Li-zi Zhao

CYP4F2 rs2108622: a minor significant genetic factor of warfarin dose in Han Chinese patients with mechanical heart valve replacement

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * Genetic polymorphisms of VKORC1 and CYP2C9 are known to influence warfarin dosage. * Recent studies among Caucasians showed that polymorphisms of CYP4F2 also play a role in warfarin pharmacogenetics. * The contribution of CYP4F2 variants to the variability inwarfarin dose requirement in Chinese subjects remains to be investigated. WHAT THIS STUDY ADDS * This research was to study the effect of CYP4F2 variants on warfarin requirements in the Han Chinese population. * This study developed a multiple regression model including CYP2C9, VKORC1 3673G>A, CYP4F2 genotypes and age, weight, combination use of amiodarone which could explain 56.1% of the individual variability in warfarin dose CYP4F2 could explain 4% of the variance in warfarin dose. * We found that one novel genotypic polymorphism 5417G>T for Asp36Tyr, which was identified as an important marker of warfarin resistance, was absent in the Han Chinese population in our study. AIMS The objective of this study was to assess the effect of the CYP4F2 on the daily stable warfarin dose requirement in Han Chinese patients with mechanical heart valve replacement (MHVR). METHODS From March 2007 to November 2008, 222 Han Chinese MHVR patients were recruited in our study. VKORC1 3673G>A, 5417G>T, CYP2C9*3 and CYP4F2 rs2108622 were genotyped by using the polymerase chain reaction restriction fragment length polymorphism method (PCR-RFLP). Polymorphisms of VKORC1 9041G>A were detected by direct sequencing. Multiple linear regression analysis was used to investigate the contribution of CYP4F2. RESULTS The CYP4F2 rs2108622 CT/TT group took a significantly higher stable warfarin dose (3.2 mg day(-1)) than the CC group (2.9 mg day(-1), 95% CI 0.2, 1.0, P= 0.033). The multiple linear regression model included VKORC1 3673G>A, CYP2C9, CYP4F2 genotypes and clinical characteristics. The model could explain 56.1% of the variance in stable warfarin dose in Han Chinese patients with MHVR. CYP4F2 contributed about 4% to the variance in the warfarin dose. There was no variation in the SNPs of VKORC1 5417G>T. CONCLUSION CYP4F2 is a minor significant factor of individual variability in the stable warfarin dose in Han Chinese patients with MHVR. The effect of CYP2C9 and VKORC1 genotypes on variability in the stable warfarin dose had also been confirmed.

Induction of cytochrome P450s by terpene trilactones and flavonoids of the Ginkgo biloba extract EGb 761 in rats

1. Ginkgo biloba is one of the most popular herbal medicines worldwide due to its memory-enhancing and cognition-improving effects. The current study was designed to investigate the effects of five major constituents (bilobalide, ginkgolide A, B, quercetin, and kaempferol) in the standardized G. biloba extract EGb 761 on various cytochrome P450s (CYPs) in rats. 2. The activity of CYP450 was measured by the quantification of six metabolites from multiple cytochrome P450 probe substrates using a validated liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) method. The levels of messenger RNA (mRNA) and protein of various CYPs were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting analysis, respectively. 3. Bilobalide significantly induced the activity, protein, and mRNA expression of CYP3A1 and 1A2, and increased CYP2E1 activity and CYP2B1/2 protein expression in a dose-dependent manner. 4. Ginkgolide A, B, quercetin, and kaempferol did not affect CYP3A1, but induced CYP1A2 in a dose-dependent manner. EGb 761 and the five individual constituents had no effects on rat CYP2D2, 2C11 and 2C7. 5. The results indicate that bilobalide, and to a lesser extent ginkgolide A, B, quercetin, and kaempferol, play a key role in the effects of EGb 761 on CYP induction. Further study is needed to elucidate the mechanism of CYP3A induction by EGb 761 and bilobalide.

Preclinical factors affecting the pharmacokinetic behaviour of tanshinone IIA, an investigational new drug isolated from Salvia miltiorrhiza for the treatment of ischaemic heart diseases

Tanshinone IIA (TSIIA) is a major active triterpenoid isolated from Salvia miltiorrhiza. The purposes of this study were to investigate various preclinical factors that determined the pharmacokinetics of TSIIA. After oral dosing at 6.7, 20, and 60 mg kg−1, TSIIA was detected mainly as glucuronidated conjugate (TSIIAG) with only small amounts of the unchanged in the plasma. TSIIA was predominantly excreted into the bile and faeces as TSIIAG, and urine to a minor extent. The Cmax and AUC0−t of TSIIAG after i.p. administration were significantly lower than those after intragastric administration. The plasma concentration–time profiles of TSIIA following oral dosing of TSIIA showed multiple peaks. The Cmax and AUC0−t of TSIIA and its glucuronides in rats with intact bile duct were significantly lower than those of rats with bile duct cannulation. Studies from the linked-rat model and intraduodenal injection of bile containing TSIIA and its metabolites indicate that TSIIA glucuronides underwent hydrolysis and the aglycone was reabsorbed from the gut and excreted into the bile as conjugates. TSIIA had a wide tissue distribution, with a very high accumulation in the lung, but very limited penetration into the brain and testes. TSIIA was metabolized by rat CYP2C, 3A and 2D, as ticlopidine, ketoconazole and quinidine all inhibited TSIIA metabolism in rat liver microsomes. Taken collectively, these findings indicate that multiple factors play important roles in determining the pharmacokinetics of TSIIA.

The potential influence of 5-aminosalicylic acid on the induction of myelotoxicity during thiopurine therapy in inflammatory bowel disease patients.

Objective To investigate the potential influence of 5-aminosalicylic acid (5-ASA) on the induction of myelotoxicity during thiopurine therapy in inflammatory bowel disease patients. Methods (a) The retrospective study included inflammatory bowel disease patients treated with azathioprine (AZA)/6-mercaptopurine (6-MP). Thiopurine methyltransferase (TPMT) activity and 6-thioguanine nucleotide (6-TGN) levels were detected at stable medication points. (b) The prospective study was performed in active disease patients: 4 weeks of AZA 50 mg/day followed by concomitant 5-ASA 3 g/day for another 4 weeks. 6-TGN was analyzed at weeks 4 and 8. Results (a) Of the 139 retrospective study patients, 45 were on AZA/6-MP+5-ASA and 94 on AZA/6-MP alone. The myelotoxicity rates were 47 and 16%, respectively. Multivariates regression analysis indicated that the administration of concomitant 5-ASA was the only risk factor associated with myelotoxicity (odds ratio=3.45, 95% confidence interval 1.31–9.04, P=0.01). (b) Thiopurine methyltransferase activity was not significantly different between patients on AZA/6-MP+5-ASA and patients on AZA/6-MP alone (P=0.78). (c) 6-TGN levels were significantly higher in samples on AZA/6-MP+5-ASA than those on AZA/6-MP (P=0.003) alone. (d) Sixteen patients completed the prospective study. After 4 weeks on AZA 50 mg/day, 6-TGN levels of 13 patients were less than 230 pmol/8×108 RBC. After another 4 weeks’ cotreatment with mesalazine 3 g/day, 12 patients had 6-TGN levels at least 230 pmol/8×108 RBC, five patients had 6-TGN levels at least 420 pmol/8×108 RBC, and two of these five patients developed myelotoxicity. Conclusion The risk of thiopurine-induced myelotoxicity markedly increases in patients treated with combined 5-ASA and 2 mg/kg/day AZA therapy, which may be correlated to the increase in 6-TGN. 50 mg daily AZA when concomitant 5-ASA might help maintain an effective 6-TGN level without increasing the risk of myelotoxicity.

Induction of Cytochrome P450 3A by the Ginkgo biloba Extract and Bilobalides in Human and Rat Primary Hepatocytes

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. The study aimed to investigate the activity and expression of cytochrome P450 (CYP) 3A in human and rat primary hepatocytes treated with standardized G. biloba extract (100, 500, and 2500 ng/ml) for 72 hr, and to measure the protein expression of CYP3A in human and rat primary hepatocytes treated with bilobalide (2, 10, and 50 ng/ml) and ginkgolides B (2, 10, and 50 ng/ml). The activity of CYP3A was measured by the quantification of dehydronifedipine formation using a validated tandem liquid chromatography mass spectrometry (LC/MS/MS) method. The levels of mRNA and protein of CYP3A were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blotting analysis, respectively. The G. biloba extract at 100-2,500 ng/ml significantly induced the activity, protein and mRNA expression of CYP3A in a dose-dependent manner in human and rat primary hepatocytes. Bilobalide at 2-50 ng/ml significantly increased CYP3A protein expression in a dose-dependent manner in human and rat primary hepatocytes. However, ginkgolide B did not affect CYP3A protein expression in vitro. The results indicate that G. biloba extract pretreatment significantly induced the expression of CYP3A protein and mRNA and increased CYP3A activity, and there was no significant species difference between human and rat. G. biloba may cause potential interactions with substrate drugs of CYP3A. Bilobalide might play a key role in the enzyme-inducing effects of G. biloba extract. Further study is needed to identify the substances in GBE that induce CYPs in vivo, and elucidate the molecular mechanism of CYP3A induction by GBE and bilobalides.

Hypoxanthine guanine phosphoribosyltransferase activity is related to 6-thioguanine nucleotide concentrations and thiopurine-induced leukopenia in the treatment of inflammatory bowel disease.

Background: Thiopurine drugs are widely used in the treatment of inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S‐methyltransferase (TPMT) is of importance for thiopurine metabolism and adverse events occurrence. The role of other thiopurine‐metabolizing enzymes is less well known. This study investigated the effects of TPMT and hypoxanthine guanine phosphoribosyltransferase (HPRT) activities on 6‐thioguanine nucleotides (6‐TGNs) concentrations and thiopurine‐induced leukopenia in patients with IBD. Methods: Clinical data and blood samples were collected from 120 IBD patients who were receiving azathioprine (AZA)/6‐mercaptopurine (6‐MP) therapy. Erythrocyte TPMT, HPRT activities and 6‐TGNs concentrations were determined. HPRT activity and its correlation with TPMT activity, 6‐TGNs level, and leukopenia were evaluated. Results: The HPRT activity of all patients ranged from 1.63–3.33 (2.31 ± 0.36) &mgr;mol/min per g Hb. HPRT activity was significantly higher in patients with leukopenia (27, 22.5%) than without (P < 0.001). A positive correlation between HPRT activity and 6‐TGNs concentration was found in patients with leukopenia (r = 0.526, P = 0.005). Patients with HPRT activity > 2.70 &mgr;mol/min per g Hb could have an increased risk of developing leukopenia (odds ratio = 7.47, P < 0.001). No correlation was observed between TPMT activity and HPRT activity, 6‐TGNs concentration, or leukopenia. Conclusions: High levels of HPRT activity could be a predictor of leukopenia and unsafe 6‐TGN concentrations in patients undergoing AZA/6‐MP therapy. This could partly explain the therapeutic response or toxicity that could not be adequately explained by the polymorphisms of TPMT. (Inflamm Bowel Dis 2011;)

Induction of propranolol metabolism by Ginkgo biloba extract EGb 761 in rats

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. When used in the elderly, Ginkgo has a high potential for interactions with cardiovascular drugs. This study aimed to investigate the effects of the standard Ginkgo biloba extract (EGB 761) treatment on the pharmacokinetics of propranolol and its metabolism to form N-desisopropylpropranolol (NDP) in rats. We also examined the activity and expression of cytochrome P450 (CYP) 1A and other CYPs in rats treated with EGb 761 at 10 and 100 mg/kg/day for 10 days. A single oral dose of propranolol (10 mg/kg) was administered on day 11 and the concentrations of both propranolol and NDP were determined using validated liquid chromatography-mass spectrometry (LC-MS) methods. The levels of mRNA and protein of various CYPs were determined by RT-PCR and Western blotting analysis, respectively. Pretreatment of EGb 761 at 100 mg/kg, but not 10 mg/kg, for 10 days significantly reduced the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of propranolol, whereas those values of NDP were significantly increased. CYP1A1, 1A2, 2B1/2, and 3A1 activities and gene expression in the rat liver were significantly increased in a dose-dependent manner by pretreatment with EGb 761. The ex-vivo formation of NDP in liver microsomes from rats pretreated with EGb 761 was markedly enhanced. The formation of NDP from propranolol in liver microsomes was significantly inhibited by alpha-naphthoflavone (ANF, a selective CYP1A2 inhibitor), but not by quinidine (a CYP2D inhibitor). These results indicated that EGb 761 pretreatment decreased the plasma concentrations of propranolol by accelerated conversion of parental drug to NDP due to induction of CYP1A2. EGb 761 pretreatment also significantly induced CYP2B1/2 and CYP3A1, suggesting potential interactions with substrate drugs for these two enzymes. Further study is needed to explore the potential for gingko-drug interactions and the clinical impact.

Effect of Wuzhi tablet (Schisandra sphenanthera extract) on the pharmacokinetics of paclitaxel in rats.

Wuzhi tablet (WZ, registration no. in China: Z20025766) is a preparation of an ethanol herb extract of Wuweizi (Schisandra sphenanthera) containing 7.5 mg Schisantherin A per tablet. It was reported recently that WZ could significantly increase the blood concentrations of tacrolimus, which might be due to the inhibitory effect of WZ and its ingredients on P‐gp and/or CYP450 activity. Paclitaxel is a substrate of the efflux transporter P‐gp, and is mainly metabolized by CYP450 enzymes in the liver. Therefore, the purpose of this study was to investigate whether and how WZ affects the pharmacokinetics of paclitaxel in rats. After pretreatment with WZ, there were significant increases in the AUC0‐24h of oral paclitaxel (from 280.8 ± 97.3 to 543.5 ± 115.2 h ng/mL; p < 0.05) and Cmax (from 44.6 ± 16.4 to 86.8 ± 16.1 ng/mL; p < 0.05). The pharmacokinetic data for i.v. paclitaxel with WZ showed a relatively small (when compared against oral paclitaxel) but still significant increase in AUC0‐24h (from 163.6 ± 22.1 to 212.7 ± 17.7 h ng/mL; p < 0.05) and a decrease in clearance (from 3.2 ± 0.6 to 2.2 ± 0.3 L/h/kg; p < 0.05). Thus, the presence of WZ improved the systemic exposure of paclitaxel in rats. The herb–drug interaction between WZ and paclitaxel should be taken into consideration in clinical use. Copyright

Enhancement of oral bioavailability of paclitaxel after oral administration of Schisandrol B in rats

Paclitaxel is a substrate of the efflux transporters such as P-glycoprotein, and is mainly metabolized by the liver. Schisandrol B (Sch B), one of the active components in Schisandra, has been reported to be able to inhibit the activity of P-gp and CYP3A. It might be possible that Sch B would alter the pharmacokinetic behavior of paclitaxel. Therefore, the purpose of this study was to investigate the effect of Sch B on the pharmacokinetics of paclitaxel administered orally and intravenously in rats. Paclitaxel were administered to rats orally (30 mg/kg) or intravenously (0.5 mg/kg) with or without the concomitant administration of Sch B (10 or 25 mg/kg). Oral pharmacokinetic parameters of paclitaxel were significantly altered when pretreated with Sch B. There were significant increases in AUC(0-24h) (from 297.7+/-110.3 to 838.9+/-302.1 h*ng/ml; p<0.05) and C(max) (from 51.7+/-20.1 to 136.4+/-35.5 ng/ml; p<0.05) in the presence of Sch B (25 mg/kg). The pharmacokinetic parameters for i.v. paclitaxel were not significantly affected by Sch B in contrast to that of oral administration. Since the presence of Sch B enhanced the systemic exposure of paclitaxel, their pharmacokinetic interaction should be taken into consideration. As the oral bioavailability of paclitaxel was increased about 3-fold in the presence of Sch B, the concomitant use of Sch B may provide a benefit in the oral delivery of paclitaxel.

Pharmacokinetic characterization of hydroxylpropyl-β-cyclodextrin-included complex of cryptotanshinone, an investigational cardiovascular drug purified from Danshen (Salvia miltiorrhiza)

1. The study aimed to investigate the pharmacokinetics of cryptotanshinone in a hydroxylpropyl-β-cyclodextrin-included complex in dogs and rats. 2. Animals were administrated the inclusion complex of cryptotanshinone and the concentrations of cryptotanshinone and its major metabolite tanshinone IIA were determined by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. 3. Cryptotanshinone in inclusion complex was absorbed slowly after an oral dose, and the Cmax and AUC0–t were dose-proportional. The bioavailability of cryptotanshinone in rats was (6.9% ± 1.9%) at 60 mg kg−1and (11.1% ± 1.8%) in dogs at 53.4 mg kg−1. The t1/2 of the compound in rats and dogs was 5.3–7.4 and 6.0–10.0 h, respectively. Cryptotanshinone showed a high accumulation in the intestine, lung and liver after oral administration, while the lung, liver and heart had the highest level following intravenous dose. Excretion data in rats showed that cryptotanshinone and its metabolites were mainly eliminated from faeces and bile, and the dose recovery rate was 0.02, 2.2, and 14.9% in urine, bile, and faeces, respectively. 4. The disposition of cryptotanshinone in an inclusion complex was dose-independent and the bioavailability was increased compared with that without cyclodextrin used to formulate the drug. Cryptotanshinone was distributed extensively into different organs. Excretion of cryptotanshinone and its metabolites into urine was extremely low, and they were mainly excreted into faeces and bile.