Activity Of Drug-metabolizing Enzymes Research Articles

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.

CYP and non-CYP drug metabolizing enzyme families exhibit differential sensitivities towards pro-inflammatory cytokine modulation.

Compromised hepatic drug metabolism in response to pro-inflammatory 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) like the flavin monooxygenases (FMOs), carboxylesterases (CESs) and UDP glucuronosyltransferases (UGTs) remains unclear. This study aimed to decipher the impact of physiologically relevant concentrations of pro-inflammatory cytokines on expression and activity of phase I and phase II enzymes, in order to establish a hierarchy of their sensitivity as compared to the CYPs. Hereto, HepaRG cells were exposed to interleukin-6 and interleukin-1β to measure alterations in DME gene expression (24h) and activity (72h). Sensitivity of DMEs towards pro-inflammatory cytokines was evaluated by determining IC50 (potency) and Imax (maximal inhibition) values from the concentration-response curves. Pro-inflammatory 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 towards interleuking-6 induced inhibition in terms of potency, with IC50 values that were 4.3-7.4 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, we have shown that FMOs, CES and UGTs enzymes are less impacted by inflammation as compared to CYP enzymes. Significance Statement While the impact of pro-inflammatory 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 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 to the CYPs.

Prediction of the drug intestinal absorption and drug-induced intestinal toxicity with the use of cultured human/animal crypt-derived intestinal stem cells

Prediction of intestinal drug absorption and drug-induced intestinal toxicity is critical for the development of orally-administered drugs. However, it is difficult to accurately predict these events because of large species differences and a lack of appropriate in vitro assay. Then, we proposed the use of human crypt-derived intestinal cells for the prediction of intestinal absorption and the risk of intestinal toxicity. 3D human intestinal spheroids were established from fresh surgical specimens of proximal jejunum and terminal ileum using the conditioned media containing Wnt3a, R-spondin 3, and noggin. To generate 2D monolayer, spheroids were enzymatically dissociated into single cells and plated onto Matrigel-precoated culture plates/inserts. We have confirmed the activities of typical drug-metabolizing enzymes and uptake/efflux transporters in human jejunal spheroid-derived differentiated cells. Intestinal availability (Fg) estimated from the apical-to-basal permeation clearance across the jejunal monolayer showed a good correlation with in vivo human Fg values for five CYP3A substrate drugs. As for the prediction of intestinal toxicity, we found that the degree of ATP decreases in intestinal spheroids incubated with different EGFR-TKIs varied greatly depending on the drugs and the rank order of the extent of ATP decrease corresponded with that of frequency of clinically-observed diarrhea. We also constructed enterochromaffin (EC) cell-rich spheroids and quantified serotonin release from EC cells upon exposure to drugs for the prediction of drug-induced nausea and vomiting. As a result, we found that the serotonin release was related to the high/low risk of nausea and vomiting of each ALK/ROS1 kinase inhibitors.

Development of radioiodine-labeled mequitazine for evaluation of hepatic CYP2D activity.

Background: As drug-metabolizing enzyme activities are affected by a variety of factors, such as drug-drug interactions, a method to evaluate drug-metabolizing enzyme activities in real time is needed. In this study, we developed a novel SPECT imaging probe for evaluation of hepatic CYP2D activity. Methods: Iodine-123- and 125-labeled 4-iodobenzylmequitazine (123/125I-BMQ) was synthesized with high labeling and purity. CYP isozymes involved in the metabolism of 125I-BMQ in mouse liver microsomes were evaluated, and the utility of 123/125I-was assessed from biological distribution and SPECT imaging evaluation in normal and CYP2D-inhibited mice. Results: In vitro metabolite analysis using mouse liver microsomes showed that 125I-BMQ is specifically metabolized by CYP2D. Biological distribution and SPECT imaging of 123/125I-BMQ in normal mice showed that injection 123/125I-BMQ accumulated early in the liver and was excreted into the gallbladder and intestines. In CYP2D-inhibited mice, accumulation in the liver was increased, but accumulation in the gallbladder and intestines, the excretory organ, was delayed. Since only metabolites of 125I-BMQ are detected in bile, visualization and measuring of the accumulation of metabolites over time in the intestine, where bile is excreted, could predict the amount of metabolites produced in the body and evaluate CYP2D activity, which would be useful in determining the dosage of various drugs metabolized by CYP2D. Conclusion: 123/125I-BMQ is useful as a SPECT imaging probe for comprehensive and direct assessment of hepatic CYP2D activity in a minimally invasive and simple approach.

In Situ Self-Assembling Liver Spheroids with Synthetic Nanoscaffolds for Preclinical Drug Screening Applications.

Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure and a major reason for the withdrawal of medications from the market. There is a growing need for advanced in vitro liver models that can effectively recapitulate hepatic function, offering a robust platform for preclinical drug screening applications. Here, we explore the potential of self-assembling liver spheroids in the presence of electrospun and cryomilled poly(caprolactone) (PCL) nanoscaffolds for use as a new preclinical drug screening tool. This study investigated the extent to which nanoscaffold concentration may have on spheroid size and viability and liver-specific biofunctionality. The efficacy of our model was further validated using a comprehensive dose-dependent acetaminophen toxicity protocol. Our findings show the strong potential of PCL-based nanoscaffolds to facilitate in situ self-assembly of liver spheroids with sizes under 350 μm. The presence of the PCL-based nanoscaffolds (0.005 and 0.01% w/v) improved spheroid viability and the secretion of critical liver-specific biomarkers, namely, albumin and urea. Liver spheroids with nanoscaffolds showed improved drug-metabolizing enzyme activity and greater sensitivity to acetaminophen compared to two-dimensional monolayer cultures and scaffold-free liver spheroids. These promising findings highlight the potential of our nanoscaffold-based liver spheroids as an in vitro liver model for drug-induced hepatotoxicity and drug screening.

Oral Drug Dosing After Gastric Bypass and Diet-Induced Weight Loss: Simpler Than We Think? Lessons Learned From the COCKTAIL Study.

This article summarizes the lessons learned from the COCKTAIL study: an open, three-armed, single-center study including patients with obesity scheduled for treatment with Roux-en-Y gastric bypass (RYGB) or nonsurgical calorie restriction, and a normal- to overweight control group. The clinical implications of the results from multiple peer-reviewed articles describing the effects of RYGB, severe caloric restriction, weight loss, and type 2 diabetes on the in vivo activity and protein expression of drug-metabolizing enzymes (cytochrome P450 (CYP) 1A2, 2C9, 2C19, and 3A) and transporters (DMETs; organic anion-transporting polypeptide (OATP) 1B1 and P-glycoprotein (P-gp)) are discussed in the perspective of three clinically relevant questions: (1) How should clinicians get the dose right in patients after RYGB? (2) Will drug disposition in patients with obesity be normalized after successful weight loss? (3) Are dose adjustments needed according to obesity and diabetes status? Overall, RYGB seems to have a lower impact on drug disposition than previously assumed, but clinicians should pay close attention to drugs with a narrow therapeutic range or where a high maximum drug concentration may be problematic. Whether obesity-related alterations of DMETs normalize with substantial weight loss depends on the DMET in question. Obesity and diabetes downregulate the in vivo activity of CYP2C19 and CYP3A (only obesity) but whether substrate drugs should be dose adjusted is also dependent on other factors that influence clearance, that is, liver blood flow and protein binding. Finally, we recommend frequent and individualized follow-up due to high inter- and intraindividual variability in these patients, particularly following RYGB.

Non-alcoholic fatty liver disease changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, which can cause serious complications and gradually increase the mortality rate. However, the effects of NAFLD on drug-metabolizing enzymes and transporters remain unclear, which may cause some confusion regarding patient medication. In this study, a NAFLD rat model was constructed by feeding rats with methionine and choline deficiency diets for 6 weeks, and the mRNA and protein levels of drug-metabolizing enzymes and transporter were analyzed by real-time fluorescent quantitative PCR and Western blot, respectively. The activity of drug-metabolizing enzymes was detected by cocktail methods. In the NAFLD rat model, the mRNA expression of phase I enzymes, phase II enzymes, and transporters decreased. At the protein level, only CYP1A1, CYP1B1, CYP2C11, and CYP2J3 presented a decrease. In addition, the activities of CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP3A2, UGT1A1, UGT1A3, UGT1A6, and UGT1A9 decreased. These changes may be caused by the alteration of FXR, HNF4α, LXRα, LXRβ, PXR, and RXR. In conclusion, NAFLD changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats, which may affect drug metabolism and pharmacokinetics. In clinical medication, drug monitoring should be strengthened to avoid potential risks.

Effects of Calamintha incana (Sm.) Helder Ethanolic Extract on the mRNA Expression of Drug-metabolizing cyp450s in the Mouse Livers.

Alteration in the expression and activity of drug-metabolizing enzymes (DMEs) can alter the pharmacokinetics and hence the response of the drug. Some chemicals found in herbs and fruits affect the expression of DMEs. Calamintha incana is commonly used in Middle Eastern Arabic countries. There is no report regarding the influence of Calamintha incana on the hepatic expression of DMEs. The current investigation aimed to investigate the effect of Calamintha incana consumption on the mRNA expression of major hepatic drug-metabolizing cytochrome (cyp) P450 genes in mice. The chemical composition of the ethanoic extract was analyzed using liquid chromatography/ mass spectrometry. Then, 21 BALB/c mice were used for the in vivo experiment. The mice were divided into three groups, each consisting of seven mice. The first group (low-dose group) was treated with 41.6 mg/kg of Calamintha incana extract and the second group was administered the high-dose (125 mg/kg) of the extract for one month. The mice in the third "control" group administrated the vehicle 20% polyethylene glycol 200. Then, the expression of cyp3a11, cyp2c29, cyp2d9, and cyp1a1 was analyzed using the real-time polymerase chain reaction. The relative liver weights of the mice and the hepatic pathohistological alterations were assessed. The ethanolic extract of Calamintha incana contained 27 phytochemical compounds. The most abundant compounds were linolenic acid, myristic acid, and p-cymene. It was found that the low dose of Calamintha incana extract upregulated significantly (P < 0.05) the expression of cyp3a11 by more than ten folds in the liver of treated mice. Furthermore, the histological analysis showed that low- and high-dose administration of the C. incana did not cause pathological alterations. It can be concluded from these findings that consumption of low doses of Calamintha incana upregulated the mRNA expression of mouse cyp3a11 without causing histopathological alterations in the livers. Further studies are needed to determine the influence of Calamintha incana on the pharmacokinetics and response of drugs metabolized by cyp3a11.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

In Vitro Studies on the Effects of Water Miscible Organic Co-Solvents on Select Drug Metabolizing Enzyme Activities

In Vitro Studies on the Effects of Water Miscible Organic Co-Solvents on Select Drug Metabolizing Enzyme Activities

Air–liquid interface culture and modified culture medium promote the differentiation of human induced pluripotent stem cells into intestinal epithelial cells

An in vitro system that evaluates pharmacokinetics in the small intestine is crucial for the development of oral drugs. We produced human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) with high drug metabolizing enzyme and drug transporter activities. However, the gene expression of our hiSIECs partially differed from that of the human small intestine, with low drug metabolizing enzyme activities. Therefore, we used air–liquid interface (ALI) culture and 5-aza-2′-deoxycytidine (5AZA)-free medium to generate hiSIECs (novel hiSIECs). Novel hiSIECs showed enhanced gene expression of drug metabolizing enzymes, such as cytochrome P450 (CYP)3A4, CYP2C9, CYP2C19, and carboxylesterase 2 that are highly expressed in the small intestine. In addition, the expression of genes involved in nutrient absorption—one of the major functions of the small intestine—also increased. The novel hiSIECs expressed ZO-1 and E-cadherin. Moreover, the novel hiSIECs exhibited a barrier function that allowed low lucifer yellow permeation. The novel hiSIECs showed high activities of CYP3A4, CYP2C9, and CYP2C19, which are abundantly expressed in the small intestine. In conclusion, the novel hiSIECs have great potential as an in vitro system to evaluate pharmacokinetics in the small intestine.

Exploring Novel Variants of the Cytochrome P450 Reductase Gene (POR) from the Genome Aggregation Database by Integrating Bioinformatic Tools and Functional Assays.

Cytochrome P450 oxidoreductase (POR) is an essential redox partner for steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR lead to metabolic disorders, including congenital adrenal hyperplasia, and affect the metabolism of steroids, drugs, and xenobiotics. In this study, we examined approximately 450 missense variants of the POR gene listed in the Genome Aggregation Database (gnomAD) using eleven different in silico prediction tools. We found that 64 novel variants were consistently predicted to be disease-causing by most tools. To validate our findings, we conducted a population analysis and selected two variations in POR for further investigation. The human POR wild type and the R268W and L577P variants were expressed in bacteria and subjected to enzyme kinetic assays using a model substrate. We also examined the activities of several cytochrome P450 proteins in the presence of POR (WT or variants) by combining P450 and reductase proteins in liposomes. We observed a decrease in enzymatic activities (ranging from 35% to 85%) of key drug-metabolizing enzymes, supported by POR variants R288W and L577P compared to WT-POR. These results validate our approach of curating a vast amount of data from genome projects and provide an updated and reliable reference for diagnosing POR deficiency.

The deleterious effects of cadmium on oxidative stress markers, drug-metabolizing, and antioxidant enzyme activities: Role of Silymarin and Garlic as Antioxidants.

Exposure to cadmium has been related to liver and kidney diseases such as polycystic and nephrotic syndrome. It is still unclear how cadmium contributes to these diseases. It is believed that the induction of oxidative stress resulting from the inhibition of antioxidant enzyme activities and changes in drug-metabolizing enzymes in the liver could explain the role of cadmium in the development of different diseases in the kidney and probably other organs. Changes in oxidative stress markers, antioxidant enzymes, and drug-metabolizing enzyme activities were assessed in the liver of male rats exposed to cadmium chloride. Additionally, the protective effects of silymarin and garlic extract against cadmium toxicosis were evaluated. Rats were randomly divided into eight groups as follows, groups 1, 2, 3, 4, and 5, received orally saline, CdCl2 (1mg/kg), garlic extract [800mg/kg], silymarin (25mg/kg) and silymarin plus garlic extract respectively for 28 consecutive days. Rats in groups 6, 7, and 8 were pretreated with the same doses of garlic, silymarin, and garlic plus silymarin, respectively for two hours before cadmium administration. The Western immunoblotting technique was used to investigate the protein expression of cytochrome P450 isozymes. Spectrophotometric methods were used to assess the activity of both antioxidant- and drug-metabolizing enzymes. Free radical levels [measured as thiobarbituric acid reactive substances (TBARS)], catalase, superoxide dismutase, and glutathione peroxidase activities increased whereas the levels of glutathione and the activities of glutathione S-transferase, glutathione reductase, and glutamyl transferase, cytochrome P450, aryl hydrocarbon dehydrogenase (AHH), dimethylnitrosamine-N-demethylase I (DMN-dI), 7-ethoxycoumarine-O-deethylase (ECOD), cytochrome b5 and NADPH-Cytochrome-c-reductase enzyme activities decreased after cadmium treatment. Furthermore, Western immunoblotting data revealed that glutathione peroxidase protein expression increased following cadmium exposure, but cytochrome P450 2E1 and 3A4 expressions were downregulated. However, pretreatment of rats with silymarin or garlic extract or both before cadmium administration was found to restore the protein expression of cytochrome P450 2E1 and 3A4, the level of free radicals, antioxidant enzymes, drug-metabolizing enzyme activities to their normal levels. Similarly, histological studies revealed that silymarin and/or garlic extract reduced the liver damage caused by cadmium. Silymarin and/or garlic extract reduced the adverse effects of cadmium on the activity of both drug-metabolizing and antioxidant enzymes activity. These antioxidants could be provided to those who work in cadmium-based sectors to help them cope with the adverse effects of cadmium on their kidneys. In addition, Inhibiting drug-metabolizing enzyme activity should be considered when administering therapeutic medications to persons exposed to cadmium because most therapeutic drugs and many endogenous substances are largely metabolized by these enzymes.

Peptide-dendrimer-reinforced bioinks for 3D bioprinting of heterogeneous and biomimetic in vitro models.

Despite 3D bioprinting having emerged as an advanced method for fabricating complex in vitro models, developing suitable bioinks that fulfill the opposing requirements for the biofabrication window still remains challenging. Although naturally derived hydrogels can better mimic the extracellular matrix (ECM) of numerous tissues, their weak mechanical properties usually result in architecturally simple shapes and patchy functions of in vitro models. Here, this limitation is addressed by a peptide-dendrimer-reinforced bioink (HC-PDN) which contained the peptide-dendrimer branched PEG with end-grafted norbornene (PDN) and the cysteamine-modified HA (HC). The extensive introduction of ethylene end-groups facilitates the grafting of sufficient moieties and enhances thiol-ene-induced crosslinking, making HC-PDN exhibits improved mechanical and rheological properties, as well as a significant reduction in reactive oxygen species (ROS) accumulation than that of methacrylated hyaluronic acid (HAMA). In addition, HC-PDN can be applied for the bioprinting of numerous complex structures with superior shape fidelity and soft matrix microenvironment. A heterogeneous and biomimetic hepatic tissue is concretely constructed in this work. The HepG2-C3As, LX-2s, and EA.hy.926s utilized with HC-PDN and assisted GelMA bioinks closely resemble the parenchymal and non-parenchymal counterparts of the native liver. The bioprinted models show the endothelium barrier function, hepatic functions, as well as increased activity of drug-metabolizing enzymes, which are essential functions of liver tissue in vivo. All these properties make HC-PDN a promising bioink to open numerous opportunities for in vitro model biofabrication. STATEMENT OF SIGNIFICANCE: In this manuscript, we introduced a peptide dendrimer system, which belongs to the family of hyperbranched 3D nanosized macromolecules that exhibit high molecular structure regularity and various biological advantages. Specifically, norbornene-modified peptide dendrimer was grafted onto PEG, and hyaluronic acid (HA) was selected as a base material for bioink formulation because it is a component of the ECM. Peptide dendrimers confer the following advantages to bioinks: (a) Geometric symmetry can facilitate construction of bioinks with homogeneous networks; (b) abundant surface functional groups allow for abundant crosslinking points; (c) the biological origin can promote biocompatibility. This study shows conceptualization to application of a peptide-dendrimer bioink to extend the Biofabrication Window of natural bioinks and will expand use of 3D bioprinting of in vitro models.

Unraveling a Combined Inactivation Mechanism of Cytochrome P450s by Genipin, the Major Reactive Aglycone Derived from Gardeniae Fructus.

Genipin (GP) is the reactive aglycone of geniposide, the main component of traditional Chinese medicine Gardeniae Fructus (GF). The covalent binding of GP to cellular proteins is suspected to be responsible for GF-induced hepatotoxicity and inhibits drug-metabolizing enzyme activity, although the mechanisms remain to be clarified. In this study, the mechanisms of GP-induced human hepatic P450 inactivation were systemically investigated. Results showed that GP inhibited all tested P450 isoforms via distinct mechanisms. CYP2C19 was directly and irreversibly inactivated without time dependency. CYP1A2, CYP2C9, CYP2D6, and CYP3A4 T (testosterone as substrate) showed time-dependent and mixed-type inactivation, while CYP2B6, CYP2C8, and CYP3A4 M (midazolam as substrate) showed time-dependent and irreversible inactivation. For CYP3A4 inactivation, the kinact/KI values in the presence or absence of NADPH were 0.26 or 0.16 min-1 mM-1 for the M site and 0.62 or 0.27 min-1 mM-1 for the T site. Ketoconazole and glutathione (GSH) both attenuated CYP3A4 inactivation, suggesting an active site occupation- and reactive metabolite-mediated inactivation mechanism. Moreover, the in vitro and in vivo formation of a P450-dependent GP-S-GSH conjugate indicated the involvement of metabolic activation and thiol residues binding in GP-induced enzyme inactivation. Lastly, molecular docking analysis simulated potential binding sites and modes of GP association with CYP2C19 and CYP3A4. We propose that direct covalent binding and metabolic activation mediate GP-induced P450 inactivation and alert readers to potential risk factors for GP-related clinical drug-drug interactions.

Extracellular Vesicles as Surrogates for Drug Metabolism and Clearance: Promise vs. Reality.

Drug-metabolizing enzymes (DMEs) and transporters play a major role in drug efficacy and safety. They are regulated at multiple levels and by multiple factors. Estimating their expression and activity could contribute to predicting drug pharmacokinetics and their regulation by drugs or pathophysiological situations. Determining the expression of these proteins in the liver, intestine, and kidney requires the collection of biopsy specimens. Instead, the isolation of extracellular vesicles (EVs), which are nanovesicles released by most cells and present in biological fluids, could deliver this information in a less invasive way. In this article, we review the use of EVs as surrogates for the expression and activity of DMEs, uptake, and efflux transporters. Preliminary evidence has been provided for a correlation between the expression of some enzymes and transporters in EVs and the tissue of origin. In some cases, data obtained in EVs reflect the induction of phase I-DMEs in the tissues. Further studies are required to elucidate to what extent the regulation of other DMEs and transporters in the tissues reflects in the EV cargo. If an association between tissues and their EVs is firmly established, EVs may represent a significant advancement toward precision therapy based on the biotransformation and excretion capacity of each individual.

Factors Determining the Susceptibility of Fish to Effects of Human Pharmaceuticals.

The increasing levels and frequencies at which active pharmaceutical ingredients (APIs) are being detected in the environment are of significant concern, especially considering the potential adverse effects they may have on nontarget species such as fish. With many pharmaceuticals lacking environmental risk assessments, there is a need to better define and understand the potential risks that APIs and their biotransformation products pose to fish, while still minimizing the use of experimental animals. There are both extrinsic (environment- and drug-related) and intrinsic (fish-related) factors that make fish potentially vulnerable to the effects of human drugs, but which are not necessarily captured in nonfish tests. This critical review explores these factors, particularly focusing on the distinctive physiological processes in fish that underlie drug absorption, distribution, metabolism, excretion and toxicity (ADMET). Focal points include the impact of fish life stage and species on drug absorption (A) via multiple routes; the potential implications of fish's unique blood pH and plasma composition on the distribution (D) of drug molecules throughout the body; how fish's endothermic nature and the varied expression and activity of drug-metabolizing enzymes in their tissues may affect drug metabolism (M); and how their distinctive physiologies may impact the relative contribution of different excretory organs to the excretion (E) of APIs and metabolites. These discussions give insight into where existing data on drug properties, pharmacokinetics and pharmacodynamics from mammalian and clinical studies may or may not help to inform on environmental risks of APIs in fish.

Mitochondrial Lipid Peroxidation and Microsomal Drug-metabolizing Enzyme Activity of Rat Hepatotoxicity under Heavy Metals from Slag Waste Exposure

Heavy metals from slag waste (HMSWs) have attracted much attention because of their serious toxicity to the environment and human organs, especially hepatotoxicity. The aim of this study was to explore the effects of different HMSWs exposure on mitochondrial lipid peroxidation, microsomal drug metabolizing enzyme activities as well as their relationship in the rat liver injury. Based on toxicogenomic analysis, heavy metals including iron, copper, cobalt, nickel and manganese, might interfere with pathophysiological processes such as oxidative stress, cell death, and energy metabolism regulation in vivo, and participate in the regulation of HIF-1 signaling pathway, peroxisomes, drug metabolism-cytochrome P450, ferroptosis, and other signaling pathways. HMSWs exposure caused weight loss, and significantly increased lactate dehydrogenase (LDH), malondialdehyde (MDA), alanine transaminase (ALT), and aspartate transaminase (AST) in different groups of rat liver, suggesting the presence of mitochondrial lipid peroxidation damage. In addition, the ratios of AST/ALT and ALT/LDH were down-regulated, especially the ALT/LDH ratios were less than 1, indicating that hepatic ischemic injury occurred in the process of liver injury. The superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) activities in rats also showed significant decreases, indicating the occurrence of hepatic oxidative/antioxidant dysfunction imbalance. Further decision tree analysis of live biochemical abnormalities suggested that AST > 58.78 U/gprot and MDA > 173.2 nmol/mgprot could be used for hepatotoxicity warning. Liver microsomal cytochrome P4501A2 (CYP1A2) and 3A1 (CYP3A1) enzymes were also involved in the hepatotoxic process of heavy metals. These results suggest that lipid peroxidation damage and metabolic damage in liver mitochondria and peroxisomes, may be one of the key events in heavy metal-induced liver injury.

Establishment of LC–MS/MS method for quantifying chlorpromazine metabolites with application to its metabolism in liver and placenta microsomes

Chlorpromazine has sedative and antiemetic pharmacological effects and is widely used in clinic. Its main metabolites include 7-hydroxychlorpromazine, N-monodesmethylchlorpromazine and chlorpromazine sulfoxide, which affect the therapeutic efficacy. To support metabolism research, the quantitative analysis method of 7-hydroxychlorpromazine, N-monodesmethylchlorpromazine and chlorpromazine sulfoxide in microsomal enzymes was established for the first time by LC-MS/MS. This method has been fully validated in rat liver microsomes, and partially verified in human liver microsomes and human placenta microsomes. The intra-day and inter-day accuracy and precision of the analytes were all within ± 15%. The extraction recovery was good, and no matrix effect was detected. This accurate and sensitive method was successfully applied to chlorpromazine metabolism in different microsomal enzymes. In particular, the biotransformation of chlorpromazine in human placenta microsomes was detected for the first time. The metabolites detected in human liver and placenta microsomes presented different formation rates, indicating the wide distribution and different activities of drug-metabolizing enzymes.

The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyze warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C. 2. Compared with that seen at 37 °C, the intrinsic clearance rates (V max/K m) of CYP2C9.1 and .2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1, .2, and .3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and .10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and .23 were increased (130 ∼ 134%). At 40 °C, the clearance rates of CYP2C19.1A, .1B, .10, and .23 remained unaffected, while that of CYP2C19.8 was decreased (74%). At 34 °C, the clearance rates of CYP3A4.1 and .16 were decreased (79 ∼ 84%), those of CYP3A4.2 and .7 were unchanged, and that of CYP3A4.18 was slightly increased (112%). At 40 °C, the clearance rate of CYP3A4.1 remained unaffected, while those of CYP3A4.2, .7, .16, and .18 were decreased (58 ∼ 82%). 3. These findings may be clinically useful for dose optimization in patients with hypothermia or hyperthermia.