gs-7340 and tenofovir-diphosphate

Tenofovir alafenamide (TAF), in combination with FTC, was recently approved for PrEP in the United States. The objective of this study was to assess the relationship between tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) in dried blood spots (DBS) with adherence to TAF/FTC.. TAF-DBS was a randomized, crossover clinical study of TFV-DP in DBS, following directly observed dosing of 33%, 67%, or 100% of daily TAF (25 mg)/FTC (200 mg). Healthy volunteers were randomized to 2 different, 12-week dosing regimens, separated by a 12-week washout. DBS were collected weekly. TFV-DP and FTC-TP were extracted from two 7-mm punches and assayed with LC-MS/MS.. Thirty-seven participants (17 female, 7 African American, and 6 Hispanic) were included. TFV-DP exhibited a mean half-life of 20.8 days (95% confidence interval: 19.3 to 21.3). The slope for TFV-DP versus dosing arm was 1.14 (90% confidence interval: 1.07 to 1.21). The mean (SD) TFV-DP after 12 weeks was 657 (186), 1451 (501), and 2381 (601) fmol/2 7-mm punches for the 33%, 67%, and 100% arms. The following adherence interpretations are proposed: <450 fmol/punches, <2 doses/wk; 450-949 fmol/punches, 2-3 doses/wk; 950-1799 fmol/punches, 4-6 doses/wk; and ≥1800 fmol/punches, 7 doses/wk. FTC-TP was quantifiable for 1 week after drug cessation in 50%, 92%, and 100% of participants in the 33%, 67%, and 100% arms, respectively.. TFV-DP in DBS after TAF/FTC exhibited a long half-life and was linearly associated with dosing, similar to its predecessor tenofovir disoproxil fumarate. FTC-TP was quantifiable for up to 1 week after drug cessation. Together, these moieties provide complementary measures of cumulative adherence and recent dosing for TAF/FTC.

Topics: Adenine; Adolescent; Adult; Alanine; Anti-HIV Agents; Cross-Over Studies; Drug Combinations; Emtricitabine; Female; HIV Infections; Humans; Male; Medication Adherence; Middle Aged; Organophosphates; Polyphosphates; Pre-Exposure Prophylaxis; Prospective Studies; Tenofovir; Young Adult

Tenofovir alafenamide, a prodrug of tenofovir, produces higher PBMC concentrations of tenofovir diphosphate (tenofovir-dp) than tenofovir disoproxil fumarate. To understand tenofovir alafenamide's mucosal tissue distribution and its implications for pre-exposure prophylaxis, we characterized tenofovir-dp in female genital tract (FGT) and lower gastrointestinal (GI) tissues.. Healthy seronegative women were given 5, 10 or 25 mg of tenofovir alafenamide ( n  =   8/group). Each participant provided plasma, PBMC and cervical, vaginal and rectal tissue samples over 14 days. Plasma, cell lysate and tissue homogenate concentrations were analysed by LC-MS/MS. Dose proportionality was declared in plasma and PBMCs if the natural log AUC versus natural log dose regression line 90% CI was within 0.57-1.43. In vitro tenofovir-dp formation was assessed in PBMCs and ectocervical (Ect1/E6E7) and vaginal (VK2/E6E7) cells incubated in 0.5 and 10 μM tenofovir alafenamide or tenofovir. clinicaltrials.gov: NCT02357602.. Following single doses of 5, 10 and 25 mg, median (IQR) tenofovir plasma AUC 0-14 days was 52.8 (49.5-59.6), 78.1 (68.2-86.9) and 169.7 (131.2-211.4) ng·h/mL and tenofovir-dp PBMC AUC 0-14 days was 2268 (1519-4090), 4584 (3113-5734) and 9306 (6891-10785) fmol·h/10 6 cells, respectively. Tenofovir was quantifiable in 52% and 92% of FGT and GI tissues, whereas tenofovir-dp was quantifiable in only 5% and 19% of FGT and GI tissues, respectively. Plasma tenofovir and PBMC tenofovir-dp were dose proportional (90% CI = 0.87-1.15 and 0.62-1.02, respectively). In vitro tenofovir-dp was 1.7-17-fold higher in epithelial cells than PBMCs.. After tenofovir alafenamide dosing in vivo , tenofovir-dp was unquantifiable in most tissues (91%) although cervical and vaginal epithelial cells efficiently formed tenofovir-dp from tenofovir alafenamide in vitro . These findings warrant further investigation of tenofovir alafenamide's pharmacology.

Topics: Adenine; Adult; Alanine; Cervix Uteri; Drug Administration Schedule; Epithelial Cells; Female; Gastrointestinal Tract; Humans; Intestinal Mucosa; Leukocytes, Mononuclear; Middle Aged; Mucous Membrane; Organophosphates; Pre-Exposure Prophylaxis; Rectum; Tenofovir; Tissue Distribution; Vagina; Young Adult

To evaluate the antiviral activity, safety, pharmacokinetics, and pharmacokinetics/pharmacodynamics of short-term monotherapy with tenofovir alafenamide (TAF), a next-generation tenofovir (TFV) prodrug.. A phase 1b, randomized, partially blinded, active- and placebo-controlled, dose-ranging study.. Treatment-naive and experienced HIV-1-positive adults currently off antiretroviral therapy were randomized to receive 8, 25, or 40 mg TAF, 300 mg tenofovir disoproxil fumarate (TDF), or placebo, each once daily for 10 days.. Thirty-eight subjects were enrolled. Baseline characteristics were similar across dose groups. Significant reductions in plasma HIV-1 RNA from baseline to day 11 were observed for all TAF dose groups compared with placebo (P < 0.01), with a median decrease of 1.08-1.73 log10 copies per milliliter, including a dose-response relationship for viral load decrease up to 25 mg. At steady state, 8, 25, and 40 mg TAF yielded mean TFV plasma exposures [area under the plasma concentration-time curve (AUCtau)] of 97%, 86%, and 79% lower, respectively, as compared with the TFV exposures observed with 300 mg TDF. For 25 and 40 mg TAF, the mean intracellular peripheral blood mononuclear cell tenofovir diphosphate AUCtau was ∼7-fold and ∼25-fold higher, relative to 300 mg TDF.. Compared with 300 mg TDF, TAF demonstrated more potent antiviral activity, higher peripheral blood mononuclear cell intracellular tenofovir diphosphate levels, and lower plasma TFV exposures, at approximately 1/10th of the dose. This may translate into greater antiviral efficacy, a higher barrier to resistance, and an improved safety profile relative to TDF, supporting further investigation of TAF dosed once daily in HIV-infected patients.

Topics: Adenine; Adult; Alanine; Anti-Retroviral Agents; Area Under Curve; Dose-Response Relationship, Drug; Fatigue; Female; HIV Infections; HIV-1; Humans; Male; Middle Aged; Nausea; Organophosphates; Organophosphonates; RNA, Viral; Single-Blind Method; Statistics, Nonparametric; Tenofovir; Viral Load; Young Adult

Human immunodeficiency virus (HIV) continues to be a major health concern. AIDS-related deaths (acquired immunodeficiency syndrome) have decreased recently, but chronic liver disease is now a major cause of mortality among HIV patients. Widespread alcohol use is recognized to be a major contributing factor. Tenofovir alafenamide fumarate (TAF), one of the most used HIV drugs, requires hydrolysis followed by phosphorylation to produce tenofovir diphosphate, the ultimate anti-HIV metabolite. Carboxylesterase-1 (CES1), established to hydrolyze TAF, is known to catalyze transesterification in the presence of ethanol. The aim of the study was to test the hypothesis that metabolism-based interactions between TAF and ethanol negatively impact both efficacy and safety of TAF. To test this hypothesis, the metabolism of TAF was determined in human primary hepatocytes and with a large number of human liver samples (S9 fractions) in the presence or absence of ethanol. The metabolism was monitored by LC-MS/MS (liquid chromatography with tandem mass spectrometry) and the level of CES1 or CES2 was determined by Western blotting. Consistent with the hypothesis, TAF underwent transesterification in the presence of ethanol accompanied by decreased hydrolysis. The formation of tenofovir diphosphate (the therapeutically active metabolite) was significantly decreased. In addition, TAF but not its hydrolytic metabolite, was found to increase intracellular lipid retention, and the increase was enhanced by ethanol. These findings conclude that alcohol consumption, beyond commonly accepted poor adherence to HIV medications, directly impacts the efficacy and safety of TAF.

Topics: Adenine; Alanine; Alcohol Drinking; Anti-HIV Agents; Carboxylic Ester Hydrolases; Chromatography, Liquid; Ethanol; Fumarates; HIV; HIV Infections; Humans; Lipids; Organophosphates; Tandem Mass Spectrometry; Tenofovir

Treatment and prevention of human immunodeficiency virus type one (HIV-1) infection was transformed through widespread use of antiretroviral therapy (ART). However, ART has limitations in requiring life-long daily adherence. Such limitations have led to the creation of long-acting (LA) ART. While nucleoside reverse transcriptase inhibitors (NRTI) remain the ART backbone, to the best of our knowledge, none have been converted into LA agents. To these ends, we transformed tenofovir (TFV) into LA surfactant stabilized aqueous prodrug nanocrystals (referred to as NM1TFV and NM2TFV), enhancing intracellular drug uptake and retention. A single intramuscular injection of NM1TFV, NM2TFV, or a nanoformulated tenofovir alafenamide (NTAF) at 75 mg/kg TFV equivalents to Sprague Dawley rats sustains active TFV-diphosphate (TFV-DP) levels ≥ four times the 90% effective dose for two months. NM1TFV, NM2TFV and NTAF elicit TFV-DP levels of 11,276, 1,651, and 397 fmol/g in rectal tissue, respectively. These results are a significant step towards a LA TFV ProTide.

Topics: Adenine; Alanine; Animals; Anti-HIV Agents; Drug Stability; Female; HIV Infections; HIV-1; Humans; Male; Nanoparticles; Organophosphates; Prodrugs; Rats, Sprague-Dawley; Tenofovir; Therapeutic Equivalency

The toxicity of tenofovir alafenamide (TAF) hemifumarate (HF) was evaluated when administered by continuous subcutaneous (s.c.) infusion via an external infusion pump for 28 days to rats and dogs. The toxicokinetics of TAF and two metabolites, tenofovir (TFV) and tenofovir diphosphate (TFV-DP) were also evaluated. After administration of TAF HF in rats and dogs, primary systemic findings supported an inflammatory response that was considered minimal to mild. Gross pathology and histopathologic evaluation of tissue surrounding the s.c. infusion site revealed signs of inflammation, including edema, mass formation, fibrosis, and mononuclear cell inflammation in groups receiving ≥300 μg/kg/day in rats and ≥25 μg/day in dogs. Although these changes were observed in animals receiving vehicle, the severity was greater in animals receiving TAF HF. Changes in the local tissue were considered a TAF HF-mediated exacerbation of an inflammatory response to the presence of the catheter. In rats, systemic and local findings were considered not adverse due to their low severity and reversibility; therefore, the "no observed adverse effect level" (NOAEL) was set at 1,000 μg/kg/day. Because none of the systemic findings were related to systemic exposure to TAF, the systemic NOAEL was set at 250 μg/kg/day in dogs. Due to the severity of the observations noted, a NOAEL for local toxicity could not be established. Although these results might allow for exploration of tolerability and pharmacokinetics of s.c. administered TAF HF in humans, data suggest a local reaction may develop in humans at doses below a clinically relevant dose.

Topics: Adenine; Alanine; Animals; Anti-HIV Agents; Dogs; Edema; HIV Infections; HIV-1; Infusions, Subcutaneous; Male; Organophosphates; Pre-Exposure Prophylaxis; Rats; Tenofovir

Topics: Adenine; Alanine; Cell Count; Chromatography, High Pressure Liquid; DNA; Fluorescent Dyes; Genomics; Humans; Image Cytometry; Intercalating Agents; Leukocytes, Mononuclear; Organophosphates; Prodrugs; Tandem Mass Spectrometry; Tenofovir

HIV pre-exposure prophylaxis (PrEP) strategies have the potential to prevent millions of incident HIV infections each year. However, the efficacy of PrEP strategies has been plagued by issues of non-adherence, likely because of the difficulty in motivating otherwise healthy people to adhere to treatment regimens that require significant behavioral changes and daily discipline. An alternative approach to PrEP is to focus on strategies that fit in to normal, and even desirable, sexual behaviors, such as the use of cleansing enemas by men who have sex with men (MSM) prior to receptive anal intercourse (RAI). Here, we describe preclinical efforts toward optimizing a tenofovir (TFV)-based enema formulation for rectal PrEP. Using a murine model, we compared the plasma and tissue pharmacokinetics of TFV and various TFV prodrugs, including tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), and hexadecyloxypropyl tenofovir (CMX157), after dosing as enema formulations with varying osmolality and ion content. We observed that the enema vehicle composition played a more important role than the TFV prodrug properties in achieving rapid and therapeutically relevant tenofovir diphosphate (TFV-DP) concentrations in mouse colorectal tissue. Our results support the next steps, which are further preclinical (non-human primate) and clinical development of a hypo-osmolar TFV enema product for rectal PrEP.

Topics: Adenine; Administration, Rectal; Alanine; Animals; Anti-HIV Agents; Anti-Infective Agents; Enema; HIV Infections; HIV-1; Homosexuality, Male; Male; Mice; Organophosphates; Organophosphonates; Pre-Exposure Prophylaxis; Prodrugs; Rectum; Sexual and Gender Minorities; Tenofovir

Tenofovir (TFV) treatment of female reproductive tract (FRT) cells results in differential accumulation of intracellular Tenofovir diphosphate (TFV-DP) in different cell types, with greater concentrations in epithelial cells (100-fold) and fibroblasts (10-fold) than in CD4+ T cells. The possibility that TFV-DP accumulation and retention in epithelial cells and fibroblasts may alter TFV availability and protection of CD4+ T cells against HIV infection, prompted us to evaluate TFV and/or Tenofovir alafenamide (TAF) release from FRT cells. Endometrial, endocervical and ectocervical polarized epithelial cells and fibroblasts were pre-loaded with TFV or TAF, and secretions tested for their ability to inhibit HIV infection of activated blood CD4+ T cells. Epithelial cell basolateral secretions (1, 2 and 3 days post-loading), but not apical secretions, suppressed HIV infection of CD4+ T cells, as did secretions from pre-loaded fibroblasts from each site. Intracellular TFV-DP levels in epithelial cells following preloading with TFV or TAF correlated directly with ARV protection of CD4+ T cells from HIV infection. When added apically to epithelial cells, TFV/TAF was released basolaterally, in part through Multidrug Resistant Protein transporters, taken up by fibroblasts and released into secretions to partially protect CD4+ T cells. These findings demonstrate that epithelial cells and fibroblasts release TFV/TAF for use by CD4+ T cells and suggest that the tissue environment plays a major role in the sustained protection against HIV infection.

Topics: Adenine; Adult; Alanine; Anti-HIV Agents; CD4-Positive T-Lymphocytes; Cervix Uteri; Drug Resistance, Multiple; Endometrium; Epithelial Cells; Female; Fibroblasts; Genitalia, Female; HIV Infections; Humans; Middle Aged; Organophosphates; Tenofovir; Vagina

In vitro evaluation of tenofovir disproxil fumarate (TDF) and tenofovir alafenamide (TAF) revealed comparable antiviral effects with respect to the tenofovir-diphosphate (TFV-DP) level in human peripheral blood mononuclear cells (PBMCs), despite the EC

Topics: Adenine; Alanine; Anti-HIV Agents; Cytoplasm; Databases, Factual; Dose-Response Relationship, Drug; HIV Infections; HIV-1; Humans; Leukocytes, Mononuclear; Meta-Analysis as Topic; Organophosphates; Prodrugs; Tenofovir; Viral Load; Virus Replication

Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP) in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore, simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide (TAF) as a representative of TFV prodrugs. A sensitive LC-MS/MS method was developed, and TAF, TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6mm×150mm, 3.5μm, Waters) with gradient elution after protein precipitation. The method provided good linearity for all the compounds (2-500nM for TFV and TAF; 20-5000nM for TFV-DP) with the correlation coefficients (r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE<10.4%) and precisions (in terms of coefficient of variation, CV<14.1%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.

Topics: Adenine; Alanine; Anti-HIV Agents; Cell Line, Tumor; Chromatography, Liquid; DNA Replication; Half-Life; Hep G2 Cells; Hepatocytes; HIV Infections; HIV-1; Humans; Organophosphates; Prodrugs; Tandem Mass Spectrometry; Tenofovir; Virus Replication

HIV prevention research is focused on combining antiretrovirals (ARV) and progestin contraceptives to prevent HIV infection and pregnancy. The possibility that progestins compromise ARV anti-HIV activity prompted us to evaluate the effects of progestins on tenofovir (TFV) and TFV-alafenamide (TAF) on HIV infection and intracellular TFV-diphosphate (TFV-DP) concentrations in blood and genital CD4+ T cells. Following incubation of blood CD4+ T cells with TFV or TAF, Medroxyprogesterone acetate (MPA), but not Levonorgestrel, Norethisterone or progesterone, suppressed the anti-HIV effect of TFV by reducing intracellular TFV-DP, but had no effect on TAF inhibition of infection or TFV-DP. In contrast, with genital CD4+ T cells, MPA suppressed TAF inhibition of HIV infection and lowered of TFV-DP concentrations without affecting TFV protection. These findings demonstrate that MPA selectively compromises TFV and TAF protection in blood and genital CD4+ T cells and suggests that MPA may decrease ARV protection in individuals who use ARV intermittently for prevention.

Topics: Adenine; Adult; Alanine; Anti-HIV Agents; Anti-Retroviral Agents; CD4-Positive T-Lymphocytes; Cells, Cultured; Contraceptive Agents; Contraceptives, Oral, Hormonal; Female; Genitalia, Female; HIV Infections; HIV-1; Humans; Middle Aged; Organophosphates; Progestins; Tenofovir

Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection.

Topics: Adenine; Alanine; Animals; Cells, Cultured; Dogs; Hepatitis B virus; Hepatocytes; Humans; Liver; Male; Organophosphates; Tenofovir