The Effects of Special Patient Population Plasma on Pharmacokinetic Quantifications Using LC-MS/MS.

BACKGROUND: Clinical development of lesinurad, a selective uric acid reabsorption inhibitor, required analysis of lesinurad in plasma from special patient populations. METHODS: EMA and FDA bioanalytical method validation guidance have recommended studying matrix effects on quantitation if samples from special patient populations are to be analyzed. In addition to lesinurad (plasma protein binding 98.2%), the matrix effects from special population plasma on the quantitation of verapamil (PPB 89.6%), allopurinol and oxypurinol (PPB negligible) were also investigated. RESULTS: The plasma from special population patients had no matrix effects on the three quantification methods with stable isotope labeled internal standard, protein precipitation extraction, and LC-MS/MS detection. The validated lesinurad plasma quantification method was successfully applied for the pharmacokinetic evaluations to support the clinical studies in renal impaired patients. CONCLUSION: Special population plasma did not affect quantitation of drugs with a wide range of plasma protein binding levels in human plasma. With the confirmation that there is no impact on quantification from the matrix, the bioanalytical method can be used to support the pharmacokinetic evaluations for clinical studies in special populations.

The Effect of Lesinurad in Combination With Allopurinol on Serum Uric Acid Levels in Patients With Gout.

The objective of the study was to evaluate the effect of lesinurad, a selective uric acid uptake inhibitor, alone and in combination with the xanthine oxidase inhibitor allopurinol, on serum uric acid and urinary urate excretion in patients with gout and hyperuricemia. A phase 1b, multicenter, open-label, multiple-dose study was carried out in patients with gout with serum uric acid ≥8 mg/dL following washout of urate-lowering therapy. Patients were treated with allopurinol 300 mg/day alone in week 1; lesinurad 400 or 600 mg/day was added in week 2, followed by lesinurad 400 or 600 mg/day alone in week 3. Serum uric acid and urine uric acid were evaluated each week. Safety was assessed throughout the study. Lesinurad 400 or 600 mg/day added to allopurinol 300 mg/day reduced serum uric acid by 60% and 72%, respectively, versus allopurinol alone (37%) or lesinurad 400 mg/day (44%) or 600 mg/day (47%) alone. A 100% response rate of serum uric acid <6 mg/dL was achieved by all combinations (serum uric acid <5 mg/dL by 50%-90%). Mean 24-hour urate excretion compared with baseline was -35% with allopurinol, +36% and +56.5% with lesinurad 400 mg/day and 600 mg/day, respectively, and -11.6% and -7.1% with the respective combination therapies. Treatments were well tolerated. In this phase 1 trial, lesinurad added to allopurinol resulted in greater serum uric acid reduction than did allopurinol or lesinurad monotherapy.

Lesinurad, a novel, oral compound for gout, acts to decrease serum uric acid through inhibition of urate transporters in the kidney.

BACKGROUND: Excess body burden of uric acid promotes gout. Diminished renal clearance of uric acid causes hyperuricemia in most patients with gout, and the renal urate transporter (URAT)1 is important for regulation of serum uric acid (sUA) levels. The URAT1 inhibitors probenecid and benzbromarone are used as gout therapies; however, their use is limited by drug-drug interactions and off-target toxicity, respectively. Here, we define the mechanism of action of lesinurad (Zurampic®; RDEA594), a novel URAT1 inhibitor, recently approved in the USA and Europe for treatment of chronic gout. METHODS: sUA levels, fractional excretion of uric acid (FEUA), lesinurad plasma levels, and urinary excretion of lesinurad were measured in healthy volunteers treated with lesinurad. In addition, lesinurad, probenecid, and benzbromarone were compared in vitro for effects on urate transporters and the organic anion transporters (OAT)1 and OAT3, changes in mitochondrial membrane potential, and human peroxisome proliferator-activated receptor gamma (PPARγ) activity. RESULTS: After 6 hours, a single 200-mg dose of lesinurad elevated FEUA 3.6-fold (p < 0.001) and reduced sUA levels by 33 % (p < 0.001). At concentrations achieved in the clinic, lesinurad inhibited activity of URAT1 and OAT4 in vitro, did not inhibit GLUT9, and had no effect on ABCG2. Lesinurad also showed a low risk for mitochondrial toxicity and PPARγ induction compared to benzbromarone. Unlike probenecid, lesinurad did not inhibit OAT1 or OAT3 in the clinical setting. CONCLUSION: The pharmacodynamic effects and in vitro activity of lesinurad are consistent with inhibition of URAT1 and OAT4, major apical transporters for uric acid. Lesinurad also has a favorable selectivity and safety profile, consistent with an important role in sUA-lowering therapy for patients with gout.

In Vitro and In Vivo Interaction Studies Between Lesinurad, a Selective Urate Reabsorption Inhibitor, and Major Liver or Kidney Transporters.

BACKGROUND AND OBJECTIVES: Lesinurad is a selective uric acid reabsorption inhibitor (SURI) under investigation for the treatment of gout. This study elucidated the interaction of lesinurad with major liver and kidney transporters in vitro and evaluated the drug-drug interactions (DDIs) of lesinurad and atorvastatin, metformin, and furosemide in clinical studies. METHODS: Lesinurad interaction with membrane transporters was evaluated in validated transporter-expressing cell systems and analyzed by liquid scintillation counting. Healthy male subjects (ages 18-65 years; body mass index 18-32 kg/m(2)) received atorvastatin (40 mg; n = 28) with or without lesinurad 200 or 400 mg, or received metformin (850 mg; n = 12) or furosemide (40 mg; n = 11) with or without lesinurad 400 mg. Plasma concentrations of each concomitant drug were determined by validated liquid chromatography with tandem mass spectrometry methods. RESULTS: Lesinurad interacted in vitro with OATP1B1, OCT1, and OAT1/3 transporters. Co-administration of lesinurad 200 mg did not significantly alter plasma exposure (maximum concentration [C max] and area under the concentration-time curve [AUC]) of total atorvastatin (atorvastatin + hydroxyl-metabolites) or atorvastatin, while co-administration of lesinurad 400 mg increased the C max of total atorvastatin and atorvastatin by 17-26 %, but had no effect on AUC. Co-administration of lesinurad 400 mg had no effect on the plasma exposure of metformin. Furosemide plasma AUC was reduced by 31 % in the presence of lesinurad 400 mg, but furosemide renal clearance and diuretic activity were unchanged. CONCLUSIONS: No clinically relevant DDIs were observed between lesinurad and substrates of major liver or kidney transporters.

A Phase I Study of the Safety, Pharmacokinetics, and Pharmacodynamics of Combination Therapy with Refametinib plus Sorafenib in Patients with Advanced Cancer.

PURPOSE: To assess the safety and tolerability of the small-molecule allosteric MEK inhibitor refametinib combined with sorafenib, in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: This phase I dose-escalation study included an expansion phase at the maximum tolerated dose (MTD). Patients received refametinib/sorafenib twice daily for 28 days, from a dose of refametinib 5 mg plus sorafenib 200 mg to a dose of refametinib 50 mg plus sorafenib 400 mg. Plasma levels of refametinib, refametinib metabolite M17, and sorafenib were measured for pharmacokinetic assessments. Tumors were biopsied at the MTD for analysis of MEK pathway mutations and ERK phosphorylation. RESULTS: Thirty-two patients were enrolled in the dose-escalation cohort. The MTD was refametinib 50 mg twice daily plus sorafenib 400 mg twice daily. The most common treatment-related toxicities were diarrhea and fatigue. Refametinib was readily absorbed following oral administration (plasma half-life of ∼16 hours at the MTD), and pharmacokinetic parameters displayed near-dose proportionality, with less than 2-fold accumulation after multiple dosing. Another 30 patients were enrolled in the MTD cohort; 19 had hepatocellular carcinoma. The combination was associated with significantly reduced ERK phosphorylation in 5 out of 6 patients biopsied, with the greatest reductions in those with KRAS or BRAF mutations. Disease was stabilized in approximately half of patients, and 1 patient with colorectal cancer achieved a partial response at the MTD lasting approximately 1 year. CONCLUSIONS: In this phase I study, refametinib plus sorafenib was well tolerated, with good oral absorption, near-dose proportionality, and target inhibition in a range of tumor types. Clin Cancer Res; 22(10); 2368-76. ©2015 AACR.

Pharmacokinetics, pharmacodynamics, and safety of lesinurad, a selective uric acid reabsorption inhibitor, in healthy adult males.

Lesinurad is a selective uric acid reabsorption inhibitor under investigation for the treatment of gout. Single and multiple ascending dose studies were conducted to evaluate pharmacokinetics, pharmacodynamics, and safety of lesinurad in healthy males. Lesinurad was administered as an oral solution between 5 mg and 600 mg (single ascending dose; N=34) and as an oral solution or immediate-release capsules once daily (qday) between 100 mg and 400 mg for 10 days under fasted or fed condition (multiple ascending dose; N=32). Following single doses of lesinurad solution, absorption was rapid and exposure (maximum observed plasma concentration and area under the plasma concentration-time curve) increased in a dose-proportional manner. Following multiple qday doses, there was no apparent accumulation of lesinurad. Urinary excretion of unchanged lesinurad was generally between 30% and 40% of dose. Increases in urinary excretion of uric acid and reductions in serum uric acid correlated with dose. Following 400 mg qday dosing, serum uric acid reduction was 35% at 24 hours post-dose, supporting qday dosing. A relative bioavailability study in healthy males (N=8) indicated a nearly identical pharmacokinetic profile following dosing of tablets or capsules. Lesinurad was generally safe and well tolerated.

Pharmacodynamic, pharmacokinetic and tolerability evaluation of concomitant administration of lesinurad and febuxostat in gout patients with hyperuricaemia.

OBJECTIVE: The aim of this study was to evaluate the pharmacodynamics (PDs), pharmacokinetics (PKs) and safety of lesinurad (selective uric acid reabsorption inhibitor) in combination with febuxostat (xanthine oxidase inhibitor) in patients with gout. METHODS: This study was a phase IB, multicentre, open-label, multiple-dose study of gout patients with serum uric acid (sUA) >8 mg/dl following washout of urate-lowering therapy with colchicine flare prophylaxis. Febuxostat 40 or 80 mg/day was administered on days 1-21, lesinurad 400 mg/day was added on days 8-14 and then lesinurad was increased to 600 mg/day on days 15-21. sUA, urine uric acid and PK profiles were evaluated at the end of each week. Safety was assessed by adverse events, laboratory tests and physical examinations. RESULTS: Initial treatment with febuxostat 40 or 80 mg/day monotherapy resulted in 67% and 56% of subjects, respectively, achieving a sUA level <6 mg/dl. Febuxostat 40 or 80 mg/day plus lesinurad 400 or 600 mg/day resulted in 100% of subjects achieving sUA <6 mg/dl and up to 100% achieving sUA <5 mg/dl. No clinically relevant changes in the PKs of either drug were noted. The combination was well tolerated. CONCLUSION: The clinically important targets of sUA <6 mg/dl and <5 mg/dl are achievable in 100% of patients when combining lesinurad and febuxostat.

Multicenter phase I trial of the mitogen-activated protein kinase 1/2 inhibitor BAY 86-9766 in patients with advanced cancer.

PURPOSE: To evaluate the safety, pharmacokinetics, and pharmacodynamics of BAY 86-9766, a selective, potent, orally available, small-molecule allosteric inhibitor of mitogen-activated protein kinase 1/2 in patients with advanced solid tumors. EXPERIMENTAL DESIGN: BAY 86-9766 was administered orally daily in 28-day courses, with doses escalated to establish the maximum-tolerated dose (MTD). An expanded cohort was evaluated at the MTD. Pharmacokinetic and pharmacodynamic parameters were assessed, with extracellular signal-regulated kinase (ERK) phosphorylation evaluated in paired biopsies from a subset of the expanded MTD cohort. Tumor specimens were evaluated for mutations in select genes. RESULTS: Sixty-nine patients were enrolled, including 20 patients at the MTD. The MTD was 100 mg given once-daily or in two divided doses. BAY 86-9766 was well-tolerated. The most common treatment-related toxicities were acneiform rash and gastrointestinal toxicity. BAY 86-9766 was well-absorbed after oral administration (plasma half-life ~12 hours), and displayed dose proportional pharmacokinetics throughout the tested dose range. Continuous daily dosing resulted in moderate accumulation at most dose levels. BAY 86-9766 suppressed ERK phosphorylation in biopsied tissue and tetradecanoylphorbol acetate-stimulated peripheral blood leukocytes. Of 53 evaluable patients, one patient with colorectal cancer achieved a partial response and 11 patients had stable disease for 4 or more courses. An ocular melanoma specimen harbored a GNAQ-activating mutation and exhibited reduced ERK phosphorylation in response to therapy. CONCLUSION: This phase I study showed that BAY 86-9766 was well-tolerated, with good oral absorption, dose proportional pharmacokinetics, target inhibition at the MTD, and some evidence of clinical benefit across a range of tumor types.

6-Benzylamino 4-oxo-1,4-dihydro-1,8-naphthyridines and 4-oxo-1,4-dihydroquinolines as HIV integrase inhibitors.

SAR studies on the quinolone carboxylic acid class of HIV-1 integrase inhibitors focused on improving the metabolic stability and led to the discovery of 27 and 38.

Phase 2a randomized controlled trial of short-term activity, safety, and pharmacokinetics of a novel nonnucleoside reverse transcriptase inhibitor, RDEA806, in HIV-1-positive, antiretroviral-naive subjects.

RDEA806 is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with potent in vitro activity against wild-type and NNRTI-resistant HIV-1. A phase 2a randomized, double-blind, placebo-controlled, dose-escalating study evaluated the short-term antiviral activity, safety, and pharmacokinetics (PKs) of RDEA806 monotherapy in antiretroviral-naïve, HIV-1-infected subjects. The subjects were randomized to four cohorts comprising four dosage regimens and two formulations of RDEA806 or placebo in a 3:1 ratio within each cohort. The investigators were blinded to the results for each cohort. The subjects received RDEA806 or placebo for 7 days. The primary end point was the change in the HIV RNA load from the baseline to day 9 for each of the four RDEA806 dose regimens compared to that achieved with placebo. The RDEA806 PKs and the immune response to RDEA806 were evaluated along with the safety and tolerability of each dose. Of a total of 48 enrolled subjects, 36 subjects (9 in each cohort) were randomized to RDEA806 study drug, and 12 (3 in each cohort) took placebo. A statistically significant decrease in the viral load from the baseline to day 9 was observed for all RDEA806 treatment groups (P<0.001). On day 9, the mean changes in the HIV RNA load from that at the baseline were -1.95 log10 copies/ml (400 mg twice a day), -1.39 log10 copies/ml (600 mg once a day [q.d.]), -1.62 log10 copies/ml (800 mg q.d.), and -1.70 log1) copies/ml (1,000 mg q.d.). The pharmacokinetics were linear and dose proportional. Treatment with RDEA806 was well tolerated, and there were no discontinuations due to adverse events. In conclusion, all doses of RDEA806 were safe and well tolerated and exhibited robust antiretroviral activity in this short-term monotherapy study with antiretroviral-naïve HIV-infected subjects. RDEA806 is a potent and promising novel NNRTI.

RDEA119/BAY 869766: a potent, selective, allosteric inhibitor of MEK1/2 for the treatment of cancer.

The RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway provides numerous opportunities for targeted oncology therapeutics. In particular, the MEK enzyme is attractive due to high selectivity for its target ERK and the central role that activated ERK plays in driving cell proliferation. The structural, pharmacologic, and pharmacokinetic properties of RDEA119/BAY 869766, an allosteric MEK inhibitor, are presented. RDEA119/BAY 869766 is selectively bound directly to an allosteric pocket in the MEK1/2 enzymes. This compound is highly efficacious at inhibiting cell proliferation in several tumor cell lines in vitro. In vivo, RDEA119/BAY 869766 exhibits potent activity in xenograft models of melanoma, colon, and epidermal carcinoma. RDEA119/BAY 869766 exhibits complete suppression of ERK phosphorylation at fully efficacious doses in mice. RDEA119/BAY 869766 shows a tissue selectivity that reduces its potential for central nervous system-related side effects. Using pharmacokinetic and pharmacodynamic data, we show that maintaining adequate MEK inhibition throughout the dosing interval is likely more important than achieving high peak levels because greater efficacy was achieved with more frequent but lower dosing. Based on its longer half-life in humans than in mice, RDEA119/BAY 869766 has the potential for use as a once- or twice-daily oral treatment for cancer. RDEA119/BAY 869766, an exquisitely selective, orally available MEK inhibitor, has been selected for clinical development because of its potency and favorable pharmacokinetic profile.

P450 phenotyping of the metabolism of selegiline to desmethylselegiline and methamphetamine.

Although there is evidence in the literature of the participation of CYP2B6 in the metabolism of selegiline, it is not clear which other CYP isoforms contribute to its metabolism. The aim of this study was to investigate the P450 isozymes (CYPs) involved in the metabolism of selegiline to desmethylselegiline (DMS) and methamphetamine (MA) using four assays: incubation of selegiline with cDNA expressed CYPs, inhibition of DMS and MA formations in human liver microsomes by CYP-selective chemical inhibitors or CYP-specific antibodies, and correlation analysis. Correlation analysis, performed in a bank of 15 individual human liver microsomes, yielded correlation coefficients for DMS and MA formation of 0.769 and 0.792, respectively, for CYP2B6 (p<0.0001) and 0.333 and 0.349, respectively, for CYP3A4 (p<0.05). These results were supported by chemical/specific antibody inhibition assays. The results of correlation analysis and chemical inhibition also indicated that CYP2A6 seems to play a small role in the metabolism of selegiline. These findings confirm that CYP2B6 plays a major role in the metabolism of selegiline and also suggest the involvement of CYP3A4 and CYP2A6.

Determination of N-acetyl retigabine in dog plasma by LC/MS/MS following off-line microElution 96-well solid phase extraction.

A high throughput off-line microElution 96-well solid phase extraction (SPE) followed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for the determination of N-acetyl retigabine in dog plasma has been developed and validated. The method involves the use of microElution 96-well SPE for the simultaneous extraction of N-acetyl retigabine and rapid removal of its N-glucuronide metabolite that has shown to be problematic due to its instability using other clean-up methods. The microElution SPE technology eliminates the need for post-extraction solvent evaporation and greatly reduces sample preparation time consequently improving assay efficiency.

Cyclic monophosphate prodrugs of base-modified 2'-C-methyl ribonucleosides as potent inhibitors of hepatitis C virus RNA replication.

A new series of heterobase-modified 2'-C-methyl ribonucleosides was synthesized and tested as inhibitors of hepatitis C virus (HCV) RNA replication. The nucleosides showed a weak inhibitory activity in a HCV replicon system (EC(50)=92 microM) and did not exhibit any cytotoxicity (CC(50)>300 microM). Cyclic monophosphate (cMP) prodrugs of the same nucleosides were synthesized and also tested in the HCV replicon system. Prodrugs exhibited strong potency (EC(50)=0.008 microM) without significant cytotoxicity (CC(50)>50 microM).

A novel nonnucleoside analogue that inhibits human immunodeficiency virus type 1 isolates resistant to current nonnucleoside reverse transcriptase inhibitors.

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance, and serious side effects can compromise the benefits of the two current drugs in this class (efavirenz and nevirapine). In this study, we report a novel and potent NNRTI, VRX-480773, that inhibits viruses from efavirenz-resistant molecular clones and most NNRTI-resistant clinical HIV-1 isolates tested. In vitro mutation selection experiments revealed that longer times were required for viruses to develop resistance to VRX-480773 than to efavirenz. RT mutations selected by VRX-480773 after 3 months of cell culture in the presence of 1 nM VRX-480773 carried the Y181C mutation, resulting in a less-than-twofold increase in resistance to the compound. A virus containing the double mutation V106I-Y181C emerged after 4 months, causing a sixfold increase in resistance. Viruses containing additional mutations of D123G, F227L, and T369I emerged when the cultures were incubated with increasing concentrations of VRX-480773. Most of the resistant viruses selected by VRX-480773 are susceptible to efavirenz. Oral administration of VRX-480773 to dogs resulted in plasma concentrations that were significantly higher than those required for the inhibition of wild-type and mutant viruses. These results warrant further clinical development of VRX-480773 for the treatment of HIV infection in both NNRTI-naive and -experienced patients.

LC-MS/MS method for simultaneous determination of viramidine and ribavirin levels in monkey red blood cells.

A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determinations of total viramidine (viramidine, viramidine monophosphate, viramidine diphosphate, and viramidine triphosphate) and total ribavirin (ribavirin, ribavirin monophosphate, ribavirin diphosphate, and ribavirin triphosphate) in monkey red blood cells (RBC). The method involves the addition of internal standards and perchloric acid, conversion of viramidine or ribavirin phosphorylated metabolites to viramidine or ribavirin, purification with an aminopropyl (NH(2)) solid phase extraction (SPE) cartridge, and LC-MS/MS analysis. The MS/MS is selected to monitor m/z 245-->113, 250-->113, 244-->112, and 249-->112 for ribavirin, [(13)C]ribavirin, viramidine, and [(13)C]viramidine, respectively, using positive electrospray ionization. The calibration curves are linear over a concentration range of 100-10,000 ng/mL (0.412-41.2 microM) with a lower limit of quantification (LLOQ) of 100 ng/mL for both compounds. Mean inter-assay recoveries for ribavirin are 101%, 98.9%, and 96.0%, with coefficient of variance (%CV) values between 1.95 and 4.50% for 100, 1000, and 10,000 ng/mL quality control (QC) samples, respectively. Mean inter-assay recoveries for viramidine are 96.3%, 101%, and 102%, with coefficient of variation (%CV) values between 3.61 and 7.22%, for 100, 1000, and 10,000 ng/mL QC samples, respectively. Over-curve dilution QC at 400 microg/mL (1639 microM) for both viramidine and ribavirin are used to ensure the dilution accuracy (25 X dilutions) for monkey samples. The method has been used to simultaneously determine the total concentrations of ribavirin and viramidine in monkey RBC following 5, 15, and 36 weeks dosing of viramidine or ribavirin (60 mg/kg). The concentrations of total ribavirin following ribavirin dosing are 1242 microM at week 5, 1257 microM at week 15, and 1146 microM at week 36. The concentrations of total ribavirin following viramidine dosing are 634 microM at week 5, 716 microM at week 15, and 683 microM at week 36. Only small amounts of viramidine are detected in RBC following viramidine dosing, 7.80 microM at week 5, 6.63 microM at week 15, and 10.4 microM at week 36. The results suggest that ribavirin levels in RBC were at steady state at week 5 of ribavirin or viramidine dosing. At steady state, ribavirin levels in RBC are approximately 2x after ribavirin dosing than viramidine dosing. The relatively small percentage of viramidine in RBC suggests that viramidine either poorly penetrated into RBC or was extensively converted to ribavirin following entry into RBC.

Metabolic activation of pradefovir by CYP3A4 and its potential as an inhibitor or inducer.

Metabolic activation of pradefovir to 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was evaluated by using cDNA-expressed CYP isozymes in portal vein-cannulated rats following oral administration and in human liver microsomes. The enzyme induction potential of pradefovir was evaluated in rats following multiple oral dosing and in primary cultures of human hepatocytes. The results indicated that CYP3A4 is the only cDNA-expressed CYP isozyme catalyzing the conversion of pradefovir to PMEA. Pradefovir was converted to PMEA in human liver microsomes with a K(m) of 60 microM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 359 ml/min. Addition of ketoconazole and monoclonal antibody 3A4 significantly inhibits the conversion of pradefovir to PMEA in human liver microsomes, suggesting the predominant role of CYP3A4 in the metabolic activation of pradefovir. Pradefovir at 0.2, 2, and 20 microM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes. In rats, the liver was the site of metabolic activation of pradefovir, whereas the small intestine did not play a significant role in the metabolic conversion of pradefovir to PMEA. Daily oral dosing (300 mg/kg of body weight) to rats for 8 days showed that pradefovir was not an inducer of P450 enzymes in rats. Furthermore, pradefovir at 10 microg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.

Absorption, metabolism, and excretion of [14C]viramidine in humans.

Absorption, metabolism, and excretion of [14C]viramidine, a prodrug of ribavirin, were studied in humans following a single oral dose (600 mg). Viramidine was rapidly absorbed, with a time to maximum concentration of the drug in plasma of 1.5 h. Viramidine and ribavirin accounted for only 4.3% and 42% of plasma area under the concentration-time curve (AUC) for radioactivity, respectively, indicating extensive conversion of viramidine to ribavirin, followed by further metabolism of ribavirin. The drug was largely trapped in red blood cells (RBC), with an RBC-to-plasma radioactivity AUC0-infinity ratio of 108. Excretion of total radioactivity in urine and feces accounted for 50.8% and 26.1% of the dose, respectively. The metabolic profile in urine (0 to 24 h) indicated that viramidine was excreted primarily as triazole carboxamide (TCONH2), triazole carboxylic acid nucleoside (TCOOH), and ribavirin with a small amount of unchanged viramidine, which each accounted for 64.1%, 17.0%, 15.7%, and 3.2% of urinary radioactivity, respectively. The amounts of unchanged viramidine (3.4% of dose) and ribavirin (10% of dose) in urine were small after oral administration of viramidine.

Conversion of viramidine to ribavirin in vivo by adenosine deaminase and its inhibition by 2'-deoxycoformycin.

Previously we reported that viramidine is a prodrug of ribavirin and that adenosine deaminase catalyses viramidine deamination to ribavirin in vivo. This in vivo study explores this prodrug conversion in rats and inhibition by a potent adenosine deaminase inhibitor, 2'-deoxycoformycin. We found that conversion of viramidine to ribavirin was viramidine dose-dependent in rat plasma. A single intravenous dose of 0.25 mg/kg 2'-deoxycoformycin suppressed orally administered viramidine conversion to ribavirin in plasma by 50%. The inhibition was 2'-deoxycoformycin dose-dependent and a single dose of 2 mg/kg decreased the ribavirin/viramidine area under the concentration-time curve between 0 h and 6 h ratio by 2.5-fold. These findings provide strong evidence that adenosine deaminase plays a major role in converting viramidine to ribavirin in vivo.