Category: Formulation and Quality
Purpose: Various epidemiological reports discuss about risk factors associated with HIV infection. Recurrent vulvovaginal candidiasis is claimed to manifest HIV transmission in women. Reports prove that 38 percent of HIV infected women had recurrent and persistent vaginal candidiasis as their first symptom. Conversely, women with vulvovaginal candidiasis have a high risk of getting HIV transmitted during intercourse. Common treatments for such conditions include combined antiretroviral and antifungal therapy. Drug-Drug interaction is a major problem due to azole and antiretroviral common CYP450 pathway. Ebselen (EB), novel class of selenium compound has demonstrated promising anti-HIV and antifungal activity in the past. This dual acting nature would serve as an effective treatment against vulvovaginal candidiasis and offer pre-prophylactic (PrEP) treatment to HIV. Scientific and medical evidence generated in recent years supports that topical pre-exposure prophylaxis (PrEP) treatment is a promising approach that can reduce sexual transmission of the virus. But, EB being lipophilic, has demonstrated poor kinetic solubility and stability in water. Therefore, the main objectives of this study are; 1) To develop and characterize highly soluble inclusion complex of EB and incorporate it into polymeric film 2) Perform in-vitro antifungal and anti-HIV activity.
Methods: Preliminary screening of various cyclodextrins were performed. Phase-solubility techniques were used to assess optimum concentration for formation of inclusion complex of EB and β-cyclodextrin polymer. Binary complex of EB: β-CD polymer was prepared by co-precipitation method. Final batch with Soluplus® as ternary compound was dispersed in 20% w/v of PVA solution and film was casted using elcometer® 4350. Particle size and polydispersity index were measured using dynamic light scattering method. Film evaluations were performed using texture analyzer and microstructural characterization. The formulated EB film was compared to marketed VCF® contraceptive film. Cytotoxicity of film and excipients on HeLa cell line were done using MTT and LDH assay. Transepithelial electrical resistance (TEER) values of VCF and EB film were compared on Hec1A cell line. Drug release was carried out in simulated vaginal fluid (SVF) and in-vitro antifungal assay was performed using resazurin assay. Anti-HIV activity of optimized formulation was performed on HIV transfected HeLa (cancerous cervical) cell line. Compatibility of EB film with E-coli bacteria was also assessed.
Results: The optimum ternary complex batch was prepared in 1:10:10 ratio of EB/ β-CD polymer/Soluplus®. Ternary and binary complex enhanced solubility of EB in water to 100- and 25- folds respectively. Ternary complex showed a particle size of 87.98 nm with polydispersity index of 0.174. Significance of the ternary complex was clearly distinguished using microstructural characterization where Soluplus® and cyclodextrin free films showed distinct precipitation. EB film disintegrated within 2mins and released 90% of the drug within 15mins in SVF media. Tensile strength of film was found to 31.96 Nm-2. Cytotoxicity assays in HeLa confirmed > 85% cell viability when treated with EB film and excipients used. VCF® film showed > 80% LDH release while EB film gave similar release of LDH to the control proving VCF® to be highly toxic. TEER value for EB film and EB in DMSO remained constant even after 3hrs. VCF® drastically reduced the TEER to 50% within 15mins. In-vitro antifungal activity of EB gave an MIC of 20µM in Candida species. Anti-HIV activity was found to be 0.44µM for EB film. EB film didn’t show any toxicity towards Ecoli even at 400 µM indicating compatibility of formulation to vaginal microflora.
Conclusion: Highly soluble, vaginally biocompatible and rapidly dissolving transparent microbicidal film of EB was formulated using ternary inclusion complex. The film was found to be safe to vaginal mucosa and bacterial microflora. A potent antifungal activity in micromolar and anti-HIV activity in nanomolar concentration of EB film proved it to be an effective pre-exposure prophylactic treatment to HIV infection which can also inhibit vulvovaginal candidiasis associated with it.
Suvidha Menon– St.John's university, Queens, New York
Manali Patki– student, St.John's university, Queens, New York
Susana Valente– Jupiter, Florida
Joseph Jablonski– Jupiter, Florida
Sonia Mediouni– Jupiter, Florida
Blase Billack– St.John's university, Queens, New York
Ketan Patel– Jamaica, New York