An Investigation of Instability in Dried Blood Spot Samples for Pharmacokinetic Sampling in Phase 3 Trials of Verubecestat.

In-clinic dried blood spot (DBS) pharmacokinetic (PK) sampling was incorporated into two phase 3 studies of verubecestat for Alzheimer's disease (EPOCH [NCT01739348] and APECS [NCT01953601]), as a potential alternative to plasma PK sampling for improved logistical feasibility and decreased blood volume burden. However, an interim PK analysis revealed verubecestat concentrations in DBS samples declined with time to assay in both trials. An investigation revealed wide variation in implementation practices for DBS sample handling procedures resulting in insufficient desiccation which caused verubecestat instability. High-resolution mass spectrometry evaluations of stressed and aged verubecestat DBS samples revealed the presence of two hydrolysis degradants. To minimize instability, new DBS handling procedures were implemented that provided additional desiccant and minimized the time to analysis. Both verubecestat hydrolysis products were previously discovered and synthesized during active pharmaceutical ingredient stability characterization. A liquid chromatography-mass spectrometry assay to quantitate the dominant verubecestat degradant in DBS samples was developed and validated. The application of this method to stressed and aged verubecestat DBS samples confirmed that degradant concentrations accounted for the observed decreases in the verubecestat concentration. Furthermore, after increasing desiccant amounts, degradant concentrations accounted for approximately 7% of the verubecestat concentration in DBS clinical samples, indicating that issues with sample handling were minimized with new storage and shipping conditions. This case study illustrates the challenges with employing new sampling techniques in large, global trials, and the importance of anticipating and mitigating implementation risks.

Making Better Dose Decisions: Using Exposure-Response Modeling to Integrate Efficacy Outcome of Two Phase IIb Clinical Trials of Ubrogepant for Migraine Treatment.

Ubrogepant (MK-1602) is a novel, oral, calcitonin gene-related peptide receptor antagonist in clinical development with positive phase III outcomes for acute treatment of migraine. This paper describes the population exposure-response (E-R) modeling and simulations, which were used to inform the phase III dose-selection rationale, based on ~ 800 participants pooled across two phase IIb randomized dose-finding clinical trials. The E-R model describes the placebo and ubrogepant treatment effects based on migraine pain end points (2-hour pain relief and 2-hour pain freedom) at various dose levels. Sensitivity analyses were conducted to evaluate various assumptions of placebo response in light of the high placebo response observed in one phase II trial. A population pharmacokinetic model describing the effect of formulations was included in the E-R simulation framework to assess potential dose implications of a formulation switch from phase II to phase III. Model-based simulations predict that a dose of 25 mg or higher is likely to achieve significantly better efficacy than placebo with desirable efficacy levels. The understanding of E-R helped support the dose selection for the phase III clinical trials.

Toward highly sensitive and reproducible LC-MS/MS analysis of MK-8591 phosphorylated anabolites in human peripheral blood mononuclear cells.

Aim: MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC-MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Materials and methods: Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC-MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Results: Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5-106% and precision was 0.7-12.1% for a linear curve range of 0.1-40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Conclusion: Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.

Leveraging Digital Health Technologies and Outpatient Sampling in Clinical Drug Development: A Phase I Exploratory Study.

Merck & Co, Inc (Kenilworth, NJ) is investing in approaches to enrich clinical trial data and augment decision making through use of digital health technologies, outpatient sampling, and real-time data access. As part of this strategy, a phase I study was conducted to explore a few technologies of interest. In this fixed-sequence two-period trial, 16 healthy subjects were administered 50-mg once-daily sitagliptin packaged in a bottle that electronically captured the date and time study medication was dispensed (period 1) and in a traditional pharmacy bottle (period 2). Dried blood spot samples were collected for sitagliptin concentration analysis on select study days, both in clinic and at home, with collection time recorded using an electronic diary in period 1 and by clinic staff in period 2. Study results demonstrated the feasibility and subject acceptance of collecting digital adherence data and outpatient dried blood spot samples in clinical trials and highlighted areas for future improvements.

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.

Extractability-mediated stability bias and hematocrit impact: High extraction recovery is critical to feasibility of volumetric adsorptive microsampling (VAMS) in regulated bioanalysis.

Volumetric absorptive microsampling (VAMS), a new microsampling technique, was evaluated for its potential in supporting regulated bioanalysis. Our initial assessment with MK-0518 (raltegravir) using a direct extraction method resulted in 45-52% extraction recovery, significant hematocrit (Ht) related bias, and more importantly, unacceptable stability (>15% bias from nominal concentration) after 7-day storage. Our investigation suggested that the observed biases were not due to VAMS absorption, sampling techniques, lot-to-lot variability, matrix effect, and/or chemical stability of the compound, but rather the low extraction recovery. An effort to improve assay recovery led to a modified liquid-liquid extraction (LLE) method that demonstrated more consistent performance, minimal Ht impact (Ht ranged from 20 to 65%), and acceptable sample stability. The same strategy was successfully applied to another more hydrophilic model compound, MK-0431 (sitagliptin). These results suggest that the previously observed Ht effect and "instability" were in fact due to inconsistent extractability, and optimizing the extraction recovery to greater than 80% was critical to ensure VAMS performance. We recommend adding Ht-independent recovery as part of feasibility assessment to de-risk the long-term extractability-mediated stability bias before implementing VAMS in regulated bioanalysis.

Population PK Analyses of Ubrogepant (MK-1602), a CGRP Receptor Antagonist: Enriching In-Clinic Plasma PK Sampling With Outpatient Dried Blood Spot Sampling.

Merck & Co., Inc. (Kenilworth, New Jersey) has recently published an integrated strategy for implementation of dried blood spots (DBS) in late-stage trials for population pharmacokinetic (PK) modeling. We applied this strategy for another late-stage clinical program: ubrogepant (MK-1602), a novel oral calcitonin gene-related peptide receptor antagonist for acute treatment of migraine. At the time of implementation, ubrogepant was entering phase 2 development. DBS was implemented to acquire PK information proximal to an acute migraine event to enable exposure-response modeling. The clinical endpoint was a spontaneous event, which generally occurs outside a clinic visit. Thus, an innovative feature of this trial was facilitating DBS in an outpatient setting. In vitro and bioanalytical tests established initial method feasibility and suitability for further evaluations in the clinic. A quantitative relationship was developed between blood and plasma concentrations from concurrently collected samples in a phase 1 (healthy subjects) and phase 2 (target patient population) study using graphical and population PK approaches. This integrated information was presented to the Food and Drug Administration for regulatory input. Following regulatory concurrence, DBS was poised for use in further clinical studies. Population PK modeling was used to dissect sources of variability contributing to DBS collection in the outpatient setting. What has been learned from this program has informed the broader integrated strategy of Merck & Co., Inc. (Kenilworth, NJ) for DBS implementation in clinical trials and research to improve the precision of PK data collected in an outpatient setting.

Suitability of a minipig model in assessing clinical bioperformance of matrix and multiparticulate extended-release formulations for a BCS class III Drug development candidate.

Preclinical studies in dogs are often employed as part of formulation screening process. However, the shorter transit time and colonic length in dogs may result in underestimation of absorption, especially for extended-release (ER) formulations. In the recent years, minipigs have attracted attention as an alternative animal model. However reports on studies with ER formulations are limited. In this manuscript, we report the first comprehensive comparison of minipig and clinical data for two types of ER formulations. Matrix tablets and multiparticulates in capsules of a BCS Class III compound were tested in the Yucatan minipig model. The relative performance of the formulations in minipigs in the fasted state was reasonably aligned with the clinical observations. The minipig model was able to rank order the formulations, reflecting the targeted release rate, in a manner consistent with the clinical data. Minipigs also reflected the loss of bioavailability relative to the immediate-release formulation. A level C in vitro/in vivo correlation was demonstrated for both the minipig and clinical data. However, an assessment of food effect in the minipig model appeared challenging, especially for the matrix formulation for which a negative food effect was observed in minipigs compared with the positive food effect in the clinic.

Automated calibration of TECAN genesis liquid handling workstation utilizing an online balance and density meter.

With robotics widely used in bioanalytical assays, accurate system performance is essential to ensure the quality and productivity of the robotics. In our lab, an automated calibration procedure has been developed to evaluate the precision and accuracy of the TECAN (Research Triangle Park, NC, U.S.A.) Genesis liquid handling system in a bioanalytical laboratory setting. The calibrations were performed by transferring and weighing the solvents automatically on a microbalance controlled by a Gemini program. From the data acquired, calibration reports were generated using a template. The novel aspect of this approach is the use of an on-line balance and a density meter, both of which combine to make the calibration process simple, efficient, and precise. For quantitative bioanalysis, a variety of solvents, including methanol, water, mixed solvents, and plasma, are typically used to prepare standards and unknown samples. Density information is usually unknown for the mixed solvents, and the density of plasma can vary from species to species. However, with the use of a universal density meter, the density could be obtained in seconds. The issue of solvent evaporation during the calibration process was also addressed. Calibration curves were set up for various liquid classes. Pipetting volumes ranged from 10 microL to 900 microL. Precision and accuracy results obtained from the semiannual performance evaluations showed this procedure to be reliable and user-friendly. Using the automated calibration procedure, the calibration and performance evaluation of the robotic system is considerably more efficient, and the incidence of unacceptable precision and accuracy is greatly reduced.