Human Liver Microsomes Research Articles

Non-additivity of the functional properties of individual P450 species and its manifestation in the effects of alcohol consumption on the metabolism of ketamine and amitriptyline

To explore functional interconnections between multiple P450 enzymes and their manifestation in alcohol-induced changes in drug metabolism, we implemented a high-throughput study of correlations between the composition of the P450 pool and the substrate saturation profiles (SSP) of amitriptyline and ketamine demethylation in a series of 23 individual human liver microsomes preparations from donors with a known history of alcohol consumption. The SSPs were approximated with linear combinations of three Michaelis-Menten equations with globally optimized KM (substrate affinity) values. This analysis revealed a strong correlation between the rate of ketamine metabolism and alcohol exposure. For both substrates, alcohol consumption caused a significant increase in the role of the low-affinity enzymes. The amplitudes of the kinetic components and the total rate were further analyzed for correlations with the abundance of 11 major P450 enzymes assessed by global proteomics. The maximal rate of metabolism of both substrates correlated with the abundance of CYP3A4, their predicted principal metabolizer. However, except for CYP2D6 and CYP2E1, responsible for the low-affinity metabolism of ketamine and amitriptyline, respectively, none of the other potent metabolizers of the drugs revealed a positive correlation. Instead, in the case of ketamine, we observed negative correlations with the abundances of CYP1A2, CYP2C9, and CYP3A5. For amitriptyline, the data suggest inhibitory effects of CYP1A2 and CYP2A6. Our results demonstrate the importance of functional interactions between multiple P450 species and their decisive role in the effects of alcohol exposure on drug metabolism.

An Ultra-Fast Green UHPLC-MS/MS Method for Assessing the In Vitro Metabolic Stability of Dovitinib: In Silico Study for Absorption, Distribution, Metabolism, Excretion, Metabolic Lability, and DEREK Alerts.

Background and Objectives: Dovitinib (DVB) is a pan-tyrosine kinase inhibitor (TKI) that can be administered orally. In September 2023, the FDA granted Oncoheroes approval to proceed with an Investigational New Drug (IND) application for dovitinib. This application is intended for the treatment of relapsed or advanced juvenile solid tumors, namely, osteosarcoma. Materials and Methods: The target of the present study was to develop a rapid, green, accurate, and sensitive UHPLC-MS/MS method for measuring DVB levels in human liver microsomes (HLMs). The validations of the HLMs were performed via the established UHPLC-MS/MS approach, as stated in the US FDA reported guidelines for the standards of bioanalytical method validation protocol. The StarDrop in silico software package (version 6.6), which involves the DEREK and WhichP450 in silico modules, was used to check the DVB structure for hazardous alerts and metabolic instability. The DVB and encorafenib (EFB), internal standard, and chromatographic peaks were successfully separated using a reversed phase column (an Eclipse Plus Agilent C8 column) and an isocratic mobile phase. The production of DVB parent ions was accomplished by utilizing the positive ionization mode of an ESI source. The identification and measurement of DVB daughter ions were conducted using the MRM mode. Results: The inter-day accuracy and precision exhibited a spectrum of values in the range of -0.56% to 9.33%, while the intra-day accuracy and precision showcased a range of scores between 0.28% and 7.28%. The DVB calibration curve showed a linear relationship that ranged from 1 to 3000 ng/mL. The usefulness of the currently validated UHPLC-MS/MS method was approved by the lower limit of quantification (LLOQ) of 1 ng/mL. The AGREE findings demonstrate that the UHPLC-MS/MS method had a noteworthy degree of ecological greenness. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of DVB were calculated to be 15.48 min and 52.39 mL/min/kg, respectively, which aligned with the findings from the WhichP450 software (version 6.6). Conclusions: Via the usage of in silico software, it has been observed that making small changes to the structure of the aryl piperazine ring and quinolinone moieties, or replacing these groups in the drug design process, shows potential for enhancing the metabolic safety and stability of newly developed derivatives compared to DVB.

Metabolic profile characterization of voxelotor in human urine based on in vivo and in vitro models for doping control.

Voxelotor was approved for the treatment of sickle cell anemia as a potent hemoglobin S polymerization inhibitor. Owing to its ability to affect blood components and its potential to enhance athletic performance, voxelotor was included in the prohibited list issued by the World Anti-Doping Agency in 2023, banning its use both in and out of competition. This study aimed to comprehensively investigate the metabolic profile of voxelotor in human urine and identify suitable metabolites for long-term analytical retrospectivity in doping control. A novel strategy for metabolite identification was established by combining in vivo human administration with isotope labeling-based in vitro metabolism analysis. A single microdose of voxelotor was administered orally to five volunteers, and urine samples were collected for up to 28 days post-administration. Concurrently, in vitro incubation of human liver microsomes with voxelotor and D3-voxelotor was conducted, and the microsomal incubates were analyzed via liquid chromatography-high-resolution mass spectrometry. Targeted metabolite searches in human urine samples and automated nontargeted screening of isotope metabolite ion pairs in incubation samples led to the discovery of 9 phase I metabolites and 23 phase II metabolites. Analysis of the urine excretion curves revealed that 4 metabolites, along with voxelotor, were suitable for long-term anti-doping monitoring, with a detection window exceeding 20 days. Using both in vivo and in vitro metabolic models, this study provides comprehensive insight into the metabolic profile of voxelotor in human urine for the first time, enhancing the capacity for doping screening and extending the retrospectivity of voxelotor detection.

Truncated NPY-based NPY(Y1)R-specific radiopeptides: Improved in vivo PET tumor imaging by application of peptidase inhibitors

The neuropeptide Y receptor subtype 1 (NPY(Y1)R) exhibits high expression rates on human breast cancer and is therefore an important target structure for the sensitive and specific visualization and characterization of the disease by Positron Emission Tomography (PET). However, imaging of this receptor type has been of limited success so far due to the low stability of peptide-based NPY-derived NPY(Y1)R-specific radiotracer candidates due to in vivo degradation. Given the challenges in stabilizing these agents, our study sought to explore whether the stability of NPY(Y1)R-specific radiopeptides could be enhanced. We aimed to achieve this by either modifying the peptide structure with various molecular scaffolds or by applying peptidase inhibitors. This evaluation aimed to identify the optimal approach for achieving effective NPY(Y1)R-specific imaging in the following. To validate our approach, we systematically investigated four new truncated 68Ga-labeled analogs of NPY and the reference compound [68Ga]Ga-[Lys4(Nε-DOTA)]BVD15, bearing different molecular scaffolds such as a DOTA or NODA-GA chelator, a 4-APipAc linker, a Bip unit, and an N-terminal Lys(lauryl) group. The four new radiotracers as well as the reference compound were obtained in high chemical and radiochemical yields with molar activities of 33–39 GBq/μmol. The radiopeptides exhibited varying logD7.4 values, ranging from −3.37 ± 0.09 to +0.35 ± 0.11, and showed different levels of stability in human serum and liver microsomes, depending on their molecular structure. Subsequently, the influence of the peptidase inhibitors actinonin, phosphoramidon, captopril and E−64 on the in vitro stability of the radiotracers was investigated. In these studies, only actinonin demonstrated a positive effect on the stability of all radiopeptides. In contrast, phosphoramidon yielded variable results, and neither captopril nor E−64 showed a significant stabilizing effect.Consequently, the effect of actinonin administration on the in vivo PET/CT imaging results of the most promising ligand [68Ga]Ga-[Lys4(Nε-NODA-GA)]BVD15 ([68Ga]Ga-2) was investigated in a T47D tumor-bearing xenograft mouse model, followed by ex vivo biodistribution studies. In these experiments, the administration of 250 μg actinonin resulted in a significantly increased uptake of [68Ga]Ga-2 in the T47D tumor (5.9 ± 1.0 % ID/g (with actinonin) instead of 3.1 ± 0.9 % ID/g (without actinonin) at 2h p.i.), and an increase in tumor-to-muscle ratios from 1.8 to 4.0 upon co-administration of the inhibitor.The results impressively demonstrate the positive influence of actinonin on the in vivo stability of the NPY(Y1)R-specific radiopeptide [68Ga]Ga-2, resulting in an increased tumor accumulation and improved tumor-to-background ratios. These findings thus provide important incentive for further advancement of NPY(Y1)R-specific tumor imaging using PET.

Preclinical metabolism and metabolic drug-drug interaction profile of pedunculoside and rotundic acid.

Pedunculoside and rotundic acid, the most abundant components in plants of the genus Ilex L. (Aquifoliaceae), exhibit biological and pharmacological significance in the treatment of cardiovascular diseases. However, there have been few studies on their metabolism. This study performed a systematic metabolism study of pedunculoside and rotundic acid and evaluated their potential for herb-drug interaction. Pedunculoside or rotundic acid was incubated with human liver microsomes and recombinant human metabolic enzymes, and analyzed using LC-Q-TOF/MS and LC-MS/MS. Pedunculoside was found to be the most stable in human liver microsomes, whereas rotundic acid was easily metabolized. Eight pedunculoside metabolites and six rotundic acid metabolites were detected and tentatively identified through hydroxylation, glucuronidation, acetylation, and glucose conjugation. Hydroxylation of pedunculoside is mainly catalyzed by CYP3A4/5 and partly by CYP2C8. Hydroxylation of rotundic acid is almost exclusively catalyzed by CYP3A4/5, and its glucuronidation reaction is mediated by UGT1A4. Neither pedunculoside nor rotundic acid showed CYP inhibition (IC50 values > 50 μM) with the probe substrates of major CYP isoforms during incubation with human liver microsomes. This study is the first investigation into the invitro metabolism of pedunculoside and rotundic acid using human liver microsomes. It also aims to assess their potential as perpetrators of drug-drug interactions involving CYP enzymes. The comprehensive metabolism and drug interaction studies of pedunculoside and rotundic acid enable us to evaluate and manage potential risks with their use in pharmacotherapy.

Evidence for the formation of 6PPD-quinone from antioxidant 6PPD by cytochrome P450

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) as a rubber antioxidant has attracted global concern, since its ozone-oxidation product 6PPD-quinone (6PPDQ) was found to be the primary toxicant responsible for urban runoff mortality syndrome in coho salmon. However, the biotransformation fate and associated toxicological mechanism of 6PPD have not received much study yet. In this work, the in vitro assays showed 6PPD can be transformed into 6PPDQ by cytochromes P450 (CYP450) in human liver microsomes (HLMs) with 0.98 % production rate, and the adducts of 6PPDQ with calf thymus DNA and the N-N coupling product between 6PPD and 6PPDQ were further identified after 6PPD incubation in HLMs. Further evidence for the 6PPDQ formation can be obtained from the in vivo assays that the 6PPDQ-DNA adducts and 6PPD-N-N-6PPDQ dimer were detected in mice by oral gavage with 6PPD, and the latter dimer species was detected as well in 6PPD exposure to zebrafish larvae. Especially, the bioaccumulation property and high reactivity of 6PPDQ result in the continuous formation of the significant DNA adducts and 6PPD-N-N-6PPDQ dimer even in case of low production rate of biotransformation of 6PPD to 6PPDQ, which may provide potentially effective biomarkers for such process. DFT computations revealed the formation mechanism of 6PPDQ is the (N)H-abstraction of 6PPD by CYP450, followed by amino radical rebound at the nearby ortho-carbon, yielding a quinol intermediate due to spin delocalization, that might readily undergo further oxidation by CYP450 into 6PPDQ.

Probe substrates assay estimates the effect of polyphyllin H on the activity of cytochrome P450 enzymes in human liver microsomes.

Cytochrome P450 enzymes (CYPs) play a crucial role in phase I metabolic reactions. The activity of CYPs would affect therapeutic efficacy and may even induce toxicity. Given the complex components of traditional Chinese medicine, it is important to understand the effect of active ingredients on CYPs activity to guide their prescription. This study aimed to evaluate the effect of polyphyllin H on the activity of CYPs major isoforms providing a reference for the clinical prescription of polyphyllin H and its source herbs. The effects of polyphyllin H were evaluated in pooled human liver microsomes using probe substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to determine their activities. The Lineweaver-Burk was used to model the inhibition, and a time-dependent inhibition experiment was performed to understand the characteristics of the inhibition. Polyphyllin H significantly suppressed the activity of CYP1A2, 2D6, and 3A4 with IC50 values of 6.44, 13.88, and 4.52 μM, respectively. The inhibition of CYP1A2 and 2D6 was best fitted with a competitive model, yielding the inhibition constant (Ki) values of 3.18 and 6.77 μM, respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with the Ki value of 2.38 μM. Moreover, the inhibition of CYP3A4 was revealed to be time-dependent with the inhibition parameters inhibition constant (KI) and inactivation rate constant (Kinact) values of 2.26 μM-1 and 0.045 min-1. Polyphyllin H acted as a competitive inhibitor of CYP1A2 and 2D6 and a non-competitive and time-dependent inhibitor of CYP3A4.

Fragment Addition-Based Design of Heteroaromatic-Biphenyl-DAPYs as Potent and Orally Available Non-nucleoside Reverse Transcriptase Inhibitors Featuring Significantly Enhanced Safety.

Our previously disclosed biphenyl-DAPY 3 emerged as a potent inhibitor against WT HIV-1 and various mutant strains. Yet, its journey toward clinical application was thwarted by pronounced cytotoxicity and low selectivity (CC50 = 6 μM, SI = 3515). The safety improvement approach we employed in this work entailed the incorporation of diverse heteroaromatic substituents at the C5 position to exploit the tolerant regions of the NNRTIs' binding pocket through fragment addition-based drug design strategy, ultimately leading to the identification of a series of novel heteroaromatic-biphenyl-DAPYs. The exemplary compound 10d revealed a striking reduction in cytotoxicity (CC50 > 272.81 μM), nearly 45.5 times lower than 3, while showcasing 15-fold increase in selectivity (SI > 52632). This analog sustained exceptional anti-HIV-1 activity against both WT HIV-1 (EC50 = 5 nM) and various mutant strains. Compared to 3, a markedly slower rate of metabolism in human liver microsomes of 10d was observed. Its pharmacokinetic profile was equally captivating, featuring excellent oral bioavailability (F = 57.4%). Moreover, 10d exhibited a delicate sensitivity toward CYP, minimal inhibition of hERG, and no detectable acute toxicity in vivo. These enchanting findings illuminated the potential of 10d as a promising candidate for HIV-1 therapy.

Developing Robust Human Liver Microsomal Stability Prediction Models: Leveraging Inter-Species Correlation with Rat Data.

Objectives: Pharmacokinetic issues were the leading cause of drug attrition, accounting for approximately 40% of all cases before the turn of the century. To this end, several high-throughput in vitro assays like microsomal stability have been developed to evaluate the pharmacokinetic profiles of compounds in the early stages of drug discovery. At NCATS, a single-point rat liver microsomal (RLM) stability assay is used as a Tier I assay, while human liver microsomal (HLM) stability is employed as a Tier II assay. We experimentally screened and collected data on over 30,000 compounds for RLM stability and over 7000 compounds for HLM stability. Although HLM stability screening provides valuable insights, the increasing number of hits generated, along with the time- and resource-intensive nature of the assay, highlights the need for alternative strategies. One promising approach is leveraging in silico models trained on these experimental datasets. Methods: We describe the development of an HLM stability prediction model using our in-house HLM stability dataset. Results: Employing both classical machine learning methods and advanced techniques, such as neural networks, we achieved model accuracies exceeding 80%. Moreover, we validated our model using external test sets and found that our models are comparable to some of the best models in literature. Additionally, the strong correlation observed between our RLM and HLM data was further reinforced by the fact that our HLM model performance improved when using RLM stability predictions as an input descriptor. Conclusions: The best model along with a subset of our dataset (PubChem AID: 1963597) has been made publicly accessible on the ADME@NCATS website for the benefit of the greater drug discovery community. To the best of our knowledge, it is the largest open-source model of its kind and the first to leverage cross-species data.

Idiosyncratic Hepatocellular Drug-Induced Liver Injury by Flucloxacillin with Evidence Based on Roussel Uclaf Causality Assessment Method and HLA B*57:01 Genotype: From Metabolic CYP 3A4/3A7 to Immune Mechanisms.

Idiosyncratic drug-induced liver injury (iDILI) by flucloxacillin presents as both cholestatic and hepatocellular injury. Its mechanistic steps are explored in the present analysis as limited data exist on the cascade of events leading to iDILI in patients with an established diagnosis assessed for causality by the Roussel Uclaf Causality Assessment Method (RUCAM). Studies with human liver microsomes showed that flucloxacillin is a substrate of cytochrome P450 (CYP) with ist preferred isoforms CYP 3A4/3A7 that toxified flucloxacillin toward 5'-hydroxymethylflucloxacillin, which was cytotoxic to human biliary epithelial cell cultures, simulating human cholestatic injury. This provided evidence for a restricted role of the metabolic CYP-dependent hypothesis. In contrast, 5'-hydroxymethylflucloxacillin generated metabolically via CYP 3A4/3A7 was not cytotoxic to human hepatocytes due to missing genetic host features and a lack of non-parenchymal cells, including immune cells, which commonly surround the hepatocytes in the intact liver in abundance. This indicated a mechanistic gap regarding the clinical hepatocellular iDILI, now closed by additional studies and clinical evidence based on HLA B*57:01-positive patients with iDILI by flucloxacillin and a verified diagnosis by the RUCAM. Naïve T-cells from volunteers expressing HLA B*57:01 activated by flucloxacillin when the drug antigen was presented by dendritic cells provided the immunological basis for hepatocellular iDILI caused by flucloxacillin. HLA B*57:01-restricted activation of drug-specific T-cells caused covalent binding of flucloxacillin to albumin acting as a hapten. Following drug stimulation, T-cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL25 and secreted interferon-γ and cytokines. In conclusion, cholestatic injury can be explained metabolically, while hepatocellular injury requires both metabolic and immune activation.

Assessment of the in vitro metabolic stability of CEP-37440, a selective FAK/ALK inhibitor, in HLMs using fast UPLC-MS/MS method: in silico metabolic lability and DEREK alerts screening.

CEP-37440 was synthesized and supplied by the research and development division of Teva Branded Pharmaceutical Products (West Chester, PA, United States). CEP-37440 represents a newly developed compound that exhibits selectivity inhibition of Focal Adhesion Kinase and Anaplastic Lymphoma Kinase FAK/ALK receptors, demonstrating novel characteristics as an orally active inhibitor. The simultaneous inhibition of ALK and FAK can effectively address resistance and enhance the therapeutic efficacy against tumors through a synergistic mechanism. The objective of this research was to create an LC-MS/MS method that is precise, efficient, environmentally friendly, and possesses a high level of sensitivity for the quantification of CEP-37440 in human liver microsomes (HLMs). The aforementioned approach was subsequently employed to evaluate the metabolic stability of CEP-37440 in HLMs in an in vitro setting. The validation procedures for the LC-MS/MS analytical method in the HLMs were performed following the bio-analytical method validation guidelines set out by the US-FDA. The AGREE program was utilized to assess the ecological impacts of the current LC-MS/MS methodology. The calibration curve linearity was seen in the range of 1-3000ng/mL. The inter-day accuracy (% RE) exhibited a range of -2.33% to 3.22%, whilst the intra-day accuracy demonstrated a range of -4.33% to 1.39%. The inter-day precision (% RSD) exhibited a range of 0.38% to 3.60%, whilst the intra-day precision demonstrated a range of 0.16% to 6.28%. The determination of the in vitro half-life (t1/2) and moderate intrinsic clearance (Clint) of CEP-37440 yielded values of 23.24min and 34.74mL/min/kg, respectively. The current manuscript is considered the first analytical study for CEP-37440 quantification with the application to metabolic stability assessment. These results suggest that CEP-37440 can be categorized as a pharmaceutical agent with a moderate extraction ratio. Consequently, it is postulated that the administration of CEP-37440 to patients may not lead to the accrual of dosages within the human organs. According to in silico P450 metabolic and DEREK software, minor structural alterations to the ethanolamine moiety or substitution of the group in drug design have the potential to enhance the metabolic stability and safety profile of novel derivatives in comparison to CEP-37440.

Inhibitory effects of cuminaldehyde on human liver cytochrome P450 enzymes

This work explored the influence of cuminaldehyde on the metabolic activity of four primary human cytochrome P450 (CYP) enzymes. Human liver microsomes (HLM) with and without cuminaldehyde were incubated with specific substrates of different CYP enzymes. The influence of cuminaldehyde on “CYP1A2, CYP2C9, CYP2D6 and CYP3A4” activities was examined. The formation of specific metabolites was analyzed by HPLC analytical methods. It was observed that cuminaldehyde appears to have a negligible influence on CYP1A2 activity. However, CYP2C9 activity was significantly and potently inhibited by cuminaldehyde at all investigated concentrations. It was found that cuminaldehyde (1 µM) inhibited the most CYP2C9 enzyme activity with 64.87 % inhibition followed by CYP3A4 (38.90 %), CYP2D6 (25.76 %) and CYP1A2 (4.99 %). Inhibition of both CYP2D6 and CYP3A4 enzyme activity was observed in response to cuminaldehyde concentrations. Findings of the current investigation indicate that herb-drug interactions are very possible when cuminaldehyde is concurrently taken with medicines predominantly metabolized by CYP2C9 and CYP3A4. Cuminaldehyde should be tested further to examine how it affects CYP2C9 and CYP3A4-metabolized drugs.

Discovery of Arylpiperazines with Broad-Spectrum Antimicrobial Activity and Favorable Pharmacokinetic Profiles.

Microorganisms can induce diseases with significant clinical implications for human health. Multidrug-resistant microorganisms have been on the rise worldwide over the past few decades, and no new antibiotics have been introduced to the market in a considerable amount of time. Such situation highlights the urgency of discovering new antimicrobial drugs to address this pressing issue. Therefore, the objective of this study was to identify bioactive compounds against 15 species of bacteria and 5 species of fungi of clinical relevance through in vitro screening of 58 synthetic compounds from four chemical classes of our internal library of synthetic compounds. Our findings highlight arylpiperazines 18, 20, 26, 27, and 29, and the aminothiazole 50, as potent broad-spectrum antimicrobials (MICs = 12.5 - 15.6 mg.mL-1) against clinically relevant bacteria and fungi. Additionally, these compounds displayed low cytotoxicity against various host cells and a favorable in vitro pharmacokinetic profile for oral administration. Indeed, all six showed adequate lipophilicity, high gastrointestinal permeability, metabolic stability in human and mouse liver microsomes, and satisfactory aqueous solubility. Thus, they emerge as promising starting points for hit-to-lead studies towards new antibacterial and antifungal agents, especially against Staphylococcus epidermidis, Staphylococcus aureus, Lactobacillus paracasei and Candida orthopsilosis.

UGT2B10 is the Major UDP-Glucuronosyltransferase 2B Isoform Involved in the Metabolism of Lamotrigine and is Implicated in the Drug-Drug Interaction with Valproic Acid.

Lamotrigine is a phenyltriazine anticonvulsant that is primarily metabolized by phase II UDP-glucuronosyltransferases (UGT) to a quaternary N2-glucuronide, which accounts for ~ 90% of the excreted dose in humans. While there is consensus that UGT1A4 plays a predominant role in the formation of the N2-glucuronide, there is compelling evidence in the literature to suggest that the metabolism of lamotrigine is catalyzed by another UGT isoform. However, the exact identity of the UGT isoform that contribute to the formation of this glucuronide remains uncertain. In this study, we harnessed a robust reaction phenotyping strategy to delineate the identities and its associated fraction metabolized (fm) of the UGTs involved in lamotrigine N2-glucuronidation. Foremost, human recombinant UGT mapping experiments revealed that the N2-glucuronide is catalyzed by multiple UGT isoforms. (i.e., UGT1A1, 1A3, 1A4, 1A9, 2B4, 2B7, and 2B10). Thereafter, scaling the apparent intrinsic clearances obtained from the enzyme kinetic experiments with our in-house liver-derived relative expression factors (REF) and relative activity factors (RAF) revealed that, in addition to UGT1A4, UGT2B10 was involved in the N2-glucuronidation of lamotrigine. This was further confirmed via chemical inhibition in human liver microsomes with the UGT1A4-selective inhibitor hecogenin and the UGT2B10-selective inhibitor desloratadine. By integrating various orthogonal approaches (i.e., REF- and RAF-scaling, and chemical inhibition), we quantitatively determined that the fm for UGT1A4 and UGT2B10 ranged from 0.42 - 0.64 and 0.32 - 0.57, respectively. Finally, we also provided nascent evidence that the pharmacokinetic interaction between lamotrigine and valproic acid likely arose from the in vivo inhibition of its UGT2B10-mediated pathway.

Absorption, Distribution, Metabolism, and Excretion of Icenticaftor (QBW251) in Healthy Male Volunteers at Steady State and In Vitro Phenotyping of Major Metabolites.

Icenticaftor (QBW251) is a potentiator of the CFTR protein and is currently in clinical development for the treatment of chronic obstructive pulmonary disease and chronic bronchitis. An absorption, distribution, metabolism, and excretion (ADME) study was performed at steady state to determine the pharmacokinetics, mass balance, and metabolite profiles of icenticaftor in humans. In this open-label study, six healthy men were treated with unlabeled oral icenticaftor (400 mg b.i.d.) for 4 days. A single oral dose of [14C]icenticaftor was administered on Day 5, and unlabeled icenticaftor was administered b.i.d. from the evening of Day 5 to Day 12. Unchanged icenticaftor accounted for 18.5% of plasma radioactivity. Moderate to rapid absorption of icenticaftor was observed (median Tmax: 4 hours), with 93.4% of the dose absorbed. It exhibited moderate distribution (Vz/F: 335 L) and was extensively metabolized, principally through N-glucuronidation, O-glucuronidation, and/or O-demethylation. The metabolites M8 and M9, formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively, represented the main entities detected in plasma (35.3% and 14.5%, respectively) in addition to unchanged icenticaftor (18.5%). The apparent mean T1/2 of icenticaftor was 15.4 hours in blood and 20.6 hours in plasma. Icenticaftor was eliminated from the body mainly through metabolism followed by renal excretion, and excretion of radioactivity was complete after 9 days. In vitro phenotyping of icenticaftor showed that cytochrome P450 and uridine diphosphate glucuronosyltransferase were responsible for 31% and 69% of the total icenticaftor metabolism in human liver microsomes, respectively. This study provided invaluable insights into the disposition of icenticaftor. Significance Statement The ADME of a single radioactive oral dose of icenticaftor was evaluated at steady state to investigate the nonlinear pharmacokinetics observed previously with icenticaftor. [14C]Icenticaftor demonstrated good systemic availability after oral administration and was extensively metabolized and moderately distributed to peripheral tissues. The most abundant metabolites, M8 and M9, were formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively. Phenotyping demonstrated that [14C]icenticaftor was metabolized predominantly by UGT1A9 with a remarkably low Km value.

Coumarin-Based Aldo-Keto Reductase Family 1C (AKR1C) 2 and 3 Inhibitors.

A series of 7-substituted coumarin derivatives have been characterized as pan-aldo-keto reductase family 1C (AKR1C) inhibitors. The AKR1C family of enzymes are overexpressed in numerous cancers where they are involved in drug resistance development. 7-hydroxy coumarin ethyl esters and their corresponding amides have high potency for AKR1C3 and AKR1C2 inhibition. Coumarin amide 3 a possessed IC50 values of 50 nM and 90 nM for AKR1C3 and AKR1C2, respectively, and exhibits 'drug-like' metabolic stability and half-life in human and mouse liver microsomes and plasma. Compound 3 a was employed as a chemical tool to determine pan-AKR1C2/3 inhibition effects both as a radiation sensitizer and as a potentiator of chemotherapy cytotoxicity. In contrast to previously reported pan-AKR1C inhibitors, 3 a demonstrated no radiation sensitization effect in a radiation-resistant prostate cancer cell line model. Pan-AKR1C inhibition also did not potentiate the in vitro cytotoxicity of ABT-737, daunorubicin or dexamethasone, in two patient-derived T-cell ALL and pre-B-cell ALL cell lines. In contrast, a highly selective AKR1C3 inhibitor, compound K90, enhanced the cytotoxicity of both ABT-737 and daunorubicin in the T-cell ALL cell line model. Thus, the inhibitory profile required to enhance chemotherapeutic cytotoxicity in leukemia may be AKR1C isoform and drug specific.

Inhibition of human UDP-glucuronosyltransferase (UGT) enzymes by darolutamide: Prediction of in vivo drug-drug interactions

Darolutamide is a potent second-generation, selective nonsteroidal androgen receptor inhibitor (ARI), which has been approved by the US Food and Drug Administration (FDA) in treating castrate-resistant, non-metastatic prostate cancer (nmCRPC). Whether darolutamide affects the activity of UDP-glucuronosyltransferases (UGTs) is unknown. The purpose of the present study is to evaluate the inhibitory effect of darolutamide on recombinant human UGTs and pooled human liver microsomes (HLMs), and explore the potential for drug-drug interactions (DDIs) mediated by darolutamide through UGTs inhibition. The product formation rate of UGTs substrates with or without darolutamide was determined by HPLC or UPLC-MS/MS to estimate the inhibitory effect and inhibition modes of darolutamide on UGTs were evaluated by using the inhibition kinetics experiments. The results showed that 100 μM darolutamide exhibited inhibitory effects on most of the 12 UGTs tested. Inhibition kinetic studies of the enzyme revealed that darolutamide noncompetitively inhibited UGT1A1 and competitively inhibited UGT1A7 and 2B15, with the Ki of 14.75 ± 0.78 μM, 14.05 ± 0.42 μM, and 6.60 ± 0.08 μM, respectively. In particular, it also potently inhibited SN-38, the active metabolite of irinotecan, glucuronidation in HLMs with an IC50 value of 3.84 ± 0.46 μM. In addition, the in vitro-in vivo extrapolation (IVIVE) method was used to quantitatively predict the risk of darolutamide-mediated DDI via inhibiting UGTs. The prediction results showed that darolutamide may increase the risk of DDIs when administered in combination with substrates of UGT1A1, UGT1A7, or UGT2B15. Therefore, the combined administration of darolutamide and drugs metabolized by the above UGTs should be used with caution to avoid the occurrence of potential DDIs.

Translational Preclinical PET Imaging and Metabolic Evaluation of a New Cannabinoid 2 Receptor (CB2R) Radioligand, (Z)-N-(3-(2-(2-[18F]Fluoroethoxy)ethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropane-1-carboxamide.

We have previously developed seven fluorinated analogues of A-836339 as new PET tracers for cannabinoid type 2 receptor (CB2R) imaging, among which (Z)-N-(3-(2-(2-[18F]fluoroethoxy)ethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropane-1-carboxamide ([18F]FC0324) displayed high affinity and selectivity for CB2R in healthy rats. In the present study, we have further evaluated the imaging and metabolic properties of [18F]FC0324 in a rat model of human CB2R overexpression in the brain (AAV-hCB2) and in non-human primates (NHPs). Autoradiography with AAV-hCB2 rat brain sections exhibited a signal of [18F]FC0324 8-fold higher in the ipsilateral region than in the contralateral region. Blocking with NE40, a CB2R-specific agonist, resulted in a 91% decrease in the radioactivity. PET experiments showed a signal 7-fold higher in the ipsilateral region, and the specificity of [18F]FC0324 for hCB2R in vivo was confirmed by the 80% decrease after blocking with NE40. In NHPs, brain time-activity curves displayed a fast and homogeneous distribution followed by a rapid washout, in accordance with the low amount of CB2Rs in healthy brain. Whole-body PET-CT suggested a high and specific uptake of the radiotracer in the spleen, a CB2R-rich organ, and in the organs involved in metabolism and excretion, with a low bone uptake. In vitro metabolism with monkey liver microsomes (MLMs) led to the formation of six main hydroxylated metabolites of FC0324. Five of them were produced by human liver microsomes, being much less active than MLMs. In vivo, in NHPs, the main radiometabolite was likely to result from further oxidation of hydroxylated compounds, and parent [18F]FC0324 accounted for 8 ± 3% of plasma radioactivity (at 120 min p.i.) with a low level of potential interfering radiometabolites. Furthermore, this metabolism should be significantly reduced in humans due to species differences. In conclusion, [18F]FC0324 appears to be a promising candidate for further human studies with suitable kinetics, selectivity, and metabolic profile for CB2R PET imaging.

Inhibitory effects of calcium channel blockers nisoldipine and nimodipine on ivacaftor metabolism and their underlying mechanism.

Ivacaftor is the first potentiator of the cystic fibrosis transmembrane conductance regulator (CFTR) protein approved for use alone in the treatment of cystic fibrosis (CF). Ivacaftor is primarily metabolized by CYP3A4 and therefore may interact with drugs that are CYP3A4 substrates, resulting in changes in plasma exposure to ivacaftor. The study determined the levels of ivacaftor and its active metabolite M1 by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). We screened 79 drugs and 19 severely inhibited ivacaftor metabolism, particularly two cardiovascular drugs (nisoldipine and nimodipine). In rat liver microsomes (RLM) and human liver microsomes (HLM), the half-maximal inhibitory concentrations (IC50) of nisoldipine on ivacaftor metabolism were 6.55μM and 9.10μM, respectively, and the inhibitory mechanism of nisoldipine on ivacaftor metabolism was mixed inhibition; the IC50 of nimodipine on ivacaftor metabolism in RLM and HLM were 4.57μM and 7.15μM, respectively, and the inhibitory mechanism of nimodipine on ivacaftor was competitive inhibition. In pharmacokinetic experiments in rats, it was observed that both nisoldipine and nimodipine significantly altered the pharmacokinetic parameters of ivacaftor, such as AUC(0-t) and CLz/F. However, this difference may not be clinically relevant. In conclusion, this paper presented the results of studies investigating the interaction between these drugs and ivacaftor in vitro and in vivo. The objective is to provide a rationale for the safety of ivacaftor in combination with other drugs.

In Vitro Metabolism of Quaternary Ammonium Compounds and Confirmation in Human Urine by Liquid Chromatography Ion-Mobility High-Resolution Mass Spectrometry.

Quaternary ammonium compounds (QACs) are high-production chemicals used as cleaning and disinfecting agents. Due to their ubiquitous presence in the environment and several toxic effects described, human exposure to these chemicals gained increasing attention in recent years. However, very limited data on the biotransformation of QACs is available, hampering exposure assessment. In this study, three QACs (dimethyl dodecyl ammonium, C10-DDAC; benzyldimethyl dodecylammonium, C12-BAC; cetyltrimethylammonium, C16-ATMAC) commonly detected in indoor microenvironments were incubated with human liver microsomes and cytosol (HLM/HLC) simulating Phase I and II metabolism. Thirty-one Phase I metabolites were annotated originating from 19 biotransformation reactions. Four metabolites of C10-DDAC were described for the first time. A detailed assessment of experimental fragmentation spectra allowed to characterize potential oxidation sites. For each annotated metabolite, drift-tube ion-mobility derived collision cross section (DTCCSN2) values were reported, serving as an additional identification parameter and allowing the characterization of changes in DTCCSN2 values following metabolism. Lastly, eight metabolites, including four metabolites of both C12-BAC and C10-DDAC, were confirmed in human urine samples showing high oxidation states through introduction of up to four oxygen atoms. This is the first report of higher oxidized C10-DDAC metabolites in human urine facilitating future biomonitoring studies on QACs.