Cytochrome P450 Research Articles

Association of polymorphisms in CYP2C8 and CYP2C9 with susceptibility to type 2 diabetes mellitus in a Chinese population

AimsCYP2C8 and CYP2C9 are cytochrome P450 epoxygenases responsible for metabolizing arachidonic acid into epoxyeicosatrienoic acids (EETs). These EETs play a crucial role as lipid mediators with numerous beneficial effects in type 2 diabetes mellitus (T2DM). In this study, we aimed to investigate the association of CYP2C8 and CYP2C9 genetic variants with T2DM in a Chinese population. MethodsWe conducted genotyping for 9 tag single nucleotide polymorphisms (SNPs) in CYP2C8 and 10 tag SNPs in CYP2C9 based on HapMap Chinese and Japanese data. Subsequently, we genotyped these SNPs in a Chinese cohort comprising 3410 individuals with T2DM and 3401 healthy controls. Statistical analyses were performed to assess the association between these SNPs and T2DM. ResultsIn our study population, we observed that rs1819173, located within the CYP2C9 gene region, was significantly associated with T2DM. Notably, the presence of the A allele was found to be protective against T2DM, as indicated by an odds ratio of 0.840 (95 % confidence interval: 0.780–0.904, P = 3.04 × 10−6). Furthermore, specific haplotypes (GT and AT) involving rs2071426 and rs6583967 in CYP2C8 exhibited associations with T2DM (P = 0.049 and 0.038, respectively). Subsequently, we conducted an analysis of the association between rs1819173 and non-alcoholic fatty liver disease (NAFLD), revealing a significant correlation (OR = 0.764, 95 % CI: 0.629–0.928, P = 0.007). ConclusionOur research identified a genetic variants rs1819173 within CYP2C8 are significantly associated with T2DM susceptibility in the Chinese population.

Cisplatin induces kidney damage through the down-regulation of Prx I by autophagic degradation

In this study, we investigated the potential role of PrxI in cis-diamminedichloroplatinum (cisplatin)-induced renal damage in mice. The anticancer drug cisplatin is a chemotherapeutic agent that is widely used to treat solid tumors. Cisplatin-induced nephrotoxicity is a serious dose-limiting side effect, primarily caused by oxidative stress. The oxidative stress further damages DNA, membranes, and mitochondria, and increases endoplasmic reticulum (ER) stress. Cisplatin produces reactive oxygen species (ROS) through Cytochrome P450 2E1 (CYP2E1) and localizes to the surface of the ER, where CYP2E1 is located.Among the six Prx isoforms, Prx I was selectively degraded in cisplatin-treated kidneys during severe renal function damage. Prx I degradation is blocked in mouse proximal tubular cells treated with 3-methyladenine, an autophagy inhibitor, and in MEF lacking ATG7. Moreover, increased ROS levels on the ER surface due to CYP2E1 overexpression further accelerated Prx I degradation. These results suggest that Prx I degradation is largely mediated through autophagy, which is promoted by cisplatin-induced ER stress. Ablation of Prx I exacerbated cisplatin-induced nephrotoxicity and significantly increased the abundance of oxidative stress, ER stress, and inflammatory markers in the kidney, indicating that Prx I plays a protective role against cisplatin-induced nephrotoxicity.

Di(2-ethylhexyl) phthalate disrupts circadian rhythm associated with changes in metabolites and cytochrome P450 gene expression in Caenorhabditis elegans

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is a widespread environmental pollutant due to its extensive use. While circadian rhythms are inherent in most living organisms, the detrimental effects of DEHP on circadian rhythm and the underlying mechanisms remain largely unknown. This study investigated the influence of early developmental exposure to DEHP on circadian rhythm and explored the possible relationship between circadian disruption and DEHP metabolism in the model organism Caenorhabditis elegans. We observed that DEHP disrupted circadian rhythm in a dose-dependent fashion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that DEHP-induced circadian disruption accompanies with altered proportions of DEHP metabolites in C. elegans. RNA sequencing data demonstrated that DEHP-induced circadian rhythm disruption caused differential gene expression. Moreover, DEHP-induced circadian disruption coincided with attenuated inductions of DEHP-induced cytochrome P450 genes, cyp-35A2, cyp-35A3, and cyp-35A4. Notably, cyp-35A2 mRNA exhibited circadian rhythm with entrainment, but DEHP exposure disrupted this rhythm. Our findings suggest that DEHP exposure disrupts circadian rhythm, which is associated with changes in DEHP metabolites and cytochrome P450 gene expression in C. elegans. Given the ubiquitous nature of DEHP pollution and the prevalence of circadian rhythms in living organisms, this study implies a potential negative impact of DEHP on circadian rhythm and DEHP metabolism in organisms.

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

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

Gene-environment interactions and colorectal cancer risk: A case-control study on xenobiotic metabolizing enzyme polymorphisms in the Jammu& Kashmir, India population

BackgroundCancer remains a significant global health concern, with colorectal cancer (CRC) showing a rising incidence, particularly among younger populations. Most CRC cases are linked to a complex interplay of genetic and environmental factors. The cytochrome P450 (CYP) superfamily, including enzymes like CYP2A13 and CYP2A6, plays a vital role in metabolizing environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and nitrosamines. Polymorphisms in these genes, alongside phase II glutathione-S-transferases (GSTs) involved in detoxification, can influence individual cancer risk. This study focuses on the association between these genetic polymorphisms and CRC risk in the Jammu & Kashmir, population, a region with high exposure to dietary and lifestyle-related carcinogens. MethodologyThis hospital-based case-control study was conducted at the Sher-I-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, J&K, India between March 2019 and March 2022. The study included 246 histopathologically confirmed colorectal cancer (CRC) cases, and an equal number of matched controls based on age (±5 years), gender, and place of residence. Blood samples were collected for DNA extraction, followed by genotyping of xenobiotic-metabolizing enzyme (XME) genes, including CYP2A13, CYP2A6, and GSTs using standard PCR-RFLP and multiplex PCR methods. Statistical analyses were performed using STATA software to assess the association between gene polymorphisms and CRC risk. ResultsThe study revealed key associations between genetic and environmental factors and CRC risk. The analysis demonstrated that cases had significantly lower education levels than controls. Family history of cancer, smoking, and dietary factors like red meat and salt tea consumption were more prevalent among cases. Genetic analysis identified significant interactions between various CYP and GST genotypes, and environmental factors such as smoking, pesticide exposure, and diet, with varying effects on CRC risk. ConclusionThe research underscores the influence of genetic and environmental factors on colorectal cancer (CRC) risk. Lower educational was associated with a heightened risk of CRC. Certain genotype variants of xenobiotic metabolizing enzymes (XME) were found to increase CRC risk, particularly in conjunction with smoking, pesticide exposure, and sun-dried vegetable consumption. A family history of cancer, especially CRC, further amplified the risk. These findings emphasize the importance of developing personalized CRC prevention and screening strategies that account for gene-environment interactions.

Cytochrome P450 Is a Metabolic Regulator of Diabetic Kidney Disease (DKD) Progression

Cytochrome P450 Is a Metabolic Regulator of Diabetic Kidney Disease (DKD) Progression

Roles of cytochromes P450 and ribosome inhibition in the interaction between two preoccupying mycotoxins, aflatoxin B1 and deoxynivalenol

Mycotoxins are a threat to human and animal health. Climate change increases their occurrence and our dietary exposure. Although humans and animals are concomitantly exposed to several mycotoxins, their combined effects are poorly characterised. This study investigated the interaction between aflatoxin B1 (AFB1), the most potent natural carcinogen, and deoxynivalenol (DON), which is among the most prevalent mycotoxins. AFB1 is associated with hepatocellular carcinoma through its bioactivation by cytochrome P450 (CYP450) enzymes; while DON induces ribotoxic stress leading to an alteration of intestinal, immune and hepatic functions. Analysis of DNA damage biomarkers γ-H2AX and 53BP1 revealed that DON reduces the genotoxicity of AFB1. This effect was mimicked with cycloheximide, another ribosome inhibitor; moreover DOM-1, a DON-derivative lacking ribosome inhibition, did not affect DNA damage. Exposure to DON, alone or in combination with AFB1, decreased the protein levels and/or activities of CYP1A2 and CYP3A4 in a time- and dose-dependent manner. Altogether, these results revealed an original interaction between DON and AFB1, DON inhibiting the genotoxicity of AFB1. The underlying mechanism involves ribosome inhibition by DON and the subsequent impairment of CYP450s, responsible for the bioactivation of AFB1. This work highlights the importance of studying mycotoxins not only individually but also in mixture and of considering food contaminants as part of the exposome.

Assessing Cold Plasma's Impact on Banana Growth and Fusarium Wilt Control.

Bananas (Musa spp.), which serve millions of people worldwide, face a serious threat from Fusarium wilt (FW) disease caused by Fusarium oxysporum f. sp. cubense (Foc). Developing disease-resistant varieties particularly through breeding is challenging due to banana's seedless nature (parthenocarpic). As an alternative, cold plasma (CP) technology, has the potential to be used for crop improvement. Our study demonstrates a favourable impact of CP on the growth performance of banana (Berangan cultivar, AAA) in terms of height, leaf number and stem diameter. CP-treated plants also displayed delayed disease progression as well as lower disease severity indicated by slightly lower value of leaf symptoms index and rhizome discoloration index compared to the control plants. Additionally, quantitative real-time polymerase chain reaction analysis revealed differential expression of several defence (PR1, WRKY22, PAL, and CEBiP) and growth (Cytochrome P450, NAC68, and CAT) related genes in CP-treated plants, particularly in conjunction with Foc infection. These findings shed light on the potential use of CP in managing FW in banana and offer insights into possible mechanism behind improved traits.

Protective effects of cilostazol via the HNF1α/FXR signalling pathway and anti-apoptotic mechanisms in a rat model of estrogen-induced intrahepatic cholestasis

Currently, there is a lack of targeted medications for estrogen-induced intrahepatic cholestasis (EIC) and the primary objective in managing this condition is to safeguard liver function. Consequently, this study was conducted to examine the pharmacological efficacy of cilostazol (CTZ) in the management of EIC and explore its underlying mechanisms through the use of an animal model. Thirty female Sprague-Dawley rats were divided into five groups of six animals each: Normal group, 17-ethinylestradiol (EE)-induced intrahepatic cholestasis group, EE + ursodeoxycholic acid (UDCA)-treated group, EE + CTZ (5 mg/kg)-treated group, and EE + CTZ (10 mg/kg)-treated group. It was found that the therapeutic efficacy of UDCA and low dosage of CTZ (5 mg/kg) was comparable. Nevertheless, when CTZ was administered at a dose of 10 mg/kg, it resulted in the normalization of all liver function parameters, oxidative stress, and pro-inflammatory markers, together with improvement in the histopathological derangements and hepatocytic apoptosis. These effects were mediated through the activation of the hepatocyte nuclear factor-1 alpha (HNF1α)/Farnesoid X receptor (FXR) pathway with subsequent down-regulation of the bile acids (BAs) synthesis enzyme; cholesterol 7α-hydroxylase (CYP7A1), and up-regulation of the BAs-metabolizing enzyme; cytochrome P450 (CYP)3A1 and the bile salt export pump; BSEP. Therefore, the administration of CTZ in a dose-dependent manner can protect against EIC through regulating the HNF1α/FXR pathway and anti-apoptotic mechanisms. This implies that CTZ exhibits considerable promise as a therapeutic agent for the treatment of cholestatic liver disorders.

Targeted LC-MS/MS method of oxylipin profiling reveals differentially expressed serum metabolites in type 2 diabetes mice with panaxynol

Panaxynol is a bioactive polyacetylene in food plants; however, its specific benefits in diabetes and metabolic disorders remain unclear. Previous studies have mainly focused on biochemical indicators and clinical evaluations. Limited research has systematically elucidated the beneficial effects of panaxynol from the oxylipins perspective. In this study, we employed an oxylipin analysis platform we previously established using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based on the multiple reaction monitoring (MRM) method for the profiling of oxylipins. After a 7-week administration of panaxynol to db/db mice, significant alterations in serum oxylipins and potential benefits to hyperglycemia, insulin resistance, and hepatic steatosis were observed. Our analysis also revealed correlations among epoxygenase products derived from arachidonic acid (AA), linoleic acid (LA), and α-linolenic acid (ALA) via cytochrome P450 (CYP) pathways. Furthermore, six potential oxylipins were identified, as offering insights into the mechanisms by which panaxynol may modulate diabetes. These results provide the first in vivo evidence of the impact of panaxynol on oxylipin metabolism and lay the foundation for developing panaxynol as a nutraceutical for diabetes management.

Synthesis, Characterization, Biological Activity, Computational Evaluation and Molecular Docking Simulation of Dimeric Cu(II) Carboxylate Complexes

Copper(II) carboxylate complexes are important in biological fields. In this study, five new dinuclear Cu(II) complexes having the general formulae [Cu2(Cl-PAA)2(DMSO)2] (1), [Cu2(Cl-PAA)2(phen)2] (2), [Cu2(Cl-PAA)2(bipy)2] (3), [Cu2(Cl-PAA)2(py)2] (4), [Cu2(Cl-PAA)2(Cl-Py)2] (5) were synthesized and characterized by FTIR and UV-Vis spectrophotometry. FTIR study supported the monodentate nature of the carboxylate in complexes 2 and 3 while bidentate bridging behavior of carboxylate was observed in complexes 1, 4 and 5. UV-Vis study revealed the presence of d-d transition in visible region, while charge transfer band was observed in ultraviolet region. Antimicrobial activities of complexes 1-5 were studied among which complexes 1, 2 and 4 showed good activities. The results of antioxidant assay demonstrated that free radical scavenging activity by Cu(II) complex containing 1,10-phenanthroline was higher compared to other complexes. All the synthesized complexes were optimized by DFT-D-based DMol3 module in Material Studio. After that, the quantum chemical parameters containing hardness, softness, and electrophilicity were calculated. Based on value of (ω), complex 2 showed the best biological activity that supported experimental studies. All complexes were investigated by molecular docking with cytochrome P450 14 alpha-sterol demethylase (1ea1), the docking findings demonstrated that complex 2 had the highest interaction with cytochrome P450 due to the biggest interaction energy of complex 2 as compared with other complexes. In addition, according to corrosion studies, complex 2 showed highest coating on the iron (110) surface protecting the iron from corrosion due to high adsorption value.

Enzymatic oxidation of polyethylene by Galleria mellonella intestinal cytochrome P450s

Polyethylene is widely used but highly resistant to biodegradation, owing to its composition of only a hydrocarbon backbone. For biodegradation to occur, oxidation within the polymer needs to be initiated. Galleria mellonella was the first insect discovered to autonomously oxidize polyethylene without the aid of gut microbes. However, the specific enzyme remains unidentified. Here, we identified for the first time two polyethylene oxidation enzyme candidates of cytochrome P450 (CYP), 6B2-GP04 and CYP6B2-13G08, from the G. mellonella midgut. Both candidate clones oxidized polyethylene efficiently, generating short-chain aliphatic compounds, with CYP6B2-GP04 exhibiting higher activity than CYP6B2-13G08 in yeast and insect cells. In silico structural modeling approaches revealed that the CYP6B2-GP04 Phe118 was essential for interacting with hydrocarbons, which was further validated by mutating it to glycine. Furthermore, directed enzyme evolution led to the identification of an enzyme variant with significantly increased oxidation efficiency. Our findings offer promising enzyme-based solutions for polyethylene biodegradation, potentially mitigating polyethylene-driven plastic pollution.

Cross-resistance patterns in field strains of the sheep blowfly following laboratory-based selection pressure with dicyclanil or imidacloprid

Control of the sheep blowfly relies largely on the use of insecticides applied prophylactically in advance of expected fly activity. However, the blowfly has shown an ability to develop resistance to some of these insecticides. Recent reports of the co-occurrence of resistance to both dicyclanil and imidacloprid in in vitro bioassays with field-collected fly strains has raised the possibility that the two resistances may represent cross-resistance linked by a common mechanism. We investigated this by imposing insecticide selection pressure on larvae of two insecticide-resistant field strains over a number of generations using either dicyclanil or imidacloprid and then measuring changes in sensitivity to both the selecting chemical and the alternate chemical. Larvae selected over six generations with each chemical showed significant increases in resistance to the selecting chemical: resistance ratios at the IC50 5.5 - 8.1-fold higher for dicyclanil, and 3.1 - 3.8-fold for imidacloprid. The larvae also showed significant increases in levels of resistance towards the alternate chemical: resistance ratios 2.6 - 3.1-fold higher towards dicyclanil following selection with imidacloprid, and 2.2 - 3.2-fold higher towards imidacloprid following selection with dicyclanil. The increases in resistance to both chemicals after exposure to either suggests a common mechanism of resistance, at least in our laboratory-selected populations. Assays with the cytochrome P450 inhibitor aminobenzotriazole showed that this synergist was able to remove the increased resistance to both compounds in strains selected with either compound, suggesting that cytochrome P450 is responsible for the resistance observed to both chemicals. Our results confirm that cross-resistance exists between dicyclanil and imidacloprid in the sheep blowfly, and hence the two compounds should be considered as related entities in insecticide rotation strategies for flystrike control.

Mutagenicity and genotoxicity evaluation of 15 nitrosamine drug substance-related impurities in human TK6 cells

Nitrosamine drug substance-related impurities (NDSRIs) are a sub-category of N-nitrosamine drug impurities that share structural similarity to the corresponding active pharmaceutical ingredient. The mutagenicity of NDSRIs is poorly understood. We previously tested a series of NDSRIs using the Enhanced Ames Test (EAT). In this follow-up study, we further examined the genotoxicity and mutagenicity of 15 of these NDSRIs in human TK6 cells. Seven EAT-positive NDSRIs, including N-nitroso-nortriptyline, N-nitroso-fluoxetine, N-nitroso-desmethyl-diphenhydramine, N-nitroso-duloxetine, N-nitroso-lorcaserin, N-nitroso-varenicline, and N-nitroso-sertraline, induced concentration-dependent increases in micronuclei after bioactivation with hamster liver S9. These NDSRIs were also mutagenic in the TK and HPRT gene mutation assays, consistent with their positive EAT results. In the presence of hamster liver S9, the eight EAT-negative NDSRIs were negative in the micronucleus assay and negative for mutation induction. Using TK6 cells endogenously expressing a single human cytochrome P450 (CYP), we found that CYP2C19, CYP2B6, CYP2A6, and CYP3A4 are key enzymes activating the genotoxicity and mutagenicity of these NDSRIs. Overall, the hamster S9-mediated TK6 cell mutagenicity results agreed with those observed in the EAT, indicating consistency in the mutagenic responses produced by NDSRIs across different testing systems. These data support the use of EAT for hazard identification and safety assessment of NDSRIs.

Induction of drug metabolizing enzyme and drug transporter expression by antifungal triazole pesticides in human HepaSH hepatocytes

Triazole pesticides are widely used fungicides, to which humans are rather highly exposed. They are known to activate drug-sensing receptors regulating expression of hepatic drug metabolizing enzymes and drug transporters, thus suggesting that the hepatic drug detoxification system is modified by these agrochemicals. To investigate this hypothesis, the effects of 9 triazole fungicides towards expression of drug metabolizing enzymes and transporters were characterized in cultured human HepaSH cells, that are human hepatocytes deriving from chimeric humanized liver TK-NOG mice. Most of triazoles used at 10 μM were found to act as inducers of cytochrome P-450 (CYP) 1A1, CYP1A2, CYP2B6, CYP3A4 and UDP-glucuronosyltransferase 1A1 mRNA levels and of CYP3A4 protein; some triazoles also enhanced mRNA expression of the canalicular transporters P-glycoprotein/MDR1, multidrug resistance-associated protein 2 and breast cancer resistance protein. Triazoles however concomitantly inhibited CYP2B6 and CYP3A4 activities and thus appeared as dual regulators of these CYPs, being both inducers of their expression and inhibitors of their activity. The inducing effect however predominated, at least for bromuconazole, propiconazole and tebuconazole. Bromuconazole was moreover predicted to enhance CYP2B6 and CYP3A4 expression in humans exposed to this fungicide in a chronic, acute or occupational context. These data demonstrate that key-actors of the human hepatic detoxification system are impacted by triazole pesticides, which may have to be considered for the risk assessment of these agrochemicals. They additionally highlight that the use of human HepaSH cells as surrogates to primary human hepatocytes represents an attractive and promising way for studying hepatic effects of environmental chemicals.

RNA-seq analysis of LPS-induced immune priming in silkworms (Bombyx mori) and the role of cytochrome P450 detoxification system in the process

While immune priming has been identified in many invertebrates, the intricate mechanisms that drive this process in insects continue to be a subject of mystery. In this study, we exposed silkworm larvae to varying doses of lipopolysaccharide (LPS) to induce immune priming and assessed their survival upon challenge with Bacillus thuringiensis (Bt). Transcriptome analysis was performed to identify differentially expressed genes (DEGs) associated with immune priming. The role of CYP450 genes in this process was further explored using RNA interference (RNAi) to knockdown CYP9E2 and CYP6K1, followed by measurements of detoxification enzyme activities and reactive oxygen species (ROS) levels. We found that LPS exposure significantly increased silkworm survival rates upon Bt challenge, indicating the induction of immune priming. Transcriptome analysis revealed 549 DEGs, including a large number involved in detoxification, immunity, and metabolism, suggesting a complex regulatory network that encompasses immune responses and metabolic pathways. Functional enrichment and gene set enrichment analysis (GSEA) highlighted the activation of immune signaling pathways and the involvement of detoxification processes. Knockdown of CYP9E2 and CYP6K1 resulted in increased ROS levels, decreased detoxification enzyme activities, and reduced survival rates post-Bt challenge, implicating the critical role of these genes in immune priming and detoxification. Our findings demonstrate that LPS-induced immune priming in silkworms involves the upregulation of CYP450 genes, which play a critical role in detoxification and immune response modulation. The study provides insights into the molecular mechanisms of immune priming in insects and highlights the potential of CYP9E2 and CYP6K1 as targets for enhancing disease resistance and pest management in insects.

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.

Dosing of Venetoclax in Pediatric Patients with Relapsed Acute Myeloid Leukemia: Analysis of Developmental Pharmacokinetics and Exposure-Response Relationships

This work aimed to characterize the pharmacokinetics and exposure-response relationships of venetoclax in pediatric patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) to identify venetoclax doses to be administered to pediatric patients in the phase 3 study. Data from 121 patients across three phase 1 studies enrolling pediatric patients with R/R malignancies were utilized to develop a population pharmacokinetic model to describe venetoclax pharmacokinetics in pediatric patients. Individual patient average venetoclax plasma concentration up to the event of interest, derived based on the population pharmacokinetics analysis, was used to evaluate the exposure-response relationships to efficacy (complete response) and safety (neutropenia and thrombocytopenia) endpoints for patients with AML who received venetoclax in combination with azacitidine, decitabine, or cytarabine (n = 36). The population pharmacokinetic model was then used to simulate exposures in pediatric age- and weight-based subgroups to identify the venetoclax doses for pediatric patients. The pharmacokinetic data were adequately described by the two-compartment population pharmacokinetic model with first-order absorption and elimination. The model accounted for cytochrome P450 3A developmental changes using a maturation function and incorporated allometric scaling to account for growth and body size effect. Weight was identified as a statistically significant covariate on clearance and volume of distribution and retained in the final model. Population pharmacokinetic estimates were comparable to previously reported estimates in adults. Exposure-response analyses suggested that the clinical efficacy of venetoclax in combination with high-dose cytarabine (HDAC) is maximized at 600 mg adult-equivalent, and higher doses are unlikely to enhance clinical efficacy. Venetoclax 600 mg adult-equivalent was selected for further development in combination with HDAC. Additionally, venetoclax 400 mg adult-equivalent was selected for bridging/maintenance therapy in combination with azacitidine. Flat exposure-response relationships were observed with Grade ≥3 neutropenia and thrombocytopenia. Doses were selected based on weight (allometric scaling) for children aged ≥2 years old and based on weight and CYP3A ontogeny for children aged <2 years. The selected age- and weight-based dosing scheme of venetoclax is projected to achieve venetoclax exposures in pediatric subgroups comparable to those observed in adults receiving venetoclax 400 mg or 600 mg. This work characterized the pharmacokinetics and exposure-response relationships of venetoclax in pediatric patients and guided the selection of pediatric dosing regimens in support of the venetoclax phase 3 trial in pediatric AML (NCT05183035). NCT03236857, NCT03181126, and NCT03194932.

A carbamazepine metabolite activates NLRP3 and controls skin homing of CD8+ T-cells in SJS/TEN

BackgroundStevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe adverse drug reactions with extensive keratinocyte death. Carbamazepine (CBZ), the most commonly implicated drug in SJS/TEN, is metabolized by the cytochrome P450 enzyme 3A4 (CYP3A4) into carbamazepine-10,11-epoxide (CBZE) in the liver. While CD8+ cytotoxic T cells play an important role in SJS/TEN, the underlying mechanism of exuberant immune response by CD8+ T cells in these conditions remains incompletely understood. ObjectivesTo examine the expression of NLRP3 inflammasome and their skin migration in CBZE-induced SJS/TEN. MethodsThe expression of the NLRP3 inflammasome complex in skin lesions, sera, and blister fluids of SJS/TEN patients were analyzed by immunohistochemistry and enzyme-linked immunosorbent assay. NLRP3 formation and CD8+ T cell activation status and their functions were examined by immunoblotting, immunofluorescence, and chemotaxis assays. ResultsThe expression of the NLRP3 inflammasome complex was greatly increased in skin lesions of SJS/TEN patients. Moreover, IL-1β and IL-18 levels in sera and blister fluids of SJS/TEN patients were approximately 3-fold higher than those in healthy individuals, with a linear correlation between IL-1β levels and disease activity. CBZE induced NLRP3 inflammasome formation, upregulated CXCL9/CXCL10 levels, and activated CD8+ cytotoxic T cell functions via IL-1β/IL-1R or IL-18/IL-18R signaling in SJS/TEN keratinocytes, which promoted CD8+ cytotoxic T cell migration in SJS/TEN patients. ConclusionThis study showed that CBZE promoted NLRP3 inflammasome formation and strengthened the activation and function of CD8+ cytotoxic T cells in the skin, which contributed to the initiation and progression of SJS/TEN.

Effect of CYP3A5*3 genotype on exposure and efficacy of quetiapine: A retrospective, cohort study

BackgroundThe involvement of cytochrome P450 3A5 (CYP3A5) in the metabolism of quetiapine has been proposed, though conclusive evidence is lacking. This study aimed to quantitatively assess the impact of CYP3A5 genetic variability on quetiapine exposure in a Chinese patient population. MethodsPatient data were retrospectively collected from the database of the Mental Health Centre at the First Hospital of Hebei Medical University, covering the period from September 1, 2019, to July 1, 2023. The study included patients genotyped for CYP3A5 who were treated with quetiapine. Inclusion criteria for the analysis of pharmacokinetic parameters, such as serum concentrations of the drug and its metabolites, included oral administration of quetiapine, availability of information on the prescribed daily dose and concomitant medications, and the determination of steady-state blood levels at the time of sampling (after at least 3 days of continuous administration at the same dose). Exclusion criteria comprised polypharmacy with known CYP3A4 inducers or inhibitors, as well as patients with hepatic or renal insufficiency. The primary endpoint was the exposure to quetiapine and N-dealkylquetiapine, measured using dose-corrected concentrations (C/D). The secondary endpoint was the metabolism of quetiapine to N-dealkylquetiapine, assessed by the ratio of metabolite to parent drug concentrations. The third endpoint is the differences in adverse reactions, QTc intervals, and biochemical parameters among patients with different CYP3A5 genotypes. ResultBased on the inclusion and exclusion criteria, clinical data from 207 patients were ultimately included in the study. Of these, 20 patients had the CYP3A5*1/*1 genotype, 78 had the CYP3A5*1/*3 genotype, and 109 had the CYP3A5*3/*3 genotype.The CYP3A5*3 variant was found to significantly impact the metabolism of quetiapine. The C/D values for both quetiapine and dealkylated quetiapine were notably higher in individuals with the *3/*3 genotype compared to those with the *1/*1 and *1/*3 genotypes (P1 <0.001 and P2 = 0.002, respectively). A comparison of the variability in metabolic ratios among different genotype groups revealed no significant difference (P = 0.067). However, a post hoc analysis indicated that the metabolic ratio in poor metabolizers was significantly lower than that in intermediate metabolizers (P = 0.021). The analysis of adverse reaction incidence and QTc intervals among different genotypes showed no statistically significant differences (P = 0.652, P = 0.486). However, comparison of biochemical parameters across different genotype groups revealed that alanine aminotransferase, uric acid, hemoglobin, and gamma-glutamyl transferase levels were significantly higher in patients with the CYP3A5*3/*3 genotype compared to those with the CYP3A5*1/*1 and CYP3A5*1/*3 genotypes. ConclusionThe results indicated that the genetic polymorphism of CYP3A5*3 significantly influences the metabolism of quetiapine. Specifically, carriers of the CYP3A5*3/*3 genotype exhibited higher blood levels of quetiapine, with a greater likelihood of these levels exceeding the therapeutic range. This finding underscores the need for clinicians to carefully monitor and potentially adjust the dosage for patients with this genotype to avoid adverse effects. This finding underscores the need for clinicians to pay special attention to the efficacy and occurrence of adverse reactions when prescribing quetiapine to patients carrying the CYP3A5*3/*3 genotype.