NOVEL IN VITRO AND IN VIVO MODEL SYSTEMS TO STUDY VAGINAL MICROBICIDE UPTAKE

Novel in vitro and in vivo model systems to study intravaginal microbicide uptakeAnti-HIV microbicides are compounds that are vaginally or rectally administered prior to intercourse to prevent viral transmission. In the search for an effective product, insufficient drug permeation across the vaginal mucosa may cause failed protection in case of microbicides that are active at the level of the HIV host cells, which are mainly localized in the lamina propria. To evaluate the tissue penetration ability of vaginally applied drugs, we established a simple in vitro set up, consisting of an apical and basal chamber that are separated by a layer of HEC-1A cells. This procedure was validated by means of the model microbicides tenofovir, darunavir, saquinavir and dapivirine. The compound s solubility and tissue permeability were identified as key determinants for vaginal uptake. Saquinavir and dapivirine permeation across HEC-1A was low because of poor permeability and solubility, respectively. In contrast, tenofovir showed the highest transport attributable to its good aqueous solubility. In addition, the impact of formulation factors on the permeation potential was investigated. The inclusion of solubilizing cyclodextrins or polyethylene glycol resulted in an enhanced permeation of the hydrophobic compound dapivirine. An excipient-induced reduction in the permeability may however counteract the solubilizing effect, as demonstrated for the emulsion formulation of darunavir. The optimization of drug uptake by inclusion of formulation excipients must take into account this delicate balance. In a next step, the described in vitro approach was applied for the biopharmaceutical assessment of the diaryltriazine compound series which resulted in the selection of UAMC01398 as the lead microbicide candidate, owing to its relatively high aqueous solubility in addition to its beneficial safety and activity profile. Two stable and safe aqueous-based gel formulations were identified for UAMC01398 including a non-solubilizing gel and a gel containing the solubilizer sulfobutyl ether-ß-cyclodextrin (SBE-ßCD, 5%); in addition, a film delivery system, consisting of the excipients hydroxypropylmethylcellulose and polyethylene glycol 400 with UAMC01398 in the amorphous state, was developed. Compared to the non-solubilizing gel, the SBE-ßCD gel and the film formulation increased the UAMC01398 uptake both in vitro across HEC-1A cell layers and in vivo in rabbits. The developed formulations are suitable for the vaginal delivery of UAMC01398 and allow further in vivo evaluation of the microbicide potential of this compound.The possible use of a supersaturation strategy to overcome solubility issues of hydrophobic microbicides was demonstrated for dapivirine. Despite being in a thermodynamically unstable state, supersaturated dapivirine showed not to precipitate in the formulation vehicle as such and in biorelevant fluids in the presence of several excipients including hydroxypropylmethylcellulose, polyethylene glycol 1000 and cyclodextrins. Dapivirine transport across HEC-1A cell layers was higher for supersaturated gels compared to suspension gels. The supersaturated dapivirine (500 µM) gel containing 2.5% of SBE-ßCD significantly increased vaginal drug uptake in rabbits compared to a non-solubilizing suspension gel and an SBE-ßCD suspension gel. The supersaturation approach thus allows the formulation of hydrophobic microbicides at concentrations above their solubility, thereby enhancing their vaginal permeation.Vaginal uptake may also be influenced by drug transporters expressed in the vaginal epithelium. We confirmed the protein expression of the efflux transporters Pgp, BCRP and MRP-2 in endocervical and vaginal tissue of premenopausal women. Several microbicide candidates including darunavir, saquinavir and maraviroc could be categorized as Pgp and MRP-2 substrates and their disposition may thus be affected by these transporters. The Pgp transporter was observed to significantly reduce the vaginal uptake of the model Pgp substrate talinolol in vivo in rabbits when formulated in a neutral, but not in an acidic gel. Consequently, the expression of efflux transporters may limit the vaginal permeation of their substrates, including certain microbicides. In conclusion, we propose the implementation of in vitro solubility and permeability evaluation, including the assessment of transporter interactions, to estimate the vaginal tissue permeation potential of microbicides prior to progressing into animal or clinical studies. This procedure may contribute to the selection of promising microbicide candidates, as illustrated for the diaryltriazines, and to successful formulation development, as shown for UAMC01398 and dapivirine. The in vitro drug transport across HEC-1A cell layers correlated well with the in vivo vaginal permeation as determined in rabbits. Formulation approaches that install solubilization or supersaturation are indicated to enhance vaginal drug uptake of poorly soluble microbicides.status: publishe