A phase I study of ATR inhibitor gartisertib (M4344) as a single agent and in combination with carboplatin in patients with advanced solid tumours.

BACKGROUND: Gartisertib is an oral inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), a key kinase of the DNA damage response. We aimed to determine the safety and tolerability of gartisertib ± carboplatin in patients with advanced solid tumours. METHODS: This phase I open-label, multicenter, first-in-human study comprised four gartisertib cohorts: A (dose escalation [DE]; Q2W); A2 (DE; QD/BID); B1 (DE+carboplatin); and C (biomarker-selected patients). RESULTS: Overall, 97 patients were enroled into cohorts A (n = 42), A2 (n = 26), B1 (n = 16) and C (n = 13). The maximum tolerated dose and recommended phase II dose (RP2D) were not declared for cohorts A or B1. In cohort A2, the RP2D for gartisertib was determined as 250 mg QD. Gartisertib was generally well-tolerated; however, unexpected increased blood bilirubin in all study cohorts precluded further DE. Investigations showed that gartisertib and its metabolite M26 inhibit UGT1A1-mediated bilirubin glucuronidation in human but not dog or rat liver microsomes. Prolonged partial response (n = 1 [cohort B1]) and stable disease >6 months (n = 3) did not appear to be associated with biomarker status. Exposure generally increased dose-dependently without accumulation. CONCLUSION: Gartisertib was generally well-tolerated at lower doses; however, unexpected liver toxicity prevented further DE, potentially limiting antitumour activity. Gartisertib development was subsequently discontinued. CLINICALTRIALS: GOV: NCT02278250.

Challenges and Opportunities for In Vitro-In Vivo Extrapolation of Aldehyde Oxidase-Mediated Clearance: Toward a Roadmap for Quantitative Translation.

Underestimation of aldehyde oxidase (AO)-mediated clearance by current in vitro assays leads to uncertainty in human dose projections, thereby reducing the likelihood of success in drug development. In the present study we first evaluated the current drug development practices for AO substrates. Next, the overall predictive performance of in vitro-in vivo extrapolation of unbound hepatic intrinsic clearance (CLint,u) and unbound hepatic intrinsic clearance by AO (CLint,u,AO) was assessed using a comprehensive literature database of in vitro (human cytosol/S9/hepatocytes) and in vivo (intravenous/oral) data collated for 22 AO substrates (total of 100 datapoints from multiple studies). Correction for unbound fraction in the incubation was done by experimental data or in silico predictions. The fraction metabolized by AO (fmAO) determined via in vitro/in vivo approaches was found to be highly variable. The geometric mean fold errors (gmfe) for scaled CLint,u (mL/min/kg) were 10.4 for human hepatocytes, 5.6 for human liver cytosols, and 5.0 for human liver S9, respectively. Application of these gmfe's as empirical scaling factors improved predictions (45%-57% within twofold of observed) compared with no correction (11%-27% within twofold), with the scaling factors qualified by leave-one-out cross-validation. A road map for quantitative translation was then proposed following a critical evaluation on the in vitro and clinical methodology to estimate in vivo fmAO In conclusion, the study provides the most robust system-specific empirical scaling factors to date as a pragmatic approach for the prediction of in vivo CLint,u,AO in the early stages of drug development. SIGNIFICANCE STATEMENT: Confidence remains low when predicting in vivo clearance of AO substrates using in vitro systems, leading to de-prioritization of AO substrates from the drug development pipeline to mitigate risk of unexpected and costly in vivo impact. The current study establishes a set of empirical scaling factors as a pragmatic tool to improve predictability of in vivo AO clearance. Developing clinical pharmacology strategies for AO substrates by utilizing mass balance/clinical drug-drug interaction data will help build confidence in fmAO.

Evobrutinib pathway to its major metabolite M463-2 and insights from a biotransformation and DDI perspective.

Evobrutinib is a highly selective, covalent, central nervous system-penetrant Bruton's tyrosine kinase (BTK) inhibitor, currently in Phase III trials for the treatment of relapsing multiple sclerosis. One major circulating metabolite of evobrutinib has been previously identified as the racemic dihydro-diol M463-2 (MSC2430422) in a Phase I human mass balance study.Phenotyping experiments were conducted to confirm the metabolic pathway of evobrutinib to M463-2. Ratio of the enantiomers was determined by enantioselective liquid chromatography with tandem mass spectrometry analysis of plasma samples from humans and preclinical species. Drug-drug interaction (DDI) characterisation, evaluation of pharmacological activity on BTK, and off-target screening experiments followed assessing safety of the metabolite.The biotransformation of evobrutinib to M463-2 was determined to be a two-step process with a CYP-mediated oxidation acting to form an epoxide intermediate, which was further hydrolysed by soluble and mitochondrial epoxide hydrolase. Only the (S)-enantiomer was determined to be a major metabolite, the (R)-enantiomer was minor. In vitro studies demonstrated the (S)-enantiomer lacked clinically relevant pharmacological activity, off-target effects and DDIs.The biotransformation of evobrutinib to its major metabolite has been elucidated, with the major (S)-enantiomer being shown to pose no on/off target or DDI risks.

Absorption, Distribution, Metabolism, and Excretion of Therapeutic Proteins: Current Industry Practices and Future Perspectives.

Therapeutic proteins (TPs) comprise a variety of modalities, including antibody-based drugs, coagulation factors, recombinant cytokines, enzymes, growth factors, and hormones. TPs usually cannot traverse cellular barriers and exert their pharmacological activity by interacting with targets on the exterior membrane of cells or with soluble ligands in the tissue interstitial fluid/blood. Due to their large size, lack of cellular permeability, variation in metabolic fate, and distinct physicochemical characteristics, TPs are subject to different absorption, distribution, metabolism, and excretion (ADME) processes as compared with small molecules. Limited regulatory guidance makes it challenging to determine the most relevant ADME data required for regulatory submissions. The TP ADME working group was sponsored by the Translational and ADME Sciences Leadership Group within the Innovation and Quality (IQ) consortium with objectives to: (1) better understand the current practices of ADME data generated for TPs across IQ member companies, (2) learn about their regulatory strategies and interaction experiences, and (3) provide recommendations on best practices for conducting ADME studies for TPs. To understand current ADME practices and regulatory strategies, an industry-wide survey was conducted within IQ member companies. In addition, ADME data submitted to the U.S. Food and Drug Administration was also collated by reviewing regulatory submission packages of TPs approved between 2011 and 2020. This article summarizes the key learnings from the survey and an overview of ADME data presented in biologics license applications along with future perspectives and recommendations for conducting ADME studies for internal decision-making as well as regulatory submissions for TPs. SIGNIFICANCE STATEMENT: This article provides comprehensive assessment of the current practices of absorption, distribution, metabolism, and excretion (ADME) data generated for therapeutic proteins (TPs) across the Innovation and Quality participating companies and the utility of the data in discovery, development, and regulatory submissions. The TP ADME working group also recommends the best practices for condu-cting ADME studies for internal decision-making and regulatory submissions.

Metabolism and Disposition of [14C]Pevonedistat, a First-in-Class NEDD8-Activating Enzyme Inhibitor, after Intravenous Infusion to Patients with Advanced Solid Tumors.

Metabolism and disposition of pevonedistat, an investigational, first-in-class inhibitor of the NEDD8-activating enzyme (NAE), were characterized in patients with advanced solid tumors after intravenous infusion of [14C]pevonedistat at 25 mg/m2 (∼60-85 µCi radioactive dose). More than 94% of the administered dose was recovered, with ∼41% and ∼53% of drug-related material eliminated in urine and feces, respectively. The metabolite profiles of [14C]pevonedistat were established in plasma using an accelerator mass spectrometer and excreta with traditional radiometric analysis. In plasma, unchanged parent drug accounted for approximately 49% of the total drug-related material. Metabolites M1 and M2 were major (>10% of the total drug-related material) circulating metabolites and accounted for approximately 15% and 22% of the drug-related material, respectively. Unchanged [14C]pevonedistat accounted for approximately 4% and 17% of the dose in urine and feces, respectively. Oxidative metabolites M1, M2, and M3 appeared as the most abundant drug-related components in the excreta and represented approximately 27%, 26%, and 15% of the administered dose, respectively. Based on the unbound plasma exposure in cancer patients and in vitro NAE inhibition, the contribution of metabolites M1 and M2 to overall in vivo pharmacological activity is anticipated to be minimal. The exposure to these metabolites was higher at safe and well tolerated doses in rat and dog (the two preclinical species used in toxicology evaluation) plasma than that observed in human plasma. Reaction phenotyping studies revealed that CYP3A4/5 are primary enzymes responsible for the metabolic clearance of pevonedistat. SIGNIFICANCE STATEMENT: This study details the metabolism and clearance mechanisms of pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, after intravenous administration to patients with cancer. Pevonedistat is biotransformed to two major circulating metabolites with higher exposure in nonclinical toxicological species than in humans. The pharmacological activity contribution of these metabolites is minimal compared to the overall target pharmacological effect of pevonedistat. Renal clearance was not an important route of excretion of unchanged pevonedistat (∼4% of the dose).

Phase I study assessing the mass balance, pharmacokinetics, and excretion of [14C]-pevonedistat, a NEDD8-activating enzyme inhibitor in patients with advanced solid tumors.

Pevonedistat (TAK-924/MLN4924) is an investigational small-molecule inhibitor of the NEDD8-activating enzyme that has demonstrated preclinical and clinical activity across solid tumors and hematological malignancies. Here we report the results of a phase I trial characterizing the mass balance, pharmacokinetics, and clearance pathways of [14C]-pevonedistat in patients with advanced solid tumors (NCT03057366). In part A (n = 8), patients received a single 1-h intravenous infusion of [14C]-pevonedistat 25 mg/m2. In part B (n = 7), patients received pevonedistat 25 or 20 mg/m2 on days 1, 3, and 5 in combination with, respectively, docetaxel 75 mg/m2 or carboplatin AUC5 plus paclitaxel 175 mg/m2 on day 1 every 3 weeks. Following the single dose of [14C]-pevonedistat 25 mg/m2 in part A, there was a parallel log-linear decline in plasma and whole blood pevonedistat concentration, with systemic exposure of unchanged pevonedistat representing 41% of drug-related material (i.e., unchanged pevonedistat and its metabolites). The mean terminal half-life of pevonedistat and drug-related material in plasma was 8.4 and 15.6 h, respectively. Pevonedistat distributed preferentially in whole blood with a mean whole-blood-to-plasma ratio for pevonedistat AUC∞ of 40.8. By 1 week post dose, the mean recovery of administered radioactivity was 94% (41% in urine and 53% in feces). The pevonedistat safety profile during both study parts was consistent with previous clinical experience, with no new safety signals observed. In part B, pevonedistat in combination with docetaxel or carboplatin plus paclitaxel was generally well tolerated. ClinicalTrials.gov identifier: NCT03057366 .

Metabolism of bioconjugate therapeutics: why, when, and how?

Bioconjugation of therapeutic agents has been used as a selective drug delivery platform for many therapeutic areas. Bioconjugates are prepared by the covalent linkage of active compounds (small or large molecule) to a carrier molecule (lipids, proteins, peptides, carbohydrates, and polymers) through a chemical linker. The linkage of the active component to a carrier molecule enhances the therapeutic window through a targeted delivery and by reducing toxicity. Bioconjugates also possess improved pharmacokinetic properties such as a long half-life, increased stability, and cleavage by intracellular enzymes/environment. However, premature cleavage of the bioconjugates and the resulting metabolites/catabolites may produce undesirable toxic effects and, hence, it is critical to understand cleavage mechanisms, metabolism of bioconjugates, and translatability to human in the discovery stages. This article provides a comprehensive overview of linker cleavage pathways and catabolism/metabolism of antibody-drug conjugates, glycoconjugates, polymer-drug conjugates, lipid-drug conjugates, folate-targeted small molecule-drug conjugates, and drug-drug conjugates.

Pharmacokinetics and Catabolism of [3H]TAK-164, a Guanylyl Cyclase C Targeted Antibody-Drug Conjugate.

TAK-164 is an antibody-drug conjugate (ADC) comprising human anti-guanylyl cyclase C (GCC) monoclonal antibody conjugated to indolinobenzodiazepine DNA alkylator IGN-P1 through a cleavable alanine-alanine dipeptide linker. TAK-164 is currently being evaluated for the treatment of gastrointestinal cancers expressing GCC. The catabolism of TAK-164 was studied using 3H-labeled ADC using GCC-expressing HEK-293 (GCC-HEK-293) cells, rat tritosomes, cathepsin B, and tumor-bearing mice. Time- and target-dependent uptake of [3H]TAK-164 was observed in GCC-HEK-293 cells with approximately 12% of radioactivity associated with DNA after 24 hours of incubation. Rat liver tritosomes and cathepsin B yielded IGN-P1 aniline, sulfonated IGN-P1 (s-IGN-P1) aniline, and a lysine conjugate of IGN-P1 (IGN-P1-Lys) aniline as catabolites. In tumor-bearing mice, [3H]TAK-164 exhibited a terminal half-life of approximately 41 and 51 hours in plasma and blood, respectively, with low plasma clearance (0.75 ml/h per kilogram). The extractable radioactivity in plasma and tumor samples revealed the presence of s-IGN-P1 aniline and IGN-P1 aniline as payload-related components. The use of a radiolabeled payload in the ADC in tumor uptake investigations provided direct and quantitative evidence for tumor uptake, DNA binding, and proof of mechanism of action of the payload. SIGNIFICANCE STATEMENT: Since payload-related species are potent cytotoxins, a thorough characterization of released products of ADCs, metabolites, and their drug interaction potential is necessary prior to clinical investigations. This study characterized in vitro and in vivo DNA binding mechanisms and released products of TAK-164. The methodologies described here will be highly useful for characterization of payload-related products of ADCs in general.

Prediction of Human Disproportionate and Biliary Excreted Metabolites Using Chimeric Mice with Humanized Liver.

The PXB-mouse is potentially a useful in vivo model to predict human hepatic metabolism and clearance. Four model compounds, [14C]desloratadine, [3H]mianserin, cyproheptadine, and [3H]carbazeran, all reported with disproportionate human metabolites, were orally administered to PXB- or control SCID mice to elucidate the biotransformation of each of them. For [14C]desloratadine in PXB-mice, O-glucuronide of 3-hydroxydesloratadine was observed as the predominant metabolite in both the plasma and urine. Both 3-hydroxydesloratadine and its O-glucuronide were detected as major drug-related materials in the bile, whereas only 3-hydroxydesloratadine was detected in the feces, suggesting that a fraction of 3-hydroxydesloratadine in feces was derived from deconjugation of its O-glucuronide by gut microflora. This information can help understand the biliary clearance mechanism of a drug and may fill the gap in a human absorption, distribution, metabolism, and excretion study, in which the bile samples are typically not available. The metabolic profiles in PXB-mice were qualitatively similar to those reported in humans in a clinical study in which 3-hydroxydesloratadine and its O-glucuronide were major and disproportionate metabolites compared with rat, mouse, and monkey. In the control SCID mice, neither of the metabolites was detected in any matrix. Similarly, for the other three compounds, all human specific or disproportionate metabolites were detected at a high level in PXB-mice, but they were either minimally observed or not observed in the control mice. Data from these four compounds indicate that studies in PXB-mice can help predict the potential for the presence of human disproportionate metabolites (relative to preclinical species) prior to conducting clinical studies and understand the biliary clearance mechanism of a drug. SIGNIFICANCE STATEMENT: Studies in PXB-mice have successfully predicted the human major and disproportionate metabolites compared with preclinical safety species for desloratadine, mianserin, cyproheptadine, and carbazeran. In addition, biliary excretion data from PXB-mice can help illustrate the human biliary clearance mechanism of a drug.

Catabolism of antibody drug conjugates and characterization methods.

Antibody drug conjugates (ADCs) are large molecule therapeutics in which a cytotoxic payload is conjugated to a monoclonal antibody (mAb) via a linker. The molecules are designed to selectively bind to target-expressing cells, thus delivering therapeutic agents directly to the tumor. Chemical and enzymatic stability prior to reaching the target is an important factor for ADCs since it impacts their safety, efficacy, and pharmacokinetics (PK). One of the main reasons for off-target effects of ADCs is premature release of cytotoxic agents, either in the blood stream or at non-specific sites. Once an ADC is internalized by target-expressing cells, the cytotoxic payload and/or related catabolites are released through chemical or enzymatic cleavage within the cells. In some cases, the released payload and/or catabolites are effluxed into the systemic circulation and follow a small molecule disposition path. Since doses of ADCs are low, the concentration of cytotoxic payload and related catabolites/metabolites range from ng to µg levels in systemic circulation or tumors in clinical studies. Hence, it is challenging to identify these species without prior knowledge of the pathways of catabolism. The current review summarizes the mechanism of cleavage/catabolism of various types of linkers and available in vitro, in vivo, and bioanalytical methods for evaluation of catabolism of ADCs.

Carbon-carbon bond cleavage and formation reactions in drug metabolism and the role of metabolic enzymes.

Elimination of xenobiotics from the human body is often facilitated by a transformation to highly water soluble and more ionizable molecules. In general, oxidation-reduction, hydrolysis, and conjugation reactions are common biotransformation reactions that are catalyzed by various metabolic enzymes including cytochrome P450s (CYPs), non-CYPs, and conjugative enzymes. Although carbon-carbon (C-C) bond formation and cleavage reactions are known to exist in plant secondary metabolism, these reactions are relatively rare in mammalian metabolism and are considered exceptions. However, various reactions such as demethylation, dealkylation, dearylation, reduction of alkyl chain, ring expansion, ring contraction, oxidative elimination of a nitrile through C-C bond cleavage, and dimerization, and glucuronidation through C-C bond formation have been reported for drug molecules. Carbon-carbon bond cleavage reactions for drug molecules are primarily catalyzed by CYP enzymes, dimerization is mediated by peroxidases, and C-glucuronidation is catalyzed by UGT1A9. This review provides an overview of C-C bond cleavage and formation reactions in drug metabolism and the metabolic enzymes associated with these reactions.

Biotransformation and bioactivation reactions of alicyclic amines in drug molecules.

Aliphatic nitrogen heterocycles such as piperazine, piperidine, pyrrolidine, morpholine, aziridine, azetidine, and azepane are well known building blocks in drug design and important core structures in approved drug therapies. These core units have been targets for metabolic attack by P450s and other drug metabolizing enzymes such as aldehyde oxidase and monoamine oxidase (MAOs). The electron rich nitrogen and/or α-carbons are often major sites of metabolism of alicyclic amines. The most common biotransformations include N-oxidation, N-conjugation, oxidative N-dealkylation, ring oxidation, and ring opening. In some instances, the metabolic pathways generate electrophilic reactive intermediates and cause bioactivation. However, potential bioactivation related adverse events can be attenuated by structural modifications. Hence it is important to understand the biotransformation pathways to design stable drug candidates that are devoid of metabolic liabilities early in the discovery stage. The current review provides a comprehensive summary of biotransformation and bioactivation pathways of aliphatic nitrogen containing heterocycles and strategies to mitigate metabolic liabilities.

Implementation of whole-exome sequencing for pharmacogenomics profiling and exploring its potential clinical utilities.

Whole-exome sequencing (WES) is widely used in clinical settings; however, the exploration of its use in pharmacogenomic analysis remains limited. Our study compared the variant callings for 28 core absorption, distribution, metabolism and elimination genes by WES and array-based technology using clinical trials samples. The results revealed that WES had a positive predictive value of 0.71-0.92 and a sensitivity of single-nucleotide variants between 0.68 and 0.95, compared with array-based technology, for the variants in the commonly targeted regions of the WES and PhamacoScan™ assay. Besides the common variants detected by both assays, WES identified 200-300 exclusive variants per sample, totalling 55 annotated exclusive variants, including important modulators of metabolism such as rs2032582 (ABCB1) and rs72547527 (SULT1A1). This study highlights the potential clinical advantages of using WES to identify a wider range of genetic variations and enabling precision medicine.

Asia-inclusive drug development leveraging principles of ICH E5 and E17 guidelines: Case studies illustrating quantitative clinical pharmacology as a foundational enabler.

With the International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) E17 guidelines in effect from 2018, the design of Asia-inclusive multiregional clinical trials (MRCTs) has been streamlined, thereby enabling efficient simultaneous global development. Furthermore, with the recent regulatory reforms in China and its drug administration joining the ICH as a full regulatory member, early participation of China in the global clinical development of novel investigational drugs is now feasible. This would also allow for inclusion of the region in the geographic footprint of pivotal MRCTs leveraging principles of the ICH E5 and E17. Herein, we describe recent case examples of model-informed Asia-inclusive global clinical development in the EMD Serono portfolio, as applied to the ataxia telangiectasia and Rad3-related inhibitors, tuvusertib and berzosertib (oncology), the toll-like receptor 7/8 antagonist, enpatoran (autoimmune diseases), the mesenchymal-epithelial transition factor inhibitor tepotinib (oncology), and the antimetabolite cladribine (neuroimmunological disease). Through these case studies, we illustrate pragmatic approaches to ethnic sensitivity assessments and the application of a model-informed drug development toolkit including population pharmacokinetic/pharmacodynamic modeling and pharmacometric disease progression modeling and simulation to enable early conduct of Asia-inclusive MRCTs. These examples demonstrate the value of a Totality of Evidence approach where every patient's data matter for de-risking ethnic sensitivity to inter-population variations in drug- and disease-related intrinsic and extrinsic factors, enabling inclusive global development strategies and timely evidence generation for characterizing benefit/risk of the proposed dosage in Asian populations.

Establishing a physiologically based pharmacokinetic framework for aldehyde oxidase and dual aldehyde oxidase-CYP substrates.

Aldehyde oxidase (AO) contributes to the clearance of many approved and investigational small molecule drugs, which are often dual substrates of AO and drug-metabolizing enzymes such as cytochrome P450s (CYPs). As such, the lack of established framework for quantitative translation of the clinical pharmacologic correlates of AO-mediated clearance represents an unmet need. This study aimed to evaluate the utility of physiologically based pharmacokinetic (PBPK) modeling in the development of AO and dual AO-CYP substrates. PBPK models were developed for capmatinib, idelalisib, lenvatinib, zaleplon, ziprasidone, and zoniporide, incorporating in vitro functional data from human liver subcellular fractions and human hepatocytes. Prediction of metabolic elimination with/without the additional empirical scaling factors (ESFs) was assessed. Clinical pharmacokinetics, human mass balance, and drug-drug interaction (DDI) studies with CYP3A4 modulators, where available, were used to refine/verify the models. Due to the lack of clinically significant AO-DDIs with known AO inhibitors, the fraction metabolized by AO (fmAO) was verified indirectly. Clearance predictions were improved by using ESFs (GMFE ≤1.4-fold versus up to fivefold with physiologically-based scaling only). Observed fmi from mass balance studies were crucial for model verification/refinement, as illustrated by capmatinib, where the fmAO (40%) was otherwise underpredicted up to fourfold. Subsequently, independent DDI studies with ketoconazole, itraconazole, rifampicin, and carbamazepine verified the fmCYP3A4, with predicted ratios of the area under the concentration-time curve (AUCR) within 1.5-fold of the observations. In conclusion, this study provides a novel PBPK-based framework for predicting AO-mediated pharmacokinetics and quantitative assessment of clinical DDI risks for dual AO-CYP substrates within a totality-of-evidence approach.

Building Confidence in Physiologically Based Pharmacokinetic Modeling of CYP3A Induction Mediated by Rifampin: An Industry Perspective.

Physiologically-based pharmacokinetic (PBPK) modeling offers a viable approach to predict induction drug-drug interactions (DDIs) with the potential to streamline or reduce clinical trial burden if predictions can be made with sufficient confidence. In the current work, the ability to predict the effect of rifampin, a well-characterized strong CYP3A4 inducer, on 20 CYP3A probes with publicly available PBPK models (often developed using a workflow with optimization following a strong inhibitor DDI study to gain confidence in fraction metabolized by CYP3A4, fm,CYP3A4, and fraction available after intestinal metabolism, Fg), was assessed. Substrates with a range of fm,CYP3A4 (0.086-1.0), Fg (0.11-1.0) and hepatic availability (0.09-0.96) were included. Predictions were most often accurate for compounds that are not P-gp substrates or that are P-gp substrates but that have high permeability. Case studies for three challenging DDI predictions (i.e., for eliglustat, tofacitinib, and ribociclib) are presented. Along with parameter sensitivity analysis to understand key parameters impacting DDI simulations, alternative model structures should be considered, for example, a mechanistic absorption model instead of a first-order absorption model might be more appropriate for a P-gp substrate with low permeability. Any mechanisms pertinent to the CYP3A substrate that rifampin might impact (e.g., induction of other enzymes or P-gp) should be considered for inclusion in the model. PBPK modeling was shown to be an effective tool to predict induction DDIs with rifampin for CYP3A substrates with limited mechanistic complications, increasing confidence in the rifampin model. While this analysis focused on rifampin, the learnings may apply to other inducers.

Glycyrrhiza uralensis flavonoids present in anti-asthma formula, ASHMI™, inhibit memory Th2 responses in vitro and in vivo.

Allergic asthma is associated with Th2-mediated inflammation. Several flavonoids were isolated from Glycyrrhiza uralensis, one of the herbs in the anti-asthma herbal medicine intervention. The aim of this investigation was to determine whether Glycyrrhiza uralensis flavonoids have inhibitory effects on memory Th2 responses in vitro and antigen-induced Th2 inflammation in vivo. The effects of three Glycyrrhiza uralensis flavonoids on effector memory Th2 cells, D10.G4.1 (D10 cells), were determined by measuring Th2 cytokine production. Isoliquiritigenin, 7, 4'-dihydroxyflavone (7, 4'-DHF) and liquiritigenin significantly suppressed IL-4 and IL-5 production in a dose-dependent manner, 7, 4'-DHF being most potent. It was also evaluated for effects on D10 cell proliferation, GATA-3 expression and IL-4 mRNA expression, which were suppressed, with no loss of cell viability. Chronic treatment with 7, 4'-DHF in a murine model of allergic asthma not only significantly reduced eosinophilic pulmonary inflammation, serum IgE levels, IL-4 and IL-13 levels, but also increased IFN-γ production in lung cell cultures in response to antigen stimulation.

Identification of metabolites in Withania sominfera fruits by liquid chromatography and high-resolution mass spectrometry.

RATIONALE: Withania somnifera is a rich source of biologically active secondary metabolites. Our earlier investigations on the fruits of this plant have yielded novel compounds, withanamides, with potent antioxidant activity and protective effect on ß-amyloid-induced cytotoxicity in vitro. However, several minor compounds present in the fruits have not been characterized previously which may contribute to the observed biological activities. METHODS: Liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) with data-dependent and targeted MS/MS experiments were conducted to elucidate the structure of observed metabolites. RESULTS: A total of 62 metabolites identified included 32 withanamides, 22 withanolides, 3 steroidal saponins, 2 lignanamides, feruloyl tyramine, methoxy feruloyl tyramine and a diglucoside of hydroxyl palmitic acid. The structures of these compounds were proposed based on accurate masses of the molecular and fragment ions. Several of these new compounds identified from the profile were derived from withanamides with variations in aliphatic and/or glycosyl moieties. In addition, six new withanolides, a new hydroxy fatty acid diglucoside and several known compounds in the extract were identified. CONCLUSIONS: The current study revealed the presence of several new and known compounds in Withania somnifera fruits. High-resolution MS and MS/MS experiments provide an extremely effective approach to derive the structures of plant secondary metabolites including isomeric compounds.

Alternatives to rifampicin: A review and perspectives on the choice of strong CYP3A inducers for clinical drug-drug interaction studies.

N-Nitrosamine (NA) impurities are considered genotoxic and have gained attention due to the recall of several marketed drug products associated with higher-than-permitted limits of these impurities. Rifampicin is an index inducer of multiple cytochrome P450s (CYPs) including CYP2B6, 2C8, 2C9, 2C19, and 3A4/5 and an inhibitor of OATP1B transporters (single dose). Hence, rifampicin is used extensively in clinical studies to assess drug-drug interactions (DDIs). Despite NA impurities being reported in rifampicin and rifapentine above the acceptable limits, these critical anti-infective drugs are available for therapeutic use considering their benefit-risk profile. Reports of NA impurities in rifampicin products have created uncertainty around using rifampicin in clinical DDI studies, especially in healthy volunteers. Hence, a systematic investigation through a literature search was performed to determine possible alternative index inducer(s) to rifampicin. The available strong CYP3A inducers were selected from the University of Washington DDI Database and their in vivo DDI potential assessed using the data from clinical DDI studies with sensitive CYP3A substrates. To propose potential alternative CYP3A inducers, factors including lack of genotoxic potential, adequate safety, feasibility of multiple dose administration to healthy volunteers, and robust in vivo evidence of induction of CYP3A were considered. Based on the qualifying criteria, carbamazepine, phenytoin, and lumacaftor were identified to be the most promising alternatives to rifampicin for conducting CYP3A induction DDI studies. Strengths and limitations of the proposed alternative CYP3A inducers, the magnitude of in vivo CYP3A induction, appropriate study designs for each alternative inducer, and future perspectives are presented in this paper.

Current Practices, Gap Analysis, and Proposed Workflows for PBPK Modeling of Cytochrome P450 Induction: An Industry Perspective.

The International Consortium for Innovation and Quality (IQ) Physiologically Based Pharmacokinetic (PBPK) Modeling Induction Working Group (IWG) conducted a survey across participating companies around general strategies for PBPK modeling of induction, including experience with its utility to address various questions, regulatory interactions, and regulatory acceptance. The results highlight areas where PBPK modeling is used with high confidence and identifies opportunities where confidence is lower and further evaluation is needed. To enhance the survey results, the PBPK-IWG also collected case studies and analyzed recent literature examples where PBPK models were applied to predict CYP3A induction-mediated drug-drug interactions. PBPK modeling of induction has evolved and progressed significantly, proving to have great potential to accelerate drug discovery and development. With the aim of enabling optimal use for new molecular entities that are either substrates and/or inducers of CYP3A, the PBPK-IWG proposes initial workflows for PBPK application, discusses future trends, and identifies gaps that need to be addressed.