Frequency of pharmacogenomic variants affecting safety and efficacy of immunomodulators and biologics in a South Asian population from Sri Lanka.

BACKGROUND: Immunomodulators are important for management of autoimmune diseases and hematological malignancies. Significant inter-individual variation in drug response/reactions exists due to genetic polymorphisms. We describe frequency of identified genetic polymorphisms among Sri Lankans. METHODS: Sri Lankan data were obtained from an anonymized database of 670 participants. Data on variants and global distribution of Minor Allele frequency (MAF) of other populations (South Asian, Ashkenazi-Jewish, East-Asian, European-Finnish, European-non-Finnish, Latino-American, African/African-American) were obtained from pharmGKB online database. RESULTS: SLC19A1 (rs1051266) variant had a MAF (95% CI) of 63.3% (60.7-65.9). Other common variants included FCGR3A (rs396991), MTHFR (rs1801133), ITPA (rs1127354), CYP2C9*3 (rs1057910) and NUD15*3 (rs116855232), with MAFs of 35.3% (32.7-37.9), 12.2% (10.4-13.9), 10.9% (9.2-12.6), 9.8% (8.2-11.4), 8.3% (6.8-9.8) respectively. Less commonly present variants included CYP2C9*2 (rs1799853) (2.5%[1.7-3.4]), TPMT*3C (rs1142345) (1.9%[1.1-2.6]), TPMT*3B (rs1800460) (0.2%[0-0.5]), CYP3A5*6 (rs10264272) (0.2%[0-0.4]) and CYP3A4*18 (rs28371759) (0.1%[0-0.2]). The SLC19A1 (rs1051266), NUD15*3 (rs116855232), CYP2C9*3 (rs1057910), FCGR3A (rs396991), and ITPA (rs1127354) showed significantly higher frequencies in Sri Lankans compared to many other populations, exceptions include FCGR3A in Ashkenazi-Jewish and ITPA in East-Asians. Conversely, MTHFR (rs1801133), TPMT*3B (rs1800460), and CYP2C9*2 (rs1799853) were significantly less prevalent among Sri Lankans than in  many other populations. Sri Lankans exhibited lower prevalence of TPMT*3C (rs1142345) compared to European-non-Finnish, Latino-Americans, and African/African-Americans; CYP3A4*18 (rs28371759) compared to East-Asians; and CYP3A5*6 (rs10264272) compared to African/African-Americans and Latino-Americans. CONCLUSION: Sri Lankans exhibit higher frequencies in variants reducing methotrexate efficacy (SLC19A1), increasing azathioprine myelotoxicity (NUDT15), and lower frequencies in variants linked to increased azathioprine toxicity (TPMT*3B, TPMT*3C), reduced tacrolimus efficacy (CYP3A4*18), and methotrexate toxicity risk (MTHFR). Beneficial variants enhancing rituximab efficacy (FCGR3A) are more prevalent, while those reducing tacrolimus dosage (CYP3A5*6) are less common. This highlights need for targeted medication strategies to improve treatment outcomes.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 µM) and low (Kd,2 > 10 µM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 µM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

The molecular basis of dapsone activation of CYP2C9-catalyzed nonsteroidal anti-inflammatory drug oxidation.

Positive heterotropic cooperativity, or "activation," results in an instantaneous increase in enzyme activity in the absence of an increase in protein expression. Thus, cytochrome P450 (CYP) enzyme activation presents as a potential drug-drug interaction mechanism. It has been demonstrated previously that dapsone activates the CYP2C9-catalyzed oxidation of a number of nonsteroidal anti-inflammatory drugs in vitro. Here, we conducted molecular dynamics simulations (MDS) together with enzyme kinetic investigations and site-directed mutagenesis to elucidate the molecular basis of the activation of CYP2C9-catalyzed S-flurbiprofen 4'-hydroxylation and S-naproxen O-demethylation by dapsone. Supplementation of incubations of recombinant CYP2C9 with dapsone increased the catalytic efficiency of flurbiprofen and naproxen oxidation by 2.3- and 16.5-fold, respectively. MDS demonstrated that activation arises predominantly from aromatic interactions between the substrate, dapsone, and the phenyl rings of Phe114 and Phe476 within a common binding domain of the CYP2C9 active site, rather than involvement of a distinct effector site. Mutagenesis of Phe114 and Phe476 abrogated flurbiprofen and naproxen oxidation, and MDS and kinetic studies with the CYP2C9 mutants further identified a pivotal role of Phe476 in dapsone activation. MDS additionally showed that aromatic stacking interactions between two molecules of naproxen are necessary for binding in a catalytically favorable orientation. In contrast to flurbiprofen and naproxen, dapsone did not activate the 4'-hydroxylation of diclofenac, suggesting that the CYP2C9 active site favors cooperative binding of nonsteroidal anti-inflammatory drugs with a planar or near-planar geometry. More generally, the work confirms the utility of MDS for investigating ligand binding in CYP enzymes.

Association between CYP2C9 and VKORC1 genetic polymorphisms and efficacy and safety of warfarin in Chinese patients.

OBJECTIVES: Genetic variation has been a major contributor to interindividual variability of warfarin dosage requirement. The specific genetic factors contributing to warfarin bleeding complications are largely unknown, particularly in Chinese patients. In this study, 896 Chinese patients were enrolled to explore the effect of CYP2C9 and VKORC1 genetic variations on both the efficacy and safety of warfarin therapy. METHODS AND RESULTS: Univariate analyses unveiled significant associations between two specific single nucleotide polymorphisms rs1057910 in CYP2C9 and rs9923231 in VKORC1 and stable warfarin dosage ( P  < 0.001). Further, employing multivariate logistic regression analysis adjusted for age, sex and height, the investigation revealed that patients harboring at least one variant allele in CYP2C9 exhibited a heightened risk of bleeding events compared to those with the wild-type genotype (odds ratio = 2.16, P  = 0.04). Moreover, a meta-analysis conducted to consolidate findings confirmed the associations of both CYP2C9 (rs1057910) and VKORC1 (rs9923231) with stable warfarin dosage. Notably, CYP2C9 variant genotypes were significantly linked to an increased risk of hemorrhagic complications ( P  < 0.00001), VKORC1 did not demonstrate a similar association. CONCLUSION: The associations found between specific genetic variants and both stable warfarin dosage and bleeding risk might be the potential significance of gene detection in optimizing warfarin therapy for improving patient efficacy and safety.

Comparative performance of pharmacogenetics-based warfarin dosing algorithms in Chinese population: use of a pharmacokinetic/pharmacodynamic model to explore dosing regimen through clinical trial simulation.

OBJECTIVE: Warfarin has a narrow therapeutic window and large variability in dosing that are affected by clinical and genetic factors. To help guide the dosing of warfarin, the Clinical Pharmacogenetics Implementation Consortium has recommended the use of pharmacogenetic algorithms, such as the ones developed by the International Warfarin Pharmacogenetics Consortium (IWPC) and by Gage et al. when genotype information is available. METHODS: In this study, simulations were performed in Chinese cohorts to explore how dosing differences between Western (by IWPC and Gage et al.) and Chinese algorithms (by Miao et al.) would mean in terms of anticoagulation effect in clinical trials. We first tried to replicate a published clinical trial comparing genotype-guided dosing to routine clinical dosing in Chinese patients. We then made simulations where Chinese cohorts received daily doses recommended by Gage, IWPC, and Miao algorithms. RESULTS: We found that in simulation conditions where dosing specifications were strictly followed, genotype-guided dosing by IWPC and Lenzini formulae was more likely to overshoot the upper limit of the therapeutic window by day 15, and thus may have a lower % time in therapeutic range (%TTR) than that of clinical dosing group. Also, in comparing Gage, IWPC, and Miao algorithms, we found that the Miao dosing cohort has the highest %TTR and the lowest risk of over-anticoagulation by day 28. CONCLUSION: In summary, our results confirmed that algorithms developed based on data from local patients may be more suitable for achieving therapeutic international normalized ratio window in Chinese population.

Influence of CYP2C9 phenotypes on phenytoin plasma concentration in neurosurgical Brazilian patients.

AIMS: To investigate the association of CYP2C9 metabolic phenotypes with phenytoin plasma concentration ([PTH]) in neurosurgical patients from the Brazilian Public Health System. METHODS: Patients (n = 170) were treated with phenytoin (300 mg/day) perioperatively as prophylaxis for postoperative seizures. Two to 10 days after surgery, a blood sample was collected for quantification of [PTH] and genotyping of CYP2C9*2 and *3 alleles. CYP2C9 metabolic phenotypes, NM (normal), IM (intermediate), and PM (poor) metabolizer, were inferred from CYP2C9 diplotypes. Linear regression modeling was applied to identify predictors of [PTH]. RESULTS: Wide (22-fold) interindividual variation in [PTH] was observed (2.2-47.5 mg/l). [PTH] associated significantly (Kruskal-Wallis P < 0.005) with CYP2C9 phenotypes and there was a significant trend (Jonckheere-Terpstra test, P < 0.0001) for [PTH] increase in the order NM < IM < PM. [PTH] was within the target therapeutic range (10-20 mg/l) in 34.7% of patients, while 39.4% and 25.9% had [PTH] below and above the range, respectively. CYP2C9 phenotypes associated significantly (chi-square P = 0.004) with the distribution of patients in [PHT] therapeutic categories and the Cramér's V test pointed to moderate magnitude of the effect of CYP2C9 phenotypes (V = 0.211). CONCLUSION: Diplotype-predicted CYP2C9 metabolic phenotypes are associated significantly with [PTH] in neurosurgical Brazilian patients receiving phenytoin for postsurgery seizure prophylaxis. [PHT] increased progressively in the phenotype order NM < IM < PM, and all PM patients had [PHT] above the target therapeutic range, consistent with the CPIC guideline 'strong' recommendation for phenytoin dosing adjustments in PMs.

Massively parallel characterization of CYP2C9 variant enzyme activity and abundance.

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.

Investigating the Correlation between Genotypes and Hepatic Protein Expression of CYP2C9, CYP2C19, CYP2D6, and CYP3A5 Using Postmortem Tissue from a Danish Population.

The cytochrome P450 (CYP) family of enzymes plays a central role in the metabolism of many drugs. CYP genes are highly polymorphic, which is known to affect protein levels, but for some low frequent CYP genotypes the correlation between genotype and CYP protein expression is less established. In this study, we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes of 250 Danish individuals included in a postmortem study. For 116 of the individuals, the hepatic CYP protein levels were investigated by a proteomics approach. Overall, we found the postmortem genetic and proteomic data to be in agreement with those of other studies performed on fresh hepatic tissue, showing the usability of postmortem hepatic tissue for this type of investigation. For less investigated genotypes, we could corroborate previously found results: 1) statistically significantly lower levels of hepatic CYP2C9 protein in individuals carrying the CYP2C9*3 variant compared with individuals with two wild type (wt) alleles; 2) comparable levels of CYP2C19 in CYP2C19*2/*17 and CYP2C19*1/*2 individuals; 3) reduced CYP2D6 protein levels in heterozygous individuals with the CYP2D6*3, CYP2D6*4, and CYP2D6*5 gene deletion variants; and 4) significantly lower levels of CYP3A5 protein in CYP3A5*3 homozygous individuals compared with individuals who were heterozygous for the CYP3A5*3 allele or homozygous individuals for the wt alleles. In conclusion, the use of postmortem tissue significantly increases the access to human specimens for research purposes, and postmortem proteomics can be used to investigate the link between CYP genotypes and hepatic protein expression. SIGNIFICANCE STATEMENT: In tissue samples from a large postmortem cohort (n = 250) we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes. Hepatic CYP protein levels were investigated in 116 individuals using a proteomics approach. For common genotypes, we found results similar to previous knowledge, pointing toward the usability of postmortem tissue. For the less investigated genotypes, we were able to corroborate genotype/protein expression correlations. It is a novel approach to use a large postmortem cohort to investigate genetic/protein expression correlations.

Novel Cytochrome P450 2C119 Enzymes in Cynomolgus and Rhesus Macaques Metabolize Progesterone, Diclofenac, and Omeprazole.

Cynomolgus and rhesus macaques are used in drug metabolism studies due to their evolutionary and phylogenetic closeness to humans. Cytochromes P450 (P450s or CYPs), including the CYP2C family enzyme, are important endogenous and exogenous substrate-metabolizing enzymes and play major roles in drug metabolism. In cynomolgus and rhesus macaques, six CYP2Cs have been identified and characterized, namely, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2C76, and CYP2C93. In this study, CYP2C119, a new CYP2C, was identified and characterized in cynomolgus and rhesus macaques. Cynomolgus and rhesus CYP2C119 contained open reading frames of 489 amino acids with high sequence identities to human CYP2C8 and to cynomolgus and rhesus CYP2C8. Phylogenetic analysis showed that cynomolgus and rhesus CYP2C119 were closely related to cynomolgus and rhesus CYP2C8. In cynomolgus and rhesus genomes, CYP2C genes, including CYP2C119, form a cluster. Among the tissues analyzed, cynomolgus CYP2C119 mRNA was predominantly expressed in liver. Hepatic expressions of CYP2C119 mRNA in four cynomolgus and two rhesus macaques varied, with no expression in one rhesus macaque. Among the CYP2C mRNAs, CYP2C119 mRNA was expressed less abundantly than CYP2C8, CYP2C9, CYP2C19, and CYP2C76 mRNAs but more abundantly than CYP2C18 mRNA. Recombinant cynomolgus and rhesus CYP2C119 catalyzed progesterone 16α-, 17α-, and 21-hydroxylation and diclofenac and omeprazole oxidations, indicating that CYP2C119 is a functional enzyme. Therefore, the novel CYP2C119 gene, expressed in macaque liver, encodes a functional enzyme that metabolizes human CYP2C substrates and is likely responsible for drug clearances. SIGNIFICANCE STATEMENT: Cytochrome P450 2C119 was found in cynomolgus and rhesus macaques, in addition to the known P450 2C8, 2C9, 2C18, 2C19, 2C76, and 2C93. Cynomolgus and rhesus CYP2C119 contain open reading frames of 489 amino acids with high sequence identity to human CYP2C8. Cynomolgus CYP2C119 mRNA is predominantly expressed in the liver. Recombinant CYP2C119 catalyzed progesterone hydroxylation and diclofenac and omeprazole oxidations. Therefore, the novel CYP2C119 gene expressed in the macaque liver encodes a functional enzyme that metabolizes human CYP2C substrates.

Global distribution of functionally important CYP2C9 alleles and their inferred metabolic consequences.

BACKGROUND: Genetic variability in the cytochrome P450 CYP2C9 constitutes an important predictor for efficacy and safety of various commonly prescribed drugs, including coumarin anticoagulants, phenytoin and multiple non-steroidal anti-inflammatory drugs (NSAIDs). A global map of CYP2C9 variability and its inferred functional consequences has been lacking. RESULTS: Frequencies of eight functionally relevant CYP2C9 alleles (*2, *3, *5, *6, *8, *11, *13 and *14) were analyzed. In total, 108 original articles were identified that included genotype data from a total of 81,662 unrelated individuals across 70 countries and 40 unique ethnic groups. The results revealed that CYP2C9*2 was most abundant in Europe and the Middle East, whereas CYP2C9*3 was the main reason for reduced CYP2C9 activity across South Asia. Our data show extensive variation within superpopulations with up to tenfold differences between geographically adjacent populations in Malaysia, Thailand and Vietnam. Translation of genetic CYP2C9 variability into functional consequences indicates that up to 40% of patients in Southern Europe and the Middle East might benefit from warfarin and phenytoin dose reductions, while 3% of patients in Southern Europe and Israel are recommended to reduce starting doses of NSAIDs. CONCLUSIONS: This study provides a comprehensive map of the genetic and functional variability of CYP2C9 with high ethnogeographic resolution. The presented data can serve as a useful resource for CYP2C9 allele and phenotype frequencies and might guide the optimization of genotyping strategies, particularly for indigenous and founder populations with distinct genetic profiles.

Concentrations of Fluoxetine Enantiomers Decline During Pregnancy and Increase After Birth.

RATIONALE: Few studies of the effect of the dynamic physiologic changes during pregnancy on plasma concentrations of fluoxetine (FLX) have been published. OBJECTIVES: We determined the change in concentration to dose (C/D) ratios of R- and S-FLX and R- and S-norfluoxetine monthly during pregnancy and postpartum, assessed their relationships to cytochrome P450 (CYP) 2D6 and CYP2C9 metabolizer phenotypes, and evaluated the course of their depressive and anxiety symptoms. METHODS: In this observational study, 10 FLX-treated pregnant individuals provided blood samples at steady state every 4 weeks during pregnancy and once postpartum for measurement of plasma FLX and norfluoxetine enantiomer concentrations. Participants were genotyped for variants in CYP2C9 and CYP2D6 using commercial assays with Taqman probes. At each assessment, depressive and anxiety symptoms were quantified. RESULTS: The C/D ratios of all FLX and norfluoxetine enantiomers, and the active moiety, decreased steadily through pregnancy and rose after birth. In the final trimester, the mean C/D ratio of the active moiety was 24.9% lower compared with the mean nonpregnant, 12-week postpartum C/D ratio. One individual with CYP2D6 ultrarapid metabolizer status was prescribed the highest FLX dose among participants. In these treated individuals, the mean depressive and anxiety symptoms remained in the mild range across the perinatal period. CONCLUSIONS: These data do not support a recommendation for routine plasma concentration monitoring or CYP2D6 pharmacogenetic testing for pregnant people treated with FLX; however, monitoring for symptom relapse is recommended because of declining plasma drug concentrations.

Pharmacogenetic guided versus standard warfarin dosing for routine clinical care with its pharmacoeconomic impact: a randomized controlled clinical trial.

BACKGROUND: Empirical use of pharmacogenetic test(PGT) is advocated for many drugs, and resource-rich setting hospitals are using the same commonly. The clinical translation of pharmacogenetic tests in terms of cost and clinical utility is yet to be examined in hospitals of low middle income countries (LMICs). AIM: The present study assessed the clinical utility of PGT by comparing the pharmacogenetically(PGT) guided- versus standard of care(SOC)- warfarin therapy, including the health economics of the two warfarin therapies. METHODS: An open-label, randomized, controlled clinical trial recruited warfarin-receiving patients in pharmacogenetically(PGT) guided- versus standard of care(SOC)- study arms. Pharmacogenetic analysis of CYP2C9*2(rs1799853), CYP2C9*3(rs1057910) and VKORC1(rs9923231) was performed for patients recruited to the PGT-guided arm. PT(Prothrombin Time)-INR(international normalized ratio) testing and dose titrations were allowed as per routine clinical practice. The primary endpoint was the percent time spent in the therapeutic INR range(TTR) during the 90-day observation period. Secondary endpoints were time to reach therapeutic INR(TRT), the proportion of adverse events, and economic comparison between two modes of therapy in a Markov model built for the commonest warfarin indication- atrial fibrillation. RESULTS: The study enrolled 168 patients, 84 in each arm. Per-protocol analysis showed a significantly high median time spent in therapeutic INR in the genotype-guided arm(42.85%; CI 21.4-66.75) as compared to the SOC arm(8.8%; CI 0-27.2)(p < 0.00001). The TRT was less in the PG-guided warfarin dosing group than the standard-of-care dosing warfarin group (17.85 vs. 33.92 days) (p = 0.002). Bleeding and thromboembolic events were similar in the two study groups. Lifetime expenditure was ₹1,26,830 in the PGT arm compared to ₹1,17,907 in the SOC arm. The QALY gain did not differ in the two groups(3.9 vs. 3.65). Compared to SOC, the incremental cost-utility ratio was ₹35,962 per QALY gain with PGT test opting. In deterministic and probabilistic sensitivity analysis, the base case results were found to be insensitive to the variation in model parameters. In the cost-effectiveness-acceptability curve analysis, a 90% probability of cost-effectiveness was reached at a willingness-to-pay(WTP) of ₹ 71,630 well below one time GDP threshold of WTP used. CONCLUSION: Clinical efficacy and the cost-effectiveness of the warfarin pharmacogenetic test suggest its routine use as a point of care investigation for patient care in LMICs.

Association between polymorphisms of the VKORC1 and CYP2C9 genes and warfarin maintenance dose in Peruvian patients.

AIMS: The aim of this study was to investigate the association between VKORC1 and CYP2C9 genes polymorphisms and the maintenance dose of warfarin in Peruvian patients. METHODS: An observational study was conducted on outpatients from the Hospital Grau ESSALUD in Lima, Peru. The participants were selected using nonprobabilistic convenience sampling. Inclusion criteria required patients to have been on anticoagulation therapy for >3 months, maintain stable doses of warfarin (consistent dose for at least 3 outpatient visits), and maintain an international normalized ratio within the therapeutic range of 2.5-3.5. DNA samples were obtained from peripheral blood for gene analysis. RESULTS: Seventy patients (mean age of 69.6 ± 13.4 years, 45.7% female) were included in the study. The average weekly warfarin dose was 31.6 ± 15.2 mg. The genotypic frequencies of VKORC1 were as follows: 7.1% (95% confidence interval, 2.4-15.9) for AA; 44.3% (32.4-56.7) for GA; and 48.6% (36.4-60.8) for GG. No deviation from the Hardy-Weinberg equilibrium was observed in the variants studied (P = .56). The mean weekly warfarin doses for AA, GA and GG genotypes were 16.5 ± 2.9, 26.5 ± 9.5 and 37.9 ± 17.1 mg, respectively (P < .001). The genotypic frequencies of CYP2C9 were as follows: 82.8% (72.0-90.8) for CC (*1/*1); 4.3% (1.0-12.0) for CT (*1/*2); and 12.9% (6.1-23.0) for TT (*2/*2). We did not find a significant association between the CYP2C9 gene polymorphism and the dose of warfarin. CONCLUSIONS: The AA genotype of the VKORC1 gene was associated with a lower maintenance dose of warfarin in Peruvian patients.

Kinetic-pharmacodynamic model of warfarin for prothrombin time-international normalized ratio in Japanese patients.

AIMS: Genotype-guided dosing algorithms can explain about half of the interindividual variability in prothrombin time-international normalized ratio (PT-INR) under warfarin treatment. This study aimed to refine a published kinetic-pharmacodynamic model and guide warfarin dosage for an optimal PT-INR based on renal function. METHODS: Using a retrospective cohort of adult patients (>20 years) who were administered warfarin and underwent PT-INR measurements, we refined the kinetic-pharmacodynamic model with age and the genotypes of cytochrome P450 2C9 and vitamin K epoxide reductase complex subunit 1 using the PRIOR subroutine in the nonlinear-mixed-effect modelling programme. We searched the significant covariates for parameters, such as the dose rate for 50% inhibition of coagulation (EDR50 ), using a stepwise forward and backward method. Monte Carlo simulation determined a required daily dose of warfarin with a target range of PT-INR (2.0-3.0 or 1.6-2.6) based on the significant covariates. RESULTS: A total of 350 patients with 2762 PT-INR measurements were enrolled (estimated glomerular filtration rate [eGFR]: 47.5 [range: 2.6-199.0] mL/min/1.73 m2 ). The final kinetic-pharmacodynamic model showed that the EDR50 changed power functionally with body surface area, serum albumin level and eGFR. Monte Carlo simulation revealed that a lower daily dose of warfarin was required to attain the target PT-INR range as eGFR decreased. CONCLUSIONS: Model-informed precision dosing of warfarin is a valuable approach for estimating its dosage in patients with renal impairment.

Nrf2 Ameliorates Atrial Fibrosis During Antithrombotic Therapy for Atrial Fibrillation by Modulating CYP2C9 Activity.

ABSTRACT: Anticoagulant therapy can significantly reduce the incidence of stroke and peripheral embolism events in patients with atrial fibrillation (AF). Although warfarin is widely used as an anticoagulant drug, a wrong dose can lead to increased risks of bleeding or blood clots. The aim of this study was to assess whether nuclear factor-erythroid-2-related factor 2 (Nrf2) can improve the efficacy of warfarin through the regulation of cytochrome P450 family 2 subfamily C member 9 (CYP2C9) using a rat model of AF. Results showed that AF significantly reduced Nrf2 in myocardial tissue of sham-operated rats. Furthermore, Nrf2 overexpression effectively reduced AF-induced atrial fibrosis by reducing collagen in the left atrium, inhibiting the expression of the fibrosis-related genes collagen I and transforming growth factor-ß1 in rats with AF. Nrf2 overexpression can activate CYP2C9, decrease the serum concentration of warfarin, and decrease prothrombin time and international normalized ratio in AF rats. In this article, Nrf2 overexpression protects against fibrosis, increased survival in AF rats, and activated CYP2C9 expression, thus broadening the therapeutic range of warfarin in AF rats.

Factors Influencing Plasma Concentrations of Valproic Acid in Pediatric Patients With Epilepsy and the Clinical Significance of CYP2C9 Genotypes in Personalized Valproic Acid Therapy.

BACKGROUND: The aim of this study was to investigate the factors affecting plasma valproic acid (VPA) concentration in pediatric patients with epilepsy and the clinical significance of CYP2C9 gene polymorphisms in personalized dosing using therapeutic drug monitoring and pharmacogenetic testing. METHODS: The medical records of children with epilepsy who underwent therapeutic drug monitoring at our institution between July 2022 and July 2023 and met the inclusion criteria were reviewed. Statistical analysis was performed to determine whether age, sex, blood ammonia, liver function, kidney function, and other characteristics affected the concentration-to-dose ratio of VPA (CDRV) in these patients. To investigate the effect of CYP2C9 polymorphisms on CDRV, DNA samples were collected from patients and the CYP2C9 genotypes were identified using real-time quantitative PCR. RESULTS: The mean age of 208 pediatric patients with epilepsy was 5.50 ± 3.50 years. Among these patients, 182 had the CYP2C9 *1/*1 genotype, with a mean CDRV (mcg.kg/mL.mg) of 2.64 ± 1.46, 24 had the CYP2C9 *1/*3 genotype, with a mean CDRV of 3.28 ± 1.74, and 2 had the CYP2C9 *3/*3 genotype, with a mean CDRV of 6.46 ± 3.33. There were statistical differences among these 3 genotypes ( P < 0.05). The CDRV in these patients were significantly influenced by age, aspartate aminotransferase, total bilirubin, direct bilirubin, globulin, albumin/globulin ratio, prealbumin, creatinine, and CYP2C9 polymorphisms. In addition, multivariate linear regression analysis identified total bilirubin, direct bilirubin, and CYP2C9 polymorphisms as independent risk factors for high CDRV. CONCLUSIONS: Liver problems and mutations in the CYP2C9 gene increase VPA levels. This underscores the importance of considering these factors when prescribing VPA to children with epilepsy, thereby enhancing the safety and efficacy of the therapy.

Prediction of Cytochrome P450 Inhibition Using a Deep Learning Approach and Substructure Pattern Recognition.

Cytochrome P450 (CYP) is a family of enzymes that are responsible for about 75% of all metabolic reactions. Among them, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 participate in the metabolism of most drugs and mediate many adverse drug reactions. Therefore, it is necessary to estimate the chemical inhibition of Cytochrome P450 enzymes in drug discovery and the food industry. In the past few decades, many computational models have been reported, and some provided good performance. However, there are still several issues that should be resolved for these models, such as single isoform, models with unbalanced performance, lack of structural characteristics analysis, and poor availability. In the present study, the deep learning models based on python using the Keras framework and TensorFlow were developed for the chemical inhibition of each CYP isoform. These models were established based on a large data set containing 85715 compounds extracted from the PubChem bioassay database. On external validation, the models provided good AUC values with 0.97, 0.94, 0.94, 0.96, and 0.94 for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. The models can be freely accessed on the Web server named CYPi-DNNpredictor (cypi.sapredictor.cn), and the codes for the model were made open source in the Supporting Information. In addition, we also analyzed the structural characteristics of chemicals with CYP450 inhibition and detected the structural alerts (SAs), which should be responsible for the inhibition. The SAs were also made available online, named CYPi-SAdetector (cypisa.sapredictor.cn). The models can be used as a powerful tool for the prediction of CYP450 inhibitors, and the SAs should provide useful information for the mechanisms of Cytochrome P450 inhibition.

Genetic variation of CYP2C9 gene and its correlation with cardiovascular disease risk factors.

BACKGROUND: The major enzyme that is responsible for Sulfonylureas (SUs) metabolism is hepatic cytochrome P-450 2C9 (CYP2C9). It is encoded by the polymorphic gene CYP2C9, which has many allelic variants, among those the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. People with diabetes mellitus type 2 (T2DM) are more likely to develop cardiovascular disease (CVD), and their risk of dying from it is more than two times higher than that of people without the condition. The purpose of this study was to evaluate the association of genetic variations in the CYP2C9 gene with cardiovascular risk factors by investigating CYP2C9*1, *2, *3, *5, *11, and *13 allelic variants. METHODS AND RESULTS: A total of 226 participants were enrolled in the current case-control study. Allele-specific amplification- PCR (ASA-PCR) was used to determine the allele of different variations and the results were confirmed by sequencing. The findings of this study showed the presence of the CYP2C9*2 allele in the T2DM group does not differ from its percentage in the control group. Also, CYP2C9*3 allele frequencies identified by Hardy-Weinberg equilibrium (HWE) analysis law were not significant, p = 0.6593 and 0.5828 in T2DM and control groups. There is no statistically significant difference between the control and diabetes groups involving the distribution of CYP2C9 alleles and CYP2C9*5, *11, and *13 polymorphisms were absent in the Iraqi population. No carrier for the CYP2C9*3 homozygous state was found in both groups. CONCLUSIONS: According to these results T2DM patients with the CYP2C9*2 and *3 variants have an increased risk of developing hypertension.

Elucidating the Mechanism of Metabolism of Cannabichromene by Human Cytochrome P450s.

Cannabichromene (CBC) is a nonpsychoactive phytocannabinoid well-known for its wide-ranging health advantages. However, there is limited knowledge regarding its human metabolism following CBC consumption. This research aimed to explore the metabolic pathways of CBC by various human liver cytochrome P450 (CYP) enzymes and support the outcomes using in vivo data from mice. The results unveiled two principal CBC metabolites generated by CYPs: 8'-hydroxy-CBC and 6',7'-epoxy-CBC, along with a minor quantity of 1″-hydroxy-CBC. Notably, among the examined CYPs, CYP2C9 demonstrated the highest efficiency in producing these metabolites. Moreover, through a molecular dynamics simulation spanning 1 µs, it was observed that CBC attains stability at the active site of CYP2J2 by forming hydrogen bonds with I487 and N379, facilitated by water molecules, which specifically promotes the hydroxy metabolite's formation. Additionally, the presence of cytochrome P450 reductase (CPR) amplified CBC's binding affinity to CYPs, particularly with CYP2C8 and CYP3A4. Furthermore, the metabolites derived from CBC reduced cytokine levels, such as IL6 and NO, by approximately 50% in microglia cells. This investigation offers valuable insights into the biotransformation of CBC, underscoring the physiological importance and the potential significance of these metabolites.

Modeled Hepatic/Plasma Exposures of Fluvastatin Prescribed Alone in Subjects with Impaired Cytochrome P450 2C9*3 as One of Possible Determinant Factors Likely Associated with Hepatic Toxicity Reported in a Japanese Adverse Event Database.

Fluvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor that competitively inhibits human cytochrome P450 (P450) 2C9 in vitro. Drug interactions between a variety of P450 2C9 substrates/inhibitors and fluvastatin can increase the incidence of fluvastatin-related hepatic or skeletal muscle toxicity in vivo. In this survey, the prescribed dosage of fluvastatin was reduced or discontinued in 133 of 164 patients receiving fluvastatin alone, as recorded in the Japanese Adverse Drug Event Report database of spontaneously reported events. The median days to onset of fluvastatin-related disorders were in the range 30-35 d in the 87 patients. Therefore, we aimed to focus on fluvastatin and, using the pharmacokinetic modeling technique, estimated the virtual plasma and hepatic exposures in subjects harboring the impaired CYP2C9*3 allele. The plasma concentrations of fluvastatin modeled after a virtual oral 20-mg dose increased in homozygotes with CYP2C9*3; the area under the plasma concentration curve was 4.9-fold higher than that in Japanese homozygotes for wild-type CYP2C9*1. The modeled hepatic concentrations of fluvastatin in patients with CYP2C9*3/*3 after virtual daily 20-mg doses for 7 d were 31-fold higher than those in subjects with CYP2C9*1/*1. However, heterozygous Chinese patients with CYP2C9*1/*3 reportedly have a limited elevation (1.2-fold) in plasma maximum concentrations. Virtual hepatic/plasma exposures in subjects harboring the impaired CYP2C9*3 allele estimated using pharmacokinetic modeling indicate that such exposure could be a causal factor for hepatic disorders induced by fluvastatin prescribed alone in a manner similar to that for interactions with a variety of co-administered drugs.