved adjustments to the number of

wash steps; quantities of protein A beads, antigen, and antibody used; incubation

periods for both antibody loading and antigen loading; amount of detergent in the

wash solution; and inclusion of cross-linking of the antibody.

For the final method, 50 μl (1.5 mg) of Dynabeads™ were washed with wash solution

(PBS with 0.01% Tween-20), followed by a 30-minute incubation on a rotating

platform with 200 μl of PBS containing 10 μg of BNP1-32 IgG antibody. Beads were

washed once with wash solution, once with 0.2 M triethanolamine (pH 8.0), and then

antibodies were cross-linked with 200 μl of 10 mM Dimethyl pimelimidate (DMP; Life

Technologies Australia Pty Ltd., Scoresby, VIC Australia) using an incubation time of

30 minutes on a rotating platform. After removing excess solution, 200 μl of 50 mM

Tris (pH 7.6) was added to the beads and mixed on a rotating platform for 15 minutes.

The tris solution was removed, and the beads were washed twice with wash solution.

300 μl of PBS containing 100 pM BNP1-32 was added to the beads and incubated at

4°C overnight (~ 12 hours) on a rotating platform. This was followed by a wash with

PBS, then three washes in water before elution with 50% (v/v) ACN and 0.1% (v/v)

FA, and MALDI-TOF analysis.

e) Nonspecific albumin and IgG depletion (ProteoPrep® Blue Albumin and IgG Depletion Kit;

Sigma Aldrich, Saint Louis, MO, USA).

Nonspecific albumin and IgG depletion was attempted using the ProteoPrep® Blue

Albumin and IgG Depletion Kit using the manufacturer’s guidelines. Frozen canine

plasma and serum samples were defrosted, vortexed, and centrifuged. Briefly, 0.4 ml

of suspended albumin and IgG medium was added to a spin column and washed

twice with 0.4 ml of the equilibration buffer. The column was centrifuged at 10,000

rpm for 20 – 40 seconds between each step. A 20 μl aliquot of plasma or serum was

101

added to the washed and dried medium and incubated at room temperature for 10

minutes. Following centrifugation for 60 seconds, the flow-through was reapplied,

and a second incubation and centrifugation step was performed. 100 μl of

equilibration buffer was used to wash the medium, and the flow through from

centrifugation was added to the twice depleted sample.

Elution of bound proteins was performed with 150 μl of 4x sample buffer consisting of

125 mM Tris (pH 6.8), 30% (v/v) glycerol, 2% (w/v) SDS, 10% (v/v) β-

Mercaptoethanol, and bromophenol blue. After centrifuging for 60 seconds, a further

150 μl of 4x sample buffer were applied to the medium and centrifuged. The eluates

were pooled.

Samples were loaded onto a 12% 1D SDS PAGE gel, and electrophoresis was

performed using 125 V for 80 minutes. The gel was stained with Coomassie Blue and

imaged using the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories Pty Ltd.,

Gladesville, NSW Australia).

Results

The predicted molecular weight for the singly-charged BNP1-32 was 3564.84 Daltons and

1782.42 for the double-charged ion. In 0.1% FA (v/v), the lowest limit of detection (LLOD) for

BNP1-32 standard using MALDI-TOF was 0.1 pM/μl (Figure 1). When BNP1-32 standard

was reconstituted in 1 x PBS, the LLOD increased to 1 pM/μl. Adjusting the concentrations

of the α-cyano-4-hydroxycinnamic acid improved appearance of the spectrum slightly

(Figure 2); however, sensitivity (as measured by LLOD) could not be improved any further.

In contrast, the sensitivity for detection of the BNP1-32 standard in 0.1% FA (v/v) was higher

using LC-ESI-MS/MS. Following optimisation of the method, the LLOD was 0.0001 pM/μl

(Figure 3). The final list of transitions, including the declustering potential and collision

energy, is listed in Table 1.

A variety of sample preparation methods were attempted for purification of BNP1-32

from plasma. Preliminary testing using BNP1-32 standard dissolved in 1 x PBS showed

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reliable capture of BNP1-32 using C8 sorbent material, some capture of BNP1-32 with C18

material, but no capture with C4 or HLB material (Figure 4). Subsequently, solvent

precipitation of BNP1-32 spiked plasma samples, followed by purification using C8 or C18

material, was trialled. However, none of these methods provided reliable recovery of

BNP1-32 from plasma. An attempt at digesting BNP1-32 using Endoproteinase Asp-N was

made; however, digestion was not successful.

A targeted approach of purification was also performed through immunoprecipitation

methods using a commercially available canine BNP1-32 antibody cross-linked to beads

coated with Protein A. However, these methods failed to recover both synthetic and

endogenous BNP1-32. This was due to a lack of adequate antigen-antibody interaction. As

an alternative, sample enrichment using a commercially available kit (ProteoPrep® Blue

Albumin and IgG Depletion Kit; Sigma Aldrich, Saint Louis, MO, USA) for selective removal

of albumin and immunoglobulins was attempted. Unfortunately, this kit did not provide

adequate removal of albumin and immunoglobulins from canine plasma samples (Figure 5).

Thus, a mass spectrometry assay for detection of clinically significant concentrations

of canine BNP1-32 in plasma could not be made.

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Figure 1: MALDI-TOF spectra of a 1 μl spot of BNP1-32 equivalent to 1 pM/μl (A), 0.1

pM/μl (B), 0.01 pM/μl (C), and 0.001 pM/μl (D) BNP1-32 standard in 0.1% FA. Arrows

point to BNP1-32 spectra (single and double charged ions).

104

Figure 2: MALDI-TOF spectra of a 1 μl spot of 2 pM/μl BNP-32 standard in PBS using

MALDI-matrix containing different concentrations of α-cyano-4-hydroxycinnamic acid

(2mg, 5mg, and 10mg per ml of matrix – A, B, and C respectively). Arrows point to

BNP1-32 spectra (single and double charged ions).

105

Figure 3: Transition list, chromatogram, and peak areas for the top 42 transitions for

BNP1-32 using the 5+ and 6+ precursor ions on a sample containing 0.0001 pM/μl

BNP1-32 standard in 0.1% FA (v/v). Data analysis was performed using Skyline

software (v3.5).

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Table 1: Top 42 transitions for BNP1-32 following collision energy optimisation and

addition of metoxidation and deamination modifications.

Precursor m/z Product m/z Dwell time Declustering Potential Collision Energy

714.174614 952.503297 30 83.2 37.4

714.174614 895.481833 30 83.2 41.4

714.174614 792.472649 30 83.2 41.4

714.174614 868.48543 30 83.2 35.4

714.174614 790.434875 30 83.2 40.4

714.174614 802.391964 30 83.2 38.4

714.174614 952.512961 30 83.2 37.4

714.174614 909.81464 30 83.2 37.4

714.174614 749.046959 30 83.2 39.4

595.313391 792.472649 30 74.5 35

595.313391 678.429721 30 74.5 34

595.313391 712.326913 30 74.5 35

595.313391 705.382111 30 74.5 35

595.313391 661.866097 30 74.5 33

595.313391 646.290853 30 74.5 31

595.313391 707.396752 30 74.5 34

595.313391 655.363048 30 74.5 33

595.313391 749.046959 30 74.5 33

720.57258 790.434875 30 83.6 41.8

720.57258 909.81464 30 83.6 39.8

720.57258 818.386879 30 83.6 41.8

600.64503 678.429721 30 74.9 34.3

600.64503 744.316743 30 74.9 35.3

600.64503 680.318458 30 74.9 32.3

714.371418 952.840966 30 83.2 36.5

714.371418 910.142645 30 83.2 37.5

714.371418 749.046959 30 83.2 41.5

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Table 1 (Continued)

Precursor m/z Product m/z Dwell time Declustering Potential Collision Energy

595.477394 678.429721 30 74.5 34

595.477394 705.874119 30 74.5 32

595.477394 749.046959 30 74.5 30

717.570401 793.456665 30 83.4 39.6

717.570401 952.840966 30 83.4 38.6

717.570401 910.142645 30 83.4 38.6

598.143213 678.429721 30 74.7 35.1

598.143213 664.323543 30 74.7 33.1

598.143213 654.28831 30 74.7 34.1

720.769384 790.926883 30 83.7 40.8

720.769384 818.386879 30 83.7 41.8

720.769384 786.387737 30 83.7 41.8

600.809032 678.429721 30 74.9 34.3

600.809032 744.316743 30 74.9 35.3

600.809032 680.318458 30 74.9 31.3

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Figure 4: MALDI-TOF spectra of a 1 μl spot of BNP1-32 eluate following C18 (A, 1

pM/μl), C8 (B, 1 pM/μl), C4 (C, 2 pM/μl) and HLB (D, 2 pM/μl) preparation. Arrows point

to BNP1-32 spectra (single and double charged ions).

109

Figure 5: 1D-SDS-PAGE of untreated plasma (P1 – P4; 90, 45, 30, and 15 μg protein

loading respectively), as well as the “depleted” flow-through (P5 – P6; 50 and 25 μg

protein loading respectively), and the eluate containing bound proteins (P7 – P8; 20

and 10 μg protein loading respectively) following treatment of plasma with

ProteoPrep® Blue Albumin and IgG Depletion Kit. There were no appreciable

differences between the untreated plasma, flow-through, and eluates.

Discussion

The aim of this part of the chapter was to create a novel assay for detection of specific

BNP1-32 in canine plasma. Unfortunately, this aim was not achieved, with several

unanticipated challenges and limitations being encountered throughout the course of assay

development.

Intact BNP1-32 could be identified with both MALDI-TOF and LC-ESI-MS/MS, with

the latter showing greater sensitivity towards its detection than the former. High sensitivity of

the assay was essential due to the extremely low concentrations of circulating peptide

expected in normal canine plasma. Reported physiological concentrations of

immunoreactive BNP-32 range from 200 – 300 pg/ml (or 0.0007 – 0.084 fM/μl)

(DeFrancesco et al., 2007; Forfia et al., 2007; Grantham et al., 1997), dictating the

110

requirement for at least 1 ml of plasma being concentrated 2 – 1,000 times before biological

concentrations of BNP1-32 would be detectable using LC-ESI-MS/MS using the existing

techniques. Yet simply concentrating the samples would be inadequate due to the presence

of substances in plasma which may interfere with the mass spectrometry through signal

suppression or by reducing the concentration of the analyte captured by the in-line liquid

chromatography (e.g. salts, proteins); some of these can also degrade or modify BNP1-32

(e.g. enzymes). As such, methods to improve both the mass spectrometry sensitivity and

sample preparation were investigated.

Of the two mass spectrometry methods used (MALDI-TOF and LC-ESI-MS/MS),

method refinement was mostly performed on the latter due to its higher sensitivity. This

involved selecting transitions with highest peak areas, collision energy optimisation, and

inclusion of methionine oxidation and deamination modifications as these were observed in

older BNP1-32 standards. From here, trimming of this transition list, increasing the dwell

time for each transition, and altering some of the mass spectrometer settings such as the ion

spray voltage, curtain gas, and entrance/exit potentials could have also been attempted. In

addition, the liquid chromatography conditions such as the gradient and column material

could have been altered. However, it was deemed that these would be best performed

following optimisation of sample preparation techniques.

Sample preparation plays a large role in peptide mass spectrometry using plasma

samples due to the extreme dynamic range of proteins, which span over eleven orders of

magnitude (R. Cunningham et al., 2012). Furthermore, the top fourteen proteins, including

albumin and immunoglobulins, comprise over 95% of the plasma proteins and are present in

concentrations of 55 – 75 mg/ml; in contrast, proteins and peptides of interest can often be

present at the far lower end of the spectrum, in concentrations closer to pg/ml. In order to

overcome this, several techniques for plasma preparation including non-specific enrichment

or depletion techniques, separation techniques (such as gel electrophoresis or liquid

chromatography), and specific immunoprecipitation techniques (Luque-Garcia & Neubert,

2007) were explored.

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Non-specific enrichment or depletion techniques offered the advantage of being easy,

inexpensive, and readily accessible. The simplest form of this was the use of solid-phase

extraction (SPE) cartridges for de-salting, extracting smaller peptides, and concentrating the

sample. BNP1-32 is basic, with mild to moderate hydrophilic properties as suggested by the

gravy index of -0.39 (Gasteiger et al., 2003; Kyte & Doolittle, 1982). At around 3566 Daltons,

it is also a small- to medium-sized peptide. These properties suggested that reverse-phase

SPE using materials such as C18, C8, or HLB would be ideal. Of these, C8 provided less

retention of non-polar compounds, and as such, appeared most appropriate. This was

confirmed through the finding that C8 material provided the most consistent capture of

BNP1-32. However, this non-specific approach was likely to result in inadequate depletion of

impurities and interfering substances; therefore, this technique was deemed most

appropriate to use in combination with other techniques of sample preparation (for example,

as a final de-salting step, or using C8 material with reversed-phase chromatography for the

in-line LC prior to mass spectrometry).

Options for non-specific depletion included solvent precipitation and use of

substances that bind albumin and non-specific immunoglobulins. Solvent precipitation

involves addition of organic solvents to precipitate larger plasma proteins whilst leaving

smaller peptides in the solution. In this process, smaller peptides bound to larger proteins

can also be dissociated and retrieved. Although this method was appealing, the recovery of

BNP1-32 using this technique was inconsistent or poor. Potential reasons for this include

loss of the peptide via precipitation or loss in the recovery, drying, reconstitution, or final

purification of the solution. Binding and depletion of albumin and non-specific

immunoglobulins was also attempted with a commercially available kit that utilised a blue

dye-based technology for albumin and protein G agarose for immunoglobulins. Unfortunately,

this kit did not work on canine plasma samples, and a canine-specific kit was not available.

With these simple techniques proving to be ineffective, and because prior

publications utilised immunoprecipitation techniques for sample preparation (Hawkridge et

al., 2005; Miller et al., 2011; Niederkofler et al., 2008), the latter avenue was explored.

112

Immunoprecipitation offers the unique advantage of targeted peptide purification.

For BNP1-32, this technique would result in capture of both the active BNP1-32 and its

less/inactive fragments and proform; however, coupled with a mass spectrometer for

detection, only the specific BNP1-32 would be identified. This technique was also

unsuccessful due to the lack of adequate antibody-antigen interaction. There are numerous

potential factors contributing towards this result, including the temperature, pH, and choice of

antibody (He, 2013). In addition, non-specific binding to the antibodies can reduce capture of

the desired analyte. In one publication, this was overcome through the use of another

immunoprecipitation step to remove non-specific immunoglobulins prior to targeting

BNP1-32 (Niederkofler et al., 2008). It is important to note that antibodies used in these

publications were raised in-house, as opposed to the commercially available antibodies used

in the present study. The degree of trial and error involved in the development of this

technique necessitated time and budgets that were not within the scope of this project. Yet

given the low concentrations of peptide present and the high degree of complexity of plasma,

this is likely the most promising technique for achieving adequate sample preparation for

detection of BNP1-32 using mass spectrometry.

Other forms of sample preparation may or may not provide successful recovery of

BNP1-32. With the low concentrations of endogenous BNP1-32 and its low molecular weight,

gel electrophoresis was deemed to be an inaccurate and unreliable method of separation of

peptides of interest. Various techniques of liquid chromatography could have been applied,

with some of these likely to facilitate a reasonable degree of fractionation or purification of

BNP1-32; however, the lack of readily available resources and expertise, as well as the

anticipated time and costs involved in developing this technique, ruled out this option. One

option that could have been explored further was the use of a centrifugal filter. Some

problems with this technique include filter failure or clogging and the possibility of peptide

loss if they become entrapped in the captured protein pellet or are bound to larger proteins

that are captured. Nevertheless, centrifugal filters or chromatography techniques may be

another step in the workflow which could be explored in future projects.

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Had adequate sample preparation and optimisation of mass spectrometry been

achieved, absolute quantitation of BNP1-32 would have been performed through the use of

a heavy isotope-labelled BNP1-32 as an internal standard. Validation of the final assay

would have involved an assessment of accuracy, precision, quantification limits, linearity,

and range within our laboratory. Yet ultimately, the utility and practicality of such an assay

are important factors to weigh against the time and resources required for ongoing assay

development. With the degree of sample preparation involved, the duration of time required

to run each sample (~ 1 – 1.5 hours for the LC-ESI-MS/MS method), and the maintenance

involved in the smooth operation of the LC and MS, it is likely that this type of assay would

not be practical for large-scale commercial or diagnostic purposes. Despite this, there is

growing interest in mass spectrometry as a diagnostic tool in veterinary research and

medicine. Though assay development was not completed during the course of this project, it

is hoped that the information acquired through this project may contribute towards any future

attempts.

Part 2: Evaluation of the BNP system and its effects in healthy dogs receiving short-

term intravenous fluids

Materials and Methods

Animals

This study was conducted over two weeks in November, 2015 on eight healthy adult dogs,

which were sourced from a contract research organisation. Prior to enrolment, each dog

underwent a physical examination, standard echocardiography, three-view chest

radiographs, and clinical pathology (PCV, TP, electrolytes, BUN, Creatinine, and USG) to

ensure no significant physical, cardiorespiratory, or renal abnormalities were present. Six

Beagles and two Kelpie cross breeds were enrolled. There was an even distribution of sexes,

with three entire males and females and one desexed male and female. The median age

was 6.5 years (range 2 – 7 years), and the median weight was 13.9 kg (range 12.6 –

114

21.7 kg). The protocol for this study was approved by the University of Sydney Animal

Ethics Committee (project number 2015/873).

Schedule of events

On each day of sampling, two dogs (one male and one female) underwent procedures. All

dogs were fasted for a minimum of 10 hours prior to sampling.

Dogs were sedated with 0.2 mg/kg of methadone subcutaneously. Anaesthesia was

induced with propofol intravenously to effect and maintained with isoflurane and 100%

oxygen delivered through a cuffed endotracheal tube. Continuous manual ventilation using a

ventilator (Merlin Small Animal Ventilator, Vetronics Services Ltd, Abbotskerwell, Devon,

TQ12 5NF, UK) set at a respiratory rate of 15 breaths per minute, tidal volume of

10 – 15 ml/kg, and peak inspiratory pressure of ~ 10 mmHg was performed throughout

anaesthesia in all dogs. Dogs were placed in right lateral recumbency and catheterised with

two 18 g 32 mm intravenous catheters placed in the cephalic veins, a double-lumen jugular

catheter (B.Braun Certofix Duo; B. Braun Australia Pty Ltd, Bella Vista, NSW Australia)

placed in the left jugular vein, a 24 g 19 mm arterial catheter placed in an auricular artery,

and an 8 Fr 55 cm indwelling urinary catheter (Surgivet Premium Foley Catheter; Smiths

Medical, St Paul, MN USA). The arterial catheter was connected to a pressure transducer

(Datex-Ohmeda S/5 Monitor, Helsinki, Finland) via an extension line filled with saline for

evaluation of direct arterial blood pressure measurement. The distal (open) lumen of the

jugular catheter was connected to a manometer via an extension line. This was filled with

saline, and the zero mark on the manometer was placed at the level of the right atrium for

evaluation of the central venous pressure (CVP). The urinary catheter was connected to an

extension line with a 60 mL syringe attached to the end for collection of urine.

The timeline of study events is depicted in Figure 6. Following acquisition of baseline

values, Hartmann's solution was administered at 90 ml/kg/h for 40 minutes via the two

peripheral intravenous catheters. Blood (8 – 10 ml) and urine (5 – 10 ml) samples were

collected for evaluation of plasma NT-proBNP, specific BNP1-32, immunoreactive BNP-32,

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and cyclic guanosine monophosphate (PcGMP) concentrations; biochemistry (PCV, TP,

BUN, creatinine, electrolytes), and urinary cGMP (UcGMP) and specific gravity (USG). Upon

completion of sampling, all catheters were removed, and dogs were recovered from

anaesthesia.

Figure 6: Timeline of events. Abbreviations: Premed – premedication; GA – general

anaesthesia; CVP – central venous pressure; HR – heart rate; BP – blood pressure;

Echo – echocardiogram; UOP – urine output; biochem – biochemistry including PCV,

TP, BUN, creatinine, electrolytes; USG – urine specific gravity.

Echocardiography, blood pressure, heart rate, and CVP

Echocardiography was performed as previously described in Chapter 2. The left atrial (LA)

and aortic (Ao) diameters were measured, and the left ventricular indices consisting of the

thickness of the interventricular septum in systole and diastole (IVSs, IVSd), left ventricular

internal diameter in systole and diastole (LVIDs, LVIDd), and thickness of the left ventricular

posterior wall in systole and diastole (LVPWs, LVPWd) were obtained and normalised to the

body weight (Cornell et al., 2004). The left atrium to aorta ratio was calculated through

dividing LA by Ao (LA:Ao), and fractional shortening (FS) was calculated using the formula:

(LVIDd – LVIDs) / LVIDd. End systolic (ESV) and end diastolic (EDV) volumes were

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calculated using the Teichholz formula (Teichholz et al., 1976). Stroke volume (SV) was

calculated as EDV – ESV, and cardiac output (CO) was calculated as (SV * Heart rate) /

body weight.

Blood pressure (systolic – SBP; mean – MBP; diastolic – DBP) and heart rate (HR)

were recorded off the pressure transducer and pulse oximetry monitor respectively. Three

consecutive or near-consecutive readings were recorded and averaged at each time point.

For CVP, the catheter was flushed and the manometer was primed with saline each time a

reading was taken. The lowest measurement, which was stable for at least 5 seconds, was

recorded at each time point.

Sample handling and storage

All blood samples were obtained via the proximal (side) lumen of the jugular catheter.

Following acquisition, they were placed into chilled plastic blood tubes containing EDTA and

centrifuged immediately in a cooled centrifuge (at or below 4°C) at 5000 RCF for 5 minutes.

For NT-proBNP analysis, the plasma was transferred into another blood tube containing

EDTA; for immunoreactive BNP-32 analysis, it was mixed with a pre-prepared mixture of

4-(2-Aminoethyl) Benzenesulfonyl Fluoride (AEBSF) and Benzamidine (Sigma Aldrich,

Castle Hill, NSW Australia) using a previously reported protocol (Niederkofler et al., 2008) for

protease inhibition (final concentration of 5 mM AEBSF and 10 mM Benzamidine). All

samples were placed into a freezer without delay and stored at or below -30°C until the time

of analysis.

Blood samples used for biochemistry were placed into plastic blood tubes containing

lithium heparin and centrifuged for analysis, which occurred within four hours of collection.

PCV and biochemistry were performed using the in-house laboratory (ABL800 Basic blood

gas analyser; Radiometer Pacific Pty. Ltd., Mt Waverley, VIC Australia. Catalyst One®

Chemistry Analyser; IDEXX Laboratories Inc., Rydalmere, NSW Australia) at the University

Veterinary Teaching Hospital, Sydney.

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Urine samples were obtained from the urinary collection system after quantification of

urine production. The samples were aliquoted and stored frozen at or below -30°C until the

time of analysis.

NT-proBNP, BNP-32, and cGMP immunoassays

Plasma NT-proBNP concentrations were measured at a commercial laboratory using a

validated (Cahill et al., 2015) enzyme-linked immunosorbent assay (Cardiopet® proBNP

test; IDEXX Laboratories Inc., Brisbane, QLD Australia). Plasma immunoreactive BNP-32

concentrations were measured at a commercial laboratory using a validated (Schellenberg

et al., 2008) radioimmunoassay kit made by the company (Canine BNP-32

Radioimmunoassay RK-011-22; Phoenix Pharmaceuticals Inc., Burlingame, CA, USA). The

samples were transported frozen to the laboratory, and each sample was assayed in

duplicate. The analysis was completed over two days. Specific BNP1-32 concentrations

could not be quantified as assay development using mass spectrometry was unsuccessful.

Plasma and urine cGMP concentrations were assayed in triplicate using a

commercially available competitive enzyme-linked immunosorbent assay kit using the

manufacturer’s guidelines (ParameterTM cGMP Assay SKGE003; R&D Systems Inc.,

Minneapolis, MN, USA). Briefly, urine and plasma samples were thawed at room

temperature, and 25 μl of each sample was mixed with 475 μl of the provided diluent. In

some samples, final concentrations exceeded the range of the assay. These were further

diluted 1:4 using the diluent provided. 100 μl of each standard and sample were mixed with

50 μl of the cGMP conjugate, followed by 50 μl of the primary antibody solution. After

incubating for three hours on a microplate shaker at 450 rpm at room temperature, wells

were washed four times, and 200 μl of substrate solution were added to each well. This was

incubated for 30 minutes in the dark, and 50 μl of stop solution were added. The plate was

read using a TECAN Infinite® M1000 PRO microplate reader set at 450 and 540 nm once

uniform colour change was achieved.

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Readings made at 540 nm were subtracted from those at 450 nm. A standard curve

was generated using a 4-parameter logistic curve fit using Graphpad Prism V7.02

(GraphPad Software Inc., La Jolla, CA, USA). Measured concentrations were derived using

this standard curve and multiplied by the dilution factor. The results of each triplicate were

averaged to obtain the final result. All standard curves had R2 > 0.99. The %CV of triplicate

standards and samples were < 10%, and the %CV of standards between plates was < 10%

for all but the highest concentration of standard (16%). The accuracy of standards ranged

from 90 – 115% at concentrations in the relevant range of the samples.

Statistical analysis

Analysis of data was performed using GraphPad Prism and GenStat 16th Edition (VSN

International Ltd., Hemel Hempstead,UK).

Descriptive analysis of cardiorenal, endocrine, and biochemical variables was

performed through visual inspection of graphs displaying the median and interquartile range

of effects over time.

Prior to statistical testing, normality of all measured variables was assessed through

evaluation of the Q-Q plot. Correlations between measured variables were assessed through

visual inspection of correlation plots. Where a monotonic relationship appeared to exist,

Spearman’s rank-order correlation was performed. For items which were only measured at

baseline and 60 minutes (PCV, USG, Na, K, Cl, BUN, Crea), paired t-tests were performed

to evaluate observed changes.

Where the data were not normally distributed, log (Ao, BNP-32, DBP, MBP,

NT-proBNP, TP, UOP, urinary cGMP, urinary excretion of cGMP, CO, weight-normalised

IVSd and LVIDd) and power (LA:Ao) transformations were performed to resemble normality.

A linear mixed model with restricted maximal likelihood estimation (REML) was performed to

assess for significant deviations of the variables from baseline for all those in which > 2

readings were taken during the study period. For this purpose, the following model was

used:

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Ŷ = Time (fixed effect) + Dog ID (random effect) + ε

Where the overall effect of time was significant (p < 0.05), least square differences (LSD)

were further evaluated. The significance level was set to 5% for the LSD. Assumptions of the

model were assessed through visual inspection of residual plots.

Results

Cardiorenal effects of fluid administration

Measures of preload and intravascular volume included CVP, LA size (absolute and ratio

between LA:Ao), and LVIDd. IVF loading caused an immediate and significant rise in CVP

which gradually increased over the 40-minute duration of fluid loading (Figure 7). The CVP

reduced upon cessation of fluid administration but remained significantly elevated compared

to baseline. This was mirrored by the sequential TP concentrations, which decreased

precipitously during the 40-minute duration of fluid loading, then partially recovered upon

cessation of fluid loading (Figure 7). There was a strong and significant negative correlation

between the CVP and serial TP values (Spearman’s r = -0.78, 95% CI -0.86 to -0.66;

p < 0.0001).

There was a small but significant increase in LVIDd which persisted for 20 minutes

after discontinuation of fluid administration (Figure 7). The LA:Ao ratio did not increase

significantly, despite a moderate and positive correlation between the CVP and LA:Ao values

(Spearman’s r = 0.52, 95% CI 0.32 – 0.67; p < 0.0001). A monotonic relationship did not

exist between the CVP and wnLVIDd or between the LA:Ao and wnLVIDd.

Changes in intravascular volume were also expected to influence plasma

concentrations of PCV, BUN, and Crea. These reduced between baseline and 60 minutes; in

contrast, plasma electrolytes (Na, K, Cl) remained static (Figure 8).

Measures of left ventricular systolic function included LVIDs and FS. Neither these,

nor other measures of left ventricular function (LVPW and IVS), were observed to change

from baseline (Figure 8).

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Figure 7: Change in CVP, TP, LVIDd, and LA:Ao ratio over time. The median and

interquartile range were plotted. * denotes significant deviations from baseline

(p < 0.05). Abbreviations: CVP = central venous pressure; TP = total plasma protein;

LVIDd = left ventricular internal diameter in diastole; LA:Ao = left atrial to aorta ratio.

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Figure 8: Change in LVIDs, FS, LVPWs, LVPWd, IVSs, IVSd, PCV, BUN, Crea, Na, K,

and Cl over time. The median and interquartile range were plotted. * denotes

significant deviations from baseline (p < 0.05). Abbreviations: LVIDs = left ventricular

internal diameter in systole; FS = fractional shortening; LVPWs/d = left ventricular

posterior wall thickness in systole/diastole; IVSs/d = interventricular septum

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thickness in systole/diastole; PCV = packed cell volume; BUN = blood urea nitrogen;

Crea = creatinine; Na = sodium; K = potassium; Cl = chloride.

There was a small drop in SBP that was noted when fluid loading was commenced

(Figure 9). However, overall, BP and HR were not significantly affected by fluid loading.

CO was also calculated as a measure of overall cardiovascular performance. This

was observed to increase significantly from baseline between 20 – 60 minutes, after which it

declined back to baseline levels (Figure 9). Although statistical significance was reached,

there was a large degree of inter-dog variability in this measurement.

Measures of urinary effects included urine output (UOP) and USG. UOP increased

throughout the duration of fluid administration, reaching maximal production at 60 minutes

(Figure 9). UOP remained significantly elevated at the end of the study period. A drop in

USG was observed between baseline and 60 minutes (Figure 9).

There was no obvious linear correlation between UOP and CVP, MBP, FS, or CO.

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Figure 9: Change in BP, HR, CO, UOP, and USG over time. The median and

interquartile range were plotted. * denotes significant deviations from baseline.

Abbreviations: SBP, MBP, DBP = systolic, mean, and diastolic blood pressure;

HR = heart rate; CO = cardiac output; UOP = urine output; USG = urine specific gravity.

Immunoassays of NT-proBNP, BNP-32, and cGMP

Plasma NT-proBNP concentrations were below the lowest limit of detection throughout the

entire sampling period in one dog (Dog F). However, levels of plasma immunoreactive

BNP-32, plasma cGMP, and urinary cGMP were similar to concentrations detected in other

dogs in the study. In one dog (Dog C), plasma concentrations of cGMP were around 10-fold

higher than in others. Plasma concentrations of immunoreactive BNP-32 were marginally

higher than those observed in other individuals, but concentrations of NT-proBNP and

urinary cGMP were similar to concentrations detected in other dogs in the study. PCV, TP,

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USG, BUN, electrolytes, and Crea, physical exam, and haemodynamic measurements in

these two individuals were not different to those observed in other dogs in the study. As

such, only the NT-proBNP concentrations for Dog F and plasma cGMP concentrations for

Dog C were removed from general analysis.

In plasma, concentrations of NT-proBNP were around 10-fold greater than that of

BNP-32. NT-proBNP decreased significantly from baseline during the period of fluid

administration (Figure 10). In contrast, concentrations of BNP-32 did not change during or

after fluid administration (Figure 10). There was a weak, positive correlation between

concentrations of NT-proBNP and BNP-32 (Spearman’s r = 0.35, 95% CI 0.11 – 0.56;

p = 0.0049).

Urinary concentrations of cGMP (UcGMP) were 10 to 20-fold greater than that of

plasma cGMP (PcGMP). Concentrations were highest in the first 30 minutes of fluid loading

and reduced from around 40 minutes (Figure 10). Urinary excretion of cGMP (UEcGMP -

taking into account urine output and body weight) was higher than baseline levels between

20 – 80 minutes, with peak levels reached at 30 minutes, followed by a gradual reduction

back to baseline levels over the remainder of the study period (Figure 10). PcGMP reduced

from baseline, reaching lowest concentrations at 30 minutes (Figure 10). Significantly lower

concentrations remained at the end of the study period. The PcGMP and UEcGMP showed

no correlation.

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Figure 10: Change in NT-proBNP, BNP-32, UcGMP, UEcGMP, and PcGMP over time.

The median and interquartile range were plotted. * denotes significant deviations from

baseline (p < 0.05). Abbreviations: NT-proBNP = plasma N-terminal proBNP

concentrations; BNP-32 = plasma immunoreactive BNP-32 concentrations;

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UcGMP = urinary cGMP concentrations; UEcGMP = urinary excretion of cGMP;

PcGMP = plasma cGMP concentrations.

Measures of cardiovascular effects (CVP, FS, and CO) and measured urinary

variables (UOP, PcGMP, UEcGMP) were not obviously correlated with NT-proBNP and

BNP-32. A moderately strong, non-linear association between UEcGMP and CVP was

present (Spearman’s r = 0.53, 95% CI 0.33 – 0.68; p < 0.0001); in contrast, UEcGMP

showed negligible correlations with FS, CO, and MBP. There was a moderate, negative

correlation between PcGMP and CVP (Spearman’s r = -0.41, 95% CI -0.60 to -0.17,

p = 0.0009), but no obvious correlations were appreciable between PcGMP and MBP, FS, or

CO.

Discussion

The aim of this part of the chapter was to utilise a novel mass spectrometry assay to

evaluate the BNP system in normal dogs receiving short-term, high-rate, intravenous fluids.

In the absence of this specific BNP1-32 assay, a complete evaluation of the BNP system

was not possible. Immunoassays of BNP-32 and NT-proBNP were performed; however, it

was not possible to test for cross-reactivity with inactive or less active fragments of BNP-32

or the precursor molecule, proBNP1-108. Nevertheless, interesting observations were made

regarding the cardiorenal and endocrine effects of rapid fluid administration.

Administration of intravenous fluids caused measurable changes to the

cardiovascular system as anticipated. Following initiation of therapy, an increase in CVP,

and therefore, an increase in right atrial and ventricular preload, was detected. The resulting

rise in intravascular volume and ensuing haemodilution were well-represented by the strong,

negative correlation between CVP and TP. There was also an increase in wnLVIDd, which

supports the presence of increased preload to the left ventricle; however, this was not

proportional to CVP, as evidenced by the lack of an obvious correlation. In contrast, the

LA:Ao showed a moderate and positive monotonic relationship to CVP; however, it did not

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significantly increase from baseline. These echocardiogram-derived factors are prone to

higher degrees of error and variability than direct measurements such as CVP; nevertheless,

the overall combination of rise in CVP and wnLVIDd, as well as the trend seen in LA:Ao,

would suggest that a global increase in preload was achieved with intravenous fluids in this

study.

The increase in intravascular volume was expected to cause a temporary rise in BP

and reduction in HR due to activation of the baroreceptor reflex. Surprisingly, the BP

appeared to reduce slightly in the first 10 minutes of fluid loading, and the HR appeared to

increase throughout the duration of loading. These effects may have been related to

anaesthesia or the use of positive pressure ventilation. In addition, the rise in preload was

expected to increase systolic function due to Starling’s law; unfortunately, this was difficult to

assess as measures of systolic function in this study (FS, LVIDs) were highly preload-

dependent. Even so, the overall cardiac performance, as measured by CO, increased –

likely due to the combination of nonsignificant trends observed in HR, BP, and FS.

Despite evidence of appropriate increases in preload, the reductions in NT-proBNP

and BNP-32 concentrations observed during the study were unexpected and raise the

question as to whether the BNP system was adequately stimulated in the present study. This

was in contrast to findings of previous reports using a similar model of challenging the BNP

system (Hori et al., 2010; Lobo et al., 2010), in which increases in NT-proBNP and BNP-32

concentrations were observed. A potential cause for the initial reduction seen in this study is

haemodilution due to the administration of intravenous fluids. Indeed, the period of decline in

NT-proBNP concentrations coincided with the period of fluid administration (the first 40

minutes of the study), and a reduction in concentrations of other substances in plasma such

as the total protein, PCV, BUN, Creatinine, and PcGMP concentrations were also observed.

However, an increase in NT-proBNP and BNP-32 concentrations were seen despite the

effect of haemodilution in the aforementioned studies. This discrepancy prompts a review of

the various aspects of this study design.

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For example, it is possible that the difference in duration and amount of fluid

administered between studies contributed towards divergent results. In this study, a 40-

minute fluid loading period was used as opposed to a 60 minute protocol in the other two

studies. The basis for the decision to use 40 instead of 60 minutes was the finding of a

significant rise in NT-proBNP concentrations from 40 minutes into fluid administration in the

study by Hori et al., (2010). In addition, as little as 12.5 ml/kg/h of isotonic saline

administered over an hour was shown to cause an increase in plasma immunoreactive

BNP-32 concentrations in healthy people (Lobo et al., 2010). At the other end of the

spectrum, however, dogs receiving 100 ml/kg/h of intravenous fluids for 1.5 h experienced

no observable changes to plasma BNP-32 concentrations in another study (Borgeson et al.,

1998). Put together, the amount of fluid and duration of administration required for adequate

stimulation of the BNP system remain somewhat of a mystery.

In contrast, it is interesting to note that studies have consistently observed stimulation

of the ANP system following acute intravenous fluid administration (Borgeson et al., 1998;

Hori et al., 2010; Kamp-Jensen et al., 1990; Watenpaugh et al., 1992; Weil et al., 1985). In

the absence of obvious rises in BNP-32 and NT-proBNP, quantification of atrial natriuretic

peptides (ANP) would have provided a more complete understanding of the NPS, and this is

a limitation of the present study.

Although ANP was not measured, concentrations of its effector molecules were

quantified as an intermediate marker of biological effects of the NPS in this study. Binding of

active ANP and BNP to their receptor (NPR-A) results in synthesis of particulate guanylate

cyclase-mediated cGMP, which facilitates their downstream effects. As such, augmentation

of the NPS (both ANP and BNP production) would be expected to result in an increase in

PcGMP and UEcGMP (Brenner et al., 1990), and numerous studies have documented

higher concentrations of plasma and urinary cGMP in response to administration of synthetic

natriuretic peptides (Cataliotti et al., 2004; H. H. Chen et al., 2000; H. H. Chen et al., 2004;

Forfia et al., 2007; Ichiki et al., 2015; Ishikawa et al., 2005). In prior studies where high rates

of intravenous fluids were administered, an increase in PcGMP accompanied the stimulation

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of the NPS (Borgeson et al., 1998; Weil et al., 1985). However, an increase in UEcGMP was

not observed despite presence of higher PcGMP in one of these studies (Borgeson et al.,

1998). In the present study, PcGMP did not increase, but rather, decreased – likely due to

haemodilution. The UEcGMP initially increased; however, this peaked at 30 minutes into

fluid therapy and declined thereafter despite persistent increases in UOP. This raises the

suspicion that a true increase in cGMP production was not experienced, as UEcGMP would

have been expected to follow the course of the increase in UOP. Together with trends in

measured natriuretic peptide concentrations, these findings support the overt lack of

stimulation of the NPS in the present study.

Is it possible, then, to conclude that the amount of fluid administered here was

inadequate to stimulate the BNP system? Though the majority of findings were in favour of

this conclusion, it is important to note that the initial hydration state of the dogs was not

regulated. Some objective measures of hydration such as the PCV, TP, and Creatinine at

baseline did not suggest overt dehydration, but three of the dogs had USG > 1.040 at

baseline. If the dogs in this study were also relatively dehydrated at baseline compared to

subjects in other studies, the BNP system may not have been similarly activated by the

administration of this volume of intravenous fluids.

It is interesting to note that despite the lack of overt cardiovascular or BNP

stimulation in this study, a substantial increase in UOP and a corresponding decrease in

USG were experienced in the dogs. This is likely a consequence of increased renal blood

flow and reduced plasma osmolarity and the ensuing pressure diuresis, inhibition of RAAS

activity, and reduction in tubular reabsorption of sodium and water (Hall, 2016). Thus, the

multiple mechanisms involved in fluid homeostasis were highlighted here.

The vast majority of endocrine, cardiovascular, and urinary variables measured in

this study did not display a simple, linear correlation. This was expected, given the complex

nature of the relationships between these systems. With advancements in computer

technology and statistical methods, the creation of mathematical models to describe

physiological effects has become more accessible. Such a model could have been

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developed to describe the effect of fluid administration on the NPS and cardiorenal systems

in this study. However, this avenue was not pursued, primarily due to limitations relating to

time and resources. Had the study been designed with this type of analysis in mind, data

collection would likely have incorporated other variables used in prior models evaluating

cardiorenal systems (Hallow & Gebremichael, 2017; Karaaslan et al., 2005; Moss & Thomas,

2014) to facilitate model building.

That the timing of analysis for NT-proBNP, BNP-32, and cGMP was not standardised

due to the challenges encountered during mass spectrometry assay development was

another limitation of this study. This resulted in a prolonged duration of storage (around 1.5

years prior to analysis) for the samples used in BNP-32 and cGMP analysis. The protease

inhibitors used in the protocol have been shown to provide stability of BNP1-32 for 6 months

at -70°C (Niederkofler et al., 2008), and cGMP has been noted to be stable in EDTA

anticoagulated plasma at -20°C for 6 months (Vorderwinkler et al., 1991); however, the

effects of longer storage on these and urine samples is unknown. In spite of this, the lack of

a rising trend in NT-proBNP (which was assayed promptly) provided support for the

conclusions drawn from the data.

Overall Conclusions

We sought to develop a canine specific BNP1-32 mass spectrometry assay and utilise this

for evaluation of the BNP system in healthy, anaesthetised dogs receiving a short-term,

high-volume, intravenous fluid bolus. Assay development was unsuccessful, primarily due to

challenges encountered with sample preparation. For future attempts, an

immunoprecipitation technique is recommended for sample concentration, followed by use of

an LC-ESI-MS/MS set up for further purification and detection of the peptide. The fluid

loading protocol in this study did not produce measurable simulation of the BNP system;

furthermore, the resultant increase in intravascular volume, haemodilution, and diuresis

posed an additional challenge to the interpretation of plasma and urinary concentrations of

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endocrine markers. Alternate methods of ventricular loading would therefore be

recommended for future studies.

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Chapter 6:

Cardiorenal and endocrine effects of synthetic canine BNP1-32

in dogs with compensated congestive heart failure

due to myxomatous mitral valve disease: A pilot study

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Author contribution statement

This chapter is under review for publication by the Journal of Veterinary Internal Medicine.

Mariko Yata was the primary investigator and author of the work reported in this manuscript

and was involved in all aspects of study design, data collection, analysis, reporting, and

cGMP immunoassay performance.

Niek Beijerink provided guidance on the study design, assisted with data collection, and

critically reviewed this manuscript.

Hans Kooistra provided guidance with assay development and critically reviewed this

chapter.

Mariko Yata Date__ 14/8/2018___

Niek Beijerink Date__19/08/2018___

Hans Kooistra Date__14/08/2018___

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Introduction

Congestive heart failure (CHF) due to myxomatous mitral valve disease (MMVD) is the

leading cause of cardiac death in older, small-breed dogs (Borgarelli & Buchanan, 2012).

Medical therapy of CHF remains the predominant form of treatment in veterinary medicine,

with current guidelines recommending a combination therapy consisting of a diuretic,

angiotensin converting enzyme inhibitor (ACE-I), and inodilator (Atkins et al., 2009). Though

survival times using this treatment regimen have not yet been published, an estimation of

around one year is considered standard amongst experts, based on extrapolations from

clinical trials and retrospective studies evaluating survival outcomes on components of this

treatment regimen (Borgarelli et al., 2008; De Madron et al., 2011; Häggström et al., 2008;

Lombard et al., 2006). The reasonably poor prognosis associated with this condition has

prompted ongoing efforts in the discovery of additional or alternative therapeutics, including

various inhibitors of the renin-angiotensin-aldosterone system (RAAS) (Bernay et al., 2010;

Ovaert et al., 2010), newer diuretics (Chetboul et al., 2017; Peddle et al., 2012), and surgical

repair (T. Mizuno et al., 2013; Uechi, 2012; Uechi et al., 2012).

In these research efforts, a somewhat neglected therapeutic target is the natriuretic

peptide system (NPS). Brain natriuretic peptide (BNP1-32) is an active component of this

system. Binding to the A-type natriuretic peptide receptor (NPR-A) causes activation of

guanylyl cyclase, which converts the cytosolic nucleotide, GTP, into cGMP. From there,

cGMP acts as an intracellular secondary messenger and causes diuresis, natriuresis,

vasodilation, and RAAS antagonism. These are desirable properties to combat the

progression of CHF, and indeed, experimental animal studies (Cataliotti et al., 2004; H. H.

Chen et al., 2000; H. H. Chen, Schirger, et al., 2006; Forfia et al., 2007; Grantham et al.,

1997; Thireau et al., 2014; Yamamoto et al., 1997) and early clinical trials in humans (H. H.

Chen et al., 2012; H. H. Chen et al., 2004; Colucci et al., 2000; Publication Committee for

the VMAC Investigators, 2002) have shown benefits of BNP1-32 in the treatment of CHF.

This led to the registration in the United States of Nesiritide (Natrecor, Scios Inc., Mountain

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View, CA, USA), a recombinant human BNP1-32 product, for intravenous use in people with

decompensated CHF displaying symptoms of dyspnea at rest.

Despite its initial success, an ultimate reduction in the rate of mortality or re-

hospitalization was not observed in humans with CHF (O'Connor et al., 2011). This caused

Nesiritide to fall out of favor in human medicine and likely contributed towards reduced

interest for use in veterinary patients. Another limiting factor is its formulation – an injectable

preparation, which has traditionally resulted in use as an intravenous drug. Yet there is

growing evidence of its safety and efficacy when administered subcutaneously (H. H. Chen

et al., 2012; H. H. Chen et al., 2000; H. H. Chen, Huntley, et al., 2006; H. H. Chen et al.,

2004; H. H. Chen, Schirger, et al., 2006; Oyama et al., 2017). Subcutaneous administration

facilitates its use in chronic therapy, and its potential role in treatment of subclinical heart

disease (McKie et al., 2016) and in humans with diastolic dysfunction and right-sided CHF

(H. H. Chen et al., 2012; Clarkson et al., 1995; Kwon et al., 2003; Tong et al., 2004;

Yamamoto et al., 1997; Zakir et al., 2005) have also been explored. Although no solid

conclusions to support its benefits in these contexts have been reported to date, there is

enough theoretical and experimental data to support further investigation into its use in dogs

with naturally occurring CHF due to MMVD.

In this pilot study, the cardiorenal and endocrine effects of subcutaneous doses of

synthetic canine BNP1-32, given with or without frusemide, were evaluated in dogs with

compensated CHF due to MMVD.

Materials and Methods

Animals

Seven client-owned geriatric (10.5 to 17 years old) male dogs with compensated ACVIM

class C (chronic) CHF due to MMVD were recruited into this study. Ethics approval was

obtained from the University of Sydney Animal Ethics Committee prior to commencement of

the study (approval number 2016/1113).

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Study design

This study was a single dose, cross-over, pilot study evaluating the efficacy of synthetic

canine BNP1-32, given with or without frusemide, in the target population. It was conducted

at the University Veterinary Teaching Hospital, Sydney between May – September, 2017.

Enrolment Criteria

Prior to enrolment, each dog had a screening examination which consisted of a review of the

history, physical examination, and oscillometric blood pressure (BP) measurements

(VetHDO, Memo Diagnostic, Darmstadt, Germany). Thoracic radiographs, an

echocardiogram, electrocardiography (ECG), and select blood tests (alanine

aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, albumin,

globulin, total protein, PCV, electrolytes) were also performed.

The inclusion criteria consisted of a diagnosis of CHF due to MMVD and good control

of clinical signs with pimobendan (Vetmedin®, Boehringer Ingelheim Pty Ltd, North Ryde,

NSW, Australia), benazepril (Fortekor; Elanco Australasia Pty Ltd, West Ryde, NSW,

Australia), and frusemide (APO Frusemide; Apotex Pty Ltd, Macquarie Park, NSW,

Australia). The exclusion criteria included previously diagnosed or suspected renal disease,

adrenal disease, diabetes mellitus, systemic hyper/hypotension, primary or concurrent right-

sided CHF, and hemodynamically significant arrhythmias. In addition, dogs were not enrolled

if concurrent treatments with sildenafil, spironolactone, amlodipine, thiazide diuretics, or

other medications which may interfere significantly with cGMP metabolism, blood pressure

regulation, or renal function were required.

The diagnosis of CHF due to MMVD was made on characteristic history and physical

examination findings (dyspnea and/or tachypnea responsive to treatment with frusemide;

presence of abnormal lung sounds, at least a grade 4/6 systolic heart murmur loudest at the

left apical region, and tachycardia on examination); echocardiographic evidence of severe

MMVD and cardiac remodeling due to volume overload (i.e. characteristic lesions of the

mitral valves, severe mitral valve regurgitation, significant left atrial and ventricular

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enlargement) on an echocardiogram performed by a board-certified veterinary cardiologist

(Niek Beijerink); and radiographic evidence of cardiomegaly with left atrial enlargement,

pulmonary edema, and pulmonary vasodilation. Two dogs did not have radiographs

performed prior to commencement of treatment for CHF. However, serial radiographs

depicting pulmonary edema which resolved with commencement of therapy were available

for the remainder of dogs enrolled.

Study outline

Following enrolment, dogs presented to the University Veterinary Teaching Hospital, Sydney

on three days, each two weeks apart, for this study. Benazepril was dosed once daily at

night, including on the night prior to each visit. The morning doses of pimobendan and

frusemide were withheld on each study day. On the morning of each visit, history and

physical examinations were performed to ensure well-being of the dogs, and peripheral IV

and indwelling urinary catheters were placed. At the end of a one hour control period, heart

rate (HR), BP, and urine output (UOP) were measured; blood and urine samples were

collected for endocrine, electrolyte, biochemistry, and cGMP testing. The amount of blood

collected was replaced with 7 ml saline. One of the following treatments was given:

• BNP: 5 μg/kg synthetic canine BNP1-32 (Canine BNP1-32 (011-22), Phoenix

Pharmaceuticals Inc, Burlingame, California, USA) subcutaneously (visit one)

• FRUS: 2 mg/kg frusemide (Ileum Frusemide, Troy Laboratory Ltd., Glendenning,

NSW, Australia) subcutaneously (visit two)

• BNP/FRUS: 5 μg/kg synthetic canine BNP1-32 and 2 mg/kg frusemide

subcutaneously (visit three)

Treatment order was not randomized, and all dogs received all three treatments in the order

listed above.

Measurement of HR and BP; collection of 7 ml blood; and IV administration of 7 ml

saline were repeated at one and three hours following treatment. Measurement of UOP and

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collection of 5 ml urine were performed at one, two, and three hours following treatment.

PCV and total protein (TP) were monitored for safety purposes throughout. At the completion

of this sampling period, IV and urinary catheters were removed, and dogs were treated with

their usual morning medications (pimobendan and frusemide, if frusemide had not been

given as a part of the treatment), then fed. Dogs were then discharged home to continue

routine medical therapy with pimobendan, benazepril, and frusemide, and owners were

advised to monitor for any abnormalities to the injection site, changes in behavior, or

development of signs of decompensated CHF until the next scheduled visit.

HR and systolic, diastolic, and mean BP (SBP, DBP, MBP) were measured in

triplicate and averaged prior to statistical analysis.

Sample processing and storage

Blood samples were obtained via jugular venipuncture and immediately placed into plastic

blood tubes containing EDTA or lithium heparin. Urine samples were collected into sterile

plastic urine tubes. EDTA anticoagulated blood samples and urine samples were

immediately centrifuged at or below 4°C at 5500 RCF for 10 minutes for collection of plasma

and sediment-free urine. After obtaining a sample for PCV and TP, the lithium heparin

anticoagulated blood samples were centrifuged at 4000 RCF for 4 minutes at room

temperature for collection of plasma. For BNP1-32 testing, additional protease

inhibition was achieved by adding 1 mL EDTA plasma to a pre-prepared mixture of

4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and Benazmidine (Sigma

Aldrich, Castle Hill, NSW Australia) to give a final concentration of 5 mM AEBSF and 10 mM

Benzamidine (Niederkofler et al., 2008). The remaining plasma and urine samples were also

transferred into sterile, plain tubes and frozen for later analysis. Prior to analysis, samples

were stored at -80°C for up to 136 days for BNP-32; at or below -30°C for up to 144 days for

cGMP; at -80°C for up to 125 days for sodium, potassium, and creatinine; and at or below

-40°C for up to 270 days for aldosterone assays. Stability testing was not performed in this

study; however, samples treated and held under similar conditions were reportedly stable in

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human plasma samples for six months for BNP-32 (Niederkofler et al., 2008) and plasma

and urinary cGMP (W.-c. Liao et al., 2003; Vorderwinkler et al., 1991; Yanhua Zhang et al.,

2009). Furthermore, aldosterone concentrations in human serum control samples stored at

-30°C at the particular laboratory consulted for aldosterone assays were noted to be stable

for at least 9 months, with no drift observed at 0.12 nM/L and 0.68 nM/L (n = 78).

Blood and urine analysis

Plasma immunoreactive BNP-32 concentrations were measured at a commercial laboratory

(Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) using a validated (Schellenberg et al.,

2008) radioimmunoassay kit made by the company (BNP-32 Canine RIA kit, RK-011-22,

Phoenix Pharmaceuticals Inc., Burlingame, CA, USA). The samples were transported frozen

to the laboratory, and each sample was assayed in duplicate and averaged for analysis.

Plasma and urine cGMP concentrations were assayed using a commercially

available competitive enzyme-linked immunosorbent assay kit using the manufacturer’s

guidelines (ParameterTM cGMP Assay, KGE003, R&D Systems Inc., Minneapolis, MN, USA).

Low, medium, and high controls (QC54 cGMP controls, R&D Systems Inc., Minneapolis, MN,

USA) were added to each plate. Briefly, urine and plasma samples were thawed at room

temperature, and 25 μl of each sample was mixed with 475 μl of diluent. Samples were

further diluted 1:2 using the diluent provided if final concentrations exceeded the range of

the assay. 100 μl of each standard, sample, and control were mixed with 50 μl of the cGMP

conjugate, followed by 50 μl of the primary antibody solution. After incubating for three hours

on a microplate shaker at 450 rpm at room temperature, wells were washed four times, and

200 μl of substrate solution were added. This was incubated for 30 minutes in the dark, and

50 μl of stop solution were added. The plate was read using a microplate reader (TECAN

Infinite® M1000 PRO, Männedorf, Switzerland) set at 450 and 540 nm.

Readings made at 540 nm were subtracted from those at 450 nm. A standard curve

was generated using a 4-parameter logistic curve fit using Graphpad Prism V7.02 (La Jolla,

CA, USA). Measured concentrations were derived using this standard curve and multiplied

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by the dilution factor. All samples and standards were assayed in triplicate, all controls in

duplicate, and results were averaged for analysis. All standard curves had R^2 > 0.987.

The %CV of standard, sample, and control replicates were <10%, and the %CV of standards

between plates were <10%. Each control fell within the expected concentration range.

Plasma concentrations of cGMP were reported as pM/ml. In order to account for differences

in concentration due to rate of urine production, urinary excretion of cGMP (UEcGMP;

pM/kg/min) was also calculated by multiplying the UOP by the urinary concentration of

cGMP.

Plasma and urine sodium (Na), potassium (K), and creatinine concentrations were

measured at a commercial veterinary diagnostic laboratory (Veterinary Pathology Diagnostic

Service, Camperdown, NSW, Australia). Fractional excretion of sodium (FENa) and

potassium (FEK) were calculated using the formula:

FEx = 100 * (Urinary x * Plasma creatinine) / (Plasma x * Urinary creatinine)

Where X = sodium or potassium

Plasma concentrations of aldosterone were determined at the Radboud University

Medical Centre laboratory (Nijmegen, Netherlands) using a validated aldosterone assay

involving ultra-high performance liquid chromatography (UHPLC) coupled with tandem mass

spectrometry (MS/MS) (Rotteveel-de Groot et al., 2018 manuscript in preparation). Briefly,

500 μl of plasma was prepared with protein precipitation, supported liquid extraction

(Isolute® SLE+, Biotage, Uppsala, Sweden) and solid phase extraction (Oasis HLB, Waters,

Etten-Leur, Netherlands). Eluates were injected on an Agilent Technologies 1290 Infinity VL

UHPLC system (Agilent Technologies, Santa Clara, California, USA) equipped with a CSH

Phenyl Hexyl (1.7 μm, 2.1 X 100mm) analytical column (Waters, Etten-Leur, Netherlands)

and coupled to an Agilent 6490 tandem mass spectrometer (Agilent Technologies, Santa

Clara, California, USA). Settings included a capillary voltage of 3.0 kV, fragmentor voltage of

380 V, sheath gas temperature of 350°C, and gas temperature of 200°C. N2 was used as the

collision gas. Aldosterone was quantified using both aldosterone-d4 and aldosterone-d7

internal standards (IsoSciences, Pennsylvania, USA). Two transitions (qualitative and

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quantitative) were measured for each analyte. The transitions (Q1 > Q3) were m/z 366.1 >

338.2 (15 kEV) and m/z 366.1 > 194.1 (15 kEV) for aldosterone-d7; m/z 363.1 > 335.2 (15

kEV) and m/z 363.1 > 190.1 (15 kEV) for aldosterone-d4; m/z 359.1 > 331.2 (15 kEV) and

m/z 359.1 > 189.1 (15 kEV) for aldosterone. The dwell time was 60 milliseconds. Sample

concentrations were derived using a 7-point calibration curve generated from aldosterone

reference standards (Batch H158, Steraloids, Rhode Island, USA) using Microsoft Excel and

MassHunter (Agilent Technologies, Santa Clara, California, USA). All samples and

standards were assayed in duplicate and averaged. The lowest limit of quantification (LLOQ)

was estimated at 0.070 nM/L, with total %CV of 9.0 and 6.9 at 0.12 nM/L and 0.68 nM/L,

respectively (n = 78).

Statistical analysis

Descriptive analysis consisted of visual inspection of trends over time and summary

statistics. The median and range were reported. Genstat 16th Edition (VSN International Ltd,

Hemel) was used for statistical analysis. Data were evaluated for normality via inspection of

Q-Q plots. Where the data were not normally distributed, transformations (loge for UOP,

plasma immunoreactive BNP-32 concentrations, FEK, urinary cGMP concentrations, urinary

excretion of cGMP; Logit transformation for FENa) were performed to approximate normality

prior to statistical analysis.

Between- and within-treatment effects were evaluated via linear mixed modelling with

restricted maximum likelihood estimation using the following equation:

Ŷ = Treatment + Time + Treatment*Time + Dog ID (random effect) + ε

Significance was set at α of 0.05. If the interaction term was significant, significant

interactions were identified using the least square difference method post hoc. If this term

was not significant, it was dropped from the model. Model assumptions were assessed

through visual inspection of residual plots.

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Results

Breeds represented in the study cohort included the Cavalier King Charles Spaniel (n = 2),

Kelpie, Maltese cross, Jack Russel Terrier cross, and Miniature Poodle cross (n = 2).

Baseline characteristics and doses of medications have been summarized in Table 1. At the

time of the first study day (visit one), dogs had been on treatment with frusemide for a

median of 59 days (range 18 - 133), pimobendan for 63 days (range 26 - 252), and

benazepril for 33 days (range 16 - 62). The median number of days between diagnosis of

CHF and the first study day was 59 days (range 18 - 133).

A deviation from the protocol occurred during visit one (BNP treatment) in one dog

when urine samples were erroneously collected from the end of an extension line holding

10 ml of volume which was attached to the urinary catheter instead of emptying the contents

of the line each time. As the line was filled with urine prior to collection of data, the

measurement of urine output was not affected. However, the volume held in this line

exceeded the amount of urine production in each hour; thus, urinary analyte concentrations

did not accurately reflect expected concentrations for the period in which the sample was

taken. As such, urinary endocrine, cGMP, and fractional excretions at visit one for this dog

were not calculated, and these data were excluded from the overall analysis.

Synthetic canine BNP1-32 was well-tolerated by all dogs in the study. There were no

systemic or local adverse effects encountered during the study.

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Table 1: Baseline characteristics at the time of screening in seven dogs enrolled in

the study. Abbreviations: LA:Ao = left atrium to aorta ratio; wnLVIDd/s = weight-

normalised left ventricular internal diameter in diastole/systole; FS = fractional

shortening; HR = heart rate; SBP/DBP/MBP = systolic/diastolic/mean blood pressure;

BG = blood glucose; BUN = blood urea nitrogen; Crea = creatinine; TP = total protein;

ALT = alanine aminotransferase; ALKP = alkaline phosphatase; Na = sodium;

K = potassium; Cl = chloride; PCV = packed cell volume.

Median Range

Weight at screening (kg) 8.9 5.2 - 14.4

Age (years) 12 10.5 - 17

Daily pimobendan dose (mg/kg/d) 0.45 0.32 - 0.56

Daily frusemide dose (mg/kg/d) 3.85 1.94 - 5.06

Daily benazepril dose (mg/kg/d) 0.32 0.17 - 0.56

LA:Ao 2.3 1.8 - 2.5

wnLVIDd 2.16 1.84 - 2.44

wnLVIDs 0.95 0.83 - 1.45

FS (%) 53 35.3 - 56.9

HR (bpm) 120 114 - 150

SBP (mmHg) 138 120 - 148

DBP (mmHg) 80 66 - 85

MBP (mmHg) 100 92 - 105

BG (mg/dL) 104.5 82.88 - 120.72

BUN (mg/dL) 33.33 24.65 - 48.74

Crea (mg/dL) 1.14 0.74 - 1.67

TP (g/dL) 7.3 5.1 - 7.8

Albumin (g/dL) 3.2 2.5 - 4.2

Globulin (g/dL) 3.6 2.7 - 4.5

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Table 1 (Continued).

Parameters Median Range

ALT (U/L) 95 24 - 293

ALKP (U/L) 101 12 - 341

Na (mEq/L) 149 145 - 153

K (mEq/L) 3.6 2.7 - 4.2

Cl (mEq/L) 112 106 - 120

PCV (%) 47 37 - 54

Effect of treatments on endocrine variables, cGMP, and fractional excretions

The effect of each treatment on endocrine, cGMP, and FENa/K are depicted in Figures 1, 2,

and 3.

Plasma concentrations of immunoreactive BNP-32 rose significantly within an hour of

BNP and FRUS/BNP treatments, but not with FRUS (Figure 1). Immunoreactive BNP-32

levels returned to baseline by three hours following treatment.

Significant between-treatment or within-treatment effects were not observed with

regard to plasma concentrations of cGMP, as indicated by the lack of a significant

treatment*time interaction (Figure 1). In contrast to plasma concentrations of cGMP, a

significant rise in UEcGMP occurred following BNP and FRUS/BNP treatments, with

observable differences arising between these treatments and FRUS (Figure 2). Maximal

effects were reached one hour post treatment and decreased to baseline levels by three

hours post treatment. In contrast, UEcGMP reduced following FRUS treatment.

Both FENa and FEK increased when dogs were given any treatment containing

frusemide (Figure 2). However, administration of BNP did not cause a significant change in

these variables, and the addition of BNP did not result in significant changes in the excretion

of these electrolytes when compared to treatment with frusemide alone.

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Around 60% of plasma samples had aldosterone concentrations < LLOQ. Due to this

large number, only a descriptive analysis was performed. Traditional approaches for

handling such data in bioanalytics include reporting values as missing, “rounding down”

values to zero, or estimating these values to be half the LLOQ (Keizer et al., 2015). As the

data was not missing, and as aldosterone concentrations would not be expected to be zero

from a biological perspective, the value of half the LLOQ was assigned where concentrations

< LLOQ were obtained, and plasma aldosterone concentrations were graphically depicted

(Figure 3).

Effect of treatments on cardiorenal variables

The UOP increased significantly from baseline following FRUS and FRUS/BNP and

remained elevated at the end of the study; in contrast, BNP alone did not result in changes

to UOP (Figure 4). Maximal effects were reached at 1 hour post treatment in the FRUS and

FRUS/BNP groups, with a 1246% and 887% increase in median UOP compared to baseline,

respectively. Though these treatments resulted in significantly higher UOP compared to BNP,

there were no differences between the two frusemide containing treatments. None of the

treatments resulted in significant changes in HR or BP (Figure 5).

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Figure 1: Plasma concentrations of immunoreactive BNP-32 (pg/ml) and cGMP

(pM/ml) following administration of BNP1-32 (5 μg/kg, solid black line), Frusemide

(2 mg/kg, solid grey line), or BNP1-32 + Frusemide (5 μg/kg + 2 mg/kg, dashed black

line) subcutaneously in seven dogs with chronic congestive heart failure due to

myxomatous mitral valve disease. The median and range were plotted.

Asterisks denote significant (p < 0.05) differences between BNP1-32 and Frusemide

(*); and BNP1-32 + Frusemide and Frusemide (***).

Hats denote significant (p < 0.05) differences from baseline for BNP1-32 (^) and

BNP1-32 + Frusemide (^^^).

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Figure 2: Urinary excretion of cGMP (pM/kg/min) and fractional excretions of sodium

(FENa, %) and potassium (FEK, %) following administration of BNP1-32 (5 μg/kg, solid

black line), Frusemide (2 mg/kg, solid grey line), or BNP1-32 + Frusemide (5 μg/kg +

2 mg/kg, dashed black line) subcutaneously in seven dogs with chronic congestive

heart failure due to myxomatous mitral valve disease. The median and range were

plotted.

Asterisks denote significant (p < 0.05) differences between BNP1-32 and Frusemide

(*); BNP1-32 and BNP1-32 + Frusemide (**); and BNP1-32 + Frusemide and Frusemide

(***).

Hats denote significant (p < 0.05) differences from baseline for BNP1-32 (^);

Frusemide (^^); and BNP1-32 + Frusemide (^^^).

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Figure 3: Plasma concentrations of aldosterone (nM/L) following administration of

BNP1-32 (5 μg/kg, solid black line), Frusemide (2 mg/kg, solid grey line), or BNP1-32 +

Frusemide (5 μg/kg + 2 mg/kg, dashed black line) subcutaneously in seven dogs with

chronic congestive heart failure due to myxomatous mitral valve disease. The LLOQ

of the assay was 0.070 nM/L. All concentrations < LLOQ were estimated to be half the

LLOQ.

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Figure 4: Urine output (UOP) following administration of BNP1-32 (5 μg/kg, solid black

line), Frusemide (2 mg/kg, solid grey line), or BNP1-32 + Frusemide (5 μg/kg + 2 mg/kg,

dashed black line) subcutaneously in seven dogs with chronic congestive heart

failure due to myxomatous mitral valve disease. The median and range were plotted.

Asterisks denote significant (p < 0.05) differences between BNP1-32 and Frusemide

(*); and BNP1-32 and BNP1-32 + Frusemide (**).

Hats denote significant (p < 0.05) differences from baseline for Frusemide (^^) and

BNP1-32 + Frusemide (^^^).

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Figure 5: Blood pressure (BP) and heart rate (HR) following administration of BNP1-32

(5 μg/kg, solid black line), Frusemide (2 mg/kg, solid grey line), or BNP1-32 +

Frusemide (5 μg/kg + 2 mg/kg, dashed black line) subcutaneously in seven dogs with

chronic congestive heart failure due to myxomatous mitral valve disease. The median

and range were plotted.

Discussion

This is a report of the first prospective study evaluating the cardiorenal and endocrine effects

and tolerability of single doses of synthetic canine BNP1-32 given subcutaneously to dogs

with compensated ACVIM stage C CHF due to MMVD. BNP1-32 was well-tolerated, and

following subcutaneous dosing, it was well-absorbed and resulted in an increase in

UEcGMP; however, there were no measurable effects on biological outcomes (UOP, BP, HR,

FENa/K, and plasma aldosterone concentration) in this study. These findings are in contrast to

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results from dogs with experimentally induced CHF, and thus, raise questions regarding the

potential for natriuretic peptide resistance in dogs with naturally occurring CHF.

In particular, derangements of intracellular pathways via which cGMP mediates

natriuresis and diuresis may exist in dogs with CHF due to MMVD. The rise in plasma

concentrations of immunoreactive BNP1-32 following its administration confirms successful

absorption from the site of administration. Appropriate binding to NPR-A is demonstrated

through the rise in UEcGMP accompanying any treatment containing BNP1-32 but not

frusemide alone. This is also supported by the trends in plasma concentrations of cGMP

albeit not reaching significance, likely owing to the high degree of variability across all time

points and small number of dogs in the study. The validity of urinary and plasma

concentrations of cGMP in demonstrating this interaction is evidenced by observations from

numerous previous studies relating to this matter (Boerrigter et al., 2007; Cataliotti et al.,

2004; H. H. Chen et al., 2012; H. H. Chen et al., 2000; H. H. Chen, Huntley, et al., 2006; H.

H. Chen et al., 2004; H. H. Chen, Schirger, et al., 2006; Forfia et al., 2007; Grantham et al.,

1997; Jensen et al., 1999; McKie et al., 2016; Nakamura et al., 1998; Stasch et al., 1989;

Wong et al., 1988). These changes in cGMP also make NPR-A receptor downregulation less

likely. The process of elimination thus renders downstream intracellular mechanisms a

potential source of natriuretic peptide resistance in these dogs. Although the entirety of the

pathways by which cGMP mediates cellular effects has yet to be discovered, there is

evidence to support involvement of cGMP-dependent protein kinases (PKG) (Martel et al.,

2010; Potter et al., 2006; Theilig & Wu, 2015) and amiloride-sensitive cation channels

(Martel et al., 2010) in the generation of natriuretic peptide-induced vasodilation, natriuresis,

and diuresis. In addition, phosphodiesterase 5 inhibition to reduce cGMP hydrolysis appears

to be of benefit in the face of CHF and augments effects of exogenous BNP1-32 (H. H. Chen,

Huntley, et al., 2006; Forfia et al., 2007; Guazzi et al., 2011; Katz et al., 2000; Takimoto et al.,

2005). These intracellular mechanisms remain an interesting area for future investigation

from both a pathophysiological and therapeutic perspective, and the findings arising from

this study justify the need for such a scientific quest.

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Notwithstanding this theory, it remains possible that biological effects of BNP1-32

may have been obscured in the present study due to aspects relating to its study design.

The labelled use of Nesiritide in people is for acutely decompensated CHF, to be given as an

intravenous bolus followed by a continuous rate infusion. In contrast, this study utilized a

subcutaneous mode of delivery in dogs with compensated chronic CHF in order to minimize

variability in clinical presentation and investigate the potential for its use in chronic therapy.

Despite experimental studies showing effects of BNP1-32 in chronic CHF (H. H. Chen et al.,

2012; H. H. Chen et al., 2004; H. H. Chen, Schirger, et al., 2006), it is possible that the lack

of an overt state of volume overload may have resulted in lack of diuresis and natriuresis in

this study, or that longer exposure to BNP1-32 (either as a constant rate infusion or via

multiple doses of subcutaneous BNP1-32) may have been required to produce detectable

changes to cardiorenal variables. The non-invasive techniques of measuring BP and HR

may have lacked the sensitivity to detect BNP1-32 effects in such a small group of dogs, and

only a limited number of cardiovascular variables were evaluated. Renal blood flow and

measures of renal function other than UOP and FENa/K were not measured. The small

number of sampling points may have also resulted in a failure to detect short-lived effects. A

cross-over treatment design was used so that each dog acted as its own “control”, thereby

reducing variability and increasing chances of detecting treatment effects in this small cohort

of dogs; however, the inclusion of a placebo treatment stream in which no frusemide or

BNP1-32 was given could have helped to highlight smaller treatment effects. Finally, a group

of healthy dogs undergoing the same study procedures would have strengthened the

conclusions drawn from this study and enabled direct comparisons to be made between

healthy and diseased dogs. These limitations highlight the need for further research efforts

into the role of BNP1-32 in dogs with CHF.

The effect of BNP1-32 on the spontaneous and frusemide-activated RAAS in this

population remains ill-defined due to the majority of samples containing aldosterone

concentrations below the LLOQ. With the reported range of plasma aldosterone

concentrations being anywhere from around 20 - 800 pM/L in dogs with naturally occurring

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CHF (Häggström et al., 1997; Häggström, Lord, et al., 2013), a LLOQ of 70 pM/L (equiv. to

0.07 nM/L) would appear to be adequate. Although in-house stability testing showed

prolonged stability of serum aldosterone, sample degradation in plasma remains a possible

cause of these low concentrations. Alternative explanations include the chronicity of

benazepril therapy (16 - 62 days, with median 33 days) and the possibility for reduced RAAS

activation in the chronic, stable phase of CHF (Hezzell, Boswood, Chang, Moonarmart, &

Elliott, 2012). Despite this, it is interesting to note that both treatments containing BNP1-32

appeared to demonstrate an initial suppression of aldosterone concentrations followed by an

rise in concentrations by three hours post treatment where trends could be appreciated on

Figure 3. This observation is weak but is in contrast to previous reports of aldosterone

suppression following BNP1-32 administration in people with CHF (H. H. Chen et al., 2004)

and in dogs with frusemide-induced RAAS activation (Cataliotti et al., 2004). This

unexpected effect therefore requires further investigations to elucidate.

As a final note, the cGMP and aldosterone assays employed in this study

should ideally have been fully validated for use in canine plasma and urine. Instead, formal

validation was performed by the manufacturers of the assays utilising samples from other

species (humans and rodents), and partial validation and quality control monitoring of canine

samples were employed during the course of this study. Yet with the molecular structure of

both cGMP and aldosterone being conserved between species, it is expected that the

assays will perform similarly in samples from different species. These tests were thus

deemed fit for use for the purposes of this study.

Conclusions

The cardiorenal and endocrine effects of a single dose of synthetic BNP1-32, given

subcutaneously with or without frusemide, were evaluated in dogs with compensated CHF

due to MMVD receiving chronic therapy with pimobendan, frusemide, and benazepril.

Subcutaneous BNP1-32 was well-absorbed and increased UEcGMP. However, there were

no biological effects on HR, BP, UOP, or FENa/K, and BNP1-32 did not augment effects of

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frusemide on UOP or FENa/K. These findings raise further questions regarding the role of

BNP1-32 in both the pathophysiology and treatment of CHF in dogs with MMVD. Areas of

potential future research were highlighted.

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Chapter 7:

Discussion and conclusions

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7.1) Discussion

The five chapters encompassing the body of this work served two overarching purposes – to

provide justification for current off-label practices and to provide novel insights for guidance

of future advancements in the treatment of canine and feline CHF. Each chapter set out to

fulfil the following aims:

• To evaluate the pharmacokinetics and pharmacodynamics of pimobendan and its

active metabolite of a novel nonaqueous oral solution of pimobendan in healthy dogs

(Chapter 2).

• To evaluate the pharmacokinetics and pharmacodynamics of pimobendan in healthy

cats (Chapter 3).

• To prospectively evaluate the natural course of disease in dogs with newly diagnosed

CHF due to MMVD when treated with a combination therapy of frusemide,

pimobendan, an ACE-I, and spironolactone (Chapter 4).

• To develop a mass spectrometry assay for identification and quantification of active

BNP1-32 in canine plasma (Chapter 5).

• To evaluate the BNP system and its effects in normal dogs experiencing an acute

expansion of intravascular volume via fluid therapy (Chapter 5).

• To investigate the efficacy of synthetic canine BNP1-32 given subcutaneously, with

or without frusemide, in dogs with CHF due to MMVD (Chapter 6).

Most of these aims were met, with numerous subsidiary discoveries made along the way.

The pharmacokinetics of the liquid formulation of pimobendan in healthy dogs

(Chapter 2) revealed similar overall kinetics to that of other published reports of pimobendan

in dogs. At the standard dose of 0.25 mg/kg, the solution was rapidly absorbed (Tmax 1.1 h

with Cmax 18.6 ng/ml) and converted to its active metabolite, ODMP (ODMP Tmax 1.3 h with

Cmax 16.2 ng/ml), widely distributed (V/F 15.5 L/kg), and rapidly cleared

(CL/F 176.5 ml/min/kg with t1/2 0.9 h). Pimobendan was deemed unlikely to accumulate

when given twice daily using the investigated dose. However, the magnitudes of these

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measures of kinetics have been somewhat variable across existing reports. For example, the

reported mean plasma Cmax of pimobendan following oral dosing of ~ 0.25 mg/kg ranged

from ~ 3 ng/ml (Boehringer Ingelheim Ltd., 2012b) to ~ 40 ng/ml (Bell et al., 2016), with a

Tmax of 40 min to 2 h (Bell et al., 2016; Boehringer Ingelheim Ltd., 2012b). Following

intravenous pimobendan at ~ 0.12 mg/kg, a mean volume of distribution and clearance of

~ 0.25 L/kg and ~ 0.4 L*h/kg were calculated using concentrations of the drug in whole blood

(Bell et al., 2016); in contrast, the mean values reported on the product insert for various oral

solid dose forms and injectable formulation of pimobendan were ~ 2.6 L/kg and

~ 90 ml/kg/min (Boehringer Ingelheim Ltd., 2008, 2012a, 2012b). The t1/2, however,

remained similar between all studies, indicating the presence of similarities across studies in

the slope of the terminal portion of the concentration-time curve. The metabolite displayed a

similar Cmax to that of pimobendan and had a prolonged t1/2 in the product insert of a

registered formulation (Boehringer Ingelheim Ltd., 2012b), and the findings in Chapter 2 of

this thesis were in line with this. As the metabolite was not directly administered, the

clearance and volume of distribution of ODMP were not described and remains a mystery

due to the lack of published information regarding this in dogs. Aside from these, there are

no other published reports of metabolite kinetics in dogs; however, plasma concentrations of

ODMP following high doses of intravenous pimobendan (0.25 – 2.25 mg/kg) have been

reported (Schneider et al., 1997). Unfortunately, the scarcity of published pharmacokinetic

reports; differences in methodology including number of dogs utilised, sampling points, and

analytical methods; use of various formulations; and the lack of data regarding bioavailability

from the present study make it extremely difficult to directly compare the results of these

studies. Nevertheless, the work in Chapter 2 contributed valuable pharmacokinetic data on

this novel pimobendan solution.

The study in Chapter 2 described the pharmacokinetics and pharmacodynamics of a

novel formulation of pimobendan but did not compare these against the registered

formulations of pimobendan. The reasons for this particular study design were

circumstantial; yet such a comparative study would have enabled a more accurate

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assessment of its performance in relation to existing formulations. In this case, bioavailability

would have been an essential fraction to clarify. Moreover, demonstration of bioequivalence

is often necessary for registration of alternate formulations and generic products (Australian

Pesticides and Veterinary Medicines Authority 2017). Although this study was conducted

and reported independently, the data obtained from this study, together with additional

pharmacokinetic, safety, and stability work, ultimately contributed towards successful

registration of the product through the Australian Pesticides and Veterinary Medicines

Authority in 2016 (Product No. 81552).

One of the key findings arising from the studies on pimobendan in healthy dogs and

cats (Chapters 2 and 3) was the discovery of reduced metabolism of pimobendan in cats

compared to dogs. In dogs, the AUC0-∞ of its active metabolite, ODMP, exceeded that of

pimobendan; in contrast, the AUC0-∞ of pimobendan far exceeded that of ODMP in cats. This

finding supports and extends upon the pharmacokinetic work of Hanzlicek et al. (2012) in

which reduced metabolism of pimobendan was suspected in cats, and it provides the first

published description of metabolite kinetics in cats. Although the metabolic pathways of

pimobendan in dogs and cats have yet to be characterised and remains an area for future

research efforts, this difference in its metabolism could contribute towards inter-species

variation in drug effects.

Indeed, a discrepancy in the pharmacodynamics of pimobendan was detected

between dogs and cats, with increases in echocardiographic measures of systolic function

being readily apparent in the former but not in the latter (Chapters 2 and 3). This was

surprising as the higher concentration of pimobendan in cats was expected to produce

greater cardiovascular effects (Böhm et al., 1991; Endoh et al., 1991; Verdouw et al., 1987).

As discussed previously, it is possible that the non-invasive methods of detection utilised in

Chapter 3 lacked the sensitivity required for identification of the magnitude and duration of its

effects in cats. The combination of an inherently small size of the heart, influence of patient

anxiety on the outcome parameters, and lack of effects beyond the published normal

reference ranges could have obscured treatment effects. Furthermore, measures of its

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effects on the left ventricular diastolic and left atrial systolic indices were not quantified,

therefore limiting assessment of its impact on global cardiac function. Alternatively, true

inter-species differences in the magnitude of its effects might also exist. Comparison of the

pharmacokinetics of pimobendan between dogs and cats (Chapters 2 and 3) revealed that

the Cmax appeared higher in cats than in dogs at comparable mg/kg doses; in addition, this

tended to be reached earlier than in dogs. Although the lack of absolute bioavailability and

the difference in formulations utilised were limitations of these studies, a higher

bioavailability in cats would be suggested by these two variables. The elimination rate

constants were similar, as evidenced by the similarities in t1/2. In contrast, a lower CL/F and

Vz/F of pimobendan was found in cats compared to dogs. Though somewhat speculative in

the absence of bioavailability, plasma protein, and blood or cell binding data, both the lower

CL/F and Vz/F in cats could be explained through reduced uptake of pimobendan by its

effector organs. The vasodilatory effect of pimobendan differs between species and types of

vessels (Fujimoto, 1994); as such, it is possible that an inter-species variation in

pimobendan uptake by the myocardium may also exist. This, in turn, may have resulted in

diminished cardiovascular effects in cats. In addition to being the first published work

describing the acute cardiovascular responses to oral doses of pimobendan in conscious

cats, Chapter 3 therefore served the important function of questioning the true utility of

pimobendan in this species and emphasising the need for further prospective evaluations.

The magnitude and duration of effects following oral dosing of pimobendan in cats

remain ill-defined, but the discovery of measurable echocardiographic treatment effects in

healthy dogs (Chapter 2) provides support for commonly employed off-label dosing regimen

in this species and a method by which therapeutic monitoring may be pursued in the future.

The onset of peak activity at 2 – 4 h following dosing and return to baseline by 8 h would

mean that a thrice daily dosing regimen as suggested in the ACVIM consensus report

(Atkins et al., 2009) would cause a longer duration of enhanced systolic activity. Further

increases in frequency or dose would result in accumulation of effects which, due to

physiological limits, would be mainly characterised by a higher trough systolic function. The

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impact of such alterations to standard treatment regimen, as well as any differences arising

from use of various formulations or administration methods (e.g. with food or with other

medications) can now be monitored in clinical practice using echocardiography.

Although beyond the scope of this thesis, longer-term outcomes of such alternate

dosing regimens can also be predicted through simultaneous pharmacokinetic-

pharmacodynamic modelling. Based on the findings from Chapter 2, modelling work is

currently being undertaken using an enriched pool of data from additional healthy dogs. In

order to assess its applicability to diseased dogs, further data is also being collected from

dogs with ACVIM stage B MMVD to test the utility of this model in target animals. This model

could be used to guide the design of survival studies to clarify ideal dosing regimen and

therapeutic monitoring in clinical practice. The use of multiple pharmacokinetic-

pharmacodynamic models, in combination with parametric time to event modelling, have

been advocated in the field of oncology to individualise treatment regimen in the face of

multiple drug interactions and variable courses of disease (Bender et al., 2015; Buil-Bruna et

al., 2016). However, it remains an unexplored area in the field of CHF therapeutics. For

veterinary patients, the work completed in Chapter 2 may therefore provide a foundation for

such future developments into individualisation and improvement of CHF therapeutics.

It is common knowledge amongst veterinary professionals that pimobendan is

seldom used alone for the treatment of CHF. Most often, it is given in combination with

frusemide and an ACE-I; other concomitant treatments include MRAs and afterload reducers

such as amlodipine or hydralazine (Atkins et al., 2009). Yet the benefits, if any, of such

combination therapy, as well as the expected natural course of disease utilising different

treatment regimen, remain ill-defined. This, together with the lack of available information

defining the role of the RAAS in development and propagation of CHF in MMVD, formed the

basis for the longitudinal observational study outlined in Chapter 4.

The data gathered during the course of this study led to an interesting observation

regarding survival in the cohort of enrolled dogs. When treated with combination therapy

consisting of frusemide, pimobendan, spironolactone, and benazepril, the MST in dogs with

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CHF due to MMVD was 419 days (Chapter 4). As previously identified, this finding supports

and strengthens the observations made by two retrospective studies investigating survival

outcomes in a similar population of dogs receiving similar medical therapy (430 days in 21

dogs (De Madron et al., 2011); 163 to advanced CHF + 281 days in advanced CHF in 54

dogs (Beaumier et al., 2018)). Furthermore, these values are ~ 100 days greater than the

MST (334 days) reported in 64 dogs receiving treatment with standard doses of pimobendan,

frusemide, and ACE-I in another recent retrospective study (M. Mizuno et al., 2017). The

retrospective nature of many of these studies, various global locations from which the

participants were selected, inclusion of cases from a range of years (2003 – 2016), and the

small numbers of individuals enrolled place some constraints on the interpretation of this

discrepancy. However, some credibility is redeemed as matching inclusion criteria were

used across these studies, thereby reducing clinical variability between populations. It is

therefore possible that the use of spironolactone and ACE-I to provide comprehensive RAAS

inhibition, together with pimobendan and frusemide, may offer survival benefits in dogs with

CHF due to MMVD. Evidence is provided to support larger clinical trials investigating this

matter.

In contrast, the MST observed in Chapter 4 cannot be compared to those reported in

numerous other survival studies due to critical differences in study designs. For example,

some previous survival studies included both dogs with MMVD and DCM (Ettinger et al.,

1998; Slupe et al., 2008; The BENCH Study Group, 1999), with the latter being associated

with poorer survival (M. W. S. Martin et al., 2010; O'Grady et al., 2008). In addition, there

were discrepancies in the classification schemes utilised to diagnose CHF. In the absence of

documented radiographic evidence of CHF, symptoms such as coughing or exercise

intolerance could be attributable to non-cardiac comorbidities such as airway disease or

degenerative joint disease; thus, the use of ISACHC classification (Bernay et al., 2010;

Borgarelli et al., 2008; Serres, Chetboul, Tissier, et al., 2007; The BENCH Study Group,

1999) without documented evidence of volume overload could result in over-estimation of

survival. Conversely, under-estimation of survival may have resulted in studies which did not

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strictly enrol dogs at first onset of CHF (Bernay et al., 2010; Ettinger et al., 1998; Häggström

et al., 2008; Lombard et al., 2006; The BENCH Study Group, 1999). Numerous

combinations of treatments have been utilised across these studies, with pimobendan not

being employed or only being provided to a small portion of enrolled dogs in many studies

despite its widespread use today (Bernay et al., 2010; Borgarelli et al., 2008; Ettinger et al.,

1998; Serres, Chetboul, Tissier, et al., 2007; Slupe et al., 2008; The BENCH Study Group,

1999). The criteria for diagnosis of CHF and the treatment utilised in the present study are

commonly employed in Australia; as such, the results presented in Chapter 4 contribute

important information regarding survival in this population of dogs despite the small number

of enrolled animals. In examining the context in which this study lies, the need for better

global standardisation of heart failure classification and a more systematic method of

undertaking survival studies in veterinary medicine is also highlighted.

In theory, counteracting the deleterious effects of the RAAS and providing

vasodilation and inotropic support could contribute towards positive cardiac remodelling and

haemodynamics and alter the course of disease in dogs treated with quadruple therapy;

however, this was not apparent in the study comprising Chapter 4. The most common cause

of death and euthanasia was recurrent CHF (78% of deaths), with clinically relevant

pulmonary hypertension experienced in five dogs (45%, but a sixth dog developed this

following conclusion of the study) and haemodynamically significant arrhythmias (atrial

fibrillation and supraventricular tachycardia) developing in two dogs (18%). Dogs with larger

left atrial and ventricular sizes tended to die earlier, and left atrial size appeared to increase

prior to the onset of death. These figures and trends are all consistent with existing reports of

dogs with MMVD on a variety of treatment regimens (Beaumier et al., 2018; Borgarelli et al.,

2008; De Madron et al., 2011; Häggström et al., 2008; Hezzell, Boswood, Moonarmart, et al.,

2012; Kellihan & Stepien, 2012; The BENCH Study Group, 1999). It would seem, then, that

while some treatment combinations may lengthen survival, the overall course of disease

remains largely unaffected on a population level.

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To shed further light upon this observation, the study design should have ideally

incorporated assessments of short-term treatment effects of pimobendan and RAAS

inhibitors. Such assessments could act as intermediate markers of treatment benefits which

could be directly evaluated against survival outcomes or development of certain

complications as described above. Although far larger numbers of enrolled animals would be

required for formal assessment of such relationships, Chapter 4 was, in some ways, a

missed opportunity to explore the utility and practicality of these methods.

Based on findings from Chapter 2, the original study design called for

echocardiographic assessment of systolic function at 2 – 4 h following treatment with

pimobendan. However, it did not include assessment of trough cardiac function (i.e. prior to

administration of pimobendan). Although this design was chosen to minimise handling and

time spent in hospital at each follow-up visit, it also meant the need to coordinate timing of

study visits with that of treatments administered by each owner. This, combined with the

need to dose on an empty stomach without food, various daily commitments of each owner,

and the demands of an otherwise busy clinical service, made for an impossible combination

of elements to facilitate long-term pharmacodynamic monitoring. Retrospectively, a study

design in which each dog was required to stay the day of the recheck and allocating a

dedicated investigator to perform procedures each day may have improved data collection.

Noting the exact timing of dosing and echocardiography, as well as identifying plasma

concentrations of pimobendan and ODMP, may have also enabled retrospective analysis of

treatment effects with the use of the aforementioned pharmacokinetic-pharmacodynamic

model. This experience (Chapter 4) thus highlighted some of the practical concerns that

would need to be addressed in future use of echocardiography to monitor treatment effects

of pimobendan (Chapter 2).

Contrary to the relative ease of therapeutic monitoring for pimobendan, the

assessment of short-term anti-RAAS therapy is far more challenging. The presence of both

circulating and local tissue RAAS, which may act independently, makes global RAAS

assessments through non-invasive methods difficult (Atlas, 2007). To date, plasma and urine

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concentrations of RAAS hormones have been used most commonly in the assessment of

treatment effects. However, there are disparate findings across studies. For example, a

recent pharmacokinetic-pharmacodynamic model identified an increase in plasma renin

activity, along with a reduction in plasma angiotensin II and aldosterone concentrations

following benazepril therapy in healthy dogs undergoing RAAS stimulation via a low sodium

diet (Mochel et al., 2015). Yet in dogs with CHF due to MMVD on frusemide therapy, no

significant effects of benazepril were observed in these measures or urinary aldosterone to

creatinine ratio (UAldo:C), urinary angiotensin to creatinine ratio, or urinary vasopressin to

creatinine ratio (Häggström, Lord, et al., 2013). Similarly, other studies in healthy dogs with

frusemide-induced RAAS activation saw little or no effects of ACE-I therapy on the urinary

aldosterone to creatinine ratio (UAldo:C) despite a decrease in serum and plasma ACE

activity (M. K. Ames et al., 2016; M. K. Ames et al., 2015; Lantis, Ames, Atkins, et al., 2015;

Lantis, Ames, Werre, et al., 2015). Further complicating these traditional approaches to

monitoring anti-RAAS therapy in dogs with CHF treated with MRAs (Chapter 4) is the fact

that MRAs invariably cause an increase in circulating and urinary aldosterone concentrations

due to receptor antagonism, rendering these measures useless for this purpose. In order to

overcome such issues, research into the effects of anti-RAAS therapy on novel biomarkers

such as those reflecting myocardial fibrosis (e.g. procollagens), extracellular matrix

degradation (e.g. matrix metalloproteases), and renal anti-RAAS activity (e.g. aquaporin 2,

epithelial sodium channels) is required. Indeed, a reduction in markers of fibrosis has been

associated with survival benefits in people with CHF treated with spironolactone and ACE-I

(Zannad et al., 2000); in addition, administration of ACE-I have reduced matrix

metalloproteases in experimental models of heart failure (Brower et al., 2007; Li et al., 2000).

However, the effects of anti-RAAS therapy on such measures have not been evaluated in

dogs with naturally occurring CHF. As can be appreciated, further research efforts are

required to identify the optimal method of detecting short-term effects of anti-RAAS therapy

in the face of multi-drug use.

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An alternative approach to identify treatment success might involve the use of

natriuretic peptides as a biomarker of reduced preload, afterload, and enhanced cardiac

output produced through the combined use of frusemide, pimobendan, and RAAS inhibition.

Plasma concentrations of NT-proBNP and BNP have been observed to reduce following

various treatments in people with acutely decompensated CHF (Antoni et al., 2004; Knebel

et al., 2005; Marco et al., 2007; Michtalik et al., 2011; Paterna et al., 2005) and in chronic

therapy (Anand et al., 2003; Hartmann et al., 2004; Jourdain et al., 2007; McMurray et al.,

2008; Troughton et al., 2000; Tsutamoto et al., 2001) due to various aetiologies leading to

CHF; yet despite the findings of greater morbidity and mortality in those experiencing less

reduction in these peptides in many of the aforementioned studies, larger meta-analysis

have found some conflicting evidence regarding this matter (De Vecchis et al., 2014; Felker

et al., 2009; Porapakkham et al., 2010; Savarese et al., 2013), and clear guidelines for its

use to guide therapy have not been established (Yancy et al., 2017). Similarly, reductions in

circulating NT-proBNP concentrations have been observed in dogs following initiation of

CHF therapeutics (Häggström, Lord, et al., 2013; Schober et al., 2011); in addition, a post-

treatment NT-proBNP concentration of < 965 pM/L, but not the percentage of reduction in

NT-proBNP in response to therapy, has been associated with longer survival in dogs with

MMVD in a recent study (Wolf et al., 2012). As in human medicine, however, optimal

methods of employing this measure to guide treatment regimen have yet to be developed.

Nevertheless, the inclusion of serial assessments of circulating natriuretic peptide

concentrations over time could have provided interesting longitudinal information about the

NPS and global treatment response in the cohort of dogs evaluated in Chapter 4.

Yet some concerns exist regarding the specificity of existing NT-proBNP and BNP

assays, and these formed the basis of the specific BNP1-32 assay development which took

place in Chapter 5. The primary drawback of using immunoassays to detect components of

the NPS is related to cross-reactivity with inactive or less active pro-forms and fragments.

This has been demonstrated in commonly used immunoassays for detection of both

NT-proBNP and BNP in human medicine using plasma samples spiked with ProBNP1-108,

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BNP fragments, and NT-proBNP (Heublein et al., 2007; Liang et al., 2007; Luckenbill et al.,

2008; Shimizu et al., 2002). Using western blotting and specific immunoassays for ProBNP1-

108, studies have also identified a large presence of high-molecular-weight BNP

components consistent with ProBNP1-108 in plasma samples of people with CHF (Giuliani

et al., 2006; Liang et al., 2007). Because ProBNP1-108 and fragments of BNP1-32 appear to

have reduced or no biological activity (Boerrigter et al., 2009; Boerrigter et al., 2007;

Heublein et al., 2007; Huntley et al., 2015; Liang et al., 2007), and given the presence of

some evidence to suggest altered ProBNP1-108, ProANP, and BNP processing in the face

of CHF (Gomez et al., 2012; Huntley et al., 2015; Ibebuogu et al., 2011), there is a clear

need for better detection methods of the various components of the BNP system in order to

facilitate a deeper understanding of how this counter-regulatory system of the RAAS may be

involved in the syndrome of CHF.

The finding that canine BNP1-32, the biologically active component of the BNP

system, could be detected using mass spectrometry (Chapter 5) was promising in that it

highlighted a method of detection which could be utilised in veterinary medicine to facilitate

further understanding of BNP processing in the face of CHF. In accordance with existing

studies in human medicine evaluating this technique for the detection of BNP1-32

(Hawkridge et al., 2005; Miller et al., 2011; Niederkofler et al., 2008), both MALDI-TOF and

LC-ESI-MS/MS methods were observed to reliably detect canine BNP1-32 in standard

preparations. The latter was associated with a higher sensitivity (0.0001 pM/μl vs 0.1 pM/μl

with MALDI-TOF), making it a more suitable method of BNP1-32 detection in plasma.

Unfortunately, application of this method to quantify canine BNP1-32 concentrations

in plasma was not successful, owing to the large dynamic range of protein concentrations in

plasma and the extremely low concentrations of circulating immunoreactive BNP species.

Notably, the aforementioned articles utilising mass spectrometry for the quantification of

plasma BNP1-32 levels employed immunoprecipitation techniques during sample

preparation to remove abundant protein and peptide species and concentrate each sample.

The work in Chapter 5 demonstrated that inexpensive and less time-consuming methods of

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sample preparation such as routine solid-phase extraction, solvent precipitation, and

commercially available kits for depletion of albumin and nonspecific immunoglobulins did not

provide adequate refinement of plasma samples to aid in mass spectrometry-based

detection of canine BNP1-32 in plasma. Although the potential utility of sophisticated

chromatography with or without centrifugal filters remains an unexplored avenue of sample

preparation, the available evidence to date would suggest that immunoprecipitation steps

remain a vital component of such an assay.

In a curious coincidence of events, the fluid administration protocol employed in the

second part of Chapter 5 was also unsuccessful in that it failed to demonstrate adequate

stimulation of the endogenous BNP system. It was particularly difficult to interpret the results

due to the haemodilution and pressure diuresis caused by the fluid, and this formed the

basis of the recommendations against this method of stimulating the NPS in future studies

where plasma and urinary markers are to be evaluated. This may be one of the reasons why

it has been employed infrequently for this purpose in the existing literature. Nevertheless, a

coherent relationship between the fluid administered and measures of preload was

demonstrated, and trends in both the urinary and plasma endocrine variables showed

consistency in their lack of a convincing BNP response. Even in the absence of a specific

BNP1-32 assay, the complexity of the interactions between the cardiovascular, renal and

endocrine systems in maintaining fluid balance was accentuated through this study.

In the absence of a specific BNP1-32 assay to investigate whether a BNP1-32

deficiency might be associated with the development of CHF in dogs, an alternative

approach was taken in Chapter 6 whereby the effects of augmenting the NPS through

administration of exogenous BNP1-32 was evaluated in dogs with CHF due to MMVD.

In doing so, this work described, for the first time, the use of exogenous BNP1-32 in dogs

with naturally occurring CHF due to MMVD. Based on its use in experimental models of CHF

and people with CHF, it was hypothesised that exogenous BNP1-32 would result in

additional diuresis, natriuresis, and RAAS antagonism in dogs with naturally occurring CHF.

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Subcutaneously administered BNP1-32 was readily absorbed from its administration

site and displayed evidence of successful receptor-ligand interaction (Chapter 6). These

were demonstrated through a rise in plasma immunoreactive BNP-32 and urinary cGMP

production following administration of any treatment containing BNP1-32. Although the

plasma cGMP concentrations also appeared to reflect this trend, significance was not

reached due to the larger variability observed at baseline. The reason for this baseline

variability remains unclear as there were no apparent influences of animal factors such as

hydration status, medications, BNP-32 concentrations, and blood pressure; sample

abnormalities such as haemolysis/lipaemia or processing anomalies; or assay effects such

as location of wells (i.e. no potential carry-over effects), inter-assay variability (i.e. not

associated with a particular batch of samples showing higher concentrations), or erroneous

pipetting of samples. As cGMP is a product of both membrane-bound and soluble guanylate

cyclase activation, it is possible that unmeasured processes involving the NPS or nitrous

oxide pathways may have contributed towards this finding.

It should be noted that although cGMP is primarily an intracellular messenger

molecule, there is strong evidence to support the utility of plasma and urinary cGMP

concentrations as biomarkers of natriuretic peptide effects. Numerous studies have

observed an increase in urinary and plasma cGMP concentrations, accompanied by

cardiorenal effects, following administration of ANP1-28 (Eiskjær et al., 1991; Gerzer et al.,

1985; Nakamura et al., 1998; Riegger et al., 1988; Wong et al., 1988) and BNP1-32

(Boerrigter et al., 2007; Cataliotti et al., 2004; H. H. Chen et al., 2012; H. H. Chen et al.,

2000; H. H. Chen, Huntley, et al., 2006; H. H. Chen et al., 2004; H. H. Chen, Schirger, et al.,

2006; Forfia et al., 2007; Grantham et al., 1997; Jensen et al., 1999; McKie et al., 2016;

Nakamura et al., 1998; Yamamoto et al., 1997; Yoshimura et al., 1991). These observations

are supported by in vitro cell and tissue studies in which increasing extracellular

concentrations of cGMP over time in the medium were documented upon bathing samples in

medium spiked with ANP (Stasch et al., 1989; Wong et al., 1988). Furthermore, a review of

various cellular and physiological studies concluded that cGMP efflux into the extracellular

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space is exhibited by a wide range of cells in the body and appears to be an ATP-dependent,

active process that is facilitated by transport proteins in cell membranes (Sager, 2004). The

family of multidrug resistance proteins and the SLC22A7 (also known as the hOAT2)

member of the solute carrier family are of particular importance, with the latter also providing

bidirectional movement of cGMP both in and out of cells (Cropp et al., 2008; Sager, 2004;

van Aubel et al., 2002). The OAT1 isoform, which is abundant in the basolateral membrane

of proximal tubules, also facilitates intracellular transport of cGMP (Sekine et al., 1997).

Within the kidneys, there is evidence to support the role of extracellular cGMP in facilitating

ANP1-28, nitric oxide, and pressure-induced natriuresis (Ahmed et al., 2007; Chevalier et al.,

1996; Jin et al., 2004), and it also mediates learning in the brain (Cabrera-Pastor et al.,

2016; Erceg et al., 2006; Erceg et al., 2005). Together, this body of evidence supports the

existence of transmembrane transport of cGMP and its role as an extracellular mediator of

biological effects, and they validate its use as a marker of successful binding of BNP1-32 to

its receptor in the studies conducted in Chapters 5 and 6.

Yet in spite of this, no significant biological effects of BNP1-32 were observed in dogs

with naturally occurring CHF (Chapter 6) – a finding which was contrary to much of the

available literature describing its use in dogs with induced CHF and people with naturally

occurring CHF. In particular, its renal effects, such as the increase in urinary sodium

excretion (Cataliotti et al., 2004; H. H. Chen et al., 2000; H. H. Chen, Huntley, et al., 2006; H.

H. Chen et al., 2004; H. H. Chen, Schirger, et al., 2006; Grantham et al., 1997; Jensen et al.,

1999) and urine output (Cataliotti et al., 2004; H. H. Chen et al., 2000; H. H. Chen, Huntley,

et al., 2006; H. H. Chen et al., 2004; H. H. Chen, Schirger, et al., 2006; Grantham et al.,

1997), were not apparent. It is possible that this difference may be partly attributable to the

different statistical analysis and measures of urinary sodium excretion utilised across the

studies. For example, the massive diuresis and natriuresis/kaliuresis caused by frusemide

administration may have obscured smaller changes experienced by BNP1-32 administration

in the present study. Indeed, even in these prior studies, the magnitude of the increase in

urine output due to BNP1-32 was low (around 0.2 – 1 ml/min on average in dogs weighing

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an average of ~ 20kgs – equivalent of around 0.01 – 0.05 ml/kg/min). The trends in urine

output following BNP1-32 administration in the present study showed a mild increase at 1 h

post treatment in line with the lower end of this range (~ 0.01 ml/kg/min on average). A sub-

analysis using only the urine output data following BNP1-32 administration did show a

significant overall difference over time (p = 0.022), with it being higher than baseline at 1 h

post treatment. However, the fact remains that the magnitude of effect was quite small.

Furthermore, the average urine output was lower with the frusemide + BNP1-32 combination

compared to frusemide alone in the present study – a finding that refutes that of a previous

study in dogs with induced CHF (Cataliotti et al., 2004). Trends over time for sodium and

potassium excretions were underwhelming following BNP1-32 administration, and a similar

sub-analysis using only the data from this treatment stream showed no significant changes

over time (p = 0.3 for FENa and p = 0.075 for FEK). Put together, the renal effects of BNP1-32

were not obvious in dogs with naturally occurring CHF. This lack of convincing effects in the

face of strong evidence to suggest adequate binding of BNP1-32 to its receptor raises

concerns for potential abnormalities in intracellular mechanisms which facilitate the biological

activity of cGMP in dogs with CHF due to MMVD. In this way, Chapter 6 provides grounds

for future investigations into this novel area of pathophysiology in dogs with naturally

occurring CHF.

Simultaneously, this finding prompts contemplation of whether alternate methods of

NPS augmentation would be effective in dogs with CHF due to MMVD and how study

designs examining these may be optimised. As discussed in Chapter 1 of this thesis, the

most recent advancement in CHF therapeutics in humans is the ARNI, with it demonstrating

convincing survival benefits over the use of an ACE-I (McMurray et al., 2014). It is tempting

to follow in the footsteps of such a discovery; however, it is possible that neprilysin inhibitors

may not necessarily be beneficial if the activity of BNP1-32 is being hindered by malfunction

of intracellular cGMP pathways. Any benefits of ARNI in this population of dogs will therefore

need to be differentiated from the effects of the ARB alone. This is a limitation that clouds

the findings of a very recently published pilot study investigating the use of ARNI versus

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ACE-I in dogs with ACVIM Stage B2 MMVD (Newhard et al.), and larger studies

investigating the use of ARNI may benefit from the inclusion of an ARB treatment stream to

clarify the role of the neprilysin inhibitor in these dogs. The use of virtually identical

intracellular pathways by ANP1-28 would also mean that using this as an alternative to

BNP1-32 may be similarly disappointing in these dogs. Including evaluations of cGMP-

mediated pathways in the study designs would provide further insight into this matter.

Findings from Chapter 6 can therefore be used to guide such future investigations into the

NPS in dogs with CHF due to MMVD.

The trends in plasma aldosterone concentrations and immunoreactive BNP-32

(Chapter 6), albeit not being representative of the entirety of the RAAS and NPS respectively,

provided some interesting insights regarding these systems. In general, the plasma

aldosterone concentrations were low in the dogs enrolled. The majority of plasma samples

(~ 60%) contained concentrations below the LLOQ of ~ 70 pM/L; this fit within the low end of

the reported range of concentrations in dogs with CHF (20 – 800 pM/L) (Häggström et al.,

1997; Häggström, Lord, et al., 2013). These low concentrations may have been due to

adequate ACE-I activity, sample degradation, or a lack of overt circulating RAAS activity in

compensated CHF. Conversely, the plasma concentration of immunoreactive BNP-32 was

higher than previously reported ranges (~ 5 – 250 pg/ml, with the majority being < ~ 80

pg/ml) (Asano et al., 1999; DeFrancesco et al., 2007; Häggström et al., 2000; MacDonald et

al., 2003; Prošek et al., 2007), with it averaging ~ 240 pg/ml (range 160 – 407 pg/ml) at

baseline across all treatment days. It is possible that these prior studies under-estimated

immunoreactive BNP-32 concentrations as not all studies utilised protease inhibitors or used

aprotinin, which is inferior to Benzamidine and AEBSF in the preservation of BNP1-32

(Belenky et al., 2004; Niederkofler et al., 2008). Furthermore, it was noted during BNP1-32

assay development in Chapter 5 that C18 material could not reliably concentrate BNP1-32;

thus, assays utilising this material could have produced erroneously low results. There is a

clear need for standardising these assays and establishing appropriate reference ranges in

dogs with CHF. The observation of a late rising trend in aldosterone following treatment with

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BNP1-32 also requires further clarification. In addition to highlighting the need for future

research into the NPS in dogs with naturally occurring CHF, Chapter 6 also accentuated the

importance of clarifying the interaction between the NPS and RAAS in these dogs.

The research presented in the body of this thesis uncovered numerous avenues for

further investigations in the field of veterinary CHF therapeutics. In particular, the following

areas are suggested as priorities for further evaluation:

• The systolic and diastolic effects of pimobendan in cats to clarify whether it provides

beneficial effects in this species.

• Large-scale clinical trials investigating the benefits, if any, of quadruple therapy

(consisting of frusemide, an ACE-I, pimobendan, and an MRA) in dogs with CHF.

• The role of the NPS in dogs with CHF in order to assess its suitability as a

therapeutic target.

• The utility of therapeutic monitoring of pimobendan in dogs with CHF.

In looking forward to these next steps, some limitations to the overall structure of the current

project should be considered. To start, addressing the topic of CHF therapeutics within the

confines of a PhD candidature posed a considerable challenge, primarily due to the breadth

of the topic. The approach undertaken, in which a selection of issues was explored, yielded

positive findings in a variety of areas; however, the significance of each may have been

diluted through the inability to explore deeper grounds. Focusing on a smaller aspect of this

research topic may have enhanced a limited number of outcomes. This project utilised the

expertise and resources of several laboratories and departments located within and outside

of the University of Sydney. Yet extending such collaborations may have facilitated even

greater success in the works performed in Chapters 4 and 5. Finally, the organisation and

timing of events both within and across studies could have been better optimised. For

example, commencing the BNP1-32 assay development (Chapter 5) earlier in the course of

the project would have allowed for more time to be dedicated to this work, and establishing a

more precise study protocol for the longitudinal study (Chapter 4) may have facilitated

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acquisition of data relating to the RAAS and NPS. Such aspects of project design may

contribute greatly to the outcomes of research attempts made in the face of time and

financial constraints, and thus, form important considerations for future studies.

7.2) Conclusions

This thesis reviewed current veterinary practices in CHF therapeutics and explored novel

avenues for medical therapy in this field. It began with a comprehensive literature review,

which identified knowledge gaps in the use of pimobendan in veterinary patients and clear

deficiencies in present understanding of the role and therapeutic potential of the RAAS and

NPS in canine and feline CHF. An investigation into the pharmacokinetics and

pharmacodynamics of pimobendan was performed, which uncovered inter-species variation

in metabolism of the drug and measurable cardiovascular effects. Pimobendan metabolism

was reduced in cats compared to dogs; although this was expected to produce greater

cardiovascular effects in cats, changes to echocardiogram-derived measures of systolic

function were less obvious and resulted in an inability to define the duration and magnitude

of treatment effects for the two doses investigated. This finding prompts an evaluation of the

way pimobendan is currently used in this species and highlights the need for further

prospective studies into its effects, ideal dosing regimen, and whether it truly offers treatment

benefits in cats with CHF. Conversely, the cardiovascular impacts of pimobendan were

readily apparent in dogs, with echocardiographic measures of systolic function showing

maximal treatment effects between 2 – 4 h following oral dosing. Echocardiography

therefore appears to be a useful tool for monitoring treatment effects in individual dogs. This

opens up the scope for further work evaluating the benefits of personalising therapy in

canine patients. When pimobendan was used as a part of multi-modal therapy together with

an ACE-I, MRA, and a diuretic in dogs with newly diagnosed CHF due to MMVD, the

treatment was well-tolerated. The natural course of disease was similar to that described

with other combinations of treatments utilised in the existing literature; recurrent CHF

remained the leading contributor towards morbidity and mortality, and common

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complications of clinical relevance included moderate to severe pulmonary hypertension and

supraventricular arrhythmias. Despite the small number of dogs enrolled and involvement of

investigators with varying degrees of experience, rising trends in echocardiographic

measures of cardiac remodelling (LA:Ao and weight-normalised LVIDd) were observed prior

to death – suggesting the robustness and utility of these variables in providing prognostic

information in everyday practice. Considered alongside existing literature evaluating

longitudinal outcomes of other commonly employed treatment regimens, possible survival

benefits in terms of longevity were observed with this combination of drugs, thereby

supporting the value of larger clinical trials to pursue this question. The importance of global

standardisation of heart failure classification and clinical study designs was also stressed.

In the final sections of this work, the NPS was investigated as a novel therapeutic

target for dogs with CHF due to MMVD. Although an attempt was made to develop a specific

BNP1-32 assay to investigate whether relative deficiencies in this protective mechanism

existed in dogs with CHF, assay development was unsuccessful. Standard preparations of

canine BNP1-32 could be detected using various methods of mass spectrometry; yet

challenges surrounding sample preparation limited its detection in canine plasma. With

simpler methods proving to be insufficient, immunoprecipitation or sophisticated separation

techniques such as chromatography are recommended for future attempts. In addition, using

clinical samples from animals undergoing transient preload augmentation via intravenous

fluids is not recommended when assessing new assays of the BNP system. This model does

not reliably stimulate the BNP system, and the direct increase in intravascular volume,

haemodilution, and ensuing diuresis cloud interpretation of results. Further scrutiny of this

system in dogs with CHF due to MMVD is, however, recommended, as a lack of identifiable

biological effects of exogenous BNP1-32 was found in dogs with this disease. In particular,

the intracellular downstream pathways facilitating cGMP-mediated effects in dogs with CHF

are areas for novel research.

Despite the wealth of advancements made in the understanding of canine and feline

CHF since its first description around a hundred years ago, much about its pathophysiology,

175

and therefore optimal treatment approaches, remain hidden or unresolved. It is hoped that

further work building on the discoveries shared in this thesis will be undertaken to attain the

ultimate goal of providing the best quality of life in our patients.

176

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