Advanced topical drug delivery system for the management of vaginal candidiasis

Figures & data

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.

Figure 1. Pathophysiology of vaginal candidiasis.

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,

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)

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).

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

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

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)

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)

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)

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)

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)

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)

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