Enzymatic activity of 38 CYP2C9 genotypes on ibuprofen.

BACKGROUND AND OBJECTIVE: Ibuprofen, a common non-steroidal anti-inflammatory drug, is used clinically for pain relief and antipyretic treatment worldwide. However, regular or long-term use of ibuprofen may lead to a series of adverse reactions, including gastrointestinal bleeding, hypertension and kidney injury. Previous studies have shown that CYP2C9 gene polymorphism plays an important role in the elimination of various drugs, which leads to the variation in drug efficacy. This study aimed to evaluate the effect of 38 CYP2C9 genotypes on ibuprofen metabolism. METHODS: Thirty-eight recombinant human CYP2C9 microsomal enzymes were obtained using a frugiperda 21 insect expression system according to a previously described method. Assessment of the catalytic function of these variants was completed via a mature incubation system: 5 pmol CYP2C9*1 and 38 CYP2C9 variants recombinant human microsomes, 5 µL cytochrome B5, ibuprofen (5-1000 µM), and Tris-HCl buffer (pH 7.4). The ibuprofen metabolite contents were determined using HPLC analysis. HPLC analysis included a UV detector, Plus-C18 column, and mobile phase [50% acetonitrile and 50% water (containing 0.05% trifluoroacetic acid)]. The kinetic parameters of the CYP2C9 genotypes were obtained by Michaelis-Menten curve fitting. RESULTS: The intrinsic clearance (CLint) of eight variants was not significantly different from CYP2C9*1; four CYP2C9 variants (CYP2C9*38, *44, *53 and *59) showed significantly higher CLint (increase by 35%-230%) than that of the wild-type; the remaining twenty-six variants exhibited significantly reduced CLint (reduced by 30%-99%) compared to that of the wild-type. CONCLUSION: This is the first systematic evaluation of the catalytic characteristics of 38 CYP2C9 genotypes involved ibuprofen metabolism. Our results provide a corresponding supplement to studies on CYP2C9 gene polymorphisms and kinetic characteristics of different variants. We need to focus on poor metabolizers (PMs) with severely abnormal metabolic functions, because they are more susceptible to drug exposure.

Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus-insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

Effects of ketoconazole, voriconazole, and itraconazole on the pharmacokinetics of apatinib in rats.

We investigated the effect of azole antifungal drugs (ketoconazole, voriconazole, and itraconazole) on the pharmacokinetics of apatinib in rats. The rats in ketoconazole, voriconazole, and itraconazole groups received single-dose apatinib 30 mg/kg after the oral administration of ketoconazole, voriconazole, and itraconazole, respectively. Co-administration of ketoconazole or voriconazole significantly increased the apatinib Cmax and AUC(0-t) and decreased the clearance. Co-administration of itraconazole did not significantly affect the pharmacokinetics parameters of apatinib. It could be concluded that both ketoconazole and voriconazole significantly increase the exposure of apatinib, and affect the pharmacokinetics of apatinib in rat. Apatinib can be co-administered with itraconazole, but ketoconazole and voriconazole should be avoided if possible or be underwent therapeutic drug monitoring of apatinib. A further clinical study should be conducted to investigate the inhibitory effect of azole antifungal drugs on the apatinib plasma concentration.

Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes.

Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4'-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug-drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10 µmol L(-1) diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25 µmol L(-1) and Ki = 4.473 µmol L(-1) in human liver microsomes. Curcumin's mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100 µmol L(-1). The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment.

Curcumin as a potent and selective inhibitor of 11ß-hydroxysteroid dehydrogenase 1: improving lipid profiles in high-fat-diet-treated rats.

BACKGROUND: 11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) activates glucocorticoid locally in liver and fat tissues to aggravate metabolic syndrome. 11ß-HSD1 selective inhibitor can be used to treat metabolic syndrome. Curcumin and its derivatives as selective inhibitors of 11ß-HSD1 have not been reported. METHODOLOGY: Curcumin and its 12 derivatives were tested for their potencies of inhibitory effects on human and rat 11ß-HSD1 with selectivity against 11ß-HSD2. 200 mg/kg curcumin was gavaged to adult male Sprague-Dawley rats with high-fat-diet-induced metabolic syndrome for 2 months. RESULTS AND CONCLUSIONS: Curcumin exhibited inhibitory potency against human and rat 11ß-HSD1 in intact cells with IC50 values of 2.29 and 5.79 µM, respectively, with selectivity against 11ß-HSD2 (IC50, 14.56 and 11.92 µM). Curcumin was a competitive inhibitor of human and rat 11ß-HSD1. Curcumin reduced serum glucose, cholesterol, triglyceride, low density lipoprotein levels in high-fat-diet-induced obese rats. Four curcumin derivatives had much higher potencies for Inhibition of 11ß-HSD1. One of them is (1E,4E)-1,5-bis(thiophen-2-yl) penta-1,4-dien-3-one (compound 6), which had IC50 values of 93 and 184 nM for human and rat 11ß-HSD1, respectively. Compound 6 did not inhibit human and rat kidney 11ß-HSD2 at 100 µM. In conclusion, curcumin is effective for the treatment of metabolic syndrome and four novel curcumin derivatives had high potencies for inhibition of human 11ß-HSD1 with selectivity against 11ß-HSD2.

[Zinc supplementation effects on alcoholic liver disease and the molecular mechanism].

OBJECTIVE: To examine dietary zinc supplementation could alleviate the damage of alcoholic liver disease and the relationship with the expression of hepatocyte nuclear factor 4alpha (HNF-4alpha). METHODS: 40 adult C57 BL/6 mice were randomly divided into four groups (n = 10): control, zinc, ethanol and zinc plus ethanol, which were sacrificed after fed four different diets for 6 months. Zinc sulfate was added in the drinking water of the Zinc and Zinc Plus Ethanol group and the content was 75 mg/L. Liver regeneration was assessed by immunohistochemical staining of proliferating cell nuclear antigen (PCNA), and the expression of HNF-4alpha was determined by RT-PCR and Western blot. And as to assess the status of oxidative stress of the mice, malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. RESULTS: Compared with the control group, the expression level of HNF-4alpha decreased significantly in the ethanol group (P < 0.05), and the content of MDA increased significantly in this group, while the content of SOD declined significantly (P < 0.05). Compared with the ethanol group, the number of PCNA-positive hepatocytes increased significantly, and the expression level of HNF-4alpha also increased in the zinc plus ethanol group (P < 0.05), and the content of SOD increased in this group, while MDA decreased significantly (P < 0.05). CONCLUSION: Long term ethanol exposure can lead to oxidoreduction imbalances which can be reversed by zinc supplementation. We suppose that zinc-enhanced liver regeneration is associated with an increase in HNF-4alpha, suggesting that dietary zinc supplementation may have beneficial effects in alcoholic liver disease.