Study On Drug Metabolism-based Clinical Rational Drug Use

In clinical pharmacy practice there are many pharmacotherapy-related problems such as medication therapy management of drug interactions in complicated regimen, design of patient-tailored therapy regimen, control of intra-and inter-person variation in clinical efficacy, cause analysis of poor therapeutic effect, prevention and analysis of adverse drug reactions and rational prescriptions of drugs within similar therapeutic or structural class. These problems are closely related to drug-drug interactions mediated by drug metabolizing enzymes and/or transporter, and related pharmacogenomics. Hospital pharmacists shoμld apply these knowledge to solve actual clinical problems and make the regimens safer, more effective and economic.Statins, also called HMG-CoA reductase inhibitors, are widely used in treatment of essential hypercholesterolemia and mixed dyslipidemia and can slow aortic stenosis progression. Besides lipid-lowing effects, statins have additional pharmacological actions such as anti-inflammation, antioxidation, stabilizing atheromatous plaques, etc. Statins are playing pivotal roles in prevention and treatment of cardiovascular and cerebrovascular diseases. Statins related drug metabolizing enzymes involv CYP3A4and CYP2C9. Meanwhile P-gp, BCRP and OATP1B1are major drug transporters related to statins. This project focuses on clinical and basic aspects, performs clinical pharmacy research on statin-related drug metabolizing enzymes and transporters.Part ⅠRationality analysis of prescription was performed from the perspective of statin-related CYPs (i.e., CYP3A4and CYP2C9) mediated drug interaction. Concurrent therapies of three statins (i.e., simvastatin, atorvastatin and fluvastatin) were investigated in a2300-bed comprehensive hospital. Prescription rationality in concurrent therapy of simvastatin or atorvastatin needs to be improved. The percentages of recipes containing coadministered CYP3A4substrates or inhibitors to total recipes of simvastatin concurrent therapy were47.3%and7.0%, respectively. The percentages of recipes containing coadministered CYP3A4substrates or inhibitors to total recipes of atorvastatin concurrent therapy were41.7%and3.9%, respectively. Twenty-one CYP3A4substrates and16CYP3A4inhibitors were coadministered with simvastatin or atorvastatin. Among these,9drugs (i.e., colchicine, itraconazole, gemfibrozil, diltiazem, erythromycin, verapamil, cisapride, clarithromycin and cyclosporin) have been documented to have clinically significant drug interactions with simvastatin or atorvastatin. Concomitant uses of these drugs with simavastatin or atorvastatin were detected in this survey. The combination frequencies of these drugs were0.063%,0.32%,0.096%,2.5%,0.064%,0.096%,0.22%,0.32%and0.063%, respectively. The percentages of specified recipes to total recipes were as follows:0.19%(commitantly containing two CYP3A4inhibitors),2.4%(one CYP3A4inhibitor plus one more CYP3A4substrate) and3.8%(two more CYP3A4substrates). The percentage of recipes containg coadministered CYP2C9substrates or inhibitors to total recipes in fluvastatin concurrent therapy was9.5%and2.9%, respectively. Four CYP2C9substrates and2CYP2C9inhibitors were coadministered with fluvastatin. Clinical relevance of CYP2C9medicated drug interactions might be more obvious in patients receiving high doses of fluvastatin or carriers of CYP2C9*3.The status of concurrent therapy of nine CYP3A4inhibitors and five CYP3A4inducers was also investigated. No incompatibility of drugs was detected in1919sheets of prescriptions. It was found that3.65%of recipes existed drug interactions with clinical relevance. Sort of percentage of prescriptions containing CYP3A4inhibitors and CYP3A4substrates to total prescriptions of CYP3A4inhibitors were as follows:cyclosporin (54.54%)> diltiazem (41.54%)> berberine (14.9%)>verapamil (14.28%)> fluoxetine (9.40%)> erythromycin (5.88%)>metronidazole (3.68%)>itraconazole (1.48%)> voriconazole (0). Percentage of prescriptions containing CYP3A4inducers and CYP3A4substrates to total prescriptions of CYP3A4inducers were as follows:carbamazepine (4.91%), phenytoin (19.35%), dexamethasone (3.33%), rifampicin (5.77%) and phenobarbital (12.50%).A rationality analysis was also performed on concurrent therapy of clarithromycin (a widely used inhibitors of CYP3A4and P-gp) in gastroenterological practice. About0.85%-1.80%prescriptions were irrational in concurrent therapy of clarithromycin. Clarithromycin had synergistic effects on pharmacokinetics of PPIs (omeprazole, lansoprazole, esomeprazole, etc.) by inhibition of CYP3A4-mediated drug metabolism and P-gp-mediated efflux.Part IIPharmacokinetic studies of four statins (i.e., fluvastatin, simvastatin, rosuvastatin and pitavastatin) were conducted in Chinese volunteers with standard design of clinical trial. Plasma concentrations of statins were determined by HPLC-MS/MS or HPLC with fluorescence detector. SNPs were determined by TaqMan genotyping assay. The influences of12SNPs on pharmacokinetics of statins were investigated. These SNPs were such as CYP2C9*3(1075A>C), MDR1C1236T, MDR1G2677T/A, MDR1C3435T, CYP3A5*3 g.6986A>G, BCRP c.34G> A, BCRP c.421C>A, SLCO1B1c.388A>G, SLCO1B1c.521T>C, SLCO1B1g.11187G>A, SLCO1B1c.571T>C and SLCO1B1c.597C>T. Comprehensive effects of pharmacogenomics of CYPs and transporters on statin pharmacokinetics were studied. The genetic mechanism of inter-subject variations in statin pharmacokinetics was analyzed.This study indicated that effects of SNPs on pharmacokinetics of statins might be closely dependent on their structures and physicochemical characteristics. In comparison with other statins, simvastatin exhibited the smallest intersubject variation in pharmacokinetics. CYP2C9*3(1075A>C) only affected pharmacokinetics of fluvastatin and pitavastatin. MDR1C1236T only affected pharmacokinetics of rosuvastatin. MDR1G2677T/A affected AUC or Cmax of rosuvastatin, pitavastatin acid and pitavastatin lactone whereas it had significant influences on t1/2of fluvastatin and simvastatin. MDR1C3435T only affected pharmacokinetics of fluvastatin and rosuvastatin. BCRP c.421C>A and SLCO1B1c.388A>G only affected pharmacokinetics of rosuvastatin. SLCO1B1c.521T>C and SLCO1B1g.11187G>A only affected pharmacokinetics of pitavastatin acid and pitavastatin lactone. SLCO1B1c.571T>C and SLCO1B1c.597C>T had no effects on major pharmacokinetic parameters of four statins.1. FluvastatinWe conducted a pharmacokinetic study of fluvastatin in12healthy volunteers following a single oral dose of40mg fluvastatin. Major pharmacokinetic parameters included Cmax (551.16±210.28μg·L-1), tmax (0.59±0.18h), t1/2(1.57±0.73h) and AUC(0-∞)(610.51±251.54μg·h·L-1. The pharmacokinetic parameters indicated great individual differences in fluvastatin disposition in Chinese. The genetic mechanism for great inter-subject variations of fluvastatin at least included three aspects:(1) Effects of CYP2C9*3(1075A>C) SNP on fluvastatin AUC were statistically significant (P(0.01). Heterozygous variant (C/A) carriers had higher AUC,146.2%of the value in homozygous wild type (A/A) carriers. It indicated that mutation gene C carriers(C/C+C/A) had a higher AUC compared to CYP2C9*1/*1carriers.(2) The fact that MDR1C3435T SNP had a significant effect on Cmax of fluvastatin was firstly reported by us. Mutation gene T (TT+CT) carriers had a higher Cmax,139.9%of the value in homozygous wild type (C/C) carriers(P<0.05). T gene causes decrease in transport activity of P-gp and increase in Cmax. Considering the high frequency of MDR1C3435T variant allele T, plasma concentrations of fluvastatin in T allele carriers were higher than those in non-T carriers. Thus, dose related adverse drug reactions in T allele carriers shoμld be monitored in duration of treatment.(3) There was no significant difference between MDRl2677non-G subjects and MDR12677G subjects with respect to Cmax and AUC value of fluvastatin. However, t1/2became longer in MDR12677non-G carriers than in MDR12677G carriers (2.21±0.47h vs1.25±0.62h, P<0.05). There was no significant difference between MDR11236T-2677non-G-3435T subjects and MDR11236C-2677G-3435C subjects with respect to Cmax and AUC value of fluvastatin. However, t1/2became longer in MDR11236T-2677non-G-3435T carriers than in MDR11236C-2677G-3435C carriers (2.31±0.51vs1.32±0.62h, P(0.05)Pharmacokinetics of fluvastatin was not related with SNPs such as MDR1C1236T, CYP3A5*3g.6986A>G, BCRP c.34G> A, SLCO1B1c.388A>G, c.521T>C, c.571T>C and c.597C>T.2. RosuvastatinRosuvastatin pharmacokinetic studies were conducted after single doses of5mg,10mg and20mg rosuvastatin calcium and multiple doses of10mg rosuvastatin in healthy Chinese volunteers. The results showed that pharmacokinetics of rosuvastatin was linear. The elimination half-life was about15h. Rosuvastatin disposition exhibited great inter-subject variation.Average steady state Css and AUCss were closely related with MDR1C1236T, MDR1G2677T/A and MDR1C3435T SNP, homozygous mutation gene carriers having a higher value compared to homozygous wild type or heterozygous mutation gene carriers. BCRP c.421C>A SNP had a significant influence on steady state trough concentration of rosuvastatin which was higher in homozygous or heterozygous mutation gene carriers than in homozygous wild type carriers (+107%, P=0.0064)We also performed data mining using dose normalized Cmax and AUC(0-∞) and SNP information. More information was indicated.The influence of MDR1C1236T, MDR1G2677T/A and MDR1C3435T on pharmacokinetics of rosuvastatin was statistically significant. Generally, mutation gene carriers had obvious higher Cmax and AUC(0-∞). This conclusion was not so far documented. MDR1haplotypes (1236TT-2677TT-3435TT) carriers had obvious higher Cmax and AUC(0-∞) compared to non-1236TT-2677TT-3435TT carriers.BCRP c.421C>A SNP had a significant effect on rosuvastatin pharmacokinetics. Mutation gene carriers had obvious higher Cmax and AUC(0-∞) values (AA>CA, CC). BCRP c.421C>A SNP had a significant effect on tmax of rosuvastatin derived from a single dose of5mg (P=0.0069). tmax in heterozygous mutation gene carriers was longer than that in homozygous wild type carriers.SLCO1B1c.388A>G SNP had a significant effect on rosuvastatin AUC(0-∞). Compared to A/G genotype carriers, G/G genotype carriers had an obvious AUC. G/G genotype of OATPIBI caused decrease in hepatic uptake ability, hepatobiliary elimination and increase in plasma exposure of rosuvastatin.Other SNPs such as CYP2C9*3(1075A>C), CYP3A5*3g.6986A>G, BCRP c.34G> A, SLCO1B1c.521T>C, SLCO1B1g.11187G>A, SLCO1B1c.571T>C and SLCO1B1c.597C>T had no statistically influences on rosuvastatin pharmacokinetics.Our study results provided the underlying mechanism for the fact that Asians had high AUC values compare to those in Caucasians. Frequencies of SLCO1B1c.388A>G, BCRP c.421C>A and three MDR1SNPs which exerted significant influences on rosuvastatin in Asians were higher than those in Caucasians.In addition, no sex difference was observed in rosuvastatin pharmacokinetics.3. PitavastatinA randomized cross-over clinical trial of pitavastatin calcium dispersible tablet was conducted in Chinese volunteers after a single dose of1mg,2mg and4mg and mμltiple doses of2mg pitavastatin calcium. The results showed that pitavastatin exhibited linear pharmacokinetic characteristics. Elimination of pitavastatin acid and pitavastatin lactone were5h and15h, respectively. Meanwhile, great inter-subject variability was observed in pitavastatin pharmacokinetics.CYP3A5*3g.6986A>G had a statistically significant effect on t1/2of pitavastatin acid. Heterozygous AG carriers had a longer t1/2of pitavastatin acid compared to homozygous GG carriers. CYP3A5*3g.6986A>G had a statistically significant effect on AUC(0-∞) of pitavastatin lactone. Heterozygous AG carriers had a higher AUC(0-∞) of pitavastatin lactone compared to homozygous GG carriers.SLCO1B1c.521T>C had a significant effect on pharmacokinetics of pitavastatin acid after a single dose of1mg pitavastatin calcium tablet. Compared to homozygous wild type carriers, heterozygous and homozygous mutation gene carriers had an obvious decreased CL value(-57.9%) and higher AUC(0-∞)(+116%). Homozygous mutation gene of SLCO1B1c.521T>C decreased hepatic uptake and secondary hepatobiliary elimination of rosuvastatin.We performed data mining using dose-normalised pharmacokinetic parameters and SNP information. We firstly reported that CYP2C9*3(1075A>C) SNP had a significant influence on Cmax and AUC(0-∞) of pitavastatin acid. Compared to homozygous wild type carriers, heterozygous mutation gene carriers had a higher Cmax(+97.1%) and AUC(o-∞)(+192%). CYP2C9*3(1075A>C) SNP had a significant influence on AUC(0-∞) of pitavastatin lactone. Compared to homozygous wild type carriers, heterozygous mutation gene carriers had a comparable Cmax and higher AUC(0-∞)(+72%).MDR1G2677T/A SNP had an obvious effect on Cmax and AUC(0-∞) of pitavastatin acid. Compared to homozygous wild type carriers, heterozygous mutation gene carriers had a higher Cmax(+66.3%) and AUC(0-∞)(+98.7%). MDR1G2677T/A SNP had a statistically significant effect on Cmax of pitavastatin lactone. Compared to subjects with G/T, G/A or G/G genotype, heterozygous non-G carriers had a higher Cmax(+27%).SLCOIBI c.521T>C SNP had a significant effect on Cmax and AUC(0-∞) of pitavastatin acid and very obvious gene-dose effect of SLCO1B1c.521T>C SNP was observed in homozygous wild type TT, heterozygous CT and homozygous CC carriers(CC>CT>TT). SLCOIBI c.521T>C SNP had a statistically significant influence on Cmax and AUC(0-∞) of pitavastatin lactone. Obvious gene-dose effect was observed among homozygous wild type TT, heterozygous CT and homozygous CC carriers(CC>CT, TT). Cmax and AUC(0-∞) in homozygous CC carriers increased by48.8%and80%, respectively.SLCO1B1g.11187G>A SNP had a significant effect on Cmax and AUC(0-∞) of pitavastatin acid and obvious gene-dose effect was observed in homozygous wild type GG, heterozygous AG and homozygous AA carriers (AA>AG, GG). Cmax and AUC(0-∞) in AA carriers increased by138%and225%, respectively. SLCOIBI g.11187G>A SNP had a statistically significant influence on Cmax and AUC(0-∞) of pitavastatin lactone. Obvious gene-dose effect was observed among homozygous wild type GG, heterozygous AG and homozygous AA carriers(AA>AG, GG). Cmax and AUC(0-∞) in homozygous AA carriers increased by38%and58.2%, respectively.Compared to SLCO1B1*1b (388A>G) and non-SLCOIBI*17carriers, SLCO1B1*17(c.388G-c.521C-11187A) carriers had higher Cmax and AUC(0-∞) values(1-2fold). Considering the frequency of SLCO1B1*15(c.388G-c.521C) in Chinese, compared to non-SLCO1B1*15carriers, about14%of patients with pitavastatin calcium treatment had a higher Cmax and plasma exposure(1-2fold). Clinicians should take care to prevention pitavastatin adverse drug reactions. The effects of CYP3A5*3g.6986A>G and CYP2C9*3(1075A>C) SNP on pitavastatin acid and lactone were not so far reported. Further studies need to be conducted to address the underlying mechanism. Possible explanation involve transporter-CYP interaction.We firstly observed obvious sex difference in AUC(0-∞) of pitavastatin acid, Cmax and of pitavastatin lactone in Chinese. In SLCO1B1c.521TT female subjects Cmax and of pitavastatin lactone were higher than the values in SLCO1B1c.521TT male subjects. Further studies need to be performed to address whether gender factor directly affect pharmacokinetics of pitavastatin or indirectly affect the pharmacokinetics of pitavastatin by modifying the uptake activity or phenotype of OATP1B1.4. SimvastatinMajor pharmacokinetic parameters derived from a single dose of simvastatin40mg in17seventeen Chinese healthy male volunteers were as followed: tmax(1.44±0.39h), Cmax(9.83±2.41μg·L-1), t1/2(4.85±1.23h) and AUC(0-∞)(40.32±6.82μg·h·L-1). There was minor inter-subject variation in pharmacokinetics of simvastatin.The influence of CYP3A5*3g.6986A>G on simvastatin pharmacokinetics was statistically insignificant. However, effect of MDR1G2677T/A SNP on t1/2of simvastatin was positive. There is significant difference in t1/2between subjects carrying G/A, G/T or G/G and non-G carriers (5.65±0.50h vs4.41±1.31h, P<0.05). Mutation gene A and T cause decrease in efflux pump function of P-gp. Major pharmacokinetic parameters of simvastatin showed no significant difference between heterozygous ABCG2421CA carriers and homozygous ABCG2421CC carriers. Similar to results in Caucasians, SLCO1B1polymorphism has no significant effects on pharmacokinetics of simvastatin lactone (i.e., parent drug) in Chinese.Part IIIA BCRP/OATP1B1*la double transfected cell line was established. LLC-PK1clones with high uptake activity were selected by rosuvastatin (a substrate of OATP1B1and BCRP) accumulation. Western Blot analysis indicated that OATP1B1and BCRP were expressed stably and extensively. The cell line was proved to have transport ability by estrone-3-sulfate (an endogenous substrate) accumulation. This experiment provided a platform for hepatobiliary drug transport study.Part IVWe detected14SNPs of statins related CYPs and transporters in Chinese subjects with TaqMan genotyping assay. SLCO1B1polymorphism such as SLCO1B1c.388A>G, c.521T>C, g.11187G>A, c.571T>C, c.597C>T and c.463C>A were similar with the results documented. We first reported that frequencies of SLCO1B1g.11187G>A, c.571T>C, c.597C>T and c.463C>A in male Chinese were12.4%,28.8%,46%and0, respectively. Frequency of SLCO1B1*15(c.388G-c.521C) and SLC01B1*1b in237blood samples was23.2%and55.7%, respectively. These two variants were the most common genetic variants in Chinese. SLCO1B1*15variant carriers had lower transport function of OATP1B1, resulted in significant effects on systematic exposure and elimination of OATP substrates. Frequencies of SLCO1B1c.521T>C genotype and allele were not related with gender (P>0.05) at the sample size of this study (male125vs female112). In combination with conclusion in pitavastatin study in charpter1, it indicated sex difference in transport ability of OATP1B1. It is essential to determine the phenotype of OATP1B1.Frequency of BCRP c.34G> A SNP and BCRP c.421C>A SNP were34.6%and29.3%, respectively. Near one third Chinese population have a decreased efflux function of BCRP. No sex difference was observed with frequency of CYP2C9*3(1075A>C). Frequencies of mutation allele of CYP3A5and MDR1were similar with distribution of genetic polymorphisms in healthy Chinese.