2022 White Paper on Recent Issues in Bioanalysis: ICH M10 BMV Guideline & Global Harmonization; Hybrid Assays; Oligonucleotides & ADC; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Mass Spectrometry, Chromatography and Sample Preparation, Novel Technologies, Novel Modalities, and Novel Challenges, ICH M10 BMV Guideline & Global Harmonization Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.

Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices.

Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are "living cell" therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

Comparison of Pre-existing Anti-AAV8 Total Antibody Screening and Confirmatory Assays with a Cell-Based Neutralizing Assay in Normal Human Serum.

Pre-existing adeno-associated viruses (AAV) neutralizing antibodies (NAb) can prevent AAV vectors from transducing target tissues. The immune responses can include binding/total antibodies (TAb) and neutralizing antibodies (NAb). This study is aimed at comparing total antibody assay (TAb) and cell-based NAb assay against AAV8 to help inform the best assay format for patient exclusion criteria. We developed a chemiluminescence-based enzyme-linked immunosorbent assay to analyze AAV8 TAb in human serum. The specificity of AAV8 TAb was determined using a confirmatory assay. A COS-7-based assay was used to analyze anti-AAV8 NAbs. The TAb screening cut point factor was determined to be 2.65, and the confirmatory cut point (CCP) was 57.1%. The prevalence of AAV8 TAb in 84 normal subjects was 40%, of which 24% were NAb positive and 16% were NAb negative. All NAb-positive subjects were confirmed to be TAb-positive and also passed the CCP-positive criteria. All 16 NAb-negative subjects did not pass the CCP criterion for the positive specificity test. There was a high concordance between AAV8 TAb confirmatory assay and NAb assay. The confirmatory assay improved the specificity of the TAb screening test and confirmed neutralizing activity. We proposed a tiered assay approach, in which an anti-AAV8 screening assay should be followed by a confirmatory assay during pre-enrollment for patient exclusions for AAV8 gene therapy. This approach can be used in lieu of developing a NAb assay and can be also implemented as a companion diagnostic assay for post-marketing seroreactivity assessments due to ease of development and use.

Effect of fluconazole on the pharmacokinetics of a single dose of fedratinib in healthy adults.

PURPOSE: Fedratinib is an orally administered Janus kinase (JAK) 2-selective inhibitor for the treatment of adult patients with intermediate-2 or high-risk primary or secondary myelofibrosis. In vitro, fedratinib is predominantly metabolized by cytochrome P450 (CYP) 3A4 and to a lesser extent by CYP2C19. Coadministration of fedratinib with CYP3A4 inhibitors is predicted to increase systemic exposure to fedratinib. This study evaluated the effect of multiple doses of the dual CYP3A4 and CYP2C19 inhibitor, fluconazole, on the pharmacokinetics of a single dose of fedratinib. METHODS: In this non-randomized, fixed-sequence, open-label study, healthy adult participants first received a single oral dose of fedratinib 100 mg on day 1. Participants then received fluconazole 400 mg on day 10 and fluconazole 200 mg once daily on days 11-23, with a single oral dose of fedratinib 100 mg on day 18. Pharmacokinetic parameters were calculated for fedratinib administered with and without fluconazole. RESULTS: A total of 16 participants completed the study and were included in the pharmacokinetic population. Coadministration of fedratinib with fluconazole increased maximum observed plasma concentration (Cmax) and area under the plasma concentration-time curve from time 0 to the last quantifiable concentration (AUC0-t) of fedratinib by 21% and 56%, respectively, compared with fedratinib alone. Single oral doses of fedratinib 100 mg administered with or without fluconazole were well tolerated. CONCLUSIONS: Systemic exposure after a single oral dose of fedratinib was increased by up to 56% when fedratinib was coadministered with fluconazole compared with fedratinib alone. TRIAL REGISTRY: CLINICALTRIALS.GOV: NCT04702464.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Impact of fedratinib on the pharmacokinetics of transporter probe substrates using a cocktail approach.

INTRODUCTION: Fedratinib, an oral, selective Janus kinase 2 inhibitor, has been shown to inhibit P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter (OCT) 2, and multidrug and toxin extrusion (MATE) 1 and MATE2-K in vitro. The objective of this study was to evaluate the influence of fedratinib on the pharmacokinetics (PK) of digoxin (P-gp substrate), rosuvastatin (OATP1B1/1B3 and BCRP substrate), and metformin (OCT2 and MATE1/2-K substrate). METHODS: In this nonrandomized, fixed-sequence, open-label study, 24 healthy adult participants received single oral doses of digoxin 0.25 mg, rosuvastatin 10 mg, and metformin 1000 mg administered as a drug cocktail (day 1, period 1). After a 6-day washout, participants received oral fedratinib 600 mg 1 h before the cocktail on day 7 (period 2). An oral glucose tolerance test (OGTT) was performed to determine possible influences of fedratinib on the antihyperglycemic effect of metformin. RESULTS: Plasma exposure to the three probe drugs was generally comparable in the presence or absence of fedratinib. Reduced metformin renal clearance by 36% and slightly higher plasma glucose levels after OGTT were observed in the presence of fedratinib. Single oral doses of the cocktail ± fedratinib were generally well tolerated. CONCLUSIONS: These results suggest that fedratinib has minimal impact on the exposure of P-gp, BCRP, OATP1B1/1B3, OCT2, and MATE1/2-K substrates. Since renal clearance of metformin was decreased in the presence of fedratinib, caution should be exercised in using coadministered drugs that are renally excreted via OCT2 and MATEs. TRIAL REGISTRATION: Clinicaltrials.gov NCT04231435 on January 18, 2020.

Effects of strong and moderate CYP3A4 inducers on the pharmacokinetics of fedratinib in healthy adult participants.

PURPOSE: Fedratinib is an oral and selective Janus kinase 2 inhibitor that is indicated for treatment of adults with intermediate-2 or high-risk primary or secondary myelofibrosis. Fedratinib is metabolized by cytochrome P450s (CYPs), primarily CYP3A4. The objective of this study was to determine the effects of the strong CYP3A4 inducer rifampin and moderate CYP3A4 inducer efavirenz on the pharmacokinetics of single doses of fedratinib. METHODS: This Phase 1, open-label, two-part study (Part 1 for rifampin and Part 2 for efavirenz) was conducted in healthy adult men and women. A single dose of fedratinib (500 mg) was administered on Day 1. Participants received rifampin 600 mg daily or efavirenz 600 mg daily on Days 9-18. On Day 17, a single dose of fedratinib (500 mg) was coadministered with rifampin or efavirenz. Plasma fedratinib concentrations were measured using validated liquid chromatography-tandem mass spectrometry. RESULTS: Maximum observed plasma fedratinib concentrations were lowered by approximately 70% and 30% during coadministration with rifampin or efavirenz, respectively, compared with fedratinib alone. Geometric means of fedratinib area under the plasma concentration-time curve from 0 to infinity were decreased by 81% (90% confidence interval [CI], 77-83%) and 47% (90% CI, 40-53%) during coadministration with rifampin or efavirenz, respectively. Fedratinib was generally well tolerated when administered alone or in combination with rifampin or efavirenz. CONCLUSION: Significant reductions in fedratinib exposure were observed in the presence of strong or moderate CYP3A4 inducers. These results suggest that agents that are strong or moderate inducers of CYP3A4 should be avoided when coadministered with fedratinib. TRIAL REGISTRATION NUMBER: NCT03983239 (Registration date: June 12, 2019).

Investigation into MAO B-Mediated Formation of CC112273, a Major Circulating Metabolite of Ozanimod, in Humans and Preclinical Species: Stereospecific Oxidative Deamination of (S)-Enantiomer of Indaneamine (RP101075) by MAO B.

Ozanimod, recently approved for treating relapsing multiple sclerosis, produced a disproportionate, active, MAO B-catalyzed metabolite (CC112273) that showed remarkable interspecies differences and led to challenges in safety testing. This study explored the kinetics of CC112273 formation from its precursor RP101075. Incubations with human liver mitochondrial fractions revealed K Mapp, V max, and intrinsic clearance (Clint) for CC112273 formation to be 4.8 µM, 50.3 pmol/min/mg protein, and 12 µl/min/mg, respectively, whereas Michaelis-Menten constant (K M) with human recombinant MAO B was 1.1 µM. Studies with liver mitochondrial fractions from preclinical species led to K Mapp, V max, and Clint estimates of 3.0, 35, and 33 µM, 80.6, 114, 37.3 pmol/min/mg, and 27.2, 3.25, and 1.14 µl/min/mg in monkey, rat, and mouse, respectively, and revealed marked differences between rodents and primates, primarily attributable to differences in the K M Comparison of Clint estimates revealed monkey to be ∼2-fold more efficient and the mouse and rat to be 11- and 4-fold less efficient than humans in CC112273 formation. The influence of stereochemistry on MAO B-mediated oxidation was also investigated using the R-isomer of RP101075 (RP101074). This showed marked selectivity toward catalysis of the S-isomer (RP101075) only. Docking into MAO B crystal structure suggested that although both the isomers occupied its active site, only the orientation of RP101075 presented the C-H on the α-carbon that was ideal for the C-H bond cleavage, which is a requisite for oxidative deamination. These studies explain the basis for the observed interspecies differences in the metabolism of ozanimod as well as the substrate stereospecificity for formation of CC112273. SIGNIFICANCE STATEMENT: This study evaluates the enzymology and the species differences of the major circulating metabolite of ozanimod, CC112273. Additionally, the study also explores the influence of stereochemistry on MAO B-catalyzed reactions. The study is of significance to the DMD readers given that this oxidation is catalyzed by a non-cytochrome P450 enzyme, and that marked species difference and notable stereospecificity was observed in MAO B-catalyzed biotransformation when the indaneamine enantiomers were used as substrates.

Absorption, Metabolism, and Excretion, In Vitro Pharmacology, and Clinical Pharmacokinetics of Ozanimod, a Novel Sphingosine 1-Phosphate Receptor Modulator.

Ozanimod is approved for the treatment of relapsing forms of multiple sclerosis. Absorption, metabolism, and excretion of ozanimod were investigated after a single oral dose of 1.0 mg [14C]ozanimod hydrochloride to six healthy subjects. In vitro experiments were conducted to understand the metabolic pathways and enzymes involved in the metabolism of ozanimod and its active metabolites. The total mean recovery of the administered radioactivity was ∼63%, with ∼26% and ∼37% recovered from urine and feces, respectively. Based on exposure, the major circulating components were active metabolite CC112273 and inactive metabolite RP101124, which together accounted for 50% of the circulating total radioactivity exposure, whereas ozanimod accounted for 6.7% of the total radioactive exposure. Ozanimod was extensively metabolized, with 14 metabolites identified, including two major active metabolites (CC112273 and CC1084037) and one major inactive metabolite (RP101124) in circulation. Ozanimod is metabolized by three primary pathways, including aldehyde dehydrogenase and alcohol dehydrogenase, cytochrome P450 isoforms 3A4 and 1A1, and reductive metabolism by gut microflora. The primary metabolite RP101075 is further metabolized to form major active metabolite CC112273 by monoamine oxidase B, which further undergoes reduction by carbonyl reductases to form CC1084037 or CYP2C8-mediated oxidation to form RP101509. CC1084037 is oxidized rapidly to form CC112273 by aldo-keto reductase 1C1/1C2 and/or 3ß- and 11ß-hydroxysteroid dehydrogenase, and this reversible oxidoreduction between two active metabolites favors CC112273. The ozanimod example illustrates the need for conducting timely radiolabeled human absorption, distribution, metabolism, and excretion studies for characterization of disproportionate metabolites and assessment of exposure coverage during drug development. SIGNIFICANCE STATEMENT: Absorption, metabolism, and excretion of ozanimod were characterized in humans, and the enzymes involved in complex metabolism were elucidated. Disproportionate metabolites were identified, and the activity of these metabolites was determined.

Assessment of drug-drug interactions of CC-90001, a potent and selective inhibitor of c-Jun N-terminal kinase.

CC-90001 is predominantly metabolised via glucuronidation, while oxidative metabolism is a minor pathway in human hepatocytes and liver microsomes. In vitro, CC-90001 glucuronidation was catalysed by UGT1A9, UGT1A4, and UGT1A1, while oxidative metabolism was primarily mediated by CYP3A4/5 with minor contributions from CYP1A2, CYP2C9, CYP2B6, and CYP2D6.CC-90001 in vitro inhibits CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 ≤ 55% at 100 µM, and the inhibition was negligible at ≤30 µM. CC-90001 is not a time-dependent CYP inhibitor.In human hepatocytes CC-90001 is an inducer of CYP2B6 and CYP3A, with mRNA levels increased 34.4% to 52.8% relative to positive controls.In vitro CC-90001 is a substrate of P-gp, and an inhibitor of P-gp, BCRP, OAT3, OATP1B1, OATP1B3, OCT2, MATE1, and MATE2k with IC50 values of 30.3, 25.8, 17.7, 0.417, 19.9, 0.605, 4.17, and 20 µM, respectively.A clinical study demonstrated that CC-90001 has no or little impact on the exposure of warfarin (CYP2C9), omeprazole (CYP2C19), midazolam (CYP3A) or metformin (OCT2, MATE1/2k). CC-90001 co-administration increases the AUCt and Cmax 176% and 339% for rosuvastatin (BCRP/OATP1B1/3), 116% and 171% for digoxin (P-gp), and 266% and 321% for nintedanib (CYP3A & P-gp), respectively.In conclusion, CC-90001 in unlikely to be a victim or perpetrator of clinically relevant interactions involving CYPs or UGTs. Weak to moderate interactions are expected in clinic with substrates of P-gp and OATP1B1 due to CC-90001 inhibition of these transporters.

Evaluation of iberdomide and cytochrome p450 drug-drug interaction potential in vitro and in a phase 1 study in healthy subjects.

PURPOSE: Iberdomide is a cereblon E3 ligase modulator capable of redirecting the protein degradation machinery of the cell towards the elimination of target proteins potentially driving therapeutic effects. In vitro studies demonstrated that iberdomide predominantly undergoes oxidative metabolism mediated by cytochrome P450 (CYP) 3A4/5 but had no notable inhibition or induction of CYP enzymes. Consequently, the potential of iberdomide as a victim of drug-drug interactions (DDI) was evaluated in a clinical study with healthy subjects. METHODS: A total of 33 males and 5 females with 19 subjects per part were enrolled. Part 1 evaluated the pharmacokinetics (PK) of iberdomide alone (0.6 mg) and when administered with the CYP3A and P-gp inhibitor itraconazole (200 mg twice daily on day 1 and 200 once daily on days 2 through 9). Part 2 evaluated the PK of iberdomide alone (0.6 mg) and with CYP3A4 inducer rifampin (600 mg QD days 1 through 13). Plasma concentrations of iberdomide and the active metabolite M12 were determined by validated liquid chromatography-tandem mass spectrometry assay. RESULTS: Coadministration of iberdomide with itraconazole increased iberdomide peak plasma concentration (Cmax) 17% and area under the concentration curve (AUC) approximately 2.4-fold relative to administration of iberdomide alone. The Cmax and AUC of iberdomide were reduced by approximately 70% and 82%, respectively, when iberdomide was administered with rifampin compared with iberdomide administered alone. Exploratory assessment of metabolite M12 concentrations demonstrated that CYP3A is responsible for M12 formation. CONCLUSIONS: Caution should be taken when coadministering iberdomide with strong CYP3A inhibitors. Coadministration of iberdomide with strong CYP3A inducers is not advised. CLINICAL TRIAL REGISTRATION: Clinical trial identification number is NCT02820935 and was registered in July 2016.

Physiologically based pharmacokinetic modeling to assess metabolic drug-drug interaction risks and inform the drug label for fedratinib.

PURPOSE: Fedratinib (INREBIC®), a Janus kinase 2 inhibitor, is approved in the United States to treat patients with myelofibrosis. Fedratinib is not only a substrate of cytochrome P450 (CYP) enzymes, but also exhibits complex auto-inhibition, time-dependent inhibition, or mixed inhibition/induction of CYP enzymes including CYP3A. Therefore, a mechanistic modeling approach was used to characterize pharmacokinetic (PK) properties and assess drug-drug interaction (DDI) potentials for fedratinib under clinical scenarios. METHODS: The physiologically based pharmacokinetic (PBPK) model of fedratinib was constructed in Simcyp® (V17R1) by integrating available in vitro and in vivo information and was further parameterized and validated by using clinical PK data. RESULTS: The validated PBPK model was applied to predict DDIs between fedratinib and CYP modulators or substrates. The model simulations indicated that the fedratinib-as-victim DDI extent in terms of geometric mean area under curve (AUC) at steady state is about twofold or 1.2-fold when strong or moderate CYP3A4 inhibitors, respectively, are co-administered with repeated doses of fedratinib. In addition, the PBPK model successfully captured the perpetrator DDI effect of fedratinib on a sensitive CY3A4 substrate midazolam and predicted minor effects of fedratinib on CYP2C8/9 substrates. CONCLUSIONS: The PBPK-DDI model of fedratinib facilitated drug development by identifying DDI potential, optimizing clinical study designs, supporting waivers for clinical studies, and informing drug label claims. Fedratinib dose should be reduced to 200 mg QD when a strong CYP3A4 inhibitor is co-administered and then re-escalated to 400 mg in a stepwise manner as tolerated after the strong CYP3A4 inhibitor is discontinued.

Excretion balance and pharmacokinetics following a single oral dose of [14C]-fedratinib in healthy subjects.

PURPOSE: Fedratinib is an oral and selective kinase inhibitor with activity against wild type and mutationally activated Janus kinase 2 and FMS-like tyrosine kinase 3, for the treatment of adult patients with intermediate-2 or high-risk primary or secondary myelofibrosis. This open-label mass balance study in healthy subjects investigated the excretion balance and systemic exposure of radioactivity after oral administration of [14C]-fedratinib; and the pharmacokinetics of fedratinib and its contribution to overall exposure of radioactivity. METHODS: Six healthy males received a single oral dose of 200 mg [14C]-fedratinib base (2.775 MBq, 75 µCi) as a solution. Blood, urine and feces samples were collected for up to 35 day postdose. Urine and feces samples were collected until the 24-h excretion of radioactivity fell below 0.5% of administered dose (at least 14 day postdose). Expired air was collected up to 8-h postdose. Total radioactivity (blood, plasma, urine, feces, and expired air) and fedratinib concentrations (plasma) were measured. RESULTS: Approximately 77% (23% unchanged) of fedratinib derived radioactivity was excreted in feces and 5% (3% unchanged) was excreted in urine. Excretion via expired air was negligible. The time to maximum concentration for both total radioactivity and parent drug was similar, with unchanged drug representing the majority of the circulating radioactivity. The ratio of blood to plasma concentration of radioactivity ranged from 0.615 to 0.753 indicating limited distribution of fedratinib and/or its metabolites into red blood cells. CONCLUSIONS: Fedratinib derived radioactivity was primarily excreted in feces following a single oral dose of radiolabeled fedratinib to healthy subjects.

Assessment of drug-drug interaction potential and PBPK modeling of CC-223, a potent inhibitor of the mammalian target of rapamycin kinase.

1. CC-223 was studied in vitro for metabolism and drug-drug interactions (DDI), and in clinic for interaction with ketoconazole. 2. In vitro, human metabolites of CC-223 included O-desmethyl CC-223 (M1), keto (M2), N-oxide (M3) and imine (M13), with M1 being the most prominent metabolite. 3. CC-223 was metabolized by CYP2C9 and CYP3A, while metabolism of M1 was mediated by CYP2C8 and CYP3A. Ketoconazole increased CC-223 and M1 exposure by 60-70% in healthy volunteers. 4. CC-223 (IC50 ≥ 27 µM) and M1 (IC50 ≥ 46 µM) were inhibitors of CYP2C9 and CYP2C19 in human liver microsomes. CC-223 and M1 were moderate inducers of CYP3A in human hepatocytes. 5. CC-223 was a substrate of BCRP, and M1 was a substrate of P-gp and BCRP. CC-223 was an inhibitor of P-gp (IC50 = 3.67 µM) and BCRP (IC50 = 11.7 µM), but at a clinically relevant concentration showed no inhibition of other transporters examined. M1 is a weak inhibitor of P-gp and BCRP. 6. PBPK model of CC-223 and M1 was developed and verified using clinical results. Model based predictions of DDI with ketoconazole were in agreement with observed results enabling prospective predictions of DDIs between CC-223 and CYP3A4 inhibitors.

Absorption, distribution, metabolism and excretion of an isocitrate dehydrogenase-2 inhibitor enasidenib in rats and humans.

1. The absorption, distribution, metabolism and excretion of enasidenib were studied following a single oral dose of [14C]enasidenib to rats (10 mg/kg; 100 µCi/kg) and healthy volunteers (100 mg; 318 nCi). 2. Enasidenib was readily absorbed, extensively metabolized and primarily eliminated via the hepatobiliary pathway. Enasidenib-derived radioactivity was widely distributed in rats. Excretion of radioactivity was approximately 95-99% of the dose from rats in 168 h post-dose and 82.4% from human volunteers in 504 h post-dose. In rat bile, approximately 35-42% of the administered dose was recovered, with less than 5% of the dose excreted as the parent drug. Renal elimination was a minor pathway, with <12% of the dose excreted in rat urine and <10% of the dose excreted in human urine. 3. Enasidenib was the prominent radioactive component in rat and human systemic circulation. Enasidenib was extensively metabolized in rats and human volunteers through N-dealkylation, oxidation, direct glucuronidation and combinations of these pathways. Glucuronidation was the major metabolic pathway in rats while N-dealkylation was the prominent metabolic pathway in human volunteers. All human metabolites were detected in rats.

Absorption, distribution, metabolism, and excretion of mTOR kinase inhibitor CC-223 in rats, dogs, and humans.

1. The absorption, distribution, metabolism, and excretion of CC-223 were studied following a single oral dose of [14C]CC-223 to rats (3 mg/kg; 90 µCi/kg), dogs (1.5 mg/kg; 10 µCi/kg), and healthy volunteers (20 mg; 200 nCi). 2. CC-223-derived radioactivity was widely distributed in rats. Excretion of radioactivity was rapid and nearly complete from rats (87%), dogs (78%), and humans (97%). Feces was the major excretion pathway for rats (67%) and dogs (70%), whereas urine (57.6%) was the major elimination route for humans. Urine and bile each contained approximately 20% administered radioactivity in rats, whereas bile (20%) played a more important role than urine (<10%) in the excretion of absorbed radioactivity in dogs. Based on excretion data, CC-223 had good absorption, with greater than 56%, 29%, and 57% of the oral dose absorbed in rats, dogs, and humans, respectively. 3. CC-223 was the prominent radioactive component in circulation of rats (>71% of the exposure to total radioactivity) and dogs (≥45.5%), whereas M1 (76.5%) was the predominant circulating metabolite in humans. M1 and M1-derived metabolites accounted for >66% of human dose. CC-223 was extensively metabolized in rats, dogs, and humans through glucuronidation, O-demethylation, oxidation, and combinations of these pathways.

In vitro inhibition of human nucleoside transporters and uptake of azacitidine by an isocitrate dehydrogenase-2 inhibitor enasidenib and its metabolite AGI-16903.

1. The present study investigated inhibitory effects of enasidenib and its metabolite AGI-16903 on (a) recombinant human nucleoside transporters (hNTs) in hNT-producing Xenopus laevis oocytes, and (b) azacitidine uptake in a normal B-lymphoblast peripheral blood cell line (PBC) and acute myeloid leukemia (AML) cell lines. 2. Enasidenib inhibited hENT1, hENT2, hENT3, and hENT4 in oocytes with IC50 values of 7, 63, 27, and 76 µM, respectively, but exhibited little inhibition of hCNT1-3. AGI-16903 exhibited little inhibition of any hNT produced in oocytes. 3. Azacitidine uptake was more than 2-fold higher in AML cells than in PBC. Enasidenib inhibited azacitidine uptake into OCI-AML2, TF-1 and PBC cells in a concentration-dependent manner with IC50 values of 0.27, 1.7, and 1.0 µM in sodium-containing transport medium, respectively. 4. IC50 values shifted approximately 100-fold higher when human plasma was used as the incubation medium (27 µM in OCI-AML2, 162 µM in TF-1, and 129 µM in PBC), likely due to high human plasma protein binding of enasidenib (98.5% bound). 5. Although enasidenib inhibits hENTs and azacitidine uptake in vitro, plasma proteins attenuate this inhibitory effect, likely resulting in no meaningful in vivo effects in humans.

2017 White Paper on recent issues in bioanalysis: aren't BMV guidance/guidelines 'Scientific'? (Part 1 - LCMS: small molecules, peptides and small molecule biomarkers).

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for Small Molecules, Peptides and Small Molecule Biomarkers using LCMS. Part 2 (Biotherapeutics, Biomarkers and Immunogenicity Assays using Hybrid LBA/LCMS and Regulatory Agencies' Inputs) and Part 3 (LBA: Immunogenicity, Biomarkers and PK Assays) are published in volume 9 of Bioanalysis, issues 23 and 24 (2017), respectively.

Application of a "Fit for Purpose" PBPK Model to Investigate the CYP3A4 Induction Potential of Enzalutamide.

BACKGROUND: Oncology therapy typically involves drug combinations since monotherapy seldom provides the desired outcome. But combination therapy presents the potential for drug-drug interactions (DDIs). Due to the narrow window between therapeutic concentrations and onset of toxicity often observed with oncology therapeutics, managing DDIs with combination therapy in cancer is critical. Physiologically based pharmacokinetic (PBPK) modeling can be effectively used for predicting DDIs and guiding dose-selection, but requires development of PBPK models of cancer drugs. Among various types of cancer, metastatic prostate cancer is an area of high unmet medical need with minimal therapeutic options. Recently, enzalutamide was approved for treatment of metastatic prostate cancer and is often dosed as a combination in clinical practice. Enzalutamide is a potent CYP3A inducer and a model-based approach to guide dose-selection for enzalutamide combinations that are CYP3A substrates is needed. OBJECTIVE: A "fit for purpose" PBPK model of enzalutamide was developed to illustrate the CYP3A4 induction potential, understand the kinetics of de-induction of CYP3A4 following cessation of enzalutamide dosing and guide dose-selection of a co-administered CYP3A substrate. METHOD: The population-based simulator, Simcyp, was used for model building purposes. Model input parameters were obtained from public information, primarily from the FDA summaries. RESULTS: The simulated concentration time profiles of enzalutamide in healthy male subjects were comparable to observed profiles in male patients. Model predicted enzalutamide pharmacokinetic (PK) parameters, i.e. AUC, Cmax and half-life were within 1.5-fold of observed results obtained from two reported studies, supporting verification of the PBPK model. Model application was demonstrated by simulating a drug-drug interaction between enzalutamide and midazolam, a sensitive CYP3A4 substrate. Based on simulations, the midazolam AUC ratio ranged from 0.06 to 0.16 and was comparable to the observed ratio of 0.14. Based on modeling, upon cessation of enzalutamide dosing, it is predicted that at least 8 weeks are needed to re-attain baseline CYP3A4 activity. Based on PBPK modeling, dose adjustment of up to 3-fold for a co-administered CYP3A substrate was shown to re-attain baseline exposure. CONCLUSION: A "fit for purpose" PBPK model of enzalutamide was successfully developed using public information that recapitulated it's observed pharmacokinetics, CYP3A4 induction potential and the potential need for dose-adjustment of co-administered CYP3A substrates.

The Uremic Toxin Indoxyl-3-Sulfate Induces CYP1A2 In Primary Human Hepatocytes.

Chronic kidney disease (CKD) generally impacts clearance of renally eliminated drugs but growing evidence shows that it can influence clearance of hepatically eliminated drugs and a complete mechanistic understanding of this phenomenon is still lacking. CKD leads to accumulation of uremic toxins, including indoxyl- 3-sulfate (3-INDS) and indole-3-acetic acid (3-IAA). OBJECTIVE: In this study, we evaluated the potential of 3-INDS and 3-IAA (10, 30 and 100 µM) to induce liver cytochrome P450 (CYP) enzymes CYP1A2, 2B6 and 3A4/5 using cultured primary human hepatocytes following once daily treatment for 3 days. RESULTS: 3-INDS potently induced CYP1A2 mRNA and enzyme activity in a dose-dependent manner but did not induce CYP2B6 or 3A4. At 100 µM, a concentration observed in humans under uremic conditions, 3-INDS increased CYP1A2 mRNA and activity by 93% and 292% respectively when compared with prototypical inducer omeprazole. However, 3-IAA did not induce CYP1A2, 2B6 or 3A4. CONCLUSION: These results suggest that the uremic toxin, 3-INDS, is a potent CYP1A2 inducer and lends valuable mechanistic basis for how kidney disease can affect hepatic metabolism.