Figure 1.1 Cross section of skin

1) Epidermis

It is the outermost layer of the skin, which is about 150 micrometers thick. Cell from lowers layers of the skin travel upward during their life cycle and become flat dead cell of the corneum.The source of energy for lower portions of epidermis glucose, and the end product of metabolism, lactic acid accumulates in skin. During differentiation from basal cells to stratum corneum by degradation of the existing cellular components, the entire cellular build-up changes. Specialized cellular somes contain a host lytic enzyme, which they release for intracellular lysis. The epidermis is a reservoir of such lytic enzymes. Many of these enzymes are inactivated (probably by auto catalytic processes) in upper granular layer; however, many also survive into the stratum corneum. The stratum corneum also has proteolytic enzymes involved in this desquamation.

Stratum Germinativum:

Basal cells are nucleated, columnar. Cells in this layer have high mitotic index and they constantly renew the epidermis and this proliferation in healthy skin balances the loss of dead horny cells from the skin surface.

Malpighion Layer:

The basal cell also include melanocytes which produce and distribute melanin granules to the keratinocytes required for pigmentation which is a protective measure against radiation.

Stratum Spinosum:

The cell of this layer is produced by morphological and histochemical alteration of the cells basal layers as they moved upward. These cells flatten and their nuclei shrink. They are interconnected by fine prickles and form intercellular bridge called desmosomes. These links maintain the integrity of the epidermis.

Stratum Granulosum:

This layer is above the keratinocytes. They manufacture basic staining particle, the keratinohylline granules. This keratogenous or transitional zone is a region of intense biochemical activity and morphological change.

Stratum Lucidum:

In the palm of the hand and sole of the foot this layer is present and it forms a thin, translucent layer immediately above the granule layer. The cells in this layer are non-nuclear.

Stratum corneum:

At the final stage of differentiation, epidermal cell construct the most superficial layer of epidermis called stratum corneum. At friction surface of the body like palms and soles, the stratum corneum adapt for weight bearing and membranous stratum corneum over the remainder of the body is flexible but impermeable. The horny pads of sole and palm are at least 40 times thicker than the membranous horny layer

2) Dermis

Despite its greater volume, the dermis contains far fewer cells than the epidermis and instead much of its bulk contains fibrous and amorphous extra cellular matrix interspersed between the skin's appendages, nerves, vessels, receptors and the dermal cells. The main cell type of the dermis is the fibroblast which is a heterogeneous migratory cell that makes and degrades extracellular matrix components. There is significant current interest in the factors that control the differentiation of the dermal fibroblast, particularly in the context of their increased synthetic and proliferative activity during wounding healing. The dermis is home to many cell types including multi-functional cells of the immune system like macrophages and mast cells, the latter which can trigger allergic reactions by secreting bioactive mediators such as histamine.

Subcutaneous tissue

This layer consist of sheet of fat rich areolar tissue, know as superficial fascia and it attaches the dermis to the underlying structure. Large arteries and veins are present only in the superficial region.

Skin Appendages

The skin is interspersed with hair follicle and associated sebaceous gland like regions which are two types of sweat glands i.e eccrine and apocrine. Collectively these are referred to as skin appendages.^4-7

Figure 1.2 Scheme of events for percutaneous absorption

1.3 Functions of skin:

• Containment of body fluids and tissues.

• Protection from external stimuli like chemicals, light, heat, cold and radiation.

• Reception of stimuli like pressure, heat, pain etc.

• Biochemical synthesis.

• Metabolism and disposal of biochemical wastes.

• Regulation of body temperature.

• Controlling of blood pressure.

• Prevent penetration of noxious foreign material and radiation.

• Cushions against mechanical shock.

• Interspecies identification and/ or sexual attraction.8

1.4 Absorption of drug through skin:

Absorption of drug through skin takes place by two routes:^9-11

1) Transepidermal absorption

2) Transfollicular (shunt pathway) absorption; which is shown in figure 1.2

1.4.1 Transepidermal absorption

It is now generally believed that the transepidermal pathway is principally responsible for diffusion through the skin. The resistance encountered along this pathway arises in the stratum corneum. Permeation by the transepidermal route first involves partitioning into the stratum corneum. Diffusion then takes place across this tissue. The current popular belief is that most substances diffuse across the stratum corneum via the intercellular lipoidal route. This is a tortuous pathway of limited fractional volume and even more limited productive fractional area in the plane of diffusion. However, there appears to be another microscopic path through the stratum corneum for extremely polar compounds and ions. Otherwise, these would not permeate at rates that are measurable considering their o/w distributing tendencies. When a permeating drug exits at the stratum corneum, it enters the wet cell mass of the epidermis and since the epidermis has no direct blood supply, the drug is forced to diffuse across it to reach the vasculature immediately beneath. The viable epidermis is considered as a single field of diffusion in models. The epidermal cell membranes are tightly joined and there is little to no intercellular space for ions and polar nonelectrolyte molecules to diffusionally squeeze through. Thus, permeation requires frequent crossings of cell membranes, each crossing being a thermodynamically prohibitive event for such water-soluble species. Extremely lipophilic molecules on the other hand, are thermodynamically constrained from dissolving in the watery regime of the cell (cytoplasm). Thus the viable tissue is rate determining when nonpolar compounds are involved.

Passage through the dermal region represents a final hurdle to systemic entry. This is so regardless of whether permeation is transepidermal or by a shunt route. Permeation through the dermis is through the interlocking channels of the ground substance. Diffusion through the dermis is facile and without any molecular selectivity since the gaps between the collagen fibers are far too wide to filter large molecules. Since the viable epidermis and dermis lack measure physiochemical distinction, they are generally considered as a single field of diffusion, except when penetrants of extreme polarity are involved, as the epidermis offers measurable resistance to such species.

1.4.2 Transfollicular (shunt pathway) absorption

The skin’s appendages offer only secondary avenues for permeation. Sebaceous and eccrine glands are the only appendages, which are seriously considered as shunts bypassing the stratum corneam since these are distributed over the entire body. Though eccrine glands are numerous, their orifices are tiny and add up to a miniscule fraction of the body’s surface. Moreover, they are either evacuated or so profusely active that molecules cannot diffuse inwardly against the glands output. For these reasons, they are not considered as a serious route for percutaneous absorption. However, the follicular route remains an important avenue for percutaneous absorption since the opening of the follicular pore, where the hair shaft exits the skin, is relatively large and sebum aids in diffusion of penetrants. Partitioning into sebum, followed by diffusion through the sebum to the depths of the epidermis is the envisioned mechanism of permeation by this route. Vasculature sub serving the hair follicle located in the dermis is the likely point of systemic entry. For absorption across a biological membrane, the current or flux and in terms of matter of molecules rather than electrons are the determining factors and the driving force is concentration gradient (technically, a chemical potential gradient) rather then a voltage drop. Membranes act as a “diffusional resistor.” Resistance is proportional to thickness (h), inversely proportional to the diffusive mobility of matter within the membrane or to the diffusion coefficient (D), inversely proportional to the fractional area of a route (F), and inversely proportional to the carrying capacity of a phase.

R = h/FDK

R =Resistance of diffusion resistor

F = Fractional area of diffusion membrane

H = Thickness of membrane

D = diffusivity of drug molecules

K = Relative carrying capacity of the diffusion phase

1.5 Kinetics of permeation through skin:

In the initial transient diffusion stage, drugs molecules may penetrate the skin along the hair follicles or sweat ducts and then be absorbed through the follicular epithelium and sebaceous glands. When the steady state has been reached, diffusion through stratum corneam becomes the dominant pathway.

The membrane-limited flux (J) under steady condition is described by the following expression.

J = - DAKO/W r C / h

Where:

J = Amount of drug passing through the membrane system per unit area, per unit area per unit time.

D= Diffusion coefficient of drug molecule

A= Area of the diffusion membrane

C= Concentration gradient

Ko/w= Partition coefficient of drug

h= Thickness of the membrane.

Knowledge of skin permeation is vital for the successful development of topical preparation. Permeation of a drug involves the following steps,

• Sorption by stratum corneum,

• Penetration of drug though viable epidermis,

• Uptake of the drug by the capillary network in the dermal papillary layer.

This permeation can be possible only if the drug possesses certain physicochemical properties. The rate of permeation across the skin (dQ/dt) is given by:

dQ / dt = Ps(cd-cr)

Where Cd and Cr are, the concentrations of skin penetrant in the donor compartment (e.g., on the surface of stratum corneum) and in the receptor compartment (e.g., epidermal layers of skin) respectively. Ps is the overall permeability coefficient of the skin tissues to the penetrant. This permeability coefficient is given by the relationship:

Ps = Ks Dss / Hs

Where, Ks is the partition coefficient for the interfacial partitioning of the penetrant molecule form a solution medium on to the stratum corneum, Dss is the apparent diffusivity for the steady state diffusion of the penetrant molecule through a thickness of skin tissues and Hs is the overall thickness of skin tissues. As Ks, Dss and Hs are constant under given conditions, the permeability coefficient (Ps) for a skin penetrant can be considered to be constant.

From equation (1) it is clear that a constant rate of drug permeation can be obtain when Cd >> Cr

i.e., the drug concentration at the surface of the stratum corneam (Cd) is consistently and substantially greater than the drug concentration in the epidermis of skin (Cr).

The equation (1) becomes:

dQ / dt = PsCd

And the rate of skin permeation (dQ/dt) is constant provided that the magnitude of Cd remains fairly constant throughout the course of skin permeation. For keeping Cd constant, the drug should be released from the device at a rate (Rr) that is either constant or greater than the rate of skin uptake (Ra) i.e., Rr >> Ra.^9-11,12

1.6 Factors affecting topical permeation:

Factors affecting topical permeation of drug depends upon various factors such as;

1.6.1 Physicochemical properties of drug substance

• Partition coefficient of drug

• Ionization constant of drugs

• Solubility of drug

• Concentration of drug

• Particle size of drug

• Polymorphism

• Molecular weight of drug ^13,14

1.6.2 Penetration enhancer

Percutaneous absorption can be enhancing in two ways either by chemical enhancer or by physical method.

Chemical penetration enhancer: By definition, a chemical skin penetration enhancer increase skin permeability by reversibly damaging or by altering the physicochemical nature of the stratum corneam to reduce its diffusional resistance. Among the alterations are increased hydration of stratum corneam and / or a change in the structure of the lipids and lipoproteins in the intercellular channels through solvent action or denaturation. These are classified under the following main heading:

1) Solvents: These compounds increase penetration possibly by swelling the polar pathway and/or by fluidizing lipids. Examples include water, alcohols, methanol and ethanol; alkyl methyl sulfoxide, dimethyl sulfoxide, alkyl homologs of methyl sulfoxide, dimethyl acetamide and dimethylformamide; pyrrolidones- 2 -pyrrolidone, N-methyl, 2- pyrrolidone; laurocapram (Azone), miseellancous solvents- propnylene glycol, glyeerol, silicone fluids, isopropyl palmitate.

2) Surfactant: These compounds are proposed to enhance polar pathway transport, especially of hydrophilic drugs. The ability of the surfactant to alter penetration is a function of polar head group and the hydrocarbon chain length. Commonly used surfactant are as follow

Anionic surfactant: can penetrant and interact strongly with skin. Examples include are dioctyl sulphosuccinate, sodium lauryl sulphate, decodecylmethyl sulphoxide etc.

Cationic surfactant: Cationic surfactants are reportedly more irritating than anionic surfactants and they have not been widely studied as skin permeation enhancer. Nonionic surfactant: Nonionic surfactants have least potential for irritation. Example includes are Pluronic F127, Pluronic F68 etc.

3) Bile salts: Sodium taurocholate, sodium deoxycholate, and sodium tauroglycocholate.

4) Binary system: These systems apparently open the heterogeneous multilaminated pathway as well as the continuous pathways. Examples include are prolylene glycol -oleic acid, phosphatidyl choline and 1,4-butane diol- linoleic acid.

5) Miscellaneous chemicals: These includes urea, N,N-dimethyl-m-toluamide, calcium thioglycolate etc.^15-20

1.6.3 Physicochemical properties of topical drug delivery system

• Release characteristics: The mechanism of drug release depends on whether the drug molecules are dissolved or suspended in the delivery system. The interfacial partition coefficient of drug from delivery systems to the skin and pH of the vehicle

• Composition of drug delivery system: Example polyethylene glycols of low molecular weight decrease permeation.

• Nature of vehicle: Liphophilic vehicle increase permeation where as lipophobic vehicle decrease permeation.8

1.6.4 Physiological and pathological condition of skin

• Reservoir effect of horny layer: The horny layer, depot and modify the transdermal permeation characteristics of some drugs. The reservoir effect is due to irreversible binding of a part of the applied drug with the skin. This binding can be reduced by pretreatment of skin surface with anionic surfactants.

• Lipid film: The lipid film on the skin surface acts a protective layer to prevent the removal of moisture from the skin and helps in maintaining the barrier function of the stratum corneum. The effect of delipidization in compromising the stratum corneum barrier function and alteration of solute uptake clearly implicates the involvement of the lipid pathway in the transdermal delivery of drugs. Changes in drug permeation through the skin in diseases that alter the lipid content of the skin have also substantiated the importance of lipid to drug penetration

• Skin hydration: Hydration of stratum corneum can enhance transdermal permeability. Covering or occluding the skin with plastic sheet leading to sweet and condensed water vapor can achieve skin hydration.

• Skin temperature: Raising skin temperature results in an increase in rate of skin permeation. This may be due to thermal energy required for diffusivity. Solubility of drug in skin tissues is increased due to vasodilation of blood vessels in skin.

• Regional Variation: Differences in the nature and thickness of barrier layer of skin causes variation in permeability. Rate of permeation increase in an atomic order: Plantar anterior for arm, scalp, ventral thigh, scrotum and posterior auricular area.

• Pathologic injuries to the skin: Injuries that disrupt the continuity of stratum corneum increase permeability

• Cutaneous drug metabolism: Systemic absorption and tissue distribution of transdermally applied drugs are believed to be partly influenced by cutaneous perfusion below the site of application, and the orientation and accessibility of local blood vessels and tissues. The other contributing factor is considered to be drug plasma-binding characteristics, which are dependent on the chemical nature of the drug. Catabolic Enzymes present in the viable epidermis may render a drug inactive by metabolism and thus affect topical bioavailability of the drug. Example. Testosteron is 95% metabolized

• Appendageal Pathway: Among the four appendages of the skin (sweat glands, hair follicles, and the associated sebaceous glands and nails), hair follicles and the associated sebaceous glands are the only appendageal pathways that are believed to contribute to drug permeation. The importance of this route is usually dismissed because the appendages occupy only 0.1– 1.0% of total skin area. Studies on TDD via the appendageal route are scarce; however, a few studies have demonstrated a potential contribution of this route to the overall flux of drugs. Because the distribution of appendages is not uniform across the body, it is likely for these drugs to experience site variation.^21-24

Topical delivery includes two basic types of product:

• External topicals that are spread, sprayed, or otherwise dispersed on to cutaneous tissues to cover the affected area.

• Internal topicals that are applied to the mucous membrane orally, vaginally or on anorectal tissues for local activity²⁵.

1.7 Advantages of topical drug delivery system:

1) Avoidance of first pass metabolism.

2) Convenient and easy to apply.

3) Avoidance of the risks and inconveniences of intravenous therapy and of the varied conditions of absorption, like pH changes, presence of enzymes, gastric emptying time etc.

4) Achievement of efficacy with lower total daily dosage of drug by continuous drug input.

5) Avoids fluctuation in drug levels, inter- and intrapatient variations.

6) Ability to easily terminate the medications, when needed.

7) A relatively large area of application in comparison with buccal or nasal cavity

8) Ability to deliver drug more selectively to a specific site.

9) Avoidance of gastro-intestinal incompatibility.

10) Providing utilization of drugs with short biological half-life, narrow therapeutic window.

11) Improving physiological and pharmacological response.

12) Improve patient compliance.

13) Provide suitability for self-medication.26-29

1.8 Disadvantages of topical drug delivery system:

1) They are very sticky causing uneasiness to the patient when applied.

2) Moreover they also have lesser spreading coefficient and need to apply with rubbing.

3) They also exhibit stability problems.

4) Skin irritation of contact dermatitis may occur due to drug and/or excipients.

5) Poor permeability of some drugs through the skin.

6) Possibility of allergenic reactions.

7) Can be used only for drugs which require very small plasma concentration for action.

8) Enzyme in epidermis may denature the drugs.

9) Drugs of larger particle size not easy to absorb through the skin.12,30,31

10) Due to all these factors with the major group of preparations, the use of Topical spray has expanded both in cosmetics and in pharmaceutical preparations.

1.9 Limitations of topical drug delivery system:

1) Powders are slippery and caution during application should be taken since, inhalation of powders can cause irritation of airways and lungs.

2) Plasters are heavy weighted and are to be kept for longer duration of time.

3) In semi-solids, great variation is found in composition, pH, and tolerance.

4) Sticky in nature.

5) Applied with special spatulas or gloves.

6) Formulations are messy and occlusive.

7) Rancidification of oils may occur.

8) Physical stability, sedimentation and compaction of sediment causes problems in suspension.

9) Concentration variation of emulsifier may cause breaking of emulsion.

10) Careless rubbing the formulation may cause inconvenience.⁴

Table 1.1 Classification of topical praparations:
Class	Example
Liquid preparations	Liniment, lotions, paints, topical solution
Semi solid preparations	Creams, pastes, gels, ointments
Solid preparations	Topical Powders, poultices
Miscellaneous preparations	Topical aerosol, transdermal drug delivery system, rubbing alcohols, tapes and gauze

1.10 Introduction of topical spray:

An Aerosol formulation consists of two essential components: product concentrate and propellant. The product concentrate consists of active ingredients, or a mixture of active ingredients, and other necessary agents such as solvents, antioxidants, and surfactants. The propellant may be a single propellant or a blend of various propellants; it can be compared with other vehicles used in a pharmaceutical formulation. Just as a blend of solvents is used to achieve desired solubility characteristics, or various surfactant are mixed to give the proper HLB value for an emulsion system, the propellant is selected to give the desired vapor pressure, solubility, and particle size.

Propellants can be combined with active ingredients in many different ways, producing products with varying characteristics. Depending on the type of the aerosol system utilized, the pharmaceutical aerosol may be dispensed as a fine mist, wet spray, quick-breaking foam, stable foam, semi-solid, or solid. The type of system selected depends on many factors, including the following: (1) Physical, chemical, and pharmacologic properties of active ingredients. (2) Site of application.

1.11 Fungal infections:

Fungal infections are widespread in the population, generally associated with skin and mucous membranes. A disquietening trend after 1950s is the rising prevalance of fungal infections due to the increasing use of broad spectrum antibiotics, corticosteroids, anticancer/ immunosuppresants, emergence of AIDS, indewelling catheters, implants and dentures. They lead to breakdown of host defence mechanism, so saprophytic fungi easily invade living tissue.^33-35

1.11.1 Types of fungal infections

Fungal infections can be

1) Superficial

2) Systemic

Superficial fungal infection may be further classified to

1) Dermatophytosis which includes infection of skin, hair and nails

2) Candidiasis which includes infection of mucous membranes of mouth or vagina Dermatophytes are located in the stratum corneum within the keratinocytes. The signs and symptoms that appear in infected individuals are due to acute and chronic inflammatory changes that appear in the dermis. For these reasons, antifungal agents should have the ability to penetrate the stratum corneum cells to be efficient when applied topically.

Dermatophytes may be classified according to the genera, the ecology and patterns of infection. The clinical picture forms distinct entities grouped according to the infected site, namely tinea capitis, tinea barbae, tinea favosa, tinea corporis, tinea imbricata, tinea cruris, tinea pedis, tinea manuum and tinea unguium.33-35

1.11.2 Fungal Infections-Dermatophytosis

The management of dermatophytosis begins with topical agents. These agents should penetrate the skin and remain there in order to suppress the fungus. In the last 50 years numerous drugs have been introduced for the treatment of superficial infections. The choice of treatment is determined by the site and extent of the infection, the species involved as well as by the efficacy and safety profile, and kinetics of the drugs available. For localised non-extensive lesions caused by dermatophytes topical therapies with an imidazole, allylamines, tolnaftate, morpholine derivates, etc is generally used.

According to WHO (World Health Organisation), the dermatophytes are defined as a group of molds that form three genera: Epidermophyton, Trichophyton and Microsporum They comprise about 40 different species, and have common characteristics:

1. Close taxonomic relationships

2. Keratinolytic properties (they all have the ability to invade and digest the keratin as saprophytes “ in vitro” and parasites “in vivo”, producing lesions in the living host).

3. Occurrence as etiologic agents of infectious diseases of man and/or animals.

In man they invade hairs, nails and skin (they are found in the stratum corneum - the keratinized outer layer - and within the hair follicle, in the nail folds and subungually in the nail bed). All these are extensions of the stratum corneum.

The types of fungus that cause these infections are universally present throughout the environment. They can be transferred to skin from inanimate objects, dirt, animals and other humans. Though all are exposed, not everybody will develop skin or nail infections as a result. The individual immunity also plays a role in determining whether one is affected or not. Fungus tends to like warm, moist environments. Sweating, not drying the skin well after bathing or swimming, tight shoes and clothing, and a warm climate all contribute to the condition.33-35

1.12 Antifungal drugs:

The vast majority of antifungals are fungistatic with the concentrations achieved in the skin when applied topically; the growth of dermatophytes is delayed and these are shed with the skin renewal and healing is achieved. The antifungal agents and the components incorporated on the vehicle should be non-irritant and well tolerated. The vast majority of antifungals are applied twice daily, although the latest ones introduced are applied only once daily. Attention is currently being directed towards shortening the course of therapy and applying the medication once daily in an attempt to increase patient compliance and it is generally advisable to prolong treatment for two weeks once clinical cure is achieved.

Skin lesions located on face, trunk and limbs usually require two or three weeks of treatment. Inflammatory dermatophyte infections of the feet should be treated for four or six weeks and hyperkeratotic lesions of palms and soles are best treated with oral antifungals since they are usually unresponsive to topical antifungals Topical treatment of dermatophytosis is possible with non-extensive lesions, the application of medication should be done by rubbing it in gently in the affected skin area and should exceed (surpass) one cm. of healthy skin. It is important that the patient follows the application of treatment with the schedule recommended by the doctor.

Since dermatophytes require keratin for growth, they are restricted to hair, nails, and superficial skin; therefore, most can be treated with topical antifungal medications.(18) Topical Antifungal Preparations can be used to treat dermatophyte infections such as tinea corporis, tinea cruris, tinea faciei, tinea manuum, tinea pedis, and Yeast infections such as candida intertrigo, pityriasis and mould skin infections such as tinea nigra and as an adjunct to oral therapy for tinea capitis and tinea barbae. Oral treatment is indicated in widespread skin lesions, tinea capitis, tinea barbae, tinea unguium, in skin lesions with folliculitis, and when either there is no response to topical treatment or tolerance is not adequate.

Indications of topical treatment for dermatophyte infections:

• Non widespread limited lesions.

• Cases with interactions with oral antifungals.

• Patients non-compliant to systemic treatment.

• As adjunctive for systemic treatment.

• Prophylactic use to avoid recurrences after oral treatment.

• Patients in whom oral treatment is contraindicated.

• Pregnant or breastfeeding women.

• Attempts to shorten, improve or limit systemic antifungal treatment.

1.17 Limitations of oral itraconazole preparations for treating topical infection: According to the biopharmaceuticals classification system, Itraconazole is an extreme example of a class II compound meaning that its oral bioavailability is determined by dissolution rate in the GI tract. Systemic side effects associated with oral administration of Itraconazole can turn fatal in certain cases (especially in patients having hepatic disorders)and the oral dose is administered 2-3 times a day (200-400 mg/day) for 3-6 months to treat onychomycosis which leads to patient non compliance. Also there is degradation of drug in stomach due to gastric enzymes. Due to the lipophilic nature of Itraconazole, significant concentrations are stored in fat cells, thereby reducing the amount available at the site of infection. ^33-37,40

Table 1.7 Ideal properties of drug needed for topical administration
Parameters	Ideal properties of drug	Properties of drug
Aqueous solubility	>1mg/ml	~ 1 ng/mL
Partition coefficient	-1 < LogP < 4	Log P > 5
Molecular weight	< 400 Da	705.64
Melting point	< 200°C	166°C

2. REVIEW OF LITERATURE:

2.1 Review on Itraconazole:

Aditya K. Gupta, et al³⁵studied about the update in antifungal therapy of dermatomycosis from which they provides a general picture of topical antifungal treatment using topical medications most frequently cited in medical literature. The authors cite that topical therapy may be indicated for mild to moderate infection or in combination with oral therapy and few adverse events are expected with topical therapy compared to oral therapy.

Russell E. Lewis⁴³ studied about the pharmacokinetic optimization of Itraconazole therapy from which they concluded that the role of Itraconazole in the treatment was limited by the erratic bioavailability of the oral capsule formulation in imunocompromised patients. The authors report that the improved absorption and consistent plasma levels achieved with oral solution and IV formulations has opened up the possibility of using Itraconazole in more seriously-ill patients with invasive fungal infections. However, predicting an appropriate dosing strategy for Itraconazole in the critically-ill patient remains a challenge due to the dose-dependent pharmacokinetic profile, potential for multiple drug interactions, and substantial inter- patient variability in drug metabolism. An appropriate dosage of Itraconazole for the individual patient remains problematic due to the considerable intra- and interpatient variability of ITR pharmacokinetics.

Jae-Young Jung a, et al⁴⁴studied about the enhanced solubility and dissolution rate of Itraconazole by a solid dispersion technique in which they concluded that tablets prepared using solid dispersions showed enhanced dissolution profiles of Itraconazole over the marketed product.

Mahendra Nakarani, et al⁴⁵formulated Itraconazole nanosuspension for oral delivery and they compared their formulation with marketed product and concluded that poor water solubility of Itraconazole and high food dependency leads to insufficient bioavailability and fluctuating plasma levels.

Eun-A Lee, et al⁴⁶studied about the microemulsion-based hydrogel formulation of Itraconazole for topical delivery in which they reported that oral delivery of Itraconazole is plagued with barriers that inhibit effective delivery to the affected site and topical delivery of Itraconazole is of great interest for the treatment of Onychomycosis and other skin fungal infections.

Marc Francois, et al⁴⁷formulated a mucoadhesive, cyclodextrin-based vaginal cream formulation of Itraconazole and concluded that cyclodextrin-based, emulsified wax cream containing Itraconazole was well tolerated, was not systemically bioavailable, and was effective in combating vaginal candidiasis.

Piyusha Devada, et al⁴⁸formulated gellified emulsion of Itraconazole for topical fungal diseases reported that Itraconazole has an interesting tissue distribution, which has made possible effective and rapid treatments of candidiasis, when the drug is administered topically. A topical Itraconazole containing formulation may be of use for several reasons including the opportunity to generate high local tissue levels and lower systemic exposure.

Demiana I. Nesseem, et al⁴¹studied about formulation and evaluation of Itraconazole via liquid crystal for topical delivery system and concluded that formulation of liquid crystals cream containing 1% Itraconazole is successful as topical delivery system since incorporation of the drug in liquid crystal exhibited better antimycotic activity against candida albicans in comparison with hydroxyethyl cellulose gel as control I and GMS cream as control II. So it could be concluded that an effective topical formulation of Itraconazole is desirable for topical action since oral therapy for topical treatment is giving numerous systemic side effects. Additionally an effective topical formulation of Itraconazole will give site specific action, enhance patient compliance and safety also.

2.2 Review on other topical spray delivery system:

Marie-Laure Leichtnam, et al⁴⁹studied about the identification of penetration enhancers for testosterone transdermal delivery from spray formulations in which they reported that isopropyl myristate is a suitable chemical enhancer for testosterone delivery across the skin.

Seyed Mohsen Foroutan, et al⁵⁰studied about the formulation and in-vitro evaluation of silver sulfadiazine spray in which they reported that zone of inhibition was larger in spray formulation than in cream. No degradation and crystal growth was found after one year. Provide rapid, more effective topical antimicrobial application compare to cream.

Veraldi, stefanol, et al⁵¹studied about the topical Fenticonazole in dermatology and gynaecology in which they reported that Fenticonazole exerts its unique antimycotic mechanism of action in the following three ways: (i) inhibition of the secretion of protease acid by candida albicans; (ii) damage to the cytoplasmic membrane; and (iii) by blocking cytochrome oxidases and peroxidises. Fenticonazole has also been shown to exhibit antibacterial action, with a spectrum of activity that includes bacteria commonly associated with superinfected fungal skin and vaginal infections. Topical fenticonazole is very well tolerated; adverse events are generally mild to moderate in severity and transient. The most frequent adverse events are burning sensation/cutaneous irritation and itch when applied to skin.

Albert Jen, et al⁵²formulated Fluconazole nasal spray for the treatment of allergic sinusitis and they reported that topical antifungal were proves to be successful as majority of the patients with this treatment had no progression of disease.

2.3 Review on solvents and cosolvents:

Barbara Bendas, et al⁵³studied about the influence of propylene glycol as cosolvent on mechanisms of drug transport from hydrogels in which they reported that propylene glycol acts on the drug diffusion from a vehicle into an adjacent acceptor. The rate and extent of hydrocortosone penetration increase with increasing propylene glycol contents of the formulation.

Muhammad Akram Randhawa⁵⁴studied the effect of dimethylsulfoxide on the growth of dermatophytes in which they reported that dimethylsulfoxide is frequently used as solvent for antifungal drug. The growth of dermatophytes was observed by agar-diffusion method. There was no growth of fungi in 10% dimethylsulfoxide.

D.I.J. Morrow, et al⁵⁵studied innovative strategies for enhancing topical and transdermal drug delivery in which they reported that ethanol and dimethylsulfoxide can be used as a solvent and as a penetration enhancer in topical and transdermal drug delivery system.

Amit Chaudhary⁵⁶studied enhancement of solubilization and bioavailability of poorly soluble drugs by physical and chemical modifications and they reported that ethanol, PEG 300, propylene glycol are widely used in cosolvent mixtures. Dimethylsulfoxide, DMA have been widely used as co-solvents because of their large solubilization capacity for poorly soluble drugs and their relatively low toxicity.

2.4 Review on topical spray patented technology:

Donald Edward smith, et al⁵⁷studied about the “Dermatological composition” in which they reported that the epidermal barrier to percutaneous absorption i.e stratum corneum is nearly impermeable so for penetration dimethylsulfoxide 50% or more can be used.

Hirsh Mark, et al studied spray formulation for the treatment of acute inflammatory skin condition in which they claimed that a topical spray or foam application is on formulation comprising antifungal or antibacterial active agent in an active amount to treat or reduce the symptoms associated with diseases or disorders of the skin in a pharmaceutically acceptable spray or foaming excipients.

Chen-Jivn-Ren, et al formulated topical spray for burn treatment and infection in which they claimed that topical spray preparation are better to treat the burn wound and infection as compared to other topical dosage form.

2.5 Review on liquified petroleum gas (mixture of propane and butane):

Febriyenti et al⁵⁷studied the Physical evaluations of Haruan spray for wound dressing and wound healing. Haruan extract was incorporated as an active ingredient of the aerosol concentrate for its ability to enhance the healing process.and was evaluated for the physical properties of using different propellant. The propellants evaluated were 1,1,1,2– tetrafluoroethane (HFA 134a), butane and propane. Concentrate of aerosol also contain hydroxypropyl methylcellulose (HPMC) as polymer and glycerine as plasticizer. All ingredients of the aerosol were packaged in an aluminium container fitted with continuous- spray valves. Evaluations for the Haruan spray included delivery rate, pressure, minimum fill, leakage, flammability, spray patterns and particles image. The result showed that HFA 134a reacted with the concentrate and produced white aggregates, while propane had a very high vapour pressure. From safety and economics point of view, butane was chosen as the propellant because it met the requirements for aerosol and produced Haruan spray with the expected characteristics.

_{Table 2.1: Drug Profile38,47}
Name of Drug	Itraconazole
CAS No.	84625-61-6
Official category	BP, EP
Description	One of the triazole antifungal agents that inhibits cytochrome P- 450-dependent enzymes resulting in impairment of ergosterol synthesis. It has been used against histoplasmosis, blastomycosis, cryptococcal meningitis and aspergillosis.
Molecular structure
Categories	Antifungal Agents, Antiprotozoal Agents
Weight	Average:705.633 Monoisotopic: 704.239307158
Appearance	A white or almost white powder, practically insoluble in water, freely soluble in methylene chloride, sparingly soluble in tetrahydrofuran, very slightly soluble in alcohol.
Chemical formula	^C35^H38^Cl2^N8^O4
IUPAC name	1-(butan-2-yl)-4-{4-[4-(4-{[(2R,4S)-2-(2,4-dichlorophenyl)-2- (1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4- yl]methoxy}phenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H- 1,2,4-triazol-5-one
Indication	For the treatment of the following fungal infections in immunocompromised and non-immunocompromised patients: pulmonary and extrapulmonary blastomycosis, histoplasmosis, aspergillosis, and onychomycosis.
Absorption	The absolute oral bioavailability of itraconazole is 55%, and is maximal when taken with a full meal.
V_d	796 ± 185 L
Half life	21 hours

Mechanism of action	Itraconazole interacts with 14-α demethylase, a cytochrome P- 450 enzyme necessary to convert lanosterol to ergosterol. As ergosterol is an essential component of the fungal cell membrane, inhibition of its synthesis results in increased cellular permeability causing leakage of cellular contents. Itraconazole may also inhibit endogenous respiration, interact with membrane phospholipids, inhibit the transformation of yeasts to mycelial forms, inhibit purine uptake, and impair triglyceride and/or phospholipid biosynthesis.
Water solubility	~ 1 ng/mL
Log P	>5
pKa	3.7
Melting point	166°C
Dose	100 – 200 mg per day orally
Dosage forms available	Capsule, oral solution, i.v injection

2.6 Solvent profile:

1) Isopropyl alcohol:

Synonyms: Alcohol isopropylicus; Dimethyl carbinol; IPA; Isopropanol; Petrohol; 2- propanol; Sec-propyl alcohol; Rubbing alcohol.

Chemical name: Propan-2-ol Molecular formula: C₃H₈O Molecular weight: 60.1

Functional categories: Disinfectant; Solvent.

Description: Isopropyl alcohol is a clear, colorless, mobile, volatile, flammable liquid with a characteristic, spirituous odor resembling that of a mixture of ethanol and acetone; it has a slightly bitter taste.

Application: Isopropyl alcohol is used in cosmetics and pharmaceutical formulations, primarily as a solvent in topical formulations. Although it is used in lotion, the marked degreasing properties of isopropyl alcohol may limit its usefulness in preparations used repeatedly. Isopropyl alcohol is used as a solvent both for tablet film-coating and for tablet granulation. It has also been shown to significantly increase the skin permeability of nimesulide from carbomer 934. Isopropyl alcohol has some antimicrobial activity and a 70% v/v aqueous solution is used as a topical disinfectant. Therapeutically, isopropyl alcohol has been investigated for the treatment of postoperative nausea or vomiting.

Stabilities and storage conditions: Isopropyl alcohol should be stored in an airtight container in a cool, dry place.

Safety: Isopropyl alcohol is widely used in cosmetics and topical pharmaceutical formulations. It is readily absorbed from the gastrointestinal tract and may be slowly absorbed through intact skin. Prolonged direct exposure of isopropyl alcohol to the skin may result in cardiac and neurological deficits: In neonates, isopropyl alcohol has been reported to cause chemical burns following topical application. Isopropyl alcohol is about twice as toxic as ethanol and should therefore not be administered orally; isopropyl alcohol also has as unpleasant taste. The lethal oral dose is estimated to be about 120-250ml although toxic symptoms may be produced by 20ml. Adverse effects following parenteral administration of up to 20ml of isopropyl alcohol diluted with water have included only a sensation of heat and a slight lowering of blood pressure. When applied to the eye it can cause corneal burns and eye damage.

Handling precautions: Observe normal precautions appropriate to the circumstances and quantity of material handled. Isopropyl alcohol may be irritant to the skin, eye, and mucous membranes upon inhalation. Eye protection and gloves are recommended. OSHA standards state that IPA 8-hour time weighted average airborne level in the work place cannot exceed 400 ppm. Isopropyl alcohol is flammable and produces toxic fumes on combustion.^38,58

2) Dimethylsulfoxide:

Synonyms: DMSO; Kemsol; Methylsulfinylmethane; Methyl sulfoxide

Molecular formula: (CH₃)₂SO

Molecular weight: 78.13 g mol⁻¹

Functional category: Penetration agent, solvent

Description: Dimethylsulfoxide is a colorless, viscous liquid, or as colorless crystals. Application: DMSO is predominantly used as a topical analgesic, a vehicle for topical application of pharmaceuticals, as an anti-inflammatory, and an antioxidant. Because DMSO increases the rate of absorption of some compounds through organic tissues, including skin, it can be used as a drug delivery system. It is frequently compounded with antifungal medications, enabling them to penetrate not just skin but also toe and fingernails. DMSO has the ability to reduce inflammation and pain in a wide range of conditions to penetrate into the skin after topical application of DMSO for lessening of pain and swelling of inflammation. The facility with which DMSO penetrates the skin and other membranes has spawned considerable research into the use of DMSO as a vehicle for the administration of drugs through topical application. In addition, DMSO also captures water from the skin, being a hydroxyl ion scavenger thereby dehydrating the skin.^{38, 58}

3) Propylene Glycol:

Nonproprietary Names:- BP: Propylene Glycol, JP: Propylene GlycolPhEur: Propylene Glycol, USP: Propylene Glycol

Synonyms: 1,2-Dihydroxypropane; E1520; 2-hydroxypropanol; methyl ethylene glycol; methyl glycol; propane-1,2-diol; propylenglycolum.

Chemical Name and CAS Registry Number: 1,2-Propanediol [57-55-6](_)-1,2- Propanediol [4254-14-2] þ)-1,2-Propanediol [4254-15-3]

Empirical Formula and Molecular Weight: C3H8O2 and 76.09

Functional Category: Antimicrobial preservative; disinfectant; humectant; plasticizer; solvent; stabilizing agent; water-miscible cosolvent

Description: Propylene glycol is a clear, colorless, viscous, practically odorless liquid, with a sweet, slightly acrid taste resembling that of glycerin

Applications in Pharmaceutical Formulation or Technology: Propylene glycol has become widely used as a solvent, extractant, and preservative in a variety of parenteral and nonparenteral pharmaceutical formulations. It is a better general solvent than glycerin and dissolves a wide variety of materials, such as corticosteroids, phenols, sulfa drugs, barbiturates, vitamins (A and D), most alkaloids, and many local anesthetics. As an antiseptic it is similar to ethanol, and against molds it is similar to glycerin and only slightly less effective than ethanol.^{38, 58}

4) Liquefied Petroleum Gas (LPG):

LPG consists of a mixture of propane and butane. Its use as an aerosol propellant and a refrigerant, LPG (or also called as LP gas) is increasingly replacing chlorofluorocarbons in order to reduce damage to the ozone layer. LPG is extracted from gas streams or oil as they emerge from the ground; it is also manufactured during the refining of crude oil Since LPG evaporates at normal temperatures and pressures, they can be stored in steel tanks. LPG is non-corrosive, free of lead but heavier than air. LPG also produces less air pollutants than oil, diesel, coal or wood. It emits about 20% less CO2 than heating oil and about 50% less than coal.⁵⁷

3. AIM AND OBJECTIVE OF PRESENT STUDY:

3.1 Rationale:

Oral and IV administrations of Itraconazole are associated with systemic side effect and inconvenience of administration. e.g In oral administration, there is an insufficient dissolution in the stomach before the drug is delivered to the intestinal lumen. In IV administration, it can be painful, uncomfortable, inconvenient and resulting in poor patient compliance.

Topical therapy allows direct delivery of drug to the skin with minimal risk of systemic side effects. The effectiveness of topical drugs depends on their ability to penetrate the epidermis.

There are two types of Superficial fungal infections:

a) Dermatomycoses- Infection of skin, hair, nails. (Onychomycosis)

b) Candidiasis- Infect the mucous membranes of the mouth, vagina and skin.^33,34

To treat these fungal infections various antifungal drugs as clotrimazole, econazole, enilconazole, fenticonazole, fluconazole, flucytosine, griseofulvin, etc are widely used and commercially available in different pharmaceutical dosage forms.

Itraconazole (Sporanox®) is a triazole antifungal that has been in clinical use for over a decade. Initially introduced as a capsule formulation, Itraconazole was marketed for the treatment of both superficial (onychomycosis) and systemic (blastomycosis, histoplasmosis) fungal infections. Itraconazole was also the first azole antifungal agent approved for the treatment of invasive aspergillosis (IA) in patients. Itraconazole is highly lipophilic and virtually insoluble in water. It is an extremely weak base (pKa =3.7) that is ionized only at low pH, such as that found in gastric fluid.⁴³

Some types of fungal infections (candida albicans) are ketoconazole-resistance particularly with chronic mucocutaneous candidiasis. Itraconazole is better tolerated and more effective than ketoconazole. Itraconazole binds only weakly to mammalian cytochrome P-450 and it has a much higher affinity than ketoconazole for fungal P-450 enzymes. Also Itraconazole may be effective for fluconazole-resistantant fungal infection of skin. Itraconazole pulse therapy was first approved for onychomycosis in 1993, in Finland. The safety profile of Itraconazole is established, with very few non- trivial adverse experiences noted in a large series of patients treated and monitored.

Itraconazole therefore represents a useful therapeutic advance for the management of most forms of fungal infection.^{35, 37}Topical Itraconazole can be generate high local tissue level, more rapid drug delivery, lower systemic exposure.⁴⁷

Therefore an effective topical dosage form would be desirable particularly for treatment of onychomycosis and other fungal infections of skin. Therefore purpose of the present investigation was to enhance the solubility of Itraconazole and to explore feasibility of preparing Itraconazole topical spray.^43,46

3.2 Aim of the study:

Aim: Formulation and evaluation of Itraconazole topical spray.

3.3 Objectives of the study:

• To enhance the solubility of Itraconazole by using cosolvancy method.

• To carry out compatibility study of drug with the container and other solvents.

• To formulate, optimised and characterize Itraconazole topical spray.

• In vitro and ex vivo diffusion study of optimised Itraconazole topical spray.

4.1 MATERIALS USED IN PRESENT INVESTIGATION:

List of materials used in the present investigation are shown in table 4.1.

4.2 EQUIPMENTS USED IN PRESENT INVESTIGATION:

List of equipments used in the present investigation are shown in table 4.2

Table 4.1 Materials used in the present investigation
Sr. No.	Material	Properties	Source
1	Itraconazole	API	Vadodara
2	Dimethylsulfoxide	Solubiliser	Lobachemie Pvt Ltd, Mumbai
3	Propylene Glycol	Cosolvent and humectant	Suvidhinath Laboratories Vadodara
4	Isopropyl alcohol	Solvent	Qualikems Fine Chem Pvt Ltd, Nandesari Vadodara
5	Ethanol 99.5% pure	Solvent	Baroda Chemical Industries ltd, Dabhoi
6	Methanol 99.5% Pure	Solvent	Rankem Laboratory Reagent, New Delhi
7	Disodium hydrogenphosphate	Buffer ingredient	Qualikems Fine Chem Pvt Ltd, Nandesari Vadodara
8	Potassium dihydrogen phosphate	Buffer ingredient	Qualikems Fine Chem Pvt Ltd, Nandesari Vadodara
9	Beef extract powder	Growth media	Finer Reagents Ahmedabad
10	Agar agar powder	Growth media	Finer Reagents Ahmedabad
11	Sodium chloride	Growth media	Lobachemie Pvt ltd Mumbai
12	LPG	Propellant	Vimsons Aerosols, Anand
13	Candida albicans	Fungal strain	Food and Drug Laboratory, Vadodara
14	Peptone	Growth media	Suvidhinath Laboratories Vadodara
15	Hydrochloric Acid	Solvent	Suvidhinath Laboratories Vadodara
16	Sudan Red	Dye	Sisco Research Lab pvt ltd, Mumbai

Table 4.2 Equipment used in the present investigation
Sr. No.	Instrument	Model	Manufacturer
1	Ultra Sonicator Bath	D COMPACT	EIE Instruments Pvt ltd, Ahmedabad
3	Pressure gauge	-	Vimsons Aerosols Anand
5	UV Visible Spectrophotometer	Specord 205	Analytical Jena AG (Germany)
6	Franz diffusion cell	-	Durga Scientific Ltd Vadodara
7	Digital Balance	AX200	Shimadzu Corp., Japan
8	pH meter	PM100	Welltronix
9	IR Spectrophotometer	FTIR : Model no. WQF-520	Aum Laboratories Ahmedabad
10	Water bath	-	Durga Scientific Ltd Vadodara
11	Magnetic stirrer	-	Durga Scientific Ltd Vadodara
12	Laminar air flow unit	-	Jankiimplex Pvt Ltd Ahmedabad
13	Autoclave	-	Durga Scientific Ltd Vadodara
14	B.O.D incubator	-	EIE Manufacturer Ahmedabad
15	Tissue homogeniser	CEY 11473	Universal Motor Ltd Mumbai
16	Oven	BLS-14	Bombay Labs services
17	Propellant filling machine	-	Vimsons Aerosols Anand
18	Valve crimping machine	-	Vimsons Aerosols Anand
19	Vernier caliper	-	Durga scientific Ltd Vadodara

4.3 PREFORMULATION STUDIES OF ITRACONAZOLE

4.3.1 Solubility determination of Itraconazole:

Solubility studies were performed according to the saturation method. An excess amount of pure Itraconazole was placed in 25ml volumetric flask containing 20 ml of different solvents. The content of the suspension were kept for 24 hours at 37±0.5ºC. After attainment of equilibrium of 24 hours, 5ml of supernatant was withdrawn and filtered and analyzed by UV spectrophotometer at 262nm. All solubility measurements were performed in triplicate.⁵⁹

4.3.2 Melting point determination of Itraconazole:

Melting point of Itraconazole was determined using capillary melting point apparatus. Minimum amount of drug was placed in a thin walled capillary tube 10-15 cm closed at one end. The capillary containing drug and a thermometer were then suspended in melting point apparatus and then their temperature range over which the drug melted was determined. The procedure was repeated in triplicate.

4.4 SPECTROPHOTOMETRIC ESTIMATION OF ITRACONAZOLE

4.4.1 Quantitative estimation of Itraconazole in methanol: Determination of λmax

Itraconazole was dissolved in methanol, diluted further with the same to obtain a 10ug/ml solution and was scanned between 200 to 400nm to determined λ max in UV Spectrophotometer. Procedure was repeated in triplicate.⁶⁰

4.4.2 Selection of media:

Approach: Preliminary determination of diffusion media and dilution media for formulated product has been done using various combination of solvents and buffers of different pH.

Itraconazole in 50 ml Phosphate buffer pH 7.4:

10mg of Itraconazole was dissolved and sonicated at 37±0.5ºC for 2-5 minutes in 50ml phosphate buffer pH 7.4.

Itraconazole in phosphate buffer pH 7.4+5% Methanol:

10mg of Itraconazole was dissolved and sonicated at 37±0.5ºC for 2-5 minutes in 50ml phosphate buffer pH 7.4 containing 5% methanol.

Itraconazole in phosphate buffer pH 7.4 saline:

10mg of Itraconazole was dissolved and sonicated at 37±0.5ºC for 2-5 minutes in 50ml phosphate buffer pH 7.4 saline.

Itraconazole in phosphate buffer pH 7.4 saline + 5% Methanol:

10mg of Itraconazole was dissolved and sonicated at 37±0.5ºC for 2-5 minutes in 50ml phosphate buffer pH 7.4 saline containing 5% methanol.

Itraconazole in phosphate buffer pH 5.5:

10mg of Itraconaole was dissolved completely and sonicated at 37±0.5ºC for 2-5 minutes in 50ml phosphate buffer of pH 5.5. Since the drug was completely dissolved and solution was transparent it was diluted further with the same to obtain a 10 ug/ml solution. This solution was subjected to scanning between 200 – 400 nm using UV-Visible Spectrophotometer.

4.4.3 Quantitative estimation of Itraconazole in selected media: Determination of λmax

100mg of Itraconazole was dissolved completely and sonicated at 37±0.5ºC for 2-5 minutes in 100ml phosphate buffer of pH 5.5 and diluted further with the same to obtain a 10 ug/ml solution. This solution was subjected to scanning between 200 – 400 nm using UV-Visible Spectrophotometer with spectral bandwidth of 1 cm and wavelength accuracy 0.1 nm and absorption maximum (λmax) was determined. Procedure was repeated in triplicate.

Standard plot of Itraconazole in phosphate buffer pH 5.5

Preparation of stock solution (1000µg/ml):

An accurately weighed 100mg drug was dissolved in phosphate buffer pH 5.5 to produce 100 ml solution.

Working sample solution:

Different dilutions of stock solution were made to obtain solutions having concentrations 4-36 µ g/ml. Absorbance was measured at 256 nm for phosphate buffer pH 5.5 using respective media as blank. A graph of absorbance v/s concentration (µg/ml) was plotted and standard curve was obtained.

4.5 AEROSOL CONTAINERS SELECTION

30 ml Aluminum canisters having inner wall laminated with paint were selected for packaging.⁶¹

4.6 PRELIMINARY COMPATIBILITY STUDIES WITH CONTAINER

After the selection of Aerosol containers, a trial product concentrate was filled and was stored at 37±0.5ºC for 1 m o n t h , allowing product to come in contact with container as well as valve assembly. The container was opened and the signs of corrosion, coatings dissolved by concentrate, discoloration of dip tube, elongation l of dip tube, softening of valve, cracking of valve, discoloration of drug concentrate were observed.⁶¹

4.7 PROCEDURE FOR PREPARING ITRACONAZOLE TOPICAL SPRAY

40 ml narrow mouth bottles were cleaned uniformly by blowing air. Formulation codes were given to each bottle. Dimethylsulfoxide and other solvents were added as formulation design to each batch. The mixture was stored and 200mg of Itraconazole was dissolved in each container. Prepared product concentrates were stored at 25º C till they got in to aerosol container.

PROPELLANT FILIING IN AEROSOL CAN

Aerosols canisters filled with drug concentrate were crimped with valve. After measuring their weight they were placed under propellant filling machine. Propellant measuring cylinder screw was set to the 50%, 60%, 70% volume of drug concentrate. Filled aluminum canisters were then weighed and sealed.

Table 4.3: Composition of preliminary trials of Itraconazole topical spray
Ingredients	F1	F2	F3	F4
Drug	1%	1%	1%	1%
DMSO	25%	25%	25%	25%
IPA	20%	10%	40%	30%
PG	5% (1ml)	15% (3ml)	5% (1ml)	15% (3ml)
LPG	50%	50%	30%	30%

4.8 FORMULATION OF ITRACONAZOLE TOPICAL SPRAY USING 3²FACTORIAL DESIGN

Factorial design was used in experiments in order to elucidate the effect of different factors or conditions on experimental results.

A 3²randomized full factorial design was used in the present study in preparation of batches of spray. In this design 2 independent factors were evaluated, each at 3 levels, and experimental trials were performed for all 9 possible combinations. The Amount of co-solvent (X₁) and Amount of propellant (X₂) were chosen as independent variables in 3² full factorial design. % skin retention and spray pattern were taken as dependent variables.

A statistical model incorporating interactive and poly nominal terms was used to evaluate the responses.

Y_i= b₀+ b₁X₁+ b₂X₂+ b₁₂X₁X₂+ b₁₁X₁²+ b₂₂X₂

Table 4.4: 3²full factorial design layout for Itraconazole topical spray
Variable levels in coded form
Formulation code	^X₁^(PG)	^X₂^(LPG)
F1	-1	-1
F2	0	-1
F3	+1	-1
F4	-1	0
F5	0	0
F6	+1	0
F7	-1	+1
F8	0	+1
F9	+1	+1

Where Y is the dependent variable, b₀is the arithmetic mean response of the 9 runs, and b_iis the estimated coefficient for the factor Xi. The main effects (X₁and X₂) represent the average result of changing 1 factor at a time from its low to high values. The two way interaction terms (X₁X₂) show how the response changes when two factors are simultaneously changed.

Table 4.5: Variables and levels of full factorial design 3²for Itraconazole spray
Variables	Low (-1)	Medium (0)	High (+1)
Amount of Propylene glycol X1	5%	10%	15%
Amount of Propellant X2	50%	40%	30%

4.9 EVALUATION OF ITRACONAZOLE TOPICAL SPRAY

Itraconazole topical spray was evaluated by a series of physicochemical, performance and biologic tests: ^{52, 62, 63}

4.9.1 PHYSICOCHEMICAL CHARACTERISTICS

1. Flame extension and flash back:

A burner was set to fire on laboratory platform. 1 meter scale was placed in adjoining burner over platform. Formulations F1 to F9 were sprayed for about 4 seconds in to a flame. With the help of the ruler flame extension and flame flash back was measured over scale. All measurements were taken in triplicate.

2. Pressure test:

Each container was placed in an upright position. The actuator was pressed to remove liquid from the dip tube and valve. The actuator was removed and replaced with the pressure gauge. The gauge was pressed to actuate the valve and the pressure exerted by the propellant was noted for each aerosol container with the help of pressure gauge.

3. Density:

Dried and emptied pycnometer was weighed. The sample was filled in it. Pycnometer was handled by the neck with one or two layers of paper between the fingers and the bottle to avoid expansion due to the heat of the hand. The proper value of the density was known by dividing the resultant weight of liquid by its volume in pycnometer.

4. pH:

pH meter was calibrated using two buffers (pH 4 and pH 7) for calibration. The tip of the probe after rinsing with water was dipped in to samples. The meter was allowed to equilibrate and then pH was noted.

4.9.2 PERFORMANCE

1. Delivery rate of Itraconazole topical spray:

The delivery rate of Itraconazole spray was evaluated according to procedure stated in USP. Six aerosol containers were used. Each valve was actuated for 5 seconds at a temperature of 25 °C. The test was repeated three times for each container. The average delivery rate was calculated, in grams per second.

2. Drug content per actuation:

Reproducibility of the dosage was determined as per USP. The average amount of active ingredient delivered through actuator per actuation was assayed, the amount delivered per actuation was determined.

3. Spray pattern of Itraconazole topical spray:

Spray formulation containing sudan red dye (10mg) was sprayed onto absorbent paper for 2 seconds. The distance separating the container from the target was kept constant, at 5 cm. spray pattern was evaluated by spraying the concentrate in horizontal position. Ovality ratios were determined by calculating Dmax and Dmin.⁶¹

4. Particle size and particle image of Itraconazole topical spray:

Itraconazole spray was sprayed onto separate slides and the images of particles were photographed. The formulation was sprayed on a glass slide and at least 100 particles was measured using microscope with an objective lens 10 x magnification of eye piece lens that has been fitted with a standardized micrometer along with Particle image.

5. Spray angle of Itraconazole topical spray:

Itraconazole spray was sprayed for 5 seconds and photograph was clicked in order to obtain the plume angle. The angle at which the valve delivers the content obtained from the photographed and measured for determining plume angle.

6. Minimum fills of Itraconazole topical spray:

Five filled containers were selected and weighed individually. The contents were removed from each container. The packages were opened and the residue was removed by washing with suitable solvents and rinsed with methanol. The container, the valve, and all associated parts were collected and heated to dryness at 100 ºC for 5 minutes and cooled. The weight of each container together with their corresponding parts was determined. The difference between the weight of the filled container and the weight of the corresponding empty container was the net weight of the content.

7. Leakage test of Itraconazole topical spray:

The leakage test was conducted according to the method in USP. Nine aerosol containers were selected and the date and time were recorded to the nearest half hour. Each container was weighed to the nearest mg and recorded as W1. The containers were allowed to stand in an upright position at a temperature of 25.0 ± 2.0 ºC for not less than 3 days, before the second weight was recorded as W2. The leakage rate, in mg per year, of each container was calculated using formula:

(365) (24/T) (W1 – W2), where T is the test period, in hours.

4.9.3 BIOLOGIC CHARACTERISTICS

1. Ex vivo Penetration studies of Itraconazole from Itraconazole topical spray:

Ex vivo diffusion study was performed by using modified Franz diffusion cells with an effective diffusion area of 4.9 cm². The excised skin samples (dorsal side of 6~8 weeks old rats) were treated with depilaptory agent to remove the skin hair. It was then clamped between the donor and the receptor chamber of modified diffusion cells with the stratum corneum facing the donor chamber. Then, formulations were sprayed for 5 sec. on treated skin mounted on franz diffusion cell. The receptor chamber was filled with 20 ml of phosphate buffer pH 5.5. The receptor medium was maintained at 37 ± 0.5⁰C and stirred at 600 rpm throughout the experiment. Aliquots of 3 ml were sampled from the receptor compartment at time interval of 1 hr and then the same volume of pure medium was immediately added into the receptor chamber. All samples were filtered through whattman filter paper and analyzed by UV method. After diffusion study the rat skin was washed in 10 ml of buffer to determined the amount of drug retained over skin. After that the skin was homogenized in 15 mlm of buffer to determined the amount of drug retained into skin. Cumulative corrections were made to obtain the total amount of drug release at each time interval. The cumulative amount of drug released across the rat skin and % skin retention were determined as a function of time.

2. In vitro antifungal activity of Itraconazole topical spray:

Candida albicans as one of the invasive fungi found in most of the fungal infections was used for studying in vitro antifungal activity of Itraconazole topical spray. Cup and plate assay method was used for determining the zone of inhibition for both placebo and formulated spray preparation.

The isolates were received from Food and Drugs Laboratory, Vadodara. They were transferred from 0.9 % sodium chloride saline solution to nutrient agar broth in a sterile tube with the help of sterile inoculation loop for sub culturing. Nutrient agar plates were prepared with appropriate turbidity by pouring plate method. Agar Agar powder, sodium chloride, peptone and beef extract were used for susceptibility testing of candida albicans. A sterile inoculation loop is dipped in the fungal suspension and the loop is streaked in at least three directions over the surface of the nutrient agar media to obtain uniform growth. A final sweep is made around the rim of the agar. The plates are allowed to dry for approximately five minutes. A sterile borer was used to create well in the center of agar plate. The well in the center of the plate was than filled with drug solution and compared with without Itraconazole as control. The plates were incubated within 15 minutes after applying the disks and boring the well with drug solution. The temperature was kept 35° ± 2°C for incubation and incubation time was 24 hours. After the overnight incubation, clearing zone around each of antifungal solution in well was measured with the help of ruler. The diameter was measured and recorded in millimeters (mm).⁴¹

3. Skin irritation studies of formulated Itrconazole topical spray:

Skin irritation studies were carried out using wistar rats as animal model. The optimized formulation was sprayed on the pre shaved skin and reactions if any erythema and edema were scored after 7 days.

4.10 STABILITY STUDIES

The optimized formulation was stored for stability testing as per ICH guidelines for 1 month. The chemical stability of the formulation was assessed by estimation of the percent drug remaining in the formulation, drug release pattern and physical stability was evaluated by monitoring any change in flammability, pressure, density, pH, delivery rate, spray pattern, spray angle. Biological stability was determined by performing ex vivo penetration studies of Itraconazole topical spray and in vitro antifungal activity of Itraconazole topical spray.

^{5. RESULTS AND DISCUSSION:}

^{5.1
PREFORMULATION STUDIES OF ITRACONAZOLE:}

The results of preformulation studies of Itraconazole are shown in table 5.1.

Table:-5.1.

Tests	Officially Reported	Laboratory Observation
Appearance	White powder	White powder
Melting point	166- 170c	165-168c
Solubility	Insoluble in water Slightly soluble in alcohol Freely soluble in methylene chloride.	Insoluble in water 0.1mg/ml in IPA 10mg/ml in DMSO

^{Melting
point of Itraconazole was found to be in the range of 165-168ºC as reported in
literature, thus indicating purity of the drug sample. Any impurity, if
present, will cause variation in the melting point of a given drug substance.}

^{From both
the IR data, it was found that the functional groups showed peaks at same
wavelength in procured pure drug sample of Itraconazole. Hence, it can be
concluded that the sample is Itraconazole as per reported specifications.}

^{5.2
SPECTROPHOTOMETRIC ESTIMATION OF ITRACONAZOLE:}

^{The results
of quantitative estimation of Itraconazole in methanol are shown in figure 5.2.}

^{5.2.1
Quantitative estimation of Itraconazole in methanol}

Figure 5.2 UV Scan of Itraconazole 10 µg/ml in methanol

From the UV scan of Itraconazole max. absorbance was observed at 262 nm in methanol and the reported λmax of Itraconazole in methanol is also 262 nm . Hence it can be taken as working wavelength for UV spectroscopic analysis of Itraconazole.

5.2.2 Selection of diffusion media

The results of selection of diffusion media are shown in table 5.3.

Table 5.3 Selection of Diffusion media
Phosphate buffer pH 7.4	Turbidity appeared	Indicated presence of undissolved drug particles. The diffusion media were found not suitable and hence rejected.
Phosphate buffer pH 7.4 + 5% methanol
Phosphate buffer saline pH 7.4
Phosphate buffer pH 7.4+5% methanol
Phosphate buffer pH 5.5	Clear solution appeared	Serial dilution and scanned between 200nm to 300nm. λmax was 256nm
Table 5.3 Selection of Diffusion media
Drug+ Buffer	Observation	Results
Phosphate buffer pH 7.4	Turbidity appeared	Indicated presence of undissolved drug particles. The diffusion media were found not suitable and hence rejected.
Phosphate buffer pH 7.4 +5% methanol
Phosphate buffer saline pH 7.4
Phosphate buffer pH 7.4+5% methanol
Phosphate buffer pH 5.5	Clear solution appeared	Serial dilution and scanned between 200nm to 300nm. λmax was 256nm

Preliminary determination of diffusion media and dilution media for formulated product was done using various combination of solvents and buffers of different pH. The results showed that phosphate buffer pH 5.5 was found to be clear. So it was used for Itraconazole diffusion study.

5.2.3 Quantitative estimation of Itraconazole in selected media

Figure 5.3 UV scan of Itraconazole in phosphate buffer pH 5.5

The results of UV scan of Itraconazole are shown in figure 5.3 and standard calibration of Itraconazole in phosphate buffer pH 5.5 are shown in table 5.4 and figure 5.4.

Table 5.4 Standard calibration of Itraconazole in phosphate buffer pH 5.5 at 256nm
Conc. (µg/ml)	Absorbance			Average Absorbance
Conc. (µg/ml)	R1	R2	R3	Average Absorbance
4	0.061	0.063	0.061	0.062±0.0012
8	0.121	0.124	0.125	0.123±0.0021
12	0.236	0.234	0.239	0.236±0.0025
16	0.347	0.349	0.351	0.349±0.002
20	0.425	0.423	0.428	0.425±0.0022
24	0.548	0.552	0.550	0.550±0.0022
28	0.681	0.684	0.685	0.683±0.0021
32	0.774	0.779	0.783	0.778±0.0022
36	0.833	0.833	0.835	0.834±0.0012

Figure 5.4 Standard plot of Itraconazole in phosphate buffer pH 5.5

^{From the
above result Itraconazole maximum absorbance was found to be at 256nm. The
curve was found to be linear in the range of 4-36 µg/ml at λmax 256 nm. The calculation of
the drug content,
in vitro release,
and stability studies
are based on this calibration
curve.}

^{5.3 AEROSOL CONTAINER SELECTION}

^{Following
are the aerosol container specifications used for the packaging of Itraconazole
topical spray measured using Vernier caliper:}

^{Packaging
components:}

^{Aluminium
plastic coated can was used with the following specification.}

^{1) Supplier
name: Vimson Aerosol}

^{2) Type of
material used: Aluminium plastic coated can}

Table 5.5 Aerosol container specification
Specifications of the container	Container 1	Container 2	Container 3	Container 4
Height	40mm	40mm	40mm	40mm
Width (inside)	33mm	33mm	33mm	33mm
Width (outside)	34mm	34mm	34mm	34mm
Wall thickness	1mm	1mm	1mm	1mm
Neck curvature	0.07cm^-1	0.07cm^-1	0.07cm^-1	0.07cm^-1
Bottom curvature	0.06cm^-1	0.06cm^-1	0.06cm^-1	0.06cm^-1
Capacity of container	40ml	40ml	40ml	40ml
Empty can weight	22.27g	22.15g	22.2g	22.23g

^{5.5 EVALUATION OF
PRELIMINARY TRIAL BATCHES
OF ITRACONAZOLE TOPICAL SPRAY}

^{The results
of evaluation parameters of preliminary trial batches are shown in table 5.7.}

Table 5.7 Evaluation data of preliminary batches of Itraconazole topical spray
Batch	Spray pattern (cm)	%CDR after 5 hours	% Skin retention after 5 hours
F1	2.76	25.15	20.3
F2	3.13	13.64	38.6
F3	2.14	18.03	27.3
F4	2.56	13.31	33.2

Results of preliminary trial batches showed that F1 and F2 formulation containing 50% LPG, showed more spherical and uniform spray pattern compared to F3 and F4 formulations which contained only 30% LPG. Formulation containing less amount of propylene glycol showed less skin retention and high skin diffusion which was not desired. F2 and F4 formulation containing high concentration of propylene glycol, showed less skin diffusion and high skin retention which was required for topical application. Based on these result co-solvent (propylene glycol) and propellant were selected as an independent variables for optimization of spray formulation using 3²factorial design.

^{5.6
EVALUATION OF ITRACONAZOLE TOPICAL SPRAY}

^{5.6.1
PHYSICOCHEMICAL CHARACTERISTICS}

1. Flame extension and flame flash:

The results of flame extension and flame flash of Itraconazole topical spray are shown in table 5.8

Table 5.8 Flame extension and flame flash batch of Itraconazole topical spray
Formulation	Flame extension (cm) using LPG	Flame flash back (cm) using LPG
F1	64.17	6
F2	64.5	5
F3	63.29	8
F4	48.31	10
F5	51.64	7
F6	49.13	9
F7	50.5	11
F8	47.79	8
F9	46.31	10

The flame extension is due to vapor pressure with which propellant filled in the container. Maximum flame extension was observed for F2 having highest vapor pressure. Flame flash back for all the formulations were found which indicated the minimum leakage on spontaneous release of pressure for all nine formulations.

2. Pressure test:

The results of vapor pressure of Itraconazole topical spray are shown in table 5.9.

All the formulas contained LPG as propellant, their vapor pressure was found in the range from 1.39 to 1.64kg/cm²when measured with pressure gauge.

3. Density:

The results of density of Itraconazole topical spray are shown in table 5.10.

Densities were determined using pycnometer and found to be in range from 0.9 to 0.908 for all the formulations. Densities were not significantly affected by varying the solvent system composition.

4. pH:

The results of pH of Itraconazole topical spray are shown in table 5.11.

The pH for all twelve formulations were near about same i.e., in the range from 6.8 to 7.1. Hence solvent pH was significantly not affected by varying the solvent system composition.

5.6.2 PERFORMANCE

1. Delivery rate of Itraconazole topical spray:

The results of delivery rate of Itraconazole topical spray are shown in table 5.12.

Delivery rate depends upon vapor pressure of propellants used in formulations. Here all formulations contained LPG as propellant in different amount their delivery rate was found in the range from 0.79 g/sec to 2.15 g/sec. The delivery rate of F3 was found to be maximum because of high vapor pressure of LPG filled in it.

2. Drug content of the Itraconazole topical spray:

The results of drug content per actuation of Itraconazole are shown in table 5.13.

All nine formulations contained drug concentrate and propellant in different proportion. Depending upon the vapor pressure in each can the unit content per spray was found maximum for F2 10.8 mg and minimum for F8 8.5 mg.

3. Spray pattern of the formulated Itraconazole topical spray:

The results of spray pattern of Itraconazole topical spray are shown in table 5.14.

The results of ex vivo penetration study of Itraconazole topical spray are shown in table 5.18.

Table 5.16 Minimum fill of Itraconazole topical spray
	Minimum fill (%)
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	100.3	100.1	100	100.1	100.3	100.2	100.5	100.1	100

Table 5.17 Leakage test of Itraconazole topical spray
	Leakage test
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	0.15	0.33	0.11	0.34	0.37	0.27	0.14	0.38	0.21

Table 5.18 (a) Ex vivo penetration study of Itraconazole topical spray
		% Cumulative drug release
Hours		1	2		3		4	5
F1		1.157	4.62		9.50		15.91	23.81
F2		0.94	3.76		7.54		12.10	17.41
F3		0.912	4.06		8.95		14.94	21.97
F4		0.89	4.03		9.28		15.97	23.63
F5		0.83	3.24		6.41		10.40	15.49
F6		0.66	2.61		5.42		9.21	14.06
F7		1.11	4.65		9.96		16.66	24.60
F8		0.77	3.03		6.27		10.61	16.05
F9		1.03	4.34		9.03		14.71	21.55
Table 5.18 (b) Ex vivo penetration study of Itraconazole topical spray
Batch	Total amount of drug diffused through rat skin in 5 hrs (%)=Y			% drug skin retention at the end of 5 hrs		% drug remained over skin at the end of 5 hrs			% loss during handling
F1	23.81			20.3		52.69			3.2
F2	17.41			27.1		52.78			2.71
F3	21.97			33.6		42.13			2.03
F4	23.63			17.6		55.79			2.98
F5	15.49			20.6		60.79			3.12
F6	14.06			18.3		63.96			3.68
F7	24.60			27.3		45.86			2.24
F8	16.05			27.8		55.16			0.99
F9	21.55			26.2		48.32			3.93

Figure 5.7 Ex vivo penetration study for F1, F2, F3

Figure 5.8 Ex vivo penetration study for F4, F5, F6

Figure 5.7 Ex vivo penetration study for F7, F8, F9

From the above stability studies, the optimized formulation was found to be stable in terms of physicochemical parameters as well as biologic parameters under all the storage conditions after 1 month.

^{6. CONCLUSION:}

^{Topical
therapy allows directly delivery of drug to the skin with minimum risk of
systemic side effects. Topical Itraconazole can be generate high local tissue
level, More rapid drug delivery, Lower systemic exposure. Spray can form the
film that is quick drying, easy to use, well tolerated and non occlusive.}

^{Oral and IV
administrations of Itraconazole are associated with systemic side effect and
inconvenience of administration. e.g In oral administration, there is an
insufficient dissolution in the stomach before the drug is delivered to the
intestinal lumen. In IV administration, it can be painful, uncomfortable,
inconvenient and resulting in poor patient compliance.}

^{Itraconazole
an imidazole antifungal is used topically to relieve the symptoms of
superfacial candidiasis, dematophytosis, pityriasis,
versicolor and skin infection.
Itraconazole is a BCS class II drug having high molecular weight.}

^{In this
present investigation, topical spray of Itraconazole was formulated using 32
full factorial design. The result showed that when LPG used as propellant in
aluminum container, they produced Itraconazole topical spray having the
required characteristics. Results of the evaluation parameters were analyzed by
use of design expert software version 8 and an optimized formulation was
derived by using the results of statistical analysis with the software and from
the results obtained after evaluation of this optimized formulation, it could
be concluded that Itraconazole topical spray prepared with 15% propylene glycol
and 50% propellant, gave highest skin retention and highest spray diameter
along with appropriate flame extension, pH, delivery rate, drug content per
actution, spray angel and % CDR. Zone of inhibition was found to be 3.76mm. No
skin irritation was observed during the study. The optimization formulation
passed short-term stability study, carried out for 1 months at25± 2°C and 37±
2°C, with no change in performance characteristic of the products.}

^{From the
obtained values in the
present work, it
can be concluded
that the topical spray formulations for Itraconazole
can be an
innovative and promising approach for
the topical administration of
Itraconazole. The skin
retention studies indicated that
the drug concentration retained
in skin by topical diffusion from spray formulation was higher.}

^{7.
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Received on 12.05.2015 Accepted on 28.08.2015

Research J. Topical and Cosmetic Sci. 6(2): July-Dec. 2015 page 91-126

DOI: 10.5958/2321-5844.2015.00013.8

Table 1.2 Different types of Aerosol systems
Solution system	Two-phase system- vapor and liquid phase. When the active ingredients are soluble in the propellant, no other solvent is required.
Water-Based system	Three-phase system- propellant, water, and vapor Large amount of water can be used to replace all or a part of the nonaqueous solvents.
Suspension or Dispersion system	Dispersion of active ingredients in the propellant or a mixture of propellants. To decreased the rate of settling of the dispersed particles, various surfactants or suspending agents have been added.
Foam system	Consists of active ingredients, aqueous or nonaqueous vehicles, surfactant, and propellant and are dispensed as a stable or a quick- breaking foam. The liquefied propellant is emulsified and is generally found in the internal phase.

Table 1.3 Antifungal agents for the treatment of dermatophytosis
Class of drug	Examples
Azoles (Imidazoles and Triazoles)	Clotrimazole, Miconazole, Econazole, Ketoconazole, Itraconazole Bifonazole, Butoconazole, Croconazole, Eberconazole, Econazole, Fenticonazole, Flutrimazole, Isoconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole,Terconazole, Tioconazole.
Allylamines and Benzylamines	Terbinafine, Naftifine, Butenafine
Morpholine derivatives	Amorolfine
Miscellenous	Griseofulvin

Table 1.4 Marketed preparations of antifungal drugs
Drug	Dosage form	Side effect
Griseofulvin	Tablet, oral Suspension	Epigastric distress; nausea, vomiting, excessive thirst; flatulence; diarrhea; oral thrush, hypersensitivity reactions including rash, and urticaria, headache, fatigue, dizziness, insomnia
Ketoconazole	Tablet, cream, shampoo	Hepatotoxicity, hypoglycemia, nausea, vomiting, abdominal pain, constipation flatulence, GI bleeding, diarrhea, pruritis rash, dermatitis, purpura, urticaria
Fluconazole	Tablet,Oral suspension, IV formulation	Alters creatinine conc, glucose level, Nausea, vomiting, abdominal pain, diarrhea , skin rash(not active against Aspergillus)
Terbinafine	Tablet, cream	Diarrhea, dyspepsia, nausea, abdominal pain, flatulence , taste disturbance, rash , pruritis, urticaria, headache, liver enzyme abnormalities, visual disturbance
Itraconazole	Capsule, oral solution, IV formulation	Diarrhea, dyspepsia, flatulence, abdominal pain, nausea, appetite increase, constipation, gastritis, gastroenteritis and no/in rare cases cutaneous side effects.33-35

Table 1.5 Solubility profile of various antifungal drugs
Drugs	Solubility
Ketoconazole	9.31 mg/ml
Econazole	1.48 mg/ml
Miconazole	0.3 mg/ml
Fluconazole	8 mg/ml
Itraconazole	1 ng/ml
Clotrimazole	29.84 mg/mL

Table 1.6 Marketed preparations of Itraconazole available for treatment of topical fungal infections
Route of administration	Dosage Forms	Strengths	Brand Names	Manufacturer
Oral	Capsules	100 mg*	Itraconazole Capsules	Ortho-McNeil- Janssen
	Solution		Sporanox	Ortho-McNeil- Janssen
IV	Solution	50 mg/5 mL	Sporanox	Centocor Ortho Biotech

Table 4.6: Composition of Itraconazole topical spray
	F1	F2	F3	F4	F5	F6	F7	F8	F9
Drug	1%	1%	1%	1%	1%	1%	1%	1%	1%
DMSO	25%	25%	25%	25%	25%	25%	25%	25%	25%
PG	5%	10%	15%	5%	10%	15%	5%	10%	15%
LPG	50%	50%	50%	40%	40%	40%	30%	30%	30%
IPA	20%	15%	10%	30%	25%	20%	40%	35%	30%

Table 5.2 Reported and observed IR frequencies of Itraconazole
Observed frequency (cm-1)	Reported frequency (cm-1)	Assignment
1699 cm -1	1699 cm-1	C=O stretching
2879-2823 cm-1	2880-2823 cm-1	C-H stretching
1600-1585 cm-1	400-1800 cm-1	C=C stretching in aromatic ring
1224 cm-1	1600-1800 cm-1	C-O stretching or C-N stretching for aromatic amine

Table 5.6 Compatibility studies of Itraconazole with container
Sr. no	Components	Before spray	After spray
1.	Product concentrate
2.	Canister	Aerosol containers were observed before filling with no signs of corrosion, coating dissolution or leakage.	Aerosol containers were observed after filling with no signs of corrosion, coating dissolution or leakage.
3.	Dip tube	Dip tube of aerosol containers were of good quality as per specification.	Dip tube was white colored and does not cause any elongation and leaching.

Table 5.9 Vapor pressure of Itraconazole topical spray
	Vapor Pressure (Kg/cm²) using LPG
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	1.58	1.61	1.64	1.50	1.46	1.52	1.44	1.39	1.40

Table 5.10 Density of Itraconazole topical spray
	Density in g/ml
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	0.903	0.904	0.904	0.9	0.903	0.908	0.9	0.902	0.906

Table 5.11 pH of Itraconazole topical spray
	pH
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	7.1	7.2	7.0	7.0	6.9	7.1	7.0	7.2	7.1

Table 5.12 Delivery rate of Itraconazole topical spray
	Delivery rate (g/sec) using LPG
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	1.17	1.83	2.15	1.06	0.91	1.23	0.83	0.79	0.85

Table 5.13 Drug content of Itraconazole topical spray
	Drug content (mg)
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	9.7	10.8	10.2	9.5	9	9.4	8.8	8.5	9.2

Table 5.14 Spray pattern of Itraconazole topical spray
Formulations	Spray pattern (cm)			Average	Ovality ratio
F1	2.6	2.7	2.8	2.7	1.07
F2	3.1	3.2	3.1	3.13	1.04
F3	3.4	3.5	3.4	3.43	1.03
F4	2.6	2.6	2.7	2.63	1.04
F5	2.7	2.5	2.7	2.63	1.08
F6	2.8	2.7	2.9	2.8	1.07
F7	2	2.3	2.1	2.16	1.15
F8	2.6	2.6	2.8	2.66	1.07
F9	2.5	2.6	2.6	2.56	1.04

Table 5.15 Spray angle of Itraconazole topical spray
	Spray angle
Formulas	F1	F2	F3	F4	F5	F6	F7	F8	F9
Formulas	20	19	21	19	18	18	19	18	17

Table 5.19 ANOVA table for skin retention
Source	Sum of square	Degree of freedom	Mean square	F- Value	P- value	Remark
Model	218.33	5	43.67	14.32	0.0264	Significant
X1- propylene glycol	169.60	1	169.60	55.63	0.0050
X2- propellant	9.88	1	9.88	3.24	0.1696
X1X2	10.24	1	10.24	3.36	0.1642
X1²	23.81	1	23.81	7.81	0.0682
X2²	4.81	1	4.81	1.58	0.2982
Residual	9.15	3	3.05	-	-
Cor Total	227.48	8	-	-	-

Table 5.20 Statistical parameters obtained from ANOVA study
Standard deviation	1.75
Mean	24.87
C. V %	7.02
R-Squared	0.9598
Adjusted R-Squared	0.8928
Predicted R-Squared	0.5200
Adequate precision	10.568

Table 5.21 ANOVA table for spray pattern
Source	Sum of square	Degree of freedom	Mean square	F- Value	P- value	Remark
Model	0.82	2	0.41	22.96	15.54	Significant
X1-propylene glycol	0.31	1	0.31	37.17	11.70
X2-propellant	0.51	1	0.51	61.55	19.37
Residual	0.16	6	0.026	-	-
Cor Total	0.98	8	-	-	-

Table 5.22 Statistical parameters obtained from ANOVA study
Standard deviation	0.16
Mean	274
C. V %	5.93
R-Squared	0.8382
Adjusted R-Squared	0.7842
Predicted R-Squared	0.6183
Adequate precision	11.062

Table 5.23 Comparison of predicted and obtained values
Parameters	X1	X2	% Skin retention	Spray pattern (cm)
Predicted values	15	50	32.43	3.25
Obtained values (F3)	15	50	33.6	3.43

Table 5.24 In vitro antifungal activity of the optimized Itraconazole topical spray
Formulation	Zone of inhibition	Average
Itraconazole 1% spray	3.6 mm	3.76 mm
	3.9 mm
	3.8 mm

Table 5.25 Skin irritation studies of the optimized Itraconazole topical spray
Formulation	Before spray	After spray
Itraconazole topical spray (1)
Itraconazole topical spray (2)

Table 5.26 Stability studies of optimized formulation
Parameters evaluated	Initial	After 1 month
Parameters evaluated	Initial	25±2	37±2
Flame extension (cm)	63.29	62.41	64.2
Flame flash back (cm)	8	6	8
Pressure (kg/cm2)	1.64	1.62	1.59
Density ( g/ml)	0.904	0.903	0.906
pH	7	7.1	7
Delivery rate (g/sec)	2.15	2	2.13
Spray pattern	Uniform and circular	Uniform and circular	Uniform and circular
Spray angle	21	19	21
Minimum fill (%)	100	100.2	100.3
In vitro zone of inhibition (mm)	3.76	3.5	3.8
% skin retention at the end of 5 hours	33.6	30.8	32.4