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.

An innovative phase I study in healthy subjects to determine the mass balance, elimination, metabolism, and absolute bioavailability of mitapivat.

Mitapivat, a first-in-class, oral, small-molecule, allosteric activator of the red blood cell-specific form of pyruvate kinase (PKR), was approved for the treatment of hemolytic anemia in adults with pyruvate kinase (PK) deficiency. In this phase I mass balance study in healthy males, we administered a single ~120 mg oral dose of [14 C]mitapivat and a concomitant intravenous ~0.1 mg microdose of [13 C6 ]mitapivat. We determined (1) the routes of total radioactivity excretion, including the mass balance of total radioactivity in urine and feces; (2) the pharmacokinetics of mitapivat and [13 C6 ]mitapivat in plasma and total radioactivity in whole blood and plasma; (3) the absolute oral bioavailability of mitapivat; and (4) the metabolite profiles in plasma and excreta. Mean recovery of the radioactive dose was 89.1% (49.6% in urine and 39.6% in feces). [14 C]Mitapivat was rapidly absorbed and extensively metabolized as <4% of the total radioactive dose was excreted unaltered in urine and feces. Mean absolute oral bioavailability was 72.7%. A total of 17 metabolites were identified. Mitapivat accounted for 57% and 34% of plasma radioactivity in AUC0-24 and AUC0-72 pooled samples, respectively. The remaining radioactivity was attributable to several metabolites, each representing <10% of the total radioactivity in pooled samples; none were disproportionate metabolites as defined by the US Food and Drug Administration and International Conference on Harmonisation M3 guidelines. Metabolite structures suggest that the primary metabolic pathways for [14 C]mitapivat in humans include N-dealkylation of the cyclopropylmethyl moiety, oxygenation of the quinoline-8-sulfonamide, oxidation/unsaturation, scission of the piperazine moiety, and amide hydrolysis.

A phase 1 dose escalation study of the pyruvate kinase activator mitapivat (AG-348) in sickle cell disease.

Polymerization of deoxygenated hemoglobin S underlies the pathophysiology of sickle cell disease (SCD). In activating red blood cell pyruvate kinase and glycolysis, mitapivat (AG-348) increases adenosine triphosphate (ATP) levels and decreases the 2,3-diphosphoglycerate (2,3-DPG) concentration, an upstream precursor in glycolysis. Both changes have therapeutic potential for patients with SCD. Here, we evaluated the safety and tolerability of multiple ascending doses of mitapivat in adults with SCD with no recent blood transfusions or changes in hydroxyurea or l-glutamine therapy. Seventeen subjects were enrolled; 1 subject was withdrawn shortly after starting the study. Sixteen subjects completed 3 ascending dose levels of mitapivat (5, 20, and 50 mg, twice daily [BID]) for 2 weeks each; following a protocol amendment, the dose was escalated to 100 mg BID in 9 subjects. Mitapivat was well tolerated at all dose levels, with the most common treatment-emergent adverse events (AEs) being insomnia, headache, and hypertension. Six serious AEs (SAEs) included 4 vaso-occlusive crises (VOCs), non-VOC-related shoulder pain, and a preexisting pulmonary embolism. Two VOCs occurred during drug taper and were possibly drug related; no other SAEs were drug related. Mean hemoglobin increase at the 50 mg BID dose level was 1.2 g/dL, with 9 of 16 (56.3%) patients achieving a hemoglobin response of a ≥1 g/dL increase compared with baseline. Mean reductions in hemolytic markers and dose-dependent decreases in 2,3-DPG and increases in ATP were also observed. This study provides proof of concept that mitapivat has disease-modifying potential in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

Quantitation of ivosidenib in human plasma via LC-MS/MS and its application in clinical trials.

Aim: Ivosidenib is a potent and selective small molecule inhibitor of mutant isocitrate dehydrogenase 1. Accurate measurement of ivosidenib is the key to ivosidenib pharmacokinetics in clinical trials. Materials & methods: Quantitation of ivosidenib was conducted by using a stable isotope labeled compound (ivosidenib-d4) as the internal standard. Results: This assay was validated and successfully applied to support multiple clinical trials. Selected clinical samples were also tested by a chiral LC-MS/MS method against four ivosidenib isomer standards to exclude the possibility of in vivo racemization of ivosidenib. Conclusion: A robust LC-MS/MS method was validated for ivosidenib in human plasma. This is the first time for ivosidenib bioanalytical method in any human matrix to be reported.

Quantitation of 2-hydroxyglutarate in human plasma via LC-MS/MS using a surrogate analyte approach.

Aim: 2-Hydroxyglutarate (2-HG) is a target engagement biomarker in patients after treatment with inhibitors of mutated isocitrate dehydrogenase (mIDH). Accurate measurement of 2-HG is critical for monitoring the inhibition effectiveness of the inhibitors. Materials & methods: Human plasma samples were spiked with stable isotope labelled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. This method was validated following regulatory guidance and has been successfully applied in a clinical study for mIDH inhibition. Results: An LC-MS/MS method with a surrogate analyte approach was developed and validated to measure 2-HG in human plasma with acceptable intra- and inter-assay accuracy and precision. Conclusion: A sensitive and robust LC-MS/MS method was developed and validated for measuring 2-HG in human plasma.

A double surrogate approach for the quantitation of 2-Hydroxyglutarate - An oncometabolite in human brain tumors via LC-MS/MS.

We are reporting a validated LC-MS/MS assay for the quantitation of 2-Hydroxyglutarate (2-HG), an endogenous oncometabolite, in human brain tumors. To address the challenges of endogenous levels of 2-HG in biological matrix and the limited availability of human brain tumors, a surrogate analyte in conjunction with a surrogate matrix approach was used (stable isotope labeled 2-HG in monkey brain homogenate). Human brain tumor homogenate samples were spiked with stable isotope labeled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. The mass spectrometer was optimized to ensure equal molar response between 2-HG - the authentic analyte and 13C5 2-HG - the surrogate analyte. Parallelism was successfully demonstrated between human brain tumor homogenate (the authentic matrix) and monkey brain homogenate (the surrogate matrix). The linear analytical range of the assay was set at 0.15-150 µM. This validated LC-MS/MS method demonstrated excellent accuracy and precision. For 13C5 2-HG in monkey brain homogenate and human brain tumor homogenate, the overall accuracy was between 98.4 % and 102 %, and the intra- and inter-assay precision (%CV) were less than 5.7 %. 2-HG was found to be stable in human brain tumor homogenate for at least 26 h at room temperature, 454 days when stored at 70 °C and after five freeze/thaw cycles. The assay has been successfully applied to human brain tumor samples to support clinical studies. Novel Aspect: A surrogate matrix and surrogate analyte approach to quantify endogenous 2-HG concentrations in human brain tumors.

"Center punch" and "whole spot" bioanalysis of apixaban in human dried blood spot samples by UHPLC-MS/MS.

Apixaban (Eliquis™) was developed by Bristol-Myers Squibb (BMS) and Pfizer to use as an antithrombotic/anticoagulant agent and has been recently approved for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. A clinical study of apixaban, sponsored by BMS and Pfizer, included a pilot exploratory portion to evaluate the potential for future drug concentration monitoring using dried blood spot (DBS) sample collection. For DBS sample collection, a fixed blood volume was dispensed onto a DBS card by either regular volumetric pipette (venous blood collection) or capillary dispenser (finger prick blood collection). A 96-well semi-automated liquid-liquid extraction sample preparation procedure was developed to provide clean extracts for UHPLC-MS/MS quantitation. Assays using both partial-spot center punch and whole spot punch were developed and validated. The linear dynamic ranges for all the analyses were from 0.5 to 500 ng/mL. The coefficient of determination (r(2)) values was >0.9944 for all the validation runs. For the center punch approach, the intra-assay precision (%CV) was within 4.4% and inter-assay precision was within 2.6%. The assay accuracy, expressed as %Dev., was within ± 5.4% of the nominal concentrations. One accuracy and precision run was performed using the whole spot approach, the intra-assay precision (%CV) was within 7.1% and the accuracy was within ± 8.0% of the nominal concentrations. In contrast to the center punch approach, the whole spot approach eliminated the effect of hematocrit and high lipids on the analysis of apixaban in human DBS when an accurate sample blood volume was collected on DBS cards.