Adefovir accumulation in the renal interstitium triggers mast cell degranulation and promotes renal interstitial fibrosis.

Organic anion transporters 1 (OAT1) and OAT3 are responsible for transporting adefovir (ADV) into renal tubular epithelial cells. Our previous research found that ADV accumulated in the renal interstitium and caused renal interstitial fibrosis when Oat1/3 were inhibited by OATs inhibitor probenecid for long-term. Mast cells (MCs) in the interstitial space are considered to be key drivers of renal fibrosis. The current work investigated the effect of ADV on MCs in vitro and during the development of interstitial fibrosis in rats. Results indicate that ADV triggers chymase release from cultured RBL-2H3 mast cells in a time-and concentration-dependent manner. Angiotensin II (Ang II) in renal interstitium is generated mainly by chymase, renin and other products released from MCs, and has a direct effect on fibrosis through the angiotensin receptor. The concentrations of Ang II and fibrosis was significantly increased after administration of ADV alone or with probenecid for 4 weeks. The MCs membrane stabilizer sodium cromoglycate (SCG) and the angiotensin receptor antagonist Valsartan (VAL) could ameliorate ADV-induced nephrotoxicity. Additionally, SCG or VAL could reduce the accumulation of ADV in the renal interstitium by upregulating the expression of Oat1/3 and multidrug resistance-associated protein 4. Therefore, ADV accumulation in the renal interstitium could promote the degranulation of interstitial MCs and drive the development of renal fibrosis. SCG or VAL could ameliorate ADV-associated fibrosis by decreasing degranulation of MCs and accelerating renal clearance of ADV.

Influence of Renal Function on the Single-Dose Pharmacokinetics of Besifovir, a Novel Antiviral Agent for theTreatment of Hepatitis B Virus Infection.

Per the well-known resistance of hepatitis B virus to nucleoside/nucleotide analogs, alternative treatment options with higher resistance barriers have been approved for use in both treatment-naïve and lamivudine-resistant hepatitis B virus infections. This phase I study was conducted in adults with normal and impaired renal function to evaluate the effect of renal impairment on the pharmacokinetics of besifovir, a prodrug of an acyclic nucleotide phosphonate, that is mainly cleared via renal excretion. An open-label, single-dose parallel-group clinical study was conducted in subjects with normal renal function and mild, moderate, and severe renal impairment. Subjects received a single oral dose of besifovir dipivoxil 150 mg, and serial blood and urine samples were collected for up to 72 hours after dosing to assess the pharmacokinetic characteristics of besifovir. The extent of plasma exposure of besifovir, detected as its major and active metabolites, LB80331 and LB80317, respectively, increased with worsening renal function. Compared to the subjects with normal renal function, the mean areas under the concentration-time curves of LB80331 increased by 1.5-, 2.5-, and 4.5-fold in subjects with mild, moderate, and severe impairment, respectively. LB80317 showed a 1.8-, 3.2-, and 6.2-fold increase in subjects with mild, moderate, and severe renal impairment compared to those with normal function. The ratios of LB80331 renal clearance and the average estimated glomerular filtration rate of each renal impairment group with respect to the normal group were similar. The increase in plasma exposure and decrease in renal clearance suggest the need to adjust dosage regimens in patients with moderate to severe renal impairment.

Quantification of adefovir and pitavastatin in human plasma and urine by LC-MS/MS: A useful tool for drug-drug interaction studies.

As a tool to be used in transporter-mediated drug-drug interaction studies, a sensitive LC-MS/MS method for the simultaneous quantification of adefovir and pitavastatin in human plasma and adefovir in urine was developed and successfully validated. Plasma samples were processed by protein precipitation using methanol with a subsequent concentrating step. Urine samples were diluted using 0.1% formic acid. Separation was achieved on a Synergy Polar-RP reversed phase column (50 × 4.6 mm, 2.5 µm) in gradient elution using a mobile phase composed of water and 0.1% formic acid and a mixture of methanol and acetonitrile (50:50, v/v) containing 0.1% formic acid at a flow rate of 1.0 mL/min. The linear range covered concentrations from 0.273 to 52.6 ng/mL for adefovir and from 0.539 to 104.2 ng/mL for pitavastatin in human plasma, respectively. The calibration curve for adefovir in urine ranged from 0.104 to 10.0 µg/mL. The weighted linear regression (1/conc2) implied excellent linearity with correlation coefficients ≥0.999.

Voltammetric determination of adefovir dipivoxil by using a nanocomposite prepared from molecularly imprinted poly(o-phenylenediamine), multi-walled carbon nanotubes and carbon nitride.

An electrochemical sensor for adefovir dipivoxil (ADV) detection was prepared by electropolymerization of o-phenylenediamine in the presence of ADV on a glassy carbon electrode modified with multi-walled carbon nanotubes and carbon nitride. The electrode was characterized by field emission scanning electron microscopy and differential pulse voltammetry. The performance was optimized by response surface methodology. The changes in differential pulse voltammetric peak currents of the redox probe, ferricyanide, were linear to ADV concentrations in the range from 0.1 to 9.9 µmol L-1, with the detection limit of 0.05 µmol L-1 (S/N = 3). The sensor was applied to the determination of ADV in drug formulations, human serum and urine samples. It is selective due to the use of an imprinted material, well reproducible, long-term stable, and regenerable. Graphical abstract By merging the unique properties of carbon nitride with intrinsic properties of MWCNTs, and molecularly imprinted polymers, a novel electrochemical sensor with selective binding sites was prepared for determination of adefovir dipivoxil in pharmaceutical and biological samples.

Stability and Efficiency of Mixed Aryl Phosphonate Prodrugs.

A set of phosphonate prodrugs of a butyrophilin ligand was synthesized and evaluated for plasma stability and cellular activity. The mixed aryl acyloxy esters were prepared either via a standard sequence through the phosphonic acid chloride, or through the more recently reported, and more facile, triflate activation. In the best of cases, this class of prodrugs shows cellular potency similar to that of bis-acyloxyalkyl phosphonate prodrugs and plasma stability similar to that of aryl phosphonamidates. For example, {[((3E)-5-hydroxy-4-methylpent-3-en-1-yl) (naphthalen-2-yloxy)phosphoryl]oxy}methyl 2,2-dimethylpropanoate can activate BTN3A1 in K562 cells after just 15 minutes of exposure (at an EC50 value of 31 nm) and is only partially metabolized (60 % remaining) after 20 hours in human plasma. Other related novel analogues showed similar potency/stability profiles. Therefore, mixed aryl acyloxyalkyl phosphonate prodrugs are an exciting new strategy for the delivery of phosphonate-containing drugs.

Clinical Pharmacology of the Reversible and Potent P2Y12 Receptor Antagonist ACT-246475 After Single Subcutaneous Administration in Healthy Male Subjects.

ACT-246475 is a selective and reversible P2Y12 receptor antagonist inducing inhibition of platelet aggregation (IPA). A randomized, double-blind, placebo-controlled, parallel-design study was performed to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of escalating single subcutaneous doses of ACT-246475 (1, 2, 4, 8, 16, or 32 mg) in healthy male subjects (N = 8 per dose, 3:1 active:placebo ratio). Pharmacodynamic effects were assessed based on maximum platelet aggregation and P2Y12 reaction units using light transmission aggregometry and VerifyNow® assays, respectively. ACT-246475 was safe and well tolerated up to 32 mg based on adverse event data and absence of clinically relevant changes in hematology, biochemistry, vital signs, and electrocardiogram variables. Median time to reach maximum plasma concentration was 0.5-0.75 hours, and geometric mean terminal half-life ranged from 1.3 to 9.2 hours across the tested dose range. Exposure to ACT-246475 was dose proportional across all dose groups. The maximal %IPA was reached within 30 minutes after subcutaneous administration of ACT-246475. A dose-dependent duration and extent of effect were observed based on area under the effect curve and maximum effect data. Similar results were observed for maximum platelet aggregation and P2Y12 reaction units. The %IPA was ≥85% at doses ≥2 mg. This level of %IPA was extended to at least 12 hours in the 32-mg dose group. The safety and pharmacokinetic/pharmacokinetic profile with quick onset and adequate duration of IPA support further investigation in patients with coronary artery disease.

Gas chromatography-mass spectrometry with spiral large-volume injection for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum.

A sensitive method for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum was developed using gas chromatography-mass spectrometry (GCMS) with large-volume injection. The GC injection was administered using stomach-type spiral injector (LVI, AiSTI SCIENCE) enabling introduction of only target compounds from 50 µL ethyl acetate extract after purging the solvent. For GCMS analysis of sarin (GB), 670 times higher sensitivity, based on limit of detection (LOD, S/N = 3, on extracted ion chromatogram (EIC) at m/z 99), was achieved using this injection (50 µL) compared to that achieved using 1 µL split injection (ratio 20:1). Ethyl (EtGB), isopropyl (GB), n-propyl (nPrGB), isobutyl (iBuGB), pinacolyl (GD), cyclohexyl (GF) methylphosphonofluoridates, and O-ethyl N, N-dimethylphosphoramidofluoridate (GAF) were detected with low LOD (15-75 pg/mL) and sharp peak shapes (high practical plate number (defined as 5.54 x (tR/Wh)2, where tR is the retention time and Wh is the bandwidth at half-height): 1100000-2400000) in GCMS using a polar separation column, electron ionization, and quadruple mass analyzer. During the analysis of fluoridated phosphonate-spiked ethyl acetate extract of solid phase extraction (SPE, Bond Elut NEXUS) from fluoride-mediated regeneration of blank human plasma, LOD (on EIC at m/z 99 except for GAF (m/z 126)) were 25-140 pg/mL with sharp peak shapes. The reaction recoveries in fluoride-mediated regeneration of plasma, which was inhibited by GB, GD, GA, GF, VX, and Russian VX (10 ng/mL), were 49-114% except for GD (10%). The concentration levels of 0.3-1 ng/mL of nerve agents in plasma could be determined.

New analytical approach to determine organophosphorus insecticides in blood by dried matrix spots sampling and GC-MS/MS.

This work describes the optimization of a new method for the determination of five organophosphorus insecticides in whole blood. The analytes were extracted from the matrix (50 µL) using the dried blood spot (DBS) approach and were analyzed by gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS). The studied compounds (diazinon, chlorpyrifos, parathion-ethyl, chlorfenvinphos and quinalphos) were chosen based on the statistics of intoxications in Portugal, and ethion was used as internal standard. The method was fully validated, taking into account international guidelines for bioanalytical method validation, such as those of the Food and Drug Administration (FDA), International Conference on Harmonization (ICH) and Scientific Working Group for Forensic Toxicology (SWGTOX). A linear range of 0.1-25 µg/mL was obtained for all compounds, except for diazinon and quinalphos (0.05-25 µg/mL and 0.25-25 µg/mL, respectively), presenting determination coefficients above 0.99. Concerning precision, the coefficients of variation (CVs) were lower than 14% for all compounds. Those compounds were found to be stable in the samples. Although the values obtained for recovery were low (between 1 and 12%), the method proved to be sensitive, since detection limits between 0.05 and 0.1 µg/mL were obtained. The novelty is the use of the DBS approach in the extraction of these compounds, and this is the first paper reporting it: DBS is a recent technique of bioanalysis in the field of toxicology, and in addition to its simplicity and sensitivity, it is applicable routinely in both clinical and forensic toxicology situations. Graphical abstract ᅟ.

Expedient synthesis and biological evaluation of alkenyl acyclic nucleoside phosphonate prodrugs.

The importance of phosphonoamidate prodrugs (ProTides) of acyclic nucleoside phosphonate (ANPs) is highlighted by the approval of Tenofovir Alafenamide Fumarate for the treatment of HIV and HBV infections. In the present paper we are reporting an expedient, one-pot, two-steps synthesis of allyl phosphonoamidates and diamidates that offers a time saving strategy when compared to literature methods. The use of these substrates in the cross metathesis reactions with alkenyl functionalised thymine and uracil nucleobases is reported. ANPs prodrugs synthesized via this methodology were evaluated for their antiviral activities against DNA and RNA viruses. It is anticipated that the use of 5,6,7,8-tetrahydro-1-napthyl as aryloxy moiety is capable to confer antiviral activity among a series of otherwise inactive uracil ProTides.

Injectable PLGA Adefovir microspheres; the way for long term therapy of chronic hepatitis-B.

For patient convenience, sustained release Adefovir Poly-d,l-lactic-co-glycolic acid (PLGA) microspheres were formulated to relieve the daily use of the drug which is a problem for patients treated from chronic hepatitis-B. PLGA microspheres were prepared and characterized by entrapment efficiency, particle size distribution and scanning electron microscopy (SEM). In-vitro release and in-vivo studies were carried out. Factors such as drug: polymer ratio, polymer viscosity and polymer lactide content were found to be important variables for the preparation of PLGA Adefovir microspheres. Fourier transform infrared (FTIR) analysis and differential scanning calorimetry (DSC) were performed to determine any drug-polymer interactions. One way analysis of variance (ANOVA) was employed to analyze the pharmacokinetic parameters after intramuscular injection of the pure drug and the selected PLGA microspheres into rats. FTIR and DSC revealed a significant interaction between the drug and the polymer. Reports of SEM before and after 1 and 24 h release showed that the microspheres had nonporous smooth surface even after 24 h release. The entrapment efficiency ranged between 55.83 and 86.95% and in-vitro release studies were continued for 16, 31 and 90 days. The pharmacokinetic parameters and statistical analysis showed a significant increase in the Tmax, AUC0-t and MRT, and a significant decrease in the Cmax of the tested formulation (p < 0.05). Results demonstrated that PLGA Adefovir microspheres could be used for long-term treatment of chronic hepatitis-B instead of the daily dose used by the patient.

Validation and implementation of liquid chromatographic-mass spectrometric (LC-MS) methods for the quantification of tenofovir prodrugs.

BACKGROUND: The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma. METHODS: K2EDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1 × 50 mm, 3.5 µm column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1 × 50 mm, 2.6 µm column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines. RESULTS: Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25 ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.4% and %DEVs ≤ ±â€¯7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also applied to quantify in vivo concentrations of prodrugs and TFV in a preclinical study post-rectal administration. CONCLUSIONS: Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the multiplexed quantification TAF and TFV, as well as an independent assay for CMX157 quantification, in plasma. The described methods meet sufficient throughput criteria to support large research trials.

High throughput online solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry method for polyfluoroalkyl phosphate esters, perfluoroalkyl phosphonates, and other perfluoroalkyl substances in human serum, plasma, and whole blood.

A rapid, sensitive and reliable method was developed for the determination of a broad range of poly- and perfluoroalkyl substances (PFASs) in various blood matrices (serum, plasma, and whole blood), and uses only 50 µL of sample material. The method consists of a rapid protein precipitation by methanol followed by high throughput online solid phase extraction (SPE), ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), and negative electrospray ionization detection. The method was developed for simultaneous determination of twenty-five PFASs, including polyfluoroalkyl phosphate esters (PAPs; 6:2, 8:2, 6:2/6:2, and 8:2/8:2), perfluoroalkyl phosphonates (PFPAs; C6, C8, and C10), perfluoroalkyl sulfonates (PFSAs; C4, C6, C7, C8, and C10), perfluoroalkyl carboxylates (PFCAs; C5C14), and perfluoroalkyl sulfonamides (FOSAs; C8, N-methyl, and N-ethyl). High linearity of matrix-matched calibration standards (correlation coefficients, R = 0.99-0.999) were obtained in the range of 0.006-45 ng mL-1 blood. Excellent sensitivity was achieved with method detection limits (MDLs) between 0.0018 and 0.09 ng mL-1, depending on the compound and matrix. The method was validated for serum, plasma, and whole blood (n = 5 + 5) at six levels in the range 0.0180-30 ng mL-1. The accuracy (n = 5) was on average 102± 12%. The intermediate precision (n = 10) ranged from 2 to 40% with an average between-batch of analyses difference of 10± 10%. Two human serum samples from a former interlaboratory comparison were analyzed and the differences between the applied method and the consensus values were below ≤22% (n = 5). The method was also successfully applied to samples of human plasma and whole blood with coefficients of variation in the range 0.8-15.2% (n = 5).

A novel pretreatment method combining sealing technique with direct injection technique applied for improving biosafety.

AIM: People today have a stronger interest in the risk of biosafety in clinical bioanalysis. A safe, simple, effective method of preparation is needed urgently. METHODOLOGY/RESULTS: To improve biosafety of clinical analysis, we used antiviral drugs of adefovir and tenofovir as model drugs and developed a safe pretreatment method combining sealing technique with direct injection technique. The inter- and intraday precision (RSD %) of the method were <4%, and the extraction recoveries ranged from 99.4 to 100.7%. Meanwhile, the results showed that standard solution could be used to prepare calibration curve instead of spiking plasma, acquiring more accuracy result. CONCLUSION/DISCUSSION: Compared with traditional methods, the novel method not only improved biosecurity of the pretreatment method significantly, but also achieved several advantages including higher precision, favorable sensitivity and satisfactory recovery. With these highly practical and desirable characteristics, the novel method may become a feasible platform in bioanalysis.

Simultaneous determination of pradefovir, PMEA and tenofovir in HBV patient serum using liquid chromatography-tandem mass spectrometry and application to phase 2 clinical trial.

Pradefovir, a prodrug of PMEA, is under phase 2 clinical trial in China to evaluate its pharmacokinetic and pharmacodynamics after multiple-dose study, with adefovir dipivoxil and tenofovir disoproxil fumarate as positive control. A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of pradefovir, PMEA and tenofovir in HBV patient serum. Serum samples were pretreated via simple protein precipitation with methanol and entecavir was used as internal standard. Chromatographic separation was carried out on a Synergi(®) fusion-RP column (150mm×4.6mm) by gradient elution with methanol and 0.1% formic acid in water (v/v) at a flow rate of 1mL/min. The analytes were detected in multiple reaction monitoring mode with positive ion electrospray ionization at m/z 424.1/151.0, 274.1/162.2, 288.1/176.1, and 278.1/152.2for pradefovir, PMEA, tenofovir and IS, respectively. The assays were validated according to current bioanalytical guidelines including specificity, linearity (2.0-500ng/mL for pradefovir and PMEA, 4.0-1000ng/mL for tenofovir), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to the pharmacokinetic study of pradefovir, adefovir dipivoxil and tenofovir disoproxil fumarate in a set of HBV patients.

Development and Validation of a Sensitive LC-MS-MS Method for the Determination of Adefovir in Human Serum and Urine: Application to a Clinical Pharmacokinetic Study.

A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of adefovir (PMEA,9-(2-phosphonylmethoxyethyl) adenine) concentration in human serum and urine. The analysis was performed on a negative ionization electrospray mass spectrometer via multiple reaction monitoring. The monitored transitions were set at m/z 272.0 → 134.0 and m/z 276.0 → 149.8 for PMEA and internal standard, respectively. After protein precipitation, samples were separated by high-performance liquid chromatography on a reversed-phase Dikma Diamonsil C18 (250 × 4.6 mm; 5 µm) column with a mobile phase of 0.1 mM ammonium formate buffer-methanol. The calibration curves were linear over the serum concentration range 0.5-1,000 ng/mL and urine concentration range 2.0-1,000 ng/mL. The intra- and interday precision values of PMEA in both serum and urine were lower than 18.16% for low quality control and 13.70% for medium and high quality control. The accuracy, recovery, matrix factor and stability were also within the acceptable limits. The developed method was successfully applied to the pharmacokinetic study of following oral administration of single dose of pradefovir mesylate (10, 30, 60, 90 and 120 mg) and adefovir dipivoxil (10 mg) to healthy Chinese volunteers.

Effects of 1α,25-Dihydroxyvitamin D3 on Intestinal Absorption and Disposition of Adefovir Dipivoxil and Its Metabolite, Adefovir, in Rats.

The aim of this study was to investigate the effect of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), an active form of vitamin D, on the oral absorption and disposition of adefovir dipivoxil (P-glycoprotein (P-gp) substrate) and its major active metabolite, adefovir (multidrug resistance-associated protein 4 (Mrp4) substrate), in rats. The pharmacokinetics of intravenous adefovir and oral adefovir dipivoxil was evaluated in control and 1,25(OH)2D3-treated rats. The intestinal absorption of adefovir dipivoxil was investigated through an in situ closed loop study, and the tissue distribution of adefovir after oral administration of adefovir dipivoxil was evaluated in the two groups. There was no significant difference in pharmacokinetic parameters of intravenous adefovir between the two groups. Importantly, the total area under the plasma concentration-time curve from time zero to time infinity (AUC), peak plasma concentration (Cmax) and extent of absolute oral bioavailability (F) of adefovir after oral administration of adefovir dipivoxil were significantly higher in 1,25(OH)2D3-treated rats than in control rats. In the in situ closed loop study, there was no significant difference in the remaining fraction of adefovir dipivoxil in the duodenum, jejunum and ileum loops between the two groups. In the tissue distribution study after oral administration of adefovir dipivoxil, the tissue-to-plasma partition coefficients of adefovir in the liver, brain, kidney, and intestine were significantly lower in the 1,25(OH)2D3-treated rats than in control rats. The present study indicates that 1,25(OH)2D3 treatment can enhance the oral absorption of adefovir dipivoxil, likely via the induction of basolateral Mrp4 function in rat intestine. However, the impact of 1,25(OH)2D3 treatment on the pharmacokinetics of intravenous adefovir was limited. These results could lead to further studies in clinically significant P-gp and/or MRP4-mediated 1,25(OH)2D3-drug interactions.

Water-compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine.

A molecularly imprinted polymer has been synthesized to specifically extract adefovir, an antiviral drug, from serum and urine by dispersive solid-phase extraction before high-performance liquid chromatography with UV analysis. The imprinted polymers were prepared by bulk polymerization by a noncovalent imprinting method that involved the use of adefovir (template molecule) and functional monomer (methacrylic acid) complex prior to polymerization, ethylene glycol dimethacrylate as cross-linker, and chloroform as porogen. Molecular recognition properties, binding capacity, and selectivity of the molecularly imprinted polymers were evaluated and the results show that the obtained polymers have high specific retention and enrichment for adefovir in aqueous medium. The new imprinted polymer was utilized as a molecular sorbent for the separation of adefovir from human serum and urine. The serum and urine extraction of adefovir by the molecularly imprinted polymer followed by high-performance liquid chromatography showed a linear calibration curve in the range of 20-100 µg/L with excellent precisions (2.5 and 2.8% for 50 µg/L), respectively. The limit of detection and limit of quantization were determined in serum (7.62 and 15.1 µg/L), and urine (5.45 and 16 µg/L). The recoveries for serum and urine samples were found to be 88.2-93.5 and 84.3-90.2%, respectively.

Tenofovir-based preexposure prophylaxis for HIV infection among African women.

BACKGROUND: Reproductive-age women need effective interventions to prevent the acquisition of human immunodeficiency virus type 1 (HIV-1) infection. METHODS: We conducted a randomized, placebo-controlled trial to assess daily treatment with oral tenofovir disoproxil fumarate (TDF), oral tenofovir-emtricitabine (TDF-FTC), or 1% tenofovir (TFV) vaginal gel as preexposure prophylaxis against HIV-1 infection in women in South Africa, Uganda, and Zimbabwe. HIV-1 testing was performed monthly, and plasma TFV levels were assessed quarterly. RESULTS: Of 12,320 women who were screened, 5029 were enrolled in the study. The rate of retention in the study was 91% during 5509 person-years of follow-up. A total of 312 HIV-1 infections occurred; the incidence of HIV-1 infection was 5.7 per 100 person-years. In the modified intention-to-treat analysis, the effectiveness was -49.0% with TDF (hazard ratio for infection, 1.49; 95% confidence interval [CI], 0.97 to 2.29), -4.4% with TDF-FTC (hazard ratio, 1.04; 95% CI, 0.73 to 1.49), and 14.5% with TFV gel (hazard ratio, 0.85; 95% CI, 0.61 to 1.21). In a random sample, TFV was detected in 30%, 29%, and 25% of available plasma samples from participants randomly assigned to receive TDF, TDF-FTC, and TFV gel, respectively. Independent predictors of TFV detection included being married, being older than 25 years of age, and being multiparous. Detection of TFV in plasma was negatively associated with characteristics predictive of HIV-1 acquisition. Elevations of serum creatinine levels were seen more frequently among participants randomly assigned to receive oral TDF-FTC than among those assigned to receive oral placebo (1.3% vs. 0.2%, P=0.004). We observed no significant differences in the frequencies of other adverse events. CONCLUSIONS: None of the drug regimens we evaluated reduced the rates of HIV-1 acquisition in an intention-to-treat analysis. Adherence to study drugs was low. (Funded by the National Institutes of Health; VOICE ClinicalTrials.gov number, NCT00705679.).

Tenofovir disoproxil fumarate: toxicity, toxicokinetics, and toxicogenomics analysis after 13 weeks of oral administration in mice.

Tenofovir disoproxil fumarate (TDF) is a prodrug of tenofovir that exhibits activity against HIV and hepatitis B. The goals of this study were to evaluate the molecular mechanism of TDF-induced toxicity in mice after 13 weeks of daily oral administration (50-1000 mg/kg) by correlating transcriptional changes with plasma drug levels and traditional toxicology end points. Plasma levels and systemic exposure of tenofovir increased less than dose proportionally and were similar on days 1 and 91. No overt toxicity was observed following the completion of TDF administration. The kidneys of TDF-treated mice were histopathologically normal. This result is consistent with the genomic microarray results, which showed no significant differences in kidney transcriptional levels between TDF-treated animals and controls. In liver, after 4 and 13 weeks, cytomegaly was observed in mice treated with 1000 mg/kg of TDF, but mice recovered from this effect following cessation of administration. Analysis of liver transcripts on day 91 reported elevated levels of Cdkn1a in TDF-treated animals compared with controls, which may have contributed to the inhibition of liver cell cycle progression.