Drug-metabolizing Enzymes Research Articles

Artificial intelligence modeling of biomarker-based physiological age: Impact on phase 1 drug-metabolizing enzyme phenotypes.

Age and aging are important predictors of health status, disease progression, drug kinetics, and effects. The purpose was to develop ensemble learning-based physiological age (PA) models for evaluating drug metabolism. National Health and Nutrition Examination Survey (NHANES) data were modeled with ensemble learning to obtain two PA models, PA-M1 and PA-M2. PA-M1 included body composition, blood and urine biomarkers, and disease variables as predictors. PA-M2 had blood and urine-derived variables as predictors. Activity phenotypes for cytochrome-P450 (CYP) CYP2E1, CYP1A2, CYP2A6, xanthine oxidase (XO), and N-acetyltransferase-2 (NAT-2) and telomere attrition were assessed. Bayesian networks were used to obtain mechanistic systems pharmacology model structures for PA. The study included n = 22,307 NHANES participants (51.5% female, mean age 46.0 years, range: 18-79 years). The PA-M1 and PA-M2 distributions had greater dispersion across age strata with a right skew for younger age strata and a left skew for older age strata. There was no evidence of algorithmic bias based on sex or race/ethnicity. Klotho, lean body mass, glycohemoglobin, and systolic blood pressure were the top four predictors for PA-M1. Glycohemoglobin, serum creatinine, total cholesterol, and urine creatinine were the top four predictors for PA-M2. The models also performed satisfactorily in independent validation. Model-predicted PA was associated with CYP2E1, CYP1A2, CYP2A6, XO, and NAT-2 activity. Telomere attrition was associated with greater PA-M1 and PA-M2. Ensemble learning models provide robust assessments of PA from easily obtained blood and urine biomarkers. PA is associated with Phase I drug-metabolizing enzyme phenotypes.

Hypertensive Nephropathy Changes the Expression of Drug-Metabolizing Enzymes and Transporters in Spontaneously Hypertensive Rat Liver and Kidney.

Hypertensive nephropathy (HN) has become one of the main causes of end-stage renal disease. Drug combination therapy is a common clinical treatment for HN. However, the impact of HN on drug-metabolizing enzymes and transporters, which may lead to drug-drug interactions (DDIs) and even trigger toxic side effects, remains unclear. The aim of this study was to investigate changes in major drug-metabolizing enzymes and transporters in the liver and kidney of HN rats to improve the scientific foundations for the clinical treatment of HN. Spontaneously hypertensive rats (SHRs) were used as an animal HN model because their hypertension is similar to that of humans. Wistar-Kyoto rats (WKYs) were used as the control group. Body weight, blood pressure, hematoxylin-eosin (HE) staining and biochemical analysis were performed to evaluate whether the HN model was successfully constructed. Quantitative real-time polymerase chain reaction (PCR) and western blotting were used to evaluate the mRNA and protein expression of drug-metabolizing enzymes, transporters and related nuclear transcription factors. In HN rats, the mRNA expression of the drug-metabolizing enzymes cytochrome P450 (Cyp) 2b1, Cyp2c11, Cyp3a1 and Cyp7a1 was significantly upregulated. The protein level of CYP3A1 was consistent with its mRNA expression. Interestingly, the mRNA expression of the hepatic transporters organic cation transporter (Oct) 1, Oct2, organic anion transporter (Oat) 1, Oat2, multidrug resistant protein (Mrp) 2, multidrug resistance (Mdr) 1, organic anion transporting polypeptide (Oatp) 1b2 and na+/taurocholate cotransporting polypeptide (Ntcp) was also markedly upregulated. This may be directly influenced by the upregulation of the expression of the nuclear receptors farnesoid X receptor (Fxr), pregnane X receptor (Pxr), liver X-activated receptor (Lxr) and constitutive androstane receptor (Car). In the kidney of HN rats, the mRNA level of the drug-metabolizing enzyme Cyp2b1 significantly increased, while levels of Cyp1a1, Cyp2c11, Cyp3a1 and Cyp3a2 did not significantly change. The mRNA expression of the transporters multidrug and toxin extrusion (Mate) 1 and Mrp2 was obviously increased but was markedly depressed for peptide transporters (Pept) 1 and Pept2. These changes may be related to the cross effects of Pxr, Fxr and Car in kidney. HN pathological status can alter the expression of drug-metabolizing enzymes and transporters in the liver and kidney to varying degrees, thus affecting the disposition of substrate drugs in vivo. This suggests that to avoid potential risks, caution should be exercised when administering combination therapy for HN treatment.

Novel Regulatory Mechanisms for Expression of Drug Metabolism-related Factors

Interindividual differences in the expression and activity of drug-metabolizing enzymes, including cytochrome P450, UDP-glucuronosyltransferase, and esterases, cause variability of therapeutic effectiveness and side effects during drug treatment. Conventional research has focused on transcriptional regulation by transcription factors and nuclear receptors such as aryl hydrocarbon receptor, pregnane X receptor (PXR), constitutive androstane receptor, and hepatocyte nuclear factor 4α, as the major mechanisms causing the differences in the expression of drug-metabolizing enzymes. Recently, we have revealed that adenosine-to-inosine RNA editing and methylation of adenosine at the N6 position on RNA, two major types of posttranscriptional modification, play a pivotal role in the regulation of drug metabolism. In addition, switch/sucrose non-fermentable complex, a chromatin remodeler, is required for PXR-mediated transcriptional regulation of drug-metabolizing enzymes. This review article introduces the significance of these epitranscriptomic and epigenetic regulations as factors in determining drug metabolism potency. Further research on this link is expected to lead to a deeper understanding of interindividual differences in the therapeutic effectiveness and side effects of medicines.

Modulation of Multispecific Transporters by Uncaria tomentosa Extract and Its Major Phytoconstituents

Background/Objectives: One of the major risks associated with the concomitant use of herbal products and therapeutic drugs is herb–drug interactions (HDIs). The most common mechanism leading to HDIs is the inhibition and/or induction of transport proteins and drug-metabolizing enzymes by herbal ingredients, causing changes in the pharmacokinetic disposition of the victim drug. The present study aimed to determine the potential interactions of Uncaria tomentosa (UT) (cat’s claw), a popular herb due to its supposed health benefits. Methods: The effect of UT extract and its major oxindole alkaloids was investigated on multispecific solute carrier (SLC) and ATP-binding cassette (ABC) drug transporters, using SLC transporter-overexpressing cell lines and vesicles prepared from ABC transporter-overexpressing cells. Results: UT extract significantly inhibited all ABC transporters and the majority of the SLC transporters tested. Of the investigated oxindole alkaloids, isopteropodine significantly inhibited OATP, OCT1 and OCT2, OAT3, ENT4, MDR1, and BCRP transporters. OCTs, OCTN1-, ENT1-, and MDR1-mediated substrate accumulation was below 50% in the presence of mitraphylline. Conclusions: Based on the calculated intestinal concentration of UT extract, interactions with intestinal transporters, especially OATP2B1, ENTs, MRP1, MRP2, MDR1, and BCRP could be relevant in vivo. Our data can help to predict the clinical consequences of UT co-administration with drugs, such as increased toxicity or altered efficacy. In conclusion, the use of these in vitro models is applicable for the analysis of transporter-mediated HDIs similar to drug–drug interaction (DDI) prediction.

Effect of donor GSTM3 rs7483 genetic variant on tacrolimus elimination in the early period after liver transplantation.

Glutathione S-transferase mu (GSTM) belongs to the group of phase II drug-metabolizing enzymes, and the GSTM1 genetic variant has been reported to have a potential association with the metabolism of immunosuppressive drug after renal transplantation. The effect of donor and recipient GSTMs genetic variants on tacrolimus (Tac) metabolism was the focus of our investigation in this study. A total of 203 liver transplant patients were recruited for the study. In the training set (n=110), twenty-one SNPs in five genes (GSTM1-5) were genotyped by the drug-metabolizing enzymes and transporter (DMET) microarray. CYP3A5 rs776746 and GSTM3 rs7483 were genotyped using a Mass ARRAY platform in the validating set (n=93). Tac C/D ratios of donor GSTM3 rs7483 AA carriers were significantly lower than those with the G allele at weeks 1, 2, 3 and 4 after liver transplantation (LT). Multivariate analysis was conducted on the training set and validating set, donor and recipient CYP3A5 rs776746, donor GSTM3 rs7483 and total bilirubin were identified as independent predictors of Tac C/D ratios in the early period after LT. Combining CYP3A5 rs776746 and donor GSTM3 rs7483 genotypes, Tac C/D ratios were observed to be increasingly lower with increasing numbers of alleles associated with fast metabolism. Moreover, the risk of a supratherapeutic C0 (Tac > 15 ug/L) was significantly higher for poor metabolizers than the other groups at week 1 after LT. There was a significant association between the donor GSTM3 rs7483 genetic variant and Tac metabolism in the early period after LT. Genotype classification might have a better predictive ability of the initial Tac doses.

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

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

Isoform-level expression of the constitutive androstane receptor (CAR or NR1I3) transcription factor better predicts the mRNA expression of the cytochrome P450s in human liver samples.

Many factors cause inter-person variability in the activity and expression of liver cytochrome P450 (CYP) drug-metabolizing enzymes, leading to variable drug exposure and treatment outcomes. Several liver-enriched transcription factors (TFs) are associated with CYP expression, with estrogen receptor alpha (ESR1) and constitutive androstane receptor (CAR or NR1I3) being the two top factors. ESR1 and NR1I3 undergo extensive alternative splicing that results in numerous splice isoforms, but how these splice isoforms associate with CYP expression is unknown. Here, we quantified 18 NR1I3 splice isoforms and the three most abundant ESR1 isoforms in 260 liver samples derived from African Americans (AA, n=125) and European Americans (EA, n=135). Our results showed variable splice isoform populations in the liver for both NR1I3 and ESR1. Multiple linear regression analyses revealed that, compared to gene-level NR1I3, isoform-level NR1I3 expression better predicted the mRNA expression of most CYPs and three UDP-glucuronosyltransferases (UGTs), while ESR1 isoforms improved predictive models for the UGTs and CYP2D6, but not for most CYPs. Also, different NR1I3 isoforms were associated with different CYPs, and the associations varied depending on sample ancestry. Surprisingly, non-canonical NR1I3 isoforms having retained introns (introns 2 or 6) were abundantly expressed and associated with the expression of most CYPs and UGTs, whereas the reference isoform (NR1I3-205) only associated with CYP2D6. Moreover, NR1I3 isoform diversity increased during the differentiation of induced pluripotent stem cells to hepatocytes, paralleling increasing CYP expression. These results suggest that isoform-level TF expression may help to explain variation in CYP or UGT expression between individuals. Significance Statement We quantified 18 NR1I3 splice isoforms and three ESR1 splice isoforms in 260 liver samples derived from AA and EA donors and found variable NR1I3 and ESR1 splice isoform expression in the liver. Multiple linear regression analysis showed that, compared to gene-level expression, isoform-level expression of NR1I3 and ESR1 better predicted the mRNA expression of some CYPs and UGTs, highlighting the importance of isoform-level analyses to enhance our understanding of gene transcriptional regulatory networks controlling the expression of drug-metabolizing enzymes.

All Blood Brain Barrier Cell Types Demonstrate Capability to Influence Differential Tenofovir and Emtricitabine Metabolism and Transport in the Brain.

The blood brain barrier (BBB) represents a significant obstacle in brain drug penetration that challenges efforts in the treatment of neurological disorders. Therapeutically targeting the brain requires interactions with each BBB cell type, including endothelial cells, pericytes, and astrocytes. Yet, the relative contribution of these BBB cell types to the mechanisms that facilitate brain drug disposition is not well characterized. Here, we use first-line antiretroviral therapies, tenofovir (TFV) and emtricitabine (FTC), as models to investigate the mechanisms of drug transport and metabolism at the BBB that may influence access of the drug to the brain. We evaluated regional and cell-type-specific drug metabolism and transport mechanisms using rhesus macaques and in vitro treatment of primary human cells. We report heterogeneous distribution of TFV, FTC, and their active metabolites, which cerebrospinal fluid measures could not reflect. We found that all BBB cell types possessed functional drug-metabolizing enzymes and transporters that promoted TFV and FTC uptake and pharmacologic activation. Pericytes and astrocytes emerged as pharmacologically dynamic cells that rival hepatocytes and were uniquely susceptible to modulation by disease and treatment. Together, our findings demonstrate the importance of considering the BBB as a unique pharmacologic entity rather than viewing it as an extension of the liver, as each cell type possesses distinct drug metabolism and transport capacities that contribute to differential brain drug disposition. Further, our work highlights pharmacologically active pathways at the BBB that may regulate brain drug disposition and impact therapeutic efforts to alleviate neurologic disease.

Validating Disease Associations of Drug-Metabolizing Enzymes through Genome-Wide Association Study Data Analysis

Background and Objectives: Phase I and phase II drug-metabolizing enzymes are crucial for the metabolism and elimination of various endogenous and exogenous compounds, such as small-molecule hormones, drugs, and xenobiotic carcinogens. While in vitro and animal studies have suggested a link between genetic mutations in these enzymes and an increased risk of cancer, human in vivo studies have provided limited supportive evidence. Methods: Genome-wide association studies (GWASs) are a powerful tool for identifying genes associated with specific diseases by comparing two large groups of individuals. In the present study, we analyzed a GWAS database to identify key diseases genetically associated with drug-metabolizing enzymes, focusing on UDP-glucuronosyltransferases (UGTs). Results: Our analysis confirmed a strong association between the UGT1 gene and hyperbilirubinemia. Additionally, over ten studies reported a link between the UGT1 gene and increased low-density lipoprotein (LDL) cholesterol levels. UGT2B7 was found to be associated with testosterone levels, total cholesterol levels, and vitamin D levels. Conclusions: Despite the in vitro capability of UGT1 and UGT2 family enzymes to metabolize small-molecule carcinogens, the GWAS data did not indicate their genetic association with cancer, except for one study that linked UGT2B4 to ovarian cancer. Further investigations are necessary to fill the gap between in vitro, animal, and human in vivo data.

Sub- and supratherapeutic efavirenz plasma concentrations with risk for HIV therapy failure are mainly genetically explained in Ugandan children: The prospective GENEFA cohort study.

Interindividual variations in efavirenz (EFV) plasma concentrations are extensive, but paediatric data on its consequences for viral control are scarce. The aim of this study was to explore the role of genetic variation in achieving therapeutic efavirenz plasma concentrations in a cohort of Ugandan children and the linkage between genetic CYP2B6 variants, EFV plasma variability, viral resistance and viral outcome. Ninety-nine treatment-naïve children, aged 3-12 years and living with HIV, were followed for 24 weeks after ART initiation assessing mid-dose efavirenz plasma concentrations, HIV RNA, HIV drug resistance and adherence. Polymorphisms in genes coding for drug-metabolizing enzymes were genotyped. Efavirenz concentrations were determined by liquid chromatography coupled with high-resolution tandem mass spectrometry. Metabolizer phenotype was predicted from composite genotypes of CYP2B6 (c.516G>T and c.983 T>C). A mixed effects restricted maximum likelihood regression model was used to identify important factors for efavirenz exposure. Efavirenz plasma concentrations were below the therapeutic interval (1000-4000 mg/mL) in 12-17% and above in 21-24% of measurements. Eight children had persisting subtherapeutic concentrations, five of which failed virologically and three acquired at least one new resistant mutation. Multivariate modelling explained 70% of interindividual variation in plasma concentration, with treatment duration, adherence, CYP2B6c.136A>G, and metabolizer phenotype as independent predictors of EFV concentration. In univariate analysis, metabolizer phenotype explained 50% of interindividual variation. Metabolizer phenotype explained 50% of interindividual variation in efavirenz plasma concentration. Autoinduction was not confirmed and >33% of the concentrations were outside the therapeutic interval. Subtherapeutic concentrations worsened virological resistance and outcomes. Genotype-based dosing may help avert both sub- and supratherapeutic efavirenz plasma concentrations in Ugandan children.

Utilization of a Human Liver Tissue Chip for Drug-Metabolizing Enzyme Induction Studies of Perpetrator and Victim Drugs.

Polypharmacy-related drug-drug interactions (DDIs) are a significant and growing healthcare concern. An increasing number of therapeutic drugs on the market underscores the necessity to accurately assess new drug combinations during pre-clinical evaluation for DDIs. In vitro primary human hepatocytes (PHH) models are only applicable for short term induction studies due to their rapid loss of metabolic function. Though co-culturing non-human stromal cells with PHH has been shown to stabilize metabolic activity long-term, there are concerns about human specificity for accurate clinical assessment. In this study, we demonstrate a PHH-only liver microphysiological system (MPS) in the Liver Tissue Chip (LTC) is capable of maintaining long-term functional and metabolic activity of PHH from three individual donors, and thus a suitable platform for long-term DDI induction studies. The responses to rifampicin induction of three PHH donors were assessed using CYP activity and mRNA changes. Additionally, victim PK studies were conducted with midazolam (high clearance) and alprazolam (low clearance) following perpetrator drug treatment, rifampicin-mediated induction, which resulted in a 2-fold and a 2.6-fold increase in midazolam and alprazolam intrinsic clearance values respectively compared to the untreated liver MPS. We also investigated the induction effects of different dosing regimens of the perpetrator drug (rifampicin) on CYP activity levels, showing minimal variation in the intrinsic clearance of the victim drug (midazolam). This study illustrates the utility of the LTC for in vitro liver-specific DDI induction studies, providing a translational experimental system to predict clinical clearance values of both perpetrator and victim drugs. Significance Statement This study demonstrates the utility of the Liver Tissue Chip (LTC) with primary human hepatocyte (PHH)-only liver microphysiological system (MPS) for drug-drug interaction (DDI) induction studies. This unique in vitro system with continuous recirculation maintains long-term functionality and metabolic activity for up to 4 weeks, enabling the study of perpetrator and victim drug pharmacokinetics, quantification of drug-induced CYP mRNA and activity levels, investigation of patient variability, and ultimately clinical predictions.

Drug Metabolizing Enzymes: An Exclusive Guide into Latest Research in Pharmaco-genetic Dynamics in Arab Countries.

Drug metabolizing enzymes play a crucial role in the pharmacokinetics and pharmacodynamics of therapeutic drugs, influencing their efficacy and safety. This review explores the impact of genetic polymorphisms in drug-metabolizing genes on drug response within Arab populations. We examine the genetic diversity specific to Arab countries, focusing on the variations in key drug-metabolizing enzymes such as CYP450, GST, and UGT families. The review highlights recent research on polymorphisms in these genes and their implications for drug metabolism, including variations in allele frequencies and their effects on therapeutic outcomes. Additionally, the paper discusses how these genetic variations contribute to the variability in drug response and adverse drug reactions among individuals in Arab populations. By synthesizing current findings, this review aims to provide a comprehensive understanding of the pharmacogenetic landscape in Arab countries and offer insights into personalized medicine approaches tailored to genetic profiles. The findings underscore the importance of incorporating pharmacogenetic data into clinical practice to enhance drug efficacy and minimize adverse effects, ultimately paving the way for more effective and individualized treatment strategies in the region.

Loss of heterozygosity of CYP2D6 enhances the sensitivity of hepatocellular carcinomas to talazoparib

Loss of heterozygosity (LOH) diminishes genetic diversity within cancer genomes. A tumour arising in an individual heterozygous for a functional and a loss-of-function (LoF) allele of a gene occasionally retain only the LoF allele. This can result in deficiency of specific protein activities in cancer cells, creating unique differences between tumour cells and normal cells of the individual. Such differences may constitute vulnerabilities that can be exploited through allele-specific therapies. To discover frequently lost genes with prevalent LoF alleles, we mined the 1000 Genomes dataset for SNVs causing protein truncation through base substitution, indels or splice site disruptions, resulting in 60 LoF variants in 60 genes. From these, the variant rs3892097 in the liver enzyme CYP2D6 was selected because it is located within a genomic region that frequently undergoes LOH in several tumor types including hepatocellular cancers. To evaluate the relationship between CYP2D6 activity and the toxicities of anticancer agents, we screened 525 compounds currently in clinical use or undergoing clinical trials using cell model systems with or without CYP2D6 activity. We identified 12 compounds, AZD-3463, CYC-116, etoposide, everolimus, GDC-0349, lenvatinib, MK-8776, PHA-680632, talazoparib, tyrphostin 9, VX-702, and WZ-3146, using an engineered HEK293T cell model. Of these, talazoparib and MK-8776 demonstrated consistently heightened cytotoxic effects against cells with compromised CYP2D6 activity in engineered hepatocellular cancer cell models. Moreover, talazoparib displayed CYP2D6 genotype dependent effects on primary hepatocellular carcinoma organoids. Exploiting the loss of drug-metabolizing enzyme gene activity in tumor cells following loss of heterozygosity could present a promising therapeutic strategy for targeted cancer treatment. This work was funded by Barncancerfonden (T.S, PR2022-0099 and PR2020-0171, X.Z, TJ2021-0111), Cancerfonden (T.S, 211719Pj and D.G, 222449Pj), Vetenskapsrådet (T.S, 2020-02371 and D.G, 2020-04707), and the Erling Persson Foundation (T.S, 2020-0037 and T.S, 2023-0113).

Generation and application of CES1-knockout Tet-Off-regulated CYP3A4 and UGT1A1-expressing Caco-2 cells

Caco-2 cells, a human colorectal adenocarcinoma cell line, are widely used to model small intestinal epithelial cells in the drug development process because they can predict drug absorption with high accuracy. However, Caco-2 cells have several issues. First, Caco-2 cells have little expression of cytochrome P450 3A4 (CYP3A4), which is a major drug-metabolizing enzyme in the human intestine. We previously developed Caco-2 cells whose expression of CYP3A4 can be controlled using doxycycline (Dox) (CYP3A4-Caco-2 cells) (Ichikawa et al., Sci. Rep, 2021). However, since the Tet-On system was used to regulate CYP3A4 expression in these cells, there was concern about drug-drug interactions. The second issue is that in the human small intestine, carboxylesterase 2 (CES2) is more highly expressed than carboxylesterase 1 (CES1), while in Caco-2 cells CES1 is more highly expressed. The third issue is the low level expression of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a phase II drug-metabolizing enzyme. In this study, we used genome-editing technology to establish CES1-knockout Caco-2 cells whose CYP3A4 and UGT1A1 expression can be regulated by the Tet-Off system. These cell lines would be useful in pharmaceutical researches, including intestinal toxicological studies, as an in vitro model for orally administered drugs.

High-Throughput Assessment of Metabolism-Mediated Neurotoxicity by Co-Culture of Neurospheres and Liver Spheroids.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC50 values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity Basic Protocol 3: Image acquisition, processing, and data analysis.

Combination screen in multi-cell type tumor spheroids reveals interaction between aryl hydrocarbon receptor antagonists and E1 ubiquitin-activating enzyme inhibitor

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes of drug transporters and metabolic enzymes to detoxify small molecule xenobiotics. It has a complex role in cancer biology, influencing both the progression and suppression of tumors by modulating malignant properties of tumor cells and anti-tumor immunity, depending on the specific tumor type and developmental stage. This has led to the discovery and development of selective AhR modulators, including BAY 2416964 which is currently in clinical trials. To identify small molecule anticancer agents that might be combined with AhR antagonists for cancer therapy, a high-throughput combination screen was performed using multi-cell type tumor spheroids grown from malignant cells, endothelial cells, and mesenchymal stem cells. The AhR selective antagonists BAY 2416964, GNF351, and CH-223191 were tested individually and in combination with twenty-five small molecule anticancer agents. As single agents, BAY 2416964 and CH-223191 showed minimal activity, whereas GNF351 reduced the viability of some spheroid models at concentrations greater than 1 µM. The activity of most combinations aligned well with the single agent activity of the combined agent, without apparent contributions from the AhR antagonist. All three AhR antagonists sensitized tumor spheroids to TAK-243, an E1 ubiquitin-activating enzyme inhibitor. These combinations were active in spheroids containing bladder, breast, ovary, kidney, pancreas, colon, and lung tumor cell lines. The AhR antagonists also potentiated pevonedistat, a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit, in several tumor spheroid models. In contrast, the AhR antagonists did not enhance the cytotoxicity of the proteasome inhibitor bortezomib.

In Silico ADME And Molecular Docking Studies of New Thiazolyl-bipyrazole, Pyrazolopyridine and Pyrano[2,3-d]pyrazolopyridine Derivatives as Antibacterial Agents

: In this study, a series of novel pyrazole-based compounds were synthesized starting from the precursor ethyl 3-(4-amino-1-phenyl-3-((4-sulfamoylphenyl)carbamoyl)-1Hpyrazol- 5-yl)-3-oxopropanoate (2). Various synthetic routes were used to obtain pyrazolylpyrazolone 3, tricyclic dipyrazolopyridine 4a-c, thiazolyl-bipyrazoles 5 & 6, pyrazolo[4,3- b]pyridines 7 & 9, and tricyclic pyranopyrazolopyridine 10a–c. These compounds were screened for their antibacterial activity against four bacterial strains. The promising candidates 4a, 4b, 4c, 7, 9, and 10c exhibited minimum inhibitory concentrations ranging from 0.98 to 31.25 μg/mL. The in silico ADME properties for the active compounds exhibited similar physiochemical properties, with compound 9 demonstrating the best likeness and no inhibition effect on the popular drug metabolism enzyme CYP. Molecular docking simulations highlighted compounds 9 and 10c as potent antibacterial agents via DNA-gyrase inhibition

CYP P450 and non-CYP P450 Drug Metabolizing Enzyme Families Exhibit Differential Sensitivities towards Proinflammatory Cytokine Modulation.

Compromised hepatic drug metabolism in response to proinflammatory cytokine release is primarily attributed to downregulation of cytochrome P450 (CYP) enzymes. However, whether inflammation also affects other phase I and phase II drug metabolizing enzymes (DMEs), such as the flavin monooxygenases (FMOs), carboxylesterases (CESs), and UDP glucuronosyltransferases (UGTs), remains unclear. This study aimed to decipher the impact of physiologically relevant concentrations of proinflammatory cytokines on expression and activity of phase I and phase II enzymes, to establish a hierarchy of their sensitivity as compared with the CYPs. Hereto, HepaRG cells were exposed to interleukin-6 and interleukin-1β to measure alterations in DME gene expression (24 h) and activity (72 h). Sensitivity of DMEs toward proinflammatory cytokines was evaluated by determining IC50 (potency) and Imax (maximal inhibition) values from the concentration-response curves. Proinflammatory cytokine treatment led to nearly complete downregulation of CYP3A4 (∼98%) but was generally less efficacious at reducing gene expression of the non-CYP DME families. Importantly, FMO, CES, and UGT family members were less sensitive toward interleukin-6 induced inhibition in terms of potency, with IC50 values that were 4.3- to 7.4-fold higher than CYP3A4. Similarly, 18- to 31-fold more interleukin-1β was required to achieve 50% of the maximal downregulation of FMO3, FMO4, CES1, UGT2B4, and UGT2B7 expression. The differential sensitivity persisted at enzyme activity level, highlighting that alterations in DME gene expression during inflammation are predictive for subsequent alterations in enzyme activity. In conclusion, this study has shown that FMOs, CESs, and UGTs enzymes are less impacted by IL-6 and IL-1β treatment as compared with CYP enzymes. SIGNIFICANCE STATEMENT: While the impact of proinflammatory cytokines on CYP expression is well established, their effects on non-CYP phase I and phase II drug metabolism remains underexplored, particularly regarding alterations in drug metabolizing enzyme (DME) activity. This study provides a quantitative understanding of the sensitivity differences to inflammation between DME family members, suggesting that non-CYP DMEs may become more important for the metabolism of drugs during inflammatory conditions due to their lower sensitivity as compared with the CYPs.

Harnessing Pharmacogenomics in Clinical Research on Psychedelic-Assisted Therapy.

Psychedelics have recently re-emerged as potential treatments for various psychiatric conditions that impose major public health costs and for which current treatment options have limited efficacy. At the same time, personalized medicine is increasingly being implemented in psychiatry to provide individualized drug dosing recommendations based on genetics. This review brings together these topics to explore the utility of pharmacogenomics (a key component of personalized medicine) in psychedelic-assisted therapies. We summarized the literature and explored the potential implications of genetic variability on the pharmacodynamics and pharmacokinetics of psychedelic drugs including lysergic acid diethylamide (LSD), psilocybin, N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), ibogaine and 3,4-methylenedioxymethamphetamine (MDMA). Although existing evidence is limited, particularly concerning pharmacodynamics, studies investigating pharmacokinetics indicate that genetic variants in drug-metabolizing enzymes, such as cytochrome P450, impact the intensity of acute psychedelic effects for LSD and ibogaine, and that a dose reduction for CYP2D6 poor metabolizers may be appropriate. Furthermore, based on the preclinical evidence, it can be hypothesized that CYP2D6 metabolizer status might contribute to altered acute psychedelic experiences with 5-MeO-DMT and psilocybin when combined with monoamine oxidase inhibitors. In conclusion, considering early evidence that genetic factors can influence the effects of certain psychedelics, we suggest that pharmacogenomic testing should be further investigated in clinical research. This is necessary to evaluate its utility in improving the safety and therapeutic profile of psychedelic therapies and a potential future role in personalizing psychedelic-assisted therapies, should these treatments become available.

Role of skin enzymes in metabolism of topical drugs

Topical drugs have gained a lot of interest with their massive market growth and are available in various dosage forms. Prodrug compounds of transdermal delivery systems can be very different and designed to convert into the form of active pharmaceutical ingredients (APIs) through enzymatic action once they enter the body. The skin, as an interfacial barrier between the body and surroundings, has demonstrated critical roles in metabolizing, filtering, and detoxifying to minimize certain side effects and improve the medication benefits of topically administered products. It is well recognized that the drug pharmacokinetics can be altered by the presence of skin enzymes driven by biotransformation reactions. To evaluate the effectiveness of a topical generic drug product, its safety, and bioequivalence with the reference one, models assessing enzyme metabolic activity are highly required for testing the amount of drugs that are metabolized or can potentially be metabolized in both healthy and compromised skin. Thus, knowledge of skin composition and enzyme expression levels is of paramount importance in mapping the relevant metabolism that may have occurred. Regulatory authorities have also been making efforts to develop efficient and harmonizable protocols to evaluate the metabolism of transdermal products. This review is a compilation of reported skin metabolizing enzymes, including their role in both drug metabolism and homeostasis regulation, along with their localization and quantification in skin equivalents (and/or membrane layers). Various aspects that potentially affect the skin enzyme metabolism study were also discussed with respect to drug development considerations.