Metabolism of Antiretroviral Drugs Used in HIV Pre-Exposure Prophylaxis
MetadataShow full item record
HIV pre-exposure prophylaxis is the use of antiretroviral drugs, including dapivirine and tenofovir, in uninfected individuals to prevent the establishment of infection. Dapivirine is being developed as a topical microbicide for application to the vaginal and colorectal mucosa, and tenofovir is in clinical use as an oral formulation with a topical gel formulation for the vaginal and colorectal mucosa undergoing further clinical trials. The metabolism of dapivirine is unknown, and understanding the metabolism of tenofovir is particularly important because it is a prodrug that must be phosphorylated intracellularly to yield the active metabolite, tenofovir diphosphate. Dapivirine and tenofovir diphosphate must be present in the colorectal and vaginal mucosa for HIV prevention; tenofovir diphosphate must also be produced in white blood cells for oral HIV treatment and prevention and in hepatocytes for hepatitis B treatment. A comprehensive understanding of the metabolism that impacts drug exposure in these tissues is imperative for optimal drug use. The metabolism of dapivirine is not yet known, nor has a full understanding of drug metabolism in the mucosal tissues been defined. The enzymes proposed to be involved in dapivirine metabolism are the cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs). Immunoblotting revealed that CYP2B6, -2C19, -3A4, and -3A5were more highly expressed in vaginal tissues as compared to colorectal tissues. A liquid chromatography mass spectrometry assay was developed to identify and characterize 11 novel metabolites of dapivirine which were produced by the CYPs, UGTs, or both. In vitro metabolism assays were used to identify members of the CYP3A family as the primary enzymes responsible for dapivirine metabolism. The dapivirine mass spectrometry assay was then used, leveraging dapivirine as a small molecule probe, to demonstrate CYP activity in both colorectal and vaginal tissues, but UGT activity only in colorectal. While creatine kinase, adenylate kinase, and pyruvate kinase have been implicated in tenofovir’s activation, these studies were carried out in vitro and do not investigate whether these enzymes are expressed in the tissues of clinical relevance. Immunoblots revealed that the four creatine kinases and adenylate kinase 2 are more highly expressed in colorectal tissue compared to vaginal tissue and white blood cells, whereas the two pyruvate kinases displayed the opposite trend. Additionally, adenylate kinase 2, creatine kinase muscle, and both pyruvate kinases were expressed in primary human hepatocytes. An ion pairing liquid chromatography mass spectrometry assay was utilized to directly detect tenofovir phosphorylation. This assay was used to in combination with siRNA mediated knockdowns to show that AK2, PKM, and PKLR are responsible for activating tenofovir in white blood cells and vaginal tissues, whereas AK2 and CKM perform the same function in colorectal tissues. Collectively, these findings represent a fundamental shift in the current understanding of drug metabolism in the body. Not only are colorectal tissues and vaginal tissues able to biotransform drugs, but they do it in a tissue specific manner. I anticipate that these results lay the groundwork for future studies in tissue specific drug metabolism that can be leveraged to optimize future drug development and use in the colorectal and vaginal mucosa.