Cytochrome P450 3A4 Research Articles

Exploring the Regulation of Cytochrome P450 in SH-SY5Y Cells: Implications for the Onset of Neurodegenerative Diseases.

Human individual differences in brain cytochrome P450 (CYP) metabolism, including induction, inhibition, and genetic variation, may influence brain sensitivity to neurotoxins and thus participate in the onset of neurodegenerative diseases. The aim of this study was to explore the modulation of CYPs in neuronal cells. The experimental approach was focused on differentiating human neuroblastoma SH-SY5Y cells into a phenotype resembling mature dopamine neurons and investigating the effects of specific CYP isoform induction. The results demonstrated that the differentiation protocols using retinoic acid followed by phorbol esters or brain-derived neurotrophic factor successfully generated SH-SY5Y cells with morphological neuronal characteristics and increased neuronal markers (NeuN, synaptophysin, β-tubulin III, and MAO-B). qRT-PCR and Western blot analysis showed that expression of the CYP 1A1, 3A4, 2D6, and 2E1 isoforms was detectable in undifferentiated cells, with subsequent increases in CYP 2E1, 2D6, and 1A1 following differentiation. Further increases in the 1A1, 2D6, and 2E1 isoforms following β-naphthoflavone treatment and 1A1 and 2D6 isoforms following ethanol treatment were evident. These results demonstrate that CYP isoforms can be modulated in SH-SY5Y cells and suggest their potential as an experimental model to investigate the role of CYPs in neuronal processes involved in the development of neurodegenerative diseases.

The cytochrome P4501A1 (CYP1A1) inhibitor bergamottin enhances host tolerance to multidrug-resistant Vibrio vulnificus infection

PurposeVibrio vulnificus (V. Vulnificus) infection is characterized by rapid onset, aggressive progression, and challenging treatment. Bacterial resistance poses a significant challenge for clinical anti-infection treatment and is thus the subject of research. Enhancing host infection tolerance represents a novel infection prevention strategy to improve patient survival. Our team initially identified cytochrome P4501A1 (CYP1A1) as an important target owing to its negative modulation of the body's infection tolerance. This study explored the superior effects of the CYP1A1 inhibitor bergamottin compared to antibiotic combination therapy on the survival of mice infected with multidrug-resistant V. Vulnificus and the protection of their vital organs. MethodsAn increasing concentration gradient method was used to induce multidrug-resistant V. Vulnificus development. We established a lethal infection model in C57BL/6J male mice and evaluated the effect of bergamottin on mouse survival. A mild infection model was established in C57BL/6J male mice, and the serum levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase were determined using enzyme-linked immunosorbent assay to evaluate the effect of bergamottin on liver and kidney function. The morphological changes induced in the presence of bergamottin in mouse organs were evaluated by hematoxylin and eosin staining of liver and kidney tissues. The bacterial growth curve and organ load determination were used to evaluate whether bergamottin has a direct antibacterial effect on multidrug-resistant V. Vulnificus. Quantification of inflammatory factors in serum by enzyme-linked immunosorbent assay and the expression levels of inflammatory factors in liver and kidney tissues by real-time quantitative polymerase chain reaction were performed to evaluate the effect of bergamottin on inflammatory factor levels. Western blot analysis of IκBα, phosphorylated IκBα, p65, and phosphorylated p65 protein expression in liver and kidney tissues and in human hepatocellular carcinomas-2 and human kidney-2 cell lines was used to evaluate the effect of bergamottin on the nuclear factor kappa-B signaling pathway. One-way ANOVA and Kaplan-Meier analysis were used for statistical analysis. ResultsIn mice infected with multidrug-resistant V. Vulnificus, bergamottin prolonged survival (p = 0.014), reduced the serum creatinine (p = 0.002), urea nitrogen (p = 0.030), aspartate aminotransferase (p = 0.029), and alanine aminotransferase (p = 0.003) levels, and protected the cellular morphology of liver and kidney tissues. Bergamottin inhibited interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1β: p = 0.010, IL-6: p = 0.029, TNF-α: p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1β, IL-6, TNF-α in liver (IL-1β: p = 0.010, IL-6: p = 0.011, TNF-α: p = 0.037) and kidney (IL-1β: p = 0.016, IL-6: p = 0.011, TNF-α: p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistant V. Vulnificus or the bacterial load in the mouse peritoneal lavage fluid (p = 0.225), liver (p = 0.186), or kidney (p = 0.637). ConclusionBergamottin enhances the tolerance of mice to multidrug-resistant V. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies for V. Vulnificus.

Genetic variation present in the CYP3A4 gene in Ni-Vanuatu and Kenyan populations in malaria endemicity

Cytochrome P450 3A4 (CYP3A4) enzyme is involved in the metabolism of about 30 % of clinically used drugs, including the antimalarials artemether and lumefantrine. CYP3A4 polymorphisms yield enzymatic variants that contribute to inter-individual variation in drug metabolism. Here, we examined CYP3A4 polymorphisms in populations from malaria-endemic islands in Lake Victoria, Kenya, and Vanuatu, to expand on the limited data sets. We used archived dried blood spots collected from 142 Kenyan and 263 ni-Vanuatu adults during cross-sectional malaria surveys in 2013 and 2005–13, respectively, to detect CYP3A4 variation by polymerase chain reaction (PCR) and sequencing. In Kenya, we identified 14 CYP3A4 single nucleotide polymorphisms (SNPs), including the 4713G (CYP3A4∗1B; allele frequency 83.9 %) and 19382A (CYP3A4∗15; 0.7 %) variants that were previously linked to altered metabolism of antimalarials. In Vanuatu, we detected 15 SNPs, including the 4713A (CYP3A4∗1A; 88.6 %) and 25183C (CYP3A4∗18; 0.6 %) variants. Additionally, we detected a rare and novel SNP C4614T (0.8 %) in the 5′ untranslated region. A higher proportion of CYP3A4 genetic variance was found among ni-Vanuatu populations (16 %) than among Lake Victoria Kenyan populations (8 %). Our work augments the scarce data sets and contributes to improved precision medicine approaches, particularly to anti-malarial chemotherapy, in East African and Pacific Islander populations.

Pharmacokinetic drug-drug interactions of JBPOS0101 mediated by cytochrome P450 3A4 and UDP-glucuronosyltransferases.

JBPOS0101 is a new antiepileptic drug and is a substrate of UDP-glucuronosyltransferases (UGTs) in invitro test. Invitro experiments showed different results regarding whether JBPOS0101 induces (EC50 136 μM) or inhibits (IC50 95.4-386.5 μM) cytochrome P450 (CYP) 3A4. As co-medication of JBPOS0101 and carbamazepine (CBZ) is expected in clinical settings, drug-drug interactions (DDIs) between them should be determined. This study aimed to investigate pharmacokinetic (PK) interactions of JBPOS0101 influenced by CYP3A4 and UGTs using midazolam (MDZ) and CBZ. A two-cohort, open-label, fixed-sequence study was conducted in healthy Koreans. In cohort A, subjects received MDZ IV alone, and then JBPOS0101 were co-administered with MDZ after oral doses of JBPOS0101 for 7 days. In cohort B, multiple doses of JBPOS0101 and CBZ were administered respectively, and subjects received both together for 7 days. Serial blood samples were collected for PK analysis. When MDZ and JBPOS0101 were co-administered, the systemic exposure of MDZ decreased by 30%. Meanwhile, JBPOS0101 did not significantly changed the PK of CBZ. CBZ decreased the systemic exposure of JBPOS0101 at steady state by 40%, respectively. With IV administration of MDZ, JBPOS0101 acted as a weak inducer of hepatic CYP3A4 and decreased systemic exposure of MDZ. The ability of JBPOS0101 to similarly modulate gut CYP3A4 activity will require further evaluation. Co-administration of multiple doses of JBPOS0101 and CBZ did not significantly alter CBZ pharmacokinetics, but the clinical impact of decreased systemic exposure of JBPOS0101 by CBZ should be further considered.

Apiole, an important constituent of parsley, is a mixed-type inhibitor of the CYP1A subfamily

Apiole (1-allyl-2,5-dimethoxy-3,4-methylenedioxybenzene) and parsley leaves ethanolic extract containing it inhibit the rat liver microsomal ethoxy- and methoxyresorufin-O-deacetylase activities associated with cytochrome P450 (CYP) 1A1 and 1A2, respectively. Cytochrome P4501A subfamily metabolizes environmental mutagens and several drugs, leading to the formation of mutagenic metabolites. Docking analysis showed that residue Phe123 within the active site of the CYP1A1 enzyme is bound to apiole through a π/π stacking of its benzene ring. In the case of 1A2, its Phe226 interacts with the dioxolane ring of apiole. Furthermore, apiole behaves as a mixed-type inhibitor of bacterial human recombinant CYP1A1. To explore one of the possible biological implications of this inhibitory effect, we tested the capacity of apiole and the parsley ethanolic extract to interfere with the mutagenicity of the promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) metabolized by CYP1A subfamily. As expected, both apiole and the plant extract reduced the number of revertant colonies of Salmonella typhimurium TA98 Ames strain after exposure to MeIQx, reaching a 78 % and 100 % reduction, respectively. Neither apiol nor parsley extract were mutagenic to the TA98 strain. We speculate that consuming apiole, a constituent of edible herbs, in conjunction with the utilization of pharmaceuticals metabolized by the CYP1A subfamily, may result in herb-drug interactions. Furthermore, the consumption of apiole by individuals who regularly ingest fresh vegetables may contribute to the low incidence of cancer observed in those who adhere to such a dietary regimen.

The effect of itraconazole, a strong CYP3A4 inhibitor, on the pharmacokinetics of the first-in-class ACKR3/CXCR7 antagonist, ACT-1004-1239.

Cytochrome P450 (CYP) 3A4 is an enzyme involved in the metabolism of many drugs that are currently on the market and is therefore a key player in drug-drug interactions (DDIs). ACT-1004-1239 is a potent and selective, first-in-class ACKR3/CXRC7 antagonist being developed as a treatment for demyelinating diseases including multiple sclerosis. Based on the human absorption, distribution, metabolism, and excretion (ADME) study results, ACT-1004-1239 is predominantly metabolized by CYP3A4. This study investigated the effect of the strong CYP3A4 inhibitor, itraconazole, on the pharmacokinetics of single-dose ACT-1004-1239 in healthy male subjects. In the open-label, fixed-sequence DDI study, a total of 16 subjects were treated. Each subject received a single dose of 10 mg ACT-1004-1239 (Treatment A) in the first period followed by concomitant administration of multiple doses of 200 mg itraconazole and a single dose of 10 mg ACT-1004-1239 in the second period. We report a median of difference in tmax (90% confidence interval, CI) of 0.5 h (0.0, 1.0) comparing both treatments. The geometric mean ratio (GMR) (90% CI) of Cmax and AUC0-∞ was 2.16 (1.89, 2.47) and 2.77 (2.55, 3.00), respectively. The GMR (90% CI) of t1/2 was 1.46 (1.26, 1.70). Both treatments were well-tolerated with an identical incidence in subjects reporting treatment-emergent adverse events (TEAE). The most frequently reported TEAEs were headache and nausea. In conclusion, ACT-1004-1239 is classified as a moderately sensitive CYP3A4 substrate (i.e., increase of AUC ≥2- to <5-fold), and this should be considered in further clinical studies if CYP3A4 inhibitors are concomitantly administered.

Investigation of Functional Cytochrome P450 4A Enzymes in Liver and Kidney of Pigs, Cats, Tree Shrews, and Dogs in Comparison with the Metabolic Capacity of Human P450 4A11.

Pigs are sometimes used in preclinical drug metabolism studies, with growing interest, and thus their drug-metabolizing enzymes, including the cytochromes P450 (P450 or CYP; EC 1.14.14.1), need to be examined. In the present study, novel CYP4A cDNAs were isolated and characterized, namely, pig CYP4A23 and CYP4A90; cat CYP4A37 and CYP4A106; and tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g. For comparison, the following known CYP4A cDNAs were also analyzed: pig CYP4A21 and dog CYP4A37, CYP4A38, and CYP4A39. These CYP4A cDNAs all contained open reading frames of 504-513 amino acids and had high amino acid sequence identity (74%-80%) with human CYP4As. Phylogenetic analysis of amino acid sequences revealed that these CYP4As were clustered in each species. All CYP4A genes contained 12 coding exons and formed a gene cluster in the corresponding genomic regions. A range of tissue types were analyzed, and these CYP4A mRNAs were preferentially expressed in liver and/or kidney, except for pig CYP4A90, which showed preferential expression in lung and duodenum. CYP4A enzymes, heterologously expressed in Escherichia coli, preferentially catalyzed lauric acid 12-hydroxylation and arachidonic acid 20-hydroxylation, just as human CYP4A11 does, with the same regioselectivity (i.e., at the ω-position of fatty acids). These results imply that dog, cat, pig, and tree shrew CYP4As have functional characteristics similar to those of human CYP4A11, with minor differences in lauric acid 12-hydroxylation. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450, CYP) 4As are important P450s in human biological processes because of their fatty acid-metabolizing ability. Pig CYP4A21, CYP4A23, and CYP4A90; cat CYP4A37 and CYP4A106; tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g; and dog CYP4A37, CYP4A38, and CYP4A39 cDNAs were isolated and analyzed. These CYP4A cDNAs shared relatively high sequence identities with human CYP4A11 and CYP4A22. Pig, cat, tree shrew, and dog CYP4As in the liver and kidneys are likely to catalyze the ω-hydroxylation of fatty acids.

Lactobacillus murinus alleviated lung inflammation induced by PAHs in mice

ObjectiveThis study aimed to investigate the mechanism that Lactobacillus murinus (L. murinus) alleviated lung inflammation induced by polycyclic aromatic hydrocarbons (PAHs) exposure based on metabolomics. MethodsFemale mice were administrated with PAHs mix, L. murinus and indoleacrylic acid (IA) or indolealdehyde (IAId). Microbial diversity in feces was detected by 16 S rRNA gene sequencing. Non-targeted metabolomics analysis in urine samples and targeted analysis of tryptophan metabolites in serum by UPLC-Orbitrap-MS and short-chain fatty acids (SCFA) in feces by GC-MS were performed, respectively. Flow cytometry was used to determine T helper immune cell differentiation in gut and lung tissues. The levels of IgE, IL-4 and IL-17A in the bronchoalveolar lavage fluid (BALF) or serum were detected by ELISA. The expressions of aryl hydrocarbon receptor (Ahr), cytochrome P450 1A1 (Cyp1a1) and forkheadbox protein 3 (Foxp3) genes and the histone deacetylation activity were detected by qPCR and by ELISA in lung tissues, respectively. ResultsPAHs exposure induced lung inflammation and microbial composition shifts and tryptophan metabolism disturbance in mice. L. murinus alleviated PAHs-induced lung inflammation and inhibited T helper cell 17 (Th17) cell differentiation and promoted regulatory T cells (Treg) cell differentiation. L. murinus increased the levels of IA and IAId in the serum and regulated Th17/Treg imbalance by activating AhR. Additionally, L. murinus restored PAHs-induced decrease of butyric acid and valeric acid which can reduce the histone deacetylase (HDAC) level in the lung tissues, enhancing the expression of the Foxp3 gene and promoting Treg cell differentiation. Conclusionour study illustrated that L. murinus alleviated PAHs-induced lung inflammation and regulated Th17/Treg cell differentiation by regulating host tryptophan metabolism and SCFA levels. The study provided new insights into the reciprocal influence between gut microbiota, host metabolism and the immune system, suggesting that L. murinus might have the potential as a novel therapeutic strategy for lung diseases caused by environmental pollution in the future.

Interaction of CYP3A4 with the inhibitor cobicistat: Structural and mechanistic insights and comparison with ritonavir

Cobicistat is a derivative of ritonavir marketed as a pharmacoenhancer for anti-HIV therapy. This study investigated the interaction of cobicistat with the target protein, drug-metabolizing cytochrome P450 3A4 (CYP3A4), at the molecular level using spectral, kinetic, functional, and structural approaches. It was found that, similar to ritonavir, cobicistat directly coordinates to the heme via the thiazole nitrogen but its affinity and the binding rate are 2-fold lower: 0.030 μM and 0.72 s−1, respectively. The newly determined 2.5 Å crystal structure of cobicistat-bound CYP3A4 suggests that these changes arise from the inability of cobicistat to H-bond to the active site S119 and establish multiple stabilizing contacts with the F–F’ connecting fragment, which becomes disordered upon steric clashing with the bulky morpholine moiety. Nonetheless, cobicistat inhibits recombinant CYP3A4 as potently as ritonavir (IC50 of 0.24 μM vs 0.22 μM, respectively) due to strong ligation to the heme and formation of extensive hydrophobic/aromatic interactions via the phenyl side-groups. To get insights into the inhibitory mechanism, the K257 residue, known to be solely and irreversibly modified by the reactive ritonavir metabolite, was substituted with alanine. Neither this nor control K266A mutation changed the extent of time-dependent inhibition of CYP3A4 by cobicistat and ritonavir, suggesting the existence of alternative inactivation mechanism(s). More importantly, K257 was found to be functionally important and contributed to CYP3A4 allosterism, possibly by modulating protein-ligand interactions through conformational dynamics.

Physiologically-based pharmacokinetic modelling to investigate the effect of CYP3A4/3A5 maturation on tacrolimus pharmacokinetics in paediatric HSCT patients

Tacrolimus (FK506) is a cornerstone of GVHD-prophylaxis treatment in paediatrics undergoing haematopoietic stem cell transplantation (HSCT). However, due to concerns about highly inter/intra-individual variability, precision dosing of FK506 is crucial. Cytochrome P450 (CYP) 3A4 and 3A5 are considered important sources of FK506 pharmacokinetic variability. Nevertheless, the impact of age-related maturation in hepatic and intestinal CYP3A4/3A5 enzymes remains unknown in paediatric HSCT patients. Physiologically-based pharmacokinetic (PBPK) models were developed and verified in adult volunteers and adult HSCT patients using GastroPlus™ (version 9.0), and then extrapolated to paediatric HSCT patients, taking into account the maturation of CYP3A4 and CYP3A5. Default CYP3A4 and CYP3A5 ontogeny profiles were updated based on the latest reports. The paediatric PBPK model was evaluated with independent data collected from Sun Yat-sen Memorial Hospital (86 paediatric HSCT patients, 1 to 16 -year-old). Simulations were performed to evaluate a reported FK506 dosing regimen in infants and children with different CYP3A5 genotypes. Extensive PBPK model validation indicated good predictability, with the predicted/observed (P/O) ratios within the range of 0.80-fold to 1.25-fold. Blood tacrolimus concentration-time curves were comparable between the real and virtual patients. Simulations showed that the higher levels of tacrolimus in 9-month-old to 3-year-old infants were mainly attributed to the CYP3A4/3A5 ontogeny profiles, which resulted in lower clearance and higher exposure relative to dose. The oral dosage of 0.1 mg/kg/day (q12 h) is considered appropriate for paediatric HSCT patients 9 months to 15 years of age with CYP3A5 *1/*1 genotypes. Lower doses were required for paediatric HSCT patients with CYP3A5 *1/*3 (0.08 mg/kg/day, q12h) or CYP3A5 *3/*3 genotypes (0.07 mg/kg/day, q12h), and analyses demonstrated 12.5–20 % decreases in ≤3-year-old patients. The study highlights the feasibility of PBPK modelling to explore age-related enzyme maturation in infants and children (≤3-year-old) undergoing HSCT and emphasizes the need to include hepatic and gut CYP3A4/3A5 maturation parameters.

Development, validation and application of a UPLC-MS/MS method for simultaneous quantification of OPC-61815 and its metabolites tolvaptan, DM-4103 and DM-4107 in human plasma

OPC-61815 is an intravenous formulation vasopressin antagonist designed to treat heart failure patients, especially who have difficulty in oral intake. Tolvaptan together with DM-4103 and DM-4107 are considered as the major metabolites of OPC-61815 biotransformed in the liver via cytochrome P450 (CYP) 3A. An efficient and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of OPC-61815 and its three metabolites in human plasma was developed and fully validated. To our best knowledge, it was the first published method that simultaneously quantified all of these four analytes in only one run. Simple and rapid sample preparation procedure and very short UPLC-MS/MS run time (3.5 min) offered OPC-61815 and its metabolites relatively high throughput detection, which was greatly beneficial to further clinical bio-sample analysis. The method showed good linearity and sufficient sensitivity in the range of 2.00–1000 ng/mL with a low limit of quantitation (2.00 ng/mL) for each analyte. For samples with concentrations above 1000 ng/mL, 100-fold dilution with blank plasma before sample preparation was accepted. High precision and accuracy, high selectivity and satisfactory recovery of this method were demonstrated. For all of the four analytes, no significant matrix effect or carry-over was observed. The stability of analytes and internal standards under different conditions were evaluated to ensure they were stable during the whole period of storage, preparation and detection. Also, re-injection reproducibility was investigated. In addition, the conversion test showed that almost no OPC-61815 converted into DM-4103 and DM-4107 during sample processing, while attention should be paid to the concentration difference between OPC-61815 and tolvaptan in bioanalysis. The developed UPLC-MS/MS method was successfully applied to an open, single and multiple dose administration phase I trial for monitoring the pharmacokinetics of OPC-61815. This work provided a promising way for further pharmacokinetic study of OPC-61815.

Mechanism of 7H-Dibenzo[c,g]carbazole metabolism in cytochrome P450 1A1: Insights from computational studies

7H-Dibenzo[c,g]carbazole (DBC) is a prevalent environmental contaminant that induces tumorigenesis in several experimental animals. Recently, it has been utilized to develop high-performance solar cells and organic phosphorescent materials. It is imperative to strengthen investigations of DBC metabolism to understand its potential risks to human health. In this study, human CYP1A1 was employed as the metabolic enzyme to investigate the metabolic mechanism of DBC by molecular docking, molecular dynamics (MD) simulation, and quantum mechanical (QM) calculation. The results indicate that DBC binds to CYP1A1 in two modes (mode 1 and mode 2) mainly through nonpolar solvation energies (ΔGnonpolar). The formation of the two binding modes is attributed to the anchoring effect of the hydrogen bond formed by DBC with Asp320 (mode 1) or Ser116 (mode 2). Mode 1 is a "reactive" conformation, while mode 2 is not considered a "reactive" conformation. C5 is identified as the dominant site, and the pyrrole nitrogen cannot participate in the metabolism. DBC is metabolized mainly by a distinct electrophilic addition-rearrangement mechanism, with an energy barrier of 21.74 kcal/mol. The results provide meaningful insights into the biometabolic process of DBC and contribute to understanding its environmental effects and health risks.

Impact of CYP3A5 Polymorphisms on Pediatric Asthma Outcomes.

Genetic variation among inhaled corticosteroid (ICS)-metabolizing enzymes may affect asthma control, but evidence is limited. This study tested the hypothesis that single-nucleotide polymorphisms (SNPs) in Cytochrome P450 3A5 (CYP3A5) would affect asthma outcomes. Patients aged 2-18 years with persistent asthma were recruited to use the electronic AsthmaTracker (e-AT), a self-monitoring tool that records weekly asthma control, medication use, and asthma outcomes. A subset of patients provided saliva samples for SNP analysis and participated in a pharmacokinetic study. Multivariable regression analysis adjusted for age, sex, race, and ethnicity was used to evaluate the impact of CYP3A5 SNPs on asthma outcomes, including asthma control (measured using the asthma symptom tracker, a modified version of the asthma control test or ACT), exacerbations, and hospital admissions. Plasma corticosteroid and cortisol concentrations post-ICS dosing were also assayed using liquid chromatography-tandem mass spectrometry. Of the 751 patients using the e-AT, 166 (22.1%) provided saliva samples and 16 completed the PK study. The e-AT cohort was 65.1% male, and 89.6% White, 6.0% Native Hawaiian, 1.2% Black, 1.2% Native American, 1.8% of unknown race, and 15.7% Hispanic/Latino; the median age was 8.35 (IQR: 5.51-11.3) years. CYP3A5*3/*3 frequency was 75.8% in White subjects, 50% in Native Hawaiians and 76.9% in Hispanic/Latino subjects. Compared with CYP3A5*3/*3, the CYP3A5*1/*x genotype was associated with reduced weekly asthma control (OR: 0.98; 95% CI: 0.97-0.98; p < 0.001), increased exacerbations (OR: 6.43; 95% CI: 4.56-9.07; p < 0.001), and increased asthma hospitalizations (OR: 1.66; 95% CI: 1.43-1.93; p < 0.001); analysis of 3/*3, *1/*1 and *1/*3 separately showed an allelic copy effect. Finally, PK analysis post-ICS dosing suggested muted changes in cortisol concentrations for patients with the CYP3A5*3/*3 genotype, as opposed to an effect on ICS PK. Detection of CYP3A5*3/3, CYPA35*1/*3, and CYP3A5*1/*1 could impact inhaled steroid treatment strategies for asthma in the future.

Metabolic transformation of cyclopiazonic acid in liver microsomes from different species based on UPLC-Q/TOF-MS

To investigate the metabolic transformation of cyclopiazonic acid (CPA) in the liver of different species and to supplement accurate risk assessment information, the metabolism of CPA in liver microsomes from four animals and humans was studied using the ultra–high–performance liquid chromatography–quadrupole/time–of–flight method. The results showed that a total of four metabolites were obtained, and dehydrogenation, hydroxylation, methylation, and glucuronidation were identified as the main metabolic pathways of CPA. Rat liver microsomes exhibited the highest metabolic capacity for CPA, with dehydrogenated (C20H18N2O3) and glucuronic acid-conjugated (C26H28N2O10) metabolites identified in all liver microsomes except chicken, indicating significant species metabolic differences. Moreover, C20H18N2O3 was only detected in the incubation system with cytochromes P450 3A4 (CYP3A4). The hydroxylated (C20H20N2O4) and methylated (C21H22N2O3) metabolites were detected in all incubation systems except for the CYP2C9, with CYP3A4 demonstrating the strongest metabolic capacity. The “cocktail” probe drug method showed that CPA exhibited a moderate inhibitory effect on the CYP3A4 (IC50 value = 8.658 μM), indicating that the substrate had a negative effect on enzyme activity. Our results provide new insights to understand the biotransformation profile of CPA in animals and humans.

Iminium ion metabolites are formed from nintedanib by human CYP3A4

Nintedanib is used to treat idiopathic pulmonary fibrosis, systemic sclerosis, interstitial lung disease, and progressive fibrotic interstitial lung disease. It is primarily cleared via hepatic metabolism, hydrolysis, and glucuronidation. In addition, formation of the iminium ion, a possible reactive metabolite, was predicted based on the chemical structure of nintedanib. To obtain a hint which may help to clarify the cause of nintedanib-induced liver injury, we investigated whether iminium ions were formed in the human liver. To detect unstable iminium ions using liquid chromatography-tandem mass spectrometry (LC-MS/MS), potassium cyanide was added to the reaction mixture as a trapping agent. Human liver and intestinal microsomes were incubated with nintedanib in the presence of NADPH to form two iminium ion metabolites on the piperazine ring. Their formation is strongly inhibited by ketoconazole, a potent cytochrome P450 (CYP) 3A4 inhibitor. Among the recombinant P450s, only CYP3A4 formed cyanide adducts. The role of CYP3A4 was supported by the positive correlation between CYP3A4 protein abundance, as determined by LC-MS-based proteomics, and the formation of cyanide adducts in 25 individual human liver microsomes. In conclusion, we have demonstrated that iminium ion metabolites are formed from nintedanib by CYP3A4 as potential reactive metabolites.

Arsenite-Induced Drug-Drug Interactions in Rats.

Arsenite is an important heavy metal. Some Chinese traditional medicines contain significant amounts of arsenite. The aim of this study was to investigate subacute exposure of arsenite on activities of cytochrome P450 enzymes and pharmacokinetic behaviors of drugs in rats. Midazolam, tolbutamide, metoprolol, omeprazole, caffeine, and chlorzoxazone, the probe substrates for cytochrome P450 (CYP) s3A, 2C6, 2D, 2C11, 1A, and 2E, were selected as probe drugs for the pharmacokinetic study. Significant decreases in areas under the curves of probe substrates were observed in rats after consecutive 30-day exposure to As at 12 mg/kg. Microsomal incubation study showed that the subacute exposure to arsenite resulted in little change in effects on the activities of P450 enzymes examined. However, everted gut sac study demonstrated that such exposure induced significant decreases in intestinal absorption of these drugs by both passive diffusion and carrier-mediated transport. In addition, in vivo study showed that the arsenite exposure decreased the rate of peristaltic propulsion. The decreases in intestinal permeability of the probe drugs and peristaltic propulsion rate most likely resulted in the observed decreases in the internal exposure of the probe drugs. Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. SIGNIFICANCE STATEMENT: Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. The present study, we found that P450 enzyme probe drug exposure was reduced in arsenic-exposed animals (areas under the curve) and the intestinal absorption of the drug was reduced in the animals. Subacute arsenic exposure tends to cause damage to intestinal function, which leads to reduced drug absorption.

3D spheroid HepaRG and fluorescent biphasic tracer for CYP3A4-mediated antibiotic interaction monitoring in sepsis.

Cytochrome P450 3A4 (CYP3A4) is a crucial enzyme in the metabolism of xenobiotics, particularly in drug metabolism interactions (DDIs), making it a significant factor in clinical drug use. However, current assay techniques are both laborious and costly, making it difficult to construct a high-throughput monitoring method that can be used in conjunction with the clinic. This poses certain safety hazards for drug combination. Therefore, it is crucial to develop a synchronized monitoring method for the inhibition and induction of CYP3A4. In this study, we utilized 3D culture technology to develop a HepaRG cells spheroid model. The CYP450 and transporter expression, the albumin secretion, and urea synthesis capacity characteristics were analyzed. The NEN probe was utilized as a tracer molecule for CYP3A4. The fluorescence intensity of metabolites was characterized by laser confocal technique to determine the inhibition and expression of CYP3A4 in the HepaRG cell spheroid model by the antibiotics for sepsis. The results indicate that the HepaRG sphere model successfully possessed the physiological phenotype of the liver, which could be used for drug interaction monitoring. Through positive drug testing, NEN probe was able to achieve bidirectional characterization of CYP3A4 induction and inhibition. The monitoring method described in this paper was successfully applied to drug interaction monitoring of commonly used antibiotics in sepsis patients, which is a convenient and rapid monitoring method. The proposed method offers a new strategy for monitoring CYP3A4-mediated drug-drug interactions with a high-throughput assay, which will help to improve the safety of clinical drug combination.

Effect of triptolide on reproductive toxicity in female rats with Ⅱ type collagen induced arthritis

In order to study the toxic effect and mechanism of triptolide(TP) on the reproductive system of female rats with Ⅱ type collagen induced arthritis(CIA), 50 SD rats were randomly divided into normal control group, CIA model group, and three groups receiving TP tablets at clinically equivalent doses of 0. 5, 1, and 2 times, respectively(with TP dosages of 3. 75, 7. 5, and 15 μg·kg~(-1)·d~(-1)), each comprising 10 rats. Intragastric administration was started on the day after the first immunization, once a day, for 42 days.The results were taken on the 21st and 42nd days to calculate the uterine and ovarian organ indexes; pathological and morphological changes in uterus and ovaries were observed under a light microscope; and the levels of estradiol(E_2) and cytochrome P450A1(aromatase,CYP19A1) in ovarian homogenate were detected by ELISA. Furthermore, immunohistochemistry was employed to detect the expression levels of transforming growth factor β3( TGFβ3) pathway-related proteins, mothers against decapentaplegic homolog 3(Smad3) and steroidogenic factor-1(SF-1) in ovarian tissues. In vitro, the mouse Chinese hamster ovary(CHO) cell line was established, and after 24 hours of TP administration(30, 60, 120 nmol·L~(-1)), cell proliferation was detected by the thiazolyl blue tetrazolium bromide(MTT) method, apoptosis by the flow cytometry, and TGFβ3, Smad3 and SF-1 protein expression in cells by the Western blot method, and the nuclear entry of SF-1 was detected by immunofluorescence. The results showed that compared with the CIA model group, all TP administration groups showed decreased number of uterine glands, total follicles, mature follicles, and corpus luteum on days 21 and 42 of administration, but there was no statistical difference, and only the administration of 2 times the clinically equivalent dose of TP could significantly increase the number of atretic follicles at 42 days of administration. TP at 3. 75 μg·kg-1·d-1significantly reduced the level of E_2 at 21 days of administration and the expression of TGFβ3 and Smad3 factors in ovarian tissues,but had no significant effect on the rate-limiting enzyme in estrogen synthesis CYP19A1. TP at 7. 5 and 15 μg·kg~(-1)·d~(-1) significantly reduced the expression of SF-1 regardless of administration for 21 days or 42 days. TP can significantly promote ovarian cell apoptosis in vitro, with apoptosis mainly concentrated in the late stage of apoptosis after 24 hours of administration. In addition, 60 nmol·L~(-1) TP significantly reduced the protein expression of TGFβ3, Smad3 and SF-1 in a dose-dependent manner. In summary, intragastric administration of TP at less than 2 times the clinically equivalent dose for 21 days and 42 days did not cause obvious reproductive damage to the uterus and ovarian tissues of CIA rats, and the number of atretic follicles changed significantly only when the 2 times the clinically equivalent dose was administered for 42 days. TP exerted reproductive toxicity in vivo on reproductive target organs and in vitro on ovarian cells by inhibiting the expression of TGFβ3/Smad3/SF-1 pathway.

Retrospective evaluation of drug to drug interactions with paclitaxel use in military health.

e24118 Background: Paclitaxel is an antineoplastic agent used in the management of multiple metastatic and non-resectable tumor types approved for ovarian, breast, AIDS-related Kaposi sarcoma, non-small cell lung, pancreatic, cervical, endometrial, and thymoma or thymic carcinoma cancer by the US Food and Drug Administration. Cytochrome P450 (CYP) 3A4 or 2C8 inducers, inhibitors, or substrates may alter paclitaxel metabolism. Because DDIs can have an effect on clinical outcomes, we aimed to identify drugs that interact with paclitaxel and describe their clinical manifestations. Methods: A retrospective analysis was performed on the health records of patients in the US Department of Defense Cancer Registry (retrieved March 2022), Comprehensive Ambulatory/Professional Encounter Records, and Pharmacy Data Transaction Service database (both retrieved October 2022). Patients’ medical history, diagnoses, and demographics were extracted and analyzed for differences in adverse effects when these agents were used alone vs concomitantly with other prescription drugs. Patients’ diagnoses and prescription drug use were extracted to compare completed vs discontinued treatment groups, identify medications commonly co-administered with paclitaxel, and evaluate DDIs with antidepressants. Results: Of 702 patients using paclitaxel, 338 completed treatments. The P value for paclitaxel discontinuation when used alone vs concomitantly or 1 drug (alone or before or after) vs combination (2 to 4 drugs) was &lt; .001. Patients who took paclitaxel concomitantly with a greater number of prescription drugs had a higher rate of treatment discontinuation than those who received fewer medications. Patients in the completed group received 9 to 56 prescription drugs, and those in the discontinued group were prescribed 6 to 70. Those who discontinued treatment had more diagnosed medical issues than those who completed treatment. Conclusions: This review cannot conclude that concomitant use with prescription drug(s) resulted in paclitaxel discontinuation. There were no significant DDIs determined between paclitaxel and antidepressants.[Table: see text]

DCC-3116 in combination with ripretinib for patients with advanced gastrointestinal stromal tumor: A phase 1/2 study.

TPS11587 Background: Advanced cancers driven by mutations at or downstream of receptor tyrosine kinases (RTKs), including KIT- and PDGFRA-mutant gastrointestinal stromal tumor (GIST), activate autophagy as an escape mechanism in response to RTK pathway inhibitors. ULK1/2 kinases are key regulators that initiate autophagy in response to stress, such as KIT/PDGFRA inhibition in KIT- and PDGFRA-mutant GIST. Therefore, ULK1/2 inhibition represents a potential targeted approach to selectively inhibit autophagy and is an attractive strategy for combination with RTK pathway inhibitors, including ripretinib. Ripretinib is a switch-control KIT/PDGFRA tyrosine kinase inhibitor approved as a ≥fourth-line therapy for advanced GIST. In the INTRIGUE trial, ripretinib demonstrated comparable efficacy and more favorable safety compared with sunitinib in second-line advanced GIST. DCC-3116 is an investigational, selective, potent, switch-control kinase inhibitor of ULK1/2 in development for combination with targeted therapies that activate autophagy, such as ripretinib. In a GIST xenograft model, animals treated with ripretinib demonstrated stable tumor burden while treatment with ripretinib + DCC-3116 resulted in complete regressions. Here, we describe the design of a phase 1/2, multicenter, open-label study evaluating the safety, recommended phase 2 dose (RP2D), and preliminary efficacy of DCC-3116 in combination with ripretinib in advanced GIST (NCT05957367). Methods: This phase 1/2 study includes safety/dose-finding (part 1) and expansion (part 2). In part 1, patients (pts) ≥18 years must have pathologically confirmed GIST with a KIT or PDGFRA mutation and disease progression on or intolerance to ≥1 systemic regimen. Escalating doses of DCC-3116 will be assessed with ripretinib 150 mg once daily. Part 1 will use safety, pharmacokinetics, pharmacodynamics, preliminary efficacy, and a Bayesian optimal interval design to identify the RP2D for part 2 and the maximum tolerated dose. In part 2, second-line pts with confirmed GIST with a KIT exon 11 mutation and disease progression on or intolerance to first-line imatinib will be enrolled. Part 2 will use a two-stage design with non-binding futility and efficacy analyses after stage 1 to explore efficacy of the combination dose selected in part 1. Exclusion criteria for both parts include use of other anticancer treatments, such as ripretinib, and any investigational therapies within 14 days, use of moderate or strong inhibitors or inducers of cytochrome P450 3A4 or P-glycoprotein, symptomatic central nervous system metastases or leptomeningeal disease, and active infections. Primary objectives in part 1 are to identify adverse events and the RP2D. The primary objective of part 2 is to determine the objective response rate per modified Response Evaluation Criteria in Solid Tumors version 1.1. The study is currently enrolling. Clinical trial information: NCT05957367 .