pyrophosphate and HIV-Infections

Multipurpose prevention technologies (MPTs), which prevent sexually transmitted infection(s) and unintended pregnancy, are highly desirable to women. In this randomized, placebo-controlled, phase I study, women used a placebo or tenofovir (TFV) and levonorgestrel (LNG) intravaginal ring (IVR), either continuously or cyclically (three, 28-day cycles with a 3 day interruption in between each cycle), for 90 days. Sixty-eight women were screened; 47 were randomized to 4 arms: TFV/LNG or placebo IVRs used continuously or cyclically (4:4:1:1). Safety was assessed by adverse events and changes from baseline in mucosal histology and immune mediators. TFV concentrations were evaluated in multiple compartments. LNG concentration was determined in serum. Modeled TFV pharmacodynamic antiviral activity was evaluated in vaginal and rectal fluids and cervicovaginal tissue ex vivo. LNG pharmacodynamics was assessed with cervical mucus quality and anovulation. All IVRs were safe with no serious adverse events nor significant changes in genital tract histology, immune cell density or secreted soluble proteins from baseline. Median vaginal fluid TFV concentrations were >500 ng/mg throughout 90d. TFV-diphosphate tissue concentrations exceeded 1,000 fmol/mg within 72hrs of IVR insertion. Mean serum LNG concentrations exceeded 200 pg/mL within 2h of TFV/LNG use, decreasing quickly after IVR removal. Vaginal fluid of women using TFV-containing IVRs had significantly greater inhibitory activity (87-98% versus 10% at baseline; p<0.01) against HIV replication in vitro. There was a >10-fold reduction in HIV p24 antigen production from ectocervical tissues after TFV/LNG exposure. TFV/LNG IVR users had significantly higher rates of anovulation, lower Insler scores and poorer/abnormal cervical mucus sperm penetration. Most TFV/LNG IVR users reported no change in menstrual cycles or fewer days of and/or lighter bleeding. All IVRs were safe. Active rings delivered high TFV concentrations locally. LNG caused changes in cervical mucus, sperm penetration, and ovulation compatible with contraceptive efficacy. Trial registration: ClinicalTrials.gov #NCT03279120.

Topics: Anovulation; Antiviral Agents; Contraceptive Agents; Contraceptive Devices, Female; Diphosphates; Female; HIV Core Protein p24; HIV Infections; Humans; Levonorgestrel; Male; Semen; Tenofovir

Tenofovir alafenamide (TAF) preferentially loads peripheral blood mononuclear cells (PBMCs), resulting in higher PBMC tenofovir-diphosphate (TFV-DP) vs. tenofovir disoproxil fumarate (TDF). No studies have yet compared TFV-DP in PBMC from lower than daily dosing between prodrugs, which has potential implications for event-driven preexposure prophylaxis and pharmacologic forgiveness.. Two separate randomized, directly observed therapy (DOT) crossover studies (DOT-DBS and TAF-DBS) were conducted to mimic low, medium and high adherence.. HIV-negative adults were randomized to two 12-week DOT regimens of 33, 67 or 100% of daily dosing with emtricitabine (F)/TAF 200 mg/25 mg (TAF-DBS) or F/TDF 200 mg/300 mg (DOT-DBS), separated by a 12-week washout. PBMC steady-state concentrations (Css) of TFV-DP and FTC-TP were estimated using nonlinear mixed models and compared between F/TAF and F/TDF.. Thirty-five participants contributed to 33% (n = 23), 67% (n = 23) and 100% (n = 23) of daily F/TAF regimens. Forty-four contributed to 33% (n = 15), 67% (n = 16) and 100% (n = 32) of daily F/TDF regimens. PBMC TFV-DP Css were 7.3 [95% confidence interval (95% CI): 6.4-8.2], 7.1 (5.9-8.2) and 6.7- (4.4-8.9) fold higher (P < 0.0001) following F/TAF vs. F/TDF; 593 vs. 81.7, 407 vs. 57.4, and 215 vs. 32.3 fmol/106 cells, respectively. TFV-DP was 2.6 (2.1-3.1) fold higher with 33% F/TAF vs. 100% F/TDF. Estimated half-lives (95% CI) of TFV-DP in PBMC were 2.9 (1.5-5.5) days for F/TAF and 2.1 (1.5-2.9) days for F/TDF. FTC-TP was similar in both studies (P = 0.119).. F/TAF produced 6.7 to 7.3-fold higher TFV-DP in PBMC vs. F/TDF across adherence levels, supporting increased potency and pharmacologic forgiveness with F/TAF in the PBMC compartment.

Topics: Alanine; Anti-HIV Agents; Diphosphates; Emtricitabine; HIV Infections; Humans; Leukocytes, Mononuclear; Tenofovir

Tenofovir (TFV; prescribed as TFV disoproxil fumarate and TFV alafenamide prodrugs) is currently used for HIV prevention and treatment. TFV must be phosphorylated twice into TFV-diphosphate (TFV-DP) to become pharmacologically active. Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. Of note, TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Thus, it was hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this hypothesis, recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, 6, and 8) were incubated, individually, with TFV-DP to determine their abilities to dephosphorylate TFV-DP in vitro. Following incubations, TFV-DP dephosphorylation was determined using both malachite green phosphate assays and ultrahigh-performance liquid chromatography tandem mass spectrometry. From these, NTPDase 1 exhibited the highest activity toward TFV-DP. Further, enzyme kinetic analysis revealed Michaelis-Menten kinetics for NTPDase 1-mediated TFV-DP dephosphorylation. Next, immunoblot analyses were conducted to confirm the expression of NTPDase 1 protein in human colorectal tissue. Liquid chromatography coupled to mass spectrometry proteomics analysis was used to measure the relative abundance of NTPDases in human colorectal tissue among healthy adult individuals (

Topics: Adenosine Triphosphate; Adult; Anti-HIV Agents; Colorectal Neoplasms; Diphosphates; HIV Infections; Humans; Kinetics; Nucleosides; Nucleotides; Tenofovir

In sub-Saharan Africa (SSA), adolescent girls and young women (AGYW) ages 15 to 24 years represent <10% of the population yet account for 1 in 5 new HIV infections. Although oral pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) can be highly effective, low persistence in PrEP programs and poor adherence have limited its ability to reduce HIV incidence among women.. A total of 336 AGYW participating in the PEPFAR-funded DREAMS PrEP program in western Kenya were enrolled into a study of PrEP use conducted between 6/2019 to 1/2020. AGYW, who used daily oral TDF/FTC, completed interviews and provided dried blood spots (DBS) for measurement of tenofovir-diphosphate (TFV-DP) concentrations at enrollment and 3 months later, and 176/302 (58.3%, 95% confidence interval [95% CI 52.3 to 63.8]) met our definition of PrEP persistence: having expressed intention to use PrEP and attended both the second interview and an interim refill visit. Among AGYW with DBS taken at the second interview, only 9/197 (4.6%, [95% CI 1.6 to 7.5]) had protective TFV-DP levels (≥700 fmol/punch) and 163/197 (82.7%, [95% CI 77.5 to 88]) had levels consistent with no recent PrEP use (<10 fmol/punch). Perception of being at moderate-to-high risk for HIV if not taking PrEP was associated with persistence (adjusted odds ratio, 10.17 [95% CI 5.14 to 20.13], p < 0.001) in a model accounting for county of residence and variables that had p-value <0.1 in unadjusted analysis (age, being in school, initiated PrEP 2 to 3 months before the first interview, still active in DREAMS, having children, having multiple sex partners, partner aware of PrEP use, partner very supportive of PrEP use, partner has other partners, AGYW believes that a partner puts her at risk, male condom use, injectable contraceptive use, and implant contraceptive use). Among AGYW who reported continuing PrEP, >90% indicated they were using PrEP to prevent HIV, although almost all had non-protective TFV-DP levels. Limitations included short study duration and inclusion of only DREAMS participants.. Many AGYW persisted in the PrEP program without taking PrEP frequently enough to receive benefit. Notably, AGYW who persisted had a higher self-perceived risk of HIV infection. These AGYW may be optimal candidates for long-acting PrEP.

Topics: Adenine; Adolescent; Adult; Anti-HIV Agents; Child; Contraceptive Agents; Diphosphates; Emtricitabine; Female; HIV Infections; Humans; Infant; Kenya; Male; Medication Adherence; Organophosphates; Pre-Exposure Prophylaxis; Prospective Studies; Tenofovir; Young Adult

Event-driven pre-exposure prophylaxis (edPrEP) with oral tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) is highly effective for preventing HIV acquisition in men who have sex with men (MSM) and is preferred over daily PrEP by some MSM. However, it is largely unknown how well MSM adhere to edPrEP. We then aimed to assess PrEP protection during CAS among MSM using edPrEP and participating in the Amsterdam PrEP demonstration project (AMPrEP).. We analysed data from participants enrolled in AMPrEP who were taking edPrEP. We measured adherence through (1) a mobile application in which sexual behaviour and PrEP-use were recorded daily, (2) three-monthly self-completed questionnaires and (3) dried blood spot (DBS) samples collected around six, twelve and twenty-four months after PrEP initiation. We assessed the proportion of days with condomless anal sex (CAS) acts that were protected by PrEP, per partner type (i.e. steady partners, known casual partners, unknown casual partners), and the proportion of three-month periods during which PrEP was correctly used. Intracellular TFV-diphosphate (TFV-DP) concentrations were determined from DBS. Good adherence was defined as at least one tablet before and one tablet within 48 hours after a CAS act.. Between 11 September 2015 and 6 October 2019, 182 of 376 MSM (48.4%) used edPrEP for at least one three-month period. Of the 8224 CAS days that were reported in the app during edPrEP-use, we observed good protection for most CAS days involving steady partners (n = 1625/2455, 66.9%), known casual partners (n = 3216/3472, 92.6%) and unknown casual partners (n = 2074/2297, 90.3%). Men reported consistently correct PrEP-use in 851 (81.4%) of the 1046 three-month periods of edPrEP-use. The median TFV-DP concentration was 591 fmol/sample (interquartile range = 270 to 896).. Adherence to edPrEP was high as determined from the online app and questionnaire. DBS measurements were consistent with two to three tablets per week on average.

Topics: Anti-HIV Agents; Diphosphates; Emtricitabine; HIV Infections; Homosexuality, Male; Humans; Male; Medication Adherence; Netherlands; Pre-Exposure Prophylaxis; Sexual and Gender Minorities; Sexual Behavior; Surveys and Questionnaires; Transgender Persons

In a pre-exposure prophylaxis program for Kenyan women, we detected tenofovir-diphosphate in 61% (125/201) of randomly selected dried blood spots collected at the first follow-up visit. Tenofovir-diphosphate was detected more frequently among women who had partners living with human immunodeficiency virus, who were not pregnant, and who were ≥24 years.

Topics: Anti-HIV Agents; Diphosphates; Emtricitabine; Female; HIV Infections; Humans; Kenya; Medication Adherence; Pre-Exposure Prophylaxis; Pregnancy; Tenofovir

Enzymatic reactions usually occur in several steps: a step of substrate binding to the surface of the protein, a step of protein reorganization around the substrate and conduction of a chemical reaction, and a step of product release. The release of inorganic phosphate-PPi-from the matrix of the protein HIV reverse transcriptase is investigated computationally. Atomically detailed simulations with explicit solvent are analyzed to obtain the free energy profile, mean first passage time, and detailed molecular mechanisms of PPi escape. A challenge for the computations is of time scales. The experimental time scale of the process of interest is in milliseconds, and straightforward molecular dynamics simulations are in sub-microseconds. To overcome the time scale gap, we use the algorithm of Milestoning along a reaction coordinate to compute the overall free energy profile and rate. The methods of locally enhanced sampling and steered molecular dynamics determine plausible reaction coordinates. The observed molecular mechanism couples the transfer of the PPi to positively charged lysine side chains that are found on the exit pathway and to an exiting magnesium ion. In accord with experimental findings, the release rate is comparable to the chemical step, allowing for variations in substrate (DNA or RNA template) in which the release becomes rate determining.

Topics: Diphosphates; HIV Infections; HIV Reverse Transcriptase; HIV-1; Humans; Lysine; Magnesium; Molecular Dynamics Simulation; Substrate Specificity; Thermodynamics

The coformulation of the nucleos(t)ide analogs (NA) tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC) is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP). Such complexities manifest in nonlinear intracellular pharmacokinetics (PK). In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP) at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD) study among forty subjects receiving daily TDF/FTC (300 mg/200 mg) from the first-dose to pharmacological intracellular steady-state (30 days). TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC) were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM). Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV)) of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) production were 1020 fmol/106 cells (130%) and 44.4 pmol/106 cells (82.5%), resulting in (90% prediction interval) 11% (0.45%, 53%) and 14% (2.6%, 35%) reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP). Simulation-based intracellular operational multiple dosing half-lives of TFV-DP and FTC-TP were 6.7 days and 33 hours. This model described the formation of intracellular TFV-DP/FTC-TP and the interaction with dNTPs, and can be used to simulate analog:dNTP time course for various dosing strategies.

Topics: Adult; Anti-HIV Agents; Computer Simulation; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Diphosphates; Emtricitabine; Female; HIV; HIV Infections; Humans; Male; Middle Aged; Models, Biological; Polyphosphates; Prospective Studies; Tenofovir; Young Adult

The active metabolites of tenofovir (TFV) and emtricitabine (FTC) in peripheral blood mononuclear cells (PBMCs) have been used as markers of long-term antiretroviral (ARV) adherence. However, the process of isolating PBMCs is expensive, complex, and not feasible in many settings. We compared concentrations of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in the upper layer packed cells (ULPCs) obtained after whole blood centrifugation to isolated PBMCs as a possible alternative marker of adherence.. Ten HIV+ adults with HIV RNA <50 copies/mL on a TDF/FTC-containing regimen provided 5 paired PBMC and ULPC samples over 6 hours. TFV-DP and FTC-TP concentrations were analyzed by liquid chromatography/mass spectrometry. Partial areas under the curve were calculated using noncompartmental methods and Spearman Rank Correlations (rho) between PBMC and ULPC were determined.. The median (25th-75th percentile) concentration of TFV-DP in PBMCs was 143 (103-248) fmol/10(6) cells and in ULPC was 227 (160-394) fmol/10(6) cells (rho = 0.65; P < 0.0001). The concentration of FTC-TP in PBMCs was 6660 (5650-10,000) fmol/10(6) cells and in ULPC was 19.0 (12.0-27.8) fmol/10(6) cells (rho = 0.55; P < 0.0001). Compared to PBMCs, ULPC TFV-DP was 64% higher and FTC-TP was 99.7% lower. ULPC concentrations of TFV-DP and FTC-TP in one additional subject receiving a single dose of TDF/FTC were only 0.05% and 25%, of the other 10 subjects, respectively.. ULPC concentrations significantly correlated with PBMC concentrations. Preliminary single-dose data suggest some discrimination between intermittent versus consistent dosing. ULPC concentrations of TFV-DP and FTC-TP should be further investigated as a simply collected surrogate measure of ARV adherence.

Topics: Adenine; Adult; Anti-HIV Agents; Blood Cells; Deoxycytidine; Diphosphates; Emtricitabine; Female; HIV Infections; Humans; Leukocytes, Mononuclear; Male; Medication Adherence; Middle Aged; Organophosphonates; Polyphosphates; Tenofovir

We present 2 human immunodeficiency virus-infected patients with tenofovir disoproxil fumarate-induced Fanconi syndrome, leading to osteomalacia. Intracellular tenofovir diphosphate levels were measured in 1 patient and were found to be very high, with plasma tenofovir levels just slightly elevated. Fibroblast growth factor-23, a phosphaturic hormone, was decreased in both patients and is therefore unlikely to have a pathophysiological role in this pathology. The different potential factors contributing to the development of tenofovir-related kidney proximal tubular dysfunction are discussed and the data presented may help to further elucidate its pathogenesis.

Topics: Adenine; Adult; Anti-HIV Agents; Diphosphates; Fanconi Syndrome; HIV Infections; Humans; Male; Middle Aged; Organophosphonates; Osteomalacia; Radionuclide Imaging; Tenofovir; Whole Body Imaging

GS-9131 is a phosphonoamidate prodrug of the novel ribose-modified phosphonate nucleotide analog GS-9148 that demonstrates potent anti-human immunodeficiency virus type 1 (HIV-1) activity and an excellent resistance profile in vitro. Prodrug moieties were optimized for the efficient delivery of GS-9148 and its active diphosphate (DP) metabolite to lymphoid cells following oral administration. To understand the intracellular pharmacology of GS-9131, incubations were performed with various types of lymphoid cells in vitro. The intracellular accumulation and antiviral activity levels of GS-9148 were limited by its lack of cellular permeation, and GS-9131 increased the delivery of GS-9148-DP by 76- to 290-fold relative to that of GS-9148. GS-9131 activation was saturable at high extracellular concentrations, potentially due to a high-affinity promoiety cleavage step. Once inside the cells, GS-9148 was efficiently phosphorylated, forming similar amounts of anabolites in primary lymphoid cells. The levels of GS-9148-DP formed in peripheral blood mononuclear cells infected with HIV-1 were similar to that in uninfected PBMCs, and approximately equivalent intracellular concentrations of GS-9148-DP and tenofovir (TVF)-DP were required to inhibit viral replication by 90%. Once it was formed, GS-9148-DP was efficiently retained in activated CD4(+) cells, with a half-life of 19 h. In addition, GS-9131 showed a low potential for drug interactions with other adenine nucleoside/nucleotide reverse transcriptase inhibitors, based on the lack of competition for anabolism between suprapharmacologic concentrations of GS-9148 and TVF and the lack of activity of GS-9131 metabolites against purine nucleoside phosphorylase, an enzyme involved in the clearance of 2',3'-dideoxyinosine. Together, these observations elucidate the cellular pharmacology of GS-9131 and illustrate its efficient loading of lymphoid cells, resulting in a prolonged intracellular exposure to the active metabolite GS-9148-DP.

Topics: Adenine; Anti-HIV Agents; Diphosphates; Guanosine; HIV Infections; HIV-1; Humans; Leukocytes, Mononuclear; Lymphocyte Activation; Organophosphonates; Prodrugs; Reverse Transcriptase Inhibitors; Tenofovir

Vgamma9(+)Vdelta2(+) gammadelta T cells (Vdelta 2(+) T cells) are activated by Mycobacterium tuberculosis and secrete interferon (IFN)-gamma. Vdelta 2(+) T cells recognize phosphoantigens, such as bromohydrin pyrophosphate (BrHPP), and link innate and adaptive immunity.. A whole-blood assay was developed that used IFN-gamma secretion in response to BrHPP as a measurement of Vdelta2(+) T cell function.. Peak IFN-gamma levels were detected after stimulating whole blood with BrHPP for 7-9 days. IFN- gamma production in whole blood in response to BrHPP paralleled IFN-gamma production and Vdelta2(+) T cell expansion of peripheral-blood mononuclear cells. The assay was used to evaluate Vdelta2(+) T cell function in subjects in the United States (n = 24) and Uganda (n = 178) who were or were not infected with M. tuberculosis and/or human immunodeficiency virus (HIV) type 1. When 50 micromol/L BrHPP was used, 100% of healthy subjects produced IFN-gamma. The Vdelta2(+) T cell response was independent of the tuberculin skin test response. In Uganda, Vdelta2(+) T cell responses were decreased in patients with tuberculosis (n = 73) compared with responses in household contacts (n = 105). HIV-1-positive household contacts had lower responses than did HIV-1-negative household contacts. HIV-1-positive patients with tuberculosis had the lowest V delta 2(+) T cell responses.. Tuberculosis and HIV-1 infection are associated with decreased Velta2(+) T cell function. Decreased Vdelta2(+) T cell function may contribute to increased risk for tuberculosis in HIV-1-positive patients.

Topics: Adolescent; Adult; Cells, Cultured; Diphosphates; Female; HIV Infections; HIV-1; Humans; Interferon-gamma; Lymphocyte Activation; Male; Middle Aged; Mycobacterium tuberculosis; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes; Tuberculosis; Uganda; United States

Nucleoside analog chain terminators such as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.

Topics: Adenosine Triphosphate; Anti-HIV Agents; Base Sequence; Diphosphates; DNA, Viral; Drug Resistance, Microbial; HIV Infections; HIV Reverse Transcriptase; HIV-1; Humans; Lamivudine; Molecular Sequence Data; Mutation; Reverse Transcriptase Inhibitors; Templates, Genetic; Zidovudine