Cell and Tissue Specific Metabolism of Nucleoside and Nucleotide Drugs: Case Studies and Implications for Precision Medicine.

Many clinically used antiviral drugs are nucleoside or nucleotide analog drugs, which have a unique mechanism of action that requires intracellular phosphorylation. This dependence on intracellular activation presents novel challenges for the discovery and development of nucleoside/nucleotide analog drugs. Contrary to many small molecule drug development programs that rely on plasma pharmacokinetics and systemic exposures, the precise mechanisms that result in efficacious intracellular nucleoside triphosphate concentrations must be understood in the process of nucleoside/nucleotide drug development. The importance is highlighted here, using the following as case studies: the herpes treatment acyclovir, the cytomegalovirus therapy ganciclovir, and human immunodeficiency virus (HIV) treatments based on tenofovir, which are also in use for HIV prophylaxis. For each drug, the specificity of metabolism that results in its activation in different cells or tissues is discussed, and the implications explored. Acyclovir's dependence on a viral enzyme for activation provides selective pressure for resistance mutations. Ganciclovir is also dependent on a viral enzyme for activation, and suicide gene therapy capitalizes on that for a novel oncology treatment. The tissue of most relevance for tenofovir activation depends on its use as treatment or as prophylaxis, and the pharmacogenomics and drug-drug interactions in those tissues must be considered. Finally, differential metabolism of different tenofovir prodrugs and its effects on toxicity risk are explored. Taken together, these examples highlight the importance of understanding tissue specific metabolism for optimal use of nucleoside/nucleotide drugs in the clinic. SIGNIFICANCE STATEMENT: Nucleoside and nucleotide analogue drugs are cornerstones in current antiviral therapy and prevention efforts that require intracellular phosphorylation for activity. Understanding their cell and tissue specific metabolism enables their rational, precision use for maximum efficacy.

Discovery of Genetic Variants of the Kinases That Activate Tenofovir in a Compartment-specific Manner.

Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention. TFV requires two phosphorylation steps to become pharmacologically active; however, the kinases that activate TFV in cells and tissues susceptible to HIV infection have yet to be identified. Peripheral blood mononuclear cells (PBMC), vaginal, and colorectal tissues were transfected with siRNA targeting nucleotide kinases, incubated with TFV, and TFV-monophosphate (TFV-MP) and TFV-diphosphate (TFV-DP) were measured using mass spectrometry-liquid chromatography. Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues. Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue. In addition, next-generation sequencing of the Microbicide Trials Network MTN-001 clinical samples detected 71 previously unreported genetic variants in the genes encoding these kinases. In conclusion, our results demonstrate that TFV is activated in a compartment-specific manner. Further, genetic variants have been identified that could negatively impact TFV activation, thereby compromising TFV efficacy in HIV treatment and prevention.

Dissimilarities in the metabolism of antiretroviral drugs used in HIV pre-exposure prophylaxis in colon and vagina tissues.

Attempts to prevent HIV infection through pre-exposure prophylaxis (PrEP) include topical application of anti-HIV drugs to the mucosal sites of infection; however, a potential role for local drug metabolizing enzymes in modulating the exposure of the mucosal tissues to these drugs has yet to be explored. Here we present the first report that enzymes belonging to the cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) families of drug metabolizing enzymes are expressed and active in vaginal and colorectal tissue using biopsies collected from healthy volunteers. In doing so, we discovered that dapivirine and maraviroc, a non-nucleoside reverse transcriptase inhibitor and an entry inhibitor currently in development as microbicides for HIV PrEP, are differentially metabolized in colorectal tissue and vaginal tissue. Taken together, these data should help to guide the optimization of small molecules being developed for HIV PrEP.