Challenges in exploring the cytochrome P450 system as a source of variation in canine drug...

2013

http://informahealthcare.com/dmrISSN: 0360-2532 (print), 1097-9883 (electronic)

Drug Metab Rev, Early Online: 1–13! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03602532.2013.765445

REVIEW ARTICLE

Challenges in exploring the cytochrome P450 system as a sourceof variation in canine drug pharmacokinetics

Marilyn N. Martinez1, Leposava Antonovic2, Michael Court3, Mauro Dacasto4, Johanna Fink-Gremmels5,Butch Kukanich6, Chuck Locuson7, Katrina Mealey3, Michael J. Myers1, and Lauren Trepanier8

1Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, Maryland, USA, 2Center for Devices and Radiological Health,

U.S. Food and Drug Administration, Silver Spring, Maryland, USA, 3Department of Veterinary Clinical Sciences, Washington State University,

Pullman, Washington, USA, 4Dipartimento di Biomedicina Comparata ed Alimentazione, Universita degli Studi di Padova, Padova, Italy,5Department of Veterinary Pharmacology, Pharmacotherapy, and Toxicology, Utrecht University, Utrecht, The Netherlands, 6Department of

Anatomy and Physiology, Kansas State University, Manhatten, New York, USA, 7Pharmaprogressio Consulting, Rochester, Minnesota, USA, and8Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

Abstract

The cytochrome P450 (CYP) superfamily constitutes a collection of enzymes responsible for themetabolism of a wide array of endo- and xenobiotic compounds. Much of the knowledge onsubstrate specificity and genetic identification of the various CYP isoforms is derived fromresearch in rodents and humans and only limited information has been captured in the dog.Currently, there exist many gaps in our knowledge of canine CYP diversity as a result of thepaucity of studies focusing on canine CYPs, canine CYP polymorphisms, and the therapeuticconsequences of these genetic variants. Challenges engendered by this lack of informationis further amplified by inter- and intraspecies differences in the specificity and affinity ofsubstrates and inhibitors, prohibiting a simple extrapolation of probe substances used inhuman CYP research. This creates a need to develop and validate canine-specific CYP probes.Failure to understand this potential metabolic and pharmacogenomic diversity can alsoinfluence the interpretation of data generated in dogs to support human drug development.It is with these objectives in mind that we provide an overview of what is currently knownabout canine CYPs with the hope that it will encourage further exploration into this importantarea of research.

Keywords

Canine, drug metabolism, enzyme inhibitors,enzyme substrates, interspeciesdifferences, orthologs, pharmacogenomics

History

Received 20 November 2012Accepted 8 January 2013Published online 20 February 2013

Introduction

The cytochrome P450 (CYP) superfamily has received a great

deal of attention because of its involvement in the metabolism

of a vast number of lipophilic compounds. Across animal

species, CYPs often share similar DNA sequences, with many

of the species-specific genes demonstrating more than 80%

sequence homology to the corresponding human CYP gene

[for specific examples, refer to the individual animal species

FASTA files (http://drnelson.uthsc.edu/CytochromeP450.

html); Nelson, 2009]. In contrast to paralogs (genes related

by duplication within a single genome), which often differ in

function, these orthologs (homologous genes across animal

species) tend to retain a similarity of function (Larhammar

et al., 2009). Nevertheless, despite these similarities,

orthologs can exhibit differences in substrate affinity, inhib-

ition, magnitude of expression, and/or enzyme kinetics

(Antonovic & Martinez, 2011), leading to interspecies

variations in drug pharmacokinetics (PK). A comparison of

the relative amounts of the various CYPs in dog versus human

liver based upon estimates obtained using an enzyme-linked

immonusorbent assay is provided in Figure 1. It should be

noted that Heikkinen et al. (2012) recently evaluated the

expression of seven CYP hepatic and intestinal enzymes

(Beagle) using mass spectrometry (MS). With the exception

of substantially lower CYP2C21 concentrations, relative

hepatic expression values were similar to the Beagle values

presented in Figure 1(B).

Human variation in drug metabolism is an area of active

research. Similarly, with recent efforts to enhance the

therapeutic arsenal for dogs, there has been a growing need

to understand the attributes of drug metabolism across the

canine population. However, there exist many gaps in our

knowledge of canine CYP diversity as a result of the

following challenges:

(1) Human-canine differences in the specificity and affinity

of substrates and inhibitors

(2) Lack of validated in vivo drug probes for canine CYP

enzymes

(3) Lack of adequate identification of the canine CYP

polymorphisms and their clinical consequences

Address for correspondence: Marilyn N. Martinez, Center for VeterinaryMedicine, U.S. Food and Drug Administration, 7520 Standish Place,HFV-100, Rockville, MD 20855, USA. Fax: 240-276-9538. E-mail:[email protected]

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(4) Obstacles in obtaining an adequate type and amount of

tissue sample to characterize breed-specific polymorph-

isms (including the inability to obtain appropriately

processed liver samples from veterinary hospitals and

the lack of the facilities and equipment necessary to

appropriately process and transport the necessary liver

samples)

(5) Lack of canine-specific high-throughput genotyping

platforms to characterize the distributional frequency of

specific polymorphisms across canine breeds

(6) Lack of canine-specific gene chips to support individu-

alization of dosing regimens. In this regard, chips

developed for human sequence assessments cannot be

readily applied to the polymorphic forms of other species.

(7) Inadequate funds available to support these research

efforts

(8) A need for better communication among researchers,

drug development scientists, and regulators as to the

important therapeutic implications of this work, the

creation of mechanisms for international scientific

harmonization to maximize through shared expertise

and resources, and the need to avoid a ‘‘one size fits

all’’ approach when establishing canine dosage

recommendations.

Phenotypic variations in CYP activity can have profound

clinical consequences. In humans, it has been estimated

that more than 90% of medications are metabolized by CYPs

(Nebert & Russell, 2002) and that human polymorphic

drug metabolism is a leading cause of adverse drug

events (Zhang et al., 2012). The polymorphisms most

frequently correlated to clinically significant effects are

those associated with CYP2D6, 2C9, and 2C19, with

variations frequently segregating in relation to ethnic

background (Scott, 2011).

The consequence of polymorphic variation in drug

metabolism is frequently classified as one of four major

phenotypes (Ingelman-Sundberg, 2004): (1) extensive metab-

olizers having normal activity; (2) poor metabolizers with

minimal to no enzyme activity; (3) intermediate metabolizers

having one functional allele, and (4) ultrarapid metabolizers

usually having more than one gene (from gene duplication)

encoding active enzyme.

Table 1. mRNA levels of canine CYPs across various tissues in normal healthy Beagles and cross-bred dogs in canine control tissues.

Gene Liver Bone Lymph node Skin Mammary gland

CYP1A1 1.13� 1.49 0.24� 0.09 1.03� 1.37 2.85� 1.19 3.30� 2.11CYP1A2 113.34� 134.32 0.29� 0.11 1.44� 1.81 4.67� 1.13 5.53� 1.80CYP1B1 0.03� 0.02 2.40� 0.56 1.55� 0.76 0.58� 0.33 0.78� 0.41CYP2A13 5.01� 4.60 0.11� 0.06 2.40� 3.82 0.12� 0.12 0.20� 0.24CYP2A25 925.83� 369.40 ND 2.75� 1.63 1.38� 2.90 0.35� 0.30CYP2B11 90.68� 5.77 0.08� 0.11 1.33� 1.60 1.85� 2.43 0.33� 0.47CYP2C21 508.82� 414.46 0.71� 0.05 0.76� 0.81 1.23� 0.20 1.02� 0.25CYP2D15 931.91� 626.96 0.87� 0.57 2.77� 1.32 2.58� 3.77 0.52� 0.38CYP2E1 957.36� 544.68 ND 1.23� 1.52 1.46� 2.62 0.41� 0.37CYP3A12 168.12� 87.18 0.43� 0.33 1.01� 0.37 2.62� 2.04 4.31� 0.70

mRNA relative quantification was performed by using the DDCt method (Livak & Schmittgen, 2001). Data (arbitrary units) are expressedas means� standard deviation.

ND, not determined.

Figure 1. Relative, mean hepatic CYP proteinexpression values as determined by immu-nochemical analysis. The amount of nonspe-cified ‘‘other’’ P450 represents the differenceof spectrally quantified P450 minus the sumof the specific immunoquantified isoforms.Other P450 content for Beagles was esti-mated by adding immunoquantified P450from multiple sources (Eguchi et al., 1996;Sakamoto et al., 1995) and subtracting fromthe spectrally quantified P450 reported byEguchi et al. (1996). Human protein expres-sion values were acquired from a meta-analysis study to represent the largest pos-sible sample size (Rowland-Yeo et al., 2004).(A) Relative human hepatic CYP proteinexpression. (B) Relative canine hepatic CYPprotein expression. (C) Donut graph to visu-ally compare the relative contribution of thevarious CYP families within the human liverversus within the canine liver. Note that in(C), several of the CYPs identified in thehuman liver (as noted in A) have beenlumped into the category of ‘‘other’’. Thiswas done to maintain consistency with theCYP subfamilies identified in the dog.

2 M. N. Martinez et al. Drug Metab Rev, Early Online: 1–13

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The aims of this article are to review the information

and tools currently available with regard to the role of, and

variability in, CYP activity in canine drug metabolism and

to identify remaining gaps that need to be resolved to support

the evolving canine therapeutic landscape. From the perspec-

tive of veterinary clinical pharmacology, it would be very

useful to have the insights needed to predict the likely

variability in drug metabolism that can occur across the

canine population and to have data to support conclusions

regarding any correlation that may exist between this

variability, drug response (i.e. effectiveness and adverse

drug responses), and canine breed. Further, when the dog is

used as a preclinical species to support human drug devel-

opment, this information is invaluable to better appreciate

potential interspecies differences in drug metabolism and

to improve our ability to extrapolate data (from human to

dog or dog to human) pertaining to potential drug–drug

interactions (DDIs).

What do we already know: human-canine orthologs

Much of what we currently know about canine CYPs has been

facilitated by the development of in vitro methods for

obtaining recombinant expressed enzymes. One of the first

successful efforts in this regard involved the scientists at

Merck who heterologously expressed seven canine CYPs

in baculovirus-Sf21 insect cells (Shou et al., 2003). The use

of these recombinant (rCYP or rP450) enzymes provided a

mechanism for systematically evaluating selective substrates

and inhibitors of the individual CYPs and for comparing these

canine results to those obtained in humans. As noted below,

many of the selective human CYP substrates and inhibitors

are not similarly specific for the canine orthologs. This

underscores the importance of identifying appropriate

markers for studying canine drug metabolism and for

understanding potential breed-related differences that are

likely to affect drug PK.

Canine CYP tissue distribution

Giantin et al. (2010, 2013) examined the messenger RNA

(mRNA) levels of canine CYPs across various tissues in

normal healthy Beagles or cross-bred dogs. CYP1A2, 2B11,

2C21, 2D15, and 3A12 were mostly expressed in the liver. In

contrast, CYP1A1 and 1B1 had greater relative expression in

extrahepatic versus hepatic tissues (Table 1). Similar hepatic

relative expression levels of CYP2A12, 3A26, and 2D16 have

recently been reported (Haller et al., 2012). Further, using

mass spectroscopy, Heikkinen et al. (2012) noted that

CYP2B11 and 3A12 appeared to be the major canine

intestinal isoforms. This is in contrast with the negligible

amounts of the human CYP2B isoform identified in the

human intestine. Therefore, despite slight differences for

certain CYPs, the general trend is that the canine CYP

expression pattern mirrors that which has been described for

humans (Oyama et al., 2004; Yokose et al., 1999). It should be

noted that information on the various intestinal segments are

absent from the list included in Table 1. Considering its

importance in first-pass drug metabolism, corresponding data

obtained from canine intestinal tissues would be highly

informative.

An additional point of note is that when comparing the

relative CYP expression in Figure 1 (protein) to that of Table

1 (mRNA), the rank order of canine CYP mRNA and protein

expressions are not entirely consistent. There are several

potential reasons for this apparent discrepancy, but differ-

ences in methodological approaches seem to be the most

relevant. Examples of challenges associated with attempts to

correlate mRNA to protein expression are reviewed elsewhere

(Greenbaum et al., 2003). Ohtsuki et al. (2012) examined this

apparent discrepancy relative to membrane transporters,

CYPs, and the UDP-glucuronyltransferase enzymes (human

liver).

Sources of variation in drug metabolism

Similar to the utility of applying ethnic background to predict

the likelihood for encountering a specific polymorphic variant

in human medicine, it would be extremely helpful to have

the information necessary to support the use of breed as an

analogous prognostic indicator for canine therapeutics.

According to the American Heritage� Dictionary, breed is

defined as a group of organisms having common ancestors

and certain distinguishable characteristics developed by

artificial selection and maintained by controlled propagation.

There are over 400 recognized canine breeds worldwide, with

156 recognized by the American Kennel Club (http://

www.akc.org/reg/dogreg_stats.cfm). Based upon comparison

of their respective DNA, the ancestral alignment of these

breeds has been described (Parker et al., 2004). The

pharmacogenetic and metabolic differences observed across

canine breeds have been reviewed elsewhere (Fleischer et al.,

2008). For example, in addition to the CYP-associated

polymorphic drug metabolism discussed below, there are

breed differences in thiopurine methyltransferase (which

can lead to thiopurine toxicity; Salavaggione et al., 2002),

hepatic copper storage problems in terriers resulting from

deletion of the MURR1 gene (van de Sluis et al., 2003),

defects in P-glycoprotein transporter function in collies and

related breeds (Mealey, 2008), and physiological and ana-

tomical differences that can influence such variables as

gastrointestinal (GI) transit time and intestinal mucosal

permeability (e.g. Weber, 2006) and creatinine clearance

(Craig et al., 2006).

The potential for dog breed differences in the in vivo PK of

drugs was first investigated as a result of the prolonged

recovery from thiopental anesthesia in Greyhounds. Although

initially attributed to a smaller thiopental volume of distribu-

tion in this lean breed (Sams et al., 1985), subsequent studies

demonstrated that the prolonged thiopental recovery time

was primarily the result of breed-specific differences in

CYP-associated drug metabolism (Sams & Muir, 1988). This

same study also demonstrated significant differences in the

Greyhound clearance of methohexital and thiamylal, but not

of phenobarbital.

Recently, a population PK model of mavacoxib (European

Medicines Agency European Public Assessment Report:

Trocoxil) demonstrated that German Shepherds and

Labrador Retrievers had significantly higher mavacoxib

clearance (expressed as a function of bioavailability), as

compared with that observed in other canine patients of

DOI: 10.3109/03602532.2013.765445 Variations in canine cytochrome P450 superfamily 3

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comparable age (Cox et al., 2011). Several canine CYP

isoforms are capable of metabolizing mavacoxib, although the

primary route of mavacoxib elimination is as the intact

drug in the feces. Thus, this variability may reflect poly-

morphic membrane transporter systems and/or breed-specific

differences in drug metabolism.

Although most of the focus to date has been on exonic

genetic variants, there also exist numerous other sources

of variation in drug metabolism that should not be ignored.

As a cell responds to its environment, there exists a multitude

of factors that can influence gene transcription, mRNA

translation, and, ultimately, protein synthesis. In addition to

these transcriptional and translational regulators, mutations

in sites other than those associated with the exonic portion

of the CYP gene itself can be important sources of population

variability in drug metabolism. For example, in humans,

population variability in drug metabolism has been attribut-

able to variation in nuclear receptors (NRs) (Kliewer et al.,

2002; Lamba et al., 2005; Savkur et al., 2003) and epigenetic

factors (Gomez & Ingelman-Sundberg, 2009; Ingelman-

Sundberg et al., 2007; Kouzmenko et al., 2010; Rodriguez-

Antona et al., 2010).

Although canine examples of metabolism-related popu-

lation variability in drug PK are limited, it is evident that the

principles and clinical consequences of these polymorphisms

are comparable to that observed in people.

CYP1A1 and 1A2

Using Beagle liver, Uchida et al. (1990) isolated two isoforms

of the CYP1A subfamily (CYP1A1 and 1A2). The canine

CYP1A1 (524 residues) and 1A2 (512 residues) amino acid

sequences were found to be 75% identical to each other. Both

enzymes are 81% identical to their human orthologs, CYP1A1

and 1A2 (Shou et al., 2003). Together, the two dog CYP1A

enzymes constitute an average of 4% of total liver CYP

content across male and female Beagles (Eguchi et al., 1996).

mRNA transcripts for canine CYP1A1 and 1A2 can be

induced in canine liver, kidney, and lung tissue upon

treatment with polychlorinated biphenyl b-napthoflavone

(b-NF) (Eguchi et al., 1996; Graham et al., 2002; Ohta

et al., 1990). Canine CYP1A1 and 1A2 substrates and

inhibitors, as discussed in this section, are summarized in

Table 2.

At low substrate concentrations, 7-ethoxyresorufin (7-ER)

dealkylation (Shou et al., 2003) and phenacetin dealkylation

(Locuson et al., 2009) are catalyzed primarily by CYP1A1

according to a panel of canine enzymes, including 1A1,

2B11, 2C21, 2D15, and 3A12. More recently, canine

hepatic CYP2A13 and 2A25 were also found to metabolize

phenacetin efficiently, and canine CYP2A25 was shown to be

capable of metabolizing 7-ER (Zhou et al., 2010a), suggesting

that additional studies are needed to confirm the selectivity

of these commonly used human CYP1A probe substrates.

For instance, if the extent of CYP2A metabolism in dogs is

found to be significant, this could explain why phenacetin

clearance in vivo appear to be minimally influenced by

CYP1A2 (Whiterock et al., 2012). Methylxanthines, such as

caffeine, have also been examined as CYP1A substrates,

although the relative contribution to intrinsic clearance

appears to be lower than that noted with the human CYP1A

ortholog (Locuson et al., 2009; Mise et al., 2008). The

endogenous human CYP1A2 substrates melatonin, 9-cis-

retinal, and estradiol do not appear to be substrates of canine

CYP1A2 (Mise et al., 2008).

Reported inhibitors of canine CYP1A enzymes in liver

microsomes include a-NF (Mise et al., 2004b), furafylline

(Azuma et al., 2002; Chauret et al., 1997), ondansetron,

metronidazole, and omeprazole (Aidasani et al., 2008),

although the significance of the findings ultimately rely

on the corresponding choice of substrate. Fluvoxamine is

the only published CYP1A inhibitor tested against canine

recombinant enzyme (CYP1A1), but its potency was roughly

an order of magnitude lower than that described for humans

(Mills et al., 2010). Of these drugs, ondansetron and

fluvoxamine are also substrates and/or inhibitors of human

CYP1A2 (Zhou et al., 2009).

Clinically relevant substrates and inhibitors have been

examined in vivo. For example, enrofloxacin (a veterinary-

approved antimicrobial whose active metabolite, ciprofloxa-

cin, is a human CYP1A inhibitor) was found to decrease the

plasma clearance of theophylline in dogs (Intorre et al.,

1995). In a similar manner, the apparent clearance of

caffeine in dogs was reduced by fluvoxamine (Mills et al.,

2010). Kamimura (2006) reviewed the striking effect that the

canine CYP1A2 variant, C1117T, has on decreasing the

in vivo clearance of several investigational drugs. These

results suggest that pharmacogenetic strategies may be

important when evaluating drugs metabolized by canine

CYP1A enzymes to ensure that the therapeutic index is not

exceeded.

Based upon the work of Tenmizu et al. (2004), there are

22 novel single-nucleotide polymorphisms (SNPs) associated

with canine CYP1A2. This is a widely studied canine CYP,

owing undoubtedly to its involvement in the metabolism of

several important human drugs (most notably, the

Table 2. Substrates and inhibitors of CYP1A1 and 1A2 in dogs and humans.

Substrates (incomplete list) Inhibitors (incomplete list) Inducers (incomplete list)

Human CYP 1A2 Ondansetron, fluvoxamine, phenacetin,caffeine, 7-ER, and theophylline (Zhouet al., 2009, 2010b)

a-NF, fluoroquinolones (ciprofloxa-cin), and furafylline (Zhou et al.,2009)

b-NF and 3-methylcholanthrene(Zhou et al., 2009)

Canine CYP 1A1/2 Phenacetin (also substrate for CYP2A;Zhou et al., 2010a; Lu et al., 2005),7-ER (also substrate for CYP2A;Zhou et al., 2010a; Shou et al., 2003),caffeine (Mills et al., 2010), andtheophylline (Intorre et al., 1995)

Ondansetron (Aidasani et al., 2008),omeprazole (Aidasani et al., 2008),fluvoxamine (Mills et al., 2010),enrofloxacin (Intorre et al., 1995),and furafylline (Chauret et al.,1997)

Polychlorinated biphenyl and b-NF(Eguchi et al., 1996; Grahamet al., 2002)


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phosphodiesterase type 4 inhibitors) (Tenmizu et al., 2006). It

is believed that these polymorphisms may be responsible for

the variability in the metabolism of CYP1A2 substrates in

dogs, with at least one of the SNPs (1117 C4T) resulting in a

nonfunctional protein (Mise et al., 2004a; Tenmizu et al.,

2004). In this case, a C4T SNP at nucleotide 1117 leads to

the substitution of Arg with a stop codon at position 373,

resulting in the formation of a truncated, nonfunctional form

of the enzyme (Mise et al., 2004a, 2004b; Tenmizu et al.,

2004). Thus, the T/T variant is associated with a protein that

is less than 75% the size of the wild-type (WT) protein, and

dogs carrying this trait are phenotypically classified as poor

metabolizers (Tenmizu et al., 2004). The incidence of the

aforementioned polymorphisms in the Beagle overall popu-

lation has been reported to be 4–15% (Tenmizu et al., 2004;

Whiterock et al., 2007).

The 1117 T/T polymorphism has also been noted in

13 other breeds of dogs; Australian Shepherds, Bearded

Collies, Collies, Dalmatians, Berger Blanc Suisse, Border

Collies, Deerhound, German Shepherds, Greyhound, Irish

Wolfhound, Jack Russell Terriers, Shetland Sheepdogs, and

Whippets (Aretz & Geyer, 2010). The wide preponderance of

this mutation raises an interesting dilemma; many PK and

target animal safety studies supporting canine drug approvals

are based upon studies using Beagles as the test subjects. Very

few studies are conducted in other breeds of dogs, potentially

affecting both those dog studies supporting human drug

development and those where the dog is the targeted

veterinary species. In either case, predictive in silico tools

(e.g. physiologically based PK software programs) would be

invaluable for improving the prediction of population dose-

exposure–response relationships in dogs or for predicting

how preclinical study outcomes may be affected by the use of

a closed population as canine subjects.

Despite their relatively low hepatic expression, canine

CYP1A enzymes are likely to influence drug metabolism.

However, their role and the effect on canine population

diversity necessitate the identification of selective probe

substrates and inhibitors. In fact, even though probe sub-

strates, such as 7-ER, have been reported on numerous times,

it is apparent that these compounds cannot, as yet, be assumed

to reliably distinguish between CYP1A1 and 1A2 activity

in liver microsomes. To this end, the recent commercial

availability of canine rCYP1A1 and 1A2 should prove

beneficial.

In summary, characterizing the relative expression of

canine CYP1A (and CYP2A) enzymes would help clarify the

validity of the existing liver microsome data and would

facilitate an interpretation of in vivo data. Methylxanthines

show promise as CYP1A probes, but their selectivity requires

confirmation. In vitro, the relative amount of caffeine

metabolized by canine CYP1A enzymes is lower than it is

for human CYP1A2, possibly explaining the weak nature of

the caffeine drug interaction in vivo.

CYP2B11

Human CYP2B6 is a primarily hepatic enzyme, representing

2–10% of total human hepatic CYP content (Mo et al., 2009;

Wang & Tompkins, 2008). This CYP is involved in the

metabolism of approximately 8% of clinically used human

drugs (Mo et al., 2009; Wang & Tompkins, 2008). Although

CYP2B6 was originally presumed to be of minor importance

in human drug metabolism, recent work has shown it to be

critical to the efficient clearance of several clinically import-

ant drugs, including (1) efavirenz (Ward et al., 2003) and

nevirapine (Erickson et al., 1999), both used in the treatment

of HIV infection, (2) bupropion (Hesse et al., 2000),

an atypical antidepressant and aid to smoking cessation,

and (3) methadone (Gerber et al., 2004), an opioid analgesic

and addiction treatment.

CYP2B11 is also largely responsible for the activation

of the antineoplastic drug, cyclophosphamide (CPA) (Raccor

et al., 2012), to metabolites that exhibit DNA-alkylating

properties.

Human CYP2B6 is characterized by very high interindi-

vidual variability (Mo et al., 2009; Wang & Tompkins, 2008).

A substantial proportion of this variability is explained by a

common polymorphism (CYP2B6 c.516 G4T) that results in

aberrant gene splicing and a truncated inactive enzyme

(Hofmann et al., 2008). However, it has also been observed

that population variability in CYP2B6 activity can be the

result of enzyme induction occurring by ligands that interact

with and activate NRs, such as the constitutive androstane

receptor (CAR) (e.g. phenobarbital; PB) and the pregnane X

receptor (PXR) (e.g. rifampicin; RIF) (Goodwin et al., 2001;

Sueyoshi et al., 1999). With regard to enzyme inhibition,

clopidogrel, an antiplatelet drug, is commonly used as a

potent, relatively specific CYP2B6 inhibitor (Walsky &

Obach, 2007).

CYP2B11 is the canine ortholog of CYP2B6, with 78% of

the 494 amino acid residues being identical to that observed

in humans (Graves et al., 1990; Shou et al., 2003). Similar to

humans, the expression of this canine enzyme is primarily in

the liver, with little information regarding CP2B11 expression

in extrahepatic tissues (Table 1). CYP2B11 was originally

identified as a major PB inducible CYP in canine liver

and immunochemical studies suggest that it constitutes

approximately 10% of total canine liver CYP content

(Eguchi et al., 1996).

Known pharmaceutical substrates, inhibitors and inducers

of canine CYP2B11 and of the human 2B6 ortholog, are

provided in Table 3. Although the majority of the listed

substances were identified as substrates by showing nicotina-

mide adenine dinucleotide phosphate–dependent oxidative

metabolism (using recombinant CYP2B11), others (propofol,

methadone, pentobarbital, and phenytoin) have only been

indirectly identified based either upon inhibition of metabol-

ism by liver microsomes (Hay Kraus et al., 2000) or in vivo

by coadministering chloramphenicol with these drugs

(Adams & Dixit, 1970; Kukanich et al., 2011; Mandsager

et al., 1995; Sanders et al., 1979; Teske & Carter, 1971).

Chloramphenicol is purported to be a highly selective

inhibitor of CYP2B11 (Ciaccio et al., 1987). Propofol

oxidation in canine liver microsomes was also inhibited by

a CYP2B-selective antibody (Ab) (Hay Kraus et al., 2000).

rCYP2B11 also metabolizes various endogenous substrates,

including testosterone (Shou et al., 2003), androstenedione

(Duignan et al., 1988), and progesterone (Duignan et al.,

1988), as well as toxic environmental chemicals, such as


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polychlorinated biphenols (Ariyoshi et al., 1995; Waller

et al., 1999).

In vitro, propofol (Hay Kraus et al., 2000) and temazepam

(Shou et al., 2003) were previously considered to be highly

selective CYP2B11 substrates. However, these probes may be

less-sensitive indicators of in vivo CYP2B11 metabolism

than previously believed because of alternate metabolic

pathways (glucuronidation, both drugs; sulfation, propofol)

and high drug clearance (propofol). In contrast, methadone

could potentially be a selective in vivo probe of CYP2B11

based upon significant inhibition of its clearance by the

coadministration of chloramphenicol (Kukanich et al., 2011).

However, there currently is no information on the ability

of methadone to be metabolized by rCYP2B11. Further, based

upon studies conducted with human liver microsomes, metha-

done metabolism is stereoselective, with CYP2B6 primarily

metabolizing the S-enantiomer, whereas CYP2C19 mainly

metabolizes the R-enantiomer (Chang et al., 2011). Previous

studies in dogs used the methadone racemate as a probe.

To date, chloramphenicol appears to be the most selective

CYP2B11 inhibitor, with demonstrated efficacy in vivo

(Kukanich et al., 2011) and in vitro (Ciaccio et al., 1987;

Hay Kraus et al., 2000). Other potentially selective inhibitors

based on in vitro studies include propofol (Aidasani et al.,

2008), S-medetomidine (Baratta et al., 2010), and atipame-

zole (Baratta et al., 2010), although the usefulness of these

agents as inhibitors of CYP2B11 substrate metabolism in vivo

has not yet been established.

No genetic polymorphisms within the canine CYP2B11

gene family have, as yet, been described. Nevertheless,

Greyhounds are known to have a reduced capacity to

eliminate propofol. In vitro studies on propofol hydroxylation

demonstrated a significant decrease in drug clearance in

Greyhounds, compared to Beagles, but not as compared to

mixed-breed dogs (Court et al., 1999). The PK of antipyrine

was significantly different in Greyhounds and Beagles

(Kukanich et al., 2007). This deficiency is related to a

reduced capacity of liver microsomes from Greyhounds to

metabolize propofol by CYP2B11 (Hays Kraus et al., 2000).

Whether this difference is the result of an unrecognized

polymorphism in CYP2B11 is not known.

CYP2C21 and 2C41

Two distinct hepatic CYP2C isoforms, CYP2C21 and 2C41,

are expressed in dogs (Blaisdell et al., 1998; Komori et al.,

1989; Uchida et al., 1990). The CYP2C subfamily is

extensively expressed by the liver, and the CYP2C21 gene

is constitutively expressed in extrahepatic tissues (Table 1).

In terms of protein expression, its relative hepatic protein

expression (as determined by immunochemical analysis)

appears to exceed that of CYP2D15 (Figure 1). Based upon

complementary DNA cloning of hepatic tissues from multiple

Beagle dogs, CYP2C21 contains 487 amino acids and 2C41

contains 489 amino acids (Blaisdell et al., 1998).

Human CYP2C9, 2C18, and 2C19 are the most closely

related orthologs to canine CYP2C21 (69.1–70.1% amino

acid similarity) and 2C41 (74.9–75.5% similarity). Eguchi

et al. (1996) estimated that, on average, the hepatic CYP2C

enzyme content of male and female Beagles comprised 35%

of total hepatic CYP content. Because these data were based

upon the use of primary Abs raised against CYP2C21, it is not

known whether this relative CYP content reflects CYP2C21

alone or the combination of CYP2C21 and 2C41. Of the

common CYP inducers (PB, RIF, and b-NF), only PB exhibits

a modest induction of CYP2C21 protein levels in Beagles

(Eguchi et al., 1996).

Early studies with CYP2C21 purified from canine liver

demonstrated C17 alcohol oxidation (i.e. androstenedione

formation) and 16a-hydroxylase activity with testosterone,

21- and 16a-hydroxylase activity with progesterone, benz-

phetamine N-demethylation, aminopyrine N-demethylation,

7-ethoxycoumarin O-deethylation, and aniline hydroxylation

(Komori et al., 1989). However, it is currently recognized that

several of these metabolic pathways are catalyzed by multiple

CYPs. Therefore, the recombinant enzyme systems

(Baculosomes� or Bactosomes) will likely be important in

characterizing the selectivity of probe substrates and

Table 3. Examples of experimentally demonstrated pharmaceutical substrates and inhibitors of human CYP2B6 and canine CYP2B11.

Human CYP2B6 Substrates (Wang & Tompkins, 2008)Inhibitors (Wang & Tompkins,

2008)Inducers (Wang & Tompkins,

2008)

Efavirenz, nevirapine, bupropion, methadone,artemisinin, CPA, ifosphamide, ketamine,propofol, selegiline

Thiotepa, clopidogrel, ticlopidine PB barbiturates, phenytoin, efavir-enz, artemisinin, RIF, clotrim-azole, carbamazepine

Canine CYP2B11 Substrates Inhibitors InducersPropofol (Hay Kraus et al., 2000), diazepam

(to nordiazepam) (Shou et al., 2003),temazepam (Shou et al., 2003), midazolam(Baratta et al., 2010; Mills et al., 2010),ketamine (Baratta et al., 2010),Atipamezole (Baratta et al., 2010),S-medetomidine (Baratta et al., 2010),R-medetomidine (Baratta et al., 2010),CPA (Chen et al., 2004), methadone(Kukanich et al., 2011), pentobarbital(Adams & Dixit 1970; Teske & Carter,1971), warfarin (Hasler et al., 1994),phenytoin (Sanders et al., 1979)

Chloramphenicol (Ciaccio et al.,1987), S-medetomidine (Barattaet al., 2010), atipamezole(Baratta et al., 2010),ketoconazole (Aidasani et al.,2008), miconazole (Aidasaniet al., 2008), propofol (Aidasaniet al., 2008)

PB (Graves et al., 1990; Grahamet al., 2002; Jayyosi et al., 1996)bergamottin (Sahi et al., 2002)


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inhibitors specific for the canine CYP2C subfamily of

enzymes (Locuson et al., 2009; Shou et al., 2003).

To date, only a limited number of compounds have

been evaluated as potential probe substrates for CYP2C21.

The recombinant enzyme systems have shown that canine

CYP2C21 exhibits moderate selectivity for 40-hydroxylation

of diclofenac. However, canine CYP2B11 also catalyzes this

reaction (Locuson et al., 2009; Shou et al., 2003). Considering

that diclofenac is directly glucuronidated and conjugated

to taurine in vivo, the sensitivity of diclofenac as a canine

CYP2C probe is probably limited (Stierlin et al., 1979).

Further, it has been discouraging to learn that other human

CYP2C probes, such as mephenytoin (Shou et al., 2003),

tolbutamide (Chauret et al., 1997), and omeprazole

(Turpeinen et al., 2007), all suffer from either poor activity

or poor selectivity upon examination either with canine liver

microsomes or recombinant dog CYP enzymes.

Although there is a dearth of information available on

CYP2C21, even less is known about the drugs metabolized by

canine CYP2C41. In vitro studies comparing recombinant

CYP2C41 to the other putative major canine drug-metaboliz-

ing isoforms indicated negligible enzymatic activity when

recombinant CYP2C41 was incubated in the presence of

typical human CYP substrates, including those compounds

readily metabolized by canine CYP2C21 (Shou et al., 2003).

One of the intriguing properties of CYP2C41 is that its

expression appears to be polymorphic, and the gene encoding

this enzyme is present in some dogs, but is absent in others

(Blaisdell et al., 1998). These investigators noted that in

28 genotyped animals, only 14% of dogs possessed the

CYP2C41 gene. In a separate study, 45% of the 11 Beagles

examined possessed the 2C41 gene (Graham et al., 2003).

Considering the limited number of breeds evaluated and the

small sample size used in these studies, definitive conclusions

regarding gene frequency and breed relationship to the

expression of the CYP2C41 gene cannot be made at this

time and further studies are needed.

The search for potential CYP2C21/41 inhibitors, and

therefore potential perpetrators of DDIs with clinically

important veterinary pharmaceuticals, has primarily been

limited to the use of liver microsomes. Using the 40-OH

diclofenac reaction as a marker for CYP2C21 activity, only

two drugs (ketoconazole and vincristine) were found to have

a half-maximal inhibitory concentration 525 mM (substrate

concentration equal to its Km). Neither compound was

considered to be a highly potent inhibitor of CYP2C21

(Aidasani et al., 2008). Several other human CYP2C enzyme

inhibitors, such as sulfaphenazole (e.g. 2C9) (Chauret et al.,

1997), tranylcypromine (e.g. 2C19), and quercetin (e.g. 2C8)

(Van Beusekom et al., 2010), showed little or no inhibition of

dog CYP2C in microsomes. Subsequently, using midazolam

as a substrate (Mills et al., 2010), ketoconazole was confirmed

to be a moderate inhibitor of rCYP2C21 (Ki¼ 7.25 mM).

Other inhibition data acquired with recombinant CYP2C are

not yet available.

One of the reasons why some of the common human

CYP2C probe substrates and inhibitors do not appear to be

suitable as canine CYP2C probes is that dogs (and several

other animal species) appear to lack the anion-binding site,

which is present in the major human CYP2C9 isoform

(Locuson et al., 2011). Hence, analogs of sulfaphenazole were

studied to characterize a new canine CYP2C21 substrate,

4-methyl-N-methyl-(2-phenyl-2H-pyrazol-3-yl)benzenesulfo-

namide. In in vitro studies, this compound, which is

distinguished by the lack an ionizable center and the presence

of a tolyl functional group (which is highly reactive to

CYP), was found to be rapidly metabolized and appeared

to be specific for CYP2C21. Thus, 4-methyl-N-methyl-

(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide may, in fact,

be a sensitive in vivo probe for 2C21; however, this requires

further validation.

A summary of our current knowledge pertaining to human

versus canine CYP2C substrates and inhibitors is provided in

Table 4. A systematic analysis of canine medications that

are substrates for the commercially available 2C21 and 2C41

recombinant enzymes could provide much of the needed

information regarding canine metabolic pathways. It would

also uncover the differences in metabolic pathways between

humans and dogs for drugs that are used in both species.

Characterizing the precise expression levels of 2C21 relative

to 2C41 and defining the genetic cause of the presence or

absence of the 2C41 gene will help establish the role of the

CYP2C subfamily in canine drug disposition.

CYP2D15

Human CYP2D6 is an important enzyme involved in the

metabolism of approximately 25% of clinically used drugs.

These include beta-blockers, antiarrhythmics, antidepressants,

and opioid derivatives (Zhou, 2009; Table 5). CYP2D6 also

bioactivates codeine to morphine. This reaction is inhibited

by quinidine in humans, leading to decreased efficacy of

codeine (Caraco et al., 1999). Other inhibitors of CYP2D6

include metoclopramide, cimetidine, and fluoxetine (Desta

Table 4. Inhibitors and substrates of CYP2C enzymes in humans and dogs.

Humana CYP2C9 Substrates Celecoxib, diclofenac, ibuprofen, tolbutamide, phenytoin,warfarin, flurbiprofen

Tracy et al., 1996

Inhibitors Sulfaphenazole, Fluconazole, benzbromarone Takahashi et al., 1999Inducers RIF

CYP2C19 Substrates Omeprazole, mephenytoin, clopidogrel Desta et al., 2002bInhibitors Proton pump inhibitorsInducers RIF

Canine 2C21/2C41 Substrates Diclofenac (also substrate for CYP2B11), midazolam,4-methyl-N-methyl analog of sulfaphenazole

Shou et al., 2003; Mills et al., 2010; Locusonet al., 2011

Inhibitors Ketoconazole, vincristine Locuson et al., 2011; Aidasani et al., 2008Inducers PB Eguchi et al., 1996

aUnless specified, all human enzyme probes can be reviewed at http://medicine.iupui.edu/clinpharm/ddis/table.aspx.


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et al., 2002a; Madeira et al., 2004; Zhou, 2009). One of the

fundamental challenges associated with drugs metabolized

largely by CYP2D6 is the tremendous variability in activity

known to occur across the human population. CYP2D6

polymorphisms are responsible for a number of significant

drug-related adverse reactions (Johansson & Ingelman-

Sundberg, 2011; Niwa et al., 2011).

The canine ortholog, CYP2D15, encodes a 500-amino-acid

protein that is 74.6% homologous to human CYP2D6

(Roussel et al., 1998; Sakomoto et al., 1995). CYP2D15 is

expressed predominantly in canine liver, but the degree of

expression is unclear; it has been reported to constitute

between 3 and 20% of total canine hepatic P450 expression

(Sakomoto et al., 1995; Tasaki et al., 1998) (Table 1). This

CYP isoform is also expressed at moderate levels in bladder,

with lower levels in kidney, lung, and brain, and no detectable

expression in small or large intestine (Roussel et al., 1998).

CYP2D15 shares a number of therapeutic drug substrates

with human CYP2D6 (Table 5), although much less is known

about the substrate range in dogs (Shou et al., 2003; Tasaki

et al., 1998). CYP2D15 is inhibited by ketoconazole,

clomipramine, and loperamide in dog liver (Aidasani et al.,

2008). The antiemetic, maropitant, which is approved for use

in dogs, has a nonlinear PK profile that is attributed to the

saturation of CYP2D15 (Benchaoui, 2007). Considering

published reports generated to date, DDIs associated with

CYP2D15 may have substantial clinical relevance (Aidasani

et al., 2008).

Given the range of clinically important substrates for

human CYP2D6, additional work is needed to understand the

potential role of canine CYP2D15 in the clearance of drugs

commonly used by veterinarians. Of particular clinical

interest are the known CYP2D15 substrates, clomipramine,

fluoxetine, metoclopramide, and tramadol. In addition, con-

sidering the structural similarities between celecoxib and the

veterinary drug, deracoxib, it is important to know whether

CYP2D15 is responsible for the clearance of deracoxib in

dogs. Such information would be helpful in interpreting the

variability in deracoxib efficacy and toxicity that has been

observed across the canine population (Case et al., 2010,

Lascelles et al., 2005; McMillan et al., 2011). Because

ketoconazole and loperamide are also commonly used in

dogs, in vivo drug-interaction studies are needed to determine

whether these in vitro inhibitors lead to clinically relevant

changes in clearance of CYP2D15 substrates.

Studies involving CYP2D15 can lead to biased data

interpretations if the human-canine differences in potential

probe substances are not adequately recognized. Although

celecoxib is a CYP2D15 substrate in dogs (Paulson et al.,

1999), in humans celecoxib is biotransformed by the CYP2C

subfamily and not by CYP2D6. In addition, debrisoquine,

which is a standard probe drug for CYP2D activity in humans,

is a poor CYP2D substrate in dogs (Tasaki et al., 1998).

These are just two examples of potential errors that can occur

when attempting to assess metabolic pathways through the

use of extrapolated CYP probe substrates that have not been

validated in the specific targeted animal species.

In humans, the existence of polymorphic variants of

CYP2D6 has resulted in clinically significant alterations in

the PK of numerous compounds. Similarly, polymorphic

CYP2D15 variants have been identified in dogs. As shown

in Table 6, there are seven variants of CYP2D15 that have

been identified (Paulson et al., 1999; Roussel et al., 1998;

Sakamoto et al., 1995). CYP2D15� is missing exon 3

(as defined by using the human 2D6 gene sequence) and

has no activity toward bufuralol or celecoxib (Paulson et al.,

1999). CYP2D15*2 and 2D15*3 both have amino acid

substitutions at positions 186, 250, and 307 (Paulson et al.,

1999) and have approximately 80% of the enzymatic activity

toward bufuralol or celecoxib, as compared to CYP2D15wt.

CYP2D15wt2 also with amino acid substitutions at positions

186, 250, and 307 and has approximately 38 and 75% of

the WT activity, respectively, toward these two substrates.

The apparent bufuralol Vmax values for CYP2D15 wt,

CYP2D15*2, CYP2D15*3, and CYP2D15wt2 were all

similar.

CYP3A12 and 3A26

It has been estimated that the CYP3A subfamily is responsible

for the biotransformation of nearly 50% of all pharmaceutical

agents in humans (Komura & Iwaki, 2008). Four members

of the human CYP3A subfamily have been identified:

CYP3A4, 3A5, 3A7, and 3A43. Among those, CYP3A4

is believed to have the greatest overall effect on drug

metabolism.

Table 5. Substrates and inhibitors of CYP2D15 (dogs) and CYP2D6 (humans) (see legend for citations).

Human CYP2D6 Substratesa Amitriptylinea bufarolol, chlorpromazine, chlorphe-niramine, clomipramine, codeine, dextromethor-phan diltiazem, dolasetron, fluoxetine (one ofseveral CYPs), imipramine, methamphetamine,metoclopramide, metoprolol, mexiletine, pro-pranolol, quinidine, tramadol, Substrates used asprobe drugs Debrisoquine, dextromethorphan

Desta et al., 2002a; Pedersen et al., 2005

Inhibitors Cimetidine, fluoxetine, metoclopramide, quinidine Madeira et al., 2004; Brown et al., 2006; Desta et al.,2002a; McLaughlin et al., 2005

Canine CYP2D15 Substrates Bufaralol, bunitrolol, celecoxib, desipramine, dex-tromethorphan (O-demethylation), imipramine,metoprolol, propranolol (4-,5-hydroxylations),maropitant Poor substrates Debrisoquine

Roussel et al., 1998; Shou et al., 2003; Tasaki et al.,1998; Nakamura et al., 1995; Paulson et al., 1999;Sakomoto et al., 1995; Benchaoui et al., 2007

Inhibitors Clomipramine, fluoxetine, ketoconazole, loperamide(potent), quinidine

Aidasani et al., 2008; Roussel et al., 1998

aInformation on human enzyme probes reviewed in Zhou (2009), unless noted.


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In dogs, two CYP3A enzymes have been described:

CYP3A12 (Ciaccio et al., 1991) and 3A26 (Fraser et al.,

1997). It has been suggested that others may also exist (Fraser

et al., 1997). The vast majority of canine CYP3A research has

involved CYP3A12, for which the deduced amino acid

sequence is 79.8% identical to human CYP3A4 (Ciaccio

et al., 1991). To date, there are only three reports describing

canine CYP3A26 (Chen et al., 2009; Fraser et al., 1997;

Mealey et al., 2008). The remaining literature either describes

canine CYP3A as a blanket term for all members of the

subfamily or describes CYP3A12 exclusively (Komura &

Iwaki, 2008; Kukanich et al., 2005; Liu et al., 2007; Marathe

& Rodrigues, 2006; Nishibe et al., 1998).

CYP3A12 and 3A26 isoforms are 96% identical to each

other at the amino acid level (Fraser et al., 1997). CYP3A26

differs from canine CYP3A12 in 22 of 503 amino acid

positions (Fraser et al, 1997), with nine of the amino acid

differences occurring at substrate recognition sites. Despite

their close identity, there are notable differences in their

capacity to metabolize steroids, with CYP3A12 demonstrat-

ing higher metabolic activity than CYP3A26. Expressed

isoforms also differ in their respective capacity to metabolize

midazolam, a CYP3A substrate in other animal species

(Locuson et al., 2009).

The CYP3As are the most abundant of the CYP superfamily

in human liver and intestine (Paine et al., 2006). Interestingly,

CYP3A12 constitutes a substantially lower relative proportion

of total hepatic CYP content, as compared to that reported

for humans (Table 1). CYP3A12 was shown to account for

approximately 15% of total P450 expression in canine liver

(Eguchi et al., 1996). However, because this study was

completed before the discovery of CYP3A26, it is unclear

as to whether or not this estimate truly represents total CYP3A

or rather if it is specific for CYP3A12. By comparison, the

CYP3A subfamily accounts for 30% of the total CYP content

in human liver (Komura & Iwaki, 2008). A more recent study

reported on the differential expression of CYP3A12 and 3A26

in canine liver and intestine (Mealey et al., 2008). Total

CYP3A mRNA expression was greater in hepatic tissue than

that in duodenal tissue. Hepatic expression of CYP3A26 was

greater than that of CYP3A12 in all dogs studied, with

CYP3A26 comprising 75.2% of the hepatic mRNA CYP3A

pool. Conversely, duodenal expression of CYP3A12 was

greater than that of CYP3A26 in all dogs, with CYP3A12

comprising 99.8% of the duodenal mRNA CYP3A pool.

There are distinct tissue differences in the relative

amounts of NRs in the liver versus segments of the GI

tract. These differences have been correlated to the expression

of CYP3A12 (Greger et al., 2006). Using liver slices from

male Beagles and a series of 22 different xenobiotics, it was

shown that CYP3A12 and 3A26 were consistently coinduced

by these agents (Chen et al., 2009). However there was no

correlation between the compounds that induce human

CYP3A4 and those that induce canine CYP3A12. Analysis

of human and canine PXRs demonstrated two amino acid

differences in the ligand-binding domain of canine PXR.

These two amino acids are hypothesized to be the basis for the

observed differences in the induction of canine human

CYP3A4 versus 3A12.

There are numerous known substrates, inhibitors, and

inducers for human CYP3A4 (reviewed in Pal & Mitra, 2006;

Zhou, 2008). Table 7 contains a selected list of these com-

pounds. In contrast, information available on canine CYP3A

enzymes is sparse. Few substrates for canine CYP3A12 have

been documented to date. Table 7 also includes a list of the

known CYP3A12 substrates, inhibitors, and inducers.

Although CYP3A12 has been labeled as the canine ortholog

of human CYP3A4, it is not clear that we can extrapolate

information from CYP3A4 and apply it to CYP3A12. For

example, whether all CYP3A4 substrates are also substrates

for CYP3A12 is not known. Although it certainly warrants

further investigation, information for the other CYP isoforms

suggests that such canine-human extrapolations are likely to

be fraught with error.

Genetic polymorphisms have been identified for

CYP3A12. The variant, CYP3A12*2, has been described

by Paulson et al. (1999) and differs by five amino acids

(SKEKT), respectively, at positions 309, 421, 422, 423,

and 452 from CYP3A12 (TRKNM). However, testosterone

6b-hydroxylation by CYP3A12*2 was approximately 90% of

that observed with CYP3A12, suggesting that this variant

may have effectively the same metabolic capacity as com-

pared to the WT isoform, at least for this substrate.

Given the role of the dog as a preclinical model for human

drug development studies, it is important to determine the

specific canine enzymes involved in metabolizing drugs

known to be human CYP3A4 substrates and to better

appreciate differences in the activities of these human

versus canine orthologs. From the perspective of veterinary

medicine, knowing the specific CYP enzyme responsible for

Table 6. Sequence variation in canine CYP2D15.

Residue no. Exon 3Res 121

splice variantRes186

Res250

Res307

Res338

Res407

Significantlyaltered activity Comments

Nucleotide no. G362-G515 deleted A556G

(S!G)

A758T(I! F)

A919G(I!V)

A1012G(I!V)

A1219G(K!E)

2D15WT þ S I I (Control) Sakamoto et al., 19952D15WT2 þ G F V Not for bufuralol Roussel et al., 1998

2D15d � G121-A172 deleted G I I Yes; very low activity Paulson et al., 19992D15V2 � G121-A172 deleted G F V Yes, almost undetectable Roussel et al., 19982D15V1 þ G F V V E Yes; bufuralol velocity

versus WT2Roussel et al., 1998

2D15*2 þ G I I Not for bufuralol Paulson et al., 19992D15*3 þ S F V Not for bufuralol Paulson et al., 1999


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metabolizing therapeutic agents is important both to avoid

adverse DDIs and for predicting the sources of variability

that can influence product safety and effectiveness across

the canine population. An example of a clinically relevant

DDI involving canine CYP3A12 is the substrate-inhibitor

DDI involving cyclosporine and ketoconazole, respectively.

Coadministration of these two compounds results in

decreased cyclosporine hepatic metabolism and, conse-

quently, higher cyclosporine plasma concentrations

(D’mello et al., 1989). As observed in humans, another

potential cause of adverse drug reactions involving CYP3A

substrates could be the presence of polymorphic forms

of CYP3A12 that can either increase or decrease enzymatic

activity. Further investigation, particularly in different

dog breeds, may yield additional polymorphisms that may

be relevant to the correlation between drug dose, safety, and

effectiveness across a canine population.

Conclusion

Physiological and metabolic differences across canine breeds

can lead to breed-associated disparities in the dose-exposure

relationship of pharmaceutical products. In turn, these

disparities have the potential to affect drug-product safety

and effectiveness. Clearly, much more research is needed to

identify appropriate probe substrates and inhibitors that can

be used for identifying sources of population variability

in CYP activity. The commercial availability of canine rP450

bactosomes should help much in this endeavor (Locuson

et al., 2009). It is evident that we cannot simply extrapolate

probes and substrates from those appropriate for humans.

Further, it is evident that assessments of sources of pheno-

typic variation should not be limited to polymorphisms

in specific CYP genes, but should also include the effect of

disease, as well as other genetic and epigenetic sources of

variation.

Ultimately, we are left with the question of how important

is it to understand CYP-related variability in dogs from both a

preclinical and clinical perspective. Clearly, CYPs are only

a small (but important) component of population diversity.

As canine therapeutics progresses in its similarity to human

medicine, an appreciation of canine population diversity in

drug clearance and the identification of covariates (such as

breed) for risk of altered drug metabolism is increasing

in importance.

Numerous obstacles exist in the in vitro and in vivo

assessment of intra- and interbreed differences in canine

metabolism. However, considering the technological

advances that have been utilized to understand ethnic and

racial differences in human drug metabolism, the tools are

available for continuing these studies in dogs and in other

veterinary species. Such studies are not only critical to define

and prevent adverse drug effects in dogs, but also need to be

considered when using the dog as a preclinical species to

support human drug discovery and development.

Given the importance of the CYP superfamily in drug

metabolism (and therefore drug-product safety and effective-

ness), additional research is greatly needed, both from the

perspective of the dog as a preclinical species in human drug

development and the dog as the target species in veterinary

medicine. In this regard, research priorities include (1) studies

to identify appropriate probe molecules for evaluating the

activities of canine-specific CYPs, (2) identification of

existing CYP variants and their metabolic consequences,

(3) development of high-throughput tools for assessing the

presence of specific CYP genetic polymorphisms within the

canine population, (4) use of these tools to assess breed-

related probabilities associated with clinically relevant

CYP variants, (5) development of canine-specific tools to

facilitate individualized dosage regimens for narrow ther-

apeutic-window drugs (such as cancer therapies), and (6)

development of collaborative scientific networks for infor-

mation and technology exchange that will optimize our ability

to achieve the scientific breakthroughs necessary to support

the evolving canine therapeutic landscape, as well as improve

predictions for human medicine, when the dog is used as a

preclinical species during drug development.

Our hope is that this review will serve as a springboard

for encouraging additional dialog on canine polymorphisms

to promote our understanding of within- and between-breed

Table 7. CYP3A4 and 3A12 substrates, inhibitors and inducers.

Substrates (incomplete list) Inhibitors (incomplete list) Inducers (incomplete list)

Human CYP 3A4 Anticancer agents (irinotecan, vincris-tine); immunosuppressants (cyclospor-ine, tacrolimus); HIV-1 proteaseinhibitors (indinavir, ritonavir, andsaquinavir); drugs affecting the CNS(midazolam, fexofenadine); calciumchannel blockers (verapamil, nifedi-pine); warfarin;Steroid molecules(testosteronea)

Ketoconazole, clarithromycin,saquinavir, diltiazem, verap-amil, cimetidine, fluoxetine

Nicardipine, RIF, rifabutin, nitredi-pine, clitrimazole, ritonavir, lacidi-pine, efavirenz, lansoprazole,troglitazone, nifedipine, troleando-mycin, pioglitazone

Canine CYP 3A12 Diazepam midazolam, eplerenone,diclofenac

Ketoconazole, dexamethasone Efavirenz, lacidipine, clotrimazole,felodipine, phenytoin, ritonavir,RIF, netredipine, RU-486, sulfin-pyrazole, lansoprazole, PB

Based upon information in the reviews by Pal & Mitra (2006) and Zhou (2008).aCYP3A26 may be another ortholog or may be an ortholog of CYP3A5 (CYP3A26 is the only other identified canine CYP3A enzyme). Please note that

because of the extensive nature of the information on the CYP3A family of enzymes, the list is truncated. For more information, refer to: human:Ohno et al. (2007), Pal & Mitra (2006), and Zhou (2008); dog: Chen et al. (2009), Cook et al. (2002), Graham et al. (2006), Locuson et al. (2009), andZhang et al. (2006a, 2006b).

CNS, central nervous system.


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variations in drug response, support efforts for further study

of the therapeutic implications of these genetic variations, and

enhance opportunities to establish research collaborations.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

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Challenges in exploring the cytochrome P450 system as a source of variation in canine drug...

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