Advanced topical drug delivery system for the management of vaginal candidiasis

Abstract

Vaginal candidiasis or vulvovaginal candidiasis (VC) is a common mucosal infection of vagina, mainly caused by Candida species. The major symptoms of VC are dyspareunia, pruritis, itching, soreness, vagina as well as vulvar erythema and edema. Most common risk factors that lead to the imbalance in the vaginal micro biota are the use of antibiotics, pregnancy, diabetes mellitus, immuno suppression as in AIDS or HIV patients, frequent sexual intercourse, spermicide and intra-uterine devices and vaginal douching. Various anti-fungal drugs are available for effective treatment of VC. Different conventional vaginal formulations (creams, gels, suppositories, powder, ointment, etc.) for VC are available today but have limited efficacy because of lesser residence time on vaginal epithelium due to self-cleansing action of vagina. So to overcome this problem, an extended and intimate contact with vaginal mucosa is desired; which can be accomplished by utilizing mucoadhesive polymers. Mucoadhesive polymers have an excellent binding capacity to mucosal tissues for considerable period of time. This unique property of these polymers significantly enhances retention time of different formulations on mucosal tissues. Currently, various novel formulations such as liposomes, nano- and microparticles, micro-emulsions, bio-adhesive gel and tablets are used to control and treat VC. In this review, we focused on current status of vaginal candidiasis, conventional and nanotechnology inspired formulation approaches.

• Bio-adhesive polymers
• liposomes
• nanotechnology
• novel drug delivery systems
• vaginal candidiasis

Introduction

Vaginal candidiasis (VC) often referred to as vulvovaginal candidiasis, is a common mucosal infection of vagina, mainly caused by Candida species (Alexander et al., 2004) and alleged to be the second most prevalent mucosal infection after bacterial vaginosis. It is a far-flung infectious disease affecting about 75% of women of reproductive age (Song et al., 2004). In the United States alone, annually 13 millions of cases of VC are observed which further results in 10 million gynecologic office visits (Francois et al., 2003). In 2002 in United States, women spend over half a billion dollars on the medication for the treatment of VC, and about half of this amount was spend on over the counter medicines (Jyotsana et al., 2010). This is despite the fact that most of the women may wrongly diagnose VC as bacterial vaginosis (De Blaey & Polderman, 1980). The major symptoms of VC are dyspareunia, pruritis, itching, soreness, signs of vagina and vulvar erythema and edema (Lee, 1990; Francois et al., 2003). Candida species, especially Candida albicans is responsible for VC. It is a dimorphic commensal organism that domiciliation on skin, mucosa and gastrointestinal tract of 30–50% of normal healthy individual. Candida albicans is not a pathogen, but when local or systemic defense mechanism of the host got afflicted, Candida spp. can induce oropharyngeal, esophageal or VC (Woolfson et al., 2000). Under normal healthy conditions, lactobacillus in vagina produces lactic acid, which act as buffer and maintains the pH of vagina in the range 4–5 (acidic) and bacteriocins and hydrogen peroxide (H₂O₂), which resist the overgrowth of pathogenic microbes. In certain ill conditions, when this balance gets disturbed, there occurs excessive overgrowth of Candida sp. and diminution or depletion Lactobacillus spp. Following the overgrowth, there are two crucial elements responsible for the developments of VC are vaginal epithelium colonization and transformation of asymptomatic (saprophytic phase) to symptomatic (pathogenichyphal phase). Most common risk factors that lead to the imbalance in the vaginal micro biota are the use of antibiotics, pregnancy, diabetes mellitus, immuno suppression as in AIDS or HIV patients, frequent sexual intercourse, vaginal douching, spermicide and intra-uterine devices (Gagandeep et al., 2014). Most commonly used drugs for VC are Fluconazole, Clotrimazole, Metronidazole, Miconazole, Econazole, Ticonazole, Voriconazole, and Isoconazole. In pharmaceutical literature, vagina is described as slightly S-shaped fibro muscular, collapsible tubular organ of approximately 6–10 cm length that extends from cervix of the uterus to the vestibule of the external genitalia (Washington et al., 2000; Woolfson et al., 2000) and has two main functions: (1) Serves as receptacle for penis during sexual intercourse and carries sperm to the uterus and fallopian tubes. (2) As a birth canal for the passage of the baby during labor. The vagina comprises of three different cell layers: epithelial layer (superficial layer), lamina propria or tunica, muscular coat (D Amati et al., 2003). Overall of 10–15 layer cell turnover is expected in the time period of 7 d (Sjöberg et al., 1988). A brief description of vaginal anatomy and physiology is presented in Table 1.

Table 1. Anatomy and physiology of vagina.

Vaginal physiology	Characteristics	Description
Epithelium	Stratified, Non-keratinized squamous	• Thickness is higher in postmenopausal women than premenopausal women • 25 layered thick estrogen content act as a dominant physical barrier • Have numerous folds, known as “Rague” (Hussain & Ahsan, 2005); which helps in easy incorporation of different formulations and enhances absorption of drugs by providing distentibility, support, increasing surface area (Choudhury et al., 2011).
Vaginal secretion	Vagina does not possess any gland (Paavonen, 1982). Vaginal fluid comprises of exudates from blood vessels, secretion from fallopian tubes, peritoneal, uterine, Bartholin's and Scene's gland (Francois et al., 2003)	• Fluid provide moisture • Solubilize solid dosage formulations in vagina • Volume and composition of vaginal fluid varies with age, infection, sexual arousal (Masters & Johnson, 1966)
pH	Range of 3.5–4.5 (average 4.2)	• Lactobacillus sp. produces lactic acid from glycogen, which helps in maintaining healthy acidic conditions in vagina • Varies with age (new born = 4–5, pre-puberty = 7, puberty = 5–7, child bearing = 4–5, pregnancy = 3–5, menopause = 6–7 and post menopause = 7–7.4). • Ionization of ionic drug alter with slight shift in vaginal pH; thus changes stability, solubility and absorption of drugs
Micro flora	Vagina has a complex micro-ecological system	• Lactobacillus is the predominant flora in vagina • It produces lactic acid, H2O2, bacteriocins, thus maintains acidic vaginal environment and also resist growth of pathogenic micro-organisms • Composition of vaginal flora varies with menstrual cycle, gestation, use of contraceptives, frequency of sexual intercourse, etc. • Candida sp. concentration reaches peak level in pre-menstrual period.

Pathophysiology of vaginal candidiasis

A normal healthy micro floral balance of Lactobacillus sp. and Candida sp. exists in vagina. When this balance gets disturbed due to certain risk factors, there occurs excessive growth of Candida sp. (Virulent). This is followed by cascade of reactions that finally leads to damage to vaginal epithelium and then symptoms get precipitated and VC occurs (Figure 1).

Figure 1. Pathophysiology of vaginal candidiasis.

Adhesion to vaginal epithelia

The initial and critical step toward fungal infections is the adhesion of Candida to epithelial cells. Candida albicans interacts by colonization and proliferation on epithelial cells followed by invasion, dissemination and damage. Cell wall components of Candida play a key role in adhesion process. Different cell wall protein adhesion candidates are:

• The Als (agglutinin-like sequence) family: Till date 8 ALS genes have been recognized ALS 1–ALS 7 and ALS 9, that are involved in adhesion. N terminus of ALS protein is involved in ligand binding (Loza et al., 2004; Rauceo et al., 2006; Liu & Filler, 2011). Study of ALS-deleted mutants have variable effect on adhesion like expression of C. albicans ALS1 or ALS5 genes in non-adhesive, ALS4 deletion decreases C. albicans adherence to endothelial cell.

• Hypha-associated genes: Hyphal wall protein (Hwp 1), major protein on hyphal cell wall. Its N-terminal domain serves as a substrate for epithelium transglutaminases. Thus, a strong covalent linking occurs between Hwp and epithelium proteins. Eap1, and Int1, also involved in adhesion but their binding ligands are unknown.

• Integrin α_m β₂-like adhesins: Different ligands, including iC3b, fibrinogen, factor X, urokinase receptor, CD14, CD23, CD54 (ICAM-1), CD102 (ICAM-2), CD242 (ICAM-4), heparin, haptoglobin, kininogen, and various microbial proteins (Haas & Plow, 1994). Out of these molecules, only ICAM-1 and -2 are widely expressed on endothelial cells.

• Integrin α_v β₃ and αvβ5-like adhesins: α_v β₃ like adhesion has been shown to bind to vitronectin (Spreghini et al., 1999; Santoni et al., 2001), but other ligands for α_v β₃ include CD31 (PECAM-1), Fibronectin, fibrinogen, thrombospondin, von Willebrand factor, and RGD sequence peptides (Haas & Plow, 1994). CD31 is expressed by endothelial cells and could act as a direct ligand for Candida adhesion αvβ5 ---- vitronectin, RGD sequence peptides but lack epithelium specific binding ligand (Jouault et al., 2006).

Transmigration vaginal epithelium

After adhesion, next step is the migration across vaginal epithelium. Different mechanism through which Candida migrates is:

• Induced endocytosis: Two Candida invasions, ALS 3 and SSA 1 (SSA 1 is a member of the heat Shock protein (HSP) 70 family that is expressed on the cell surface). Present in hyphal cell wall and induce endocytosis. These binds to E- cadherin, then tyrosine phosphorylation occurs that leads to microfilament rearrangement and then leads to pseudopod formation and subsequent engulfment into the cell through clathrin mediated actin-dependent mechanism.

• Active penetration: Fungi must be viable and changes to hyphal form during or after the penetration. Sap enzymes primarily contribute to active penetration. Sap (Secreted aspartic proteinases) 5 degrade E- cadherin of epithelial cell and violate integrity of vaginal epithelium, thereby enabling hyphal penetration into epithelial cells.

• Damage: Once Candida goes across vaginal epithelium, it cusses severe damage by apoptosis and necrosis. However, exact mechanism behind this is yet to be revealed.

Figure 2 represents diagrammatic description of vaginal colonization of Candida Spp. Table 2 discusses the different ligand specific adhesion and invasions involved in adhesion and transmigration across vaginal epithelium.

Figure 2. Diagrammatic description of vaginal colonization of Candida spp.

Table 2. Ligand specific adhesion and invasions involved in adhesion and transmigration across vaginal epithelium.

Interaction of Candida with epithelium	Candidates	Ligand
Adhesion	Als 1-7, 9	N- cadherin
	Hwp 1	transglutaminases
	Eap1	Unknown
	Int1	Unknown
	Integrin αm β2-like adhesins	ICAM-1 and -2
	Integrin αv β3 like adhesins	CD31 (PECAM-1),
	N-linked mannosyl residues	Mannose receptor (MR)
	O-linked mannosyl residues	Toll-like receptor 4 (TLR-4)
	Phospholipomannan	TLR-2
	Mannosides	galectin-3
Transmigration	ALS 3 and SSA 1	E- cadherin,
	Sap enzymes	E- cadherin,

Prevalence of vaginal candidiasis

VC has a wide geographical distribution all over the world (Table 3). On the basis of various research papers it can be concluded that C. albicans is the most dominant and prevalent sp. in females with VC as it has an excellent binding capacity for mucus membrane. Non-albicans species are contributing to VC for a far lesser extent. VC with positive samples was observed to occur on higher levels on USA and UK while other countries had significant lesser number of positive samples. Among all positive samples, occurrence of C. albicans sp. was much higher than non-albicans sp. Among non-albicans sp., C. glabrata was the most common fungi found in subject's vagina. This vast variation was observed in females which are illiterate, high school education, married, having diabetes mellitus (Alli et al., 2011; Faraji et al., 2012), frequent sexual intercourse, oral contraceptives, spermicides (Alli et al., 2011), etc.

Table 3. Worldwide distribution of various Candida sp. responsible for vaginal candidiasis.

		Candida
Country	Total no. of subjects	Albicans (%)	Glabrata (%)	Parasilosis (%)	Krusei (%)	Tropicalis (%)	Other sp. (%)	References
IOWA	593	70	18.8	5	2	–	1.6	(Richter et al., 2005)
USA	93 775	88.9	7.9	1.7	1.4	0.008	–	(Vermitsky et al., 2008)
Nigeria	106	36.8	–	5.6	1.88	10.3	–	(Ogunshe et al., 2008)
Iran	605	26.28	0.82	0.33	4.29	0.33	4.13	(Shafik et al., 2007; Faraji et al., 2012)
Pakistan	250	12	3.2	4	1.2	8.4	3.2	(Khan & Baqai, 2010)
India	350	17.42	2.5	0.5	0.8	1.4	0.5	(Jindal et al., 2007)
Australia	275	15.63	4.3	1.09	–	0.3	–	(Pirotta & Garland, 2006)
UK	548	86.86	2.7	0.5	0.1	0.7	0.9	(El-Din et al., 2001; Dias et al., 2011)
Brazil	404	33.16	1.2	1.2	1.2	1.2	–-	(Sobel et al., 2004)

A concerning trend was observed in prevalence of Candida sp. in different age groups. With increasing age, a drastic fall in albicans sp. was observed; however, there is a significant increase in distribution of non-albicans sp. among which C. glabrata was the dominant one. Increase in number of C. glabrata was also observed in elderly diabetic patients (Vermitsky et al., 2008). This can be justified, as continuous use of azole agents leads to development of resistance in C. glabrata, which is characterized by higher colonization of C. glabrata in vaginal epithelium as compared to C. albicans. Thus for effective treatment of VC, proper microscopic identification of virulent sp. should be done. Non-azole anti-fungal drugs like Boric acid and Flucytosine can be used for non-albicans species (Ogunshe et al., 2008; Vermitsky et al., 2008; Abruquah, 2012).

Factor affecting vaginal drug absorption (Stewart-Tull, 1964; Hussain & Ahsan, 2005; Mathiowitz et al., 2013)

Like other mucosal routes, drug administrated via vaginal route is absorbed by three major ways: (1) transcellularly; mediated via concentration-dependent gradient (2) paracellularly; through tight junctions present in between the cells (3) vesicular or receptor-mediated transport as remarked by Ilium and Richardson. Absorption of drug from vagina follows two main steps: drug dissolution in vaginal lumen and membrane penetration. So any factor influencing physiology of vagina and formulation aspects like drug dissolution and membrane transport will potentially alter the absorption profile of drug from vaginal drug delivery systems (Garg et al., 2014a). Different vaginal physiological factors that influence drug absorption in vaginal cavity are discussed in Table 4.

Table 4. Factor affecting vaginal drug absorption.

Factors	Sub-types	Drug-related features
Physiological factors	Vaginal epithelium thickness Vaginal fluid Cervical mucus pH	• Higher the epithelium thickness lesser will be permeability and vice versa; thus absorption varies. • Example: as in case of steroids (Vermesh et al., 1988) and estrogen. • In guinea pigs, in early disastrous stage, Vidarabine has shown a 5–100 times more permeability coefficient as compared to that in oestrous stage (Hwang et al., 1977). • Poorly water soluble drugs are more frequently absorbed when fluid volume is high. • However this condition may remove drug from vaginal cavity thus reducing drug absorption (Owen et al., 1999). • Thick mucus is less permeable and vice versa. • It act as permeability barrier for most of the drugs (Johnson et al., 1992). • It can be exploited for bioadhesive delivery systems • For pH-sensitive drugs and drugs which are weak electrolyte, alteration in vaginal pH may alter drug ionization, solubility, stability and subsequent drug release(Katz & Dunmire, 1993).
Physicochemical factors	Lipophilicity Molecular weight Solubility Degree of ionization	• Lipophilic steroids like progesterone and estrone have higher permeability as compared to hydrophilic steroids i.e. hydrocortisone and testosterone (Robinson & Bologna, 1994). • Lipophilic drugs of low molecular weight are readily absorbed then high molecular weight hydrophilic or lipophilic drugs. • As vaginal fluid have some water content so it favors absorption of drugs having certain solubility in water (Hwang et al., 1976). • Drugs like peptide, weak electrolyte are frequently absorbed in their unionized form (Brannon-Peppas, 1993).

Available therapies for vaginal candidiasis

Various anti-fungal drugs are available for effective treatment of VC. The treatment is initiated in symptomatic women because they have 80% of Candida colonization (Syed & Braverman, 2004). The endeavor of the treatment is to prevent over-growth of Candida that precipitates symptoms. Almost 3–7 d are sufficient for effective results. Formulation other than oral, are usually administered at night to prevent any leakage or removal from vagina. There is no evidence available favoring any specific formulation or any particular azole agent. However in case of severe infection, oral preparations may not provide symptomatic relief. In that case low-potency steroids as topical formulations should be used (Cejtin & Mason, 2000). Different oral and topical therapies available for VC are given in Table 5.

Table 5. Different available therapies for VC (Faro, 1994; Carr et al., 1998; Watson & Pirotta, 2011; Newson, 2013).

Agent	Formulation	Dose
Clotrimazole	1% cream	5 g, intravaginal for 7 to 14 d
	100 mg vaginal tablet	100 mg × 7 d
	100 mg vaginal tablet	200 mg × 3 d
	500 mg vaginal tablet	500 mg × single dose
Miconazole	2% cream	5 g, intravaginal for 7 d
	100 mg, vaginal suppository	100 mg 7 d
	200 mg, vaginal suppository	200 mg × 3 d
	1200 mg vaginal suppository	1200 mg × single dose
Terconazole	80 mg vaginal suppository	80 mg × 3 d
	0.4% cream, 5 g,	5 g × 7 d
	0.8% cream, 5 g,	5 g × 3 d
Ticonazole	6.5% ointment, 5 g,	5g × single dose
	2% cream	5 g × 3 d
Nystatin	100 000 IU, vaginal tablet,	100 000 IU, × 14 d
Butoconazole	2% cream,	5 g, × 3 d
Ketoconazole	200 mg oral tablet	400 mg × 5 d
Fluconazole	150 mg oral capsule	150 mg × single dose
Itraconazole	100 mg oral capsule	200 mg × 3 d

While these therapies are quite effective, but still associated with number of limitations like side effects, drug interaction, contraindication, etc., as presented in Table 6.

Table 6. Drug associated limitations for anti-fungal therapy.

Anti-fungal drug	Side-effect	Drug interaction	Contra-indications
Fluconazole	• Nausea, vomiting, abdominal pain, and diarrhea, have been reported in approximately 5% of patients (Ernest, 1992). • Skin rash, acne, itch, headache, GI upset(Sobel et al., 1995) • Abnormal liver function in 5.1% of patients, Hepatotoxicity in AIDS patient(Gearhart, 1994)	• Cisapride • Erythromycin, non-sedating antihistamines, diuretics raise fluconazole levels. • Dilantin, oral hypo-glycemic drugs benzodiazepines; theophylline and warfarin level can get elevated. • Cimetidine may limit efficacy of fluconazole.	Renal and hepatic dysfunction Pregnancy
Itraconazole	• Nausea, headache, dizziness(Stein & Mummaw, 1993) increased level of transaminase enzyme • Reversible peripheral neuropathy (Hay, 1993) and reversible changes in liver function with low frequency	• Quinidine • Pimozide • Dofetilide • Modazolam • Nisoldipine • Ergotamine	Heart disease
Clotrimazole	• Vaginal burning, sensitive clitoris in (5%) Patients(Fong, 1992)	• Cholecalciferol • Acetaminophen • Montelukast • Gabapentin • Furosemide • Diphenhydramine • Aspirin	Hepatic dysfunction
Ticonazole	• Local pruritis, local burning, vaginal irritation. recurrent candidiasis (30%)(Stein et al., 1986)	• Cyclosporine, • Methotrexate • Prednisone	Hypersensitivity, diabetes
Ketoconazole	• Hepatotoxicity, alcohol intolerance, anorexia, increased appetite, headache, dizziness, insomnia hepatitis, jaundice,	• Alprazolam • Midazolam • Triazolam • Quinidine • Amlodipine • Felodipine • Nicardipin • Phenytoine • Nifedipine • Cyclosporine • Tacrolimus	Liver disease, hypersensitivity

Conventional topical intravaginal delivery systems

Creams and gels

Creams and gels as intravaginal delivery systems are used to deliver contraceptives and anti-bacterial agents (Garg & Goyal, 2014a). However these systems are messy in use, uncomfortable and because of non-uniformity and leakage, exact dose can never be provided. The worthy properties of vaginal creams and gels are acceptability; feasibility and non-toxic, non-irritant nature towards vaginal mucosa. Vaginal creams of metronidazole and clindamycin are found to be as efficacious as the orally administered drugs for treatment of bacterial vaginosis (Mcgregor et al., 1998). Oxytocin, dinoprostone and misoprostol used for cervical ripening and labor induction, can be administered in gel form. Shetty et al. studied efficacy of dinoprostone vaginal gel against oral tablet for induction of labor and observed significant difference there. Several researchers are comparing efficacy of vaginal gel with oral products for misoprostone, and the results obtained leads to conflicting outcome. Hall et al. reported that orally given misoprostone is far more safe and effective in labor induction against when it is vaginally administered. However, Shetty et al. concluded that among vaginally and orally administered misoprostone, vaginal delivery was the most effective. Vaccines can also be delivered intravaginal in the form of gel.

Pesseries and suppositories

A variety of vaginal medications are available in form of pesseries and suppositories. They are designed in such a way to melt in vaginal cavity and release active medicament in controlled manner. Suppositories are used for localized delivery of drugs like anti-septic, anti-fungal and contraceptives. Primarily, they are used to deliver drugs like dehydroepiandrosterone Sulphate (Yamashita et al., 1991) for cervical ripening, prior to birth; miconazole for VC (Vukovich et al., 1977; Abrams & Weintraub, 1983) and progesterone for hormonal replacement therapy. Different techniques for preparation of suppositories are hand-molding, pour-molding or by automatic machine (Brannon-Peppas, 1993; Hussain & Ahsan, 2005) where drug is dispersed in suppository base, e g. cocoa butter. Pesseries are known to deliver prostaglandin E₂ (PGE₂) for cervical ripening and labor induction. A semi-crystalline hydrogel of cross linked polyethylene oxide swells in saturated solution of PGE₂ to give final product, pesseries.

Vaginal tablets, powder and ointment

Vaginal tablets contain same components as that of conventional oral tablets like binders, disintegrants and other excipients. These are advantageous over the other dosage form as have ease of manufacture and insertion. Usually deliver prostaglandins and anti-fungal drugs like Itraconazole, Clotrimazole, etc. Highly hydrophobic drugs are not suitable candidates for vaginal tablets as they have poor absorption. However, use of penetration enhancers like surfactants, bile salts can overcome this problem. Sometimes mucoadhesive polymers can be incorporated to enhance vaginal residence time. Polystyrene sulfonate (PSS) when formulated as vaginal tablet, have higher anti-microbial effect against HIV and HSV and is neither cytotoxic nor it inhibit vaginal flora (Kast et al., 2002). Vaginal powder is prepared by dissolving Hydroxypropyl cellulose in water with continuous heating. This mixture is then slightly cooled and bisphosphonate was added. This final mixture was then lyophilized. Vaginal ointment comprises of an aqueous phase and oil phase. Drug was dissolved in the aqueous phase and the oil phase was incorporated into it with mixing (Kaur et al., 2014).

Vaginal ring

Vaginal rings are circular device inserted in the vagina to achieve controlled release of the active medicament. These are approximately 5 cm in diameter and have 4–5 mm of cross-sectional diameter. These are generally polymeric rings in which the drug is homogeneously dispersed. These offer several advantages: user controlled, deliver drug continuously and do not interfere with coitus. From the surface of the ring, drug release at faster rate as compared to the inner layer of ring. This may provide an initial burst release of the drug followed by sustained release for several days. In order to achieve constant release, two types of system are developed for vaginal rings: sandwich and reservoir type. In sandwich type, a narrow layer of drug is placed between non-medicated central core and non-medicated outer band. In reservoir type, central core having the drug is encapsulated with drug-free polymer layer (Garg & Goyal, 2012). Commonly used polymers are poly (dimethylsiloxane) or silicone devices. Moreover in the recent years, elastomeric polymer like ethylene vinyl acetate and styrene are extensively used, as it have increased flexibility, improved optical properties, greater adhesion and increased impact and punch resistance (Novak et al., 2003). Vaginal rings are most commonly employed for hormonal replacement therapy and contraceptives delivery. To deliver contraceptive, rings are placed in vagina for 21 d followed by 1 week ring free for menstrual cycle to take place. NuvaRing is a common example of vaginal ring available in U.S market to deliver contraceptives. It is transparent, flexible ring containing etonogestrel and ethinyl estradiol and releases 120 mg/d of former and 15 mg/d of later one over a period of 3-week. Femring and Estring are employed for the hormonal replacement therapy. Dapivirine also known as TMC 120 is given in the form of ring acting as potent microbicide against transmission of STIs and HIV. Plastic rings are sometimes used to hold and support suppositories in position in vagina.

Limitations of conventional vaginal formulations

These conventional vaginal delivery systems are somewhat effective; however, they still offer several disadvantages which we need to encounter in order to deliver anti-fungal therapy in an efficacious way. Disadvantages associated are:

• Leakage and messiness as in case of creams and gel

• Uncomfortable

• Efficacy is quite low as gels may not provide an exact dose because of non-uniformity and leakage

• Low retention to the vaginal epithelium

• Poor patient compliance

• Frequent administration of drug is required

• Prolonged duration of therapy

• Low bioavailability

• Drug release pattern is inappropriate (Parnami et al., 2013; Singh et al., 2014).

Novel approaches in vaginal formulations

Different conventional vaginal formulations for VC are available today but have limited efficacy because of lesser residence time on vaginal epithelium due to self-cleansing action of vagina. This leads to frequent administration of the formulation, which ultimately cause inconvenience to the user. So to overcome this problem, an extended and intimate contact with vaginal mucosa is desired; which can be accomplished by utilizing mucoadhesive polymers. Mucoadhesive polymers have an excellent binding capacity to mucosal tissues for considerable period of time (Kataria et al., 2014). This unique property of these polymers significantly enhances retention time of different formulations on mucosal tissues. Thus, controlled release can be fruitfully achieved and in turn frequent administration of dosage forms is prevented. Several bio-adhesive polymers are available like polycarbophil, hydroxypropylcellulose, polyacrylic acid, chitosan, carbopol, etc.

Vaginal bio-adhesive tablets

Method for preparation of vaginal bio-adhesive tablets is similar to those of normal tablet; however, they differ in composition of excipients as the former have a single or combination of bio-adhesive polymer and the latter one is devoid of it. In case of evaluation of these tablets, additional parameters are included like swelling index, bio-adhesion time and bio-adhesive strength. The very first bio-adhesive tablet prepared was of Bleomycin, antibiotic; containing polymers like hydroxy propyl cellulose (HPC) and poly acrylic acid (PAA) or Carbopol-934. It was observed that with increasing amount of HPC, in-vitro release rate increases and increment in concentration of PAA, water absorption property rises. 5-Flurouracil and Carbaquinone, potent anti-cancer drugs were also formulated in the tablet form (Brannon-Peppas, 1993). Various anti-fungal agents are formulated in form of bio-adhesive tablets are mentioned in Table 7.

Table 7. Vaginal bio-adhesive tablets loaded with anti-fungal agent.

Active drug	Bio-adhesive polymer system	Comments	Reference
Clotrimazole	Chitosan-Thioglycolic acid Conjugate (TGA)	The polymeric conjugates have 26-time longer adhesion time as compared to unmodified polymer.	(Kast et al., 2002)
Econazole nitrate	Carbopol 941/ NaCMC (1:1)	Moderate swelling Good bio-adhesion for much longer duration Retarded release profile of the drug	(Ameen)
Clotrimazole	Carbopol 934P/ Sodium alginate (2:1)	Releases the drug for extended period of 24 h Significant bio-adhesion property	(Sharma et al., 2006)
Ketoconazole (bio-adhesive effervescent tablet)	Carbopol 934P/ HPC (1:9)	Excellent swelling, Controlled release of drug(95% for 24 h) In-vivo study in rats showed high vaginal residence time (17% drug still retained after 24 h.)	(Wang & Tang, 2008)
Clotrimazole	Mixture of NaCMC and HPMC	Slowly released 72% of the drug over 12 h. Good swelling Excellent bio-adhesiveness	(Bhat & Shivakumar, 2010a)
Ketoconazole effervescent tablet	HPMC K4M: Chitosan (1:1) effervescent (sodium bicarbonate and citric acid at the mole ratio of 3:1)	High in-vitro anti-fungal activity Bio-adhesion time more than 12 h Sustained release (more than 94% in 10 h.)	(Patel & Patel, 2010)
Sertaconazole effervescent tablet	Combination of HPMC K4M:Carbopol 934P Effervescent mixture (sodium bicarbonate and citric acid at the mole ratio of 3:1)	Controlled release (more than 80%) in 12 h. Excellent bio-adhesive strength High in-vitro anti-fungal activity	(Patel et al., 2012)
Clotrimazole	Combination Chitosan: HPMC K15 M(3:1)	Extended release profile( 98% for 30 h) Good bio-adhesive strength Good swelling index	(Dangi et al., 2011)
Clotrimazole	Eudragit RL-100	Excellent in-vivo bio-adhesive strength and time Sustained release profile of 98% after 24 h. Excellent in-vitro anti-fungal activity	(Gupta et al., 2013)

Vaginal liposome

Liposomes are the spherical vesicle, characterized on their lipid composition, size, number of lamellae, and inner/outer phases (Garg & Goyal, 2014b). Because of biocompatibility, stability and structural versatility, these have been extensively used for different therapies (Goyal et al., 2013). For having high stability with good mucoadhesive strength, positively charged vesicles are preferred over negative ones; as mucus membrane is negatively charged (Garg et al., 2014b). Before 1990, liposomes were used for parenteral and skin delivery but later on there was a drastic shift in their use in vaginal drug delivery. Jain et al. utilized liposomes for vaginal delivery of progesterone (1997). Foldvari et al. developed interferon alpha liposomes for treating genital papilloma virus infections. Pavelić et al. developed Lecithin liposomes of Clotrimazole, metronidazole and chloramphenicol for treating fungal infections; then tested for in-vitro stability in pre- and post- menopausal environment, as well as for in-situ stability in cow vaginal mucosa. In order to enhance stability, better release characteristics and overall applicability of these drugs, author incorporated these liposomes in bio-adhesive carbopol hydrogels. In-vitro release testing performed in vaginal fluid stimulant ensured controlled release of all three drugs (Pavelić et al., 1999). Ning et al. also reported controlled release of Clotrimazole from proliposomes for vaginal therapy. Poorly soluble anti-fungal drug, Amphotericin B was successfully administered in vagina when formulated as thermo-sensitive gel of poloxamers 407 and 188 having drug-loaded cationic liposomes (Kang et al., 2010). Curcumin, a well-known anti-oxidant and anti-inflammatory agent when formulated in form of liposomal gel against vaginal inflammation; overall anti-inflammatory activity was significantly enhanced as revealed by in-vitro studies (Basnet et al., 2012). Liposomal preparations loaded with anti-fungal agents are represented in Table 8.

Table 8. Anti-fungal agents formulated as vaginal liposomes.

Active drug	Bioadhesive polymer system	Comments	Reference
Clotrimazole	Carbopol	Sustained release profile Significant in-vivo activity in rats	(Ning et al., 2005)
	Carbopol	Controlled release was achieved High in-situ stability in cow's vaginal mucosa	(Pavelić et al., 2001)
Amphotericin B	Poloxamer 407 and 188	Good in-vitro anti-fungal activity	(Kang et al., 2010)
Metronidazole (Elastic liposomes)	Carbopol	Controlled release for 24 h	(Vanic et al., 2013)
	Carbopol	Controlled release was achieved High in-situ stability in cow's vaginal mucosa	(Pavelić et al., 2001)

Vaginal micro-emulsions

In recent times, micro emulsion serves as an efficient candidate for vaginal delivery of proteins, peptides and anti-fungal drugs, because of their long term stability, ease of preparation and high solubilization capacity. Micro-emulsion based vaginal gel system has been efficiently used to deliver different anti-fungal drugs as given in Table 9.

Table 9. Micro-emulsion based vaginal gel loaded with anti-fungal agents.

Drug	Formulation	Bio-adhesive polymer	Animal model	Comments	References
Clotrimazole	Micro emulsion based vaginal gel	Carbopol ETD-2020	In-vitro	High in-vitro bio-adhesion time Excellent in-vitro anti-fungal activity as compared to marketed formulation Controlled release profile of drug (more than 85% in 12 h.)	(Bachhav & Patravale, 2009)
Sertaconazole	Micro emulsion based vaginal gel	Carbopol 940	In-vitro	Excellent anti-fungal activity Good bio-adhesive and retention properties Controlled release of 99% in 8 h.	(Patel & Patel, 2012)
Miconazole nitrate	Micro emulsion based gel	Polycarbophil	In-vitro	High bio-adhesive strength Excellent in-vivo anti-fungal activity in mice Good in-vitro anti-fungal activity	(Bhalekar et al., 2009)

Vaginal bio-adhesive suppositories

Another novel approach towards successful vaginal delivery is the concept of bio-adhesive suppositories. To deliver anti-miotic agent, Clotrimazole into vagina, suppositories of semi-synthetic solid triglycerides were prepared having bio-adhesive polymers viz. polycarbophil, hydroxypropylmethylcellulose and hyaluronic sodium salt. The author reported that these polymer increased residence time of suppositories in vagina by modifying adhesion force, liquefaction time and permanence of drug at expected site without any adverse effect. The developed formulation showed controlled release profile.

Vaginal bio-adhesive gel

A marketed bio-adhesive gel, Replens R of polycarbophil; used to lubricate and to retain moisture of vagina. The formulation maintains healthy acidic pH in vagina and remains over there for 3–4 d (Lee et al., 1996; Hwang et al., 1977). Another bio-adhesive gel named Prochieve TM is used in hormonal replacement therapy. Later on, concept of hydrogel was introduced which provides excellent controlled release profile of most drugs. Hydrogels can be defined as a three-dimensional, cross-linked hydrophilic polymeric network which can absorb significant amount of water (Singh et al., 2010). These are insoluble in water because of cross-linked structure and can imbibe water up to 10–20 times of its molecular weight and become swollen (Kim et al., 1992; Peppas et al., 2000). Hydrogel swells under the influence of different stimuli-like temp, magnetic field, sound, electric field, etc. and then drug releases from swelled hydrogel in controlled manner (Garg et al., 2013). Different anti-fungal drugs formulated in form of bioadhesive gel are given in Table 10.

Table 10. Bio-adhesive gel loaded with different anti-fungal agents.

Active Drug	Delivery system	Bioadhesive Polymer	Comments	Reference
Metronidazole	Bio-adhesive gel	Combination of chitosan and xanthan gum	Controlled release profile Good in-vitro anti-fungal activity	(Yellanki et al., 2010)
	Bio-adhesive gel	Combination of Xanthan gum and HPMC-K4M	Sustained release for 6 h. Excellent in-vitro anti-fungal activity	(Ahmad et al., 2008)
Nonoxynol-9	Bio-adhesive gel	Carbopol934P	Initial high burst for 2min followed by sustained release for 7 h	(Lee et al., 1996)
Clotrimazole	Bio-adhesive gel	Combination of Xanthan gum and HPMC-K4M	Sustained release for 6 h. Good in-vitro anti-fungal activity	(Ahmad et al., 2008)
Itraconazole	Thermo-sensitive vaginal gel	Poloxamer 407- HPMC	Improved treatment of vaginal candidiasis	(Karavana et al., 2012)
Miconazole nitrate	Thermo-sensitive vaginal gel	Pluronic F127, carbopol 934 and polycarbophil	Sustained release for 12 h.	(Hani & Shivakumar)
Miconazole nitrate	Thermo-sensitive gel	PEG-4000	Serves as controlled release carrier	(Bhat & Shivakumar, 2010b)
Clotrimazole	Ion-sensitive gel	Carbopol 934, HPMC and sodium alginate	Excellent in-vivo anti-fungal activity in mice and zero order release for 8 h	(Dhanaraj)
Clotrimazole:β Cyclodextrin complex	Thermo-sensitive gel	Pluronic F127- HPMC	Sustained release for 92 h	(Bilensoy et al., 2006)

Vaginal micro particles (microspheres, microcapsules)

In the recent time, micro particles systems have also employed for designing vaginal delivery system. With addition of mucoadhesive polymers, these systems were made bio-adhesive so as to gain intimate prolonged contact with vaginal mucosa for controlled drug delivery (Garg et al., 2012). Ketoconazole was formulated as bio-adhesive microcapsules and incorporated in tablet for vaginal delivery. Dissolution studies of these microcapsules attest sustained release of the drug. Different anti-fungal drugs formulated in form of microparticles are given in Table 11.

Table 11. Different anti-fungal vaginal micro particle formulations.

Drug	Formulation type	Bio-adhesive polymer	Animal model	Comments	References
Clotrimazole	Microspheres based vaginal gel	Carbopol 934P.	In-vitro	Good control release pattern (99% in 12 h) Higher bio-adhesion and retention time in vagina Excellent in-vitro anti-fungal activity	(Hani et al.)
Metronidazole	Microencapsulated bio-adhesive vaginal gel	Carbopol 974	In-vivo New Zealand rabbits	Extended release of drug (100% release for 36 h) Formulation was non-irritant to vagina of New Zealand rabbits Significant vaginal bio-adhesion time	(Bhowmik et al., 2009)
Clotrimazole	Spray dried microspheres as bio-adhesive vaginal tablet	Combination of HPMC and Carbopol	In-vitro	Sufficient bio-adhesive strength with controlled release up to 24 h	(Gupta et al., 2013)

Cyclodextrin in vaginal therapy

Cyclodextrin complexation was primarily used to increase bioavailability of poorly soluble drugs by incorporating them in cyclodextrin complexes (Patel & Rajesh). However, several studies advocate the use of Cyclodextrin for improving vaginal drug delivery. Hydroxypropyl-β-cyclodextrin formulation having Itraconazole was found to have good drug solubility and excellent mucoadhesive property. Vaginal cream having Itraconazole complexed with β-cyclodextrin was well tolerated and remained in vagina for several days. Hydroxypropyl-β-cyclodextrin complex was reported to increase solubility of Amphotericin B; when both were formulated as thermo-sensitive, pH-sensitive gel, controlled release was successfully achieved. Cyclodextrin complexes were also successfully employed in anti-viral therapy to deliver anti-HIV agents (Yang et al., 2008). Chang Yun et al (2002) fabricated Clotrimazole-loaded cyclodextrin complex by using combination of poloxamers (P) 407, 188, and polycarbophil (PC). The results showed that controlled release of drug was achieved and exhibit excellent in-vivo anti-fungal activity in female rats (Yun Chang et al., 2002).

Other novel approaches against VC

With continuous use of anti-fungal agents for VC, subsequent failure of therapy was observed. This is due to development of resistant by Candida sp., so further prolongation of the therapy will be ineffective. In spite of that, a new concept of genetically engineered antibody was introduced, which successfully encounter this limitation (Garg et al., 2011). Different vaccines having modified antigens are introduced that produce C. albicans specific antibodies that either have fungicidal activity or inhibit adhesion of Candida to epithelial cells as described in Table 12. Different techniques employed for generating monoclonal antibodies are Hybridoma cell production, Recombinant antibody engineering technique, complementary-determining region (CDR) engraftment, Cambridge Antibody Technology (CAT) (De St Groth & Scheidegger, 1980), etc. Vaccine consists of diseases causing micro-organism either in dead or partly killed form which will stimulate immune system to recognize it as foreign microbe and act against it by producing antibodies. Antibodies are Y-shaped protein produced by plasma cells and utilized by immune system to recognize and neutralize foreign particles like bacteria or fungi. Monoclonal antibodies are the mono-specific antibodies produced from single parent immune cell by different techniques like hybridoma, recombinant technique, etc. Idiotypic is a shared characteristic of immunoglobulin or T cell receptors (TCR). Idiotypic describes distinctive sequence and region that makes any immunoglobing/TCR unique from others of the same type which is its variable region. Variable region has a specific amino-acid sequence that determines its antigen binding affinity and therefore the idiotope of the molecule. IgG or T cell receptor having shared idiotope is the same idiotype (Miller et al., 1982). The main component of these vaccines can be whole Candida cell or its cell extract, antibodies, idiotypes, glycoconjugate and its subunits, human peptide, antigen-pulsed cell, etc. Method for production these different antigens are discussed below

• Human Peptide Hepcidin 20: Human liver derived Hepticin-20 has a significant anti-fungal activity and is either purchased or extracted from human source. This peptide release β-glycosidase that cleaves β-Glycoside linking in fungal cell wall.

• Candida surface mannan complex: Candida surface mannan was obtained by extracting yeast cell with β-mercaptoethanol and then encapsulated in multilamillar liposomescomposed of phosphatidylcholine and cholesterol in ratio 3.2:1. After immunization antibodies specific to β 1, 2-mannotriose (cell wall component of Candida) is produced, and exerts their anti-fungal activity (Han et al., 1998). This mannan extract may complexed with Bovine serum albumin, and immunization leads to production of IgG and IgM antibodies that degrade Candida cell wall mannan (mannoprotein; Han et al., 1999).

• Dendritic cells pulsed with fungal RNA: Candida cells were ruptured through repeated thawing and freezing on liquid nitrogen. Hot extraction buffer (a 1:1 mixture of phenol and 0.1 M LiCl, 100 mM Tris-HCl (pH 8), 10 mM EDTA, and 1% SDS at 80 °C) and then a mixture ((24:1, v/v) of chloroform and isoamyl alcohol) was added to the cells. Followed by centrifugation at 10 000 rpm at 4 °C and water phase was mixed with equal volume of 4M lithium chloride. This mixture was again centrifuged at 10 000 at 4 °C and the RNA gets precipitated. Obtained RNA pellet was dissolved in water and precipitated using sodium acetate and ethanol at −20 °C. Dendritic cells (DCs) were either extracted from bone-marrow of spleen. Spleen cells were subjected to overnight plastic adherence to remove macrophages, then reacted with 100 µl of anti-mouse CD11c mAbs (against CD11c present on macrophage surface) conjugated with Micro Beads followed by magnetic separation. Bone marrow DCs, were obtained from femur of mice and seeded for 6 d in six-well plates in 3 ml IMDM (Iscove's Modified Dulbecco's Medium) with 10% FCS (fetal calf serum), 50 mM 2-ME(2-mercaptoethanol), 50 µg/ml gentamicin sulfate, 2000 U/ml GM-CSF (granulocyte-macrophage colony-stimulating factor), and 1 × 10³ U/ml IL-4. On day 3, non-adherent cells were replaced with mixture of GM-CSF and interleukin-4. On day 6, DCs were isolated from non-adherent cells and incubated at 37 °C for 3 h. RNA (25 µg in 250 µl Opti-MEM medium) and DOTAP (50 µg in 250 µl Opti-MEM medium (minimum essential media)) was mixed in 12 × 75-mm polystyrene tubes and then 2 ml of it was added to DC and incubated for 37 °C for 24 h. IL-4 Immunization leads to activation of T-helper 1 thus initiating immune response against Candida by releasing cytokines IL-4,IL-6,IL-10,IL-12 P70 (Bacci et al., 2002).

• Human domain antibodies: A complex procedure was adopted to yield Human domain antibodies against virulent traits of Candida (De Bernardis et al., 2007). In brief, the author uses genetically modified Antibody variable domains (domain antibodies [DAbs]) that have individual heavy-chain (VH) or k-chain (Vk) variable domains and lacks the Fc region. From Phage expression libraries, Human DAbs against 65-kDa mannoprotein (MP65) or the secretory aspartyl proteinase (SAP)–2 of C. albicans (mono-specific DAbs) or against both fungal antigens (heterodimeric, bispecific DAbs) were generated. A significant inhibition of fungal adherence (mediated by MP65 and SAP)–2) and complete clearance of vaginal infection of fungus was observed using both mono- and bi-specific DAbs in rat vagina.

• B-glucan-conjugate vaccine for VC: Donatella et al. formulated β-glucan-conjugate in human compatible MF59 adjuvant and anti-fungal activity was assessed in murine model (Pietrella et al., 2010). The infection was monitored using genetically engineered, luminescent C. albicans strain and then Cfu was measured. The mice were immunized with this conjugate and then a prominent fall in Cfu of C. albicans was observed. This anti-fungal activity was due to production of serum and vaginal anti-β-glucan IgG antibodies. This antibody recognizes octa-β- 1, 3-glucan epitope which is present in hyphal cell wall protein that mediates fungal adhesion and invasion (Torosantucci et al., 2009). Then in-vivo imaging techniques confirm excellent anti-fungal activity. Antonella et al. reported good protection against VC in mice by formulating β-glucan (preparation from the brown alga Laminaria digitata) conjugate with diphtheria toxoid CRM197 (carrier protein). This conjugate produces anti-β-glucan IgG antibodies which provide protection against Candida sp. (Torosantucci et al., 2005).

• Recombinant ALS vaccine for VC: Ibrahim et al. developed vaccine of recombinant N-terminus of Als 1p (rAls 1p) for protection of mice against disseminated and mucosal candidiasis. The vaccine enhances cell-mediated immunity rather than humoral and improves survival of mice during candidiasis (Ibrahim et al., 2005). Latter on they formulated another vaccine of recombinant N-teminus of Als 3p (rAls 3p) against disseminated and mucosal Candidiasis. The vaccine proved to as effective as rAls 1p in disseminated Candidiasis and more effective in Mucosal (vaginal) Candidiasis (Spellberg et al., 2006).

• Candida albicans mannan extract–protein conjugates: Vaccine having C. albicans Mannan (fungal cell wall constituent) extract- Bovine serum albumin conjugate was assessed for its anti-fungal activity in mice. The vaccine was administered intraperitoneal (i.p) followed by i.v. administration of viable Candida spp. Mice developed both IgG and IgM antibodies specific for the cell surface of Candida yeast cells and exerts its anti-fungal activity (Han et al., 1999). Table 12 represents the recently developed vaccines against VC.

Table 12. Recently developed vaccines against VC.

Category/source	Antigen	Animal model	Underlying immunity	Mechanism of action	References
Subunits and glyco conjugates	65-kDa mannoprotein (MP65)	Rat	Anti-MP65 antibodies	Inhibit fungal adhesion to epithelial cells mediated by MP65 and SAP 2	(Sandini et al., 2007)
	Secretory aspartyl proteinase (SAP)–2	Rat	Anti- SAP 2 antibodies		(De Bernardis et al., 2002, 2007, 2012)
	Recombinant N-teminus of Als 3p (rAls 3p)	Mice	Anti- rAls 3p antibodies	Inhibit fungal adhesion mediated by Als 3	(Spellberg et al., 2006)
	Candida surface mannan	Mice	MAb B6 and MAb B6.1	Degrade β-1, 2-mannotriose (cell wall component of fungus)	(Han et al., 1998)
	Octa-β- 1, 3-glucan epitope	Mice	Serum and vaginal anti-β-glucan IgG antibodies	Degrade β-1, 2-mannotriose (cell wall component of fungus)	(Torosantucci et al., 2005, 2009; Pietrella et al., 2010)
Peptide	Hepcidin 20 (Hep-20)	In-vitro	Antimicrobial activity	Release β-glactosidase that cleave β-glycoside linking in fungal cell wall	(Del Gaudio et al., 2013)
Idiotypes	Killer-toxin neutralizing m Ab KT4	Rat	Fungicidal antibodies	Unknown	(Polonelli et al., 1997)
Antibodies	Mycograb (anti-Hsp-90 antibodies)	Human	Fungicidal	Degrade Hsp-90 (cell wall component of fungus)	(Pachl et al., 2006)
Antigen-pulsed cells	Dendritic cell pulsed with Candida yeasts or yeast RNA	Mice	Activation of T-helper 1	Immunity provided by IL-4,IL-6,IL-10, IL-12 P70	(Bacci et al., 2002)
Whole cell or cell extract	Heat killed Candida cells with novel mucosal adjuvant LT(R192G)	Mice	Delayed hypersensitivity responses and Increased levels of Immunoglobulin G (IgG)	Release of cytokines that degrade fungal cells	(Cárdenas-Freytag et al., 1999)
	C. albicans Mannan extract- Bovine serum albumin conjugate	Mice	IgG and IgM antibodies	Degrade fungal cell wall mannan (mannoprotein)	(Han et al., 1999)

Conclusion

Although much of research work has been done to deliver anti-fungal drugs safely and effectively for VC, various conventional dosage forms are available like creams, gel, suppositories, etc. But have numerous limitations like systemic side-effects, lesser residence time, etc. To overcome these limitations, a novel concept of bio-adhesive formulations was introduced. While this delivery system successfully encountered most of the disadvantages of conventional dosage forms, but there continuous use has led to significant resistance in Candida against azole agents. However now a day, vaccines are employed for anti-fungal therapy and they have been proved to be a good and potential alternative for VC. But this delivery system still needs to be exploited, in order to develop a novel, ideal, effective delivery system against all Candida spp. and to protect and maintain integrity of epithelial cells.

Declaration of interest

The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.

Acknowledgement

Authors Amit K Goyal (under IYBA scheme; BT/01/IYBA/2009 dated 24/05/2010) thankful to Department of Biotechnology (DBT), New Delhi, India.