Highly sensitive liquid chromatography-mass spectrometry method for the quantitative analysis of mometasone furoate in human plasma: Method validation and application to clinical pharmacokinetic studies.

We report the development and the validation of a sensitive liquid chromatography-mass spectrometry (LC-MS/MS) method for mometasone furoate (MF) analysis in human plasma. Plasma samples were processed through liquid-liquid extraction and analyzed using LC-MS/MS operating in positive mode using multiple reaction monitoring of transitions m/z 520.9 â†’ 355.0 and m/z 525.8 â†’ 355.0 for MF and the internal standard (IS), respectively. Separation was achieved at 1.0 mL/min on a C18 column using a gradient elution of mobile phase of 0.05% ammonia in water (phase A) and acetonitrile (phase B). The assay range was 0.250-100 pg/mL and proved to be accurate and precise MF. Normalized recoveries were consistent and reproducible with a coefficient of variation (CV%) value of 6.0. The CV (%) of the IS normalized matrix factor was not observed in normal, lipemic, and hemolyzed plasmas. Dilutions of 1:10 were accurately quantified. A cycle of three freeze and thaw and stabilities at room temperature and on the autosampler were demonstrated. In addition, MF in the presence of indacaterol and glycopyrronium was proven to be stable at -70°C for at least 157 days. The present method was successfully applied to quantify MF in patients receiving MF, indacaterol, and glycopyrronium as a fixed-dose combination.

Use of Pharmacokinetic and Pharmacodynamic Data to Develop the CDK4/6 Inhibitor Ribociclib for Patients with Advanced Breast Cancer.

Ribociclib is an orally bioavailable, selective cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor. CDK4/6 inhibition by ribociclib leads to retinoblastoma tumor suppressor protein (Rb) reactivation, thereby restoring Rb-mediated cell cycle arrest. Ribociclib is approved for the treatment of patients with hormone receptor-positive/human epidermal growth factor receptor-2-negative (HR+/HER2-) advanced breast cancer (ABC), at the dose of 600 mg once daily (QD) during cycles of 21 days on/7 days off, with optional dose reduction to 400 mg and 200 mg. Ribociclib is rapidly absorbed with a median time to reach maximum plasma concentration of 2.4 h, mean half-life of 32.0 h and oral bioavailability of 65.8% at 600 mg. It is eliminated mainly by hepatic metabolism (~ 84% of total elimination), mostly by cytochrome P450 (CYP) 3A4. Age, body weight, race, baseline Eastern Cooperative Oncology Group status, food, mild hepatic impairment, mild-to-moderate renal impairment, proton pump inhibitors, and combination partners (non-steroidal aromatase inhibitors or fulvestrant) have no clinically relevant impact on ribociclib exposure. Ribociclib inhibits CYP3A at 600 mg leading to increased exposure of CYP3A substrates. Strong CYP3A inhibitors or inducers increase or decrease, respectively, ribociclib exposure. Exposure-safety and exposure-efficacy analyses support the clinical benefit of the 600 mg QD starting dose, with potential individualized dose reductions to 400 mg and 200 mg for effective management of the adverse events neutropenia and QTcF interval prolongation, while maintaining efficacy, in patients with HR+/HER2- ABC. Overall, these clinical pharmacology data informed ribociclib dose justification and clinical development, as well as its prescribing information for clinical use in advanced breast cancer patients.

Metabolite Bioanalysis in Drug Development: Recommendations from the IQ Consortium Metabolite Bioanalysis Working Group.

The intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development Metabolite Bioanalysis Working Group on the fit-for-purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from investigational new drug (IND) enabling studies, and phase I, phase II, and phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development.

Supported liquid extraction combined with liquid chromatography tandem mass spectrometry for the quantitative analysis of a TLR7 agonist imiquimod LFX453 in plasma at low picograms per milliliter: Method validation and its application to a pharmacokinetic study in minipig.

Sample preparation is essential for low-level compound determination. In the present work, supported liquid extraction (SLE) was used as sample preparation for the low-level determination of a new TLR7 agonist imiquimod compound, LFX453. Samples were extracted on ISOLUTE® SLE 96-well plates using tert-butyl-methyl ether followed by evaporation and dry residue reconstitution with 150 µl of a mixture of 0.1% formic acid in acetonitrile-water (50/50, v/v). Samples were eluted using a flow rate of 0.750 ml/min on a C18 column (50 × 2.1 mm, 2.7 µm) with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Tandem mass spectrometry was used to analyze the samples in positive mode. The method run time was 6.5 min, and the low limit of quantification was 1.00 pg/ml with 0.100 ml of minipig plasma. Intra-run and inter-run precision and accuracy were within the acceptance criteria at four concentration levels over a concentration ranging from 1.00 to 200 pg/ml. There was no matrix effect and recovery, three freeze-thaw cycles and incurred samples reanalysis were validated. The method was successfully applied for measuring LFX453 in minipig plasma after application on minipig skin.

Development and validation of an LC-MS/MS method for the quantitative analysis of the adenosine A2a receptor antagonist NIR178 and its monohydroxy metabolite in human plasma: Application to clinical pharmacokinetics.

We report a selective LC-MS/MS method for the simultaneous quantitative determinations of the adenosine A2a receptor antagonist NIR178 (NIR178) and its major metabolite NJI765 in human plasma. Sample preparation steps involved protein precipitation, sample evaporation and reconstitution using a plasma sample volume of 0.1 ml plasma. Separation was achieved in 10 min on an Acquity UPLC BEH C18 1.7 µm, 2.1 × 50 mm column heated at 60°C with a gradient elution at 0.6 ml/min mobile phase made of water and acetonitrile both acidified with 0.1% formic acid. The detection was performed in positive ion mode and quantification based on multiple reaction monitoring. The linear response range was 1.00-1,000 ng/ml using a 1/x2 weighting factor. The intra- and inter-day accuracies (bias %) and intra- and inter-day precisions (CV, %) obtained for NIR178 and NJI765 were within the acceptance criteria. The normalized NIR178 and NJI765 matrix factor calculated from six lots of normal, lipemic and hemolyzed plasmas ranged from 0.97 to 1.05. The normalized recoveries of both NIR178 and NJI765 compared with their internal standards were consistent and reproducible with a CV ≤8.0. This method was successfully applied to support pharmacokinetic studies in adult patients with cancer.

Relative bioavailability of diclofenac potassium from softgel capsule versus powder for oral solution and immediate-release tablet formulation.

The oral bioavailability of diclofenac potassium 50 mg administered as a soft gelatin capsule (softgel capsule), powder for oral solution (oral solution), and tablet was evaluated in a randomized, open-label, 3-period, 6-sequence crossover study in healthy adults. Plasma diclofenac concentrations were measured using a validated liquid chromatography-mass spectrometry/mass spectrometry method, and pharmacokinetic analysis was performed by noncompartmental methods. The median time to achieve peak plasma concentrations of diclofenac was 0.5, 0.25, and 0.75 hours with the softgel capsule, oral solution, and tablet formulations, respectively. The geometric mean ratio and associated 90%CI for AUCinf, and Cmax of the softgel capsule formulation relative to the oral solution formulation were 0.97 (0.95-1.00) and 0.85 (0.76-0.95), respectively. The geometric mean ratio and associated 90%CI for AUCinf and Cmax of the softgel capsule formulation relative to the tablet formulation were 1.04 (1.00-1.08) and 1.67 (1.43-1.96), respectively. In conclusion, the exposure (AUC) of diclofenac with the new diclofenac potassium softgel capsule formulation was comparable to that of the existing oral solution and tablet formulations. The peak plasma concentration of diclofenac from the new softgel capsule was 67% higher than the existing tablet formulation, whereas it was 15% lower in comparison with the oral solution formulation.

Effect of food on the oral bioavailability of amlodipine/valsartan and amlodipine/valsartan/hydrochlorothiazide fixed dose combination tablets in healthy subjects.

A double fixed dose combination of amlodipine/valsartan and triple fixed dose combination of amlodipine/valsartan/HCTZ tablets have been developed to treat patients with moderate-to-severe hypertension. Here, we present the effect of food on the oral bioavailability of these two fixed dose combination tablets from two separate clinical studies in healthy subjects. Single oral doses of amlodipine/valsartan (10/160 mg) and amlodipine/valsartan/HCTZ (10/320/25 mg were administered under fasted or fed conditions. Blood samples were collected in both studies to determine the pharmacokinetic parameters of amlodipine, valsartan, and/or HCTZ using non-compartmental analysis. Following amlodipine/valsartan administration, the geometric mean ratios (GMRs, 90% CI) of AUC0-∞ and Cmax were 1.09 (1.05-1.13) and 1.03 (0.97-1.09) for amlodipine, and 0.94 (0.81-1.10) and 0.86 (0.73-1.02) for valsartan, respectively. Following amlodipine/valsartan/HCTZ administration, the GMRs (90%CI) of AUC0-∞ and Cmax were 1.09 (1.04-1.15) and 1.11 (1.05-1.08) for amlodipine, 1.14 (0.99-1.31) and 1.12 (0.98-1.29) for valsartan, and 1.09 (1.02-1.16) and 0.86 (0.79-0.93) for HCTZ, respectively. Considering the sample size and pharmacokinetic variability associated with analytes, these study results indicate that food effect is minimal or none when fixed dose combination tablets are administered with food. In conclusion, both fixed dose combination tablets can be administered without regards to meals.

Validation of an LC-MS/MS method for the quantitative determination of mavoglurant (AFQ056) in human plasma.

A simple, sensitive, and selective liquid chromatography/tandem mass spectrometry method was validated for the identification and quantification of mavoglurant (AFQ056) in human plasma. The chromatographic separation was performed using a Cosmosil 5 C18 (150 × 4.6 mm, 5 µm) column at 40 ± 0.5 °C with a mobile phase consisting of acetic acid in water (0.1%, v/v)/methanol (10:90, v/v) with a flow rate of 1.0 mL/min followed by quantification with tandem mass spectrometry, operating with electrospray ionization in positive ion mode and applying multiple reaction monitoring. The validated method described in this paper presents high absolute recovery with precision and accuracy meeting the acceptance criteria. The method was precise and accurate for 2- and 10-fold dilution of samples. The method was validated using sodium heparin as specific anticoagulant, and the anticoagulant effect was tested by lithium heparin and K(3)EDTA. The method was successfully cross-validated between two bioanalytical sites. The method was specific for mavoglurant within the given criteria for acceptance (apparent peak area at the retention time of mavoglurant in zero samples was less than 20% compared with the mean peak area at LLOQ) in human plasma. The method was fully validated for the quantitative determination of mavoglurant in human plasma between the range of 2.00 and 2,500 ng/mL.

In silico prediction for the investigation of comedication interferences in quantitative LC-MS detection in the SRM mode.

BACKGROUND: LC-SRM/MS method validation in quantitative bioanalysis requires screening for potential interferences caused by the coelution of comedications or their metabolites. Current approaches are time-consuming, difficult to transfer to other experimental systems and not comprehensive. We propose an in silico strategy based on predicted LC retention time and MS precursor interferences to rank compounds that could potentially interfere with the analyte of interest, followed by a more focused experimental verification. RESULTS: The suggested screening strategy was applied to investigate 129 potential comedications in everolimus patient samples analyzed with a validated LC-SRM/MS assay. A mixture of analytes with the same nominal mass was also investigated to illustrate the interference issues in SRM method development. CONCLUSION: A strategy was developed that allows the rapid screening of comedications, which is scalable to any analyte and transferable to any other LC-MS system.

Enantiomeric separation of verapamil and its active metabolite, norverapamil, and simultaneous quantification in human plasma by LC-ESI-MS-MS.

A simple, selective and high-throughput liquid chromatography-tandem mass spectrometry method has been developed and validated for the chromatographic separation and quantification of (R)- and (S)-enantiomers of verapamil and its active metabolite, norverapamil, in human plasma. All four analytes along with deuterated internal standards (D(6)-verapamil and D(6)-norverapamil) were extracted from 50 µL human plasma by liquid-liquid extraction. Separation was achieved on a Chiralcel OD-RH (150 × 4.6 mm, 5 µm) analytical column with resolution factors of 1.4 and 1.9 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. A mobile phase consisting of 0.05% trifluoroacetic acid in water-acetonitrile (70:30, v/v) afforded capacity factors of 2.45, 3.05, 2.27 and 3.13 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. Detection was carried out on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion modes. The method was validated over the concentration range of 1.0-250.0 ng/mL for all four analytes. Absolute recovery for the analytes ranged from 91.1 to 108.1%. Matrix factors calculated at three quality control levels varied from 0.96-1.07. The method was successfully applied to a pharmacokinetic study in 18 healthy Indian males after oral administration of a 240-mg verapamil tablet formulation under fasting conditions.

Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood.

A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method was validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin in human blood. The validation of the analytical procedure was performed according to the latest Food and Drug Administration (FDA) "Guidance for Industry, Bioanalytical Method Validation". Chromatographic separation was performed using an RP C18 (50 mm × 4.6 mm, 5 µm) column at 40±3.0 °C with a mobile phase consisted of 2 mM ammonium acetate in water (pH 4.5):methanol:acetonitrile (25:15:60, v/v) of a flow rate of 1 mL/min followed by quantification with tandem mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery, with a sensitivity of 3.00 ng/mL as lower limit of quantitation using a sample volume of 300 µL, low inter-run bias and variability (for Sotrastaurin, -4.4 to 0.4% and 1.8 to 2.5% and for N-desmethyl-sotrastaurin, ranged from 1.6 to 2.3% and 2.7 to 3.9%, respectively) with a short runtime of 3.5 min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethyl-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethyl-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3.00 ng/mL and 1200 ng/mL.

Sotrastaurin and cyclosporine drug interaction study in healthy subjects.

INTRODUCTION: Sotrastaurin is an immunosuppressant that inhibits protein kinase C and blocks T-lymphocyte activation. The authors determined the effect of combining sotrastaurin with the calcineurin inhibitor cyclosporine on the pharmacokinetics and biomarker responses to both drugs. METHODS: This was a randomized, 4-period, crossover study in 20 healthy subjects who received single oral doses of (1) sotrastaurin 100 mg, (2) cyclosporine 400 mg, (3) 100 mg sotrastaurin with 100 mg cyclosporine and (4) 100 mg sotrastaurin with 400 mg cyclosporine. Blood samples were collected to measure drug levels and biomarkers of T-lymphocyte activation (interleukin-2 and tumor necrosis factor producing T-cells and interleukin-2 messenger RNA levels) and of T-lymphocyte proliferation (thymidine uptake). RESULTS: Sotrastaurin did not alter cyclosporine AUC; however, low-dose and high-dose cyclosporine increased sotrastaurin AUC by 1.2-fold [90% confidence interval, 1.1-1.4] and 1.8-fold [1.6-2.1], respectively. Adding high-dose cyclosporine to a low-therapeutic dose of sotrastaurin significantly enhanced the inhibition of cytokine production by 31% [95% confidence interval, 25-36%], of interleukin-2 messenger RNA levels by 13% [7-19%], and of thymidine uptake by 37% [32-42%] compared with sotrastaurin alone. Addition of low-dose cyclosporine elicited slightly lower enhancements in inhibition by 21% [14-28%], 6% [-4-16%], and 26% [21-30%], respectively, compared with sotrastaurin alone. CONCLUSIONS: Sotrastaurin did not alter the pharmacokinetics of cyclosporine, but cyclosporine increased sotrastaurin AUC up to 1.8-fold. The combined drugs elicited a significantly greater inhibition of T-cell activation and proliferation than sotrastaurin alone.

Sotrastaurin and tacrolimus coadministration: effects on pharmacokinetics and biomarker responses.

Sotrastaurin is an immunosuppressant that inhibits protein kinase C. In the prevention of acute rejection in organ transplantation, sotrastaurin might be combined with tacrolimus. A drug interaction study was performed in 18 healthy subjects who received single oral doses of sotrastaurin 400 mg, tacrolimus 7 mg, and the drug combination. Drug blood levels and lymphocyte activation and proliferation were measured. Tacrolimus did not alter the pharmacokinetics of sotrastaurin; however, sotrastaurin increased tacrolimus area under the concentration-time curve by 2.0-fold (90% confidence interval, 1.8-2.1). Production of interleukin-2 and tumor necrosis factor by T cells activated via calcium-independent pathways was inhibited by 75% ± 22% from baseline by sotrastaurin. Interleukin-2 messenger RNA levels were decreased by 90% ± 9% from baseline by sotrastaurin. Addition of tacrolimus to sotrastaurin had minimal or no effect on these biomarkers, consistent with tacrolimus' mechanism of action. Lymphocyte proliferation induced via calcium-dependent pathways was decreased from baseline by 82% ± 9% by sotrastaurin, 76% ± 11% by tacrolimus, and 96% ± 2% for the drug combination. How sotrastaurin and tacrolimus could be partnered in an immunosuppressive regimen will need to be established in the context of controlled clinical trials in organ transplant patients, taking into account the pharmacokinetic interaction on tacrolimus and the potentially enhanced immunosuppressive activity of this drug combination.