Pei-Pei Pan

Effect of 36 CYP2C9 variants found in the Chinese population on losartan metabolism in vitro

Abstract 1. CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, with 57 CYP2C9 allelic variants being previously reported. Among these variants, we recently identified 21 novel alleles (*36–*56) in the Han Chinese population. The aim of this study was to assess the catalytic activities of 36 CYP2C9 variants found in the Chinese population toward losartan in vitro. 2. Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.5–25 μM losartan for 30 min at 37 °C. Next, the products were extracted, and signal detection was performed using high-performance liquid chromatography. 3. Compared with wild-type CYP2C9.1, the intrinsic clearance (Vmax/Km) values of all variants except for CYP2C9.56 were significantly altered. One variant exhibited markedly increased values (>250%), whereas 33 variants exhibited significantly decreased values (from 20 to 96%) due to increased Km and/or decreased Vmax values. 4. These findings suggest that more attention should be paid to subjects carrying these infrequent CYP2C9 alleles when administering losartan in the clinic.

Determination of metoprolol and its two metabolites in human plasma and urine by high performance liquid chromatography with fluorescence detection and its application in pharmacokinetics.

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol and its two metabolites in human plasma and urine. Separation of metoprolol, α-hydroxymetoprolol, O-desmethylmetoprolol and esmolol (internal standard) was achieved on an Agilent XDB-C18 column (150mm×4.6mm, 5μm) using fluorescence detection with Ex 216nm and Em 312nm. The mobile phase consisted of ACN-H2O-0.1%TFA. The analysis was performed in less than 16min with a flow rate of 0.8mL/min. The assay was linear over the concentration range of 5-600ng/mL and 2.5-300ng/mL for metoprolol and its metabolites, respectively. The LOQ were 5.0 and 2.5ng/mL for plasma and urine, respectively. Good precision and accuracy for metoprolol and its two metabolites were obtained. The extraction recoveries were found to be more than 86.91% both in plasma and urine. At the same time, the method was successfully applied to nine healthy volunteers who had been given an oral tablet of 100mg metoprolol.

Effect of CYP2C9 genetic polymorphism on the metabolism of flurbiprofen in vitro

Abstract CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, and 57 cytochrome P450 2C9 alleles have been previously reported. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for 4′-hydroxyflurbiprofen were determined using recombinant human P450s CYP2C9 microsomes from insect cells Sf21 carrying wild-type CYP2C9*1 and other variants. The results showed that the enzyme activity of most of the variants decreased comparing with the wild type as the previous studies reported, while the enzyme activity of some of them increased, which were not in accordance with the previous researches. Of the 36 tested CYP2C9 allelic isoforms, two variants (CYP2C9*53 and CYP2C9*56) showed a higher intrinsic clearance value than the wild-type protein, especially for CYP2C9*56, exhibited much higher intrinsic clearance (197.3%) relative to wild-type CYP2C9*1, while the remaining 33 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.6 to 83.8%) compared to CYP2C9*1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used non-steroidal anti-inflammatory drug, flurbiprofen (FP). The results indicated that most of the tested rare alleles decreased the catalytic activity of CYP2C9 variants toward FP hydroxylation in vitro. This is the first report of all these rare alleles for FP metabolism providing fundamental data for further clinical studies on CYP2C9 alleles for FP metabolism in vivo.

Effects of Cytochrome P450 2C9 Polymorphism on Bosentan Metabolism

Cytochrome P450 (P450) 2C9 is an important member of the P450 enzyme superfamily, with 58 CYP2C9 allelic variants previously reported. Genetic polymorphisms of CYP2C9 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to assess the catalytic activities of 38 human CYP2C9 alleles, including 24 novel alleles (*36–*60) found in the Han Chinese population, toward bosentan (BOS) in vitro. Insect microsomes expressing the 38 CYP2C9 alleles were incubated with 10–625 μM bosentan for 30 minutes at 37°C and terminated by cooling to −80°C immediately. BOS and hydroxyl bosentan, the major metabolite of BOS, were analyzed by ultra-performance liquid chromatography–tandem mass spectrometry system. Thirty-eight defective alleles can be classified into three categories according to the relative clearance value compared with wild type: nine alleles exhibited significantly increased intrinsic clearance values (Vmax/Km) compared with the wild type (1.5-fold–∼4.9-fold relative clearance); nine alleles exhibited significantly reduced intrinsic clearance values compared with the wild type (0.6–28.9% relative clearance). The remaining 20 alleles exhibited no significant difference (1-fold) in enzyme activity compared with the wild type. These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2C9 alleles when administering BOS in the clinic. This is the first report of all these rare alleles for BOS metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles.

In vitro assessment of 39 CYP2C9 variants found in the Chinese population on the metabolism of the model substrate fluoxetine and a summary of their effects on other substrates

Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme that is responsible for clearing approximately 15% of clinically important drugs. The objective of this study was to assess the catalytic characteristics of 39 CYP2C9 isoforms found in the Chinese population and their effects on the metabolism of the model substrate fluoxetine in vitro.

Drug-drug interaction of losartan and glimepiride metabolism by recombinant microsome CYP2C9*1, 2C9*3, 2C9*13, and 2C9*16 in vitro

OBJECTIVE Co-administration of anti-hypertension and anti-diabetic drugs is common in clinical settings. METHODS In this study, we characterized the drug-drug interactions of losartan (LOS) and glimepiride (GLP) using recombinant cytochrome P450 (CYP) 2C9 enzymes (CYP2C9*1, CYP2C9*3, CYP2C9*13, and CYP2C9*16). RESULTS Metabolism of losartan by recombinant CYP2C9* 1, CYP2C9*3, CYP2C9*13, and CYP2C9* 16 was inhibited by glimepiride competitively with IC50 values of 0.669 ± 0.055 µM, 0.424 ± 0.032 µM, 2.557 ± 0.058 µM, and 0.667 ± 0.039 µM, respectively. The inhibitory effect of glimepiride on losartan metabolism by CYP2C9*13 was marginal. The apparent Ki value of glimepiride with CYP2C9*3 (0.0416 ± 0.0059 µM) was significantly lower than with CYP2C9*1 (0.1476 ± 0.0219 µM) and CYP2C9*16 (0.2671 ± 0.0456 µM). On the other hand, losartan weakly inhibited the hydroxylation of glimepiride by P450 2C9 enzymes competitively. The potencies for inhibition of glimepiride hydroxylation were determined to be CYP2C9*1~CYP2C9*3~CYP2C9*16 > CYP2C9*13 by 4 µM losartan. No significant inhibition was observed when 0.5 µM losartan was used. CONCLUSIONS Given these results, the potential inhibition of losartan metabolism by CYP2C9*3, CYP2C9*13, and CYP2C9*16 in vivo by glimepiride deserves further investigation. These results may provide valuable information for optimizing the anti-hypertension efficacy of losartan when glimepiride is co-administered to patients.

In vitro and in vivo drug-drug interaction of losartan and glimepiride in rats and its possible mechanism.

Background: Losartan and glimepiride are commonly used drugs to treat chronic diseases of hypertension and diabetes; they are both substrates of CYP2C9. The aim of the present study was to investigate the possible interaction of losartan and glimepiride both in vitro (rat liver microsomes) and in vivo (healthy Sprague-Dawley rats). Methods: In rat liver microsomes, 1-10 μmol/l losartan and glimepiride were coincubated, and the inhibitory effect was analyzed. In the subsequent pharmacokinetic study, 15 healthy Sprague-Dawley rats received administrations of 5 mg/kg losartan or 1 mg/kg glimepiride or a coadministration. Results: In the rat liver microsome system, glimepiride showed a slight inhibition of losartan at concentrations of 1-10 μmol/l, whereas losartan exhibited no inhibitory effect on glimepiride. In vivo, glimepiride did not modify the plasma concentration of losartan and its metabolite E-3174. The alteration of an increased AUC and Cmax was observed in the pharmacokinetic parameters of glimepiride and hydroxy glimepiride. Conclusions: Glimepiride did not affect losartan pharmacokinetics in rats, while losartan potently altered glimepiride metabolism; this result was inconsistent with the in vitro outcome. The mechanism requires further investigation. In clinical settings, attention should be paid to the interaction of these two drugs in the human body as well as the possible adverse reactions of glimepiride.

In Vitro and in Vivo Characterization of 13 CYP2C9 allelic variants found in Chinese Han population

Our previous study detected totally 35 CYP2C9 allelic variants in 2127 Chinese subjects, of whom 21 novel alleles were reported for the first time in Chinese populations. The aim of the present study was to characterize the 13 CYP2C9 allelic variants both in vitro and in vivo. Different types of CYP2C9 variants were highly expressed in COS-7 cells, and 50 μM tolbutamide was added as the probing substrate to evaluate their metabolic abilities in vitro. Subsequently, the concentrations of tolbutamide and its metabolite in the plasma and urine within individuals with different types of genotypes were determined by HPLC to evaluate the catalytic activity of the 13 mutant CYP2C9 proteins in vivo. Our results showed that compared with *1/*1 wild-type subjects, subjects with *1/*40 genotype showed increased oral clearance (CL/F), whereas individuals with *1/*3, *1/*13, *3/*3, *3/*13, *1/*16, *1/*19, *1/*34, *1/*42, *1/*45, *1/*46, and *1/*48 genotype exhibited significantly decreased CL/F, and those with *1/*27, *1/*29, *1/*40, and *1/*41 genotype presented similar CL/F value. When expressed in COS-7 cells, the CYP2C9 variants showed similar pattern to the results in clinical study. The study suggests that, besides two typical defective alleles, *3 and *13, seven CYP2C9 allelic variants (*16, *19, *34, *42, *45, *46, and *48) cause defective effects on the enzymatic activities both in vitro and in vivo. In clinic, patients with these defective alleles should be paid close attention to.

Impact of CYP2C9 Polymorphism Found in the Chinese Population on the Metabolism of Propofol in Vitro

The microsomal CYP2C9 alleles involved in the biotransformation of propofol, a widely used anesthetic agent, were investigated in vitro. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for propofol 4-hydroxylation were determined in recombinant human P450s CYP2C9 microsomes from Sf21 insects cells carrying CYP2C9*1 and other variants. Some of the variants showed decreased enzyme activity compared with the wild type, as previously reported. Two variants (CYP2C9*36 and *56) were found substantially to increase intrinsic clearance relative to the wild type variant. Most variants significantly (p<0.05) decreased intrinsic clearance of propofol compared with the wild type, except *11, *47, and *54. This study is the first to report these rare alleles for propofol metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles for propofol metabolism in vivo.

Effects of Methoxychlor and 2,2-bis (p-Hydroxyphenyl)-1,1,1-Trichloroethane on Cytochrome P450 Enzyme Activities in Human and Rat Livers

Cytochrome P450 (CYP) enzymes are involved in the metabolism of endogenous and exogenous compounds. Human and rat liver microsomes were used to investigate the inhibitory effects of methoxychlor (MXC) and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on the activities of corresponding human and rat CYPs. Probe drugs were used to test the inhibitory effects of MXC and HPTE on human and rat CYPs. The results showed that MXC and HPTE inhibited both human CYP2C9 and rat liver CYP2C11 activity, with half-maximal inhibitory concentration (IC50) values of 15.47 ± 0.36 (MXC) and 8.87 ± 0.53 μmol/l (HPTE) for human CYP2C9, and of 22.45 ± 1.48 (MXC) and 24.63 ± 1.35 μmol/l (HPTE) for rat CYP2C11. MXC and HPTE had no effects on human CYP2C19 activity but inhibited rat CYP2C6 activity with IC50 values of 14.84 ± 0.04 (MXC) and 8.72 ± 0.25 μmol/l (HPTE). With regard to human CYP2D6 and rat CYP2D2 activity, only HPTE potently inhibited human CYP2D6 activity, with an IC50 value of 16.56 ± 0.69 μmol/l. Both chemicals had no effect on human CYP3A4 and rat CYP3A1 activity. In summary, MXC and HPTE are potent inhibitors of some human and rat CYPs.