蔡卫民 论文题目：CYP2D6遗传多态性对普罗帕酮药动学与药效学影响的研究 作者简介：蔡卫民，男，1958年04月出生，1997年9月师从于南京医科大学张银娣教授，于2000年07月获博士学位。
摘要
细胞色素P450酶(cytochrome
P450, CYP)是人体内参与内源性物质和外源性物质如药物代谢的重要代谢酶。已知异喹胍羟化酶CYP2D6存在着显著的遗传多态性，不同种族和不同个体代谢药物的能力有着较大的差异。普罗帕酮是临床上常用的抗心律失常药物，在体内代谢受CYP2D6介导，在药动学和药效学上存在的个体差异是否与CYP2D6遗传多态性有关有待探讨。因此，进行中国人普罗帕酮药动学与药效学结合CYP2D6表型和基因型的研究，将有助于了解国人在普罗帕酮体内过程与作用方面存在较大个体差异的原因所在，为相关药物的合理用药提供理论依据。为此，本文进行了以下五个部分的研究：
第一部分 采用右美沙芬为探针测定健康受试者CYP2D6表型
1. 人尿中右美沙芬及代谢物的测定：采用反相高效液相色谱法以荧光检测分析人尿中右美沙芬(DM)及代谢物右啡烷(DX)浓度。DM和DX的最低检测浓度分别为0.027
mg×L-1和0.031 mg×L-1，平均回收率为105.3%和104.5%，日内、日间相对标准差(RSD)均小于5%。
2. 中国人CYP2D6表型分析：120名健康中国人口服右美沙芬片剂20mg后留8h尿，用HPLC测定尿DM和DX浓度，并计算代谢比值。结果发现120名受试者中有1名慢代谢者(PM)(0.8%)，在余下的快代谢者(EM)中采用数理统计方法可进一步区分为76名(63%)极快代谢者(VEM)和43名(36%)中速代谢者(IM)。CYP2D6表型分析为中国人异喹胍多态性提供了新的信息。
第二部分 手性药物普罗帕酮对映体的人体药动学研究
1. 普罗帕酮对映体血浆浓度分析：采用高效液相色谱法加柱前衍生化分析血浆中普罗帕酮对映体浓度，得S-和R-普罗帕酮的最低检测浓度分别为18.6 mg × L-1和15.7 mg × L-1，平均回收率分别为101.9%和102.4%，日内、日间RSD均小于6%。
2. CYP2D6表型对普罗帕酮对映体药动学的影响：17名健康受试者(7名VEM，9名IM和1名PM)口服单剂量盐酸普罗帕酮400mg后，于0~15小时抽取静脉血，并用HPLC测定血浆中普罗帕酮对映体浓度(S-PPF和R-PPF)，计算对映体药动学参数。结果表明，S-PPF体内代谢速率显著大于R-PPF，具有明显的立体选择性；而且，CYP2D6表型在普罗帕酮对映体代谢中起重要作用，IM组的Cl只有VEM组的一半(P<0.01)。CYP2D6酶活性(lgMR)与普罗帕酮对映体药动学参数(Cmax，AUC和Cl)之间存在着良好的相关性。因此，CYP2D6表型决定了普罗帕酮对映体的药动学差异，IM的存在也许与中国人CYP2D6活性下降有关。
第三部分 CYP2D6抑制剂对右美沙芬表型和普罗帕酮对映体药动学的影响
1. 氟西汀和特比萘芬对右美沙芬表型的影响：健康年轻受试者19名(氟西汀组)和10名(特比萘芬组)，男女兼有，肝肾功能正常，不嗜烟酒，参加试验前两周禁用任何药物。受试者两次口服右美沙芬及留尿试验同第一部分，其间服用氟西汀20mg连续10天或特比萘芬250mg连续14天。19名口服氟西汀受试者前后的右美沙芬MR值分别为0.03 ± 0.04和0.09 ± 0.07(P<0.01)，增加三倍，说明氟西汀对CYP2D6有一定的抑制作用。10名受试者连续口服14天特比萘芬后，右美沙芬MR值从0.016 ± 0.011增加到0.321 ± 0.333
(P<0.01)，增加总幅度约高达20倍，有4人从EM转变为PM，说明特比萘芬较氟西汀对右美沙芬代谢的抑制作用更强。另外，氟西汀和特比萘芬对VEM的抑制作用均较IM更强。
2. 氟西汀对普罗帕酮对映体药动学的影响：9名健康受试者在应用氟西汀(20mg/天)10天前后单剂量口服400mg普罗帕酮，定时抽取静脉血并用HPLC测定血浆中普罗帕酮对映体浓度，计算对映体药动学参数。结果表明，普罗帕酮两对映体在合用氟西汀之后，t1/2，Cmax和AUC有显著增加(P<0.01)，而Cl则有明显下降(P<0.05)。而且氟西汀对S-PPF
Cmax增加幅度较R-PPF小(39% vs
71%，P<0.05)，Tmax的延长也仅在R-PPF有显著差异(P<0.05)，说明氟西汀不仅造成普罗帕酮对映体代谢的减慢，而且这种作用有立体选择性。
第四部分 中国人CYP2D6基因多态性的分子机制研究
119名健康汉族志愿者，男女兼有，肝肾功能正常，不嗜烟酒，右美沙芬表型测定区别为76名VEM，42名IM和1名PM。采取受试者全血，用经改良的酚/氯仿抽提法提取DNA。采用PCR-RFLP法测定受试者CYP2D6基因型，PCR反应特异扩增产物经1.2%琼脂糖凝胶电泳分析之后，再经HphI酶切反应，最后用3%琼脂糖凝胶电泳分析缺陷等位基CYP2D6*10B。
结果表明，CYP2D6*10B的基因频率为58.4%，其中13名(10.9%)为野生型(w/w)纯合子，33名(27.7%)为突变型(m/m)纯合子，其余73名(61.3%)为杂合子(m/w)。将右美沙芬表型与基因型作比较，发现76名VEM中有63人(约83%)为CYP2D6*10B的杂合子，42名IM中高达29人(近70%)是由CYP2D6*10B m/m造成的，而1名PM亦为m/m型。另外，10名受试者不存在与白种人PM相关的6种CYP2D6缺陷等位基因(CYP2D6*3，*4，*5，*6，*7和*9)。我们的实验提示，CYP2D6*10B等位基因的存在，可在很大程度上解释中国人右美沙芬氧化代谢酶活性降低的分子机理。
第五部分 普罗帕酮的临床药效学研究
1. 普罗帕酮药效动力学研究：健康志愿者10名，男女各半，所有受试者经右美沙芬表型测定分为5名VEM和5名IM。受试者口服普罗帕酮400mg后，于给药后1，2，3，4，6，8和15h取血并测定心电图指标PR间期。血浆中普罗帕酮浓度系采用HPLC测得的S-PPF和R-PPF浓度之和。采用CAPP软件对普罗帕酮血药浓度及PR间期延长百分率进行药动-药效结合计算，符合一级吸收二房室加效应室模型。实验表明，普罗帕酮的药效学过程符合Sigmoid Emax模型，效应与时间之间存在着较好的相关性。CYP2D6表型对药动学和药效学参数亦有影响，IM组的AUC明显高于VEM组，而Ce50 IM组也比VEM组大(P<0.05)。10名受试者的平均药效学参数Keo为1.17h-1，Emax为43.7%，Ce50为477.7mg × L-1，g为1.85。
2. 室性早搏病人口服普罗帕酮达稳态后的药效观察：17例室性早搏病人(VPCs³1000次/日)，男女不限，肝、肾功能、血尿常规检查正常。每位受试者停用所有心脏活性药物5个半衰期之后，口服普罗帕酮150~200mg/次，3次/日共7日，于给药前描记12导心电图和做24h动态心电图，并在给药7天后同上做两种心电图，并抽取用药前和用药后2h血，用HPLC测定血浆中普罗帕酮浓度。所有病例还测定了CYP2D6基因型。结果表明，17例病人室早搏的总抑制率(VPCs%)达65.3%，PR间期从用药前的0.15 ± 0.02秒增加到0.16 ± 0.02秒(P<0.05)。然而普罗帕酮血浆浓度与VPCs%和PR%等指标相关性不大。CYP2D6基因型对普罗帕酮血浆浓度和疗效有明显作用，*10/*10组病人不仅Cmax约两倍于*1/*1组病人，而且VPCs%也约两倍于后者，说明CYP2D6*10B与右美沙芬中速代谢缺陷高度相关，血药浓度升高与药效增加一致。
关键词 细胞色素P450，异喹胍羟化酶(CYP2D6)，遗传(基因)多态性，右美沙芬，氧化代谢，表型，高效液相色谱法，普罗帕酮，对映体，立体异构，药代动力学，抑制剂，基因型，聚合酶链反应(PCR)，限制性片段长度多态性(RFLP)，药效动力学，药动-药效结合模型，室性早搏
Effect of genetic polymorphism of CYP2D6 on pharmacokinetics and pharmacodynamics of propafenone
ABSTRACT
Cytochrome P450 is one of
the most important metabolic enzymes, which are responsible for many endogenous
and exogenous substances such as drugs in human body. It has been well known
that debrisoquine hydroxylase (CYP2D6) exists significant genetic polymorphism
with great differences of drug metabolizing ability in different races and
individuals. Propafenone, a commonly used antiarrhythmic agent, is given
clinically as a racemate. It is biotransformed mainly through CYP2D6 to the
active metabolite (5-OH propafenone). There is great pharmacokinetic and
pharmacodynamic variability of propafenone. Whether this variability in Chinese
is related to CYP2D6 genetic polymorphism needs to be clarified. Therefor, the
purpose of this paper is to determine the effect of CYP2D6 genetic polymorphism
on pharmacokinetics and pharmacodynamics of propafenone in Chinese.
Part I. Determination of
CYP2D6 phenotype by using dextromethorphan as a probe drug
1.
Analysis of dextromethorphan (DM) and dextrophan (DX) in human urine: A reverse-phase
high performance liquid chromatographic (HPLC) method was established for the
determination of DM and DX. The lowest detection levels of DM and DX were 0.027
mg×L-1
and 0.031 mg×L-1,
respectively. The within-day and between-day relative standard deviations (RSD)
were all below 5%. The average recoveries of DM and DX were 105.3% and 104.5%,
respectively.
2.
Phenotyping of CYP2D6 in Chinese subjects: 120 healthy Chinese subjects took
dextromethorphan tablets (20mg) orally and 8h urine was collected overnight.
The concentrations of DM and DX were assayed by HPLC and molar metabolic ratios
(MR) were calculated. The incidence of poor metabolizers (PM) was 0.8% (one in
120 subjects). There were distinct bimodal distributions, which divided
extensive metabolizers (EM) into 43 intermediate metabolizers (IM), and 76 very
extensive metabolizers (VEM). Dextromethorphan metabolic phenotyping provides a
new information for debrisoquine 4-hydroxylase (CYP2D6) polymorphism in native
Chinese.
Part II. Clinical pharmacokinetics
of propafenone enantiomers
1.
Analysis of plasma concentrations of propafenone enantiomers: A HPLC method
with precolumn derivatization was used to quantitate plasma concentrations of
propafenone enantiomers. The lowest detective levels of S-propafenone (S-PPF)
and R-propafenone (R-PPF) were 18.6 mg×L-1
and mg×L-1,
respectively. The average recoveries of S-PPF and R-PPF were 101.9% and 102.4%,
respectively. The within-day and between-day RSD were all less than 6%.
2.
Influences of CYP2D6 phenotypes on pharmacokinetics of propafenone enantiomers:
A single dose of propafenone hydrochloride (400mg) was given orally to 17
healthy Chinese subjects (7 VEM, 9 IM and 1 PM). Plasma concentrations of
propafenone were measured by HPLC during 0~15h after administration.
Pharmacokinetic parameters were calculated thereafter. The results showed that
S-PPF was less metabolized and had higher plasma concentrations than R-PPF in
both CYP2D6 phenotypes. Besides, the T1/2
of R-PPF was larger than that of S-PPF in IM, but not in VEM. However,
there were significant differences in the metabolism of PPF enantiomers between
VEM and IM. The Cmax and
AUC of both isomers in the IM group were higher than those in the VEM group (P < 0.01). The Cl of PPF enantiomers in IM group were only about half of that in
VEM group (67.3±18.6 vs 124.8±26.1 L × h-1 for
S-PPF, 90.2±23.6 vs 186.6±70.2 L × h-1 for
R-PPF, P < 0.01). The S/R
ratio of T1/2 , Cmax, Cl and AUC
was not significantly different ( p
> 0.05). The correlation between dextromethorphan MR and pharmacokinetic
parameters (Cmax，AUC and Cl) were
highly significant.
Part III. Influences of
CYP2D6 inhibitors on dextromethorphan phenotypes and pharmacokinetics of
propafenone enantiomers
1. Effects
of fluoxetine and terbinafine on dextromethorphan: 19 (fluoxetine group) and 10
(terbinafine group) young healthy subjects were recruited, with normal hepatic
and kidney functions. All were non-smokers and drug free for at least 2 weeks
before and during the study. Fluoxetine hydrochloride 20 mg was given once daily to all 19 subjects for
10 days. Terbinafine hydrochloride 250mg was given once daily to all 10
subjects for 14 days. Dextromethorphan phenotyping tests were performed before
and after pretreatment of CYP2D6 inhibitors. There were significant differences
of mean dextromethorphan MR values before and after fluoxetine therapy (0.03 ± 0.04 vs 0.09 ± 0.07, P<0.01), indicating a strong
inhibition of the CYP2D6 activity by fluoxetine in Chinese subjects. For 10
subjects treated with terbinafine for 14 days, mean MR values were increased
from 0.016 ± 0.011 to
0.321 ± 0.333 (P<0.01) with a 20 fold raise in
extent. Four out of 10 subjects were converted to PM of CYP2D6, indicating a
stronger inhibitive effect of terbinafine on dextromethorphan metabolism compared to fluoxetine. Besides, the
inhibitory effects of fluoxetine and terbinafine on VEM were stronger than on
IM.
2.
Influence of fluoxetine on pharmacokinetics of propafenone enantiomers: Nine
healthy subjects administered fluoxetine hydrochloride 20mg once daily for 10
days. Pharmacokinetics of propafenone enantiomers after a single dose of 400 mg
propafenone hydrochloride was performed before and after fluoxetine
pretreatment. The t1/2, Cmax and AUC0~¥ of two
enantiomers after fluoxetine therapy were significantly increased compared to
those at baseline (P < 0.01),
whereas, oral clearance decreased from 75.01 ± 17.69 L/h to 49.36 ± 8.62 L/h for S-propafenone (P = 0.005) and from 107.62 ± 33.82 L/h to 70.60 ± 12.42 L/h for R-propafenone (P = 0.027). Besides, fluoxetine increased the peak concentration of
S-propafenone by 39% and that of R-propafenone by 71% (P < 0.05). A significant increase of
Tmax was seen only in the R-enantiomer,
not the S-enantiomer of propafenone
after fluoxetine therapy. Our results suggest that fluoxetine not only impairs
propafenone metabolism significantly, but also raises its effect
stereoselectively.
Part IV. Molecular
mechanism of genetic polymorphism of CYP2D6 in Chinese subjects
119 healthy HAN volunteers
with normal hepatic and kidney functions and both genders were recruited.
Dextromethorphan phenotyping showed that there were 76 VEM, 42 IM and one PM of
CYP2D6. DNA was extracted from peripheral blood by a modified phenol /
chloroforms method. In order to determine CYP2D6 genotype, polymerase chain
reaction (PCR) and restriction fragment length polymorphism (RFLP) were use to
analyze CYP2D6*10B alleles. The gene frequency of CYP2D6*10B was 58.4%, including
13 (10.9%) homozygous wild type (w/w), 33 (27.7%) of homozygous mutant (m/m),
and 73 (61.3%) of heterozygous genotype (m/w). Twenty-nine subjects out of 42
IM (69%) of dextromethorphan were homozygous for CYP2D6*10B. One PM subject
also showed a m/m genotype. Besides, 10 Chinese subjects were tested and
excluded for the presence of any for the six mutant alleles associated with
poor metabolism of CYP2D6 in Caucasians. It is concluded that the CYP2D6*10B
allele containing the C188®T mutation is the major cause of CYP2D6 polymorphism in relation to
diminished dextromethorphan oxidative capacity in Chinese subjects.
Part V. Clinical
pharmacodynamics of propafenone
1.
Simultaneous modeling of pharmacokinetics and pharmacodynamics of propafenone: Ten
healthy volunteers with each gender were recruited. Dextromethorphan
phenotyping showed that there were 5 VEM and 5 IM of CYP2D6. After oral
administration of 400 mg of propafenone hydrochloride, blood collection and PR
interval monitoring were performed at 1, 2, 3, 4, 5, 6, 8 and 15 h. Total
propafenone concentrations as a sum of S-PPF and R-PPF were determined as
before. A computer aid pharmacokinetic & pharmacodynamic program (CAPP) was
used to simulate plasma concentrations of propafenone and percentage of PR
interval prolongation by using a model of first rate, two compartment plus
effect compartment. It has been shown that pharmacodynamic course of
propafenone accord with sigmoid Emax model in Chinese subjects.
There were good relationship between drug effect and time. CYP2D6 phenotype
also played a role in pharmacokinetic and pharmacodynamics of propafenone. AUC
of IM group was significantly higher than that of VEM group. Whereas, Ce50
of IM group was also greater than that of VEM group (P<0.05). The average pharmacokinetic parameters in 10 subjects
were as following: Keo = 1.17h-1, Emax =
43.7%, Ce50 = 477.7mg × L-1,
g = 1.85.
2.
Pharmacodynamic observation of steady-state propafenone in patients with
ventricular premature contractions: 17 patients with ventricular premature
contractions (VPCs ³ 1000/d)
were recruited from hospitals. They were normal in routine laboratory testing.
Every patient was free of cardioactive drugs for at least 5 half-life. The
patient administered propafenone hydrochloride 450mg~ 600mg per day in three
divided doses. Twelve-lead cardiogram and Holter monitoring were performed
before and after propafenone administration for 7 days. Peak and trough levels
of propafenones were drawn after drug treatment for 7 days and were measured by
HPLC as before. CYP2D6 genotypes were assayed for each patient. Our result
showed that total inhibitory rate of ventricular premature contractions (VPCs%)
was 65.3%. PR interval prolongation was increased from 0.15 ± 0.02 s to 0.16 ± 0.02 s after drug
treatment (P<0.05). However, there
were no significant relationship between plasma concentrations of propafenone
and pharmacodynamic index such as VPCs% and PR%. CYP2D6 genotypes played an
important role in plasma levels and effect of propafenone. Patients with
homozygous mutant of CYP2D6*10B not only had a Cmax two times as
high as wild-type, but also had VPCs% two times as high as wild-type. It
suggests that CYP2D6*10B is highly related to IM and elevated plasma level is
consistent with better efficiency of propafenone.
Conclusion: Genetic
polymorphism of CYP2D6 contributes to the variability in pharmacokinetics and
pharmacodynamics of propafenone in Chinese. It provides useful information for
safe and rational use of propafenone and related drugs clinically.
KEY WORDS: Cytochrome P450, debrisoquine hydroxylase (CYP2D6), genetic polymorphism, dextromethorphan, oxidative metabolism, phenotype, high performance liquid chromatography, propafenone, enantiomer, stereoisomerism, pharmacokinetics, inhibitor, genotype, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), pharmacodynamics, simultaneous modeling of pharmacokinetics and pharmacodynamics, ventricular premature contraction
