A Thesis

entitled

Ultrasound-mediated Topical Delivery of Econazole nitrate for Treating Raynaud’s

Phenomenon

by

Saloni Daftardar

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in

Pharmaceutical Sciences, Industrial Pharmacy

_________________________________________

Sai Hanuman Sagar Boddu, PhD., Committee Chair

_________________________________________

Jerry Nesamony, PhD., Committee Member

_________________________________________

Caren L.Steinmiller, PhD., Committee Member

_________________________________________

Gabriella Baki, PhD., Committee Member

_________________________________________

Dr. Amanda Bryant-Friedrich, Dean

College of Graduate Studies

The University of Toledo

August 2017

Copyright 2017, Saloni Daftardar

This document is copyrighted material. Under copyright law, no parts of this document

may be reproduced without the expressed permission of the author.

iii

An Abstract of

Ultrasound-mediated Topical Delivery of Econazole nitrate for Treating Raynaud’s

Phenomenon

by

Saloni Daftardar

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in

Pharmaceutical Sciences Industrial Pharmacy

The University of Toledo

August 2017

Objectives: The focus of this investigation was to assess the ultrasound-assisted

econazole nitrate (EN) permeation from topically applied formulations for treating

Raynaud’s phenomenon. The aims of this study were i) to identify the optimal ultrasound

parameters for the percutaneous absorption of EN, ii) to assess the in vitro percutaneous

absorption of EN and toxicity in the porcine ear skin from different topical formulations

following ultrasound application.

Methods: Optimization of ultrasound parameters such as the distance of the horn,

application time and amplitude were performed. In vitro percutaneous absorption studies

were performed using different EN formulations (F1_solution, F2_HPMC dispersion,

F3_Lipoderm®

Activemax™ Cream and F4_Versabase®

cream) across ultrasound-treated

porcine skin and were compared with the control group (skin samples without ultrasound

treatment). Histological evaluation using hematoxylin and eosin stain was carried out to

assess the dermal toxicity of formulations and ultrasound exposure. To further support

the findings of ultrasound-assisted drug permeation studies, ATR-FTIR was performed to

iv

investigate the effect of ultrasound on the conformational changes in stratum corneum

lipids characterized by the changes in spectral shifts.

Results: A constant frequency (20 kHz) ultrasound application with 40% amplitude,

0.5cm distance between ultrasound horn and skin surface for 2 minutes was optimized.

The permeation of EN was found to be higher from ultrasound- treated skin samples than

the control group. Drug permeation from F2_HPMC dispersion was found to be higher as

compared to other formulations and the marketed cream. The lag time of ultrasound-

treated skin samples were found to be significantly lower than the control. Histological

evaluation confirmed that HPMC dispersion showed no signs of toxicity. ATR-FTIR

studies revealed a slight increase in the -CH2- stretching vibrations (~ 2920 cm-1

and

2850 cm-1

) in ultrasound-treated skin samples as compared with control. This confirms

the reversible conformational changes in the stratum corneum lipids, thereby enhancing

the drug permeabilization.

Conclusions: The ultrasound-assisted topical delivery of F2_HPMC dispersion could be

explored as a new strategy for treating Raynaud’s phenomenon.

Dedicated to my Family

vi

Acknowledgements

I would like to acknowledge and express my gratitude to all those who have directly

or indirectly helped me in this research as this project is an outcome of collaboratively

shared ideas and experiences. In the words of Brad Henry, “A good teacher can

inspire hope, ignite the imagination and instill a love of learning.” I would like to

express my deepest appreciation to my advisor, Dr. Sai HS. Boddu, for his valuable

guidance and suggestions for the planning and execution of this research. His unique

research insight has been the stepping stone for the development of this research

work. His constructive criticism and brain-storming sessions have urged me to work

diligently.

I would like to thank my committee members, Dr. Jerry Nesamony and Dr. Caren

Steinmiller for their support and constant encouragement over the past two years. I

would like to express my sincere gratitude to Dr. Gabriella Baki for serving as my

graduate faculty representative for my thesis defense. I would also like to thank Allen

Schroering for helping me with histology, Dr. Andrea Kalinoski and Dr. David

Weaver for allowing me to use their facility and training me. I would like to thank Dr.

Joseph Lawrence & Dr. Lidia Rodriguez for providing the facility and expertise on

ATR FTIR spectroscopy. I would like to thank Kastel's Slaughter House & Processing

Center (Riga, MI) for providing porcine ears for my study. I am thankful to my

friends and colleagues who have given me their unconditional support. My deepest

gratitude goes to my family for their constant support throughout my life.

vii

Table of Contents

Abstract .............................................................................................................................. iii

Acknowledgements ............................................................................................................ vi

Table of Contents .............................................................................................................. vii

List of Tables .................................................................................................................. xii

List of Figures .................................................................................................................. xiii

List of Abbreviations ....................................................................................................... xvi

List of Symbols .............................................................................................................. xviii

1 Introduction ..........................................................................................................1

1.1 Raynaud’s phenomenon ..........................................................................................1

1.1.1 Classification and epidemiology...................................................................1

1.1.2 Diagnosis…...................................................................................................2

1.1.3 Treatment of Raynaud’s phenomenon ..........................................................4

1.2 Topical drug delivery ..............................................................................................5

1.2.1 Skin as a barrier: Biggest challenge to topical drug delivery .......................7

1.2.2 Structure of the skin ......................................................................................7

1.3 Enhancement of skin permeability........................................................................10

1.4 Ultrasound therapy ................................................................................................11

1.4.1 Overview of ultrasound...............................................................................11

1.4.2 Wave propagation .......................................................................................11

viii

1.4.3 Ultrasound characteristics ...........................................................................12

1.4.4 Ultrasound instrumentation .........................................................................13

1.5 Applications of ultrasound in drug delivery .........................................................14

1.5.1 Topical drug delivery ..................................................................................15

1.5.2 Ultrasound in gastrointestinal drug delivery ...............................................27

1.5.3 Ultrasound in gene drug delivery ................................................................28

1.6 Synergistic effects of ultrasound ...........................................................................29

1.6.1 Effect of ultrasound with other physical techniques ...................................29

1.6. 2 Effect of ultrasound with chemical permeation enhancers ........................30

1.7 Conclusions………. ..............................................................................................31

1.8 Percutaneous absorption using Franz diffusion study ..........................................32

1.8.1 Theoretical considerations of percutaneous absorption ..............................32

1.8.2 In vitro dermal testing .................................................................................34

1.9 Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR)

spectroscopy and its use to characterize skin barrier ............................................37

1.10 Econazole nitrate .................................................................................................39

2 Significance of research .........................................................................................42

3 Ultrasound-mediated Topical Drug Delivery of Econazole nitrate for Treating

Raynaud’s Phenomenon ........................................................................................45

3.1 Abstract. …… .......................................................................................................45

3.2 Introduction .......................................................................................................47

3.3 Materials and Methods ..........................................................................................51

3.3.1 Materials ......................................................................................................51

3.3.2 Ultrasound application ................................................................................51

ix

3.3.3 Determination of ultrasound intensity .........................................................52

3.3.4 Optimization of ultrasound parameters .......................................................52

3.3.5 Transepithelial water loss (TEWL) measurement.......................................52

3.3.6 High Performance Liquid Chromatography analysis ..................................53

3.3.7 Preparation of porcine ear skin ...................................................................53

3.3.8 In vitro permeability study of econazole nitrate across porcine ear skin ....54

3.3.9 Mass balance recovery ................................................................................55

3.3.10 Skin toxicity assessment of the formulations ............................................56

3.3.11 Attenuated Total Reflectance Fourier Transform Infrared

spectroscopy ............................................................................................57

3.3.12 Statistical analysis .....................................................................................58

3.4 Results & Discussion ............................................................................................58

3.4.1 HPLC analysis .............................................................................................58

3.4.2 Ultrasound pre-treatment ............................................................................59

3.4.3 Optimization of ultrasound parameters .......................................................60

3.4.4 Percutaneous absorption .............................................................................63

3.4.5 Skin toxicity assessment of Econazole nitrate formulations .......................73

3.4.6 Attenuated Total Reflectance Fourier Transform Infrared spectroscopy ...77

3.5 Conclusions .......................................................................................................80

References ..........................................................................................................................82

x

List of Tables

1.1 Studies conducted using ultrasound to enhance the topical drug delivery ............25

1.2 Vibrational frequencies of the stratum corneum lipids ..........................................39

3.1 Apparent permeability (Papp), flux of econazole nitrate across excised porcine

skin and lag time. ...................................................................................................71

3.2 Total absorption and mass balance (as a percentage of the applied dose) of

econazole nitrate ....................................................................................................72

3.3 Important vibrational frequencies depicting stratum corneum layer .....................78

3.4 CH2- asymmetric and symmetric stretching vibration frequencies ........................78

xi

List of Figures

1-1 Structure of epidermis ..............................................................................................9

1-2 Ultrasound wave propagation ................................................................................12

1-3 Diagram of amplitude of ultrasound wave .............................................................13

1-4 Schematic diagram of ultrasound equipment .........................................................14

1-5 Cavitation leading to disordering of lipid bilayers ................................................18

1-6 Cavity bubble collapse and formation of microjet .................................................19

1-7 The cycle of bubble formation, growth and collapse .............................................20

1-8 Vertical Franz Diffusion cell ................................................................................35

1-9 Molecular organization of stratum corneum ..........................................................38

1-10 Econazole effects on menthol induced ET1 mRNA expression............................40

3-1 Percentage amount of econazole nitrate percutaneously absorbed into different

skin layers ..............................................................................................................50

3-2 Franz diffusion cell apparatus ................................................................................54

3-3 HPLC chromatogram of econazole nitrate ............................................................59

3-4 Effect of horn-to-skin distance on the permeation of econazole nitrate ................61

3-5 TEWL values for skin after ultrasound application at a horn-to-skin ...................62

3-6 TEWL measurements of porcine ear skin at 1 minute and 2 minute ultrasound

application time .....................................................................................................63

3-7 In vitro diffusion of econazole nitrate from F2_HPMC dispersion .......................65

xii

3-8 Percentage of econazole nitrate percutaneously absorbed into different skin

layers from F2_HPMC dispersion .........................................................................66

3-9 In vitro diffusion of econazole nitrate from F3_Lipoderm® Activemax™

Cream ...................................................................................................................66

3-10 Percentage of econazole nitrate percutaneously absorbed into different skin

layers from F3_Lipoderm® Activemax™ Cream ..................................................67

3-11 In vitro diffusion of econazole nitrate from F1_solution .......................................67

3-12 Percentage of econazole nitrate percutaneously absorbed into different skin

layers from F1_solution .........................................................................................68

3-13 In vitro diffusion of econazole nitrate from F4_Versabase®

cream .......................68

3-14 Percentage of econazole nitrate percutaneously absorbed into different skin

layers from F4_Versabase®

cream .........................................................................69

3-15 In vitro diffusion of econazole nitrate from marketed cream ................................69

3-16 Percentage of econazole nitrate percutaneously absorbed into different skin

layers from marketed cream ..................................................................................70

3-17 Hematoxylin and eosin staining of the control samples ........................................74

3-18 Hematoxylin and eosin staining of skin samples treated with F3_Lipoderm®

Activemax™ Cream ..............................................................................................75

3-19 Hematoxylin and eosin staining of skin samples treated with F2_HPMC

dispersion ...............................................................................................................76

3-20 Hematoxylin and eosin staining of skin samples treated with marketed

formulation ............................................................................................................77

xiii

3-21 ATR-FTIR spectra of porcine skin treated with marketed formulation,

F3_Lipoderm® Activemax™, F2_HPMC dispersion compared with skin as

such (control) .........................................................................................................79

3-22 ATR-FTIR spectra of porcine skin after ultrasound application and treatment

with marketed formulation, F3_Lipoderm® Activemax™, F2_HPMC

dispersion compared with skin as such (ultrasound- treated control) ...................79

xiv

List of Abbreviations

ANA ...........................Antinuclear antibodies

ATR-FTIR..................Attenuated Total Reflectance Fourier Transform Infrared

BSA ............................Bovine serum albumin

CCB............................Calcium channel blockers

cGMP .........................Cyclic guanosine monophosphate

EZ ...............................Econazole

EN ..............................Econazole nitrate

GIT .............................Gastrointestinal tract

HA ..............................Hyaluronic acid

HCC ...........................Hepatocellular carcinoma

HPMC ........................Hydroxypropyl methyl cellulose

HPLC .........................High Performance Liquid Chromatography

MT..............................Methanol

OECD .........................Organisation for Economic Co-operation and Development

PDE-5 .........................Phosphodiesterase type 5 inhibitors

PEG ............................Polyethylene glycol

PRP ............................Primary Raynaud’s Phenomenon

PZT ............................Lead zirconate titanate

RCS ............................Raynaud’s Condition score

RNA ...........................Ribonucleic acid

RP ...............................Raynaud’s Phenomenon

SC ...............................Stratum corneum

SD ..............................Standard deviation

SEM ...........................Standard error of mean

SEMA ........................Sonophoretic-enhanced micro-needle array

SLS .............................Sodium lauryl sulfate

SLES ..........................Sodium lauryl ether sulfate

TRP ............................Transient receptor potential cation channels

xv

TRPM8 .......................Transient Receptor Potential Melastatin 8

TEWL ........................Transepidermal water loss

TER ............................Transcutaneous Electrical Resistance

UV ..............................Ultraviolet

US ..............................Ultrasound

US FDA .....................United States Food and Drug Administration

VEGF .........................Vascular endothelial growth factor

W ................................Watts

xvi

List of Symbols

Hz ...............................Hertz

kHz .............................Kilohertz

MHz ..........................Megahertz

cm/min .......................Centimeter per minute

cm/hr ..........................Centimeter per hour

cm2/s ...........................Square centimeter per second

cm2/h ..........................Square centimeter per hour

μs ................................Microseconds

μ .................................Microns

μm ..............................Micrometers

μl ................................Microliter

nm ..............................Nanometer

mg/ml .........................Milligram per milliliter

μg/ml ..........................Microgram per milliliter

g/cm2 ..........................Grams per square centimeter

˚C................................Degrees Celsius

rpm .............................Revolutions per minute

V .................................Volts

W/cm2 ........................Watts per square centimeter

Da ...............................Dalton

kp ...............................Permeability coefficient

Km..............................Distribution coefficient

Papp .............................Apparent permeability

1

Chapter 1

Introduction

1.1 Raynaud’s phenomenon

Raynaud’s phenomenon (RP) was first diagnosed and described by Maurice Raynaud

in 1862 [1]. It is a clinical manifestation of recurrent, reversible vasospasm or

vasoconstriction of digital blood vessels upon exposure to cold temperatures. RP

mainly affects the arterioles, precapillary vessels and cutaneous arteriovenous shunts.

The phenomenon, a disorder of microcirculation and of an ischemic nature, is

characterized by pallor or cyanotic skin on the digits after exposure to cold or stressful

conditions, which eventually spread to the digits of both hands. There might also be

redness due to reopening of the blood vessels (reactive hyperemia) [2].

1.1.1 Classification and epidemiology

RP can be classified into two types: i) primary Raynaud’s phenomenon (PRP) and ii)

secondary Raynaud’s phenomenon (SRP). When the symptoms of RP are of an

unknown cause and there is no underlying disease to associate with it, then it is

termed as PRP [3]. PRP is characterized by cold exposure stimulated vasospasm,

2

bilateral involvement of extremities, absence of gangrene, and skin discoloration from

white to bluish to red [4]. When there is the presence of underlying disease associated

with RP, it is termed SRP. Its symptoms are almost similar to those of PRP, but are

associated with a disease condition, extremely dilated blood vessels, increased red

blood cell viscosity and aggregation, abnormal skin with sores and scars, and

sometimes results in gangrene of fingers or toes [5]. When the symptoms are visible

in the thumbs, it is generally a form of SRP [6].

PRP accounts for almost 80% of the diagnoses and its occurrence depends on the

geographic location. People living in colder regions have a higher risk of RP [7]. PRP

is more commonly observed in women than in men and affects around 11% women in

the United States. The occurrence of RP may increase in men due to occupational

hazards and hand trauma [8]. The onset of PRP has been reported at the median age of

14 years with only 27% of cases detected at the age of 40 years or later [9]. In PRP,

the symptoms are generally mild. There has been evidence of family history of RP in

about a quarter of the PRP patients [10]. SRP is diagnosed at the age of 30 years or

later with symptoms being more intense and painful, indicative of connective tissue

and microvascular disease [11].

1.1.2 Diagnosis

It’s important to distinguish RP from general common cold, which is associated with

cold hands and feet. Additionally, it is crucial to identify the form of RP, either

primary or secondary as the treatment options for these two classifications is different.

The following has been identified as the means to define the diagnosis of PRP: normal

3

results of capillaroscopy, lab tests such as antinuclear antibodies (ANA),

inflammatory parameters, absence of ulceration and underlying disease. On the other

hand, diagnosis criteria for SRP has been identified by abnormal capillaroscopy,

presence of pitting, scars, ulceration, reduced renal blood flow and positive ANA [12,

13]. As an aid to confirming the diagnosis of RP, a standard questionnaire has been

designed [14];

1. Are you more sensitive to cold than others?

2. Do you notice color changes of your skin?

3. Do your fingers look white or blue on cold exposure?

Sometimes, the clinician may use actual pictorial diagrams for the patients to identify

symptoms [15]. Criteria are proposed to describe the relative degrees of certainty in

the diagnosis of RP [14];

Definite RP – characterized by repeated occurrences of cold induced attacks

resulting in biphasic color changes of fingers or toes.

Possible RP - uniphasic color changes in addition to numbness or paresthesia

upon cold exposure.

No RP – no color changes

In SRP, non-specific abnormalities associated with scleroderma might be evident

along with other rheumatic conditions such as systemic lupus erythematous, mixed or

un-differentiated connective tissue disease or Sjögren’s syndrome [16].

4

1.1.3 Treatment of Raynaud’s phenomenon

The treatment intervention for RP should be clearly understood and the risk

assessment should be carried out by taking into account all significant symptoms.

There is no direct remedy for this condition, but treatment may certainly decrease the

severity and occurrence of attacks and would attempt to decrease the pain, numbness

and needle-like stinging sensations and prevent progression of the disease into more

complicated pathological conditions such as digital ulcers [17]. The people diagnosed

with PRP may not need drug treatment, as it can be managed using non-drug

management. These include: avoiding sudden temperature changes, keeping the body

warm and using chemical warmers for hands and feet. Drug treatment can be

recommended in patients with severe symptoms or where there is a considerable

chance of an underlying disease or evidence of ischemia–reperfusion, which can

cause significant tissue injury. The first-line treatment for PRP, or in some cases the

SRP, includes pharmacologic drug-related treatment with calcium channel blockers

(CCBs) [12, 17]. CCBs have been reported to reduce the frequency of the attacks.

Dihydropyridine calcium channel blockers such as nifedipine, nicardipine, amlodipine

and felodipine have been used. The dose of a CCB can be increased to achieve

maximum benefit in well-tolerated patients. When CCBs are ineffective or are not

well tolerated owing to their side effects (such as headaches, flushing and

hypotension), then phosphodiesterase type 5 (PDE-5) inhibitors such as Sildenafil,

Tadalafil may be used. PDE-5 inhibitors degrade cyclic GMP thereby resulting in

vasodilation. They have been reported to significantly decrease the Raynaud’s

condition score (RCS) and decrease the occurrence and duration of the vasospastic

events [18]. PDE-5 inhibitors can also be effective in combination with CCB.

5

Prostacyclin or epoprostenol is a vasodilator ideally used in the treatment of

pulmonary arterial hypertension. It inhibits thrombosis and stimulates the endothelial

cell protection. It has been reported to act against the SRP, reducing the severity of

attacks and prevents digital ulcers. Two intravenous analogs of prostacyclin, iloprost

and trepostinil, have found to be useful in SRP [17]. Some α - adrenergic blockers,

such as prazosin, have also been used for treating RP. Other antithrombotic agents,

such as dipyridamole, have been used to treat thrombosis and ulceration in RP

patients [19].

Nitrates could be a good treatment option for reducing the symptoms associated with

RP. These lead to the formation of nitric oxide giving rise to endothelium-derived

protective agent. However, owing to their side effects, oral nitrates are not used often.

Hence, a lot of emphasis has been given to topical nitrates as a potential treatment for

RP. Topical nitrates can cause fewer side effects with a tendency to cause vasodilation

of blood vessels [17]. It was reported that topical nitroglycerin improved the blood

flow in the digits [20]; glyceryl trinitrate patches in a sustained release action caused a

decrease in the frequency and severity of attacks [21]. Thus, topical nitrates could be

used in combination with a CCB to treat RP. As there is not much evidence to support

these findings, and the fact that topical drug delivery seems to be an ideal treatment to

deliver the drug into deeper layers of the skin, there is a need to further investigate the

potential of topical nitrates in treating primary RP.

1.2 Topical drug delivery

Topical drug delivery has been an efficacious and safe route of drug delivery across

the skin. It has been reported that the limitations of conventional (oral and parenteral)

6

routes of administration (oral and parenteral routes) namely the first-pass effect,

frequency of administration for drugs that have shorter biological half-life, poor

patient adherence and the fear of needles and pain associated with parenteral drug

delivery, can be overcome by using various advancements in the topical drug delivery

methodologies [22-25]. Several advantages offered by topical drug delivery are:

prevents drug degradation from hostile GI microenviroment and presystemic

metabolism [26]; reduces side-effects; unlike parenteral delivery systems there is no

pain associated with topical delivery [27]; better patient adherence; and reduced

frequency of drug administration and fluctuations in plasma drug concentrations.

The skin is the largest organ and can act as the model site of administration for locally

and systemically acting drugs [28]. Both topical and transdermal systems have been

widely used to treat local infections, motion sickness, analgesia, angina, hormonal

imbalance and hypertension [29]. Owing to the advancements in transdermal drug

delivery, in 2015, their market contribution accounted for $31.6 billion. This is

attributed to companies investing in this market following patent expirations,

reformulation of existing drugs, and enormous investments made in developing new

chemical entities. The high growth potential (over 12 %) of transdermal drug delivery

methods can be exemplified by the advent of newer applications, for example,

cardiovascular segment related to the treatment of angina pectoris, congestive heart

failure and hypertension. Similarly, the topical drug delivery market globally

accounted for $89.76 billion in 2016 and is expected to grow over 7% by 2022. Thus,

aforementioned factors are anticipated to widen the horizon for topical delivery

growth.

7

1.2.1 Skin as a barrier: Biggest challenge for topical drug delivery

The biggest challenge that remains for a formulator is the selective nature of the skin

barrier. The therapeutic efficacy of topical delivery depends largely on the potency of

a drug and its ability to permeate into the deeper layers of the skin. Topically applied

formulations can enter the skin transepidermally through the stratum corneum either

via the transcellular route or intercellular route. The drug can also enter the skin via

the transappendageal route, through hair follicles and eccrine ducts. Skin acts as a

robust barrier to the surrounding environment and protects body from UV radiation,

chemicals, loss of moisture, and helps regulates body temperature [28, 30]. The skin

barrier acts as a major constraint and limits the permeability of drug candidates from

various formulations across the skin. The skin is comprised of three layers: epidermis,

dermis and subcutaneous tissues. The skin acts as a protective layer mainly due to the

impermeable nature of epidermis [31].

1.2.2 Structure of the skin

Skin covers an area of around 1.7 square meters and accounts for about 10% of the

total body mass. The pH of the skin is around 3-6. Skin, along with its accessory

structures, forms the integumentary system of the body [32]. As it is a formidable

barrier to the permeation of many drugs, a thorough understanding of the physiology

and function of skin is fundamental for the advancements of methodologies to

develop innovative and robust transdermal dosage forms [31]. Skin is composed of

three regions: stratum corneum that is outermost layer of epidermis, viable epidermis,

dermis and subcutaneous tissues. (Figure 1.1) The barrier function is provided mainly

by the stratum corneum layer.

8

1.2.2.1 Stratum corneum layer (SC): It is the uppermost layer of epidermis with a

thickness of about 10-15 microns [33]. The corneocytes, contain 18-20 cell layers of

SC with no organelles, are loaded with protein keratin embedded in the matrix

structure. It is rich in lipids and forms the basis of obstruction to percutaneous drug

penetration [34]. The thickness and width of the corneocytes are found to be 0.2-0.4

microns and 40 microns, respectively. The SC layer is abundant in lipids (~ 14% by

weight lipid content) and low hydration. The lipids are composed of multi-lamelar

bilayers of ceramides, cholesterol and saturated fatty acids. Ceramides, which form

about 50% of SC lipids, consist of sphingosine and long chain mono-and di-

unsaturated omega hydroxy acids [35]. Cholesterol, 25% by weight of SC lipids,

comprises of cholesterol sulfate and cholesterol esters. Around 10% of SC lipids are

composed of free fatty acids, predominantly saturated long chain fatty acids, along

with minor quantities of unsaturated fatty acids such as oleic acid and linoleic acid

[35]. Cell differentiation is observed in the form of distinctive layers beneath SC

namely, stratum basale, stratum spinosum, stratum granulosum and stratum lucidum.

When the skin is in its constant state of renewal, cells (keratinocytes) transform from

the stratum basale to the outermost layer of SC (cornified squames or corneocytes)

through the intermediate stages of stratum spinosum, stratum granulosum, and stratum

lucidum [28].

1.2.2.2 Epidermis: The epidermis is a multi-layered region with varying thickness

from 0.06 to 0.8 mm, depending on the part of the body. It consists of 4-5 layers of

stratified epithelium [28]. The keratinocytes are the major cells of epidermis and their

crucial role is to undergo cell differentiation and lead to the formation of stratum

9

corneum hindrance layer. The epidermis consists of living cells of stratum

germinativum, stratum spinosum, stratum granulosum and stratum lucidum and dead

cells of the horny layer, SC [32].

Figure 1.1: Structure of epidermis

Source: https://opentextbc.ca/anatomyandphysiology/chapter/5-1-layers-of-the-skin/

1.2.2.3 Dermis: The dermis is located below the epidermis and consists of collagen

fibers and elastic connective tissue embedded in a mucopolysaccharide matrix.

Dermis is about 0.1 – 0.5 cm in thickness. The matrix structure of the dermis contains

fibroblasts, the cells which produce collagen, laminin and other components of the

connective tissue, mast cells and melanocytes (melanin producing pigments). The

upper structure of the dermis is a loose connective tissue which progresses into the

epidermal layer, while the lower part of the dermis is a coarse fibrous supporting

layer. Several structures are present in the dermal layer of skin such as vascular and

10

lymphatic vessels, nervous tissues, hair follicles, subcutaneous fat, sweat and

sebaceous glands [30, 36]. Dermis does not participate much in the barrier properties

of the skin in regards to the drug permeation.

1.2.2.4 Subcutaneous tissue: It is also termed hypodermis, which consists of an

internal fat layer. This layer acts as a support for all the skin layers above it and

contains blood vessels. Being a fat depository, hypodermis acts as a shock absorber

and heat padding. Subcutaneous tissue consists of loosely arranged adipose cells in

the form of lobules often interconnected with collagen and elastin fibers [30]. This

layer joins the skin with deeper muscles and tissues.

1.3 Enhancement of skin permeability

To overcome the limitation of the low permeability of the skin barrier, several

innovative techniques have been employed to reversibly alter the skin’s barrier

function and enhance the transepidermal delivery of drugs. These include chemical,

mechanical and electrical techniques. Use of permeation enhancers, ultrasound,

iontophoresis, electroporation, photomechanical waves, laser cell ablation and

microneedle arrays has demonstrated a reduction in the SC barrier properties

increasing the transepidermal transport of drugs. The use of ultrasound as a physical

method to enhance skin permeability and facilitate drug delivery has been explained

in detail.

11

1.4 Ultrasound therapy

1.4.1 Overview of ultrasound

Sound is generated by the propagation of mechanical energy through a medium. It is

defined as a source-generated wave that propagates away from the source in a

medium by the creation of alternating areas of compression and rarefaction of

molecules resulting in variable density vibrations Martini [37]. Ultrasound is a

longitudinal sound wave (acoustic wave) characterized by its frequency, wavelength

and amplitude. The frequency of sound waves determines their type. Out of the three

categories of sound waves; namely infrasonic, sonic and ultrasonic; the sound waves

with a frequency above 20 kHz are termed as ultrasonic waves. The ultrasonic waves

can be further divided into three regions namely, low-frequency (20–100 kHz),

therapeutic (0.7–3MHz) and high-frequency (>3MHz) ultrasound[38, 39].

When an ultrasound wave travels from the source through a medium there is a

specific velocity at which this occurs [37]. The equation which defines the

relationship between the velocity of ultrasound and its frequency and wavelength is

given by,

Where, c = speed of propagation or velocity

ν = frequency

λ = wavelength

1.4.2 Wave propagation

As the acoustic wave travels away from the source, there is a local displacement (no

net displacement) of particles as they oscillate around their equilibrium positions

12

resulting in the formation of regions of compression (high concentration of particles)

and rarefaction (low concentration of particles), as shown in figure 1.2.

Figure 1.2: Ultrasound wave propagation from left to right resulting in the formation

of regions of high (compression) and low (rarefaction) density, which correspond to

the acoustic wave amplitude; modified from [37]

1.4.3 Ultrasound characteristics [40][41]

1.4.3.1 Ultrasound frequency: It is defined by the number of events (oscillations)

which take place per unit time. It is constant for a particular ultrasound source. It can

be expressed in units of 1/second or Hertz (Hz).

1.4.3.2 Amplitude: It is the difference in the peak value and the average value of the

waveform, which depends on the strength of the wave. (Figure 1.3) It is expressed in

units of decibels or dB.

13

Figure 1.3: Diagram of amplitude of ultrasound wave.

Source: http://www.echopedia.org/wiki/The_principle_of_ultrasound

1.4.3.3 Wavelength: It is defined as the length of a single cycle, i.e. from one peak to

the next. It is dependent on the frequency of the wave and the medium of propagation.

It is expressed in units of nm.Wavelength is inversely related to the frequency.

1.4.3.4 Intensity: It is the measure of flow of acoustic energy. It is defined as a vector

quantity, which is the time average of the net flow of energy through a unit area or the

concentration of energy in the ultrasound beam. With the propagation of ultrasound

wave, the intensity decreases.

1.4.3.5 Power: It is defined as the rate of energy transfer and is measured in Watts.

The relationship between power and intensity can be expressed as,

1.4.4 Ultrasound instrumentation

Therapeutic ultrasound consists of a high-frequency generator connected to a

transducer (treatment head). The ultrasound transducer is the main component which

generates ultrasound waves using piezoelectric effect, a principle discovered by Pierre

14

and Jacques Curie in 1880 [42]. The transducer is comprised of quartz crystals (called

piezoelectric crystals or elements), either single or in combination. Some of the

commonly used piezoelectric crystals are lead zirconate titanate (PZT) or ceramic

[43]. A voltage is applied across these crystals. One side of the crystal is fixed to the

backing material (layer used to prevent excessive vibrations, thereby leading to short

pulsed ultrasonic waves), while the other is left free. When these crystals are

electrically stimulated, there are vibrations, these oscillations (shape changes or rapid

expansion and contraction) of the crystals lead to the generation of sound waves,

which travel across the medium. A schematic diagram of ultrasound equipment is

shown in figure 1.4.

Figure 1.4: Schematic diagram of ultrasound equipment

1.5 Applications of ultrasound in drug delivery

There are several applications of ultrasound in drug delivery; some of them are

mentioned below. However, the major focus of the following sections will be the

ultrasound-mediated topical drug delivery.

Skin

15

1.5.1 Topical drug delivery

During the last decade, one such physical method for skin permeabilization,

ultrasound or sonophoresis, has been investigated by researchers. Back in 1954,

application of ultrasound to deliver hydrocortisone for the treatment of polyarthritis of

the digits was reported. Ultrasound frequencies used for medicinal purposes can be

high, medium or low in the range of 20 kHz to 16 MHz and their effect on various

drugs have been assessed at different frequencies [44], [45, 46]. High ultrasound leads

to a typical ~ 10-fold enhancement in drug permeation, which might be sufficient for

local drug delivery. However, it might be insignificant for systemic delivery [47]. The

focus of ultrasound-assisted transdermal drug delivery shifted from high-frequency to

low-frequency in mid-1990, since then ultrasound frequencies between 20 – 100 kHz

have been explored extensively. Studies have reported that the transdermal delivery of

molecules is much more effective at low-frequency ultrasound as compared to higher

frequencies. A major reason for this has been attributed to the inverse nature of the

penetration depth of the ultrasound waved into the skin with the ultrasound frequency.

Low-frequency ultrasound was approved for transdermal drug delivery enhancement

of lidocaine by the US FDA in 2004. It was reported that ultrasound pretreatment

shortened the lag time of lidocaine thus decreasing the onset of therapeutic effect of

the drug from 60 minutes to 5 minutes [48].

1.5.1.1 Ultrasound biophysics

Ultrasound biophysics primarily involves the study of mechanisms which govern the

interaction of ultrasound on biological materials [49]. Ultrasound biophysics forms

the fundamental basis of understanding ultrasound therapeutic applications and its

16

associated risk assessment. The thermal and non-thermal effects, or mechanisms, of

ultrasound are the significant biological effects arising from ultrasound exposure.

1.5.1.1.1 Thermal mechanism

With the propagation of ultrasonic energy into a medium, a part of the ultrasound

wave is absorbed, that is, wave energy is converted to heat. Thus, there is an increase

in the temperature of the medium which can be expressed using several mathematical

expressions [49]. Ultrasound induced temperature increase of the medium is directly

proportional to the ultrasound application time and its intensity [50]. An increase in

the skin permeability due to the thermal effect of ultrasound can occur as a function of

increased diffusivity of drugs, dilation of the points of entry on the skin and

absorption of drugs [51]. The extent to which the temperature changes occur depends

upon the thermal effects exerted by the ultrasound. For a temperature rise of 1-2˚C, it

has been reported that there is no significant role of thermal effects [52], whereas for

larger temperature changes (~10˚C), the thermal mechanism seems to be predominant.

For high or intermediate frequency ultrasound, 1 MHz and 150 kHz respectively, the

increase in the flux of drugs across the skin might be attributed mainly to the

ultrasound-induced thermal effects [53]. In the case of low-frequency ultrasound, an

attempt has been made to control the rise in temperature of the medium as it can lead

to adverse effects such as necrosis, epidermis detachment and burns [54]. This is done

by periodic replacement of the coupling medium during ultrasound treatment. Thus,

in low-frequency ultrasound, the thermal effects do not exert any major impact. This

is evident by studies performed by Polat et al. in which sodium lauryl sulfate

permeation in the skin did not increase significantly with an increase in the

17

temperature of medium (solution) to 37˚C from 25˚C control after ultrasound

application time of 20 minutes [55]. On the other hand, if the temperature is allowed

to increase consistently for an extended time period, the thermal effects turn dominant

and are directly related to the increase in drug permeation.

1.5.1.1.2 Non-Thermal mechanism

Non-thermal effects of ultrasound include acoustic cavitation, shock waves and

acoustic streaming [50, 56].

1.5.1.1.2.1 Acoustic cavitation

The main contribution for non-thermal effects of ultrasound is acoustic cavitation

[57]. This is true especially in the case of low-frequency ultrasound. Cavitation is

characterized by ultrasound-induced pressure variation in the medium resulting in the

formation and oscillation of gas cavities or bubbles. Such bubbles are created from

pre-existing gas nuclei in the liquid or medium and as the pressure decreases, these

gas nuclei expand forming a microbubble [50]. Collapse of microbubbles transmit a

shock wave across the material or tissue leading to alterations in the tissue structure

[58] (Figure 1.5). The air pockets in tissues resemble gas nuclei for cavitation after

exposure to ultrasound. Cavitation is inversely related to the ultrasound frequency.

There are two types of cavitation; namely stable cavitation (non-inertial) and transient

(inertial) cavitation. Stable cavitation refers to the pulsation and slow oscillatory

movement of a bubble in an acoustic field without collapse. Inertial cavitation is

defined as the rapid growth and collapse of bubbles over many ultrasound pressure

cycles. This may result in the formation of smaller bubbles or due to the irregularity

of bubble collapse at the interface of skin; it forms a microjet [59] (Figure 1.6).

18

Figure 1.5: Cavitation leading to disordering of lipid bilayers and the formation of

microchannels; Modified from [60].

Inertial cavitation with bubble collapse at the interface resulting in microjets causes

skin permeation enhancement in low-frequency ultrasound. Several studies have

reported that the size of cavitation bubbles is inversely dependent on the ultrasound

frequency [56, 61]. Thus, there is smaller bubble radius for bubbles generated by

high-frequency ultrasound than low-frequency ultrasound. Also, the phenomenon of

inertial cavitation occurring in the coupling medium increases with decreasing

ultrasound frequency signifying the effectiveness of low-frequency ultrasound over

high-frequency ultrasound [62].

19

Figure 1.6: Cavity bubble collapse and formation of microjet; cavity appears spherical

initially followed by rapid shrinking of the bubble leading to microjet [58]

The process of growth of cavitation bubbles is called as rectified diffusion. The

rectified diffusion and collapse of cavitational bubbles under acoustic field together

result in a significant increase in the topical drug delivery [63]. In the first half of the

acoustic cycle, the negative pressure experienced by the bubble will cause it to grow

on account of gaseous expansion in the bubble. This is followed by a positive pressure

build up which causes the bubble to burst into smaller bubbles (figure 1.7). The

bubble growth is further enhanced due to two effects; namely the area effect and the

shell effect. In the expanded state, surface area of the bubble is greater than the

collapsed state. The shell effect is a result of the diffusion boundary layer (shell) at the

interface of gas or bubble and surrounding liquid phase [64]. As the bubble expands,

the thickness of the shell layer decreases; thereby increasing the diffusion

concentration gradient into the bubble. During bubble collapse, the shell layer

thickens and this causes the diffusion concentration gradient to decrease. With the

rapid progress of rectified diffusion resulting in a fast-paced bubble growth cycle, an

inertial cavitation can be expected depending on the ultrasound frequency, bubble size

and ultrasound amplitude. If rectified diffusion occurs slowly, it might lead to stable

cavitation owing to equilibrium attainment with continuous oscillation of the bubble

20

[59, 64]. Thus, the occurrence of rectified diffusion plays a crucial role in acoustic

cavitation, especially the inertial cavitation.

Figure 1.7: The cycle of bubble formation, growth and collapse [58]

1.5.1.1.2.1.1 Role of cavitation in low-frequency and high-frequency ultrasound

Cavitation, especially inertial cavitation, is the predominant mechanism of low-

frequency ultrasound. In a study performed by Tang et al, the selective suppression of

the effect of cavitation inside and outside the skin was demonstrated [57]. This was

done using castor oil as the coupling medium, thus suppressing the cavitation outside

the skin. Additionally, to suppress the cavitational effect inside the skin, a high-

pressure diffusion cell was used. Since there was no change in skin permeability as

compared to control, it was demonstrated that the primary mechanism of enhancing

the skin permeability was cavitation outside the skin. Studies performed by Tezel et

al, exhibited the critical distance between the ultrasound horn and skin surface of one

maximum bubble radius (to which the bubble expands) which is ~ 150 μm at a

constant frequency of 20 kHz [57, 63]. Due to this close critical distance from the skin

surface, it was confirmed that a microjet formation into or near the skin causing skin

21

perturbation is more likely for low-frequency ultrasound thus further confirming

inertial cavitation phenomenon. In case of high-frequency ultrasound, Mitragotri et al.

showed that skin permeation was increased due to cavitation within the stratum

corneum corneocytes [65]. This was attributed to direct contact of oscillating bubbles

leading to disorderly structure of lipids of stratum corneum [66].

1.5.1.1.2.2 Acoustic streaming

When cavitation bubbles are produced upon ultrasound application, the movement of

the ultrasound horn causes movement of the bubbles. This movement is induced by

convection. This is termed as acoustic streaming [50, 67]. This phenomenon causes

the movement of the bulk fluid towards the skin surface, that is, in the direction of the

applied acoustic field. It helps the small bubbles to move towards the skin and

prevents their buoyancy tendency. This movement of bulk fluid causes sufficient

mixing of the coupling medium, which in turn aids in enhancing the skin permeability

in regions where microjets due to cavitation do not occur.

1.5.1.2 Ultrasound process parameters affecting the skin permeability

1.5.1.2.1 Categories of ultrasound frequency

The high or low-frequency ultrasound can cause significant changes in skin

permeability. As the intensity of ultrasound is inversely related to its frequency, low-

frequency ultrasound has been more effective in skin permeabilization. Low-

frequency ultrasound can be categorized as simultaneous and pretreatment ultrasound

depending on the protocol of an experiment.

22

1.5.1.2.1.1 Simultaneous ultrasound: This involves application of ultrasound and

drug simultaneously to the skin [68-70]. The mechanisms resulting in an increase in

topical drug delivery are increased diffusion through conformational alterations in the

skin and ultrasound induced convection effects.

1.5.1.2.1.2 Pretreatment: In this method, ultrasound is applied for a short time prior

to drug delivery to permeabilize the skin for a couple of hours [71, 72]. An advantage

of this approach is that, no sophisticated ultrasonic device is required to be worn by

the patient.

1.5.1.2.2 Ultrasound intensity

Intensity of ultrasound is governed by the velocity of ultrasound waves and the

ultrasound energy. The effect of ultrasound on skin conductivity depends largely upon

the energy density of ultrasound. A threshold value of ultrasound energy is considered

to be in existence under which the skin conductivity or permeability enhancement

cannot be detected. It can be defined as the minimum energy required to generate

reversible structural changes in the SC layer of skin to produce a quantifiable change

in skin conductivity or permeability. This threshold value might depend on other

process parameters such as frequency [73].

1.5.1.2.3 Ultrasound application time and mode

As a certain threshold dose of ultrasound energy is required it is crucial to understand

the impact of actual ultrasound application time [74]. The total energy can be

expressed as,

Energy = (intensity) x (application time) x (duty cycle)

23

The higher the ultrasound application time, the higher will be the energy supplied.

Several studies have been performed to optimize the application time of ultrasound in

skin permeation experiments ranging from 1 minute to 4 h [46, 73, 75]. Other

parameters such as the intensity, frequency of ultrasound and thermal effects should

also be considered to optimize application time.

Ultrasound can be operated in a continuous mode or in a sequential (discontinuous or

pulsed) mode. Thermal effects are predominant in the case of continuous ultrasound

due to heating of the coupling medium. Such thermal effects may be avoided by

periodically changing the coupling medium. Pulsed ultrasound can be used to reduce

the heating effect. It refers to the actual time during which the ultrasound is activated

as it is followed by a period of no ultrasound. This effect of ultrasound duty cycle or

the mode can be assessed to determine its impact on skin permeability of drugs.

Studies performed by Mitragotri et al. revealed that the duty cycle did not

significantly affect the increase in skin conductivity values [74]. However, the drug

accumulation in the deeper skin layers increased with continuous ultrasound. In the

research conducted by Sachdeva et al. ketoprofen delivery via the transdermal route

by continuous and pulsed ultrasound application was fond to be equally effective [76].

1.5.1.2.4 Distance of the ultrasound transducer

The distance of ultrasound transducer or horn and the skin plays an important role in

the enhancement of skin permeability. As the ultrasound beam is emitted from the

transducer, it propagates in the medium. The intensity of the wave decreases

(attenuation) as the distance from the source increases [77]. This explains the

24

dependence of ultrasound on the distance of the horn and it should be optimized for

efficient ultrasound-enhanced drug skin permeation.

1.5.1.3 In vitro studies to gain an insight of ultrasound enhanced topical drug

delivery

The table 1.1 summarizes the some of the studies conducted to establish the

application of ultrasound to enhance topical drug permeation in vitro.

25

Table 1.1: Studies conducted using ultrasound to enhance the topical drug delivery

Drug/molecule Study

Model

Methods Results Reference

Fluorescein

isothiocyanate

conjugated to serum

albumin

Ex-vivo

rabbit eye

model

Ultrasound frequency 1 MHz

with an intensity of 0.05 W/cm2

and 30s exposure time

Sonophoresis was used to enhance intrascleral delivery

of the protein 1.6-fold without any toxic reactions to

ocular tissues.

[78]

Green fluorescent

nanoparticles and

hyaluronic acid

(HA)

Porcine skin Ultrasound frequency (643.5

kHz), 10 minute application

time and 5 minutes pretreatment

with ultrasound contrast agents.

With ultrasound exposure, skin permeation of the

markers was increased than passive diffusion controls.

Penetration of the markers increased by 4-5 folds and

drug penetration depth of over 600 µm in the presence

of ultrasound contrast agents.

[79]

Quantum dots Porcine split

thickness

skin

US conditions: frequency – 20

kHz, intensity – 7.5 W/cm2,

pulse length – 5 s on, 5 s off,

and distance between the probe

tip and the skin – 3 mm

0.01% of quantum dots penetrated the untreated skin,

whereas they fully permeate the ultrasound treated skin

[80]

Proteins – insulin,

and high molecular

weight compounds

such as

erythropoietin,

interferon gamma

Human

cadaver skin

Low-frequency ultrasound 20

kHz, pulses applied every

second for 4 h, 225 mW/cm2

Significantly increased delivery rates of the proteins

and their blood concentration was similar to that

achieved using a subcutaneous injection

[65]

Anti-inflammatory

drugs like

ibuprofen,

piroxicam and

diclofenac

Cellulose

and

rabbit skin

membranes

Modified Franz diffusion

Assembly was used for

permeation study. Ultrasound

intensity was varied between

0.5 to 3.0 W/cm2

There was a significant increase in the permeation of

model drugs upon ultrasound application and the

increase in ultrasound intensity further enhanced the

drug transport

[81]

26

sodium

Diclofenac

sodium

a 3D viable

skin

(EpiDerm)

Low-frequency ultrasound 20

kHz, PBS as coupling medium,

horn-to-skin-distance around 2

cm, variable amplitude,

application time

With increase in amplitude from 10% to 15%, increase

in application time from 3 to 5 minutes in continuous

mode, resulted in increased flux and permeability of

the drug

[82]

Ketoprofen Porcine ear

skin

Ultrasound parameters such as

application time duty cycle,

distance of horn, intensity were

optimized. Franz study was

carried out and permeability of

the drug was calculated.

Ultrasound at 20 kHz frequency, intensity of 6.9

W/cm2, application time of two minutes, 0.3 cm

distance between the probe tip and skin was optimized.

There was a significant increase in ketoprofen

permeability with these ultrasound parameters. An

increase in TEWL values was observed, suggesting

alterations in SC layer.

[76]

Ibuprofen cellulose

membrane

and rabbit

skin along

with in vivo

studies on

rabbits

Permeation studies were carried

out using Franz cells.

Ultrasound was applied at an

intensity of 1.5 W/cm2 with a

frequency of 800 kHz. Gels or

emulsions were applied to the

skin.

The transdermal transport was increased for gel

formulation over oleaginous or emulsion based

formulations with cellulose membrane resulting in

enhanced transport. This was attributed to the presence

of a greater alcohol concentration over propylene

glycol. Ultrasound enhanced the drug transport 11-

fold. This study concluded that aqueous gel

formulations can acts as better coupling agents to

enhance the drug permeation using ultrasound.

[83]

Fentanyl, caffeine Rat skin In vitro ultrasound pretreated

skin were mounted on Franz

cells. Histological evaluation

performed to assess the impact

of ultrasound exposure on skin.

Ultrasound applied in discontinuous mode 20 kHz with

intensity of 2.5 W/cm2 proved to be effective in

increasing permeation of both drugs.

[44]

27

1.5.2 Ultrasound in gastrointestinal drug delivery

The intracellular delivery of nuclei acids into gastrointestinal tissues (GIT) can be

very challenging due to their large size. Also, the pH microenvironment and enzymes

such as proteases and nucleases, make the transport of nuclei acids to GIT almost

impossible [84]. Thus, there is a need for local administration of biologics without the

need for structural modifications and extensive formulation steps to treat diseases like

inflammatory bowel disease like ulcerative colitis. To this effect, a physical method

such as ultrasound to enhance the local drug delivery of nuclei acids could bring a

paradigm shift in drug delivery [85]. Apart from its diagnostic applications for

imaging and tumor ablation and its facilitation of skin permeabilization, ultrasound

has been reported to have a potential to facilitate permeabilization of cellular

membranes, thereby enhancing intracellular drug delivery. This has been as a result of

reversible processes due to the cavitation effects temporarily altering cell membranes

to deliver biologics. In a study done by Schoellhammer et al., the effect of ultrasound

in the delivery of RNA to colonic mucosa of living mice and Yorkshire pigs (colon

tissues ex vivo) was studied [86]. It was found that on the application of 20 kHz

ultrasound, 1 minute application time increased the delivery of fluorescent permeants

7-fold compared to passive controls. An ultrasound of 40 kHz for 0.5 seconds caused

a 3-fold increase in drug delivery in living mice. In another such study, ultrasound

increased the absorption (around 2 to 10- fold) of inulin, hydrocortisone and

mesalamine in pig and mice model ex vivo [87]. The ultrasound treatment did not

show any toxic effect upon histological evaluation and no inflammatory response.

Thus, ultrasound mediated drug delivery can be a physical method to overcome

barrier-related problems in gastrointestinal drug delivery.

28

1.5.3 Ultrasound in gene drug delivery

Gene delivery into the body without the use of needles seems to be an innovative and

promising approach [88-90]. Several attempts have been made to efficiently and

selectively deliver genes to a target location in the body. Recently, ultrasound targeted

microbubble destruction has been employed as a potential strategy for the delivery of

genes by increasing the gene expression in a targeted manner in the body. The

ultrasound-induced oscillations of the cavitation nuclei, that is the microbubbles cause

an increase in cell membrane permeability via the formation of pores in the cell

membrane [91]. Thus, ultrasound promotes gene transfection efficiency and has been

found to be a potential method to treat ocular diseases. Gene therapy has been

investigated for treating cardiovascular diseases. However, low gene transfection

efficiency (cell death)is a limitation of this therapy. Ultrasound targeted microbubble

destruction serves as a non-invasive and non-viral method of targeted plasmid

delivery [92, 93]. The endothelial damage due to ultrasound induced microbubble

destruction further enhances targeted gene delivery to the myocardium. Studies have

shown that regional perfusion is improved by the transfection of VEGF gene into the

ischemic myocardium by ultrasound targeted microbubble destruction [94, 95]. In

another study, transfection of HSVtk or Timp3 gene targeting hepatocellular

carcinoma (HCC) resulted in the reduction of cancer cell viability by more than 40%.

With the co-transfection of both the genes, cell viability was decreased over 50%.

Using the combination of ultrasound and microbubble destruction, co-delivery of two

genes resulted in tumor suppression over 70%, synergistically improving their anti-

tumor activity [96].

29

1.6 Synergistic effects of ultrasound

1.6.1 Effect of ultrasound with other physical techniques

Ultrasound might increase the transport of drugs across the skin, but several studies

have exhibited that the effect of ultrasound can be synergistically enhanced using a

combination of ultrasound with other physical methods such as iontophoresis,

electroporation, laser ablation or microneedle array.

As the ultrasound exposure causes a decrease in skin resistivity, the iontophoretic

voltage to deliver a drug across the skin is also lower than that without ultrasound.

This in turn results in less skin irritation. Studies conducted by Hikima et al., stated

that a synergistic effect of combination of sonophoresis (3 MHz, 5.2W/cm2, 5.4%

duty cycle) and iontophoresis (0.32 ± 0.3 mA/cm2) was observed in hairless mice thus

enabling the delivery of macromolecules (molecular weight ~1350 Da) [97].

Recently, a combination therapy using microneedles and sonophoresis termed as

sonophoretic-enhanced micro-needle array (SEMA) has been employed to deliver

large molecules across the skin [98]. It consists of hollow microneedles, which pierce

the skin through the epidermis into the dermis. When ultrasound is applied, the flow

rate of drugs can occur and the microneedles can deliver the drugs across the skin. In

an attempt to deliver calcein and BSA, some researchers have pierced the skin with

100 μm microneedles and given ultrasound (20 kHz frequency, 0.5 W/cm2 intensity

and 20% duty cycles with 10 μsec on time) through the back of the microneedle

patch. They have reported a 9 times and 12 times increase in delivery of calcein and

BSA, respectively. [98] Solid microneedles that create holes on the skin have also

been used with cavitational effects of ultrasound. Through these holes created by

microneedles, cavitational effects can lead to enhanced permeation compared to

30

ultrasound alone since the cavitation bubbles are in contact with the deeper skin

layers.

Synergistic effects of ultrasound have also been examined in combination with laser

cell ablation. Erbium laser ablation was combined with ultrasound therapy (3 MHz)

and resulted in efficient enhancement of gold nanoparticles across rat skin in an in

vivo study [99].

Synergism of ultrasound and electroporation has been studied by Kost et al.[100]. In

that study, there was an increase in the flux of calcein and sulforhodamine upon the

combination of ultrasound (1 MHz) and electroporation (10 - 150 V) by a factor of 2

and 3 (respectively), relative to flux observed using only electroporation. Lag time

was also decreased from 15 minutes to 9 minutes for the combination therapy, which

suggested that the mechanism of this synergy was a cavitation-based effect.

1.6.2 Effect of ultrasound with chemical permeation enhancers

Chemical permeation enhancers solubilize skin lipids and cause denaturation or

corneocytes of the SC layer resulting in permeabilization of the skin. This effect

seems to be elevated using their combination with ultrasound. A study conducted by

Mitragotri et al., demonstrated that combination of ultrasound and 1% sodium lauryl

sulfate, a surfactant, synergistically enhanced the permeation of mannitol 200-fold

[101]. This might have been attributed to cavitation induced convection leading to

increased permeation. In a recent study, dendrimers were used as permeation

enhancers in a diclofenac gel formulation. Ultrasound 20 kHz was applied to the rat

skin using SLS as a coupling medium and in vitro diffusion study was performed. It

was found that an increase in the drug permeation from 4-fold to 16-fold resulted due

to a combination of ultrasound with dendrimers [102].

31

1.7 Conclusions

In addition to diagnostic applications, ultrasound has gained a lot of interest in the

field of drug delivery. Enhancement of topical drug delivery using physical methods

such as ultrasound or sonophoresis has been in use since the last decade. However, the

ultrasound process parameters and its applications to increase the skin permeation of

drugs for newer diseases are ever-evolving. This review summarizes the fundamentals

of ultrasound, its biophysics and its applications in drug delivery with a special

emphasis on the use of ultrasound for topical drug delivery. It describes the process

parameters of ultrasound, which play a significant role on skin permeabilization.

Cavitation mechanism of ultrasound is considered to be the main factor responsible

for the increase in skin permeation. It has now been established that inertial cavitation

on account of low-frequency ultrasound can deliver drugs into the deeper layers of the

skin such as epidermis and dermis through structural alterations in the barrier

properties of skin exerted by stratum corneum. In order to make continuous

improvement in topical delivery, combination treatments of ultrasound with other

physical or chemical methods to enhance drug permeation have been now employed.

Further investigation is required to prove the potential of ultrasound as an effective

strategy to enhance drug delivery by continued assessment in vivo.

1.8 Percutaneous absorption using Franz diffusion study

Percutaneous absorption or dermal penetration as per the Organization for Economic

Cooperation and Development (OECD) guideline 2004 a, is a measure to evaluate the

absorption or penetration of a drug into and across the skin [103]. A series of

partitioning steps must be performed for a chemical from a formulation to be

absorbed percutaneously into and across the skin. The dermal percutaneous

32

absorption primarily occurs by passive diffusion although metabolism of the drug

might occur in the deeper regions of skin. The properties of the formulation, vehicle

containing the chemical and the chemical itself (physicochemical properties such as

lipophilicity, concentration, molecular weight), all together govern the rate of

partitioning the chemical into the skin. In addition to these governing factors,

physiological factors such as integrity of skin, temperature, disease state, and blood

flow also affect the percutaneous absorption of a chemical. As the chemical is in

direct contact with the stratum corneum, it will first diffuse into this layer followed by

diffusion across the SC layer. Permeation can be attributed to a single route or a

combination of routes namely intercellular, intracellular and transappendageal

(through hair follicles and sweat ducts). Penetration of a drug through stratum

corneum by the transepidermal route is the major pathway [104].

1.8.1 Theoretical considerations of percutaneous absorption

Percutaneous absorption is governed by the process of diffusion. Diffusion is defined

as the process of transfer of individual particles of a substance by molecular motion

across the concentration gradient. As the diffusion coefficient of all the skin layers,

(except stratum corneum) is negligible, diffusion coefficient value for stratum

corneum layer is represented. Thus, the transport of a chemical across the stratum

corneum follows Fick’s first law of mass transport;

Eq. 1

Where J is flux i.e. amount of drug permeated through the skin per unit time measured

as (mass/area.time), C is concentration of the diffusing drug, x is the spatial

coordinate measured normal to section, therefore,

is the concentration gradient, D

is the diffusion coefficient.

33

The following equation can express the dermal permeability coefficient (kp) as,

Eq. 2

Or

Eq. 3

Equations 2 and 3 give the following expression for the dermal permeability

coefficient or permeability constant,

Eq. 4

Where, Cv is concentration of the drug under sink conditions, Jss is the steady-state

flux of the solute, D is average diffusion coefficient (cm2/s or cm

2/h), Km is

distribution coefficient between stratum corneum and the vehicle or formulation, and

h is the thickness of the skin.

The permeability constant (kp) can also be calculated from the following relationship;

[ ] Eq. 5

Where, Q is the amount of drug transported across the skin in time t, Co and Ci are

the concentrations of the drug on donor and receptor side of the skin respectively, and

A is area of the exposed skin in cm2. Kp is measured in cm/min or cm/h [105].

1.8.2 In vitro dermal testing

Since the middle of the twentieth century, the foundations of measuring the in vitro

diffusion of drugs across the skin has been established [106]. In vitro techniques of

percutaneous absorption have been popular owing to its advantages such as ease of

technique with quick results. As per the OECD guidance document for the conduct of

skin absorption studies (2004a), in vitro methods were reported to generate a broad

range of data for drug delivery systems into and across the skin and can serve as

34

predictive models for risk assessment due to percutaneous absorption in humans [25]

[107]. In this method, a piece of excised skin is placed in a diffusion chamber and

drug-containing formulation is applied to one side of the skin. The presence of drug is

analyzed on the other side in a collection vessel using a suitable analytical method

[25].

1.8.2.1 Franz diffusion cells

Franz diffusion cells consist of two chambers - donor and receptor chambers made up

of glass. A typical vertical Franz diffusion cell is shown in the Figure 1.8. In between

these two chambers, a skin sample is mounted. The chambers should provide a good

seal to the skin, facilitate proper mixing of receptor fluid and ensure easy sampling.

Temperature should be controlled to 32 ± 1˚C corresponding to normal skin

temperature. During the diffusion study, the receptor fluid is sampled at

predetermined time intervals and replaced with fresh receptor fluid. In the

percutaneous absorption studies, the amount of drug that has penetrated across the

skin in the receptor fluid per unit area is dependent on the applied concentration in

finite dose. This can be expressed as the percentage of the dose applied, which has

been percutaneously absorbed into the skin.

35

Figure 1.8: Vertical Franz diffusion cell

1.8.2.2 Receptor fluid

It is crucial to select a receptor fluid that has good solubility of the drug. Drug

stability in the receptor fluid should be examined. The receptor fluid should not affect

the skin integrity. The amount of drug present in the receptor fluid versus time should

be calculated for multiple time points (early time points, intermediate and at the end)

over a 24 h exposure period. The receptor fluid must be constantly stirred such that

there is no high concentration of the drug just below the skin, which could lead to a

falsely low diffusion rate and penetration.

1.8.2.3 Skin

Human skin or porcine ear skin, which closely resembles the human skin, is used for

in vitro permeation studies. The origin of skin, species, area (body site) from which

they are obtained and the storage conditions, for example wrapped in aluminum foil at

36

-20˚C should be taken into consideration and justified. Skin samples can be either

split-thickness (200-500 μm) or full- thickness (~1000 μm).

1.8.2.4 Skin integrity

Skin may be damaged during skin preparation in a percutaneous absorption study.

This would result in falsely increased drug concentration in the receptor chamber.

Hence, the assessment of integrity of the skin mounted in a Franz diffusion cell prior

to percutaneous absorption study is very important. The integrity measurement can be

performed using physical methods such as transepidermal water loss (TEWL), a

measure of natural water loss, or by measuring the electrical resistance of the skin,

using TER (Transcutaneous Electrical Resistance).

1.8.2.5 Application of the formulation

The application of the formulation on the skin affects lag time and flux depending on

the exposure area and its concentration in the donor chamber. For assessing the

steady-state flux, the amount of drug substance in a formulation that is applied onto

the skin should not be more than 10% of the drug absorbed during in vitro study.

1.8.2.6 Mass balance recovery

After the in vitro permeation study, a mass balance recovery of the applied dose

should be performed. Mass balance recovery is the sum of drug amount that did not

permeate through the skin (washed-off from the upper side of the skin using a

washing procedure after the exposure period), the amount of drug present in the

stratum corneum layer (measured using a sequential tape stripping method) [108], the

portion of drug present in the epidermis and dermis layers (determined using a heat

37

separation method) [107] and the drug amount permeated into the receptor fluid. The

mass balance recovery of a drug should be in the range of 100 ± 20% [103].

1.9 Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR)

spectroscopy and its application in characterizing skin barrier

ATR-FTIR has been established as a simple and effective method of acquiring IR

spectra of several samples which cannot be analyzed using conventional transmission

techniques [109]. This is a type of internal reflectance spectroscopy in which the

sample is place in contact with an internal reflectance element made up of zinc

selenide (ZnSe) or germanium (Ge) [110]. For the internal reflectance to occur, the

ATR crystal element should be made up of a material that has a higher refractive

index than the sample. As it is used for the surface analysis of sample, the sample

should be in direct (close) contact with the ATR crystal element since the evanescent

wave only travels 0.5 – 5 μm thickness into the sample [109].

Among the several biophysical methods to study the skin and its barrier properties

exerted by stratum corneum, ATR-FTIR spectroscopy is a promising, highly sensitive

and non-invasive method to analyze samples rapidly. As the depth of penetration

ranges to about 1 to 1.5 cell layers of stratum corneum, spectra have information

about the surface of the sample [111]. Sequential tape-stripping is recommended to

obtain information about the layers beneath [112]. Elias and Friend in 1975, described

stratum corneum as a “bricks and mortar model”, where the bricks symbolize

corneocytes of stratum corneum, while the mortar signifies the intercellular lipid

matrix made up of a mixture of SC lipids namely ceramides, cholesterol and free

fatty acids [113] (Figure 1.9). This peculiar molecular organization of SC lipids is

responsible for its barrier properties and plays a crucial role in understanding skin

permeabilization [114, 115]. The domain mosaic model suggested by Forslind

38

proposes the supramolecular organization of lipids in ordered domains (gel phase),

depicted as an orthorhombic or hexagonal phase [116]. The orthorhombic phase

represents tightly packed lipids with their alkyl chains arranged in all-trans

conformations. This phase is connected by disordered domains depicted by lipids in a

liquid crystalline phase. The lipids are loosely packed in a hexagonal phase and the

degree of disorder increases towards the liquid-crystalline phase in which lipids have

greatest rotational freedom and their alkyl chains are arranged in gauche conformation

[114, 117, 118].

Figure 1.9: Molecular organization of stratum corneum showing the ordered and

disordered lipid phases of the lipid matrix of SC layer, modified from [119]

The IR spectrum of the SC layer, which depicts the lipid conformation, is invented

from hydrophobic alkyl chains [120]. The vibrational frequencies of the IR spectrum

that provide information about the skin lipids are listed in the Table 1.2. Among these,

the carbon – hydrogen stretching vibrations in the asymmetric and symmetric modes

depicted by peaks at 2850 cm-1

and 2920 cm-1

respectively, are the primary

39

characteristic vibrational peaks describing the conformational order of SC lipids [121,

122]. Thus, the peak width and its wavenumber or position in the IR spectrum defines

the molecular organization of lipids. Ideally, a shift of vibrational frequencies to a

higher wavenumber is an indication of more fluid structure formation. An

orthorhombic pattern of lipids is indicative of a stretching band at or around 2850 cm-

1 and as the degree of fluidity of the lipid alkyl chains increases, this stretching

vibrational band shifts to a higher wavenumber (~ 2852 cm-1

– 2854 cm-1

) [114]

[123].

Table 1.2: Vibrational frequencies of the stratum corneum lipids

Functional group Absorption

maximum (cm-1

)

SC physiologic

parameter

[CH3] 2960, 2870 SC lipids

[CH2] 2920; 2850 SC lipids

[C=O] amide I ~ 1645 SC proteins

[N-H] amide II ~1545 SC proteins

[O-H] 3000-3500 SC water

1.10 Econazole nitrate

Econazole nitrate (EN) is a well-established anti-fungal agent, which is an imidazole

derivative. It has been used for the treatment of yeast infections and other skin

infections like tinea, athlete’s foot and ringworm [124]. It is highly effective against

Candida albicans [125]. EN is highly lipophilic in nature (solubility in water 1.48

μg/ml) with a logP and pKa value of 5.5 and 6.6, respectively [126, 127]. Due to these

physicochemical properties of the drug, it is challenging for the drug to penetrate

deeper layers of the skin following topical application.

There is evidence that Transient Receptor Potential Melastatin 8 (TRPM8), a TRP

proteins which is a calcium permeable non-selective cation transporter protein, is

directly activated by cold temperatures (below 26˚C). TRPM8 has been convincingly

40

demonstrated to be the main molecular transducer responsible for the sensitivity to

innocuous cold [128, 129]. The role of TRPM8 in mediating attacks of RP and the

antagonizing properties of EN were studied by our collaborators (attn: Dr. Bashar

Kahaleh, Professor, Department of Medicine and Nezam Altorok, Assistant Professor,

Department of Medicine). They studied the endothelin (ET1) response in

microvascular endothelial cells (MVEC) in response to TRPM8 activation following

menthol exposure with and without EN. With an increase in menthol concentration,

the expression of ET1 increased (compared to control) and was returned to control

levels by econazole in a concentration dependent manner proving it to be an effective

TRPM-8 antagonist in treating RP [130] (figure 2.0).

Figure 1.10: Graph showing econazole effects on menthol (MT) induced ET1 mRNA

expression in normal endothelial cells.

The currently available marketed topical formulation of EN is a 1% cream used for

topical skin infections [126]. It suffers from several drawbacks. Macrogols and liquid

paraffin form major constituents of the cream formulation in which the drug is

partially suspended. Physical phase separation of the cream has been observed due to

econazole salting effect. More than 90% of the topically applied drug remains on the

skin due to its low solubility and thus resulting in poor skin permeation [126].

41

Ensuring the delivery of EN to the vasculature beneath the stratum corneum and

epidermal layers is important for achieving a topical therapy for RP [17, 130, 131]. To

this effect, new formulations containing 3% EN (F1_topical solution, F2_HPMC

dispersion, F3_VersaBase® cream and F4_Lipoderm

® Activemax™ Cream) to ensure

better penetration of the drug through the skin for treating RP were designed and

characterized in our lab. All four formulations showed better percutaneous absorption

of EN compared to the 1% marketed cream (data yet to be published).

42

Chapter 2

Significance of research

Topical drug delivery offers several advantages over oral and parenteral

administration such as: prevents drug degradation from hostile GI microenviroment

and presystemic metabolism [26]; reduces side-effects; unlike parenteral delivery

systems there is no pain associated with topical delivery [27]; better patient

adherence; reduces the frequency of drug administration; and reduces fluctuations in

plasma drug concentrations. However, the stratum corneum acts as a protective

barrier that limits the permeation of topically applied drugs across the skin. In this

regard, newer techniques have been employed to reversibly alter the barrier properties

of the stratum corneum to allow the entry of drugs across the skin.

Current treatment options for treating Raynaud’s phenomenon (RP) only alleviate

some the disease symptoms such as numbness, pain, vasospasm of digits due to cold

or emotional stress, discoloration of skin and pallor. These may not provide relief

from all the events of RP. To date, there is no FDA approved treatment for RP. The

dihydropyridine class of calcium channel blockers has been used as the first-line

therapy. In severe cases of RP, prostacyclins such as iloprost have been prescribed via

intravenous delivery. Despite these drug treatments used for the management of RP,

43

there is very little or weak evidence to support the use of these drugs, or others such

as phosphodiesterase-5-inhibitors, selective serotonin reuptake inhibitors, alpha

adrenergic receptor blockers and topical nitrates against RP. Topical delivery seems to

be a better option to treat RP than oral or intravenous routes due to the associated

disadvantages such as side-effects, hepatic first-pass and less patient adherence due to

use of needles and the associated pain. In RP, delivery of the drug beneath the barrier

layer of stratum corneum and into the epidermal skin layers (transepidermal delivery)

to the vasculature should be the focus of topical therapy.

In a recent study conducted by our collaborators, EN has been identified as a TRPM8

antagonist in treating RP and was found to normalize the imbalance in vascular tone

control associated with endothelial cells cold exposure. The marketed formulation of

EN is a 1% cream. However, it has drawbacks such as salting out of the drug due to

phase separation and low permeability of the drug. New topical formulations of 3%

EN developed in our lab showed better permeability of the drug than the marketed

formulation.

As topical drug delivery still faces the challenge of the skin itself acting as a barrier to

the entry of drugs, the skin barrier properties could be manipulated to achieve

effective therapy. This study presents the “proof-of-concept” data to show that

ultrasound therapy could be used to alter the structure of SC to facilitate EN delivery

into the deeper skin layers. We hypothesized the use of low-frequency ultrasound

therapy to enhance the skin permeability of EN from the newly developed

formulations in our lab. The purpose of this research was to optimize the ultrasound

process parameters and assess its effect on the penetration and permeation of topically

44

applied EN across the skin. This was hypothesized by investigating the cavitation

mechanism of ultrasound, which could be useful for enhancing the drug

permeabilization across skin in vitro. Attenuated Total Reflectance – Fourier

transform infra-red (ATR- FTIR) spectroscopy was used to study the influence of

formulations with and without ultrasound pretreatment on the organization of lipids in

the stratum corneum layer. Spectral shifts of C-H stretching vibrations were studied

using ATR- FTIR spectroscopy. Skin toxicity of the formulations was assessed and

the effect of ultrasound exposure on the skin was also evaluated.

45

Chapter 3

3.1 Abstract

Objectives: The focus of this investigation was to assess the ultrasound-assisted

econazole nitrate (EN) permeation from topically applied formulations for treating

Raynaud’s phenomenon. The aims of this study were i) to identify the ultrasound

parameters optimal for the percutaneous absorption of the drug, and ii) to assess the in

vitro percutaneous absorption of EN and toxicity in the porcine ear skin from different

topical formulations following ultrasound application.

Methods: Optimization of ultrasound parameters such as the distance of the horn,

application time and amplitude were performed. In vitro percutaneous absorption

studies were performed using different econazole formulations (F1_solution,

F2_HPMC dispersion, F3_Lipoderm® Activemax™ Cream and F4_Versabase

®

cream) across ultrasound-treated porcine skin and were compared with the control

group (skin samples not treated with ultrasound). Histological evaluation using

hematoxylin and eosin stain was carried out to assess the dermal toxicity of

formulations and ultrasound exposure. To further support the findings of ultrasound-

assisted drug permeation studies, ATR-FTIR was performed to investigate the effect

46

of ultrasound on the conformational changes in the stratum corneum lipids

characterized by the changes in spectral shifts.

Results: A constant frequency (20 kHz) ultrasound application with 40% amplitude,

0.5cm distance between ultrasound horn and skin surface for 2 minutes was

optimized. The permeation of EN was found to be higher from ultrasound- treated

skin samples than the control group. Drug permeation from F2_HPMC dispersion was

found to be higher as compared to other formulations and the marketed cream. The

lag time of ultrasound- treated skin samples were found to be significantly lower than

the control. Histological evaluation confirmed that F2_HPMC dispersion showed no

signs of toxicity. ATR-FTIR studies revealed a slight increase in the -CH2- stretching

vibrations (~ 2920 cm-1

and 2850 cm-1

) in ultrasound-treated skin samples as

compared with control. This confirms the reversible conformational changes in the

stratum corneum lipids, thereby enhancing the drug permeabilization.

Conclusions: The ultrasound-assisted transdermal delivery of F2_HPMC dispersion

could be explored as a new treatment option for Raynaud’s phenomenon.

47

3.2 Introduction

Topical and transdermal drug delivery possess several advantages over conventional

and parenteral administration such as prevention of drug metabolism by

gastrointestinal (GI) lumen and hepatic first- pass [132], reduction of GI side-effects

resulting from prolonged exposure to certain drugs [133], and non-invasiveness with

no pain or discomfort associated with needles and better patient adherence [27, 134,

135]. However, a drug molecule has to overcome the barrier properties of the skin in

order to enter systemic circulation. Skin protects the body from the external

environment, against microbes, UV radiation, allergens and prevents the loss of water

from the body. Skin acts as the major rate-limiting factor to topical and transdermal

drug permeation [33]. The outermost layer of the skin, stratum corneum (SC) is ~20

microns thick and is made up of hexagonal, highly ordered and compact cells called

corneocytes that are embedded in the lipid bilayer matrix. This peculiar structure of

SC acts as a diffusional barrier [136]. Thus, the actual drug concentration that

permeates the skin is reduced. Several physical and chemical methods to overcome

the barrier properties of the skin and to enhance the drug permeation have been

employed. Such methods include ablation, microneedles, iontophoresis,

electrophoresis, sonophoresis or ultrasound – assisted transdermal delivery and the

use of chemical penetration enhancers [38, 132-134, 137-139].

Ultrasound has been widely investigated as a non-invasive method to enhance the

transdermal permeation of drugs. This technique was first used to deliver

hydrocortisone across the skin for treating digital polyarthritis in 1954 [140]. In 2004,

SonoPrep® (Sontra Medical) was approved by FDA for the transdermal delivery of

local anesthesia [48, 141, 142]. Ultrasound-assisted drug delivery has been tested for

48

its ability to enhance localized permeation of drugs such as anti-inflammatory agents

such as (dexamethasone, and cortisol), local anesthetics (such as lidocaine and

prilocaine) and drugs used in orthopaedic diagnosis [143-145]. Recently, many

macromolecules such as proteins and peptides have also been successfully

administered using ultrasound-assisted transdermal delivery [146].

Ultrasound is defined as a longitudinal sound wave, i.e., mechanical energy which is

transferred by oscillating neighboring particles from one point to the other. Ultrasonic

acoustic waves have frequency above 20 kHz. There are three frequencies into which

the ultrasound is categorized: low-frequency ultrasound (20–100 kHz), therapeutic

ultrasound (0.7–3 MHz) and high-frequency ultrasound (>3MHz) [38, 66, 147-149].

Studies have reported that low-frequency ultrasound is more effective in enhancing

the transdermal drug permeation than high-frequency ultrasound [66], [149]. The

extent to which the ultrasound waves can penetrate into the skin is inversely

proportional to the ultrasound frequency [150]. The exact mechanism by which

ultrasound enhances drug permeation is still unclear, although there have been several

reports on the mechanisms of ultrasound namely, acoustic and thermal streaming and

inertial cavitation [139]. Cavitation is characterized by bubble formation due to

pressure changes induced in the coupling medium by ultrasound [57], [151]. The

formed bubbles cause disruption of the stratum corneum lipids, thereby increasing the

skin permeabilization.

Raynaud’s phenomenon (RP) is characterized by the reversible vasospasm of the

digits induced by cold exposure or emotional stress [19]. The exact cause of RP is

undetermined and the current treatment modalities are largely unsatisfactory and

49

associated with significant side effects. Transient Receptor Potential Melastatin 8

(TRPM8) is one of the TRP proteins, which is a calcium permeable non-selective

cation transporter protein that is directly activated by cold temperatures below 26˚C.

TRPM8 has been convincingly demonstrated to be the main molecular transducer

responsible for the sensitivity to innocuous cold [128], [129]. Recently, the role for

TRPM8 in mediating attacks of RP has been identified. The role of TRPM8 in

mediating attacks of RP and the antagonizing properties of econazole nitrate (EN)

were studied by our collaborators (attn: Dr. Bashar Kahaleh, Professor, Department of

Medicine and Dr. Nezam Altorok, Assistant Professor, Department of Medicine)

[130], [124]. EN has been widely used as an anti-fungal drug for the treatment of skin

infections such as tinea, athlete’s foot, ringworm and yeast infections. It is marketed

as a 1% topical cream. Physical phase separation of the cream has been observed due

to econazole salting effect. More than 90% of the topically applied drug remains on

the skin due to its low solubility and thus resulting in poor skin permeation [124].

Ensuring the delivery of EN to the vasculature beneath the stratum corneum and

epidermal layers is important for achieving a topical therapy for RP. In this regard,

our lab has developed and characterized four formulations containing 3% EN:

F1_solution; F2_HPMC dispersion; F3_VersaBase cream®; and F4_Lipoderm

®

Activemax™ Cream for ensuring the penetration of econazole through the skin to the

dermal vasculature, where cold sensing receptors are expressed by microvascular

endothelial cells. These formulations demonstrated an increase in the permeability of

the drug compared to the marketed formulation (Figure 3.1).

50

Figure 3.1: Percentage amount of econazole nitrate percutaneously absorbed into

different skin layers of prepared formulations compared to the marketed cream

In this study, the effect of low-frequency ultrasound on the skin permeability of EN

from the previously developed formulations (F1_solution, F2_HPMC dispersion,

F3_Versabase® cream and F4_Lipoderm

® Activemax™ Cream) was evaluated across

pig ear skin. For the effective treatment of RP, it is imperative that the drug permeates

and penetrates the skin rapidly providing prompt therapeutic action. We hypothesize

that the use of ultrasound would further enhance the permeation of EN across the

skin. Optimization of the critical ultrasound parameters such as the distance of the

horn, application time and ultrasound amplitude was performed. The influence of

formulations and ultrasound exposure on the stratum corneum structure of the skin

was determined depending on the spectral shifts of the C-H stretching vibrations using

attenuated total reflectance-Fourier transform infra-red (ATR- FTIR) spectroscopy.

Further, skin toxicity assessment of formulations and ultrasound exposure was

investigated.

51

3.3 Materials and methods

3.3.1 Materials

Ecoanzole nitrate was purchased from Alfa Aesar, (Ward Hill, MA). Distilled

deionized water was used for the preparation of buffers. High Performance Liquid

Chromatography (HPLC) grade solvents such as methanol (Lot 165479) and

ammonium dihydrogen phthalate (ADP) (Lot 120935) were provided from Fisher

Scientific (Pittsburgh, PA). The ingredients used for the preparation of phosphate

buffer pH 6.8 as coupling medium and receptor fluid consisted of potassium

phosphate monobasic (Lot 122120), sodium hydroxide pellets (Lot 101166) and

sodium chloride (Lot 123819) were also obtained from Fisher Scientific (Pittsburgh,

PA). Polyethylene glycol 400 was purchased from Hampton Research (Aliso Viejo,

CA). Sodium laureth sulfate (Lot 16134) was procured from Nature’s Oil, OH. D-

Squame (D-100) standard sampling discs/strips were purchased from CuDerm

Corporation, (Dallas, TX) for sequential tape stripping. Pig ears were obtained from

Kastel's Slaughter House & Processing Center (Riga, MI).

3.3.2 Ultrasound application

Low-frequency ultrasound was applied using a sonicator horn at 20 kHz frequency

(VCX 750, Sonics and Materials, Newton, CT, USA). A donor chamber of the Franz

diffusion cells was placed over the skin sample. The stratum corneum side of the skin

was facing the sonicator horn. The distance of horn and skin was adjusted to 0.5 cm.

Phosphate buffer (pH 6.8) was used as the coupling medium for sonophoresis. The

sonicator horn was then immersed in the donor chamber and ultrasound was applied

for a specific time. After the ultrasound treatment, skin was mounted in vertical Franz

diffusion cells for percutaneous absorption studies.

52

3.3.3 Determination of ultrasound intensity

Calorimetric method, as described by Mason et al., was used to measure the

ultrasound intensity. Fifty grams of water were placed in a beaker. The beaker was

then kept in a temperature controlled water bath. At specific time intervals, the

ultrasound was activated at different duty cycles. Changes in temperature after

ultrasound application were recorded. Using the following equation, ultrasound

intensity was calculated as:

Mass of water = 50 g

Specific heat capacity of water = 4.18 J/g ˚C

= rate of change of temperature of water

3.3.4 Optimization of ultrasound parameters

Optimization of various ultrasound parameters such as application time, amplitude

and horn distance was performed. To estimate the application time, ultrasound was

activated for 0.5, 1 and 2 minutes. In order to determine the effect of distance between

the horn and skin, 0.3, 0.5 and 1 cm distances from the skin were evaluated.

3.3.5 Transepithelial water loss (TEWL) measurement

Transepithelial water loss (TEWL) readings were used as an indicator to study

changes in the skin barrier properties. A sharp increase in TEWL values are observed

if the skin barrier properties are altered. TEWL was used as an important indicator to

optimize the ultrasound parameters. TEWL was measured before and after the

53

exposure of skin samples to the ultrasound in Franz diffusion cells. Skin samples were

equilibrated for 45 minutes before taking TEWL measurements.

3.3.6 High Performance Liquid Chromatography (HPLC) analysis

Waters HPLC (Alliance e2695 separation module, Milford, MA) equipped with 2998

PDA detector and C18 (Hypersil™ ODS Thermo scientific, MA) reverse phase

column (150mm X 4.6mm, 5μm particle size) was used for the analysis of econazole

nitrate. The mobile phase, containing of methanol: ammonium dihydrogen phosphate

(ADP) (85:15), was pumped into the column at a flow rate of 1 ml/minute. An

isocratic elution method was performed. The injection volume was 10 μl and the

absorbance for econazole nitrate was measured at 230 nm. A stock solution of mg/ml

econazole nitrate was prepared in methanol. Calibration standards ranging from 0.39 -

50 μg/ml were prepared in the mobile phase. Each calibration standard was analyzed

in triplicate and the average peak area was plotted against the concentration.

3.3.7 Preparation of porcine ear skin

Porcine ears of 3 month old pigs were obtained from a slaughterhouse (Kastel’s

Slaughter House Processing Center, Riga MI). The hair on skin was removed gently

with a razor. The subcutaneous fat layer was then carefully removed. Thickness of the

skin samples was measured using a digital electronic micrometer. Samples with

thickness around 0.8 mm to 1.5 mm were used for the study. The TEWL

measurements were taken and skin samples with TEWL values less than 13-15 g/m2 h

were selected for the study.

54

3.3.8 In vitro permeability study of econazole nitrate across porcine ear skin

In vitro percutaneous absorption study of EN from the formulations was performed

using Franz diffusion cells. The Franz cell apparatus is shown in figure 3.2. Porcine

ear skin with stratum corneum side-up was clamped between the donor and receptor

chamber. A 0.9% normal saline and polyethylene glycol (PEG 400) in a ratio of 70:30

was used as the receptor fluid. A magnetic bead was used to stir the medium

continuously at 300 rpm. The temperature of the receptor medium was maintained at

34 ± 0.5˚C by the circulating water jacket. The air bubbles in the receptor chamber

were carefully removed. After equilibration, the skin samples were pre-treated with

the optimized ultrasound parameters using phosphate buffer (pH 6.8) as the coupling

medium. TEWL values were measured again for ultrasound treated skin samples. The

skin sample without ultrasound pre-treatment, served as a control. Five hundred

milligrams of the marketed econazole nitrate cream or equivalent drug-loaded EN

formulations were placed in the donor chamber. The donor chamber and the opening

of the receptor sampling port were sealed with parafilm to prevent any evaporation.

Five hundred microliters of the receptor fluid sample were collected at pre-determined

time intervals for up to 16 hours. An equal volume of fresh receptor fluid was

replaced after each sampling. The samples were analyzed using a validated HPLC

method for quantifying the drug, both permeated and penetrated.

Figure 3.2: Franz diffusion cell apparatus

55

3.3.9 Mass balance recovery

The mass balance recovery was obtained by adding the amount of drug removed from

the skin surface at the end of in vitro study (washing) with the drug adsorbed into the

stratum corneum, epidermis and dermis layers and the drug permeated through the

skin (receptor fluid concentration). Our current recovery limits ranged from 80-120%.

3.3.9.1 Washing procedure

A 5% solution of sodium lauryl ether sulfate (SLES) was prepared in distilled water.

After the in vitro skin exposure, the excess formulation was removed from the skin

surface using Q-tips and transferred into a container. This was followed by the

addition of 2 ml of the SLES solution to the donor chamber, rinsing the application

site and wiping off using Q-tips. This was repeated thrice such that a volume of 6 ml

SLES solution was used to wash off one diffusion cell. All Q-tips were returned to the

original container and 6 ml of methanol was added as the extraction solvent. The drug

was extracted by shaking the vials in a Thermoshaker for 16 hours at 1400 rpm. Then

they were sonicated for 15 minutes and centrifuged at 7000 rpm. Supernatants were

filtered through Waters EDGE 0.22 μm filter and analyzed using the HPLC method.

3.3.9.2 Sequential tape stripping

After the washing procedure, the skin surface was dried using Kimwipes. An adhesive

tape (D-Squam D100 strip) was placed on the skin surface and a constant pressure or

a weight of 150 g/cm2 was applied for 10 seconds. Then the strip was removed using

tweezers. The same procedure was repeated for the next strips. A maximum of 20

strips were used for a single diffusion cell. The strips were extracted with 6 ml

methanol and agitated on Thermoshaker for 16 hours at 1400 rpm. Then they were

56

sonicated for 15 minutes and centrifuged at 7000 rpm. Supernatants were filtered

through Waters EDGE 0.22 μm filter and analyzed using the developed HPLC

method.

3.3.9.3 Heat separation of epidermis and dermis

Upon removal of the stratum corneum layer, underlying epidermis was separated

using the heat treatment technique. The skin surface was heated using a hair dryer and

the epidermis was removed using a scalpel blade and separated from the dermis. The

epidermis and dermis were extracted with methanol (1ml) and agitated in a

Thermoshaker for 16 h at 1400 rpm. Then they were sonicated for 15 minutes and

centrifuged at 7000 rpm. Supernatants were filtered through Waters EDGE 0.22 μm

filter and analyzed using the HPLC method.

3.3.10 Skin toxicity assessment of the formulations

3.3.10.1 Histological evaluation

After 10 hours of in vitro study, the skin samples were wiped off with Kim-wipes to

remove the excess formulation. Sequential tape stripping was performed on the

samples to evaluate the impact of stripping on the skin integrity. Some samples were

kept intact as controls. The skin specimens were fixed in 10% neutral buffered

formalin for 24 hours. A ratio of 20:1 formalin to tissue was used to properly fix the

skin specimens. Then they were transferred into 70% ethanol. Skin specimens were

transferred to labeled cassettes (perforated baskets). Tissue processing was done by

immersing skin samples in a sequence of increasing concentrations (70%, 90% and

100%) of ethanol solutions. A clearing agent such as xylene was then used to displace

ethanol in skin samples. The tissue was infiltrated with molten paraffin wax at 60˚C

57

and then allowed to cool to 20˚C. Thus, paraffin blocks were obtained, which allowed

consistent sectioning of specimens. Thin sections of 5 microns were cut using a rotary

microtome (Reichert-Jung 2030 microtome, Reichert Technologies). Sections were

then floated on a water bath maintained at 40 - 45˚C, to get rid of any wrinkles and

then mounted on glass slides. The cut sections were colored by haematoxylin and

eosin. Glass slides were dried in an oven overnight at 37˚C. Sections were examined

by optical microscopy using Cytation™ 5 (Cell Imaging Multi-Mode Reader, Biotek

Instruments, Inc) at 4X and 20X magnification. Any histological changes in different

skin layers after application of formulations and the impact of ultrasound on the skin

specimens was examined. Appropriate control samples were also evaluated.

3.3.11 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

Valuable information about the molecular structure of the stratum corneum (SC), its

lipid, protein and water content, drug penetration in the SC region and its impact on

barrier function was evaluated by Fourier Transform Infrared (FTIR) spectroscopic

measurement by Attenuated Total Reflection (ATR) method. To determine the

modifications in the deeper regions of SC, ATR-FTIR is combined with the sequential

tape stripping method. After the end of the in vitro Franz study, the skin samples were

wiped off with Kim-wipe to remove the excess formulation. An adhesive tape (D-

Squam D100 strip) was placed on the skin surface and a constant pressure or a weight

of 150 g/cm2 was applied for 10 seconds. Then the strip was removed using tweezers.

The first strip was discarded due to possible surface contamination. The tape stripping

procedure was repeated while recording the spectrum of the skin sample for up to 18

strips. The skin samples, without application of formulations and without the

application of ultrasound, served as controls. All ATR-FTIR spectra were recorded

58

using Varian Excalibur Series FTIR spectrometer (Microscope mode) equipped with a

horizontal ZnSe crystal (45˚). Spectra were recorded between 4000 cm−1

and 400

cm−1

at 4 cm−1

spectral resolution and 144 scans were co-added. IR spectra of the pure

drug and the formulations were also recorded.

3.3.12 Statistical analysis

All the experiments were carried out in triplicate. The results were expressed as mean

± standard deviation (SD). The data was analyzed statistically using a Student’s t-test

and/or one way ANOVA and p values < 0.05 were considered to be statistically

significant.

3.4 Results and Discussion

3.4.1 HPLC analysis

A RP-HPLC method was developed for EN. The retention time of EN (λmax= 230 nm)

was found to be 7.4 minutes as shown in the figure 3.3.

Figure 3.3: HPLC chromatogram of econazole nitrate. Samples were analyzed using

a Waters Alliance e2695 separation module (Milford, MA) equipped with a 2998

PDA detector

2.616

2.961 3.8

51

7.471

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

59

3.4.2 Ultrasound pre-treatment

Ultrasound or sonophoresis is a non-invasive method to enhance the drug permeation

transdermally, though the exact mechanism is difficult to construe. This technique has

the potential to overcome the skin barrier properties, which are demonstrated as rate-

limiting for drug delivery, and facilitate drug delivery [38]. Ultrasound-assisted

transdermal delivery has been utilized to enhance the delivery of counter irritants,

anaesthetics and anti-inflammatory drugs [152]. Although, both high or therapeutic

frequency (1 MHz) and low-frequency ultrasound (20-100 kHz) are used in the

transdermal delivery of drugs, recent research has reported that low-frequency

ultrasound therapy is more effective compared to the high-frequency ultrasound [38,

50, 138]. This increased effectiveness of low-frequency ultrasound over therapeutic

frequency is due to inverse relation of ultrasound frequency to acoustic cavitation

bubbles [153]. The cavitation bubbles generated by high-frequency ultrasound are ~3

microns in size and cause some disorder in stratum corneum structure (stable

cavitation), while low-frequency ultrasound produced larger bubbles of ~150 microns

outside the skin at the liquid-solid interface and implode instantly, a phenomenon

called as inertial cavitation [58], [154]. This in turn results in the formation of

microjets, which enhance the permeability of the skin. Hence, in the present study, a

low-frequency (20 kHz) ultrasound was evaluated for the transdermal permeation of

econazole nitrate. A continuous mode, i.e. 100% duty cycle of ultrasound, was used.

Pulsed-ultrasound treatment may be effective; however, in accordance with the

studies conducted by Mitragotri et al. increase in skin permeability with respect to an

increase in skin conductivity values was independent of the duty cycle [74]. The

continuous ultrasound treatment increased accumulation of drug in skin. A possible

60

explanation for such an occurrence might be a rapid increase in thermal effects

associated with continuous ultrasound as compared to the pulsed [75].

3.4.3 Optimization of ultrasound parameters

3.4.3.1 Distance of horn

In sonophoresis, horn-to-skin distance is one of the important variables to achieve

efficient permeability. Several studies have been performed for both high-frequency

ultrasound and low-frequency ultrasound, ranging from zero tip displacement when

the ultrasound horn is in contact of skin (common with high-frequency ultrasound) to

1 cm in case of low-frequency ultrasound [55, 73, 82, 101, 155]. The distance of the

horn from skin was varied at 0.3 cm, 0.5 cm and 1.0 cm and its effect of drug

permeation was evaluated. At a distance of 0.3 cm from the skin, there was damage to

the skin evident by the complete removal of the epidermal layer. Hence, further

investigations on permeation profile were performed for distance of 0.5 cm and 1 cm.

It was found that as the horn-to-skin distance decreased from 1 cm to 0.5 cm, the EN

permeation increased as shown in Figure 3.4.

61

Figure 3.4: Effect of horn-to-skin distance on the permeation of econazole nitrate.

Ultrasound activated for 2 minutes, phosphate buffer pH 6.8 as coupling medium,

40% amplitude.

TEWL measurements were performed to evaluate the effect of distance on drug

permeation. TEWL values decreased as the horn-to-skin distance increased from 0.5

cm to 1 cm. The process of cavitation, a predominant mechanism of ultrasound, is

dependent on the distance between the ultrasound horn and skin surface. Cavitation

impact might be reduced when the distance is increased. This finding is in good

agreement with the study conducted by Terahara et al. which showed that the acoustic

pressure amplitude at the skin surface increased resulting in enhanced efficiency of

ultrasound as the distance between the horn and skin decreased. [73], [82] Therefore,

0.5 cm distance between the ultrasound horn and the skin surface was considered

optimum.

0

50

100

150

200

250

0 5 10 15 20

Aver

age

cum

ula

tive

am

ou

nt

of

dru

g

Time (hours)

1 cm

0.5 cm

62

Figure 3.5: Transepithelial water loss (TEWL) values for skin after ultrasound

application at a horn-to-skin distance of 0.5 cm and 1 cm as compared to the control

(non-ultrasound treated) skin sample, (mean ± SD, n = 3), P < 0.001

3.4.3.2 Application time

Effect of ultrasound application time on the drug permeation was studied. Application

times of 1 and 2 minutes at 40% amplitude, 0.5 cm horn-to-skin distance was applied

to the skin mounted in Franz cells. The change in TEWL values as a function of

ultrasound application time was examined. The TEWL values increased from 1

minute to 2 minute application times as compared to the control. Application of 0.5

minutes did not significantly increase the TEWL of skin. Several studies have been

performed with application times from 60s to 4h to study the effect on transdermal

delivery. This could be due to the presence of a threshold level of ultrasound energy

required to exert the cavitation effect. Below the threshold, ultrasound has no

significant effect on the drug permeation.

0.000

5.000

10.000

15.000

20.000

25.000

30.000

35.000

Control 0.5 cm 1.0 cm

Tra

nse

pit

hel

ial

wa

ter

loss

**

63

Figure 3.6: TEWL measurements of porcine ear skin at 1 minute and 2 minute

ultrasound application time; (mean + SD), n=3 P <0.0001

Increase in ultrasound exposure time resulting in increased drug permeation has been

reported to level off. Thus, an increase in exposure time beyond a cut-off value may

not necessarily enhance drug permeation. This can be attributed to a phenomenon

called acoustic decoupling, in which the total amount transported through the skin is

much less than what is actually produced at the ultrasound horn due to the formation

of cavitation exerted gaseous bubbles. [74] [76] The thermal effects attributed by an

increase in the temperature due to longer exposure times should be accounted for and

avoided.

3.4.4 Percutaneous absorption

Porcine skin mimics the human skin in various anatomical, physiological and

immunological aspects [156-158]. The thickness of the porcine stratum corneum and

epidermis is ~20-25 μm and 30-140 μm, respectively, while the thickness of human

skin stratum corneum and epidermis is ~20-25 μm and 50-120 μm, respectively. Thus,

0

5

10

15

20

25

30

35

Control 1 minute 2 minute

Tra

nse

pit

hel

ial w

ate

r loss

***

64

porcine skin can be considered as a good substitute for human skin in ex vivo

permeation studies [159, 160]. Several studies using porcine skin have been used to

evaluate transdermal permeation [161], topical drug delivery [162], assessment of

infectious skin diseases, skin toxicity assessment and skin immunology [163-165].

Hence, in this study, porcine skin being comparable with human skin was selected as

a model to evaluate the percutaneous absorption of EN formulations. As per the

OECD Guideline 2004 [103], for the conduct of skin penetration, in vitro method for

percutaneous absorption of a drug formulation has been widely accepted. This model

has manifold advantages over in vivo methods, as skin tissue of animals and humans

can be used equivalently; sample replicates can be taken, anticipated exposure

conditions can be mimicked, and the effect of skin toxicity on percutaneous

absorption can be examined. The choice of method, either in vitro or in vivo, depends

on the regulatory authority. In the present study, an in vitro method to measure the

penetration and permeation of EN across different skin layers was employed. An open

chamber design such as Franz diffusion cell was used as it has been reported to better

mimic the in vivo performance [166].

In this study, we attempted to determine the effect of ultrasound on the penetration

and permeation of topically applied EN across pig ear skin. Using the optimized

ultrasound parameters, i.e. 20 kHz frequency, 0.5cm distance between the ultrasound

horn and the skin sample and 40% amplitude in continuous mode, it was

demonstrated that the percutaneous absorption of the drug (amount penetrated and

permeated) into different skin layers, (namely stratum corneum, epidermis and

dermis) was almost twice compared to the control samples (skin samples which did

not receive ultrasound pre-treatment). The order of percent of drug permeated for the

65

ultrasound pre-treated skin samples from highest to lowest was F2_HPMC dispersion

(7.10% ±0.16%) > F4_Lipoderm® Activemax™ (3.15% ± 0.22%) > F1_solution

(2.49% ±0.49%) > F3_Versabase® cream (1.51% ± 0.41%) > marketed formulation

(1.16% ±0.36%). The control samples showed the percent of drug permeation in the

same order of formulation such as F2_HPMC dispersion (5.79% ± 0.5%) >

F4_Lipoderm® Activemax™ (1.63% ± 0.2%) > F1_solution (1.40% ± 0.19%) >

F3_Versabase® cream (0.92% ± 0.29%) > marketed formulation (0.67% ± 0.05%).

The amount of drug penetrated into different skin layers was almost twice in

ultrasound pre-treated skin samples as compared to the control (Fig. 3.8).

Figure 3.7: In vitro diffusion of econazole nitrate from F2_HPMC dispersion across

porcine ear skin; mean ± SEM, (n=3)

0

50

100

150

200

250

0 5 10 15 20

Cu

mu

lati

ve

am

ou

nt

of

dru

g r

elea

sed

per

un

it a

rea (

μg/s

q.c

m)

Time (hours)

F2_HPMC dispersion

control Ultrasound

66

Figure 3.8: Percentage of econazole nitrate percutaneously absorbed into different

skin layers from F2_HPMC dispersion; Values expressed as mean, (n=3), *p < 0.05

Figure 3.9: In vitro diffusion of econazole nitrate from F4_Lipoderm® Activemax™

cream across porcine ear skin; mean ± SEM, (n=3)

0

0.5

1

1.5

2

2.5

3

SC Epidermis Dermis

Per

cen

t ec

on

azo

le n

itra

te p

erm

eate

d i

nto

dif

fere

nt

skin

la

yer

s

F2_HPMC Dispersion

Control

Ultrasound

*

*

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20

Cu

mu

lati

ve

am

ou

nt

of

dru

g r

elea

sed

per

un

it a

rea (

μg/s

q.c

m)

Time in hours

F4_Lipoderm® Activemax™

Control Ultrasound

67

Figure 3.10: Percentage of econazole nitrate percutaneously absorbed into different

skin layers from F4_Lipoderm® Activemax™ cream; Values expressed as mean,

(n=3), *p < 0.05

Figure 3.11: In vitro diffusion of econazole nitrate from F1_Solution across porcine

ear skin; mean ± SEM, (n=3)

0

1

2

3

4

5

6

7

8

SC Epidermis Dermis

Per

cen

t ec

on

azo

le n

itra

te p

erm

eate

d w

ith

dif

fere

nt

skin

la

yer

s

F4_Lipoderm® Activemax™

Control Ultrasound

0

10

20

30

40

50

60

70

80

90

0 5 10 15 20

Cu

mu

lati

ve

am

ou

nt

of

dru

g r

elea

sed

per

un

it a

rea (

μg/s

q.c

m)

Time in hours

F1_Solution

Control Ultrasound

68

Figure 3.12: Percentage of econazole nitrate percutaneously absorbed into different

skin layers from F1_Solution; Values expressed as mean, (n=3), * p<0.05,

*** p<0.001

Figure 3.13: In vitro diffusion of econazole nitrate from F3_Versabase® cream across

porcine ear skin; mean ± SEM, (n=3)

0

1

2

3

4

5

6

SC Epidermis DermisPer

cen

t ec

on

azo

le n

itra

te p

erm

eate

d i

nto

dif

fere

nt

skin

la

yer

s

F1_Solution

Control Ultrasound

***

*

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20

Cu

mu

lati

ve

am

ou

nt

of

dru

g r

elea

sed

per

un

it a

rea (

μg/s

q.c

m)

Time in hours

F3_Versabase® cream

Control Ultrasound

69

Figure 3.14: Percentage of econazole nitrate percutaneously absorbed into different

skin layers from F3_Versabase® cream; Values expressed as mean, (n=3), *p < 0.05

Figure 3.15: In vitro diffusion of econazole nitrate from 1% marketed cream across

porcine ear skin; mean ± SEM, (n=3)

0.00

1.00

2.00

3.00

SC Epidermis Dermis

Per

cen

t ec

on

azo

le n

itra

te p

erm

eate

d

wit

hin

dif

fere

nt

skin

la

yer

s

F3_Versabase® cream

Control Ultrasound

*

0

5

10

15

20

25

30

35

40

45

0 5 10 15 20

Cu

mu

lati

ve

am

ou

nt

of

dru

g r

elea

sed

per

un

it t

ime

(μg/s

q.c

m)

Time in hours

Marketed formulation

Control Ultrasound

70

Figure 3.16: Percentage of econazole nitrate percutaneously absorbed into different

skin layers from 1% marketed cream; Values expressed as mean, (n=3), *p < 0.05

The mass balance recovery was within the acceptable range of 100 ± 20% (Table 3.2).

Permeability values of the drug from the formulations were found to be 2.57 x 10-3

,

6.87 x 10-4

, 5.52 x 10-4

, 3.79 x 10-4

and 2.53 x 10-4

cm/hour, respectively (Table 3.1)

for F2_HPMC dispersion, F4_Lipoderm® Activemax™, F1_solution, F3_Versabase

®

cream and marketed formulation for the control group. There was an increase in the

permeability value of the drug from the formulations for ultrasound pre-treated skin

samples: 3.3 x 10-3

(F2_HPMC dispersion), 9.61 x 10-4

(F4_Lipoderm®

Activemax™), 9.15 x 10-4

(F1_solution), 6.61 x 10-4

(F3_Versabase® cream) and 2.59

x 10-4

(marketed formulation), respectively. There was a decrease in the lag time

(from 4 hours to about 1 hour) for ultrasound treated skin samples for all formulations

as compared to the control group.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

SC Epidermis Dermis

Per

cen

t ec

on

azo

le n

itra

te p

erm

eate

d

wit

hin

dif

fere

nt

skin

la

yer

s

Marketed formulation

Control Ultrasound

*

71

Table 3.1: Apparent permeability (Papp), flux of econazole nitrate across excised porcine skin and lag time. Values are expressed as mean ±

SEM, n=3.

Control Ultrasound

Formulation Papp (cm/h)

Flux

(µg/cm2/h)

Lag time

(hours) Papp (cm/h)

Flux

(µg/cm2/h)

Lag time

(hours)

F2_HPMC Dispersion 2.57 x 10-3

12.858 ± 1.01 3.7 ± 0.15 3.3 x 10-3

16.49 ± 1.12 1.1 ± 0.22

F3_Lipoderm 6.87 x 10-4

3.434 ± 2.44 4.0 ± 0.00 9.61 x 10-4

4.80 ± 4.14 1.0 ± 0.00

FI_solution 5.52 x 10-4

2.760 ± 2.98 4.0 ± 0.00 9.15 x 10-4

4.57 ± 4.76 0.3±0.00

F4--Versabase cream 3.79 x 10-4

1.89 ± 0.26 7.0± 2.00 6.61 X 10-4

3.30 ± 0.45 2.7 ± 1.15

Marketed formulation 2.53 x 10-4

1.262 ± 0.08 4.0 ± 0.03 2.59 x 10-4

1.29 ± 0.25 1.0 ± 0.19

72

Table 3.2: Total absorption and mass balance results (as a percentage of the applied dose) of econazole nitrate across porcine ear skin.

Percutaneous absorption and permeation values from different formulations on control group and after ultrasound treatment, into and through the

intact porcine ear skin over 16 hours from a single application. Values expressed as mean ± standard deviation

Control (without ultrasound treatment)

Percent unpermeated drug

Formulation Stratum

Corneum Epidermis Dermis Washing

Percent drug

permeated Mass balance (%)

F2_HPMC Dispersion 1.08 ± 0.45 0.22 ± 0.11 1.10 ± 0.45 89.98 ± 1.12 5.80 ± 0.50 96.19 ± 4.72

F4_Lipoderm®

0.78 ± 0.05 0.25 ± 0.09 0.95 ± 0.14 82.81 ± 3.67 1.63 ± 0.18 86.42 ± 4.02

F1_solution 0.80 ± 0.53 0.31 ± 0.02 0.91 ± 0.04 83.45 ± 0.92 1.40 ± 0.19 86.87 ± 1.51

F3_Versabase® cream 0.25 ± 0.14 0.32 ± 0.27 0.77 ± 0.50 86.31 ± 3.64 0.92 ± 0.29 88.56 ± 4.63

Marketed formulation 0.08 ± 0.02 0.02 ± 0.00 0.16 ± 0.05 87.78 ± 4.68 0.67 ± 0.06 88.70 ± 4.71

After ultrasound treatment

Percent unpermeated drug

Formulation Stratum

Corneum Epidermis Dermis Washing

Percent drug

permeated Mass balance (%)

F2_HPMC Dispersion 1.56 ± 0.27 0.99 ± 0.46 2.32 ± 0.19 89.25 ± 2.61 7.10 ± 0.16 101.2 ± 2.65

F4_Lipoderm®

0.42 ± 0.52 0.32 ± 0.05 1.91 ± 0.35 89.76 ± 1.13 3.15 ± 0.22 95.56 ± 1.55

F1_solution 1.63 ± 0.22 0.60 ± 0.10 1.76 ± 0.04 87.29 ± 6.29 2.49 ± 0.50 93.76 ± 6.74

F3_Versabase® cream 0.64 ± 0.37 0.92 ± 0.22 1.83 ± 1.00 87.00 ± 6.53 1.51 ± 0.41 91.90 ± 6.62

Marketed formulation 0.17 ± 0.05 0.06 ± 0.02 0.33± 0.11 89.87 ± 6.76 1.16 ± 0.36 91.59 ± 6.83

73

This increase in the drug permeation can be attributed to the effect of ultrasound on

the skin. Low-frequency ultrasound therapy is known to enhance the drug permeation

via thermal effects, convection, and radiation forces and acoustic cavitation [57, 60,

151]. Acoustic cavitation phenomenon involves the formation of oscillating bubbles

in the coupling medium in an acoustic field. This bubble dynamics leads to

unidirectional flow of fluid, term named as microstreaming [167]. This, in turn gives

rise to increased shear stress and stretching on the skin surface resulting in channel

activation. This facilitates the drug permeation across the skin layers.

3.4.5. Skin toxicity assessment of econazole nitrate formulations

3.4.5.1 Histological evaluation

The skin toxicity of EN formulations was assessed in both intact skin and tape-

stripped skin. Tape-stripping was performed to evaluate the impact of tape removal on

the skin morphology. As shown in the figure 3.17, control intact skin specimen

showed a distinct, densely packed stratum corneum with 15-20 cell layers of

corneocytes. This was followed by the epidermis layer consisting of stratified-

squamous epithelium. Dermal papillae, folding of the basal layers of epithelium, were

evident. The dermis layer, consisting of thicker connective tissue, was evident below

the epidermis. In the control tape-stripped skin specimen, removal of stratum corneum

was evident (Figure 3.17 A and B). Ultrasound pre-treated intact skin sample showed

loosely attached stratum corneum, whereas tape-stripped samples showed the removal

of the stratum corneum layer (Figure 3.17 C and D). This suggested that ultrasound

had an impact on the structural conformation of stratum corneum. Ultrasound results

in loosening of the stratum corneum and temporary disruption of the lipid bilayers of

74

the stratum corneum [63, 168, 169]. The lipid bilayers of stratum corneum are

susceptible to conformational changes such as reduction in thickness and lateral

extension [63, 170, 171]. This is demonstrated by a significant reduction in the highly

ordered structure of the lipid bilayers, leading to a loss in their structural integrity.

Figure 3.17: Hematoxylin and eosin staining of the control samples; 4X

magnification; A) untreated intact; B) untreated tape stripped; C) ultrasound-treated

intact; D) ultrasound-treated tape stripped

This phenomenon facilitates drug penetration and permeability across the skin. Thus,

there is a significant increase in the skin permeability of the drug in ultrasound-

pretreated samples as compared to the control. As shown in the figure 3.17, there was

minimal damage of the epidermis with no necrosis due to the application of

ultrasound. The skin specimens, on which F4_Lipoderm® Activemax™ cream was

75

applied (figure 3.18), showed signs of skin toxicity with the presence of infiltration of

cells and necrosis.

The histology study of the formulations suggested that the F2_HPMC dispersion

formulation did not show any signs of dermal toxicity, with and without the

application of ultrasound, indicating the absence of any skin irritation or inflammatory

reaction of the formulation on the skin (Figure 3.19). Marketed formulation showed

toxic reaction on the skin samples pre-treated with ultrasound. There was a significant

inflammation characterized by erythema and infiltration of cells, mostly neutrophils,

and necrosis.

Figure 3.18: Hematoxylin and eosin staining of skin samples treated with

F4_Lipoderm® Activemax™; 4X magnification, Inset: signs of dermal toxicity, 20X

magnification; A) untreated intact; B) untreated tape stripped; C) ultrasound-treated

intact; D) ultrasound-treated tape stripped

76

Figure 3.19: Hematoxylin and eosin staining of skin samples treated with F2_HPMC

dispersion; 4X magnification; A) untreated intact; B) untreated tape stripped; C)

ultrasound-treated intact; D) ultrasound-treated tape stripped

In all of the ultrasound pre-treated skin samples, modification in the stratum corneum

layer and epidermis was evident due to the presence of voids and empty spaces. This

could be attributed to the extraction of stratum corneum lipids resulting in the loss of

moisture from the skin. This, in turn, provided evidence in support of in vitro

percutaneous skin absorption data and the literature previously reported.

77

Figure 3.20: Hematoxylin and eosin staining of skin samples treated with 1%

marketed cream formulation; 4X magnification; A) untreated intact; B) untreated tape

stripped; C) ultrasound-treated intact; D) ultrasound-treated tape stripped

3.4.6 Attenuated Total Reflectance Fourier Transform Infrared spectroscopy

FT-IR is a unique tool used to identify changes in spatial organization of lipid

components in the stratum corneum, namely ceramides, free fatty acids and

cholesterol, which primarily act as the barrier of the skin [114, 119, 172]. In this

study, ATR-FTIR was used to examine the changes in the lipid organizational

structure within the stratum corneum due to the different formulations. This was

correlated with the percutaneous absorption data. The effect of ultrasound on the

modifications of stratum corneum lipids was also studied. Different vibrational

frequencies are studied for the investigation of the barrier properties of skin lipids. In

this study, we looked at the symmetric and asymmetric stretching vibrations of

stratum corneum lipids. Such characteristic frequencies are: [114]

78

Table 3.3: Some important vibrational frequencies depicting stratum corneum layer

Frequency Absorption maximum (cm-1

) Physiologic parameter

-CH2- symmetric stretching 2847-2855 cm-1

Stratum corneum lipids

-CH2- asymmetric stretching 2915- 2924 cm-1

Figure A and B show the IR overlay of spectra of skin samples with and without

ultrasound treatment along with their respective controls. The values for asymmetric

and symmetric CH2 vibrations are mentioned in the Table 3.3.

Table 3.4: -CH2- asymmetric and symmetric stretching vibration frequencies of both,

control and ultrasound treated skin samples

Formulation Without ultrasound With ultrasound

Asymmetric

(cm-1

) Symmetric

(cm-1

) Asymmetric

(cm-1

) Symmetric

(cm-1

)

Control 2914.43 2844.99 2916.4 2848.85

F2_HPMC Dispersion 2916.36 2848.85 2920.22 2850.78

F4_Lipoderm®

Activemax™ 2916.36 2848.85 2918.22 2850.78

Marketed formulation 2914.43 2848.85 2916.36 2850.78

79

Figure 3.21: Representative ATR-FTIR spectra of porcine ear skin treated with

marketed formulation, F4_Lipoderm® Activemax™, F2_HPMC dispersion compared

with skin as such (control). Inset: shift in -CH2- stretching vibrations around 2920cm-1

and 2850cm-1

upon application of different formulations.

Figure 3.22: Representative ATR-FTIR spectra of porcine ear skin after ultrasound

application and treatment with marketed formulation, F4_Lipoderm® Activemax™,

F2_HPMC dispersion compared with skin as such (ultrasound- treated control). Inset:

shift in CH2 stretching vibrations around 2920 cm -1

and 2850 cm -1

upon application

of different formulations.

80

In both cases, there was a change in the asymmetric vibrations represented by changes

in wavenumbers (shift to higher wavenumbers) for formulations than the respective

control. All the formulations also showed a shift in symmetric stretching vibration

towards greater frequencies from 2848cm-1

to 2850cm-1

as compared to control. The

order of increase in the wavenumbers is F2_HPMC dispersion > F4_Lipoderm®

Activemax™ > marketed formulation > control. This slight increase in the vibrational

frequencies is attributed to penetration enhancers used in the formulations. The

penetration enhancers cause increased rotational motions along the alkyl lipid chains

of stratum corneum, thus, leading to a change in the alkyl lipid chains from a highly

ordered trans state (orthorhombic and/or hexagonal chain conformation) to a gauche

isomerized form characterized by loss of lateral chain packing and increase in fluidity

of the chains [114, 173]. There was a slightly greater shift in wavenumbers of the

ultrasound treated skin samples as compared to non-treated ones. This suggests that

exposure to ultrasound impacted the conformation of the stratum corneum lipids;

however, no major permanent changes were observed. Nanavaty et al. and Kost et al.

studied the effect of ultrasound on permeability of drug by inducing structural

changes on the lipoidal configuration of stratum corneum. They found that there are

no major changes related to morphology of stratum corneum layer caused by the

ultrasound [25]. Thus, this data is in good agreement with the literature findings and

in vitro permeation results.

3.5 Conclusions

In the present study, we evaluated the ultrasound-assisted transdermal delivery of EN

from different topical formulations. The ultrasound parameters such as amplitude,

application time and distance of the horn were optimized and used for the in vitro

81

percutaneous absorption study across the porcine ear skin. A constant frequency (20

kHz) ultrasound application with 40% amplitude, 0.5 cm distance between ultrasound

horn and skin surface for 2 minutes were considered optimum. Percutaneous

absorption study confirmed that all four formulations exhibit better EN absorption

compared to the marketed formulation upon ultrasound application. After ultrasound

treatment, the formulations showed a significant increase in the drug permeation. An

increase in drug permeation was also evident in the skin layers compared to the

control (without ultrasound application). Permeability studies indicated higher

penetration of the drug from the F2_HPMC dispersion followed by F4_Lipoderm®

Activemax™, F1_solution, F3_Versabase® cream and marketed formulation. Dermal

skin toxicity assessment using histological evaluation confirmed that the F2_HPMC

dispersion showed no signs of toxicity with or without ultrasound application. On the

other hand, F4_Lipoderm® Activemax™ formulation and marketed cream showed

some amount of dermal toxicity upon ultrasound application. ATR-FTIR studies

clearly indicated that there was a shift towards the higher wavenumbers of skin

samples treated with ultrasound than control samples, demonstrating the

conformational changes in stratum corneum lipids after ultrasound application. From

the results obtained in the drug permeation study, dermal toxicity assessment and

ATR-FTIR spectroscopy, it is clearly evident that the F2_HPMC dispersion is a better

formulation than the marketed formulation and that ultrasound-assisted topical

delivery of EN could be explored as a new treatment modality for RP. A better insight

of ultrasound and its impact on the drug permeation could be found by performing

permeation studies on excised human skin or by using a surfactant or an ultrasound

contrast agent. Impact of ultrasound application should be further assessed in vivo.

82

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