Investigating the Contribution of Drug-Metabolizing Enzymes in Drug-Drug Interactions of Dapivirine and Miconazole.

Dapivirine (DPV) is a potent NNRTI used to prevent the sexual transmission of HIV. In a phase 1 trial (IPM 028), the concomitant use of a DPV vaginal ring and an antifungal miconazole (MIC) vaginal capsule was found to increase the systemic exposure to DPV in women, suggesting a potential for drug-drug interactions. This study's objective was to investigate the mechanism of DPV-MIC interactions using drug-metabolizing enzymes (DMEs; CYPs and UGTs) that are locally expressed in the female reproductive tract (FRT). In vitro studies were performed to evaluate the metabolism of DPV and its inhibition and induction potential with DMEs. In addition, the impact of MIC on DPV metabolism and the inhibitory potential of DPV with DMEs were studied. Our findings suggest that DPV is a substrate of CYP1A1 and CYP3A4 enzymes and that MIC significantly decreased the DPV metabolism by inhibiting these two enzymes. DPV demonstrated potent inhibition of CYP1A1 and moderate/weak inhibition of the six CYP and eight UGT enzymes evaluated. MIC showed potent/moderate inhibition of seven CYP enzymes and weak/no inhibition of eight UGT enzymes. The combination of DPV and MIC showed potent inhibition of seven CYP enzymes (1A1, 1A2, 1B1, 2B6, 2C8, 2C19, and 3A4) and four UGT enzymes (1A3, 1A6, 1A9, and 2B7). DPV was not an inducer of CYP1A2, CYP2B6, and CYP3A4 enzymes in primary human hepatocytes. Therefore, the increased systemic concentrations of DPV observed in IPM 028 were likely due to the reduced metabolism of DPV because of CYP1A1 and CYP3A4 enzymes inhibition by MIC in the FRT.

Drug delivery strategies for management of women's health issues in the upper genital tract.

The female upper genital tract (UGT) hosts important reproductive organs including the cervix, uterus, fallopian tubes, and ovaries. Several pathologies affect these organ systems such as infections, reproductive issues, structural abnormalities, cancer, and inflammatory diseases that could have significant impact on women's overall health. Effective disease management is constrained by the multifaceted nature of the UGT, complex anatomy and a dynamic physiological environment. Development of drug delivery strategies that can overcome mucosal and safety barriers are needed for effective disease management. This review introduces the anatomy, physiology, and mucosal properties of the UGT and describes drug delivery barriers, advances in drug delivery technologies, and opportunities available for new technologies that target the UGT.

On-demand microbicide products: design matters.

Sexual intercourse (vaginal and anal) is the predominant mode of human immunodeficiency virus (HIV) transmission. Topical microbicides used in an on-demand format (i.e., immediately before or after sex) can be part of an effective tool kit utilized to prevent sexual transmission of HIV. The effectiveness of prevention products is positively correlated with adherence, which is likely to depend on user acceptability of the product. The development of an efficacious and acceptable product is therefore paramount for the success of an on-demand product. Acceptability of on-demand products (e.g., gels, films, and tablets) and their attributes is influenced by a multitude of user-specific factors that span behavioral, lifestyle, socio-economic, and cultural aspects. In addition, physicochemical properties of the drug, anatomical and physiological aspects of anorectal and vaginal compartments, issues relating to large-scale production, and cost can impact product development. These factors together with user preferences determine the design space of an effective, acceptable, and feasible on-demand product. In this review, we summarize the interacting factors that together determine product choice and its target product profile.

Increased cerebrospinal fluid levels of GABA, testosterone and estradiol in women with polycystic ovary syndrome.

STUDY QUESTION: Do cerebrospinal fluid (CSF) concentrations of gamma-aminobutyric acid (GABA), testosterone (T) and estradiol (E2) differ in women with polycystic ovary syndrome (PCOS) as compared to eumenorrheic, ovulatory women (EW)? SUMMARY ANSWER: Women with PCOS displayed higher CSF levels of GABA and E2, and possibly T, than EW. WHAT IS KNOWN ALREADY: The chronic anovulation characteristic of PCOS has been attributed to increased central GnRH drive and resulting gonadotropin aberrations. Androgens are thought to regulate GABA, which in turn regulates the neural cascade that modulates GnRH drive. STUDY DESIGN, SIZE, DURATION: This cross-sectional observational study included 15 EW and 12 non-obese women with PCOS who consented to a lumbar puncture in addition to 24 h of serum blood collection at 15-min intervals. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 27 women were studied at a the General Clinical Research Center (GCRC) at the University of Pittsburgh. Serum analytes included T, E2 and androstenedione. CSF analytes included GABA, glutamate, glucose, T and E2. MAIN RESULTS AND THE ROLE OF CHANCE: Women with PCOS had higher CSF GABA as compared to EW (9.04 versus 7.04 µmol/L, P < 0.05). CSF glucose and glutamate concentrations were similar between the two groups. CSF T was 52% higher (P = 0.1) and CSF E2 was 30% higher (P < 0.01) in women with PCOS compared to EW. Circulating T was 122% higher (P < 0.01) and circulating E2 was 75% higher (P < 0.01) in women with PCOS than in EW. LIMITATIONS REASONS FOR CAUTION: The study is limited by its small sample size and the technical limitations of measuring CSF analytes that are pulsatile and have short half-lives. WIDER IMPLICATIONS OF THE FINDINGS: Women with PCOS displayed significantly higher circulating levels of T and E2, significantly higher CSF levels of E2, and higher levels of CSF testosterone, although the latter was not statistically significant. A better understanding of the central milieu informs our understanding of the mechanisms mediating increased the GnRH drive in PCOS and lends a new perspective for understanding the presentation, pathogenesis and potential health consequences of PCOS, including gender identity issues. STUDY FUNDING/COMPETING INTEREST(S): No conflicts of interest. The study was funded by NIH grants to SLB (RO1-MH50748, U54-HD08610) and NIH RR-00056 to the General Clinical Research Center of the University of Pittsburgh. TRIAL REGISTRATION NUMBER: NCT01674426.

Formulation and characterization of polymeric films containing combinations of antiretrovirals (ARVs) for HIV prevention.

PURPOSE: To develop polymeric films containing dual combinations of anti-HIV drug candidate tenofovir, maraviroc and dapivirine for vaginal application as topical microbicides. METHODS: A solvent casting method was used to manufacture the films. Solid phase solubility was used to identify potential polymers for use in the film formulation. Physical and chemical properties (such as water content, puncture strength and in vitro release) and product stability were determined. The bioactivity of the film products against HIV was assessed using the TZM-bl assay and a cervical explant model. RESULTS: Polymers identified from the solid phase solubility study maintained tenofovir and maraviroc in an amorphous state and prevented drug crystallization. Three combination film products were developed using cellulose polymers and polyvinyl alcohol. The residual water content in all films was <10% (w/w). All films delivered the active agents with release of >50% of film drug content within 30 min. Stability testing confirmed that the combination film products were stable for 12 months at ambient temperature and 6 months under stressed conditions. Antiviral activity was confirmed in TZM-bl and cervical explant models. CONCLUSIONS: Polymeric films can be used as a stable dosage form for the delivery of antiretroviral combinations as microbicides.

Increased Dapivirine tissue accumulation through vaginal film codelivery of dapivirine and Tenofovir.

The HIV-1 replication inhibitor dapivirine (DPV) is one of the most promising drug candidates being used in topical microbicide products for prevention of HIV-1 sexual transmission. To be able to block HIV-1 replication, DPV must have access to the viral reverse transcriptase enzyme. The window for DPV to access the enzyme happens during the HIV-1 cellular infection cycle. Thus, in order for DPV to exert its anti-HIV activity, it must be present in the mucosal tissue or cells where HIV-1 infection occurs. A dosage form containing DPV must be able to deliver the drug to the tissue site of action. Polymeric films are solid dosage forms that dissolve and release their payload upon contact with fluids. Films have been used as vaginal delivery systems of topical microbicide drug candidates including DPV. For use in topical microbicide products containing DPV, polymeric films must prove their ability to deliver DPV to the target tissue site of action. Ex vivo exposure studies of human ectocervical tissue to DPV film revealed that DPV was released from the film and did diffuse into the tissue in a concentration dependent manner indicating a process of passive diffusion. Analysis of drug distribution in the tissue revealed that DPV accumulated mostly at the basal layer of the epithelium infiltrating the upper part of the stroma. Furthermore, as a combination microbicide product, codelivery of DPV and TFV from a polymeric film resulted in a significant increase in DPV tissue concentration [14.21 (single entity film) and 31.03 µg/g (combination film)], whereas no impact on TFV tissue concentration was found. In vitro release experiments showed that this observation was due to a more rapid DPV release from the combination film as compared to the single entity film. In conclusion, the findings of this study confirm the ability of polymeric films to deliver DPV and TFV to human ectocervical tissue and show that codelivery of the two agents has a significant impact on DPV tissue accumulation. These findings support the use of polymeric films for topical microbicide products containing DPV and/or TFV.

PEG-derivatized embelin as a dual functional carrier for the delivery of paclitaxel.

Embelin, identified primarily from the Embelia ribes plant, has been shown to be a natural small molecule inhibitor of X-linked inhibitor of apoptosis protein (XIAP). It is also a potent inhibitor of NF-κB activation, which makes it a potentially effective suppressor of tumor cell survival, proliferation, invasion, angiogenesis, and inflammation. However, embelin itself is insoluble in water, which makes it unsuitable for in vivo applications. In this work, we developed a novel micelle system through conjugating embelin to a hydrophilic polymer, poly(ethylene glycol) 3500 (PEG(3.5K)) through an aspartic acid bridge. The PEG(3.5k)-embelin(2) (PEG(3.5k)-EB(2)) conjugate readily forms micelles in aqueous solutions with a CMC of 0.0205 mg/mL. Furthermore, PEG(3.5k)-EB(2) micelles effectively solubilize paclitaxel (PTX), a model hydrophobic drug used in this study. Both drug-free and drug-loaded micelles were small in size (20-30 nm) with low polydispersity indexes. In vitro cytotoxicity studies with several tumor cell lines showed that PEG(3.5k)-EB(2) is comparable to embelin in antitumor activity and synergizes with PTX at much lower doses. Our results suggest that PEG-derivatized embelin may represent a novel and dual-functional carrier to facilitate the in vivo applications of poorly water-soluble anticancer drugs such as PTX.

Characterization of UC781-tenofovir combination gel products for HIV-1 infection prevention in an ex vivo ectocervical model.

HIV continues to be a problem worldwide. Topical vaginal microbicides represent one option being evaluated to stop the spread of HIV. With drug candidates that have a specific action against HIV now being studied, it is important that, when appropriate and based on the mechanism of action, the drug permeates the tissue so that it can be delivered to specific targets which reside there. Novel formulations of the nucleotide reverse transcriptase inhibitor tenofovir (TFV) and the nonnucleoside reverse transcriptase inhibitor UC781 have been developed and evaluated here. Gels with three distinct rheological properties were prepared. The three gels released both UC781 and TFV under in vitro conditions at concentrations equal to or above the reported 50% effective concentrations (EC(50)s). The drug concentrations in ectocervical tissues were well in excess of the reported EC(50)s. The gels maintain ectocervical viability and prevent infection of ectocervical explants after a HIV-1 challenge. This study successfully demonstrates the feasibility of using this novel combination of antiretroviral agents in an aqueous gel as an HIV infection preventative.

Vaginal film drug delivery of the pyrimidinedione IQP-0528 for the prevention of HIV infection.

PURPOSE: Polymeric quick-dissolving films were developed as a solid dosage topical microbicide formulation for the vaginal delivery of the highly potent and non-toxic, dual-acting HIV nonnucleoside reverse transcriptase inhibitor (NNRTI) pyrimidinedione, IQP-0528. METHODS: Formulated from approved excipients, a polyvinyl alcohol (PVA) based film was manufactured via solvent casting methods. The film formulations were evaluated based upon quantitative physicochemical evaluations defined by a Target Product Profile (TPP) RESULTS: Films dosed with 0.1% (w/w) of IQP-0528 disintegrated within 10 min with over 50% of drug released and near 100% total drug released after 30 min. The IQP-0528 films were found to be non-toxic in in vitro CEM-SS and PBMC cell-based assays and biologically active with sub-nanomolar efficacy against HIV-1 infection. In a 12 month stability protocol, the IQP-0528 films demonstrated no significant degradation at International Conference on Harmonization (ICH) recommended standard (25°C/65% relative humidity (R.H.)) and accelerated (40°C/75% R.H.) environmental conditions. CONCLUSIONS: Based on the above evaluations, a vaginal film formulation has been identified as a potential solid dosage form for the vaginal delivery of the topical microbicide candidate IQP-0528.

Development of a liposome microbicide formulation for vaginal delivery of octylglycerol for HIV prevention.

The feasibility of using a liposome drug delivery system to formulate octylglycerol (OG) as a vaginal microbicide product was explored. A liposome formulation was developed containing 1% OG and phosphatidyl choline in a ratio that demonstrated in vitro activity against Neisseria gonorrhoeae, HSV-1, HSV-2 and HIV-1 while sparing the innate vaginal flora, Lactobacillus. Two conventional gel formulations were prepared for comparison. The OG liposome formulation with the appropriate OG/lipid ratio and dosing level had greater efficacy than either conventional gel formulation and maintained this efficacy for at least 2 months. No toxicity was observed for the liposome formulation in ex vivo testing in a human ectocervical tissue model or in vivo testing in the macaque safety model. Furthermore, minimal toxicity was observed to lactobacilli in vitro or in vivo safety testing. The OG liposome formulation offers a promising microbicide product with efficacy against HSV, HIV and N. gonorrhoeae.

Degradation of naturally occurring and engineered antimicrobial peptides by proteases.

We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.

The importance of the vaginal delivery route for antiretrovirals in HIV prevention.

The HIV/AIDS pandemic continues to be a global health priority, with high rates of new HIV-I infections persisting in young women. One HIV prevention strategy is topical pre-exposure prophylactics or microbicides, which are applied vaginally or rectally to protect the user from HIV and possibly other sexually transmitted infections. Vaginal microbicide delivery will be the focus of this review. Multiple nonspecific and specific antiretroviral microbicide products have been clinically evaluated, and many are in preclinical development, The events of HIV mucosal transmission and dynamics of the cervicovaginal environment should be considered for successful vaginal microbicide delivery. Beyond conventional vaginal formulations, intravaginal rings, tablets and films are employed as platforms in the hope to increase the likelihood of microbicide use. Furthermore, combining multiple antiretrovirals within a given formulation, combining a microbicide product with a vaginal device and integrating novel drug-delivery strategies within a microbicide product are approaches to successful vaginal-microbicide delivery.

Development and Characterization of a Vaginal Film Containing Dapivirine, a Non- nucleoside Reverse Transcriptase Inhibitor (NNRTI), for prevention of HIV-1 sexual transmission.

Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is a potent and promising anti-HIV molecule. It is currently being investigated for use as a vaginal microbicide in two dosage forms, a semi-solid gel and a silicone elastomer ring. Quick-dissolving films are promising and attractive dosage forms that may provide an alternative platform for the vaginal delivery of microbicide drug candidates. Vaginal films may provide advantages such as discreet use, no product leakage during use, lack of requirement for an applicator for insertion, rapid drug release and minimal packaging and reduced wastage. Within this study the in vitro bioactivity of dapivirine as compared to the NNRTI UC781 was further established and a quick dissolve film was developed for vaginal application of dapivirine for prevention of HIV infection. The developed film was characterized with respect to its physical and chemical attributes including water content, mechanical strength, drug release profile, permeability, compatibility with lactobacilli and bioactivity. The anti-HIV activity of the formulated dapivirine film was confirmed in in vitro and ex vivo models. Importantly the physical and chemical properties of the film as well as its bioactivity were maintained for a period of 18 months. In conclusion, a vaginal film containing dapivirine was developed and characterized. The film was shown to prevent HIV-1 infection in vitro and ex vivo and have acceptable characteristics which make this film a promising candidate for testing as vaginal microbicide.

Progress in antiretroviral drug delivery using nanotechnology.

There are currently a number of antiretroviral drugs that have been approved by the Food and Drug Administration for use in the treatment of human immunodeficiency virus (HIV). More recently, antiretrovirals are being evaluated in the clinic for prevention of HIV infection. Due to the challenging nature of treatment and prevention of this disease, the use of nanocarriers to achieve more efficient delivery of antiretroviral drugs has been studied. Various forms of nanocarriers, such as nanoparticles (polymeric, inorganic, and solid lipid), liposomes, polymeric micelles, dendrimers, cyclodextrins, and cell-based nanoformulations have been studied for delivery of drugs intended for HIV prevention or therapy. The aim of this review is to provide a summary of the application of nanocarrier systems to the delivery of anti-HIV drugs, specifically antiretrovirals. For anti-HIV drugs to be effective, adequate distribution to specific sites in the body must be achieved, and effective drug concentrations must be maintained at those sites for the required period of time. Nanocarriers provide a means to overcome cellular and anatomical barriers to drug delivery. Their application in the area of HIV prevention and therapy may lead to the development of more effective drug products for combating this pandemic disease.

Nanoparticle-based vaginal drug delivery systems for HIV prevention.

IMPORTANCE OF THE FIELD: Several strategies are being investigated for the prevention of heterosexual transmission of HIV. Of these, topical vaginal drug delivery systems, microbicides, are being actively pursued. HIV prevention by means of a topical microbicide has several drug delivery challenges. These challenges include the vaginal mucosal barriers and potential degradation of the drugs in the vaginal lumen due to pH and enzymes present. Also, new drugs being evaluated as microbicides have specific mechanisms of action, which in some cases require drug targeting to a specific site of action. Nanoparticles provide a delivery strategy for targeted or controlled delivery to the vagina which can be applied in the field of HIV prevention. AREAS COVERED IN THE REVIEW: This review summarizes nanoparticulate systems and their use in mucosal delivery to date. The sexual transmission of HIV along with the various targets to prevent transmission are discussed as well as the potential opportunities, challenges and advantages in using a nanoparticle-based approach for microbicidal drug delivery. WHAT THE READER WILL GAIN: This review provides a general understanding of vaginal drug delivery, its challenges, and nanoparticulate delivery systems. Additionally, insight will be gained as to the limited existing application of this technology to the field of HIV prevention. TAKE HOME MESSAGE: To date, few studies have been published that exploit nanoparticle-based microbicidal delivery to the vagina. The use of nanoparticles for vaginal drug delivery provides an approach to overcome the existing barriers to success.

Vaginal drug delivery systems for HIV prevention.

Microbicides have become a principal focus for HIV prevention strategies. The successful design of drug delivery systems for vaginal microbicide drug candidates brings with it a multitude of challenges. It is imperative that the chemical and physical characteristics of the drug candidate and its mechanism of action be clearly understood and considered to successfully deliver and target drug candidates efficiently. In addition, an understanding of the dynamic nature of the vaginal environment, the tissue and innate barriers present, as well as patient preferences are critical considerations in the design of effective microbicide products. Although the majority of drug candidates clinically evaluated to date have been delivered using conventional semisolid aqueous-based gel dosage forms, drug delivery system design has recently been extended to include advanced delivery systems such as vaginal rings, quick-dissolve films, and tablets. Ultimately, it may be necessary to develop multiple dosage platforms for a single active agent to provide users with options that can be used within the constraints of their social environment, personal choice, and environmental conditions.

Targeted delivery of PSC-RANTES for HIV-1 prevention using biodegradable nanoparticles.

PURPOSE: Nanoparticles formulated from the biodegradable co-polymer poly(lactic-co-glycolic acid) (PLGA), were investigated as a drug delivery system to enhance tissue uptake, permeation, and targeting for PSC-RANTES anti-HIV-1 activity. MATERIALS AND METHODS: PSC-RANTES nanoparticles formulated via a double emulsion process and characterized in both in vitro and ex vivo systems to determine PSC-RANTES release rate, nanoparticle tissue permeation, and anti-HIV bioactivity. RESULTS: Spherical, monodisperse (PDI = 0.098 +/- 0.054) PSC-RANTES nanoparticles (d = 256.58 +/- 19.57 nm) with an encapsulation efficiency of 82.23 +/- 8.35% were manufactured. In vitro release studies demonstrated a controlled release profile of PSC-RANTES (71.48 +/- 5.25% release). PSC-RANTES nanoparticle maintained comparable anti-HIV activity with unformulated PSC-RANTES in a HeLa cell-based system with an IC(50) of approximately 1pM. In an ex vivo cervical tissue model, PSC-RANTES nanoparticles displayed a fivefold increase in tissue uptake, enhanced tissue permeation, and significant localization at the basal layers of the epithelium over unformulated PSC-RANTES. CONCLUSIONS: These results indicate that PSC-RANTES can readily be encapsulated into a PLGA nanoparticle drug delivery system, retain its anti-HIV-1 activity, and deliver PSC-RANTES to the target tissue. This is crucial for the success of this drug candidate as a topical microbicide product.

Permeability of tritiated water through human cervical and vaginal tissue.

The increased incidence of human immunodeficiency virus infection in women has identified an urgent need to develop a female-controlled method to prevent acquisition of human immunodeficiency virus and other sexually transmitted diseases. Women would apply the product intravaginally before intercourse. Development of such a product requires a better understanding of the permeability characteristics of the tissues with which such products would come into contact. However, limited studies have been performed in this area. In the present study, water permeability of fresh human cervical and vaginal tissue was evaluated. The average apparent permeability coefficient was found to be 8 x 10(-5) cm/s for fresh human cervical tissue and 7 x 10(-5) cm/s for fresh human vaginal tissue. Considering the lack of regularity in obtaining cervical and vaginal tissue from surgical specimens, additional tests were performed to evaluate the effect of freezing on tritiated water permeability. No statistically significant differences were observed in the permeability values obtained when comparing fresh versus frozen tissues.

Measurement of anti-HIV activity of marketed vaginal products and excipients using a PBMC-based in vitro assay.

BACKGROUND AND OBJECTIVE: Because microbicides will be applied topically in the vagina, the active agent must be formulated as a gel or cream by combining the active agent with suitable excipients. Although a number of in vitro methods have been developed for testing anti-HIV activity of microbicides, most of these methods have not been used for testing microbicides as a gel or cream. GOAL: The goal of this study was to measure anti-HIV activity of marketed vaginal gel or cream products and excipients. STUDY DESIGN: A PBMC-based in vitro method has been developed for the evaluation of anti-HIV activity of gel or cream marketed products and formulated drug delivery systems. This method includes viral exposure to test compounds followed by differential centrifugation and filtration. RESULTS: Using this methodology, a number of marketed vaginal products showed 83% to 100% inactivation of a variety of X4 and R5 HIV of different clades. Cell viability as determined by the MTT assay for all marketed products was greater than 90%. Some of the excipients also showed anti-HIV activity (20-90%) of their own. CONCLUSION: This knowledge of baseline anti-HIV activity of vaginal products (cream/gel) and excipients is useful for the final formulation and development of anti-HIV microbicides.