Formulation and Evaluation of Dermatological Products For

Home , Sertaconazole

FORMULATION AND EVALUATION OF DERMATOLOGICAL PRODUCTS FOR

TOPICAL DELIVERY

MAHIMA MANIAN

Mahima Manian

FORMULATION AND EVALUATION OF DERMATOLOGICAL PRODUCTS FOR

TOPICAL DELIVERY

MAHIMA MANIAN

Approved:

Ajay. K. Banga, Ph.D. Date Dissertation Committee Chair

Martin. J. D’Souza, Ph.D. Date Dissertation Committee Member

Grady. J. Strom, Ph.D. Date Dissertation Committee Member

Ayyappa Chaturvedula, Ph.D. Date Dissertation Committee Member

Piyush Jain, Ph.D. Date Dissertation Committee Member

Hewitt. W. Matthews, Ph.D. Date Dean, College of Pharmacy

DEDICATION

This dissertation is dedicated to my better half Sai for his unconditional love and encouragement, to my family, who have incessantly motivated me to pursue my dreams and to my besties for their constant love and support.

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ACKNOWLEDGEMENTS

Thank you God for everything!

This journey would have been incomplete without the guidance and friendship of

Dr.Ajay.K.Banga. He will always be the ‘bestest’ advisor, who continues to inspire and motivate me to be a better human being and scientist.

I would like to thank my committee members, Drs. D’Souza, Strom,

Chaturvedula and Jain for their guidance and helpful discussions. I would also like to thank the members of the Department of Pharmaceutical Sciences for providing the required technical and professional assistance throughout my Ph.D. for which I am truly grateful.

A big shout-out to my lab colleagues and friends, you have been my second family in Atlanta, our talks and laughter will always remain unforgettable.

iv Table of Contents

CHAPTER 1 ...... 1

INTRODUCTION ...... 1

CHAPTER 2 ...... 6

REVIEW OF LITERATURE ...... 6

Background ...... 6

Factors influencing percutaneous absorption: ...... 7

Vehicle effects ...... 8

In vitro permeation and drug distribution ...... 9

Modulation and enhancement of topical drug delivery ...... 11

Advantages of topical and transdermal drug delivery ...... 12

CHAPTER 3 ...... 14

INVESTIGATION OF THE DERMAL ABSORPTION AND IRRITATION POTENTIAL

OF SERTACONAZOLE NITRATE ANHYDROUS GEL ...... 14

Abstract ...... 14

Introduction ...... 15

Materials and Methods ...... 17

Results ...... 24

Discussion ...... 30

Conclusion ...... 34

CHAPTER 4 ...... 36

EVALUATION OF THE IN VITRO PERCUTANEOUS ABSORPTION AND

RHEOLOGICAL BEHAVIOR OF TOPICAL MENTHOL FORMULATIONS ...... 36

Abstract ...... 36 v Table of Contents (continued)

Introduction ...... 37

Materials and Methods ...... 40

Results ...... 46

Discussion ...... 54

Conclusion ...... 59

CHAPTER 5 ...... 60

EFFECT OF DIFFERENT VEHICLES ON THE IN VITRO RELEASE OF DICLOFENAC

SODIUM ...... 60

Abstract: ...... 60

Introduction ...... 61

Material and Methods ...... 63

Results ...... 68

Discussion ...... 79

Conclusion ...... 81

CHAPTER 6 ...... 82

FORMULATION AND EVALUATION OF A POSITIVELY CHARGED

NANOEMULSION FOR THE TOPICAL DELIVERY OF MINOXIDIL ...... 82

Abstract ...... 82

Introduction ...... 83

Materials and Methods ...... 85

Results ...... 90

Discussion ...... 96

Table of Contents (continued)

Conclusion ...... 100

CHAPTER 7 ...... 101

SUMMARY AND CONCLUSION ...... 101

REFERENCES ...... 105

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LIST OF TABLES

Table 1. Anhydrous gel composition

Table 2. Details of TS experiment and parameter estimation

Table 3. Composition (% w/w) of menthol in different vehicles

Table 4. Composition (% w/w) of menthol gels containing different percentages of carbomer, propylene glycol and ethanol

Table 5. Average damping factor values for menthol gel formulations

Table 6. Composition of different semi-solid formulations containing 1% diclofenac sodium

Table 7. Commercially available membranes and their dimensions

Table 8. Recovery of DS from standard solutions after passing through different membranes

Table 9. Comparison of flux values for different batches having the same composition

Table 10. Flux values for inter-day and intra-day reproducibility

Table 11. Comparison of flux values for different dosage strengths of all formulations containing DS

Table 12. Composition of the o/w nanoemulsion formulations containing minoxidil

Table 13. MNX permeation and skin uptake after 24h of passive diffusion using control solution or F9 NE

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LIST OF FIGURES

Figure 1.Schematic representation of the concentration-depth profile during the tape stripping experiment. Shaded region (i.e., stratum corneum) was stripped while drug concentration left in remaining stripped skin was recorded at end of experiment

Figure 2.Amount of SN in tape strips and stripped skin following unsteady-state conditions (5 min)

Figure 3.Amount of SN in tape strips and stripped skin following steady-state conditions (24 h)

Figure 4. (a) Average SN concentration-depth profile following application of the formulation for 5 min and 24 h; (b) Sample fit for application of formulation for 24 h: markers are observed data and solid line is fit; (c) Sample fit for application of formulation for 5 min: markers are observed data and solid line is fit.

Figure 5.Simulated SN concentration across the SC plotted along with MIC100 of M. furfur and yeast

Figure 6. Skin irritation testing showed anhydrous gel to be non-irritant

Figure 7.Comparison of the cumulative permeation of menthol through human skin in vitro following application of infinite dose of menthol in propylene glycol and ethanol (Mean ± SD, n=4)

Figure 8.Permeation profile of menthol through human skin in vitro following application of finite dose of menthol in propylene glycol: ethanol vehicle (Mean ± SD, n=4)

Figure 9. Skin penetration of menthol solution into epidermis and dermis at the end of 8 hours (Mean ± SD, n=4)

Figure 10. Flow curve testing of menthol gel formulations

Figure 11:Frequency sweep testing of menthol gel formulations

Figure 12.Thixotropy test using menthol gel formulations

Figure 13.Comparison of permeation profiles across human skin in vitro following application of finite dose of F4 and F7 (Mean ± SD, n=4)

Figure 14.Comparison of amount in epidermis following application of finite dose of F4 and F7 at 1h, 2h, 4h and 8h (Mean ± SD, n=4)

Figure 15.Comparison of amount in dermis following application of finite dose of F4 and F7 at 1h, 2h, 4h and 8h (Mean ± SD, n=4)

Figure 16:Microscopic observation of (a) Gel, (b) Emulgel, (c) Emulsion and (d) Ointment

Figure 17. Change in viscosity of different formulations with increasing shear rate

Figure 18. Frequency sweep test for different semi-solid formulations

Figure 19. In vitro release comparison of different semi-solid formulations, *** indicates p< 0.01 and statistically significant in comparison to all other formulations, ** indicates p< 0.01 and statistically significant in comparison to emulsion and ointment, * indicates p< 0.01 and statistically significant in comparison to ointment

Figure 20. Inter-day reproducibility for all semi-solid formulations. Data shown are mean (±SD) of 6 replicates.

Figure 21. Intra-day reproducibility for all semi-solid formulations. Data shown are mean (±SD) of 6 replicates.

Figure 22. Effect of dosage strength on release rate for all formulations. Data shown are mean (±SD) of 6 replicates

Figure 23. Particle size of NE formulations during storage for 60 days

Figure 24.Zeta potential of NE formulations during storage for 60 days

Figure 25. TEM micrograph of F9 NE showing spherical droplets

Figure 26. Percutaneous absorption and MNX recovery from SC tape strips and follicular casts following passive delivery and iontophoresis for 4h. Data shown are the mean (± SD) of four replicates. * indicates F9 delivered a significantly higher amount passively while ** indicates a significantly higher delivery using iontophoresis

Figure 27. MNX levels in SC (top) and hair follicles (bottom) as a function of time post 4h of iontophoresis. Data shown are the mean (± SD) of four replicates. * indicates a significantly higher amount of drug was delivered using the F9 formulation

ABSTRACT

FORMULATION AND EVALUATION OF DERMATOLOGICAL

PRODUCTS FOR TOPICAL DELIVERY

Under the direction of Dr. Ajay.K.Banga, Professor and Chair of the Department of Pharmaceutical Sciences, Mercer University

The use of topical route for drug delivery relies on the formulation of effective dermatological products, which maximize drug availability at the target site with minimal adverse effects. In this study, the first aim was to formulate an anhydrous gel formulation containing sertaconazole nitrate for the treatment of cutaneous fungal infections. In vitro techniques like tape stripping and cell culture model was used to evaluate the topical absorption and irritation potential of the formulation. The drug concentration-stratum corneum depth profile showed that the formulation attained sufficient levels to treat any potential fungal infections and was non-irritant in nature. In case of analgesics like menthol, many over-the

xii

counter-products are easily available in the market. However, there is limited information available on the topical absorption of this compound using human skin which could also be affected by drug-vehicle interactions. Hence, the second aim used in vitro permeation and rheological analysis of different vehicles in order to evaluate the skin absorption of menthol. Skin distribution studies showed that the drug was able to penetrate in sufficient concentrations while rheological characterization helped to understand the structural stability of the topical formulations. In addition to permeation, in vitro release testing has been used frequently as an important tool in the development and formulation of topical vehicles. The third aim involved formulation of different semi-solid dermatological vehicles and evaluation of their rheological properties and in vitro release by using diclofenac sodium as the active ingredient. The formulations were found to be easily spreadable and predominantly viscoelastic in nature. The release test was able to discriminate between the different semi-solid formulations and found to be accurate and reproducible. The fourth aim involved the formulation of a positively charged nanoemulsion which could be potentially used to enhance the topical delivery of minoxidil. This study also used an active enhancement technique like iontophoresis to evaluate its effect on drug delivery.

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Differential tape stripping showed that the formation in combination with iontophoresis effectively enhanced the topical absorption of minoxidil with reduced systemic uptake. Thus, using in vitro techniques effectively helps in the optimization of topical formulations with enhanced efficacy and safety.

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CHAPTER 1

INTRODUCTION

The skin is the largest organ of the integumentary system and plays an integral role in providing a protective barrier from the external environment as well as regulating body temperature in humans. As a result, the skin is most susceptible to physical and environmental stressors. In addition, autoimmune disorders, drug- induced hypersensitivity and other conditions may contribute to skin diseases and disorders which can be potentially treated by using topical formulations.

Depending on the physicochemical properties of the active ingredient, the target site and formulation strategies used, the drug can be incorporated in different topical formulations in order to exert its activity on the surface tissue layer, via penetration into the deeper layers of skin to reach the target site or via systemic delivery (Chang, Raw, Lionberger, & Yu, 2013). Irrespective of the site of action, any drug must pass the stratum corneum (SC) which is the main barrier for topical administration in order to exert its activity. In such cases, the vehicle used in formulation of the dermatological product can have a significant effect on the penetration of the active ingredient by altering the barrier properties of the skin.

Hence, the main aim of our study was to prepare formulations containing different active ingredients, evaluate the in vitro topical absorption and assess their suitability in the potential treatment of various topical disorders.

For azole antifungals such as sertaconazole nitrate (SN), an anhydrous topical formulation is generally preferred (B. Elewski, Ling, & Phillips, 2006) since the higher solubility of the drug in vehicle may result in higher thermodynamic activity and thus greater partitioning into the skin via the SC. Information about the dermatopharmacokinetic (DPK) profile of this topical formulation would help to assess the local bioavailability of the drug as well as provide a rationale for therapy. Tape stripping is one such relatively noninvasive method which has been used in estimating the diffusivity and partition coefficients from dermal exposures

(Jakasa, Verberk, Esposito, Bos, & Kezic, 2007; Wagner, Kostka, Lehr, &

Schaefer, 2002). These dermal absorption parameters can be used to quantify drug uptake in the SC and thus absorption rate following topical application. Hence, one goal of this study was to evaluate the in vitro topical absorption and irritation of sertaconazole nitrate anhydrous gel.

In case of topical analgesics like menthol, a survey of the scientific literature reveals limited information regarding its percutaneous absorption in human skin in spite of the fact that literally hundreds of over-the- counter topical dosage forms contain menthol. In concentrations of 1% or less, menthol depresses the

cutaneous sensory receptors while at concentrations between 1.25-16%, it stimulates the sensory receptors and exerts the counter-irritant effect (Patel,

Ishiuji, & Yosipovitch, 2007). In this case, in vitro permeation studies can be performed to provide some understanding relating to the topical absorption of menthol following ‘in use’ application. In addition, for semi-solids like gels, the rheological properties can cause significant changes in the consistency of the product, affect the application of the formulation at the treatment site and may affect the amount of drug delivered to the target site. Hence, our study has primarily attempted to address this lack of percutaneous penetration and skin distribution data for menthol by using different topical vehicles.

Nanoparticles represent an effective carrier system for drug delivery via the follicular pathway due to properties such as particle size and surface active charge for treating disorders such as androgenic alopecia (Hamishehkar et al., 2016;

Mura, Pirot, Manconi, Falson, & Fadda, 2007). Hence, in case of drugs like minoxidil, an o/w nanoemulsion (NE) possesses suitable characteristics for both topical and transfollicular delivery. In addition, differential tape stripping can be used to quantify the penetration of the drug not only in the SC but also in the hair follicles non-invasively and selectively (Teichmann et al., 2005). Although the topical route of delivery has several advantages such as improved patient compliance and ability to bypass first pass metabolism, due to some limitations of

the skin barrier, some enhancement technique may be required to increase drug diffusion and bioavailability at the target site. In this case, iontophoresis has been found to extremely suitable to drive the diffusion of ionized molecules across skin as well as via the appendageal route (Singh & Bhatia, 1996). Hence, our study has attempted to explore if the combination of iontophoresis and nanosized formulation would be effective in providing a more ‘targeted’ delivery in the treatment of follicular disorders like androgenic alopecia.

In addition to the nature of the dermatological vehicle, a number of other additional factors such as the viscosity and consistency of the product can potentially influence drug release. Historically, the FDA has recommended the use of in vitro release testing (IVRT) as part of the SUPAC-SS guidance as a means of demonstrating consistent delivery of the active ingredient from semi- solid products (Food & Drug Administration, 1997). This test can also be used to reflect the physicochemical parameters, including solubility of drug and rheological properties of the dosage form. Knowledge of the rheological properties of any formulation is also essential as the microstructural environment of the formulation may influence the subsequent drug diffusion and permeation.

Hence, it is important to understand the effect of these formulation parameters on the release of anti-inflammatory drugs which are applied topically for localized delivery.

In addition to the efficacy, an excellent safety profile is integral for the successful development of topical products. Skin irritation can have a significant influence on the therapeutic outcome and can cause adverse reactions. The in vitro

Epiderm™ model which consists of three-dimensional human epidermal keratinocytes offers a suitable alternative to conventional animal testing. This model has been validated by the European Centre for the Validation of

Alternative Methods (ECVAM) and has been used since 2009 to assess skin irritancy of any topical formulation. Thus, any of these parameters can be used to develop and optimize any dermatological formulation with enhanced therapeutic efficacy and safety.

CHAPTER 2

REVIEW OF LITERATURE

Background

The human skin has evolved to become a complex barrier which not only prevents water loss but also limits the entry of xenobiotics and harmful substances into the body. The major limitation for penetration of active compounds through the skin is overcoming the SC which is the outermost layer of skin. The SC serves as a rate-limiting lipophilic barrier due to the nature of its closely packed ‘brick and mortar structure’ which consists of flattened, dead keratinocytes embedded within multi-lamellar lipid bilayers (Michaels, Chandrasekaran, & Shaw, 1975).

The remaining layers of the skin, namely the viable epidermis and dermis offer lower resistance to the passage of active substances. The viable epidermis is about

50-100 µm thick, primarily composed of keratinocytes and is essentially devoid of blood capillaries and nerve endings. The bulk of the skin is comprised of the dermis which provides strength, elasticity and hosts a range of important structures including the blood and lymphatic vessels and base of various skin appendages. Finally, at the base of the skin, lies the hypodermis which acts as a depository for fat and contains the blood vessels that supply the skin.

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The major routes of drug transport for percutaneous absorption are the intracellular route across the corneocytes of the SC, penetration through the intercellular spaces of the SC and the appendageal route via the hair follicles, sebaceous and/or sweat glands. As the fractional area of the appendageal route is less than 0.1%, the predominant route of drug penetration is through the SC

(Scheuplein, 1967). The follicular route still plays an integral role in topical penetration and provides a reservoir comparable to that of the SC for treatment of various follicular disorders (Knorr et al., 2009). However, drug delivery into

(topical) and across (transdermal) the skin still remains a challenging area for scientists due to the excellent barrier properties of the skin. An understanding of some of the factors and mechanisms that influence skin permeability is therefore required to optimize topical formulations and provide effective targeting and therapeutic efficacy.

Factors influencing percutaneous absorption: For a chemical to pass into and/or across skin, a series of complex steps is required to occur which are influenced by the properties of both the penetrant and vehicle. An inverse relationship exists between drug diffusion and molecular weight and as such, drug molecules with molecular weight less than 500Da can diffuse passively through skin for local or systemic action (Bos & Meinardi, 2000). Due to the lipophilic nature of the SC, the partition coefficient or the log P value of the penetrant influences its partitioning into the skin. According to Williams (Williams, 2003), permeants

8 with intermediate log P value of 1-3 will traverse the skin via both lipid and aqueous pathways while those with log P greater than 3 will be transported predominantly via the intercellular pathway. In case of the applied dose, increasing concentration of the active ingredient generally implies an increase in the amount of chemical absorbed across skin. The use of an in vitro finite dose implies the application of a clinical dose of a topical formulation to the skin surface whereas an infinite dose experiment is one where the concentration of the permeant is constant and does not reduce over time. Frequency of application and duration of contact can also increase the amount of chemical absorbed topically.

Finally, the lipophilic nature of the SC suggests that unionized drug molecules are likely to permeate the skin compared to the ionized form. Studies have shown that the vehicle pH and the pKa of the penetrant can significantly affect the permeability of a molecule into skin (Chantasart, Chootanasoontorn,

Suksiriworapong, & Li, 2015; Li et al., 2012). Adjustment of the pH will therefore likely alter the extent of the ionized and unionized form of the penetrant, consequently affecting the concentration, solubility and ultimately its topical absorption into skin. In addition, physiological factors such as skin hydration, age, regional variation, ethnicity and skin temperature may influence the topical absorption of various compounds.

Vehicle effects: The rate and extent of percutaneous absorption relies on the characteristics of the skin, penetrant and the vehicle that affect the diffusion

9 process. The thermodynamic activity of the drug in the vehicle is the main factor that drives drug diffusion into and across the skin surface. The vehicle can also alter the skin permeability by affecting the barrier integrity of the SC, hence, depending on the desired therapeutic outcome, different vehicles can be used to optimize topical absorption. Aqueous vehicles are likely to provide less occlusion while ointment bases may provide more occlusion resulting in increased drug permeability. Vehicles containing ethanol have been known to increase the permeability of lipophilic drugs (Trommer & Neubert, 2006) while semi-solid formulations such as emulgels have the advantage of providing controlled release as well as being aesthetically acceptable to patients (Ajazuddin et al., 2013). In addition, it is important to understand the rheology of semi-solid vehicles since the application and delivery of the drug into the skin may be affected by the consistency of the product (Krishnaiah et al., 2014). Overall, there is not one universal vehicle for topical absorption, the formulation must be designed with not just the therapeutic end goal in mind but should also consider any potential irritation or toxicity in order to improve patient compliance and maximize the advantages of topical delivery

In vitro permeation and drug distribution: As per Guidance Document 2 established on the conduct of skin absorption studies (Organization for Economic

Co-operation and Development, 2004) , a significant number of studies substantiate direct comparisons of in vivo and in vitro methods. A good in vitro-in

10 vivo correlation has been observed on applying topical formulations to excised human or porcine skin (Franz, Lehman, & Raney, 2009; Schwarz, Klang, Hoppel,

Wolzt, & Valenta, 2012). Franz diffusion cells have been commonly used in these in vitro studies for testing of compounds into and across skin. A suitable membrane such as skin with stratum corneum side facing up is usually clamped between the donor and receiver compartments of the diffusion cells. The cells are jacketed and connected to a water bath to regulate the temperature. The temperature of the water bath is maintained at 37 °C in order to ensure a skin surface temperature of 32 °C. The receiver compartment is stirred constantly to ensure homogeneity and contains a sampling arm from which samples can be taken periodically. The receiver solvent should be selected to ensure sufficient solubility of the drug under investigation in order to maintain sink conditions throughout the experiment. Subsequent to diffusion studies, the skin deposition of topical compounds can be investigated by the tape stripping method which has been used to evaluate the bioavailability and bioequivalence of topical products clinically (Pershing et al., 2003; Puglia et al., 2008). The typical procedure involves sequential removal of the SC by placing an adhesive tape onto the skin surface, applying pressure, and then removal by a sharp upward movement. This relatively noninvasive technique is useful for determining the rate and extent of drug absorption in the skin, especially in the case of drugs whose target is the SC, such as antifungals (Plaum, Fleischer, & Hardas, 2013), keratolytics (Bashir et al.,

2005) and sunscreen agents (Roussel et al., 2015). Once the drug has been recovered and quantified from the extracted tape strips, the total amount in the SC can be determined. In case of follicular drug penetration, differential tape stripping is another powerful technique which enables the combined removal of

SC along with the cyanoacrylate surface biopsies which contains the follicular material (Teichmann et al., 2005). This allows both parts to be quantified, selectively providing comprehensive information on the penetration of topically applied substances. Thus, in vitro studies are a convenient, economic and powerful tool to evaluate topical dermatological products.

Modulation and enhancement of topical drug delivery: In comparison to the other routes of administration, drug transport across skin is limited primarily due to the barrier properties of the SC. This is also reflected by the limited number of drugs available on the market for administration across skin. Much research has therefore been undertaken to enhance drug delivery and improve the subsequent bioavailability of various drugs. One such physical enhancement technique is iontophoresis which involves the use of a physiologically acceptable electric current to drive charged and neutral molecules into and across skin. A basic iontophoretic system consists of an anode, cathode and the electrical current source. The iontophoretic principle of ‘like repels like’ implies that a positively charged formulation would use anodal delivery while negatively charged formulation would use cathodal delivery. In order to prevent electrolysis of water

12 and pH shifts, consumable electrodes such as silver/silver chloride electrode can be used for anodal iontophoresis. At the anode, the silver oxidizes to form insoluble silver chloride and an electron is released to the circuit while at the cathode, silver chloride is reduced to form silver and chloride ion (Banga, 2011).

The drug is thus driven into the skin and becomes available at the target site. In clinical practice, iontophoresis is primarily used to treat inflammatory conditions, in pain management, for the treatment of hyperhidrosis and as a diagnostic tool for cystic fibrosis (Kalia, Naik, Garrison, & Guy, 2004).

Advantages of topical and transdermal drug delivery: Drug transport across skin offers several advantages over the conventional routes of administration such as oral or parenteral. Dermatological drug products are designed to exert a local effect in diseased skin following application on skin surface. The objective is to maximize drug concentration at the target site with minimal systemic exposure.

This route offers several advantages such as specific drug delivery, avoidance of gastro-intestinal irritation and metabolic degradation associated with oral administration (Nair & Taylor-Gjevre, 2010). In addition, drug bioavailability may be improved by avoiding the first-pass hepatic metabolism and consistent drug delivery for an extended period of time (Rosen & Abribat, 2005). Clinical studies have shown that topical NSAID formulations were comparable in efficacy to their oral forms with a more favorable safety profile (Klinge & Sawyer, 2013).

Similarly, use of transdermal patches offers ease of use, non-invasiveness,

13 controlled and reversible drug delivery for a variety of clinical indications.

However, the scope of this research is mainly limited to the use of various formulations and enhancement techniques for topical drug delivery.

CHAPTER 3

INVESTIGATION OF THE DERMAL ABSORPTION AND IRRITATION

POTENTIAL OF SERTACONAZOLE NITRATE ANHYDROUS GEL

Abstract

Effective topical therapy of cutaneous fungal diseases requires the delivery of the active agent to the target site in adequate concentrations to produce a pharmacological effect and inhibit the growth of the pathogen. In addition, it is important to determine the concentration of the drug in the skin in order to evaluate the subsequent efficacy and potential toxicity for topical formulations.

For this purpose, an anhydrous gel containing sertaconazole nitrate as a model drug was formulated and the amount of the drug in the skin was determined by in vitro tape stripping. The apparent diffusivity and partition coefficients were then calculated by a mathematical model describing the dermal absorption as passive diffusion through a pseudo-homogenous membrane. The skin irritation potential of the formulation was also assessed by using the in vitro Epiderm™ model. An estimation of the dermal absorption parameters allowed us to evaluate drug transport across the stratum corneum following topical application. The estimated concentration for the formulation was found to be higher than the MIC100 at the 14

target site which suggested its potential efficacy for treating fungal infections. The skin irritation test showed the formulation to be non-irritating in nature. Thus, in vitro techniques can be used for laying the groundwork in developing efficient and non-toxic topical products.

Introduction

Cutaneous fungal infections are not only widespread, but are responsible for many diseases of the skin and mucus membrane. Up to 70% of the population worldwide are most commonly affected by a dermatophyte infection, usually tinea pedis (Faergemann, 2000; Vander Straten, Hossain, & Ghannoum, 2003).

Over the past few decades, the HIV pandemic and the increasing use of immunosuppressive agents for treating cancer, autoimmune diseases and other medical conditions have resulted in an increase in the incidence and severity of fungal infections (Croxtall & Plosker, 2009; Gandhi, Brieva, & Lacouture, 2014).

A number of azole antifungals are frequently used as a first line treatment for these superficial fungal infections. Sertaconazole nitrate (SN) is one such agent which was approved by the US FDA for treating tinea pedis in 2003. Chemically,

SN is unique from other azole antifungals as it contains a benzothiophene ring which mimics tryptophan and increases the drug’s ability to form pores in the fungal cell membrane. The mechanism of action involves the inhibition of ergosterol synthesis resulting in increased cell wall permeability or leakage of cellular constituents which may ultimately result in cell death. In addition, the

lipophilic benzothiophene ring aids in enhanced cutaneous retention (Palacin,

Tarrago, Sacristan, & Ortiz, 1995) without increasing systemic absorption. The drug has both fungistatic and fungicidal activity against a variety of micro- organisms belonging to the Dermatophytes and Candida species (Carrillo-Munoz et al., 2004; Pfaller & Sutton, 2006) . The potency of sertaconazole to inhibit strains of Malassezia has also been reported (Van Gerven & Odds, 1995). All these studies demonstrate the broad spectrum antifungal activity of SN against a variety of fungal pathogens.

Currently, various topical formulations are available on the market for treating fungal diseases. The efficiency of topical antifungal therapy depends on the delivery of the active agent to the target site in adequate concentrations in order to exert its activity. The objective of our research was to investigate the in vitro topical absorption of the model antifungal drug (SN) by using an anhydrous gel formulation. The topical absorption of the formulation was evaluated by in vitro tape stripping using both unsteady-state and steady-state experiments, similar to the methods reported in literature (Reddy, Stinchcomb, Guy, & Bunge, 2002). In addition to the bioavailability, it is also important to consider factors such as skin irritation and sensitization which may affect the subsequent safety and effectiveness of the product. Hence, the irritation potential of the formulation was also evaluated by the in vitro Epiderm™ tissue model.

Materials and Methods

Materials: SN was purchased from 2A Pharmachem (Lisle, IL, USA). PEG 400 and propylene glycol were provided by BASF (Tarrytown, NY, USA). Klucel®

(hydroxypropylcellulose) was provided by Ashland Inc. (Bridgewater, NJ, USA) while isopropyl myristate was from Croda Inc. (Edison, NJ, USA). Ascorbic acid and menthol were purchased from Sigma Aldrich (St. Louis, MO, USA) while glycerin was from Fisher Scientific (Waltham, MA, USA). Anhydrous ethanol

(200 proof) was purchased from Acros Organic (Morris Plains, NJ, USA). All other reagents used were of high purity or HPLC grade.

Formulation of anhydrous gel: The gel was prepared in a glass jar at room temperature by slowly dispersing the gelling agent (Klucel®) in a mixture of glycerin, propylene glycol and anhydrous ethanol. The dispersion was continually stirred using a Teflon™-coated magnetic stir bar until a visually homogenous gel was formed. The 2% SN was gradually dissolved in polyethylene glycol (PEG)

400. Finally, isopropyl myristate, ascorbic acid, and menthol were added and the mixture was stirred continuously till a clear gel was obtained. Throughout the procedure, the jar was covered with Parafilm® in order to prevent evaporation of ethanol. The final composition of the anhydrous gel used is shown in Table 1.

Table 1. Anhydrous gel composition

Components Formula (% w/w)

Sertaconazole Nitrate 2

Propylene glycol 20

Klucel® 2

Glycerin 15

PEG 400 20

IPM 2

Menthol 1

Ascorbic acid 0.2

Ethanol, anhydrous 37.8

In Vitro Permeation: Freshly excised porcine ear was obtained from a local abattoir no more than a few hours post-mortem and without any prior sanitization.

The outer region of the skin was separated from the subcutaneous fat and stored at

−80 °C until further use. Diffusion studies were performed using vertical Franz diffusion cells (Permegear, Hellertown, PA, USA). The thawed skin was clamped between the donor and receiver compartments of the diffusion cells. The temperature of the water bath was maintained at 37 °C in order to ensure a skin surface temperature of 32°C. The receiver compartment contained PEG 400: 10 mM PBS (phosphate buffered saline, pH 7.4) (60:40 v/v) which ensured sufficient

solubility of the drug in order to maintain sink conditions. A dose of 10 mg/cm2 of the anhydrous formulation was applied on porcine skin by using a positive displacement pipette. A minimum of four replicates was used for the entire study.

Tape Stripping: Following the diffusion studies, tape stripping was performed in vitro to quantify the amount of drug in the SC. In case of the anhydrous gel, the study was performed both for 5 min and for 24 h to establish an unsteady-state and steady-state profile, respectively. Based on the methods reported in (Reddy et al., 2002), the unsteady-state experiment was performed when 0.03 ≤ texp/tlag ≤ 0.3

(where texp is duration of the exposure and tlag is the lag time) in order to reduce large variations in estimating the lag time. The steady-state experiment was performed when texp > 1.7 tlag. Up to 20 pre-weighed adhesive tapes (3-M

Transpore tape, USA) were cut into 2 × 2 cm square pieces, pressed onto the treated skin area and removed sequentially. Each tape strip was then weighed again and the mass of SC removed was determined. The tape stripping procedure was performed rapidly (less than 5 min) for both sets of experiments. The first tape strip was discarded due to potential contamination of residual drug on the skin surface. The remaining tapes were extracted individually for drug quantification by HPLC.

Skin Extraction: After tape stripping, the remaining stripped skin was minced finely and 1 mL of suitable extraction solvent (methanol) was added. After shaking for a suitable time period of 4 h to ensure adequate extraction of the drug

from the tapes and skin, the samples were centrifuged, the supernatant was filtered and analyzed by HPLC.

Dermal Absorption Parameters Estimation: Data recorded in terms of mass of SC and mass of SN on tape strips were used to calculate concentration of SN (Cn) in the tape strip using Equation (1) of (Reddy et al., 2002):

C = m ρ / m n n sc sc,n ( 1 )

th where mn is the mass of SN in the n tape strip, ρsc is the density of the SC and

th msc,n is the mass of the SC in the n layer. The depth of the SC from which the tape strip was drawn (xn) was calculated according to Equation (2) of (Reddy et al., 2002):

n−1 1 ⎧msc,n ⎫ xn = ⎨ + msc,i ⎬ A 2 ∑ ρ sc ⎩ i=1 ⎭ ( 2 )

where A is the area being tape stripped and xn is at the center of msc,n. For this purpose, the density of the SC was assumed to be 1 g/cm3 (Pirot et al., 1997) and the area of tape stripping was calculated to be 4 cm2. It was assumed that the process of tape stripping removed the entire SC. The thickness of the SC (Lsc) was determined by Equation (3) of (Reddy et al., 2002)

M L = sc sc A ρsc ( 3 )

where Msc is the sum of the entire SC obtained through tape stripping. It was assumed that dermal absorption occurs passively through a pseudo-homogenous membrane. Since SN is a hydrophobic compound and previous work characterizing skin to be a homogenous membrane for lipids has been published

(Frasch & Barbero, 2003), this assumption was considered appropriate. These were then used to fit Equation (4) of (Reddy et al., 2002) using the curve-fit toolbox of Matlab R2010a (Natick, MA, USA) to derive estimates on the partition coefficient Ksc/v between the SC and the vehicle and the lag time tlag for chemical to penetrate the SC under necessary assumptions.

∞ 22 C xnx21 ⎛⎞ntπ exp ⎛⎞π n =1expsin− − ⎜⎟ ( 4 ) KC0 L∑ n⎜⎟6 t⎜⎟ L sc/lag v V scπ n=1 ⎝⎠⎝⎠ sc

where � is the initial concentration of the vehicle and texp is the time for which

the drug exposure is allowed. The tlag is defined as �/6� where Dsc is the effective diffusion coefficient in the SC. The parameter space used for identification of both the partition coefficient and the lag time ranged from zero to infinity. To ensure that Equation (4) remains valid, partition coefficient Ksc/v and the lag time tlag were estimated after an exposure of 5 min. The density of the gel was assumed to be 106 g/m3 and since SN concentration is 2% of the formulation,

3 3 the initial concentration of the vehicle � was considered as 20 × 10 g/m .

Diffusion Model: As seen in Figure 1, the one-layered diffusion model assumes that the SC is the rate-limiting barrier for topical absorption. With the removal of

each tape strip, the outside edge of the barrier begins to move inward until no more SC is present. The vertical axis represents the concentration of the drug while the horizontal axis indicates the depth of the SC. The striped area represents the amount of penetrant in unit area of the skin.

The parameter estimates obtained were used to solve partial differential equations using the Matlab pdepe function to generate concentration time profiles. A 2D rectangular geometry of the same thickness as the SC was used to perform the simulations. Since SC is the primary site of action for anti-fungal drugs, the one layered diffusion model was used to simulate concentrations in the stratum

corneum. The following equations as before (Pirot et al., 1997) were used to simulate dermal absorption:

2 ∂Csc ∂ Csc = Dsc 2 ∂t ∂x (5a)

C K C 0 sc ]x=0 = sc × V (5b)

Csc ] = 0 x=Lsc (5c)

Csc = 0, for t = 0; 0 ≤ x ≤ Lsc (5d)

Simulations of stratum corneum concentrations were conducted for 5 min and the concentration time profiles derived thereafter were used to examine the efficiency of SN in treating fungal infections for an exposure of 5 min. An MIC100 value of

2.695 µg/mL was used for yeasts (Drouhet & Dupont, 1992) and an MIC100 value of 52 µg/mL was used for the fungal species of M. furfur (Van Gerven & Odds,

1995).

Skin Irritation Testing: The anhydrous gel formulation was tested for skin irritation using the 3D tissue culture Epiderm™ model (MatTek Corporation,

Ashland, MA, USA). As per the protocol, the tissues were pre-incubated to overcome shipping stress and then exposed to different treatments. The positive control (PC) was 5% SDS solution while negative control (NC) was sterile

Dulbecco’s Phosphate Buffer Saline (DPBS). Tissues were exposed to 100 µL of the PC and the NC and 30 µL of the gel formulation for 1 h. Post-treatment, the tissues were incubated for 48h. MTT assay was then carried out to determine the

cell viability. The formulation was then classified as irritant/non-irritant based on the criteria in the given protocol.

HPLC Analysis: Quantification of SN was done using a validated method developed in our laboratory. The analysis was carried out on an Alliance HPLC

Water 2795 Separation Module consisting of an autosampler, a pump and a UV- diode array detector, using a Luna C18(2), 5 µm column (250 × 4.6 mm)

(Phenomenex, Torrance, CA, USA). The column temperature was set at 40 °C and injection volume was 20 µL. The mobile phase consisted of 10 mM sodium phosphate buffer (pH, 3.0): acetonitrile (40:60 v/v). The flow rate was 1 mL/min and the detection wavelength was set to 225 nm.

Results

Localization of SN in Skin: The tape stripping method was used to determine the amount of SN in the skin following in vitro permeation. After 5 min of formulation application of the gel, the skin was tape stripped immediately to simulate unsteady-state conditions. The amount of drug in the tape strips and stripped skin are shown in Figure 2. Thus, after immediate administration of the formulation, the drug had already distributed in the skin. Similarly, tape stripping was performed after 24 h to represent steady-state conditions and the results are shown in Figure 3.

The SC concentration depth profile obtained after tape stripping is shown in

Figure 4. During the tape stripping, with each successive tape strip, a gradient

from the outside to the inside was established which means that the drug penetrated throughout the layers of the SC.

18 16 14 SE

± 12 10 8 6

g) (µ g) Amount Avg. 4 2 0 Amount in Tape strips Amount in stripped skin

Figure 2.Amount of SN in tape strips and stripped skin following unsteady-state conditions (5 min)

SE 50 ±

20 g) ( µ g) Amount Avg 10

0 Amount in Tape strips Amount in stripped skin

Figure 3.Amount of SN in tape strips and stripped skin following steady-state conditions (24 h)

5 minute exposure 20 24 hour exposure

SN Concentration (g/L) SN Concentration 5

0 0.000 0.200 0.400 0.600 0.800 1.000

x/Lsc

(a)

35 R2 = 0.5085 30

10 SN Concentration (g/L) SN Concentration 5

0 0 0.2 0.4 0.6 0.8 1 x/L sc (b)

2.5 R2 = 0.8888 2

1.5

1 Concentration (g/L) 0.5

0 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 1.000 x/Lsc (c) Figure 4. (a) Average SN concentration-depth profile following application of the formulation for 5 min and 24 h; (b) Sample fit for application of formulation for 24 h: markers are observed data and solid line is fit; (c) Sample fit for application of formulation for 5 min: markers are observed data and solid line is fit.

Parameter Estimation: As seen in Table 2, the thickness of the SC estimated using

Equation (3) was found to be close to the physiologically reported SC thickness of the porcine ear skin (Popov, Lademann, Priezzhev, Myllylä, & Fitzmaurice,

2007). The SN diffusivity and partition coefficient were calculated for an exposure time (Texp) of 5 min and 24 h. However, due to the poor quality of fits for the parameters estimated for the 24 h exposure, the parameters estimated from the 5 min exposure were used for the diffusion simulations. Comparison of the fits is done in Figures 4b, 4c and the parameters estimated are mentioned in Table 2.

Table 2. Details of TS experiment and parameter estimation

Experimental details Estimated Parameters

2 Texp Lsc (µm) Ksc/v Dsc (m /s ) 5 minute 5 min 24.4 ± 3.3 0.64±0.51 (6.47±1.56)×10-14 exposure

24 hour 24 hours 23.7 ± 3.95 0.98±0.37 (8.88±6.4)×10-14 exposure

Lsc: Thickness of SC; Ksc/v: Partition coefficient of drug between SC and vehicle;

Dsc: Diffusion coefficient in the SC

Diffusion Model: Since very low amounts of the drug were found in the stripped skin for the experiment with the 5 min exposure, the single layer diffusion model, as considered neglecting diffusion to the dermis, should be a good, reasonable approximation for this period of exposure. The simulated concentration-depth profile following application of the formulation and tape stripping is shown in

Figure 5. The concentration-depth profile obtained was compared with the minimum inhibitory concentration (MIC100) of fungal pathogens of interest, namely M. furfur and yeast. The resultant profile showed that the concentration of the drug stayed above the MIC100 value for yeast throughout the depth of the stratum corneum and above the MIC100 for M. furfur for almost the entire depth of the stratum corneum. This suggests that effective concentrations of sertaconazole may be achieved at the target site in patients following application of the

formulation, thus indicating that the formulation is quite effective in treating fungal infections even after a brief exposure of 5 min.

Figure 5.Simulated SN concentration across the SC plotted along with MIC100 of M. furfur and yeast

Skin Irritation Test: The relative percent viability of the Epiderm™ tissues treated with PC, NC and the anhydrous gel are shown in Figure 6. Based on the given protocol, any substance is considered as an irritant if the percentage of cell viability is less than 50% similar to the PC. On performing the MTT assay, the cell viability for the NC was considered to be 100% while the PC and the gel showed cell viability of 7.7% and 63.4% ± 11.62%, respectively.

100 % cell viability cell% 50

0 PC NC Gel

Figure 6. Skin irritation testing showed anhydrous gel to be non-irritant

Discussion

Effective topical therapy requires sufficient amounts of the drug in the skin in order to produce maximal pharmacological activity. The vehicle used can also have a significant effect on the permeation of the active ingredient. Ethanol has already been established as an effective penetration enhancer for lipophilic drugs.

Hence, in order to enhance the cutaneous penetration of SN, which is highly lipophilic, an anhydrous gel containing ethanol was formulated. No drug crystallization was observed in the optimized formulation which remained clear and transparent, indicating the stability of the drug in the anhydrous formulation.

An anhydrous vehicle has the advantage of being well tolerated and acceptable without leaving any residue. Clinical studies using a commercially available anhydrous gel containing 2% ketoconazole have already shown the superior efficacy of this vehicle for the treatment of certain fungal diseases (B. Elewski et al., 2006). Hence, the anhydrous gel containing SN was formulated as it may

show better effectiveness in treating cutaneous fungal infections due to all these advantages.

Recent clinical trials have demonstrated the superior efficacy of SN in treating tinea pedis and other fungal infections (Sharma et al., 2011; Weinberg &

Koestenblatt, 2011). Hence, one of the main objectives of our study was to use

SN as a model drug in order to demonstrate the rate and extent of in vitro drug absorption in skin and predict its local bioavailability. Basically, the main objective is to maximize the drug concentration at the site of action (SC surface for antifungals) with minimal systemic uptake. Previous in vivo studies done using 2% SN cream have showed a similar trend with undeterminable/minimum systemic bioavailability (Susilo et al., 2005). Our study also showed minimal amounts in the receptor (data not shown), similar to the in vivo situation. This outcome is reasonable since the study aims to show the absorption of the SN formulation at the target site rather than its systemic availability.

In this study, we used porcine ear skin to perform in vitro tape stripping as it has been established as a suitable model for simulating human skin (Saeheng,

Nosoongnoen, Varothai, & Sathirakul, 2013). The gravimetric approach was then used to quantify the amount of SC removed. Although the skin is a multilayered membrane, the SC and the underlying layers such as the viable epidermis are the main barriers to topical absorption. In the case of antifungals such as SN, the SC is the principal barrier and hence all the equations used in this study are based on the assumption that the SC is the rate-limiting barrier for dermal absorption.

Hence, the one-layered diffusion model approach (Figure 1) was used in the parameter estimations. The clinical efficacy of the antifungal agent may be determined partly by the levels of the drug in the stratum corneum and also by the persistence of the drug at the target site for a suitable period which is enough to eradicate fungal growth (B. E. Elewski). Following tape stripping, as seen in

Figure 2 and Figure 3, the amount of drug in the SC and deeper layers was determined from experiments with two different times of exposure.

As seen in Figure 4a, the calculated SC concentration normalized depth profile was determined as a function of time following application of the formulation.

After 24 h, although the time required to tape strip was lesser than the lag time, it is possible that some drug may have still diffused during the tape stripping process, resulting in a non-linear profile (Figure 4b). In comparison, the profile after application for 5 min shows an inverse linear relationship between the drug concentration and SC depth (Figure 4c) in spite of the fact that drug in only 10 tape strips could be quantified potentially due to sensitivity constraints of the analytical method. However, studies have shown that for determining the SC thickness and for the interpretation of the concentration-depth profile, complete ablation of the barrier is unnecessary for determining the required information

(Herkenne, Naik, Kalia, Hadgraft, & Guy, 2007). The results of the parameter estimation following the tape stripping procedure are summarized in Table 2. The

SC thickness of porcine skin was found to be approximately 23.7 ± 4.0 µm and

24.4 ± 3.3 µm for steady-state and unsteady-state conditions, respectively, which

are close to the reported values in literature (Popov et al., 2007). It has been reported that in the case of a volatile vehicle which begins to evaporate from the moment of application, drug uptake was the same for both a short duration of exposure as well as after the chemical was allowed to stay on the skin for a more extended duration (Stinchcomb, Pirot, Touraille, Bunge, & Guy, 1999). Hence, the estimated parameters for the 5 min exposure represent a reasonable estimate of drug uptake into the skin. The partition coefficient Ksc/v determines the affinity of the drug for the SC compared to the applied formulation. The diffusivity

2 coefficient was estimated by the relationship Dsc = Lsc / (6 × tlag). Thus, the drug concentration–SC depth profile can be used to estimate both thermodynamic and kinetic parameters which are useful to characterize drug uptake in the skin following application of the formulation.

Due to the better fit of data from the 5 min exposure, the profile obtained from the 5 min exposure data was chosen for further simulation. The MIC100 represents the lowest concentration of the drug that can cause total inhibition of fungal growth. The simulated concentration-depth profile of SN (Figure 5) proved to be higher than the literature-derived MIC100 of yeasts for the entire thickness of the stratum corneum and higher than the literature-derived MIC100 of M. furfur for almost the entire thickness of the stratum corneum. Thus, the exposure levels attained by the drug may be sufficient to treat superficial fungal infections caused by these pathogens once steady-state is achieved.

In addition to the therapeutic outcome, an excellent safety profile is an integral part of any topical therapy. SN has already been well established as safe and non- toxic in the treatment of fungal infections . Since the gel contained a large percentage of ethanol, the final step of our study involved evaluating the irritation potential of the formulation. The Epiderm™ model consists of three-dimensional human epidermal keratinocytes comprised of an intact basal layer along the SC and viable epidermis. As per the given protocol, any formulation showing less than 50% cell viability following the MTT assay can be considered an irritant. As seen in Figure 6, since the percentage cell viability of the anhydrous gel was greater than 50% similar to the NC, the formulation can be classified as non-toxic and non-irritant (Kandarova et al., 2009).

Conclusion

In this study, we evaluated the dermal absorption of SN anhydrous gel by in vitro tape stripping in order to understand the local bioavailability in skin. A mathematical model was then established for estimating the partition and diffusion coefficients. These dermal absorption parameters were used to predict the topical absorption of the drug up to 5 min. The estimated concentration time profile was found to predict a greater concentration than the MIC100 for M. furfur and yeast for almost the entire depth of the stratum corneum. This suggests that the formulation may reach the target site in adequate concentrations to effectively treat fungal infections. In order to evaluate the toxicity of the formulation, the

Epiderm™ skin irritation test was then performed which categorized the

anhydrous gel formulation as non-irritating. Thus, these in vitro techniques are extremely useful surrogate tools in the development of topical formulations with improved therapeutic efficacy and safety.

CHAPTER 4

EVALUATION OF THE IN VITRO PERCUTANEOUS ABSORPTION AND

RHEOLOGICAL BEHAVIOR OF TOPICAL MENTHOL FORMULATIONS

Abstract

Menthol is available in a variety of over-the-counter products for treating mild aches and injuries due its analgesic and anti-inflammatory properties. In spite of the drug’s ubiquitousness, there is limited data available on the human skin permeation of menthol. Hence, the goal of our study was to evaluate the in vitro penetration and absorption of menthol by using liquid and gel formulations. In addition, the rheological properties of the gel formulations were also evaluated as the structural properties of the formulation may influence the drug diffusion and permeation. In vitro permeation was performed using dermatomed human skin under different dosing conditions. In case of the semi-solid formulations, rheological characterization was performed and gel formulations with the most desirable viscoelastic properties were then selected for further permeation studies.

The amount of menthol in the skin was also analyzed as a function of time up to 8 hours. A finite dose of 10 µL/cm2 of menthol solution showed flux values of

27.51 ± 6.23 µg/cm2/h after 8 hours. No significant difference was found in the amount of drug transported across human skin using different vehicles under different dosing conditions. This study also demonstrated an increase in the cumulative amounts of menthol in both the receptor and skin with time under finite dosing conditions. Overall, our findings indicate that menthol was able to permeate human skin in sufficient concentrations when applied topically and studying the rheological behavior can be useful during development of such topical semi-solid formulations.

Introduction

Menthol is a naturally occurring terpene of plant origin which imparts the typical minty smell and flavor to plants of the Mentha species. Chemically, menthol is a cyclic monoterpene alcohol and has four pairs of enantiomers. The predominant isomer is L-menthol which has the characteristic peppermint odor, exerts a cooling sensation when applied on skin and has more potent analgesic activity in comparison to the other isomers (Eccles, 1994; Galeotti, Di Cesare Mannelli,

Mazzanti, Bartolini, & Ghelardini, 2002). In animal models, oral, topical or systemic administration of menthol induced analgesic activity in case of acute and inflammatory pain (Klein et al., 2010; Pan et al., 2012) The main mechanism of action involves activation of cold-sensitive transient receptor potential cation channel (TRPM8). The tingling sensation followed by ‘cool’ feeling on topical

application of menthol is due to the stimulation of these thermosensitive receptors by speedily increasing intracellular calcium and thus inducing cold response signals at the application site (Farco & Grundmann, 2013; Yosipovitch, Szolar,

Hui, & Maibach, 1996)

Menthol is commonly used as both a cooling agent and a counterirritant especially for relieving pain in the muscles and other remote areas as well as for treating pruritis. Currently, menthol is widely available in both prescribed and over-the- counter (OTC) medications for the treatment of respiratory disorders, gastrointestinal disturbances and musculoskeletal pain.(Eccles, 1994) In concentrations of 1% or less, menthol depresses the cutaneous sensory receptors while at concentrations between 1.25-16%, it stimulates the sensory receptors and exerts the counter-irritant effect (Patel et al., 2007) One of the most important applications of this versatile compound is probably its penetration-enhancing effect and subsequent influence on percutaneous absorption of transdermal and topical formulations.(Chadha, Sathigari, Parsons, & Jayachandra Babu, 2011; Lan et al., 2015). This effect is mainly due to disruption of the lipid bilayer of the stratum corneum (Wan, Dai, Yin, Shi, & Qiao, 2015). In spite of the fact that a variety of OTC topical formulations contain menthol, there is limited information available regarding the percutaneous absorption of this compound in humans. The current lack of experimental evidence combined with the physicochemical

properties of the compound has resulted in an assumption that menthol is easily absorbed through human skin. A survey of the scientific literature revealed only four articles where human skin absorption of menthol was evaluated. These studies mainly involved studying the in vitro skin penetration of menthol in the presence of other terpenes or drug substances or the in vivo exposure from the application of commercial patches on human subjects (Cal, 2008b, 2009; Cal &

Sopala, 2008; Martin, Valdez, Boren, & Mayersohn, 2004)

Hence, our study has primarily attempted to address this lack of percutaneous penetration and skin distribution data for menthol. For this purpose, permeation studies were performed using excised human skin as it is a commonly used method for assessment of in vitro drug delivery or penetration following application of any topical formulation (Franz et al., 2009). As concentrations of up to 16% menthol have been approved by the FDA for OTC external use and the safety profile has been well established (Kamatou, Vermaak, Viljoen, &

Lawrence, 2013; NCI (National Cancer Institute), 1993) various topical formulations containing 16% menthol were evaluated in order to represent a formulation likely to maximize drug delivery and also meet the requirements of the analytical method used in the study. In this study, the percutaneous absorption of menthol in human skin was reported by using semi-solid formulations in order to gain some understanding relating to its topical absorption at different time

points by simulating actual consumer or ‘in use’ application. For semi-solids like gels, the rheological properties can cause significant changes in the consistency of the product, affect the application of the formulation at the treatment site and may affect the amount of drug delivered to the target site. Hence, the rheological properties of the gel formulations were also evaluated as changes in drug-vehicle interactions could potentially influence both the topical application and the subsequent absorption of menthol.

Materials and Methods

L-Menthol at purity of 99% was purchased from Alfa Aesar (Ward Hill, MA,

USA). Propylene glycol, sodium hydroxide (NaOH) were obtained from Fisher

Scientific (Waltham, MA, USA) while ethanol and propyl paraben was from

Sigma Aldrich (St.Louis, MO, USA). Carbopol® Ultrez 10 was a gift from

Lubrizol Corporation (Cleveland, OH, USA). All other reagents used were of

ACS or HPLC grade.

Preparation of menthol solutions: The composition of menthol solutions used in the study is given in Table 3. Menthol solutions were prepared by dissolving the drug in either propylene glycol, ethanol or a combination of both solvents (1:1 w/w).

Table 3. Composition (% w/w) of menthol in different vehicles

Components F1 F2 F3

L-menthol 16 16 16 Propylene glycol 84 - 42 Ethanol - 84 42

Preparation of menthol gels: The formulations were prepared by slowly dispersing

the carbomer in water with continuous stirring and. the dispersion was allowed to

hydrate for 60 minutes. The drug was dissolved in an appropriate mixture of

propylene glycol and ethanol and then transferred to the container containing the

dispersion with continuous agitation. NaOH solution (0.1% w/w) was then added

to neutralize the mixture and to adjust the pH between 5-6 and stirring was

continued until a clear homogenous gel was obtained. The container was sealed

tightly throughout the procedure in order to minimize any potential evaporation.

The components of the different gel formulations are shown in Table 4.

Table 4. Composition (% w/w) of menthol gels containing different percentages of carbomer, propylene glycol and ethanol

Components F4 F5 F6 F7 F8 F9 F10 F11 L-menthol 16 16 16 16 16 16 16 16 Propylene 15 20 15 20 25 15 20 25 glycol Ethanol 15 20 15 20 25 15 20 25 Carbopol® 1 1 1.5 1.5 1.5 2 2 2 Ultrez™ 10 Propyl 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 paraben Water q.s. to 100

Rheological measurements: The rheological behavior of the gel formulations was evaluated at 32°C by using the MCR 302 rheometer plate (Anton-Paar, Ashland,

VA). A parallel-plate measuring device with a diameter of 25 mm and a gap of

0.1 mm was used for performing the experiments. A peltier hood was used in conjunction with the fixture to maintain the temperature of the formulation at

32°C and to prevent any potential evaporation of the formulation. All measurements were performed in triplicate and always with a fresh sample.

Flow measurements were evaluated by subjecting samples to a shear rate starting from 1s-1 to final stress of 100s-1 and back. An amplitude sweep test was done by using a strain between 0.01 to 100% in order to determine the linear viscoelastic

(LVE) range of the samples. An oscillatory frequency sweep test was then performed over an angular frequency range of 0.1-100 rad/s. Finally, the thixotropic behavior of the formulations was also evaluated in a three-step process by (1) initially applying an oscillation at a strain amplitude of 5% and angular frequency of 10 rad/s to simulate behavior at rest, (2) followed by a rotational test with a strong shear rate of 100s-1 to simulate structural breakdown of the sample and (3) the same initial oscillatory conditions in order to simulate structural regeneration at rest.

In vitro permeation: Cryopreserved, dermatomed human skin was obtained from a skin bank (New York FireFighters, New York, NY). Prior to the experiment, the skin was thawed and cut into sections large enough to fit on 0.64 cm2 vertical

Franz diffusion cells (Permegear, Hellertown, PA). The receptor chamber was filled with phosphate-buffered isotonic solution (PBS) (10mM, pH 7.4) and the diffusion apparatus was temperature-controlled in order to maintain the surface skin temperature at 32°C. Sink conditions were maintained throughout the experiment. To assure integrity of each skin piece, the electrical resistance was

measured before application of the formulations at a frequency of 100 Hz and low voltage of 100 mV.

An infinite dose (100 µL) of F1 and F2 and a finite dose of 10 µL/cm2 of the F3 solution was applied on the surface of the skin. In case of the gel formulations, a finite dose of 10 mg/cm2 of was applied using a positive displacement pipette. At specific time intervals, samples (0.3 mL) were withdrawn from the receptor compartment and replenished with the same volume of fresh receptor. The donor compartment remained unoccluded in order to simulate actual application on skin while the sampling arm of the receptor compartment was covered to protect against the volatilization of menthol. A minimum of four replicates were used for all experiments.

Skin distribution study: Following permeation studies using finite dose of the gels, the skin was cleaned of excess formulation and removed from the Franz cells at different time intervals of 1h, 2h, 4h and 8h. The epidermis was then carefully separated from the dermis by means of forceps. Each of the skin sections were minced and placed in 2 mL of ethanol. Samples were kept on a shaker at 100 rpm for up to 4 hours. Skin extraction study was also performed by adding a known amount of drug to the skin and by using the same extraction method to determine the recovery factor. All samples were analyzed by HPLC.

HPLC Analysis: Menthol was quantified by HPLC analysis on a Waters Alliance

2795 separation module equipped with a Waters 2414 RI detector (Milford, MA,

USA). Using a method modified from the literature (Shaikh & Patil, 2010), chromatographic separation was achieved on a XBridge™ C18 column, 3.5 µm

(4.6x50 mm) (Waters, Milford, MA, USA) column using water: methanol (30:70 v/v) as mobile phase. The flow rate was 1 mL/min and the detector settings were set at positive polarity with a sensitivity of 512 RUI for an enhanced peak response. Linearity was established over a concentration range of 1-50 µg/mL.

Permeability coefficient determination: The infinite dose study was used to estimate the steady state flux (J) by linear regression of the cumulative amount versus time plot. The permeability coefficient was calculated from the equation:

J K = p C ×A where Kp is the permeability coefficient (cm/h), J is the flux at steady state

(µg/h), C is the concentration in the donor (µg/mL), and A is the area (cm2)

Data Analysis: All statistical analysis was performed using two-tailed t-test.

Significance was taken at p< 0.05. Rheological data was analyzed using the

Rheoplus software included with the Rheometer series MCR 302.

Results

Determination of permeability coefficient using infinite dose: An infinite dose of

100 µL was primarily used to prevent any depletion of the permeant in the donor chamber. After 8 hours, the average cumulative amount of drug permeation using propylene glycol and ethanol vehicle was 460.58±50.74 and 549.51±88.02

µg/cm2 respectively (Figure 7). There was no significant difference in the amount of drug in receptor or flux values for both vehicles.

Menthol in EtOH (100 µL ) Menthol in PG (100 µL) 700 600 500 400 300 200 100 0

Avg cum amt/sqcm ( µ g/sqcm) amt/sqcmcum Avg 0 1 2 3 4 5 6 7 8 Time (hrs)

Figure 7.Comparison of the cumulative permeation of menthol through human skin in vitro following application of infinite dose of menthol in propylene glycol and ethanol (Mean ± SD, n=4)

The permeability coefficient is a measure of drug transport from a vehicle across skin. This parameter was determined from the steady state flux of the infinite

dose. Values of Kp for menthol in propylene glycol was 0.302± 0.077 x 10-3 cm/h and for ethanol was 0.245±0.052 x 10-3 cm/h. However, there was no significant difference on comparison between the two vehicles.

Finite dose study of topical solutions: For the finite dose study, the vehicle chosen was a combination of propylene glycol and ethanol (1:1 w/w) in order to ensure a maximal rate of penetration of menthol across human skin. In this case, the amount of drug in the receptor after 8 hours was found to be 266.97±56.16

µg/cm2 as seen in Figure 8. Drug distribution in the epidermis and dermis was found to be 408.26 ± 65.18 and 307.12 ± 70.94 µg/cm2 respectively (Figure 9).

350.00 300.00 250.00 200.00 150.00 100.00 50.00 0.00 0 1 2 3 4 5 6 7 8 Avg cum amt/sqcm ( µ g/sqcm) amt/sqcmcum Avg Time (hrs) Figure 8.Permeation profile of menthol through human skin in vitro following application of finite dose of menthol in propylene glycol: ethanol vehicle (Mean ± SD, n=4)

500.00

400.00

300.00

200.00

100.00 Avg cum amt/sqcm ( µ g/sqcm) amt/sqcmcum Avg

0.00 Amount in epidermis Amount in dermis

Figure 9. Skin penetration of menthol solution into epidermis and dermis at the end of 8 hours (Mean ± SD, n=4)

Rheological characterization of topical gel formulations: The gel formulations were then evaluated in order to select gels with the most desirable viscoelastic properties for further in vitro permeation studies. The flow curve analysis showed that all the topical gels exhibited pseudoplastic and shear-thinning properties as shown in Figure 10.

ETA

1,200,000 cP 1,100,000

1,000,000

Menthol gel F4

900,000 Viscosity

Menthol gel F5

800,000 Viscosity Menthol gel F6

Viscosity 700,000 Menthol gel F7

Viscosity

600,000 Menthol gel F8

Viscosity

500,000 Menthol gel F9

Viscosity 400,000 Menthol gel F10 Viscosity

Menthol gel F11 300,000 Viscosity

200,000

100,000

0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 1/s 100 . Shear Rate Anton Paar GmbH

Figure 10. Flow curve testing of menthol gel formulations

The oscillatory amplitude sweep test showed that between 0.1 to 1% of strain amplitude, all samples were in the LVE range at approximately 0.5% strain which was chosen for all further measurements. The dynamic viscoelastic function of loss modulus G’’ as well as storage modulus G’ was measured at 32°C. Figure 11 shows that G’>G’’ which means that even at a low angular frequency, the samples are more elastic than viscous in nature. The average of the damping factor values

(n=16) are shown in Table 5. The value for gels F4 and F7 were closer to 0 as compared to other formulations. As seen in Figure 12, the thixotropic behavior of

the different formulations was also assessed in order to predict their structural stability. The structural recovery was then calculated using the Rheoplus software and formulations F4 and F7 showed a good structural recovery of 94% and 91% respectively. Based on the rheological data, formulations F4 and F7 were then selected for the diffusion experiments as they exhibited the most suitable viscoelastic properties amongst all the gel formulations.

Rheoplus

4 4 10 10

Pa·s Pa

Menthol gel F4 Menthol gel F8

3 | *| Complex Viscosity | *| Complex Viscosity 10 G' Storage Modulus G' Storage Modulus

3 G'' Loss Modulus G'' Loss Modulus 10 Menthol gel F5 Menthol gel F9 G' | *| Complex Viscosity | *| Complex Viscosity

G' Storage Modulus G' Storage Modulus

| *| G'' Loss Modulus G'' Loss Modulus 2 10 Menthol gel F6 Menthol gel F10 G'' | *| Complex Viscosity | *| Complex Viscosity G' Storage Modulus G' Storage Modulus

G'' Loss Modulus G'' Loss Modulus

2 Menthol gel F7 Menthol gel F11 10 | *| Complex Viscosity | *| Complex Viscosity

G' Storage Modulus G' Storage Modulus 1 10 G'' Loss Modulus G'' Loss Modulus

0 1 10 10 0.1 1 10 rad/s 100 Angular Frequency

Anton Paar GmbH

Figure 11:Frequency sweep testing of menthol gel formulations

3ITT - OSC

10,000 0.4

Pa·s

0.36

0.34 Pa

0.32 Menthol gel F4 Menthol gel F8 0.3 G' Storage Modulus G' Storage Modulus G'' Loss Modulus G'' Loss Modulus 0.28 1,000 Viscosity Viscosity 0.26 Menthol gel F5 Menthol gel F9 G' 0.24 G' Storage Modulus G' Storage Modulus G'' Loss Modulus G'' Loss Modulus 0.22 Viscosity Viscosity

0.2 Menthol gel F6 Menthol gel F10 G'' 0.18 G' Storage Modulus G' Storage Modulus G'' Loss Modulus G'' Loss Modulus 0.16 Viscosity Viscosity 0.14 100 Menthol gel F7 Menthol gel F11 0.12 G' Storage Modulus G' Storage Modulus G'' Loss Modulus G'' Loss Modulus 0.1 Viscosity Viscosity 0.08

0.06

0.04

0.02

10 0 0 100 200 300 400 500 600 700 800 900 1,000 s 1,100 Time t

Anton Paar GmbH

Figure 12.Thixotropy test using menthol gel formulations

Table 5. Average damping factor values for menthol gel formulations

Damping factor (tan δ)= G’’/G’

F4 F5 F6 F7 F8 F9 F10 F11

0.06 0.1 0.10 0.07 0.09 0.09 0.12 0.23

Finite dose study using menthol gels: After 8 hours, the amount of drug in the receptor was found to be 260.66± 19.47 µg/cm2 and 286.08± 52.88 µg/cm2 for F4 and F7 respectively (Figure 13). The amount of drug in the epidermis and dermis was analyzed at different time points for 1h, 2h, 4h and 8h and the results are shown in Figure 14 and Figure 15 respectively. In case of skin, the amount in the epidermis was 489.51±72.5 µg/cm2 and 402.75±60.59 µg/cm2 while amount in dermis was 65.53±6.65 µg/cm2 and 60.15±12.29 µg/cm2 for F4 and F7 respectively. However, there was no significant difference in the cumulative amount of drug in the receptor and skin for both formulations at all time points.

F7 F4

400.00

300.00

200.00

100.00

0.00 0 1 2 3 4 5 6 7 8

g/sqcm)µ ( amt/sqcm Avgcum Time (hrs)

Figure 13.Comparison of permeation profiles across human skin in vitro following application of finite dose of F4 and F7 (Mean ± SD, n=4)

F7 F4

700.00 600.00 500.00 400.00 300.00 200.00 100.00 g/sqcm)µ Avg( amt/sqcm 0.00 1h 2h 4h 8h

Figure 14.Comparison of amount in epidermis following application of finite dose of F4 and F7 at 1h, 2h, 4h and 8h (Mean ± SD, n=4)

F7 F4

90.00

70.00

50.00

30.00

10.00 Amt/sqcm Amt/sqcm g/sqcm) µ (

-10.00 1h 2h 4h 8h

Figure 15.Comparison of amount in dermis following application of finite dose of F4 and F7 at 1h, 2h, 4h and 8h (Mean ± SD, n=4)

Discussion

L-menthol is a counter-irritant that provides a cooling sensation on application to skin and is a common ingredient in antipruritic, antiseptic, analgesic and cooling formulations (Patel et al., 2007). A walk through the pain relief aisle in any pharmacy will reveal menthol as an ingredient in a variety of compositions and dosage forms. It is important to understand the percutaneous absorption of this compound from both a therapeutic and safety point of view. However, a review of the scientific literature shows that the focus so far has been on the permeation enhancement of various drugs by using menthol rather than evaluating menthol solely as the active ingredient. For this reason, the present study first evaluated the permeation of menthol Figure 7. The permeability coefficient which is a key parameter in dermal absorption was then used to determine the transport of menthol through human skin. No significant difference was found in the permeability coefficient using both propylene glycol and ethanol vehicle.

A combination of propylene glycol and ethanol vehicle was then used to evaluate the dermal absorption of menthol by using a finite dose of 10 µL/cm2. Our study showed that menthol was able to penetrate dermatomed human skin and be recovered in the receptor phase. Although previous findings report the absence of menthol in the receptor (Cal, 2008b), the extent of penetration of terpenes like

menthol is dependent on the source of skin, the dose of the drug applied and the vehicle (Cal, 2008a). The dose of menthol applied in this case was comparatively higher than the previous studies, also, the vehicles used in our study had a high solubilization capacity for menthol which could potentially result in greater partitioning into and across skin. In addition, alcoholic solutions in combination with propylene glycol have been reported to extract proteins and lipids resulting in impaired barrier properties of the skin and subsequently higher permeation

(Levang, Zhao, & Singh, 1999). Since the analgesic activity of menthol has been attributed to the activation of the thermoreceptors which are located in the underlying layers of skin tissue below the stratum corneum (Green, 1992;

Wasner, Schattschneider, Binder, & Baron, 2004), the topical absorption of menthol was mainly evaluated in the epidermis and dermis in the present study as seen in Figure 9. The cumulative amount in both the skin layers was found to increase gradually with time up to 8h.

In case of semi-solid dosage forms like gels, it is extremely important to understand the rheological and mechanical properties as it may affect both the application and delivery of the active compound into skin. Changes in shear stress with shear rate can be used to determine if the flow behavior of any formulation is

Newtonian or non-Newtonian. A rotational test like the flow curve analysis across a range of shear rate can provide important information about structural changes

in the formulation due to applied shear during storage, processing and application

(Herh.P, 1998). The flow behavior is also important in order to evaluate the spreading of the formulation on the application site. In this case, the test showed that the gel formulations were characteristically non-Newtonian and pseudoplastic with increasing shear rate. Oscillatory tests can be used to evaluate the viscoelastic property of the material by subjecting the sample to a sinusoidal shear stress. Such tests include the amplitude sweep test which is used to understand the deformation behavior of samples by determining the limits of linear viscoelastic range which in this case was 0.5%. This strain was then used for the frequency sweep test where structural information about the material may be obtained by studying the response of the material to different shear rates. In case of the gel formulations, the storage modulus was higher than the loss modulus which suggests that these formulations are highly elastic and organized at the applied frequency range. When the dispersion containing carbomer is neutralized with

NaOH, the swollen carbomer particles form a closely packed structure thus forming an elastic network (Nae & Reichert, 1992). Figure 11 also shows little difference in the storage modulus over different constant frequencies which substantiate the elastic nature of the gel formulations. The damping factor is measured by the magnitude of the ratio G”/G’ = tan δ where δ is the phase angle.

This dimensionless parameter compares the relative importance of the elastic

contributions to the viscous contributions in a viscoelastic material. Typically, the structural strength of the formulation is measured by this parameter where smaller the tan δ, stronger is the interaction (Herh.P, 1998). In this case, formulations F4 and F7 had values closer to 0 which substantiated the choice of formulation.

The thixotropy test or time-dependent flow measures the change in the viscosity of the formulation with time and the extent of structural recovery is dependent on the time allowed for recovery of the sample. Thus, initially after applying an increasing shear on the sample, this test is used to measure the restoration of the original structure of the formulation once the external force has been removed.

This information is valuable since the semi-solid formulation must maintain the balance between its viscous and elastic properties in order to be applied on the skin and regain its consistency to form a uniform film after application. The structural recovery was found to be 94% and 91% for F4 and F7 respectively which suggests that these formulations had a much better structural stability in comparison to the other formulations.

Formulations F4 and F7 were then used for the permeation experiments where a finite dose of 10 mg/cm2 was applied to simulate ‘in use’ application. The plateauing effect seen in Figure 13 was consistent with finite dose application which resulted in lowering of the concentration gradient across skin and decreasing permeation rate. In this study, the topical absorption of menthol in

human skin was also evaluated at different durations of exposure up to 8 hours.

The amount of menthol in the epidermis and dermis increased progressively with time. However, no significant difference was observed between the two formulations for all time points. This could potentially be due to the higher concentration of carbomer in formulation F7 resulting in lower permeation of drug. Similar results have been reported in previous findings that showed an inverse relationship between viscosity of the formulation and drug permeation

(Dragicevic-Curic et al., 2009; Teichmann et al., 2005). The degree of therapeutic efficacy depends on the proper spreading of the formulation on the skin by the patient. Hence, an optimal consistency is required to ensure that a suitable dose is applied and delivered to the target site. Although there was no statistical difference in the amount of drug delivered between the two formulations, all the above results suggest that formulation F4 had more robust mechanical and rheological properties and the amount of drug delivered in and across skin was also comparatively higher with respect to F7. Hence, by understanding the rheological behavior of a topical formulation and by performing in vitro permeation studies, a rational basis can be provided for the development of suitable topical formulations containing a local analgesic such as menthol as the active ingredient.

Conclusion

In conclusion, the present study evaluated the in vitro dermal absorption of menthol under infinite and normal ‘in use’ conditions. The cumulative amount of menthol was found to increase in both human epidermis and dermis with time.

The viscoelastic nature of the gel formulations such as flow property, structural stability and thixotropic nature was also evaluated as these properties may influence the application of these topical products. Amongst all the formulations, the F4 gel was considered to be the optimal formulation as it showed the most desirable rheological properties and also delivered a higher amount of drug into skin. Thus, performing rheological measurements and diffusion studies can not only increase the efficiency in processing but can also help in the selection of formulations that are optimal and meet the end user’s individual needs both aesthetically and therapeutically.

CHAPTER 5

EFFECT OF DIFFERENT VEHICLES ON THE IN VITRO RELEASE OF

DICLOFENAC SODIUM

Abstract:

The selection and optimization of a suitable topical vehicle is of utmost importance since the vehicle composition and rheological properties can potentially cause changes in drug-vehicle interactions and affect drug release.

Hence, the aim of this study was to investigate the influence of different formulation parameters on the rheological properties and in vitro release by using diclofenac sodium as the model drug. Different semi-solid vehicles (ointment, cream, gel and emulgel) were formulated and characterized. Rheological tests including flow curve and oscillatory shear responses were evaluated for all formulations at 32°C by using the Anton-Paar rheometer. For release study, approximately 300 mg of formulation (n=6) was applied on Nuclepore™

(polycarbonate) membrane mounted on the vertical Franz diffusion cells. Samples were taken at different time intervals up to 6 hours and replaced with fresh receptor. Validation of the in vitro release test (IVRT) developed was performed in order to confirm if the method was able to differentiate between the formulations by testing for accuracy, repeatability and effect of dosage strength. 60

All samples were analyzed by a suitable HPLC method. The pH of all the semi- solid formulations was around 7.0. No phase separation was observed for any of the formulations on visual inspection while light microscopy confirmed the absence of drug crystals. Rheological analysis showed that all the formulations tested exhibited characteristic shear-thinning and viscoelastic behavior. The in vitro release of the drug from all the formulations was found to be linear with the square root of time in accordance with Higuchi’s model. The amount of drug released from the gel formulation (785.46 ± 3.07 µg/cm2) was statistically higher in comparison to all other formulations. The release test was found to be accurate and precise with R2> 0.99 while the release rate was found to be proportional to changes in the dosage strength of all the formulations. A robust and precise in vitro release method was developed and validated for diclofenac sodium using different semi-solid formulations. Performing rheological measurements and release studies can help in the development and optimization of topical dermatological products as a quality control tool.

Introduction

The oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with side effects like gastric irritation and toxicity (Heyneman,

Lawless-Liday, & Wall, 2000). The topical delivery of NSAIDs is often preferred over the oral route of administration due to the ability to bypass first pass

metabolism. Systemic exposure of a 1% diclofenac sodium gel was found to be approximately 5 to 17 fold lower than oral diclofenac administration with minimal systemic exposure in a clinical study (Kienzler, Gold, & Nollevaux,

2010). Diclofenac is a non-selective NSAID that exerts its action via inhibition of prostaglandin synthesis. Moreover, research suggests the drug has multimodal action in alteration of IL-6 production and activating the nitric oxide-cGMP antinociceptive pathway (Gan, 2010).

When NSAIDs are applied topically, the active ingredient must be released from the vehicle before it can partition into different layers of the skin. The therapeutic efficacy of a topical formulation is dependent on the vehicle composition as well which can influence drug release and skin permeability. Hence, the selection of an appropriate vehicle is of utmost importance. In this study, different semisolid formulations containing 1% diclofenac sodium were prepared and their stability was evaluated over a period of 30 days. In addition, rheological characterization of the different semisolid formulations was also performed as it provides valuable insight on the structure and viscoelastic properties which been reported to affect drug release and diffusion (Welin-Berger, Neelissen, & Bergenstahl, 2001).

Historically, IVRT has been often used in order to assure product sameness, however, in research and development, it can be used to predict the vehicle design and performance and how rapidly the drug may be release into skin. Thus, the test

can be used to estimate the in vivo performance of a topical product (Shah, 2005).

In this study, IVRT was performed in order to determine the effect of different vehicles on the in vitro release of diclofenac. The method was then validated in order to ensure a rugged, reliable and discriminating method could be used to differentiate between the different formulations.

Material and Methods

Diclofenac sodium and benzoic acid was purchased from Sigma Aldrich

(St.Louis, MO, USA). PEG bases for ointment, cetostearyl alcohol (CSA) 50, PG were kindly provided by BASF (Tarrytown, NY, USA). Liquid paraffin was obtained from EMD Millipore (Billerica, MA, USA). Span and Tween 80 was kindly provided by Croda Inc. (Edison, NJ, USA), Klucel®

(hydroxypropylcellulose) was provided by Ashland Inc. (Bridgewater, NJ, USA) while Transcutol was provided by Gattefosse Corp. (Paramus, NJ, USA).

Preparation of semi-solid formulations: The composition of the formulations prepared is shown in Table 6. All formulations contained 1% diclofenac sodium as the active ingredient.

Table 6. Composition of different semi-solid formulations containing 1% diclofenac sodium Components Emulsion Emulgel Gel Ointment (% w/w)

Diclofenac 1 1 1 1 sodium

Propylene glycol 5 5 5 -

Transcutol P 10 10 10 -

CSA 50 2 2 - -

Liquid Paraffin 7.5 7.5 - -

Span 60 4.5 4.5 - -

Tween 60 0.5 0.5 - -

Klucel® - 1 2 -

PEG 400 - - - 59

PEG 3350 - - - 40

Benzoic acid 0.25 0.25 0.25 -

q.s. to q.s. to q.s. to Dist. Water - 100 100 100

Emulsion and emulgel: The oil phase of the emulsion was prepared by dissolving

Span 60 and CSA 50 in liquid paraffin while aqueous phase was prepared by

dissolving Tween 60 in water at 70°C. Diclofenac was dissolved in a combination of PG and Transcutol and then mixed with the aqueous phase. The oily phase was added to aqueous phase and the mixture was homogenized at 8000 rpm for 5-6 minutes using a homogenizer (Omni Inc., Kennesaw, GA, USA). The formulation was then cooled and finally stored at room temperature. For the preparation of the emulgel, 1% Klucel was dispersed in cold water and mixed until a homogeneous mixture was obtained. The same emulsion was then mixed with the gel in a 1:1 ratio with gentle stirring to obtain the emulgel.

Gel formulation: The gel formulation was prepared by dispersing Klucel® very slowly in cold water with continuous stirring and the dispersion was allowed to hydrate for 60 minutes. The drug was dissolved in an appropriate mixture of PG and Transcutol and then transferred to the container containing the dispersion with continuous agitation until a clear homogenous gel was obtained.

Ointment: For the ointment, the drug was dissolved in PEG 400 which was heated to 70°C. PEG 3350 was then added to the mixture, the mixture was stirred slowly and then cooled to room temperature until completely congealed.

All the formulations were then evaluated under microscope (40X) up to 30 days.

Rheological studies: The different formulations were evaluated at 32°C by using the MCR 302 rheometer plate (Anton-Paar, Ashland, VA). A parallel-plate measuring device with a diameter of 25 mm and a gap of 0.1 mm was used for

performing the experiments. A peltier hood was used in conjunction with the fixture to maintain the temperature of the formulation at 32°C and to prevent any potential evaporation of the formulation. All measurements were performed in triplicate.

The viscosity of the different formulations were determined at different shear rates by the flow curve test. An amplitude sweep test then performed by using a strain between 0.01 to 100% in order to determine the linear viscoelastic (LVE) range of the samples. An oscillatory frequency sweep test was then performed over an angular frequency range of 0.1-100 rad/s.

Selection of membrane for IVRT: Several different membranes (VWR, Radnor,

PA, USA) shown in Table 7 were investigated. A standard solution of diclofenac sodium in the receiving medium was prepared at a low and high concentration range. Commercially available filter cartridges were assembled using the tested membranes. Both standard solutions were passed through the membrane filters, and these solutions were then analyzed in order to determine the extent of drug binding to the membrane.

Table 7. Commercially available membranes and their dimensions

Membrane Pore size Diameter

Cellulose acetate (OE 67) 0.45µ 25 mm

Nuclepore Track-etched Polycarbonate 0.4µ 25 mm

HT Tuffryn Polysulfone 0.45µ 25 mm

Nylon 0.45µ 25 mm

In vitro release: The release study was performed using vertical Franz diffusion

cells which were maintained at a temperature of 32°C. Based on the membrane

binding studies, a suitable membrane was chosen and mounted between the donor

and receptor compartment. 10mM PBS (pH 7.4) was used as the receiver solution

while 300 mg of each semi-solid formulation (n=6) was applied on the membrane.

Receptor samples were taken at specific time points up to 6h and replaced with

equal volume of fresh receptor. Sink conditions were maintained throughout the

experiments. All samples were analyzed by HPLC.

IVRT validation: Once an IVRT method has been developed, the next step is to

validate the method based on several key parameters which include

reproducibility or cell-to-cell variability, accuracy or the ‘sameness’ among

batches of the same composition tested at different times, inter-day and intra-day

precision and effect of dosage strength on the release rate. The slope and correlation coefficient (R2) for the line described by the square root of time (X axis) versus the cumulative amount release per surface area (Y axis) was calculated and line with R2>0.98 was found to be acceptable.

HPLC analysis: HPLC analysis was carried out on Alliance HPLC Waters 2695

Separations Module attached to a Waters UV detector. The HPLC assay was performed using a C8 Luna column (250 x 4.6 mm, 5 µ) (Phenomenex, Torrance,

CA, USA). The mobile phase consisted of 66% methanol and 34% 10 mM sodium phosphate buffer (adjusted to pH 3.0 with o-phosphoric acid). The flow rate and injection volume was set to 1.2 mL/min and 20 µL, respectively while wavelength was set to 276 nm.

Statistical analysis: The data obtained was subjected to statistical analysis using one-way analysis of variance (ANOVA) following Tukey’s test. P value of less than 0.01 was considered to be of significant difference.

Results

Semi-solid formulations: All the formulations shown in Table 6 were visually inspected and while the ointment was opaque in appearance, the emulsion was white and milky in appearance. The gel formulation was clear and homogenous while the emulgel was found to be opaque in a gel base. The pH of all

formulations was found to be in the range of 7.0-7.1. On observing under microscope, no visible drug crystals were observed in any of the formulations after storage for 30 months as seen in Figure 16.

Figure 16:Microscopic observation of (a) Gel, (b) Emulgel, (c) Emulsion and (d) Ointment

Rheological characterization: For such semi-solid pharmaceutical dosage forms, it is important to understand the rheology of the formulation as both the application and delivery of therapeutic agent may be affected by the consistency of the product. Hence, studies were done to understand the effect of various processing

conditions or stresses on the rheological properties of these formulations. The flow behavior for all formulation was found to be typically non-Newtonian. As seen in Figure 17, the viscosity for the ointment was found to be the highest amongst all the formulations.

Viscosity of semisolids

Emulsion Gel Emulgel Ointment

1000000 908,667 900000 800000 700000 600000 500000

Viscosity (cP) 400000 300000 200000 101,467 77,067 94,067 27,067 885 323 100000 31,100 10,997 3,947 4,053 1,353 0 1 20 75

Shear rate (1/s)

Figure 17. Change in viscosity of different formulations with increasing shear rate

On subjecting to an increasing strain, all the formulations exhibited a typical viscoelastic behavior where the storage modulus (G’) was higher than the loss modulus (G’’) which suggests that the microstructure of the formulations was highly dominated by cohesive forces (Figure 18).

Rheoplus

6 10

5 10

Diclo emulsion

4 G'' Loss Modulus 10 G' Storage Modulus

Diclo emulgel

G'' G'' Loss Modulus G' Storage Modulus 3 10 Diclo ointment G' G'' Loss Modulus G' Storage Modulus

Diclo gel

2 G'' Loss Modulus 10 G' Storage Modulus

1 10

0 10 0.1 1 10 rad/s 100 Angular Frequency

Anton Paar GmbH

Figure 18. Frequency sweep test for different semi-solid formulations

Selection of membrane: The filtered standard solutions were analyzed in order to evaluate recovery of DS. As seen in Table 8, for both the low and high concentration of standard solutions, the Nuclepore membrane showed the highest recovery compared to all the other membranes which means that the membrane was inert and offered least resistance to the free diffusion of the drug.

Table 8. Recovery of DS from standard solutions after passing through different membranes

Drug Conc. Recovery Membrane (µg/mL) (%) SD 10 88.95 0.20 Cellulose acetate 120 96.57 0.66 10 50.19 0.69 Nylon 120 29.00 4.35 10 64.13 0.93 Tuffryn 120 97.97 1.87 10 95.78 0.07 Nuclepore 120 99.49 1.07

In Vitro release: Based on the membrane binding studies, the Nuclepore membrane was used to evaluate the in vitro release of the different formulations.

When the amount of drug diffused per cm2 was plotted against the square root of time, a linear relationship was observed with correlation coefficient greater than

0.98 for all formulations indicating Higuchi’s diffusion kinetics. The drug release rate was found to be significantly higher for the gel formulation compared to all the other formulations as seen in Figure 19.

Gel Emulgel Ointment Emulsion

900.00 800.00 700.00 600.00

500.00

400.00

300.00

200.00

100.00 Cumulative amt release (ug/sqcm) 0.00 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0

Sq.rt of time (h0.5)

Validation of IVRT: In order to validate the IVRT, certain parameters were tested on the investigational formulations in order to confirm the ability of the method to differentiate between formulations with differing compositions.

Accuracy: Different batches of each formulation with identical composition were

prepared by the same process. The flux values were found to be comparable for

all the three different batches as seen in Table 9.

Table 9. Comparison of flux values for different batches having the same composition

Flux for Batch 1 Flux for Batch 2 Flux for Batch 3

Emulsion 284.84 297.22 313.73

Gel 378.21 369.2 368.52

Emulgel 340.17 337.03 343.96

Ointment 268.22 278.11 281.17

Where flux = slope of line where t0.5 is X-axis and cumulative amount

released is the Y-axis

Reproducibility: The inter-day reproducibility was evaluated by testing the same

batch for all formulations with identical composition on three different days as

seen below.

Emulsion Gel ) 1000.00 ) 1000.00 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 sqcm /

800.00 sqcm 800.00 µg g/ ( µ ( 600.00 600.00 release

400.00 release 400.00 amt amt 200.00 200.00

0.00 0.00

Cumulative 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Sq.rt of time Cumulative Sq.rt of time

Emulgel Ointment 1000.00 1000.00 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

g/sqcm) 800.00 800.00 g/sqcm) µ µ

600.00 600.00

400.00 400.00

200.00 200.00

0.00 0.00 Cumulative amt release amt Cumulative ( release

0.0 0.5 1.0 1.5 2.0 2.5 3.0 amt Cumulative ( release 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Sq.rt of time Sq.rt of time

Figure 20. Inter-day reproducibility for all semi-solid formulations. Data shown are mean (±SD) of 6 replicates.

The intra-day reproducibility was evaluated by testing the same batch on the same day at different times. As seen in Table 10, the flux values were found to be close with R2> 0.99 indicating that the method was reproducible.

Emulsion Gel 1000.00 ) 1000.00 Batch 1A Batch 1B Batch 1C Batch 1A Batch 1B Batch 1C

sqcm 800.00 800.00 g/ g/sqcm) µ µ ( 600.00 600.00

release 400.00 400.00 amt 200.00 200.00

0.00 0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cumulative Sq.rt of time amt Cumulative ( release Sq.rt of time

Emulgel Ointment ) Batch 1A Batch 1B Batch 1C Batch 1A Batch 1B Batch 1C

sqcm 600.00 1000.00 g/ µ

g/sqcm) 500.00 800.00 µ 400.00

release ( 600.00 300.00

amt 400.00 200.00 200.00 100.00 0.00 0.00

Cumulative 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Sq.rt of time amt Cumulative ( release Sq.rt of time

Figure 21. Intra-day reproducibility for all semi-solid formulations. Data shown are mean (±SD) of 6 replicates.

Table 10. Flux values for inter-day and intra-day reproducibility Inter-day reproducibility Intra-day reproducibility

Formulations Flux on Flux on Flux on Flux for Flux for Flux for

Day 1 Day 2 Day 3 Batch 1A Batch 1B Batch 1C

Emulsion 284.84 284.87 289.14 284.84 298.83 295.92

Gel 378.21 366.06 372.42 378.21 375.25 373.49

Emulgel 340.17 340.56 345.33 340.17 340.97 346.18

Ointment 268.22 265.87 268.29 268.22 272.33 262.50

Where flux = slope of line where t0.5 is X-axis and cumulative amount released is the Y-

axis

Effect of dosage strength: All the formulations shown in Table 6 were prepared by the same process but contained 0.5% drug. The release rate or flux and amount of drug released was found to be proportional to the dosage strength for all the formulations as seen in Figure 22 and Table 11.

Emulsion (0.5% DS) Emulsion (1% DS) Gel (0.5% DS) Gel (1% DS)

800.00 900.00 700.00 800.00 600.00 700.00 600.00 500.00 500.00 400.00 400.00 300.00 300.00 200.00 200.00 100.00 100.00 0.00 0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cumulative amt release (ug/sqcm)

Sq.rt of time Cumulative amt release (ug/sqcm) Sq.rt of time

Emulgel (0.5% DS) Emulgel (1% DS) Ointment (0.5% DS) Ointment (1% DS) 900.00 700.00 800.00 700.00 600.00 600.00 500.00 500.00 400.00 400.00 300.00 300.00 200.00 200.00 100.00 100.00 0.00 0.00

0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cumulative amt release (ug/sqcm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cumulative amt release (ug/sqcm) Sq.rt of time Sq.rt of time

Figure 22. Effect of dosage strength on release rate for all formulations. Data shown are mean (±SD) of 6 replicates

Table 11. Comparison of flux values for different dosage strengths of all formulations containing DS

Formulations Flux for 0.5% DS formulations Flux for 1% DS formulations

Emulsion 160.86 284.84

Gel 185.58 378.21

Emulgel 181.73 340.17

Ointment 135.01 268.22

Where flux = slope of line where t0.5 is X-axis and cumulative amount released is the Y-axis

Discussion

The overall effectiveness of a topical formulation depends on the composition of

the semi-solid vehicle as well as the physicochemical properties of the active

substance. The semi-solid formulation used in this study were first visually

observed and then examined under the microscope up to a period of 30 days.

None of the formulations showed any presence of drug crystals or phase

separation thus indicating their stability for this period. The pH of all the

formulations was found to be acceptable for application on skin without any

potential for irritation. As seen in Figure 17, the flow curve test showed that the

ointment had the highest viscosity amongst all the formulations. All the

formulations exhibited a shear-thinning behavior with high viscosity at a low

shear rate which quickly decreased with increasing shear. This indicates that the

formulations can be easily spread on the skin. The oscillation stress sweep test showed that the G’>G’’ indicating the predominant elastic properties of the formulations. In case of the gel formulation, a crossover of the G’ and G’’ at a relatively low strain shows that the formulation becomes progressively more fluid-like and is an indication of the relaxation of the internal structure of the network formed by the polymer. This property has been found to be characteristic of gels containing hydroxypropylcellulose as polymer and indicates a greater elastic contribution which can be related to physical properties such as pourability, processability and so on (Ramachandran, Chen, & Etzler, 1999).

The FDA has recognized the IVRT to be an important tool in the development of topical semi-solid formulations as the test is based on sound scientific principles and has been originally used to assure product sameness between pre- and post- change topical dosage forms. The √t–release rate is a distinguishing property of each formulation and may be indicative of the clinical performance of the particular semi-solid formulation (Flynn et al., 1999). In this study, the Nuclepore membrane was shown to have no measurable resistance to the diffusion of DS.

The release of DS from the different vehicles was found to follow the Higuchi diffusion model and the drug release was found to be less than 30% thus satisfying the requirements of a typical IVRT (Thakker & Chern, 2003) . The gel formulation showed the highest drug release in comparison to all the formulations

probably due to the fluid-like nature of the formulation. Previous studies have shown that formulations with higher viscosity like the ointment exhibit lower release for NSAID drugs like tiaprofenic acid (Özsoy, Güngör, & Cevher, 2004).

On testing the different parameters for validation, the developed method was able to differentiate between the formulations, was found to detect changes in dosage strength and also assured a good correlation between the uniformity of same and different batches for all the formulations. Thus, the method was found to be

‘valid’ for all the parameters that were tested as part of the study.

Conclusion

The semi-solid formulations were found to be stable and exhibited typical rheological behavior that were found to be favorable for application on skin. The

IVRT method was able to differentiate changes in composition of the vehicles with different rheological properties with the gel formulation exhibiting the highest release rate amongst all the formulations. Thus, the developed method was found to be robust, precise and discriminatory and serves well as a pre- formulation tool for the development of topical formulations with good aesthetic acceptability and efficacy.

CHAPTER 6

FORMULATION AND EVALUATION OF A POSITIVELY CHARGED

NANOEMULSION FOR THE TOPICAL DELIVERY OF MINOXIDIL

Abstract

Minoxidil is commonly used in the treatment of androgenic alopecia. However, the commercially available ethanol-based solutions often cause adverse effects like scalp dryness and contact dermatitis on topical application and unwanted cardiovascular effects due to systemic absorption. The aim of this study was to develop an alcohol-free positively charged nanoemulsion which can potentially enhance the topical and transfollicular delivery of minoxidil. A series of o/w nanoemulsion formulations containing 2% w/w minoxidil were prepared by using the high-pressure homogenization method. The mean particle size and zeta potential of these formulations were then evaluated over a period of 60 days.

Short term stability was determined by centrifuging the formulations in order to examine for phase separation. Finally, in vitro permeation studies on porcine ear skin was performed in order to compare the topical delivery of the nanoemulsion with a solution as control. The formulations (500 µL) were applied on skin and samples were taken up to 24h. Differential tape stripping was performed in order to determine amount of drug in stratum corneum and hair follicles and quantified

using HPLC. Amongst all the formulations, only one formulation did not show significant change in particle size and zeta potential over time up to 60 days while the polydispersity index was less than 0.2. No phase separation was observed as well on centrifugation indicating the stability of the formulation. In vitro permeation studies showed very low amount of drug in the receptor passively.

The application of F9 in combination with iontophoresis resulted in a significantly higher amount of drug in the stratum corneum and hair follicles compared to passive delivery. This combination consistently delivered a higher amount of drug in skin compared to the solution over time. Thus, a stable, positively charged nanoemulsion was developed using high-pressure homogenization. In vitro permeation studies showed that the formulation F9 has potential in topical and transfollicular delivery of minoxidil with reduced systemic uptake.

Introduction

Androgenic alopecia or alopecia areata is a chronic inflammatory disorder that affects hair follicles and leads to hair loss. Minoxidil is one of the commonly use drugs that is employed as a first-line topical treatment and has been approved by the US FDA for the treatment of androgenic alopecia. The 2% product for hair regrowth in men was first marketed in 1986. However, it has been frequently reported that the effects of minoxidil is temporary and can also cause skin

irritation due to the solvents present in the formulation. Topically applied minoxidil is also systemically absorbed and can lead to unwanted cardiovascular effects (Messenger 2004, Sentjruc et al 1999). In order to minimize size effects and improve efficacy, these drawbacks can be resolved by the use of nanoemulsions that are suitable for localized delivery and and allow for a reduction of the dose applied on skin (Manconi, Sinico, Valenti, Lai, & Fadda,

2006; Yu, Ma, Lei, Li, & Tan, 2014). Using a positively charged nanoemulsion in this case could also present several advantages such as promoting adsorption to negatively charged skin which can potentially increase the drug bioavailability

(Piemi, Korner, Benita, & MartyJp, 1999). This study used phytosphingosine (PS) to impart the positive charge to the nanoemulsion. PS is a natural compound present in mammalian SC and indirectly influences a wide variety of functions such as inflammation of the skin, skin hydration and elasticity. It is a sphingoid base with a pKb of approximately 9, thus, at physiological pH of skin which is approximately 5.5, it is positively charged which should improve the topical absorption of minoxidil when applied topically.

Studies have shown that the transfollicular pathway plays a major role in the skin penetration of topical applied substances and serves as a reservoir for the major entry point of drugs such as minoxidil (Blume-Peytavi et al., 2010). In addition, hair follicles have been implicated as possible ion channels for localized drug

delivery in which the significant contribution of iontophoresis has been well documented (Scott, White, & Bradley Phipps, 1992). Iontophoresis uses a small electric current to increase drug delivery in and through skin and the lower appendageal resistance helps in the involvement of this route during electro- transport. Hence, a positively charged formulation would be favorable to anodal iontophoresis which can potentially result in enhanced topical absorption at the target site. Pre-clinical studies have shown that using a combination of iontophoresis and nanosized formulation enhanced the drug delivery of lipophilic drugs as well as increased the potential of transcutaneous immunization (Bernardi et al., 2016; Charoenputtakun, Li, & Ngawhirunpat, 2015)

The aim of this study was to develop and formulate a positively charged nanoemulsion (NE) containing minoxidil as active. In addition, anodal iontophoresis was used to determine whether a combination of iontophoresis and nanoemulsion would specifically target and enhance the follicular delivery of minoxidil in the treatment of androgenic alopecia.

Materials and Methods:

Minoxidil was purchased from Alfa Aesar (Haverhill, PA, USA), PS was obtained from TCI America (Portland, OR, USA) while Lipoid E80 was kindly gifted by

Lipoid LLC (Newark, NJ, USA). Tween 80 and octyldodecanol were samples from BASF (Tarrytown, NY, USA) while Transcutol was kindly provided by

Gattefosse (Paramus, NJ, USA). PG, silver wire and silver chloride electrodes were purchased from Fisher Scientific (Waltham, MA, USA).

Preparation of nanomeulsions: A series of positively charged NEs containing 2% w/w minoxidil were prepared by using the high pressure homogenizer (Nano

DeBee, BEE International, MA, USA). Different concentrations of Tween

80/Lipoid E80 and phytosphingosine were used as emulsifying and charge- inducing agents, respectively. The oil phase contained PS, minoxidil, Lipoid E80 and octyldodecanol while the aqueous phase contained Tween 80 and potassium sorbate. Both phases were heated separately to about 75°C and the pre-emulsion was obtained by homogenizing the mixture at 8000rpm for 5 minutes. The nanoemulsion was obtained by passing the mixture through the high pressure homogenizer at a pressure of 10,000 psi for 10 cycles. The pH of all the formulation was then adjusted to approximately 5.5 by using dilute hydrochloric acid solution.

Table 12. Composition of the o/w nanoemulsion formulations containing minoxidil

Ingredients F1 F2 F3 F4 F5 F6 F7 F8 F9

Minoxidil 2 2 2 2 2 2 2 2 2

Phytosphingosine 0.6 0.8 1.0 0.6 0.8 1.0 0.6 0.8 1.0

Lipoid® E80 2 2 2 3 3 3 1 1 1

Tween 80 2 2 2 3 3 3 1 1 1

Octyldodecanol 15 15 15 20 20 20 25 25 25

Transcutol 10 10 10 10 10 10 10 10 10

Propylene glycol 5 5 5 5 5 5 5 5 5

Benzoic acid 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Water q.s to 100

Characterization: Particle size and zeta potential: The particle size of the NEs was measured on Day 0. They were then stored at room temperature and both the particle size and zeta potential were measured after Days 15, 45, 30, and 60. The dynamic light scattering technique was used to measure PS where a PDI below

0.2 indicates a narrow size distribution. The surface charge or zeta potential was also determined for the same time points by using the Zetasizer Nano ZS

(Malvern Instruments, UK).

Short term stability was determined by centrifuging the formulations at 4000 rpm for 30 minutes at 40°C in order to examine for phase separation.

Transmission Electron Microscopy: A drop of the suitably diluted nanoemulsion was applied to a carbon film-covered copper grid and was stained with 1% phosphotungstic acid. The sample was then examined with a transmission electron microscope (Hitachi H-7500, Illinois) after allowing to dry for 10 minutes.

Passive permeation: Porcine ear skin was obtained from a local abattoir and was stored frozen. The skin was dermatomed to a nominal thickness of 700µm, thawed and mounted on a Franz diffusion cell. The donor compartment was filled with either 0.5 mL of nanomeulsion or minoxidil in PG:Ethanol:Water (20:20:60 v/v) which served as a control. The receiver chamber was filled with 10 mM PBS solution containing 25mM NaCl. Samples were collected at different time points up to 24h and amount of MNX in receiver, SC and hair follicles was analyzed by

HPLC.

Iontophoresis: Iontophoretic experiments were conducted in the same way as passive permeation except that a direct current of 0.5mA/cm2 was applied to the skin using Ag/AgCl electrode for 4 hours. Post-iontophoresis, the formulation was left in contact with the skin for 2h and 4h and amount of drug in receptor, SC and hair follicles was determined.

Differential tape stripping: The follicular and SC uptake of MNX was evaluated by different tape stripping which combines tape stripping with cyanoacrylate surface biopsies. The residual formulation was cleaned from the skin surface by using cotton swabs. The skin was then tape stripped 15 times using D-squame tapes (CuDerm, TX, USA) until the glistening surface was visible which confirmed complete SC removal. The amount of drug present in the tapes was evaluated after overnight extraction with methanol. After SC removal, a drop of cyanoacrylate glue was applied to the stripped skin and covered with a tape-strip.

The tape was then removed after total polymerization of the glue (after 5 minutes). This tape contained the follicular casts which was analyzed by following the same extraction procedure for MNX with methanol.

HPLC analysis was carried out on Alliance HPLC Waters 2695 Separations

Module attached to a Waters UV detector. The column used was Luna Phenyl-

Hexyl, C18 (150 x 4.6 mm, 5µ) (Phenomenex, Torrance, CA, USA). The HPLC assay was performed using an isocratic method with acetonitrile and water (0.1% acetic acid) (15:85 v/v) and flow rate of 1.0 ml/min. Wavelength of detection was set at 281 nm.

Statistical analysis: Statistical comparison was carried out using Student’s t-test to determine significant difference between the groups. A 0.05 level of probability

(p< 0.05) was taken as the level of significance.

Results

Formulation of nanoemulsions: As seen in Table 12, a series of NEs were prepared by the high pressure homogenization technique. The NEs were produced by high pressure homogenization at 25°C, 10,000 psi and 10 homogenization cycles. The effect of different concentrations of Tween 80/Lipoid E80 and PS was investigated with respect to changes in size, size distribution (polydispersity index), surface charge and physical stability. On centrifugation, only F7, F8 and

F9 did not show any visible phase separation. Hence, theses formulations were used further for physicochemical characterization.

Characterization: Particle size analysis: The mean particle size of F7. F8 and F9 respectively was evaluated on day of production (Day 0). After 60 days, the particle size for most of the formulations changed significantly while there was not much difference in the case of F9 as seen in Figure 23. The highest PDI for

F9 was less than 0.2 even after 60 days which indicates the narrow size distribution of the nanoemulsion droplets

250.00 Day 0 Day 15 Day 30 Day 45 Day 60

200.00

150.00

100.00

50.00 Particle size (nm) size Particle

0.00 F7 F8 F9

Figure 23. Particle size of NE formulations during storage for 60 days

Zeta Potential: As a general rule, any formulation with a zeta potential over 30mV is regarded as physically stable. In this case, formulations F9 consistently showed a greater value of zeta potential over a period of 60 days as seen in Figure 24.

However, in case of F9, there was no significant difference in the zeta potential compared to the other formulations over a period of 2 months indicating the stability of the formulation.

Day 0 Day 15 Day 30 Day 45 Day 60

60.00 50.00 40.00 30.00 20.00

Zeta potential (mV) potentialZeta 10.00 0.00 F7 F8 F9

Figure 24.Zeta potential of NE formulations during storage for 60 days

Morphology: TEM analysis of the F9 NE was performed and the resultant micrograph is shown in Figure 25. In general, majority of the NE droplets were spherical in shape without any visible agglomeration.

Figure 25. TEM micrograph of F9 NE showing spherical droplets

Passive and iontophoretic drug delivery: The percutaneous absorption of MNX from either a simple solution or from the NE is shown in Table 13. The amount of drug in the receptor solution increased as a function of time with almost double the quantity of drug in case of the solution as compared to the NE. As shown, the levels of drug in the SC tape strips exhibited a consistently higher and significant amount of drug for the NE as compared to the solution. Similarly, follicular delivery of MNX from the NE was consistently greater than the solution as well, almost being twofold higher at the end of 24h.

Table 13. MNX permeation and skin uptake after 24h of passive diffusion using control solution or F9 NE

Vehicle

Amount of MNX Control solution F9 NE (PG:EtOH:Water)

Receptor (µg/cm2 ) 1.85 ± 0.59 3.75 ± 0.44

SC (µg/cm2) 28.43 ± 4.95 12.12 ± 3.45

Hair follicles (µg/cm2) 2.53 ± 0.43 1.21 ± 0.09

Data shown are mean (± SD) of four replicates. MNX penetration in skin and hair follicles was significantly enhanced (p< 0.05) by application of F9 formulation.

Since the NE is positively charged, it was postulated that an anodal current would provide a more ‘targeted’ delivery and potentially enhance follicular localization.

Following 4h of iontophoresis, the passive data was then compared with the iontophoretic data as shown in Figure 26. It is clear that iontophoresis significantly enhanced the delivery of MNX into the SC and hair follicles as compared to the control. In case of the receptor, no drug was found in the receptor for the F9 NE passively at the end of 4h. However, the amount of drug in the receptor was higher with iontophoretic delivery for both formulations. For both

formulations, drug uptake into SC and hair follicles was significantly higher as compared to passive delivery.

Control Iontophoresis F9 NE Iontophoresis Control Passive F9 NE Passive

14.00 ** 12.00

10.00 * 8.00 6.00 4.00 ** 2.00 * Avg cum amt/sqcm (µg/sqcm)amt/sqcm Avgcum 0.00 Receptor SC Hair Follicles

Post-iontophoretic drug delivery: In another set of experiments, after 4h of iontophoresis, drug disposition was evaluated over the next 4h while leaving the formulation in contact with the skin. The results recorded in Figure 27 show the amounts recovered after 0h which is immediately after stopping iontophoresis, 2h and 4h. A consistent and significant increase in MNX delivery from the F9 NE was observed at different time intervals for both the SC and hair follicles.

MNX solution Iontophoresis MNX F9 NE Iontophoresis 20 * * 15 *

0 5 0h 2h 4h * Amt in SC (µg/sqcm)SC inAmt 4 * 3 * 2 1 0 Amt in hair follicles (µg/sqcm hairfollicles inAmt

Discussion

The appendageal route has been reported of significance for delivery of compounds such as MNX in the treatment of alopecia. Several studies have used nanosized formulations for increasing the bioavailability of MNX at the target site and/or to target the drug to the desired site of action (Cevc, 2004; Manconi et al.,

2006). Moreover, it has been shown that smaller particles with size up to 320nm may be retained in the hair follicle for up to 10 days (Lademann et al., 2007).

Hence, the aim of this study was to develop a nanoemulsion formulation that is

positively charged, stable and therapeutically effective with reduced systemic effects.

As seen in Figure 23, the particle size for F7 and F8 was smaller but negligible compared to F9 due to the lower concentration of PS. The zeta potential increased with increasing concentration of PS suggesting increased physical stability. Only

F9 remained physically stable when stored at room temperature with no significant change in particle size over time. This result was also confirmed by zeta potential measurements where a decrease in the zeta was observed only for

F9 nanoemulsion (Figure 24). Previous studies have shown that the amphiphilic structure of F9 serves as a co-surfactant and aids Lipoid E80 and Tween 80 in forming a stable film at the o/w interface (Baspinar, Keck, & Borchert, 2010).

TEM analysis confirmed the morphology and stability of the NE without any signs of visible agglomeration.

All the permeation studies performed used porcine skin which represents a better model compared to excise human skin for the uptake of topically applied compounds to the hair follicles (Lademann, Richter, Meinke, Sterry, & Patzelt,

2010). This is due to contraction of pig skin to a lesser extent avoiding potential artifacts in vitro. As seen in Table 13, the MNX recovery from tape strips and follicular casts were significantly higher from the F9 NE compared to the control solution passively after 24h. Previous studies have reported the recovery of MNX

from SC and hair follicles being of the same order as the cumulative penetration across skin when a finite dose of the formulation was applied and was dependent on the vehicle composition (Grice et al, 2010). Notably, the amount of drug recovered from the hair follicles was appreciable smaller in comparison to the SC.

However, as previously stated, since the follicles occupy a fractional area, it becomes clear that the NE formulation dramatically enhanced the follicular localization of MNX. If we consider human scalp skin, this finding is of significance since the scalp skin has about 10 times the amount of hair follicles as compared to porcine skin (Jacobi et al., 2007).

Previously, it has been shown that the follicular delivery of minoxidil sulfate, a hydrophilic derivative of MNX, was increased from an aqueous solution following application of iontophoresis both in vitro and in vivo (Gelfuso, Gratieri,

Delgado-Charro, Guy, & Vianna Lopez, 2013). In addition, the pH of the formulation for iontophoresis influences drug ionization as well as its subsequent transport. In this case, at a pH of 5.5, the formulation was positively charged, which was confirmed by the positive zeta potential of the formulation. Hence, it was hypothesized that anodal iontophoresis would enhance follicular localization due to the combination of electroreplusion and electroosmosis. Experiments were then performed where the passive data was compared to the results obtained following application of 4h of iontophoretic current using both formulations in

order to explore if a more ‘targeted’ delivery to the hair follicles was achievable.

After 4h of iontophoresis, amount of MNX in the SC and hair follicle was found to be significantly greater in comparison to passive permeation as seen in Figure

26. The relative enhancement in follicular uptake achieved with the use of the anodal current was twofold which implies improved bioavailability as well.

Finally, post-iontophoretic delivery was performed where both the formulations were allowed to remain in contact with the skin for 2h and 4h as seen in Figure

27. The goal of these experiments were to assess whether the apparent enhancement of follicular uptake by using the NE formulation could be sustained over a period of time. It was seen that the NE formulation consistently delivered a greater amount of drug to the SC and hair follicles (p< 0.05) at all time points compared to the solution. This outcome is desirable as the topical treatment of alopecia requires a localized effect with minimal systemic exposure. Clinically, the dose application of the marketed formulation is 1mL which is to applied on the scalp twice daily and often leads to skin irritation (Sinclair 2005). The formulation developed in this study is free from alcohol which would potentially improve the cutaneous tolerance and decrease any irritation when applied on scalp skin. However, from a practical viewpoint, the judicious use of current density and application time is required for optimal therapy.

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Conclusion

A stable, positively charged nanoemulsion was developed using high-pressure homogenization. In vitro permeation studies showed that the application of formulation F9 resulted in enhanced topical and transfollicular delivery of minoxidil with reduced systemic uptake passively compared to a simple solution.

Further, the use of iontophoresis significantly enhanced the skin and follicular uptake of MNX compared to passive delivery by almost twofold. On comparing both formulations post-iontophoresis, the NE consistently delivered a high amount of drug to the SC and follicles compared to the solution. Thus, it can be surmised that a combination of iontophoresis and nanoparticles represents a potentially useful strategy for providing a targeted therapy in the treatment of alopecia.

CHAPTER 7

SUMMARY AND CONCLUSION

Topical drug delivery has been found to offer several advantages over the conventional routes of administration (oral, parenteral) such as avoiding fluctuations in drug levels, reduced systemic effects, ability to apply formulations close to the target site and increased patient compliance due to the non- invasiveness nature and ease of use. With topical therapy, the formulation plays an equally important role as the interaction of the vehicle with the skin can alter the efficacy of the penetrant. The formulation ensures that the drug substance is delivered to the right target site and maintains the dosage integrity for the required period. For a drug to act locally or even systemically, it must first cross the stratum corneum, which is the main barrier for drug penetration into skin.

Different passive or active enhancement technologies may be used to achieve this objective (Brown, Martin, Jones, & Akomeah, 2006). Hence, the aim of this research project was to formulate different topical formulations that can be potentially used to treat a variety of indications, characterize them by using various techniques, evalute the in vitro release or penetration in skin and determine their efficiency in drug delivery to the target site.

101

102

The anhydrous gel formulation containing sertaconazole nitrate was used to estimate the amount of drug in the SC by the dermatopharmacokinetic method or skin stripping which has been used to study the penetration of topically applied substances. The one-layered diffusion model which has been used extensively

(Todo, Oshizaka, Kadhum, & Sugibayashi, 2013) was used to predict skin concentrations following topical application. Establishing a dermal absorption profile helps in optimizing the formulation and improving the therapeutic efficacy of the product. The estimated drug concentration-SC depth profile showed that the formulation may reach the target site in adequate concentrations for effective therapy. In addition, skin irritation potential of the formulation was evaluated as it can have a significant influence on therapeutic effectiveness and may result in adverse reactions. In some cases, the cause of skin irritation may be the vehicle and not the drug itself, however, in our study, the anhydrous formulation was found to be non-irritant in nature.

Topical analgesics like menthol are commonly available in a variety of topical dosage forms in the market. However, there is limited literature available on the percutanoues absorption of this compound. Hence, the main objective of this study was to evaluate the drug penetration in human skin using different topical vehicles. Besides optimizing drug bioavailability, it is important to ensure the formulation is aesthetically acceptable to the patient, is easy to spread and adheres

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to the treated area for the required time. Hence, the rheological characterization of the vehicles was also carried out and the final outcome of this study showed that the topical formulations showed a correlation between their rheological properties and drug penetration in skin. Thus, topical formulations can be optimized for better efficacy and drug delivery to the site of action.

Ideally, topical semi-solid preparations should exhibit a number of advantageous characteristics such as excellent viscoelasticity, spreadability and tolerance to the effects of stresses on the product. Formulations lacking these desirable properties may be perceived as unacceptable by the end user. Hence, our study attempted to predict the effects of physiological stresses on product performance by using the rheometer. In addition, IVRT has been recognized as an important tool in research and development for optimizing topical formulations. Hence, an IVRT method was successfully developed and validated for evaluating the release of diclofenac sodium from different semi-solid vehicles.

Finally, a combination of iontophoresis and use of a novel nanoemulsion formulation was explored in order to evaluate the topical delivery of minoxidil.

Since conventional formulations contain solvents which often leads to adverse reactions, the aim was to develop an alcohol-free formulation with increased drug retention in skin. The study showed that the formulation not only increased the bioavailability of the drug in skin, but also resulted in an increase in the follicular

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content with the use of iontophoresis. In conclusion, use of this formulation may allow a reduction of the dose applied on skin which in turn will lead to decreased systemic effects and result in improved therapeutic efficacy.

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