Ultrasensitive liquid chromatography-tandem mass spectrometric methodologies for quantification of five HIV-1 integrase inhibitors in plasma for a microdose clinical trial.

HIV-1 integrase strand transfer inhibitors are an important class of compounds targeted for the treatment of HIV-1 infection. Microdosing has emerged as an attractive tool to assist in drug candidate screening for clinical development, but necessitates extremely sensitive bioanalytical assays, typically in the pg/mL concentration range. Currently, accelerator mass spectrometry is the predominant tool for microdosing support, which requires a specialized facility and synthesis of radiolabeled compounds. There have been few studies attempted to comprehensively assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach in the context of microdosing applications. Herein, we describe the development of automated LC-MS/MS methods to quantify five integrase inhibitors in plasma with the limits of quantification at 1 pg/mL for raltegravir and 2 pg/mL for four proprietary compounds. The assays involved double extractions followed by UPLC coupled with negative ion electrospray MS/MS analysis. All methods were fully validated to the rigor of regulated bioanalysis requirements, with intraday precision between 1.20 and 14.1% and accuracy between 93.8 and 107% at the standard curve concentration range. These methods were successfully applied to a human microdose study and demonstrated to be accurate, reproducible, and cost-effective. Results of the study indicate that raltegravir displayed linear pharmacokinetics between a microdose and a pharmacologically active dose.

Inhibition of capsaicin-induced increase in dermal blood flow by the oral CGRP receptor antagonist, telcagepant (MK-0974).

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * Calcitonin gene-related peptide (CGRP) was first described as a potent vasodilator. * CGRP is also increasingly recognized as a key player in the pathophysiology of migraine, and CGRP receptor antagonists potentially offer a new approach for treating migraine. * A novel pharmacodynamic assay to measure CGRP receptor antagonist activity non-invasively in humans has been developed, which involves measuring the increase in dermal blood flow induced by topical application of capsaicin on the forearm. WHAT THIS STUDY ADDS: * This study shows that the novel oral CGRP receptor antagonist, telcagepant, inhibits the increases in dermal blood flow induced by the topical application of capsaicin on the human forearm. * This experimental medicine model may have utility to assist in dose selection for the development of CGRP receptor antagonists. AIMS: To evaluate inhibition of capsaicin-induced increase in dermal blood flow (DBF) following telcagepant (MK-0974), a potent and selective orally bioavailable calcitonin gene-related peptide (CGRP) receptor antagonist being developed for the acute treatment of migraine. METHODS: A three-period crossover study in 12 healthy adult men. Each subject received a single oral dose of telcagepant 300 mg, telcagepant 800 mg or placebo at 0 h, followed 0.5 and 3.5 h later by two topical doses of 300 and 1000 microg capsaicin per 20 microl water-ethanol mixture. Capsaicin was applied at two sites on the volar surface of the subjects' left and right forearms. DBF was assessed by laser Doppler perfusion imaging immediately before ('baseline'), and 0.5 h after each capsaicin application at 1 and 4 h. Plasma samples to determine telcagepant concentrations were collected immediately after laser Doppler perfusion imaging. A pharmacodynamic model was developed to explore the relationship between plasma concentration and inhibition of capsaicin-induced increase in DBF. RESULTS: Geometric mean plasma concentrations after dosing with 300 mg and 800 mg telcagepant were 720 and 1146 nm, respectively, at 1 h, vs. 582 and 2548 nm, respectively, at 4 h. The pharmacodynamic model suggested that the EC(90) for telcagepant inhibition of capsaicin-induced increases in DBF was 909 nm. CONCLUSIONS: Telcagepant inhibits the increases in DBF induced by the topical application of capsaicin on the human forearm. This experimental medicine model may have utility to assist in dose selection for the development of CGRP receptor antagonists.

Strategies on efficient method development of on-line extraction assays for determination of MK-0974 in human plasma and urine using turbulent-flow chromatography and tandem mass spectrometry.

On-line extraction assays using cohesive high-turbulence liquid chromatography (HTLC) coupled with tandem mass spectrometer (MS/MS) have been developed for the determination of MK-0974 in human plasma and urine. In this report, a four-step strategy for efficient method development of an on-line extraction assay was discussed. Several challenges - namely extraction recovery, carryover and analyte loss to urine container - were addressed. The assay procedures included sample preparation on a Packard MultiPROBE II liquid-handling system, direct injection on a Cohesive Flux 2300 system, on-line extraction with a Cohesive C(18) column (0.5mmx50mm, 50microm), HPLC separation on a FluophasePFP column (50mmx3mm, 5microm) under cohesive quick-elution mode and MS detection on a Sciex API4000 in multiple-reaction monitoring (MRM) mode using positive ionization and turbo ion-spray. Since 37-80% analyte loss was observed in urine QCs, 1% BSA was added to bring urine QC accuracy back to approximately 100% of nominal. Because of the nature of BSA, the urine assay was established by adapting the plasma method. Thus, the two assays were able to be validated side-by-side, which reduced the validation time by approximately two-fold. The linear dynamic ranges were 0.5-500 and 1-1000nM for the plasma and urine assays, respectively. Obtained from five standard curves constructed with five lots of human plasma or urine, the intra-day precision (%CV) was <3.14 and <2.62%, and the accuracy was 98.3-101.0 and 99.13-100.64% of nominal for plasma and urine assays, respectively. Both plasma and urine QC samples were stable when kept at room temperature for 4h, at -70 degrees C for 3 weeks, or after three freeze-thaw cycles. Both assays gave reasonable relative recovery (>88.8%) and acceptable matrix effect (<15%). The carryover from the upper limit of quantification (ULOQ) was able to be controlled at <20% of lower limit of quantification (LLOQ).

Strategy for peptide quantification using LC-MS in regulated bioanalysis: case study with a glucose-responsive insulin.

AIM: Advances in technology have led to a shift for peptide quantification from traditional ligand-binding assays to LC-MS/MS-based analysis, which presents challenges, in other assay sensitivity, specificity and ruggedness, in addition to lacking of regulatory guidance, especially for the hybrid assay format. Methodology & results: This report communicates a strategy that has been employed in our laboratories for method development and assay validation, and exemplified in a case study of MK-2640, a glucose-responsive insulin, in multiple matrices. Intact MK-2640 was monitored, while immunoaffinity purification and SPE were used to support the rat/dog GLP and clinical studies, respectively. The rationale and considerations behind our approach, as well as the acceptance criteria applied to the assay validation are discussed.

Single- and multiple-dose pharmacokinetics and tolerability of telcagepant, an oral calcitonin gene-related peptide receptor antagonist, in adults.

Telcagepant is a novel, orally active, and selective calcitonin gene-related peptide receptor antagonist being developed for acute treatment of migraine with and without aura. Three separate clinical studies were conducted to evaluate the pharmacokinetics and tolerability of telcagepant following single oral doses in healthy young and elderly men and women and multiple oral doses in men. Telcagepant was rapidly absorbed with a time to maximum concentration of approximately 1.5 hours. The terminal half-life was approximately 6 hours. A greater than dose-proportional increase was observed in the area under the plasma concentration versus time curve from zero to infinity. Following twice-daily dosing, with each dose separated by 2 hours, steady state was achieved in approximately 3 to 4 days with an accumulation ratio of approximately 2. There were no clinically meaningful pharmacokinetic differences when compared across age and gender. Telcagepant was generally well tolerated up to single doses of 1200 mg and multiple doses of 400 mg twice daily.

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry.

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5-500 nM and 5-5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C18 column (50 mm x 0.5 mm, 50 microm), followed by HPLC separation on a FluoPhase RP column (100 mm x 2.1 mm, 5 microm) and MS/MS detection. The off-line SPE assay used Waters Oasis HLB microElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm x 2.1 mm, 5 microm) for LC-MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of < 2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.