Evaluation of the single-dose pharmacokinetics and pharmacodynamics of apixaban in healthy Japanese and Caucasian subjects.

PURPOSE: This double-blind, placebo-controlled, intra-subject, dose-escalation study assessed single-dose safety, pharmacokinetics, and pharmacodynamics of apixaban in healthy Japanese and Caucasian subjects. SUBJECTS AND METHODS: Sixteen healthy male Japanese and sixteen healthy male Caucasian subjects, matched for age, weight, and smoking status were randomized to receive four sequential single oral doses of either apixaban (2.5, 10, 25, and 50 mg) or matched placebo. Doses were separated by a ≥5-day washout. Blood samples were collected for the determination of apixaban plasma concentration, clotting times (international normalized ratio [INR], activated partial thromboplastin time, and modified prothrombin time [mPT]), and ex vivo thrombin generation (TG). Urine samples were collected for the analysis of apixaban concentration. RESULTS: Ascending single doses of apixaban 2.5-50 mg were safe and well tolerated by all subjects. Apixaban exposure increased the dose proportionally up to 10 mg. Apixaban reached maximum concentrations (C max) 3-4 h postdose, with mean C max ranging from 52.5-485.0 to 44.8-494.3 ng/mL in Japanese and Caucasian subjects. The mean half-life was ~8 and ~13 h and the renal clearance was 1.1 and 0.8 L/h in Japanese and Caucasian subjects, respectively. Pharmacodynamic assessments were similar between ethnic groups, with comparable dose-related prolongation of INR and mPT and inhibition of TG. CONCLUSION: Ascending single doses of apixaban over a 20-fold dose range were safe and well tolerated in Japanese and Caucasian subjects in this study. The consistency between pharmacokinetic and pharmacodynamic measures in Japanese and Caucasian subjects indicates that apixaban may be administered as a fixed dose with no need for adjustment in Japanese patients.

Effect of renal impairment on the pharmacokinetics, pharmacodynamics, and safety of apixaban.

This open-label study evaluated apixaban pharmacokinetics, pharmacodynamics, and safety in subjects with mild, moderate, or severe renal impairment and in healthy subjects following a single 10-mg oral dose. The primary analysis determined the relationship between apixaban AUC∞ and 24-hour creatinine clearance (CLcr ) as a measure of renal function. The relationships between 24-hour CLcr and iohexol clearance, estimated CLcr (Cockcroft-Gault equation), and estimated glomerular filtration rate (modification of diet in renal disease [MDRD] equation) were also assessed. Secondary objectives included assessment of safety and tolerability as well as international normalized ratio (INR) and anti-factor Xa activity as pharmacodynamic endpoints. The regression analysis showed that decreasing renal function resulted in modestly increased apixaban exposure (AUC∞ increased by 44% in severe impairment with a 24-hour CLcr of 15 mL/min, compared with subjects with normal renal function), but it did not affect Cmax or the direct relationship between apixaban plasma concentration and anti-factor Xa activity or INR. The assessment of renal function measured by iohexol clearance, Cockcroft-Gault, and MDRD was consistent with that determined by 24-hour CLcr . Apixaban was well tolerated in this study. These results suggest that dose adjustment of apixaban is not required on the basis of renal function alone.

Pharmacokinetics, pharmacodynamics, and safety of apixaban in subjects with end-stage renal disease on hemodialysis.

An open-label, parallel-group, single-dose study was conducted to assess the pharmacokinetics, pharmacodynamics, and safety of apixaban in 8 subjects with end-stage renal disease (ESRD) on hemodialysis compared with 8 subjects with normal renal function. A single oral 5-mg dose of apixaban was administered once to healthy subjects and twice to subjects with ESRD, separated by ≥7 days: 2 hours before (on hemodialysis) and immediately after a 4-hour hemodialysis session (off hemodialysis). Blood samples were collected for determination of apixaban pharmacokinetic parameters, measures of clotting (prothrombin time, international normalized ratio, activated partial thromboplastin time), and anti-factor Xa (FXa) activity. Compared with healthy subjects, apixaban Cmax and AUCinf were 10% lower and 36% higher, respectively, in subjects with ESRD off hemodialysis. Hemodialysis in subjects with ESRD was associated with reductions in apixaban Cmax and AUCinf of 13% and 14%, respectively. The percent change from baseline in clotting measures was similar in healthy subjects and subjects with ESRD, and differences in anti-FXa activity were similar to differences in apixaban concentration. A single 5-mg oral dose of apixaban was well tolerated in both groups. In conclusion, ESRD resulted in a modest increase (36%) in apixaban AUC and no increase in Cmax , and hemodialysis had a limited impact on apixaban clearance.

Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma.

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

A randomized direct comparison of the pharmacokinetics and pharmacodynamics of apixaban and rivaroxaban.

BACKGROUND: Currently, there are no direct comparisons of apixaban and rivaroxaban, two new oral direct factor Xa inhibitors approved for management of thromboembolic disorders. OBJECTIVE: Compare the pharmacokinetics and anti-factor Xa activity (AXA) of apixaban and rivaroxaban. METHODS: In this randomized, open-label, two-period, two-treatment crossover study, healthy subjects (N=14) received apixaban 2.5 mg twice daily (BID) and rivaroxaban 10 mg once daily (QD) for 4 days with a ≥4.5-day washout. Plasma samples were obtained for pharmacokinetic and AXA assessments; parameters were calculated using noncompartmental methods. RESULTS: Median time-to-maximum concentration was 2 hours for both compounds, and the mean half-life was 8.7 and 7.9 hours for apixaban and rivaroxaban, respectively. Daily exposure, the area under the curve (AUC(0-24)), appeared similar for rivaroxaban (1,094 ng · h/mL) and apixaban (935 ng · h/mL), whereas mean peak-to-trough plasma concentration ratio was 3.6-fold greater for rivaroxaban (16.9) than apixaban (4.7). Coefficient of variation for exposure parameters (AUC0-24, Cmax, Cmin) was 20%-24% for apixaban versus 29%-46% for rivaroxaban. Peak AXA, AXA AUC(0-24), and AXA fluctuation were ~2.5-, 1.3-, and 3.5-fold higher for rivaroxaban than apixaban, respectively. Trough concentrations and AXA were lower for rivaroxaban (10 ng/mL and 0.17 IU/mL vs 17 ng/mL and 0.24 IU/mL for apixaban, respectively). Rivaroxaban exhibited a steeper concentration-AXA response (slope: 0.0172 IU/ng vs 0.0134 IU/ng for apixaban, P<0.0001). CONCLUSION: Apixaban 2.5 mg BID demonstrated less intersubject variability in exposure, lower AXA AUC, and higher trough and smaller peak-to-trough fluctuations in plasma concentration and AXA, suggesting more constant anticoagulation compared with rivaroxaban 10 mg QD. However, the clinical impact of these differences on the relative efficacy and safety of apixaban and rivaroxaban remains to be determined.

LC-MS/MS determination of apixaban (BMS-562247) and its major metabolite in human plasma: an application of polarity switching and monolithic HPLC column.

BACKGROUND: apixaban (BMS-562247) (Eliquis(®)) is a novel, orally active, selective, direct, reversible inhibitor of the coagulation factor Xa (FXa). A sensitive and reliable method was developed and validated for the measurement of apixaban (BMS-562247) and its major circulating metabolite (BMS-730823) in human citrated plasma for use in clinical testing. METHODOLOGY/RESULTS: A 0.100 ml portion of citrated plasma sample was extracted and analyzed by LC-MS/MS. Run times were approximately 3 min. The lower limit of quantification (LLOQ) was 1.00 ng/ml for BMS-562247 and 5.00 ng/ml for BMS-730823. Intra- and inter-assay precision values for replicate QC control samples were within ≤5.36% for both analytes (≤7.52% at the LLOQ). The accuracy for both analytes was within ±9.00%. CONCLUSION: The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple doses of apixaban in healthy Japanese male subjects.

OBJECTIVE: This was a randomized, placebo-controlled, double-blind, sequential, ascending-dose study to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple oral doses of apixaban in healthy Japanese male subjects. METHODS: The study was conducted using three sequential dose panels: apixaban 2.5 mg, 5 mg, and 10 mg given twice daily. For each dose panel, subjects were randomly assigned to receive oral apixaban (n = 6) or matching placebo (n = 2) for 7 days. The pharmacokinetics of apixaban and effect on pharmacodynamic variables (clotting assays and anti-Xa activity) were assessed on day 1 and day 7 of treatment. Safety was assessed throughout the study. Only after the preceding dose was confirmed to be safe and well-tolerated subjects were enrolled into the next-higher-dose panel. RESULTS: Apixaban was safe and well-tolerated in these healthy Japanese male subjects across the doses evaluated. On day 7, peak plasma concentrations were reached ~ 3 hours postdose, and increases in peak plasma concentration (C(max)), trough plasma concentration, and area under the plasma concentration-time curve across one dosing interval (12 hours) were tested dose-proportional across the dose range. A modest degree of accumulation was observed that was similar for all doses (accumulation index of 1.7 to 2.0), and renal clearance was consistent across doses (0.91 L/h - 1.07 L/h). Exposure-dependent prolongation of prothrombin time, activated partial thromboplastin time, modified prothrombin time, and increases in anti-Xa activity were observed after single and multiple doses of apixaban. CONCLUSIONS: Apixaban was safe and well-tolerated in healthy Japanese subjects. The pharmacokinetic profile of apixaban following multiple twice-daily doses was linear, and exposure parameters such as C(max), observed at ~ 3 hours post-dose, and area under the plasma concentration-time curve increased in a dose-proportional manner. Pharmacodynamic profiles closely followed the apixaban plasma concentration-time profiles.

Evaluation of the effect of naproxen on the pharmacokinetics and pharmacodynamics of apixaban.

AIM: To assess pharmacokinetic and pharmacodynamic interactions between naproxen (a non-steroidal anti-inflammatory drug) and apixaban (an oral, selective, direct factor-Xa inhibitor). METHOD: In this randomized, three period, two sequence study, 21 healthy subjects received a single oral dose of apixaban 10 mg, naproxen 500 mg or co-administration of both. Blood samples were collected for determination of apixaban and naproxen pharmacokinetics and pharmacodynamics (anti-Xa activity, international normalized ratio [INR] and arachidonic acid-induced platelet aggregation [AAI-PA]). Adverse events, bleeding time and routine safety assessments were also evaluated. RESULTS: Apixaban had no effect on naproxen pharmacokinetics. However, following co-administration, apixaban AUC(0,∞), AUC(0,t) and Cmax were 54% (geometric mean ratio 1.537; 90% confidence interval (CI) 1.394, 1.694), 55% (1.549; 90% CI 1.400, 1.713) and 61% (1.611; 90% CI 1.417, 1.831) higher, respectively. Mean (standard deviation [SD]) anti-Xa activity at 3 h post-dose was approximately 60% higher following co-administration compared with apixaban alone, 4.4 [1.0] vs. 2.7 [0.7] IU ml(-1) , consistent with the apixaban concentration increase following co-administration. INR was within the normal reference range after all treatments. AAI-PA was reduced by approximately 80% with naproxen. Co-administration had no impact beyond that of naproxen. Mean [SD] bleeding time was higher following co-administration (9.1 [4.1] min) compared with either agent alone (5.8 [2.3] and 6.9 [2.6] min for apixaban and naproxen, respectively). CONCLUSION: Co-administration of naproxen with apixaban results in higher apixaban exposure and appears to occur through increased apixaban bioavailability. The effects on anti-Xa activity, INR and inhibition of AAI-PA observed in this study were consistent with the individual pharmacologic effects of apixaban and naproxen.

Pharmacokinetics, pharmacodynamics, and immunogenicity of belatacept in adult kidney transplant recipients.

BACKGROUND AND OBJECTIVES: Belatacept is a first-in-class, selective co-stimulation blocker recently approved for the prophylaxis of organ rejection in adult kidney transplant recipients. The objective of this study was to report the pharmacokinetics, pharmacodynamics, and immunogenicity of belatacept. METHODS: The pharmacokinetics, pharmacodynamics (CD86 receptor occupancy), and immunogenicity of belatacept were studied in de novo adult kidney transplant recipients in phase II and III clinical studies. RESULTS: Following multiple doses of 5 or 10 mg/kg, the geometric mean (percentage coefficient of variation) maximum serum concentration and area under the serum concentration-time curve over one dosing interval of belatacept were 136 (20%) and 238 (27%) µg/mL, and 13,587 (27%) and 21,241 (35%) µg·h/mL, respectively. The median belatacept elimination half-life was 8-9 days. Belatacept exhibited concentration-dependent binding to CD86 receptors. The pre-dose CD86 receptor occupancy by belatacept decreased from 94 to 65% between day 5 and 1 year post-transplant, with corresponding pre-dose trough serum concentrations of belatacept decreasing from ~35 to 4 µg/mL during this period. The cumulative incidence of developing anti-belatacept antibodies was 5.3% up to 3 years post-transplant and had no impact on belatacept exposure. CONCLUSIONS: Belatacept in adult kidney transplant demonstrated linear pharmacokinetics with low variability, concentration-dependent pharmacodynamics, and a low incidence of anti-drug antibodies.

Effect of famotidine on the pharmacokinetics of apixaban, an oral direct factor Xa inhibitor.

BACKGROUND: Apixaban is an oral, selective, direct factor Xa inhibitor approved for thromboprophylaxis after orthopedic surgery and stroke prevention in patients with atrial fibrillation, and under development for treatment of venous thromboembolism. This study investigated the effect of a gastric acid suppressant, famotidine (a histamine H2-receptor antagonist), on the pharmacokinetics of apixaban in healthy subjects. METHODS: This two-period, two-treatment crossover study randomized 18 healthy subjects to receive a single oral dose of apixaban 10 mg with and without a single oral dose of famotidine 40 mg administered 3 hours before dosing with apixaban. Plasma apixaban concentrations were measured up to 60 hours post-dose and pharmacokinetic parameters were calculated. RESULTS: Famotidine did not affect maximum apixaban plasma concentration (Cmax) or area under the plasma concentration-time curve from zero to infinite time (AUC∞). Point estimates for ratios of geometric means with and without famotidine were close to unity for Cmax (0.978) and AUC∞ (1.007), and 90% confidence intervals were entirely contained within the 80%-125% no-effect interval. Administration of apixaban alone and with famotidine was well tolerated. CONCLUSION: Famotidine does not affect the pharmacokinetics of apixaban, consistent with the physicochemical properties of apixaban (lack of an ionizable group and pH-independent solubility). Apixaban pharmacokinetics would not be affected by an increase in gastrointestinal pH due to underlying conditions (eg, achlorhydria), or by gastrointestinal pH-mediated effects of other histamine H2-receptor antagonists, antacids, or proton pump inhibitors. Given that famotidine is also an inhibitor of the human organic cation transporter (hOCT), these results indicate that apixaban pharmacokinetics are not influenced by hOCT uptake transporter inhibitors. Overall, these results support that apixaban can be administered without regard to coadministration of gastric acid modifiers.

Effect of extremes of body weight on the pharmacokinetics, pharmacodynamics, safety and tolerability of apixaban in healthy subjects.

AIM: Apixaban is an oral, direct, factor-Xa inhibitor approved for thromboprophylaxis in patients who have undergone elective hip or knee replacement surgery and for prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation. This open label, parallel group study investigated effects of extremes of body weight on apixaban pharmacokinetics, pharmacodynamics, safety and tolerability. METHOD: Fifty-four healthy subjects were enrolled [18 each into low (≤50 kg), reference (65-85 kg) and high (≥120 kg) body weight groups]. Following administration of a single oral dose of 10 mg apixaban, plasma and urine samples were collected for determination of apixaban pharmacokinetics and anti-factor Xa activity. Adverse events, vital signs and laboratory assessments were monitored. RESULTS: Compared with the reference body weight group, low body weight had approximately 27% [90% confidence interval (CI): 8-51%] and 20% (90% CI: 11-42%) higher apixaban maximum observed plasma concentration (Cmax) and area under the concentration-time curve extrapolated to infinity (AUC(0,∞)), respectively, and high body weight had approximately 31% (90% CI: 18-41%) and 23% (90% CI: 9-35%) lower apixaban Cmax and AUC(0,∞) , respectively. Apixaban renal clearance was similar across the weight groups. Plasma anti-factor Xa activity showed a direct, linear relationship with apixaban plasma concentration, regardless of body weight group. Apixaban was well tolerated in this study. CONCLUSION: The modest change in apixaban exposure is unlikely to require dose adjustment for apixaban based on body weight alone. However, caution is warranted in the presence of additional factors (such as severe renal impairment) that could increase apixaban exposure.

Metabolic chiral inversion of brivanib and its relevance to safety and pharmacology.

Brivanib alaninate is an orally administered alanine prodrug of brivanib, a dual inhibitor of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. It is currently in clinical trials for the treatment of hepatocellular carcinoma and colorectal cancer. Brivanib has a single asymmetric center derived from a secondary alcohol. The potential for chiral inversion was investigated in incubations with liver subcellular fractions and in animals and humans after oral doses of brivanib alaninate. Incubations of [¹4C]brivanib alaninate with liver microsomes and cytosols from rats, monkeys, and humans followed by chiral chromatography resulted in two radioactive peaks, corresponding to brivanib and its enantiomer. The percentage of the enantiomeric metabolite relative to brivanib in microsomal and cytosolic incubations of different species in the presence of NADPH ranged from 11.6 to 15.8 and 0.8 to 3.1%, respectively. The proposed mechanism of inversion involves the oxidation of brivanib to a ketone metabolite, which is subsequently reduced to brivanib and its enantiomer. After oral doses of brivanib alaninate to rats and monkeys, the enantiomeric metabolite was a prominent drug-related component in plasma, with the percentages of area under the curve (AUC) at 94.7 and 39.7%, respectively, relative to brivanib. In humans, the enantiomeric metabolite was a minor circulating component, with the AUC <3% of brivanib. Pharmacological studies indicated that brivanib and its enantiomer had similar potency toward the inhibition of VEGF receptor-2 and FGF receptor-1 kinases. Because of low plasma concentration in humans, the enantiomeric metabolite was not expected to contribute significantly to target-related pharmacology of brivanib. Moreover, adequate exposure in the toxicology species suggested no specific safety concerns with respect to exposure to the enantiomeric metabolite.

A randomised assessment of the pharmacokinetic, pharmacodynamic and safety interaction between apixaban and enoxaparin in healthy subjects.

Following major orthopaedic surgery, guidelines usually recommend continued thromboprophylaxis after hospitalisation. The availability of an effective oral anticoagulant with an acceptable safety profile that does not require routine clinical monitoring may lead clinicians to switch patients from subcutaneous to an oral therapy either during hospitalisation or at discharge. The purpose of this study was to assess the effect of enoxaparin on the pharmacokinetics, pharmacodynamics and safety of apixaban, an oral, direct inhibitor of coagulation factor Xa. In this four-period, crossover study, 20 healthy subjects were randomised to receive single doses of apixaban 5 mg orally; enoxaparin 40 mg subcutaneously; apixaban 5 mg and enoxaparin 40 mg concomitantly; and apixaban 5 mg followed 6 hours (h) after by enoxaparin 40 mg. Pharmacokinetics of apixaban were not affected by enoxaparin. Average peak pharmacodynamic effect, measured by anti-Xa activity, was 1.36 U/ml after administration of apixaban and was 0.42 U/ml after enoxaparin. Following co-administration of apixaban and enoxaparin, peak anti-Xa activity was 42% higher than for apixaban alone. Following administration of enoxaparin 6 h after apixaban, peak anti-Xa activity was 15% higher than for apixaban alone. In conclusion, enoxaparin had no effect on the pharmacokinetics of apixaban. The increase in anti-Xa activity after co-administration was modest and appeared to be additive. Peak anti-Xa activity increases are mitigated by separating administration of subcutaneous anticoagulation and apixaban when switching between therapies; the potential for pharmacodynamic interaction may be further mitigated by transitioning at the next scheduled dose (12 h).

Metabolism and disposition of [14C]brivanib alaninate after oral administration to rats, monkeys, and humans.

Brivanib [(R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[1,2,4]triazin-6-yloxy)propan-2-ol, BMS-540215] is a potent and selective dual inhibitor of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. Its alanine prodrug, brivanib alaninate [(1R,2S)-2-aminopropionic acid 2-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-1-methylethyl ester, BMS-582664], is currently under development as an oral agent for the treatment of cancer. This study describes the in vivo biotransformation of brivanib after a single oral dose of [(14)C]brivanib alaninate to intact rats, bile duct-cannulated (BDC) rats, intact monkeys, BDC monkeys, and humans. Fecal excretion was the primary route of elimination of drug-derived radioactivity in animals and humans. In BDC rats and monkeys, the majority of radioactivity was excreted in bile. Brivanib alaninate was rapidly and completely converted via hydrolysis to brivanib in vivo. The area under the curve from zero to infinity of brivanib accounted for 14.2 to 54.3% of circulating radioactivity in plasma in animals and humans, suggesting that metabolites contributed significantly to the total drug-related radioactivity. In plasma from animals and humans, brivanib was a prominent circulating component. All the metabolites that humans were exposed to were also present in toxicological species. On the basis of metabolite exposure and activity against VEGF and FGF receptors of the prominent human circulating metabolites, only brivanib is expected to contribute to the pharmacological effects in humans. Unchanged brivanib was not detected in urine or bile samples, suggesting that metabolic clearance was the primary route of elimination. The primary metabolic pathways were oxidative and conjugative metabolism of brivanib.

Metabolism, excretion, and pharmacokinetics of oral brivanib in patients with advanced or metastatic solid tumors.

The goal of this study was to evaluate the pharmacokinetics, mass balance, metabolism, routes and extent of elimination, and safety of a single oral dose of (14)C-labeled brivanib alaninate and the safety and tolerability of brivanib after multiple doses in patients with advanced or metastatic solid tumors. This was a two-part, single-center, open-label, single oral-dose (part A) followed by multiple-dose (part B) study in patients with advanced or metastatic solid tumors. In part A, patients received a single dose of [(14)C]brivanib alaninate and in part B patients received 800 mg of nonradiolabeled brivanib alaninate every day. Four patients (two white, two black: two with non-small-cell lung cancer, one with ovarian cancer, and one with renal cell carcinoma) were treated in both parts. The median time to reach the maximal plasma concentration of brivanib was 1 h, geometric mean maximal plasma concentration was 6146 ng/ml, mean terminal half-life was 13.8 h, and geometric mean apparent oral clearance was 14.7 l/h. After a single oral dose of [(14)C]brivanib alaninate, 12.2 and 81.5% of administered radioactivity was recovered in urine and feces, respectively. Brivanib alaninate was completely converted to the active moiety, brivanib, and the predominant route of elimination was fecal. Renal excretion of unchanged brivanib was minimal. Brivanib was well tolerated; fatigue was the most frequent adverse event occurring in all patients and the most frequent treatment-related adverse event in three (75%). The best clinical response in one patient was stable disease; the other three had progressive disease. Brivanib alaninate was rapidly absorbed and extensively metabolized after a single 800-mg oral dose; the majority of drug-related radioactivity was excreted in feces.

Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry.

BMS-299897 is a gamma-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid-liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized (13)C6-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm x 50 mm, 3 microm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25-100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within +/-7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.

A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of muraglitazar, a novel diabetes drug, in human plasma.

A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of muraglitazar, a PPAR alpha/gamma dual agonist, in human plasma. The internal standard, a chemical analogue, was dissolved in acetonitrile containing 0.1% formic acid. The solvent system was also served as a protein precipitation reagent. Human plasma samples (0.1 mL) and the internal standard solution (0.3 mL) were added to a 96-well plate. The plate was vortexed for 1 min and centrifuged for 5 min. Then the supernatant layers were directly injected into the LC/MS/MS system. The chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm x 50 mm, 5 microm). The mobile phase contained 20/80 (v/v) of water and acetonitrile containing 0.1% formic acid. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 3000. The standard curve, which ranged from 1 to 1000 ng/mL, was fitted to a 1/x weighted quadratic regression model. This single-pot approach effectively eliminated three time consuming sample preparation steps: sample transfer, dry-down, and reconstitution before the injection, while it preserved all the benefits of the traditional protein precipitation. By properly adjusting the autosampler needle offset level, only the supernatant was injected, without disturbing the precipitated proteins in the bottom. As a result, the quality of chromatography and column life were not compromised. After more than 600 injections, there was only slightly increase of column back-pressure. The validation results demonstrated that this method was rugged and provide satisfactory precision and accuracy. The method has been successfully applied to analyze human plasma samples in support of a first-in-man study. This method has also been validated in monkey and mouse plasma for the determination of muraglitazar.

A simple 96-well liquid-liquid extraction with a mixture of acetonitrile and methyl t-butyl ether for the determination of a drug in human plasma by high-performance liquid chromatography with tandem mass spectrometry.

A simple 96-well plate liquid-liquid extraction (LLE), liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a basic drug candidate in human plasma. Against the wisdom of conventional approaches, an aqueous/organic miscible solvent, acetonitrile, was used for liquid-liquid extraction along with methyl t-butyl ether. The use of acetonitrile effectively eliminated the formation of the irregular emulsion between aqueous/organic interfaces and modulated the polarity of the extraction solvents to achieve the desired recovery. This approach, which solved the emulsion problem, permitted the method to be automated using standard 96-well plate technology. A practical application was demonstrated through the use of this technique in the measurement of a novel drug in human plasma samples by LC/MS/MS. Chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm x 50 mm, 5 microm). The mobile phase contained 60% of 0.1% formic acid and 40% acetonitrile. Detection was by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 1.22 to 979ng/ml, was fitted to a 1/x2 weighted quadratic regression model. The validation results show that this method was very rugged and had excellent precision and accuracy. The actual sample analysis results further demonstrated that this extraction procedure is well suited for real life applications. It is expected that with some modifications, this approach can be applied for the extraction of similar compounds from various biological fluids.

Quantitative determination of pioglitazone in human serum by direct-injection high-performance liquid chromatography mass spectrometry and its application to a bioequivalence study.

A simple, high throughput, direct-injection high-performance liquid chromatography tandem mass spectrometry method (LC/MS/MS) has been developed and validated for the quantitation of pioglitazone in human serum. After mixing the internal standard with a sample, a 10 microl portion of the mixture was directly injected into a high-flow LC/MS/MS system, which included an extraction column, an analytical column and a six-port switching valve. The on-line extraction was achieved on an Oasis HLB column (1 mm x 50 mm, 30 microm) with a 100% aqueous loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a flow rate of 4 ml/min. The extracted analyte was eluted by a mobile phase which contained 5 mM ammonium acetate and acetonitrile. The analytical column was a Luna C18 column (4.6 mm x 50 mm, 5 microm). Detection was achieved by positive ion electrospray tandem mass spectrometry. The lower limit of quantitation of the method was 9 ng/ml. The standard curve, which ranged from 9 to 1350 ng/ml, was fitted by a weighted (1/x2) quadratic regression model. The validation results demonstrated that this method had satisfactory precision and accuracy across the calibration range. There was no evidence of instability of the analyte in human serum following three freeze-thaw cycles, and samples could be stored for at least 2 weeks at -30 degrees C. This method was used to analyze pioglitazone concentrations in human serum samples from a bioequivalence study of a blinded Actos formulation (encapsulated 15 mg tablet) and an Actos 15 mg tablet. The blinded formulation was shown to be bioequivalent to an Actos 15 mg tablet.