Antiretroviral drug absorption and disposition in cervicovaginal tissue is important for the effectiveness of vaginally or orally administered drug products in preexposure prophylaxis (PrEP) of HIV-1 sexual transmission to women. Therefore, it is imperative to understand critical determinants of cervicovaginal tissue pharmacokinetics. This study aimed to examine the mRNA expression and protein localization of three efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated protein 4 (MRP4), and breast cancer resistance protein (BCRP), in the lower genital tract of premenopausal women and pigtailed macaques. Along the human lower genital tract, the three transporters were moderately to highly expressed compared to colorectal tissue and liver, as revealed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). In a given genital tract segment, the transporter with the highest expression level was either BCRP or P-gp, while MRP4 was always expressed at the lowest level among the three transporters tested. The immunohistochemical staining showed that P-gp and MRP4 were localized in multiple cell types including epithelial cells and vascular endothelial cells. BCRP was predominantly localized in the vascular endothelial cells. Differences in transporter mRNA level and localization were observed among endocervix, ectocervix, and vagina. Compared to human tissues, the macaque cervicovaginal tissues displayed comparable expression and localization patterns of the three transporters, although subtle differences were observed between the two species. The role of these cervicovaginal transporters in drug absorption and disposition warrants further studies. The resemblance between human and pigtailed macaque in transporter expression and localization suggests the utility of the macaque model in the studies of human cervicovaginal transporters.

Systemic delivery of therapeutic proteins through gene transfer approaches has been carried out mostly by ex vivo transduction of single cells or by direct in vivo injection of an expression vector. In this work an intact miniature biopsy of human dermis (microdermis) is harvested and transduced ex vivo by a viral vector encoding a gene for the therapeutic protein. The microdermis preserves its three-dimensional structure and viability during the ex vivo manipulations. Furthermore, upon transduction with adenoviral and adeno-associated viral vectors the microdermis secretes recombinant human erythropoietin (hEPO). Biochemical analysis of the secreted hEPO showed similarity to the clinically approved recombinant hEPO. Subcutaneous implantation of microdermal hEPO into SCID mice exhibited hEPO secretion in the blood circulation and preserved elevated hematocrit for several months, demonstrating the technology's potential for sustained delivery of protein therapeutics.

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4) are three efflux transporters that play key roles in the pharmacokinetics of antiretroviral drugs used in the pre-exposure prophylaxis of HIV sexual transmission. In this study, we investigated the expression, regulation, and function of these transporters in cervicovaginal tissues of a mouse model. Expression and regulation were examined using real-time RT-PCR and immunohistochemical staining, in the mouse tissues harvested at estrus and diestrus stages under natural cycling or after hormone synchronization. The three transporters were expressed at moderate to high levels compared to the liver. Transporter proteins were localized in various cell types in different tissue segments. Estrous cycle and exogenous hormone treatment affected transporter mRNA and protein expression, in a tissue- and transporter-dependent manner. Depo-Provera-synchronized mice were dosed vaginally or intraperitoneally with 3H-TFV, with or without MK571 co-administration, to delineate the function of cervicovaginal Mrp4. Co-administration of MK571 significantly increased the concentration of vaginally-administered TFV in endocervix and vagina. MK571 increased the concentration of intraperitoneally-administered TFV in the cervicovaginal lavage and vagina by several fold. Overall, P-gp, Bcrp, and Mrp4 were positively expressed in mouse cervicovaginal tissues, and their expression can be regulated by the estrous cycle or by exogenous hormones. In this model, the Mrp4 transporter impacted TFV distribution in cervicovaginal tissues.

A novel approach for sustained production of therapeutic proteins is described, using genetic modification of intact autologous micro-organ tissue explants from the subject's own skin. The skin-derived micro-organ can be maintained viable ex vivo for extended periods and is transduced with a transgene encoding a desired therapeutic protein, resulting in protein-secreting micro-organ (biopump (BP)). The daily protein production from each BP is quantified, enabling drug dosing by subcutaneous implantation of the requisite number of BPs into the patient to provide continuous production to the circulation of a known amount of the therapeutic protein. Each implanted BP remains localized and is accessible, to enable removal or ablation if needed. Examples from preclinical and clinical studies are presented, including use of associated virus vector 1 and helper-dependent adenoviral vectors producing BPs to provide long-term sustained secretion of recombinant interferon-α and erythropoietin.