Human Liver Microsomes Research Articles

Development of fluorine-substituted NH2-biphenyl-diarylpyrimidines as highly potent non-nucleoside reverse transcriptase inhibitors: Boosting the safety and metabolic stability.

Our recent studies for nonnucleoside reverse transcriptase inhibitors identified a highly potent compound JK-4b against WT HIV-1 (EC50=1.0nmol/L), but the poor metabolic stability in human liver microsomes (t 1/2=14.6min) and insufficient selectivity (SI=2059) with high cytotoxicity (CC50=2.08μmol/L) remained major issues associated with JK-4b. The present efforts were devoted to the introduction of fluorine into the biphenyl ring of JK-4b, leading to the discovery of a novel series of fluorine-substituted NH2-biphenyl-diarylpyrimidines with noticeable inhibitory activity toward WT HIV-1 strain (EC50=1.8-349nmol/L). The best compound 5t in this collection (EC50=1.8nmol/L, CC50=117μmol/L) was 32-fold in selectivity (SI=66,443) compared to JK-4b and showed remarkable potency toward clinically multiple mutant strains, such as L100I, K103N, E138K, and Y181C. The metabolic stability of 5t was also significantly improved (t 1/2=74.52min), approximately 5-fold higher than JK-4b in human liver microsomes (t 1/2=14.6min). Also, 5t possessed good stability in both human and monkey plasma. No significant invitro inhibition effect toward CYP enzyme and hERG was observed. The single-dose acute toxicity test did not induce mice death or obvious pathological damage. These findings pave the way for further development of 5t as a drug candidate.

Simplifying the Extended Clearance Concept Classification System (EC3S) to Guide Clearance Prediction in Drug Discovery.

The Extended Clearance Concept Classification System was established as a development-stage tool to provide a framework for identifying fundamental mechanism(s) governing drug disposition in humans. In the present study, the applicability of the EC3S in drug discovery has been investigated. In its current format, the EC3S relies on low-throughput hepatocyte uptake data, which are not frequently generated in a discovery setting. A relationship between hepatocyte uptake clearance and MDCK permeability was first established along with intrinsic clearance from human liver microsomes. The performance of this approach was examined by categorizing 64 drugs into EC3S classes and comparing the predicted major elimination pathway(s) to that observed in humans. As an extension of the work, the ability of the simplified EC3S to predict human systemic clearance based on intrinsic clearance generated using in-vitro metabolic systems was evaluated. The assessment enabled the use of MDCK permeability and unscaled unbound intrinsic clearance to generate cut-off criteria to categorize compounds into four EC3S classes: Class12ab, 2cd, 34ab, and 34cd, with major elimination mechanism(s) assigned to each class. The predictivity analysis suggested that systemic clearance could generally be predicted within threefold for EC3S class 12ab and 34ab compounds. For classes 2cd and 34cd, systemic clearance was poorly predicted using in-vitro systems explored in this study. Collectively, our simplified classification approach is expected to facilitate the identification of mechanism(s) involved in drug elimination, faster resolution of in-vitro to in-vivo disconnects, and better design of mechanistic pharmacokinetic studies in drug discovery.

Combined contributions of cytochrome P450s (CYPs) and non-enzymatic metabolism in the in vitro biotransformation of anaprazole, a novel proton pump inhibitor.

Anaprazole, a new proton pump inhibitor (PPI), is designed for the treatment of acid-related diseases, such as gastric ulcers and gastroesophageal reflux. This study explored the in vitro metabolic transformation of anaprazole. The metabolic stabilities of anaprazole in human plasma and human liver microsomes (HLM) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, the contribution (%) of non-enzymatic and cytochrome P450s (CYPs) enzyme-mediated anaprazole metabolism was assessed. To obtain the metabolic pathways of anaprazole, the metabolites generated in HLM, thermal deactivated HLM, and cDNA-expressed recombinant CYPs incubation systems were identified by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Results showed thatanaprazole was very stable in human plasma and unstable in HLM. The contribution (%) of non-enzymatic vs. CYPs enzyme-mediated metabolism was 49% vs. 51%. CYP3A4 was the major enzyme (48.3%), followed by CYP2C9 (17.7%) and CYP2C8 (12.3%), in responsible for the metabolism of anaprazole. Specific chemical inhibitors targeting CYP enzymes notably blocked the metabolic transformation of anaprazole. Six metabolites of anaprazole were identified in the non-enzymatic system, whereas 17 metabolites were generated in HLM. The biotransformation reactions mainly included sulfoxide reduction to thioether, sulfoxide oxidation to sulfone, deoxidation, dehydrogenation, O-dealkylation or O-demethylation of thioether, O-demethylation and dehydrogenation of thioether, O-dealkylation and dehydrogenation of thioether, thioether O-dealkylation and dehydrogenation of thioether, and O-dealkylation of sulfone. Both enzymatic and non-enzymatic metabolisms contribute to the clearance of anaprazole in human. Anaprazole is less likely to develop drug-drug interactions in clinical use compared to other PPIs.

2-Aminopyrimidine derivatives as selective dual inhibitors of JAK2 and FLT3 for the treatment of acute myeloid leukemia

Dual inhibitors of JAK2 and FLT3 can synergistically control the development of acute myeloid leukemia (AML), and overcome secondary drug resistance of AML that is associated with FLT3 inhibition. We therefore designed and synthesized a series of 4-piperazinyl-2-aminopyrimidines as dual inhibitors of JAK2 and FLT3, and improved their selectivity for JAK2. Screening cascades revealed that compound 11r exhibited inhibitory activity with IC50 values of 2.01, 0.51, and 104.40 nM against JAK2, FLT3, and JAK3, respectively. Compound 11r achieved a high selectivity for JAK2 at a ratio of 51.94, and also showed potent antiproliferative activity in HEL (IC50 = 1.10 μM) and MV4-11 (IC50 = 9.43 nM) cell lines. In an in vitro metabolism assay, 11r exhibited moderate stability in human liver microsomes (HLMs), with a half-life time of 44.4 min, and in rat liver microsomes (RLMs), with a half-life of 143 min. In pharmacokinetic studies, compound 11r showed moderate absorption (Tmax = 5.33 h), with a peak concentration of 38.7 ng/mL and an AUC of 522 ng h/mL in rats, and an oral bioavailability of 25.2%. In addition, 11r induced MV4-11 cell apoptosis in a dose-dependent manner. These results indicate that 11r is a promising selective JAK2/FLT3 dual inhibitor.

Metabolic Evaluation of Synthetic Opioids on the Example of U-47700 with the Use of In Vitro and In Vivo Methods for Forensic Toxicology Application

Legal highs present a great threat to health, especially in groups of people experimenting with psychoactive substances. The lack of available knowledge on the biotransformation of these substances necessitates symptomatic treatment in the event of intoxication, which, unfortunately, may be ineffective. Opioids, including heroin analogues, such as U-47700, constitute a special group of designer drugs. In this study, a multi-directional approach to trace the biotransformation of U-47700 in living organisms was used. For this purpose, the in silico assessment (ADMET Predictor) was used first and then followed by an in vitro study using human liver microsomes and the S9 fraction. The biotransformation was then followed in an animal model (Wistar rats). Tissues such as blood, brain and liver were collected for analysis. The study was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The obtained results were compared to those obtained from the analysis of autopsy materials (cases analysed in the Toxicology Laboratory of the Department of Forensic Medicine, Jagiellonian University Medical College in Krakow).

Involvement of multiple cytochrome P450 isoenzymes in drug interactions between ritonavir and direct oral anticoagulants

Herein, we aimed to determine the significance of drug interactions (DIs) between ritonavir and direct oral anticoagulants (DOACs) and identify the involved cytochrome P450 (CYP) isoenzymes. Using an in vitro cocktail method with human liver microsomes (HLM), we observed that ritonavir strongly inhibited CYPs in the following order: CYP3A, CYP2C8, CYP2D6, CYP2C9, CYP2C19, CYP2B6, and CYP2J2 (IC50: 0.023–6.79 μM). The degree of CYP2J2 inhibition was inconclusive, given the substantial discrepancy between the HLM and human expression system. Selective inhibition of CYP3A decreased the O-demethylation of apixaban by only 13.4%, and the involvement of multiple CYP isoenzymes was suggested, all of which were inhibited by ritonavir. Multiple CYP isoenzymes contributed also to the metabolism of rivaroxaban. Replacement of the incubation medium with phosphate buffer instead of HEPES enhanced apixaban hydroxylation. On surveying the FDA Adverse Event Reporting System, we detected that the signal of the proportional reporting ratio of “death” and found increase for “hemoglobin decreased” (12.5-fold) and “procedural hemorrhage” (201.9-fold) on administering apixaban with ritonavir; these were far less significant for other CYP3A inhibitors. Overall, these findings suggest that co-administration of ritonavir-boosted drugs with DOACs may induce serious DIs owing to the simultaneous inhibition of multiple CYP isoenzymes.

Pharmacokinetics and tissue distribution of Ramulus Mori (Sangzhi) alkaloids in rats and its effects on liver enzyme activity.

Ramulus Mori (Sangzhi) alkaloids (SZ-A) derived from twigs of mulberry (Morus alba L., genus Morus in the Moraceae family) was approved by the National Medical Products Administration in 2020 for the treatment of type 2 diabetes mellitus. In addition to excellent hypoglycemic effect, increasing evidence has confirmed that SZ-A exerts multiple pharmacological effects, such as protecting pancreatic ß-cell function, stimulating adiponectin expression, and alleviating hepatic steatosis. Importantly, a specific distribution of SZ-A in target tissues following oral absorption into the blood is essential for the induction of multiple pharmacological effects. However, there is a lack of studies thoroughly exploring the pharmacokinetic profiles and tissue distribution of SZ-A following oral absorption into the blood, particularly dose-linear pharmacokinetics and target tissue distribution associated with glycolipid metabolic diseases. In the present study, we systematically investigated the pharmacokinetics and tissue distribution of SZ-A and its metabolites in human and rat liver microsomes, and rat plasma, as well as its effects on the activity of hepatic cytochrome P450 enzymes (CYP450s). The results revealed that SZ-A was rapidly absorbed into the blood, exhibited linear pharmacokinetic characteristics in the dose range of 25-200mg/kg, and was broadly distributed in glycolipid metabolism-related tissues. The highest SZ-A concentrations were observed in the kidney, liver, and aortic vessels, followed by the brown and subcutaneous adipose tissues, and the heart, spleen, lung, muscle, pancreas, and brain. Except for the trace oxidation products produced by fagomine, other phase I or phase II metabolites were not detected. SZ-A had no inhibitory or activating effects on major CYP450s. Conclusively, SZ-A is rapidly and widely distributed in target tissues, with good metabolic stability and a low risk of triggering drug-drug interactions. This study provides a framework for deciphering the material basis of the multiple pharmacological functions of SZ-A, its rational clinical use, and the expansion of its indications.

In vitro metabolic characterization of the SARS-CoV-2 papain-like protease inhibitors GRL0617 and HY-17542.

The SARS-CoV-2 pandemic requires a new therapeutic target for viral infection, and papain-like protease (Plpro) has been suggested as a druggable target. This in-vitro study was conducted to examine the drug metabolism of the GRL0617 and HY-17542, Plpro inhibitors. Metabolism of these inhibitors was studied to predict the pharmacokinetics in human liver microsomes. The hepatic cytochrome P450 (CYP) isoforms responsible for their metabolism were identified using recombinant enzymes. The drug-drug interaction potential mediated by cytochrome P450 inhibition was estimated. In human liver microsomes, the Plpro inhibitors had phase I and phase I + II metabolism with half-lives of 26.35 and 29.53min, respectively. Hydroxylation (M1) and desaturation (-H2, M3) of the para-amino toluene side chain were the predominant reactions mediated with CYP3A4 and CYP3A5. CYP2D6 is responsible for the hydroxylation of the naphthalene side ring. GRL0617 inhibits major drug-metabolizing enzymes, including CYP2C9 and CYP3A4. HY-17542 is structural analog of GRL0617 and it is metabolized to GRL0617 through non-cytochrome P450 reactions in human liver microsomes without NADPH. Like GRL0617 and HY-17542 undergoes additional hepatic metabolism. The in-vitro hepatic metabolism of the Plpro inhibitors featured short half-lives; preclinical metabolism studies are needed to determine therapeutic doses for these inhibitors.

The effect of Resveratrol on the pharmacokinetic profile of tofacitinib and the underlying mechanism

The purpose of this study was to (i) investigate the effect of CYP3A4 variants on tofacitinib metabolism, and (ii) investigate the interaction of tofacitinib with resveratrol and its underlying mechanisms. The concentration of M9, the main metabolite of tofacitinib, was determined by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The results showed that the clearance rate of CYP3A4.18 variant was significantly decreased compared with CYP3A4.1, and the CYP3A4.28 variant was changed, but not statistically significant. In addition, the potential interaction of resveratrol with tofacitinib was determined based on rat liver microsomes (RLM), human liver microsomes (HLM), and CYP3A4 response systems. Resveratrol has an IC50 of 15.67 μM in RLM with a non-competitive mechanism. In HLM with a non-competitive mechanism, the IC50 value was 8.88 μM. The IC50 values were 6.41 μM, 10.60 μM and 27.08 μM in CYP3A4.1, .18 and .28, respectively, all with a competitive mechanism. In the in vivo study, Sprague-Dawley (SD) rats were randomized into two groups (n = 6) to receive tofacitinib with or without resveratrol. We found that the AUC(0-∞) of tofacitinib in the experimental group increased to around 207.5% compared with the control group. And Cmax increased to 260.0%. In summary, our data showed that resveratrol significantly affect the metabolism of tofacitinib, thus providing basic data for the precise clinical application of tofacitinib.

Production of a major metabolite of niclosamide using bacterial cytochrome P450 enzymes

Niclosamide has been proposed as a possible candidate for a Covid-19 drug. However, the metabolites of niclosamide are difficult to investigate because they are usually not available commercially or they are quite expensive in the commercial market. In this study, the major metabolite of niclosamide in human liver microsomes (HLMs) was confirmed to be 3-OH niclosamide. Because the production of 3-OH niclosamide using HLMs has a slow turnover rate, a new method of producing niclosamide metabolite with an easier and highly cost-efficient method was thus conducted. Bacterial CYP102A1 (BM3) is one of the bacterial cytochrome P450s (CYPs) from Bacillus megaterium that structurally show similar activities to human CYPs. Here, the BM3 mutants were used to produce niclosamide metabolites and the metabolites were analyzed using high-performance liquid chromatography and LC-mass spectrometry. Among a set of mutants tested here, BM3 M14 mutant was the most active in producing 3-OH niclosamide, the major metabolite of niclosamide. Comparing BM3 M14 and HLMs, BM3 M14 production of 3-OH niclosamide was 34-fold higher than that of HLMs. Hence, the engineering of BM3 can be a cost-efficient method to produce 3-OH niclosamide.

Rapid LC-MS/MS Bosutinib Quantification with Applications in Metabolic Stability Estimation

Bosutinib (BOS) is FDA approved drug for the treatment of chronic phase (CP) Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML). We report a fast, sensitive, and simple LC-MS/MS method, validated for the determination of BOS in human liver microsomes, utilizing tofacitinib (TOF) as the internal standard. The separation of BOS and TOF was done using a 1.8 μm C18 column (2.1 × 50 mm) at room temperature using the isocratic elution system of acetonitrile-water (30:70, v/v) containing 0.1 M formic acid at a flow rate of 0.15 mL/min, and a triple-quadrupole tandem mass spectrometer (TQD-MS) with an electrospray ionization (ESI) source that was operated in the positive ion mode. The method was validated according to the European Medicines Agency, and the rapid and specific quantification of BOS in human liver microsomes was achieved in the range of 5-200 ng/mL, with a determination coefficient of 0.999. Intra- and inter-day accuracy and precision values were <4% in all cases. The procedure is rapid, specific, reliable, and can be applied in metabolic stability evaluations since it is the first LC-MS/MS method specific to BOS quantification. The metabolic stability assessment of BOS showed high CLint (34.3 µL/min/mg) and short in vitro t1/2 values of 20.21 min, indicating that BOS may be rapidly eliminated from the blood by the liver.

Phytochemical Comparison of Medicinal Cannabis Extracts and Study of Their CYP-Mediated Interactions with Coumarinic Oral Anticoagulants

Introduction: Treatment with cannabis extracts for a variety of diseases has gained popularity. However, differences in herb-drug interaction potential of extracts from different plant sources are poorly understood. In this study, we provide a characterization of cannabis extracts prepared from four cannabis chemotypes and an in vitro assessment of their Cytochrome P450 (CYP)-mediated herb-drug interaction profiles. Methods: Plant extracts were either commercially obtained or prepared using ethanol as solvent, followed by overnight decarboxylation in a reflux condenser system. The extracts were characterized for their cannabinoid content using NMR and HPLC-PDA-ELSD-ESIMS. CYP inhibition studies with the cannabis extracts and pure cannabinoids (tetrahydrocannabinol [THC] and cannabidiol [CBD]) were performed using pooled, mixed gender human liver microsomes. Tolbutamide and testosterone were used as specific substrates to assess the inhibitory potential of the extracts on CYP2C9 and CYP3A4, and the coumarinic oral anticoagulants warfarin, phenprocoumon, and acenocoumarol were studied as model compounds since in vivo herb-drug interactions have previously been reported for this compound class. Results: In accordance with the plant chemotypes, two extracts were rich in THC and CBD (at different proportions); one extract contained mostly CBD and the other mostly cannabigerol (CBG). Residual amounts of the corresponding acids were found in all extracts. The extracts with a single major cannabinoid (CBD or CBG) inhibited CYP2C9- and CYP3A4-mediated metabolism stronger than the extracts containing both major cannabinoids (THC and CBD). The inhibition of CYP3A4 and CYP2C9 by the extract containing mostly CBD was comparable to their inhibition by pure CBD. In contrast, the inhibitory potency of extracts containing both THC and CBD did not correspond to the combined inhibitory potency of pure THC and CBD. Although being structural analogs, the three coumarin derivatives displayed major differences in their herb-drug interaction profiles with the cannabis extracts and the pure cannabinoids. Conclusion: Despite the fact that cannabinoids are the major components in ethanolic, decarboxylated cannabis extracts, it is difficult to foresee their herb-drug interaction profiles. Our in vitro data and the literature-based evidence on in vivo interactions indicate that cannabis extracts should be used cautiously when co-administered with drugs exhibiting a narrow therapeutic window, such as coumarinic anticoagulants, regardless of the cannabis chemotype used for extract preparation.

Identification of the Putative Binding Site of a Benzimidazole Opioid (Etazene) and Its Metabolites at µ-Opioid Receptor: A Human Liver Microsomal Assay and Systematic Computational Study

The synthetic benzimidazole opioid etazene (which has a 70-times higher analgesic activity than morphine), a recreational drug, has gained popularity as a novel psychoactive substance (NPS) on the illegal/darknet market; however, no experimental information is available at the molecular level on the binding mechanism and putative binding site of etazene and its metabolites at the µ-opioid receptor (MOR). In the present study, we investigated the metabolism of etazene in human liver microsomes using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). We also explored the possibilities of MOR activation by etazene and its metabolites by studying their binding mechanisms and interaction profiles at an active-state MOR model via molecular docking, binding free energy calculations, and all-atom molecular dynamics (MD) simulations. The putative metabolites of etazene were also predicted using the ADMET Predictor 10.1. The molecular docking studies and free energy calculations showed that etazene and its metabolites (M1, M2, and M5-M7) exhibited strong predicted binding affinity at MOR and showed overlapped binding orientation with MOR-bound agonist BU72, which was co-crystallized in the MOR X-ray crystal structure (PDB ID: 5C1M). MD also confirmed the stability of the MOR-etazene and MOR-M6 complexes. These results suggest that etazene and its metabolites may act as strong MOR agonists, highlighting the necessity of experimental validation. The insights from this study, such as key interactions between etazene and its metabolites and the MOR, will allow authorities to predict potential analogs and clarify the target-protein interactions associated with this illicit substance, granting advanced or rapid reactions to confiscating or banning potential emerging drugs.

Biotransformation of Penindolone, an Influenza A Virus Inhibitor.

Penindolone (PND) is a novel broad-spectrum anti-Influenza A Virus (IAV) agent blocking hemagglutinin-mediated adsorption and membrane fusion. The goal of this work was to reveal the metabolic route of PND in rats. Ultra-high-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS) was used for metabolite identification in rat bile, feces and urine after administration of PND. A total of 25 metabolites, including 9 phase I metabolites and 16 phase II metabolites, were characterized. The metabolic pathways were proposed, and metabolites were visualized via Global Natural Product Social Molecular Networking (GNPS). It was found that 65.24-80.44% of the PND presented in the formation of glucuronide conjugate products in bile, and more than 51% of prototype was excreted through feces. In in vitro metabolism of PND by rat, mouse and human liver microsomes (LMs) system, PND was discovered to be eliminated in LMs to different extents with significant species differences. The effects of chemical inhibitors of isozymes on the metabolism of PND in vitro indicated that CYP2E1/2C9/3A4 and UGT1A1/1A6/1A9 were the metabolic enzymes responsible for PND metabolism. PND metabolism in vivo could be blocked by UGTs inhibitor (ibrutinib) to a certain extent. These findings provided a basis for further research and development of PND.

Discovery of CZS-241: A Potent, Selective, and Orally Available Polo-Like Kinase 4 Inhibitor for the Treatment of Chronic Myeloid Leukemia

Recent studies demonstrate that PLK4 has emerged as a therapeutic target for the treatment of multiple cancers owing to its indispensable role in cell division. Herein, starting from previously identified effective compound CZS-034, based on rational drug design strategies, tyrosine kinase receptor A (TRKA) selectivity- and metabolic stability-guided structure-activity relationship (SAR) exploration were carried out to discover a highly potent (IC50 = 2.6 nM) and selective (SF = 1054.4 over TRKA) PLK4 inhibitor B43 (CZS-241) with acceptable human liver microsome stability (t1/2 = 31.5 min). Moreover, compound B43 effectively inhibited leukemia cells in 29 tested cell lines, especially chronic myeloid leukemia (CML) cell lines K562 and KU-812. Pharmacokinetic characteristics revealed that compound B43 possessed over 4 h of half-life and 70.8% bioavailability in mice. In the K562 cells xenograft mouse model, a 20 mg/kg/day dosage treatment obviously suppressed tumor progression. As a potential and novel PLK4-targeted candidate drug for CML, compound B43 is undergoing extensive preclinical safety evaluation.

An Insight into the Metabolism of 2,5-Disubstituted Monotetrazole Bearing Bisphenol Structures: Emerging Bisphenol A Structural Congeners.

The non-estrogenic 2,5-disubstituted tetrazole core-bearing bisphenol structures (TbB) are being researched as emerging structural congeners of Bisphenol A, an established industrial endocrine disruptor. However, there is no understanding of TbB's adverse effects elicited via metabolic activation. Therefore, the current study aimed to investigate the metabolism of TbB ligands, with in silico results serving as a guide for in vitro studies. The Cytochrome P450 enzymes (CYP) inhibitory assay of TbB ligands on the seven human liver CYP isoforms (i.e., 1A2, 2A6, 2D6, 2C9, 2C8, 2C19, and 3A4) using human liver microsomes (HLM) revealed TbB ligand 223-3 to have a 50% inhibitory effect on all the CYP isoforms at a 10 μM concentration, except 1A2. The TbB ligand 223-10 inhibited 2B6 and 2C8, whereas the TbB ligand 223-2 inhibited only 2C9. The first-order inactivity rate constant (Kobs) studies indicated TbB ligands 223-3, 223-10 to be time-dependent (TD) inhibitors, whereas the TbB 223-2 ligand did not show such a significant effect. The 223-3 exhibited a TD inhibition for 2C9, 2C19, and 1A2 with Kobs values of 0.0748, 0.0306, and 0.0333 min-1, respectively. On the other hand, the TbB ligand 223-10 inhibited 2C9 in a TD inhibition manner with Kobs value 0.0748 min-1. However, the TbB ligand 223-2 showed no significant TD inhibition effect on the CYPs. The 223-2 ligand biotransformation pathway by in vitro studies in cryopreserved human hepatocytes suggested the clearance via glucuronidation with the predominant detection of only 223-2 derived mono glucuronide as a potential inactive metabolite. The present study demonstrated that the 223-2 ligand did not elicit any significant adverse effect via metabolic activation, thus paving the way for its in vivo drug-drug interactions (DDI) studies.

The piperazine analogue para-fluorophenylpiperazine alters timing of the physiological effects of the synthetic cannabinoid receptor agonist AMB-FUBINACA, without changing its discriminative stimulus, signalling effects, or metabolism

AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA), which has been associated with substantial abuse and health harm since 2016 in many countries including New Zealand. A characteristic of AMB-FUBINACA use in New Zealand has included the observation that forensic samples (from autopsies) and drugs seized by police have often been found to contain para-fluorophenylpiperazine (pFPP), a relatively little-characterised piperazine analogue that has been suggested to act through 5HT1a serotonin receptors. In the current study, we aimed to characterise the interactions of these two agents in rat physiological endpoints using plethysmography and telemetry, and to examine whether pFPP altered the subjective effects of AMB-FUBINACA in mice trained to differentiate a cannabinoid (THC) from vehicle. Though pFPP did not alter the ability of AMB-FUBINACA to substitute for THC, it did appear to abate some of the physiological effects of AMB-FUBINACA in rats by delaying the onset of AMB-FUBINACA-mediated hypothermia and shortening duration of bradycardia. In HEK cells stably expressing the CB1 cannabinoid receptor, 5HT1a, or both CB1 and 5HT1a, cAMP signalling was recorded using a BRET biosensor (CAMYEL) to assess possible direct receptor interactions. Although low potency pFPP agonism at 5HT1a was confirmed, little evidence for signalling interactions was detected in these assays: additive or synergistic effects on potency or efficacy were not detected between pFPP and AMB-FUBINACA-mediated cAMP inhibition. Experiments utilising higher potency, classical 5HT1a ligands (agonist 8OH-DPAT and antagonist WAY100635) also failed to reveal evidence for mutual CB1/5HT1a interactions or cross-antagonism. Finally, the ability of pFPP to alter the metabolism of AMB-FUBINACA in rat and human liver microsomes into its primary carboxylic acid metabolite via carboxylesterase-1 was assessed by HPLC; no inhibition was detected. Overall, the effects we have observed do not suggest that increased harm/toxicity would result from the combination of pFPP and AMB-FUBINACA.

Synthesis and Evaluation of 2-Amine-4-oxyphosaniline Pyrimidine Derivatives as EGFR L858R/T790M/C797S Mutant Inhibitors.

Epidermal growth factor receptor (EGFR) C797S mutation leads to Osimertinib drug resistance by disturbing the covalent biding of Michael acceptor group to the Cys797 residue in the ATP biding cleft. In this manuscript, a class of 2-amine-4-oxyphosaniline pyrimidine derivatives were designed, synthesized and evaluated as new noncovalent reversible EGFR inhibitors against L858R/T790M/C797S (CTL) triple mutant. The kinases inhibitiory activity evaluation showed that four compounds exhibited significant inhibitory activities against CTL (IC50 < 30 nM). In particularly, the most promising compound 7a showed excellent enzymatic inhibitory activity against CTL with IC50 value of 9.9 nM, which was more potent than control compound Osimertinib. Moreover, cell proliferation assays indicated that 7a effectively inhibited H1975-EGFR L858R/T790M/C797S with IC50 value of 0.33 µM. Furthermore, compound 7a displayed good metabolic stabilities in human, rat and mouse liver microsomes, and the putative biding mode of compound 7a with ATP was revealed by molecular docking study. These findings strongly indicated that compound 7a was a promising L858R/T790M/C797S mutant EGFR inhibitor.

Identification of HSD17B12 as an enzyme catalyzing drug reduction reactions through investigation of nabumetone metabolism

Nabumetone, a nonsteroidal anti-inflammatory prodrug, is converted to a pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA); however, it is 11-fold more efficiently converted to 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) via a reduction reaction in human hepatocytes. The goal of this study was to identify the enzyme(s) responsible for MNBO formation from nabumetone in the human liver. MNBO formation by human liver microsomes (HLM) was 5.7-fold higher than in the liver cytosol. In a panel of 24 individual HLM samples with quantitative proteomics data, the 17β-hydroxysteroid dehydrogenase 12 (HSD17B12) protein level had the high correlation coefficient (r = 0.80, P < 0.001) among 4457 proteins quantified in microsomal fractions during MNBO formation. Recombinant HSD17B12 expressed in HEK293T cells exhibited prominent nabumetone reductase activity, and the contribution of HSD17B12 to the activity in the HLM was calculated as almost 100%. MNBO formation in HepG2 and Huh7 cells was significantly decreased by the knockdown of HSD17B12. We also examined the role of HSD17B12 in drug metabolism and found that recombinant HSD17B12 catalyzed the reduction reactions of pentoxifylline and S-warfarin, suggesting that HSD17B12 prefers compounds containing a methyl ketone group on the alkyl chain. In conclusion, our study demonstrated that HSD17B12 is responsible for the formation of MNBO from nabumetone. Together with the evidence for pentoxifylline and S-warfarin reduction, this is the first study to report that HSD17B12, which is known to metabolize endogenous compounds, such as estrone and 3-ketoacyl-CoA, plays a role as a drug-metabolizing enzyme.

Characterization of the metabolism of eupalinolide A and B by carboxylesterase and cytochrome P450 in human liver microsomes.

Eupalinolide A (EA; Z-configuration) and eupalinolide B (EB; E-configuration) are bioactive cis-trans isomers isolated from Eupatorii Lindleyani Herba that exert anti-inflammatory and antitumor effects. Although one pharmacokinetic study found that the metabolic parameters of the isomers were different in rats, metabolic processes relevant to EA and EB remain largely unknown. Our preliminary findings revealed that EA and EB are rapidly hydrolyzed by carboxylesterase. Here, we investigated the metabolic stability and enzyme kinetics of carboxylesterase-mediated hydrolysis and cytochrome P450 (CYP)-mediated oxidation of EA and EB in human liver microsomes (HLMs). We also explored differences in the hydrolytic stability of EA and EB in human liver microsomes and rat liver microsomes (RLMs). Moreover, cytochrome P450 reaction phenotyping of the isomers was performed via in silico methods (i.e., using a quantitative structure-activity relationship model and molecular docking) and confirmed using human recombinant enzymes. The total normalized rate approach was considered to assess the relative contributions of five major cytochrome P450s to EA and EB metabolism. We found that EA and EB were eliminated rapidly, mainly by carboxylesterase-mediated hydrolysis, as compared with cytochrome P450-mediated oxidation. An inter-species difference was observed as well, with faster rates of EA and EB hydrolysis in rat liver microsomes. Furthermore, our findings confirmed EA and EB were metabolized by multiple cytochrome P450s, among which CYP3A4 played a particularly important role.