Human Liver Microsomes Research Articles

1-Sulfonyl-3-amino-1H-1,2,4-triazoles as Yellow Fever Virus Inhibitors: Synthesis and Structure-Activity Relationship.

Yellow fever virus (YFV) transmitted by infected mosquitoes causes an acute viral disease for which there are no approved small-molecule therapeutics. Our recently developed machine learning models for YFV inhibitors led to the selection of a new pyrazolesulfonamide derivative RCB16003 with acceptable in vitro activity. We report that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class, which was recently identified as active non-nucleoside reverse transcriptase inhibitors against HIV-1, can also be repositioned as inhibitors of yellow fever virus replication. As compared to other Flaviviridae or Togaviridae family viruses tested, both compounds RCB16003 and RCB16007 demonstrate selectivity for YFV over related viruses, with only RCB16007 showing some inhibition of the West Nile virus (EC50 7.9 μM, CC50 17 μM, SI 2.2). We also describe the absorption, distribution, metabolism, and excretion (ADME) in vitro and pharmacokinetics (PK) for RCB16007 in mice. This compound had previously been shown to not inhibit hERG, and we now describe that it has good metabolic stability in mouse and human liver microsomes, low levels of CYP inhibition, high protein binding, and no indication of efflux in Caco-2 cells. A single-dose oral PK study in mice has a T1/2 of 3.4 h and Cmax of 1190 ng/mL, suggesting good availability and stability. We now propose that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class may be prioritized for in vivo efficacy testing against YFV.

Distinct inhibitory strength of bisphenol A analogues on human and rat 11β-hydroxysteroid dehydrogenase 1: 3D quantitative structure-activity relationship and in silico molecular docking analysis

Bisphenol A (BPA) analogues are developed to replace BPA usage. However, their effects on 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) are largely unknown. The inhibitory effects of BPA and 10 BPA analogues with the substituents on the bridge moiety on human and rat 11β-HSD1 were explored in human and rat liver microsomes. The strength of inhibiting human 11β-HSD1 was bisphenol FL (IC50, 3.87 μM) > bisphenol Z (6.86 μM) > bisphenol AF (9.42 μM) > bisphenol C (16.14 μM) > bisphenol AP (32.14 μM) = bisphenol B (32.34 μM) > 4,4′-thiodiphenol (67.35 μM) > BPA (297.35 μM) > other BPA analogues (ineffective at 100 μM). The strength of inhibiting rat 11β-HSD1 was bisphenol Z (IC50, 14.44 μM) > 4,4′-thiodiphenol (19.01 μM) > bisphenol B (20.13 μM) > bisphenol F (22.10 μM) > bisphenol E (33.04 μM) > bisphenol AF (49.67 μM) > bisphenol C > (56.97 μM) > bisphenol AP (62.71 μM) >bisphenol FL (96.31 μM) > other BPA analogues (ineffective at 100 μM). Bisphenol A, AF, AP, B, C, F, FL, Z, and 4,4′-thiodiphenol bind to the active sites of human and rat 11β-HSD1. Regression of LogP and molecular weight with IC50 values revealed distinct inhibitory pattern (negative correlation for human 11β-HSD1 vs. positive correlation for rat enzyme). Regression of the lowest binding energy with IC50 values revealed a significant positive regression. 3D QSAR pharmacophore analysis showed one hydrogen bond acceptor and two hydrogen bond donors for human 11β-HSD1. In conclusion, most BPA analogues are more potent inhibitors of human and rat 11β-HSD1 enzymes and there is structure-dependent and species-dependent inhibition.

Preclinical pharmacokinetic and in vitro metabolic stability study of lysosomotropic autophagy inhibitor, IITZ-01 in mice by using UPLC-MS/MS

The present study evaluates the pharmacokinetics and metabolic stability of a novel lysosomotropic autophagy inhibitor, IITZ-01 using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS-MS). It is required as this lead molecule awaits pre-clinical studies for development because of significant therapeutic outcomes in triple-negative breast cancer and renal cancer. A bioanalytical method for the quantitative determination of IITZ-01 in the plasma of mice was developed using the UPLC-MS/MS technique. The UPLC-MS/MS method was validated according to US-FDA bioanalytical guidance and successfully applied to study the pharmacokinetics and metabolic stability. Separation of IITZ- 01 and ZSTK474 (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved using Waters Acquity BEH C-18 column (50 mm × 2.1 mm, 1.7 μm). A gradient mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile was applied at a flow rate of 0.2 mL/min. Electrospray ionization was employed in positive ion mode for detection, while quantification utilized the multiple reaction monitoring (MRM) mode. This involved using [M+H]+fragment ions at m/z 483.19 → 235.09 for IITZ-01 and m/z 418 → 138 for the internal standard (IS). The method was validated over the calibration range of 0.5–800 ng/mL. The LLOQ of IITZ-01 was 0.5 ng/mL in mice plasma. The method demonstrated good in terms of intra- and inter-day precision and accuracy. The matrix effect was found to be negligible, and the stability data were within acceptable limits. The validated technique supports suitability, reliability, reproducibility, and sensitivity for the pre-clinical investigation of IITZ-01 pharmacokinetics in mice and metabolic stability in human liver microsomes.

The roles of CYP2C19 and CYP3A4 in the invitro metabolism of β-eudesmol in human liver: Reaction phenotyping and enzyme kinetics.

β-eudesmol is a major bioactive component of Atractylodes lancea (AL). AL has been developed as the capsule formulation of standardized AL extract for treating cholangiocarcinoma (CCA). However, the complex constituents of herbal products increase the risk of adverse drug interactions. β-eudesmol has demonstrated inhibitory effects on rCYP2C19 and rCYP3A4 in the previous research. This study aimed to identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of β-eudesmol and determine the enzyme kinetic parameters and the metabolic stability of β-eudesmol metabolism in the microsomal system. Reaction phenotyping using human recombinant CYPs (rCYPs) and selective chemical inhibitors of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was performed, and enzyme kinetics and metabolic stability were investigated using human liver microsome (HLM). The results suggest that CYP2C19 and CYP3A4 play significant roles in β-eudesmol metabolism. The disappearance half-life (t1/2 ) and intrinsic clearance (CLint ) of β-eudesmol were 17.09 min and 0.20 mL/min·mg protein, respectively. Enzyme kinetic analysis revealed the Michaelis-Menten constant (Km ) and maximum velocity (Vmax ) of 16.76 μM and 3.35 nmol/min·mg protein, respectively. As a component of AL, β-eudesmol, as a substrate and inhibitor of CYP2C19 and CYP3A4, has a high potential for drug-drug interactions when AL is co-administered with other herbs or conventional medicines.

CYP1A2 expression rather than genotype is associated with olanzapine concentration in psychiatric patients

Olanzapine is a commonly prescribed atypical antipsychotic agent for treatment of patients with schizophrenia and bipolar disorders. Previous in vitro studies using human liver microsomes identified CYP1A2 and CYP2D6 enzymes being responsible for CYP-mediated metabolism of olanzapine. The present work focused on the impact of CYP1A2 and CYP2D6 genetic polymorphisms as well as of CYP1A2 metabolizing capacity influenced by non-genetic factors (sex, age, smoking) on olanzapine blood concentration in patients with psychiatric disorders (N = 139). CYP2D6 genotype-based phenotype appeared to have negligible contribution to olanzapine metabolism, whereas a dominant role of CYP1A2 in olanzapine exposure was confirmed. However, CYP1A2 expression rather than CYP1A2 genetic variability was demonstrated to be associated with olanzapine concentration in patients. Significant contribution of − 163C > A (rs762551), the most common SNP (single nucleotide polymorphism) in CYP1A2 gene, to enhanced inducibility was confirmed by an increase in CYP1A2 mRNA expression in smokers carrying − 163A, and smoking was found to have appreciable impact on olanzapine concentration normalized by the dose/bodyweight. Furthermore, patients’ olanzapine exposure was in strong association with CYP1A2 expression; therefore, assaying CYP1A2 mRNA level in leukocytes can be an appropriate tool for the estimation of patients’ olanzapine metabolizing capacity and may be relevant in optimizing olanzapine dosage.

Construction of humanized CYP1A2 rats using CRISPR/Cas9 to promote drug metabolism and pharmacokinetic research.

Cytochrome P450 family 1 subfamily A member 2 (CYP1A2), performs an indispensable role in metabolism of both exogenous and endogenous substances. What is more, CYP1A2 functions in human diseases by regulating homeostasis of cholesterol. Despite the emergence of gene-editing animal models, genetically humanized animals that overcome species differences for further exploring the role of CYP1A2 in drug metabolism and human diseases have not yet been constructed. In this study, we inserted human CYP1A2 cDNA into the rat Cyp1a2 gene by using CRISPR/Cas9 technology. Results showed that human CYP1A2 was successfully expressed in humanized rat liver and there were no statistically significant differences of physiological symptoms compared with wild-type (WT) rats. In vitro incubation results indicated the different inhibition of furafylline on CYP1A2 activity in human liver microsomes, humanized CYP1A2 (hCYP1A2) rat liver microsomes, and WT rat liver microsomes, with IC50 values of 7.1 μM, 36.5 μM, and 285.8 μM, respectively. Meanwhile, pharmacokinetic characteristics of clozapine were conducted, and the results suggested that in hCYP1A2 rats, clozapine tended to be metabolized into norclozapine. Both the in vitro and in vivo results demonstrated the different metabolic functions of CYP1A2 in humanized and WT rats. We successfully constructed a novel humanized CYP1A2 rat model using the CRISPR/Cas9 system, providing a powerful tool for better predicting CYP1A2-mediated drug metabolism and pharmacokinetics. Significance Statement Human CYP1A2 takes active part both in the biotransformation of exogenous substances and endogenous substances. Meanwhile, it plays a regulatory role in human diseases, including hypercholesterolemia, hypertension as well as various malignant tumors. This study successfully constructed humanized CYP1A2 rat model by CRISPR/Cas9 technology, providing a powerful model for promoting drug development and safety evaluation, as well as further exploring the role of CYP1A2 in human diseases.

Metabolic Stability of Eight Airborne OrganoPhosphate Flame Retardants (OPFRs) in Human Liver, Skin Microsomes and Human Hepatocytes

The waste of electrical and electronic equipment (WEEE) is generally considered a secondary raw material for the recovery of valuable components. However, emerging issues regarding the impact of suspended particles arising from WEEE recycling operations are a concern. It was recently demonstrated that samples from three different WEEE plants were rich in organophosphate flame retardants (OPFRs). Since exposure to a xenobiotic can lead to its biotransformation through human metabolism routes, in the present study, the metabolism of eight OPFRs of interest in our sampling campaign (triphenyl phosphate (TPhP), tri-m-tolyl phosphate (TMTP), ethylhexyl diphenyl phosphate (EHDPhP), tributoxyethyl phosphate (TBOEP), diphenyl phosphate (DPhP), trichloroethyl phosphate (TCEP), tris(1,3-dichloropropan-2-yl) phosphate (TDClPP) and bisphenol A bis(diphenyl phosphate) (BDP)) was investigated. Their metabolism was studied at different time points in three matrices: human liver microsomes, human hepatocytes and human skin microsomes. This study, which was run using a common experimental setting, allowed easy comparison of results for each OPFR of interest, and a comparison with other data in the literature was performed. In particular, a number of metabolites not previously described were detected, and for the first time, it was shown that TPhP could be metabolized in human skin microsomes.

Covalent Stapling of the Cereblon Sensor Loop Histidine Using Sulfur-Heterocycle Exchange.

Site-specific modification of amino acid residues in protein binding pockets using sulfonyl exchange chemistry expands the druggable proteome by enabling the development of covalent modulators that target residues beyond cysteine. Sulfonyl fluoride and triazole electrophiles were incorporated previously into the cereblon (CRBN) molecular glue degrader EM12, to covalently engage His353 within the CRBN sensor loop, but these probes had poor human plasma stability. Attenuation of intrinsic reactivity through the development of sulfonyl pyrazoles, imidazoles, and nucleobases enhanced plasma stability, and several compounds retained efficient labeling of His353. For example, sulfonyl imidazole EM12-SO2Im covalently blocked the CRBN binding site and possessed excellent metabolic stability in human plasma, liver microsomes, and hepatocytes. These results highlight the potential suitability of sulfonyl imidazole and related sulfur(VI)-diazole exchange (SuDEx) warheads for covalent drug development and further exemplify the therapeutic promise of site-specific histidine targeting.

An Ultrafast UPLC-MS/MS Method for Characterizing the In Vitro Metabolic Stability of Acalabrutinib.

Acalabrutinib, commercially known as Calquence®, is a pharmacological molecule that has robust inhibitory activity against Bruton tyrosine kinase. The medicine in question was carefully developed by the esteemed pharmaceutical company AstraZeneca. The FDA granted authorization on 21 November 2019 for the utilization of acalabrutinib (ACB) in the treatment of small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL) in adult patients. The aim of this study was to develop a UPLC-MS/MS method that is effective, accurate, environmentally sustainable, and has a high degree of sensitivity. The methodology was specifically developed with the intention of quantifying ACB in human liver microsomes (HLMs). The methodology described above was subsequently utilized to assess the metabolic stability of ACB in HLMs in an in vitro environment. The validation procedures for the UPLC-MS/MS method in the HLMs were conducted in accordance with the bioanalytical method validation criteria established by the U.S.- DA. The utilization of the StarDrop software (version 6.6), which integrates the P450 metabolic module and DEREK software (KB 2018 1.1), was employed for the purpose of evaluating the metabolic stability and identifying potential hazardous alarms associated with the chemical structure of ACB. The calibration curve, as established by the ACB, demonstrated a linear correlation across the concentration range of 1 to 3000 ng/mL in the matrix of HLMs. The present study conducted an assessment of the accuracy and precision of the UPLC-MS/MS method in quantifying inter-day and intra-day fluctuations. The inter-day accuracy demonstrated a spectrum of values ranging from -1.00% to 8.36%, whilst the intra-day accuracy presented a range of values spanning from -2.87% to 4.11%. The t1/2 and intrinsic clearance (Clint) of ACB were determined through in vitro testing to be 20.45 min and 39.65 mL/min/kg, respectively. The analysis concluded that the extraction ratio of ACB demonstrated a moderate level, thus supporting the recommended dosage of ACB (100 mg) to be administered twice daily for the therapeutic treatment of persons suffering from B-cell malignancies. Several computational tools have suggested that introducing minor structural alterations to the butynoyl group, particularly the alpha, beta-unsaturated amide moiety, or substituting this group during the drug design procedure, could potentially enhance the metabolic stability and safety properties of novel derivatives in comparison to ACB.

Gene Polymorphisms and Drug-Drug Interactions Determine the Metabolic Profile of Blonanserin.

This study aimed to evaluate the effects of cytochrome P450 3A4 (CYP3A4) gene polymorphism and drug interaction on the metabolism of blonanserin. Human recombinant CYP3A4 was prepared using the Bac-to-Bac baculovirus expression system. A microsomal enzyme reaction system was established, and drug-drug interactions were evaluated using Sprague-Dawley rats. Ultra-performance liquid chromatography-tandem mass spectrometry was used to detect the concentrations of blonanserin and its metabolite. Compared with wild type CYP34A, the relative clearance of blonanserin by CYP3A4.29 significantly increased to 251.3%, while it decreased notably with CYP3A4.4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 23, 24, 28, 31, 33, and 34, ranging from 6.09% to 63.34%. Among 153 tested drugs, nimodipine, felodipine, and amlodipine were found to potently inhibit the metabolism of blonanserin. Moreover, the inhibitory potency of nimodipine, felodipine, and amlodipine varied with different CYP3A4 variants. The half-maximal inhibitory concentration and enzymatic kinetics assay demonstrated that the metabolism of blonanserin was noncompetitively inhibited by nimodipine in rat liver microsomes and was inhibited in a mixed manner by felodipine and amlodipine in both rat liver microsomes and human liver microsomes. When nimodipine and felodipine were coadministered with blonanserin, the area under the blood concentration-time curve (AUC)(0-t), AUC(0-∞), and C max of blonanserin increased. When amlodipine and blonanserin were combined, the C max of blonanserin C increased remarkably. The vast majority of CYP3A4 variants have a low ability to catalyze blonanserin. With combined administration of nimodipine, felodipine, and amlodipine, the elimination of blonanserin was inhibited. This study provides the basis for individualized clinical use of blonanserin. SIGNIFICANCE STATEMENT: The enzyme kinetics of novel CYP3A4 enzymes for metabolizing blonanserin were investigated. Clearance of blonanserin by CYP3A4.4, 5, 7-10, 12-14, 16-18, 23-24, 28, 31, 33, and 34 decreased notably, but increased with CYP3A4.29. Additionally, we established a drug interaction spectrum for blonanserin, in which nimodipine, felodipine, and amlodipine kinetics exhibited mixed inhibition. Moreover, their inhibitory potencies decreased with CYP3A4.4 and 5 compared to CYP3A4.1. This study provides essential data for personalized clinical use of blonanserin.

Structural Identification of Zotarolimus (ABT-578) Metabolites Generated by Human Liver Microsomes Using Ion-Trap and High-Resolution Time-of-Flight Mass Spectrometry in Combination with the Analysis of Fragmentation Patterns.

Zotarolimus (ABT-578) is a sirolimus derivative that, like sirolimus and everolimus, is an inhibitor of cell growth via inhibition of the mechanistic target of rapamycin (mTOR). Zotarolimus was developed for coating coronary stents to prevent smooth muscle cell proliferation and restenosis. Albeit zotarolimus-eluting cardiovascular devices have been on the market for years, details of zotarolimus drug metabolism in humans are still unknown. Hence, it was the goal of the present study to identify zotarolimus metabolites generated by incubation with human liver microsomes. Metabolite structures were identified using high-resolution mass spectrometry, MS/ion-trap (MSn), and comparison of fragmentation patterns of the metabolites with those of zotarolimus and other known sirolimus derivatives. Kinetic parameters such as incubation time, human liver microsomal protein concentrations, and drug concentrations were optimized before scaling up the metabolism experiments. Human liver microsomes mainly hydroxylated and/or demethylated zotarolimus. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl, and 27-O-desmethyl zotarolimus; hydroxylated metabolites: hydroxy piperidine zotarolimus, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 45/46-hydroxy, and 49-hydroxy zotarolimus; demethylated-hydroxylated metabolites: 16-O-desmethyl, 23/24-hydroxy; 39-O-desmethyl, 23/24-hydroxy; 39-O-desmethyl, 25-hydroxy zotarolimus; 39-O-desmethyl, 11-hydroxy zotarolimus; 39-O-desmethyl, hydroxy-piperidine zotarolimus; 27-O-desmethyl, 45/46-hydroxy zotarolimus; didemethylated metabolites: 16,39-O-didesmethyl zotarolimus; 16,27-O-didesmethyl zotarolimus; 27,39-O-didesmethyl zotarolimus; and dihydroxylated metabolites: 11,24-dihydroxy zotarolimus, 12,24-dihydroxy zotarolimus, and 11,47/48-dihydroxy zotarolimus. It is concluded that zotarolimus is extensively metabolized by human liver microsomes. Twenty-four of these metabolites could be structurally identified using a combination of ion-trap MSn and high-resolution mass spectrometry.

Mechanistic Static Model based Prediction of Transporter Substrate Drug-Drug Interactions Utilizing Atorvastatin and Rifampicin.

An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair. In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations. The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR. This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.

Evaluation of Alectinib Metabolic Stability in HLMs Using Fast LC-MS/MS Method: In Silico ADME Profile, P450 Metabolic Lability, and Toxic Alerts Screening.

Alectinib, also known as Alecensa®, is prescribed for the therapeutic treatment of individuals diagnosed with metastatic non-small cell lung cancer (NSCLC) who have a specific genetic mutation referred to as anaplastic lymphoma kinase (ALK) positivity. The Food and Drug Administration granted regular approval to alectinib, a drug developed by Hoffmann-La Roche, Inc. (Basel, Switzerland)/Genentech, Inc. (South San Francisco, CA, USA), on 6 November 2017. The screening of the metabolic stability and identification of hazardous alarms within the chemical structure of ALC was conducted using the StarDrop software package (version 6.6), which incorporates the P450 metabolic module and DEREK software (KB 2018 1.1). The primary aim of this investigation was to develop a high-throughput and accurate LC-MS/MS technique for the quantification of ALC in the metabolic matrix (human liver microsomes; HLMs). The aforementioned methodology was subsequently employed to assess the metabolic stability of ALC in HLMs through in vitro tests, with the obtained results further validated using in silico software. The calibration curve of the ALC showed a linear correlation that exists within the concentration range from 1 to 3000 ng/mL. The LC-MS/MS approach that was recommended exhibited accuracy and precision levels for both inter-day and intra-day measurements. Specifically, the accuracy values ranged from -2.56% to 3.45%, while the precision values ranged from -3.78% to 4.33%. The sensitivity of the established approach was proved by its ability to adhere to an LLOQ of 0.82 ng/mL. The half-life (t1/2) and intrinsic clearance (Clint) of ALC were estimated to be 22.28 min and 36.37 mL/min/kg, correspondingly, using in vitro experiments. The ALC exhibited a moderate extraction ratio. The metabolic stability and safety properties of newly created derivatives can be enhanced by making modest adjustments to the morpholine and piperidine rings or by substituting the substituent, as per computational software. In in silico ADME prediction, ALC was shown to have poor water solubility and high gastrointestinal absorption along with inhibition of some cytochrome P450s (CYP2C19 and CYP2C9) without inhibition of others (CYP1A2, CYP3A4, and CYP2D6) and P-glycoprotein substrate. The study design that involves using both laboratory experiments and different in silico software demonstrates a novel and groundbreaking approach in the establishment and uniformization of LC-MS/MS techniques for the estimation of ALC concentrations, identifying structural alerts and the assessment of its metabolic stability. The utilization of this study strategy has the potential to be employed in the screening and optimization of prospective compounds during the drug creation process. This strategy may also facilitate the development of novel derivatives of the medicine that maintain the same biological action by targeted structural modifications, based on an understanding of the structural alerts included within the chemical structure of ALC.

Quantitative Translation of Substrate Intrinsic Clearance from Recombinant CYP1A1 to Humans.

CYP1A1 is a cytochrome P450 family 1 enzyme that is mostly expressed in the extrahepatic tissues. To understand the CYP1A1 contribution to drug clearance in humans, we examined the in vitro-in vivo extrapolation (IVIVE) of intrinsic clearance (CLint) for a set of drugs that are in vitro CYP1A1 substrates. Despite being strong in vitro CYP1A1 substrates, 82% of drugs gave good IVIVE with predicted CLint within 2-3-fold of the observed values using human liver microsomes and hepatocytes, suggesting they were not in vivo CYP1A1 substrates due to the lack of extrahepatic contribution to CLint. Only three drugs (riluzole, melatonin and ramelteon) that are CYP1A2 substrates yielded significant underprediction of in vivo CLint up to 11-fold. The fold of CLint underprediction was linearly proportional to human recombinant CYP1A1 (rCYP1A1) CLint, indicating they were likely to be in vivo CYP1A1 substrates. Using these three substrates, a calibration curve can be developed to enable direct translation from in vitro rCYP1A1 CLint to in vivo extrahepatic contributions in humans. In vivo CYP1A1 substrates are planar and small, which is consistent with the structure of the active site. This is in contrast to the in vitro substrates, which include large and nonplanar molecules, suggesting rCYP1A1 is more accessible than what is in vivo. The impact of CYP1A1 on first-pass intestinal metabolism was also evaluated and shown to be minimal. This is the first study providing new insights on in vivo translation of CYP1A1 contributions to human clearance using in vitro rCYP1A1 data.

Characterization of the glucuronidating pathway of pectolinarigenin, the major active constituent of the Chinese medicine Daji, in humans and its influence on biological activities

Ethnopharmacological relevanceThe Chinese medicine Daji (the aerial part of Cirsium japonicum DC.) and its charred product (Cirsii Japonici Herba Carbonisata) have been widely used as hemostatic agents or diuretic agents to prepare a variety of Chinese herbal formula. Pectolinarigenin (PEC), one of the most abundant constituents in both Daji and its charred product, has been considered as the key effective substance responsible for the major pharmacological activities of Daji, including hemostasis, hepatoprotective, anti-tumor and anti-osteoporosis effects. However, the major metabolic pathways of PEC in humans and the influence of PEC metabolism on its biological activities are poorly understood. Aim of the studyTo characterize the main metabolic pathway(s) and key enzymes of PEC in human biological systems, as well as to reveal the influence of PEC metabolism on its biological activities. Materials and methodsThe metabolic stability assays of PEC were investigated in human liver microsomes (HLM). The O-glucuronide of PEC was biosynthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy. The key enzymes responsible for O-glucuronidation of PEC in humans were assigned by performing UGT reaction phenotyping, chemical inhibition and enzymatic kinetic assays. The agonist effects of PEC and its O-glucuronide on nuclear factor erythroid2-related factor 2 (Nrf2), Peroxisome proliferator activated receptors (PPARα and PPARβ) were tested at the cellular level. ResultsPEC could be readily metabolized to form a mono-O-glucuronide in both human liver microsome (HLM) and human intestinal microsome (HIM). The mono-O-glucuronide was bio-synthesized by mouse liver S9 and its structure was fully characterized as PEC-7-O-β-D-glucuronide (PEC-O-7-G). UGT1A1, UGT1A3 and UGT1A9 are key enzymes responsible for PEC-7-O-glucuronidation in HLM, while UGT1A1, UGT1A9 and 1A10 may play key roles in this reaction in HIM. Biological tests revealed that PEC displayed strong agonist effects on Nrf2, PPARα and PPARβ, whereas PEC-7-O-glucuronide showed relatively weak Nrf2 agonist effect and very weak PPAR agonist effects, indicating that PEC-7-O-glucuronidation strongly weaken its agonist effects on Nrf2 and PPAR. ConclusionsOur results demonstrate that 7-O-glucuronidation is the major metabolic pathway of PEC in human tissues, while UGT1A1, 1A3 and 1A9 are key contributing enzymes responsible for PEC-7-O-glucuronidation in human liver. It is also found that PEC 7-O-glucuronidation significantly weakens the Nrf2 and PPAR agonist effects. All these findings are very helpful for the pharmacologists to deep understand the metabolic rates of PEC in humans.

Enhancing drug-drug Interaction Prediction by Integrating Physiologically-Based Pharmacokinetic Model with Fraction Metabolized by CYP3A4

ABSTRACT Background Enhancing the precision of drug–drug interaction (DDI) prediction is essential for improving drug safety and efficacy. The aim is to identify the most effective fraction metabolized by CY3A4 (f m ) for improving DDI prediction using physiologically based pharmacokinetic (PBPK) models. Research Design and Methods The f m values were determined for 33 approved drugs using a human liver microsome for in vitro measurements and the ADMET Predictor software for in silico predictions. Subsequently, these f m values were integrated into PBPK models using the GastroPlus platform. The PBPK models, combined with a ketoconazole model, were utilized to predict AUCR (AUCcombo with ketoconazole/AUCdosing alone), and the accuracy of these predictions was evaluated by comparison with observed AUCR. Results The integration of in vitro f m method demonstrates superior performance compared to the in silico f m method and f m of 100% method. Under the Guest-limits criteria, the integration of in vitro f m achieves an accuracy of 76%, while the in silico f m and f m of 100% methods achieve accuracies of 67% and 58%, respectively. Conclusions Our study highlights the importance of in vitro f m data to improve the accuracy of predicting DDIs and demonstrates the promising potential of in silico f m in predicting DDIs.

Evaluation of the inhibitory effect of azoles on pharmacokinetics of lenvatinib in rats both in vivo and in vitro by UPLC-MS/MS.

Lenvatinib is a multitargeted tyrosine kinase inhibitor used in the treatment of a variety of solid tumors. This study aims to investigate the potential pharmacokinetic interactions between lenvatinib and various azoles (ketoconazole, voriconazole, isavuconazole and posaconazole) when orally administered to rats. A total of 30 Sprague-Dawley rats were randomly allocated into five groups and administered 20 mg/kg of ketoconazole, voriconazole, isavuconazole and 30 mg/kg of posaconazole and 0.5% CMC-Na, through gavage for a duration of 7 days prior to the commencement of the experiment. On the final day, the rats were given 10 mg/kg of lenvatinib. The blood concentration of lenvatinib was determined using UPLC-MS-MS. In vitro lenvatinib were incubated with azoles and rat liver microsomes (RLMs) or human liver microsomes (HLMs). Molecular docking was lastly used to examine the binding strength of the enzymes and ligands with Autodock Vina. AUC and Cmax of lenvatinib significantly increased with each of the azoles (p < 0.05), whereas CLz/F decreased 0.83-flod, 0.41-fold (p < 0.05) and 0.72-fold (p < 0.01) in voriconazole, isavuconazole and ketoconazole in rats. The IC50 of lenvatinib with the azoles were 0.237, 1.300, 0.355 and 2.403 μM in RLMs and 0.160, 1.933, 3.622 and 1.831 μM in HLMs. Molecular docking analysis suggested that azoles exhibited a strong binding ability towards the target enzymes. It is imperative to acknowledge the potential drug-drug interactions mediated by CYP3A4 between azoles and lenvatinib, as these interactions hold significant implications for their clinical utilization.

In vitro metabolic profiling of new synthetic cannabinoids, ADB-FUBIATA, AFUBIATA, CH-FUBIATA, and CH-PIATA.

In the recreational drug market, synthetic cannabinoids with a new acetamide linker structure emerged, most likely to circumvent the law. As the knowledge of drug metabolites is vital for proving drug consumption, the phase I metabolism of the newly emerging cannabinoids, ADB-FUBIATA, AFUBIATA, CH-FUBIATA, and CH-PIATA, was investigated. Each drug (10μmol/L) was incubated with human liver microsomes for 1h, and the samples, after dilution, were analyzed by liquid chromatography-high-resolution mass spectrometry. All drugs were metabolized via hydroxylation and N-dealkylation, while AFUBIATA and CH-PIATA additionally underwent ketone formation. The metabolites AF7 (hydroxylated at the indole/adjacent methylene) of ADB-FUBIATA, A16 (hydroxylated at the adamantane) of AFUBIATA, CF15 (hydroxylated at the cyclohexane) of CH-FUBIATA, and CP9 (hydroxylated at the pentane) of CH-PIATA were the most abundant metabolites by considering the peak areas on the chromatograms, and are recommended for urinalysis. The structure-metabolism relationship was also discussed, which generally agreed well with previously reported metabolic pathways of other synthetic cannabinoids. However, the preferred hydroxylation site of ADB-FUBIATA, the indole/adjacent methylene, clearly differed from that of ADB-FUBICA, the 3,3-dimethylbutanamide moiety, despite their structures differing only by a methylene group, emphasizing that metabolic predictions of new drugs should not replace in vitro experimental analyses, albeit helpful.

Inhibitory mechanism of vortioxetine on CYP450 enzymes in human and rat liver microsomes.

Vortioxetine is a novel anti-major depression disorder drug with a high safety profile compared with other similar drugs. However, little research has been done on drug-drug interactions (DDI) about vortioxetine. In this paper, the inhibitory effect of vortioxetine on cytochrome P450 (CYP450) and the type of inhibitory mechanism were investigated in human and rat liver microsomes. We set up an in vitro incubation system of 200μL to measure the metabolism of probe substrates at the present of vortioxetine at 37°C. The concentrations of the metabolites of probe substrates were all measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. It was found no time-dependent inhibition (TDI) of vortioxetine through determination of half-maximal inhibitory concentration (IC50) shift values. The enzymes and metabolites involved in this experiment in human and rats were as follows: CYP3A4/CYP3A (midazolam); CYP2B6/CYP2B (bupropion); CYP2D6/CYP2D (dextromethorphan); CYP2C8/CYP2C-1 (amodiaquine); CYP2C9/CYP2C-2 (losartan); and CYP2C19/CYP2C-3 (mephenytoin). We found that vortioxetine competitively inhibited CYP2C19 and CYP2D6 in human liver microsomes (HLMs) with inhibition constant (Ki) values of 2.17μM and 9.37μM, respectively. It was noncompetitive inhibition for CYP3A4 and CYP2C8, and its Ki values were 7.26μM and 6.96μM, respectively. For CYP2B6 and CYP2C9, vortioxetine exhibited the mixed inhibition with Ki values were 8.55μM and 4.17μM, respectively. In RLMs, the type of vortioxetine inhibition was uncompetitive for CYP3A and CYP2D (Ki = 4.41 and 100.9μM). The inhibition type was competitive inhibition, including CYP2B and CYP2C-2 (Ki = 2.87 and 0.12μM). The inhibition types of CYP2C-1 and CYP2C-3 (Ki = 39.91 and 4.23μM) were mixed inhibition and noncompetitive inhibition, respectively. The study of the above mechanism will provide guidance for the safe clinical use of vortioxetine so that the occurrence of DDI can be avoided.

In Vitro and In Vivo Evaluation of Pellotine: A Hypnotic Lophophora Alkaloid.

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1. We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT)1D, 5-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM, Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM, Emax = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.