Drug-associated dysfunction of mitochondria is believed to play a role in the etiology of the various adverse symptoms that occur in human immunodeficiency virus (HIV)-infected patients treated with the nucleoside reverse transcriptase inhibitors (NRTIs). Tenofovir, a nucleotide analog recently approved for use in the treatment of HIV infection, was evaluated in vitro for its potential to cause mitochondrial toxicity and was compared to currently used NRTIs. Treatment with tenofovir (3 to 300 M) for up to 3 weeks produced no significant changes in mitochondrial DNA (mtDNA) levels in human hepatoblastoma (HepG2) cells, skeletal muscle cells (SkMCs), or renal proximal tubule epithelial cells. The potencies of inhibition of mtDNA synthesis by the NRTIs tested were zalcitabine (ddC) > didanosine (ddI) > stavudine > zidovudine (ZDV) > lamivudine ‫؍‬ abacavir ‫؍‬ tenofovir, with comparable relative effects in the three cell types. Unlike ddC and ddI, tenofovir did not affect cellular expression of COX II and COX IV, two components of the mitochondrial cytochrome c oxidase complex. Lactate production was elevated by less than 20% in HepG2 cells or SkMCs following treatment with 300 M tenofovir. In contrast, lactate synthesis increased by >200% in the presence of 300 M ZDV. Thus, treatment of various human cell types with tenofovir at concentrations that greatly exceed those required for it both to have in vitro anti-HIV type 1 activity in peripheral blood mononuclear cells (50% effective concentration, 0.2 M) and to achieve therapeutically relevant levels in plasma (maximum concentrations in plasma, 0.8 to 1.3 M) is not associated with mitochondrial toxicity.

Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

Diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) have inherent flexibility, helping to maintain activity against a wide range of resistance mutations. Crystal structures were determined with wild-type and K103N HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278). These structures reveal a similar binding mode for TMC125 and TMC278, whether bound to wild-type or K103N RT. Comparison to previously published structures reveals differences in binding modes for TMC125 and differences in protein conformation for TMC278.

GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-propyl]adenine and 9-(R)-4-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-Tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV), an acyclic nucleotide analog of dAMP, is a potent in vitro and in vivo inhibitor of human immunodeficiency virus type 1 (HIV-1) replication (2). TFV is sequentially phosphorylated in the cell by AMP kinase and nucleoside diphosphate kinase to the active species, tenofovir diphosphate (23, 39), which acts as a potent inhibitor of HIV-1 reverse transcriptase (7,49). The presence of a nonhydrolyzable phosphonic acid moiety in tenofovir circumvents an initial phosphorylation step which can be rate limiting for the activation of nucleoside analog inhibitors of HIV reverse transcriptase (3, 4).In order to increase the cellular permeability and oral bioavailability of TFV, which is a dianion at physiological pH, neutral prodrugs of TFV have been synthesized. Tenofovir disoproxil fumarate (TDF) is a bis-isopropoxycarbonyloxymethyl ester prodrug of TFV approved for the treatment of HIV. TDF is well tolerated, with infrequent development of resistance and a favorable long-term toxicity profile (2,8, 16). The oral administration of TDF results in high systemic levels of TFV (5); however, the rapid systemic degradation of TDF to TFV limits its uptake into target cells.Investigations to develop plasma-stable prodrugs which would be selectively hydrolyzed inside cells to antiviral nucleotides led to the design of GS-7340 and GS-9131{9--fluoro-1Ј-furanyladenine, respectively}, alkylalaninyl amidate phenyl ester prodrugs of TFV and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2Ј-fluoro-2Ј,3Ј-dideoxydidehydroadenosine), respectively. Both GS-7340 and GS-9131 exhibit potent in vitro anti-HIV-1 activities, favorable resistance profiles, and low cytotoxicities (9, 25). Compared to TDF, GS-7340 is significantly more stable in plasma and delivers ϳ30-fold-greater levels of active diphosphate metabolites into peripheral blood mononuclear cells (PBMCs) in vitro and in vivo (12). Compared to what was seen for TDF, oral administration of an equal dose of GS-7340 resulted in significantly higher levels of TFV accumulation both in lymphatic tissues and in PBMCs (24). While GS-7340 and other amidate prodrugs of tenofovir successfully validated the concept of enhanced in vivo intracellular delivery of parent nucleotide, GS-9131 has been selected as a clinical development candidate based on its unique activity against HIV-1 strains resistant to approved antiretroviral nucleosides and its favorable in vivo pharmacological properties, including the ability to effectively deliver the active GS-9148 diphosphate metabolite into PBMCs (9, 36). The prodrug moieties of GS-7340 and GS-9131 are structurally similar to a class of nucleoside monophosphate pro-* Corresponding author. Mailing address: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404.

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines was prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model.