臺灣博碩士論文加值系統

研究背景
Warfarin是臨床上一個常用來治療及預防血液栓塞疾病的藥物。然而使用warfarin來治療有其風險存在，基本上是因為病患間的個體差異，可歸因於：基因變異、環境、疾病種類及嚴重程度所影響，而使得在劑量的調整相當重要，現在臨床上的作法是監測其國際標準凝血時間比值(INR, international normalized ratio)。為達個別化用藥的目的，我們將討論可能的影響因子。

研究方法
本研究共收集94年7月至94年10月台大醫院內科部有使用warfarin之門診病患，於病情穩定後，抗凝血劑warfarin之使用情形，且收集病患之性別、年齡、適應症、病患用藥、INR值，並檢測病患的基因型，包括CYP1A2、CYP2C9、CYP2C19、CYP3A4、凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ、蛋白質C、蛋白質S、EPHX1、GGCX、GSTA1和VKORC1等基因，以及檢測warfarin異構物之血中濃度，研究病患個體化的warfarin用藥與藥物基因體學的相關性。

研究結果
符合受試者條件的病患共計75位，其中73位成功鑑定出所有納入檢測基因之基因型型別。病患處方warfarin之適應症主要為深層靜脈栓塞(deep vein thrombosis)。病患的平均維持劑量為3.9毫克/天，平均之INR值為2.02。基因型與劑量使用之關連性分析發現，VKORC1基因的-1639、1173、2225位置和CYP2C19基因的681位置，與warfarin的維持劑量有高度的相關。且VKORC1基因的-1639、1173、2225三個位置，有鏈鎖不平衡(linkage disequilibrium)現象出現。另外，年齡的因素也和維持劑量的多寡有高度的負相關。
異構物濃度與維持劑量間有顯著的相關性存在，但與療效間卻沒有直接的相關，故無法以監測濃度的方式來調整病患之劑量，仍需採用臨床上使用的INR值(或PT值) 來監測。

結論
Warfarin的維持劑量，是多種因素互相影響所決定的。台灣人的平均維持劑量與西方人不同的原因，可能是因為CYP2C19與VKORC1上SNP位置變異頻率的不同所致。經由迴歸模式的建立，可根據年齡、體重及基因上的變異等因素，計算出適合台灣人所使用的warfarin藥物投與劑量，以減少副作用的產生，增加藥物使用的安全性。

Background
Warfarin is an anticoagulant that is prescribed widely for the treatment and prevention of thrombosis. However, risks caused by warfarin vary, depending on the individuals, mainly genetic variations, environment, diseases and the severity of disease. It is important to adjust the dose of warfarin in clinical use based on the International Normalized Ratio (INR) value monitoring. All the possible factors involving dose adjustment will be investigated in this study in order to approach the individualized drug use.

Methods
Outpatients receiving long-term therapy were enrolled in National Taiwan University Hospital from July to October 2005. We collected the data including sex, age, indication, concomitant agents, INR value and the genotype of certain SNP sites in CYP1A2, CYP2C9, CYP2C19, CYP3A4, coagulation factor II, factor VII, factor IX, factor X, protein C, protein S, EPHX1, GGCX, GSTA1 and VKORC1. Furthermore, we detected the concentration of warfarin enantiomers and studied the association of pharmacogenomics and personalized warfarin doses.

Results
There were 75 patients enrolled in the study but two were excluded due to the failure of identifying the genotype. Deep vein thrombosis is the main indication in all patients. The mean maintenance dose was 3.9 mg/day and the mean INR value was 2.02. Results showed the variants of the positions -1639, 1173, 2225 in VKORC1 and the position 681 in CYP2C19 were highly related with warfarin maintenance dose. Also, there was negative relationship between age and warfarin maintenance dose in our study. Linkage disequilibrium of factor IX, GGCX and VKORC1 in certain SNP sites occurred in the study. The concentration of enantiomers was positively related to the maintenance dose of warfarin, but not to drug response. It is not applicable to adjustment of the dose by monitoring the concentration of racemic warfarin, or to its enantiomers.

Conclusion
The maintenance dose of warfarin was determined by multiple factors. The ethnic effect of Taiwanese and Caucasian might be partly caused by the different frequency of SNP sites of VKORC1 and CYP2C19. According to the regression model, we can calculate the warfarin maintenance dose by age and the genotype of these SNP sites so as to decrease the adverse drug effects and increase the safety of the drug.

中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ
目錄……………………………………………………………………Ⅳ
表目錄……………………………………………………………… Ⅵ
圖目錄…………………………………………………………………Ⅶ
第一章 前言……………………………………………………………1
第二章 文獻探討………………………………………………………3
第一節 Warfarin 總論………………………………………………3
一、化學結構與物化性質……………………………………………3
二、藥理作用機轉………………………………………………………4
三、藥物動力學…………………………………………………………4
四、藥物藥效學…………………………………………………………5
五、適應症、劑量及副作用……………………………………………6
六、藥物交互作用………………………………………………………7
第二節 基因多型性……………………………………………………9
一、單核甘酸多型性(SNP)……………………………………………9
二、Cytochrome P450的基因多型性…………………………………9
三、凝血因子相關的基因多型性……………………………………10
四、與維他命K 循環有關的基因多型性……………………………10
五、基因多型性與warfarin 用藥劑量的關係……………………10
第三章 研究目的………………………………………………………13
第四章 研究方法………………………………………………………14
第一節 試驗設計與流程………………………………………………14
一、研究對象…………………………………………………………14
二、受試者選擇標準…………………………………………………14
三、研究進行流程……………………………………………………14
四、資料來源…………………………………………………………14
第二節 基因型鑑定……………………………………………………15
一、SNPstream 之鑑定……………………………………………15
二、PCR-RFLP 之鑑定………………………………………………17
第三節 Warfarin 異構物的濃度測定：HPLC 分析…………………20
一、檢體處理…………………………………………………………20
二、實驗步驟…………………………………………………………20
三、分析條件…………………………………………………………20
四、標準溶液配製……………………………………………………21
五、標準曲線(Standard curve)製作……………………………… 21
六、校正曲線(Calibration curve)製作……………………………22
七、分析方法之確效…………………………………………………22
八、萃取回收率………………………………………………………22
第四節 試劑配方………………………………………………………24
第五章 研究結果………………………………………………………29
第一節 研究對象的基本資料…………………………………………30
第二節 基因型鑑定……………………………………………………31
第三節 HPLC 異構物濃度分析………………………………………32
一、滯留時間及檢量線水準…………………………………………32
二、萃取回收率………………………………………………………32
三、確效性試驗………………………………………………………32
四、樣本數、異構物平均濃度………………………………………32
第四節 各因子間相關性分析…………………………………………33
一、維持劑量與基因型或其他因子之關聯性………………………34
二、異構物濃度與劑量或其他因子間的關聯性……………………34
第五節 Warfarin 維持劑量投與的模式建立………………………36
一、單變項分析………………………………………………………36
二、多變項分析及迴歸模式的建立…………………………………36
第六章 討論…………………………………………………………50
第一節 研究對象的基本資料…………………………………………50
第二節 基因型鑑定……………………………………………………51
第三節 HPLC 異構物濃度分析………………………………………53
第四節 各因子間相關性分析…………………………………………54
第五節 Warfarin 維持劑量投與的模式建立………………………56
第六節 研究限制………………………………………………………57
第七章 結論及建議……………………………………………………58
第八章 參考文獻………………………………………………………59
附錄一 受試者同意書…………………………………………………65
附錄二 問卷……………………………………………………………69

表目錄
表2-1 維生素K 依賴型蛋白質………………………………………12
表4-1 Primers for polymerase chain reaction-restriction fragment length
polymorphism analysis…………………………………28
表5-1 Demographic and clinical characteristics of the enrolled patients……37
表5-2 Selected SNP sites for genotyping………………………………… 38
表5-3 Genetic variants of the enrolled patients…………………………………39
表5-4 人體血漿中warfarin 異構物及diclofenac 的萃取回收率…………………………40
表5-5 偵測人體血中warfarin 異構物之方法確效參數……………40
表5-6 Warfarin daily dose and INR value in various genotypes……………………41
表5-7 Characteristics of patients with higher or lower warfarin dose requirements…43
表5-8 維持劑量與其它因子關係表………………………………………44
表5-9 影響warfarin 維持劑量之單變項迴歸分析…………………………………44
表5-10 Regression equation for modeling warfarin daily dose requirements……….46
表5-11 Regression equation for modeling warfarin weekly dose requirements
in desired INR value……………………………………………………46

圖目錄
圖2-1 The structure of racemic warfarin……..………………………….……...3
圖2-2 The vitamin K cycle…………………………………………………..…..12
圖4-1 SNPstream®作用原理之圖解………………………………………..…..26
圖4-2 The strategy of RFLP to genotype CYP2C19*2 and *3…………..….27
圖5-1 Warfarin doses for population study…………………………………….47
圖5-2 INR value of patients receiving warfarin…………….……..…………..47
圖5-3 RFLP analysis of CYP2C19……………………………………….…….48
圖5-4 Calibration curve of warfarin enantiomers……….………………........48
圖5-5 Box plot of mean warfarin doses for different age and genotype.…..49

1. Lutomski, D.M., LaFrance, R.J., Bower, R.H. & Fischer, J.E. Warfarin
absorption after massive small bowel resection. Am J Gastroenterol 80,
99-102 (1985).
2. Shetty, H.G., Fennerty, A.G. & Routledge, P.A. Clinical pharmacokinetic
considerations in the control of oral anticoagulant therapy. Clin
Pharmacokinet 16, 238-253 (1989).
3. Muller, F.O., Steyn, J.M., Hundt, H.K. & Luus, H.G. Warfarin bio-availability.
A comparison of 4 products. S Afr Med J 74, 566-567 (1988).
4. Yacobi, A. et al. Who needs individual bioequivalence studies for narrow
therapeutic index drugs? A case for warfarin. J Clin Pharmacol 40, 826-835
(2000).
5. Urien, S., Albengres, E., Pinquier, J.L. & Tillement, J.P. Role of alpha-1 acid
glycoprotein, albumin, and nonesterified fatty acids in serum binding of
apazone and warfarin. Clin Pharmacol Ther 39, 683-689 (1986).
6. O''Reilly, R.A., Welling, P.G. & Wagner, J.G. Pharmacokinetics of warfarin
following intravenous administration to man. Thromb Diath Haemorrh 25,
178-186 (1971).
7. Swan, S.K. & Bennett, W.M. Drug dosing guidelines in patients with renal
failure. West J Med 156, 633-638 (1992).
8. Holford, N.H. Clinical pharmacokinetics and pharmacodynamics of warfarin.
Understanding the dose-effect relationship. Clin Pharmacokinet 11, 483-504
(1986).
9. Lewis, R.J. & Trager, W.F. Warfarin metabolism in man: identification of
metabolites in urine. J Clin Invest 49, 907-913 (1970).
10. Rettie, A.E. et al. Hydroxylation of warfarin by human cDNA-expressed
cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug
interactions. Chem Res Toxicol 5, 54-59 (1992).
11. Fishbain, D.A. et al. Genetic testing for enzymes of drug metabolism: does it
have clinical utility for pain medicine at the present time? A structured review.
Pain Med 5, 81-93 (2004).
12. Clark, S.L., Porter, T.F. & West, F.G. Coumarin derivatives and breast-feeding.
Obstet Gynecol 95, 938-940 (2000).
13. Breckenridge, A. & Orme, M. Kinetics of warfarin absorption in man. Clin
Pharmacol Ther 14, 955-961 (1973).
60
14. Hignite, C., Uetrecht, J., Tschanz, C. & Azarnoff, D. Kinetics of R andS
warfarin enantiomers. Clin Pharmacol Ther 28, 99-105 (1980).
15. Eble JM, W.B.a.L.E. A comparison of the isomers of warfarin. Biochem
Pharmacol 15, 1003-1006 (1966).
16. Yamazaki, H. & Shimada, T. Human liver cytochrome P450 enzymes involved
in the 7-hydroxylation of R- and S-warfarin enantiomers. Biochem Pharmacol
54, 1195-1203 (1997).
17. O''Reilly, R.A. Interaction of the anticoagulant drug warfarin and its
metabolites with human plasma albumin. J Clin Invest 48, 193-202 (1969).
18. Chan, E. et al. Disposition of warfarin enantiomers and metabolites in patients
during multiple dosing with rac-warfarin. Br J Clin Pharmacol 37, 563-569
(1994).
19. Hotraphinyo, K., Triggs, E.J., Maybloom, B. & Maclaine-Cross, A. Warfarin
sodium: steady-state plasma levels and patient age. Clin Exp Pharmacol
Physiol 5, 143-149 (1978).
20. Kumar, S. et al. Effect of warfarin on plasma concentrations of vitamin K
dependent coagulation factors in patients with stable control and monitored
compliance. Br J Haematol 74, 82-85 (1990).
21. Hirsh, J. et al. Oral anticoagulants: mechanism of action, clinical effectiveness,
and optimal therapeutic range. Chest 119, 8S-21S (2001).
22. Guidelines for thromboplastin and plasma used to control oral anticoagulant
therapy. WHO Technical Report Series 889, 64-93 (1999).
23. Computerized clinical infromation from MICROMEDEX® healthcare series.
(2005).
24. Gurwitz, J.H., Avorn, J., Ross-Degnan, D., Choodnovskiy, I. & Ansell, J.
Aging and the anticoagulant response to warfarin therapy. Ann Intern Med 116,
901-904 (1992).
25. Wells, P.S., Holbrook, A.M., Crowther, N.R. & Hirsh, J. Interactions of
warfarin with drugs and food. Ann Intern Med 121, 676-683 (1994).
26. Panneerselvam, S., Baglin, C., Lefort, W. & Baglin, T. Analysis of risk factors
for over-anticoagulation in patients receiving long-term warfarin. Br J
Haematol 103, 422-424 (1998).
27. Brigden, M.L., Kay, C., Le, A., Graydon, C. & McLeod, B. Audit of the
frequency and clinical response to excessive oral anticoagulation in an
out-patient population. Am J Hematol 59, 22-27 (1998).
28. Hylek, E.M., Heiman, H., Skates, S.J., Sheehan, M.A. & Singer, D.E.
Acetaminophen and other risk factors for excessive warfarin anticoagulation.
Jama 279, 657-662 (1998).
61
29. Robinson, D.S., Benjamin, D.M. & McCormack, J.J. Interaction of warfarin
and nonsystemic gastrointestinal drugs. Clin Pharmacol Ther 12, 491-495
(1971).
30. Jahnchen, E., Meinertz, T., Gilfrich, H.J., Kersting, F. & Groth, U. Enhanced
elimination of warfarin during treatment with cholestyramine. Br J Clin
Pharmacol 5, 437-440 (1978).
31. Sellers, E.M. & Koch-Weser, J. Potentiation of warfarin-induced
hypoprothrombinemia by chloral hydrate. N Engl J Med 283, 827-831 (1970).
32. Harder, S. & Thurmann, P. Clinically important drug interactions with
anticoagulants. An update. Clin Pharmacokinet 30, 416-444 (1996).
33. Guthrie, S.K., Stoysich, A.M., Bader, G. & Hilleman, D.E. Hypothesized
interaction between valproic acid and warfarin. J Clin Psychopharmacol 15,
138-139 (1995).
34. Freedman, M.D. & Olatidoye, A.G. Clinically significant drug interactions
with the oral anticoagulants. Drug Saf 10, 381-394 (1994).
35. O''Reilly, R.A. Interaction of sodium warfarin and rifampin. Studies in man.
Ann Intern Med 81, 337-340 (1974).
36. O''Reilly, R.A., Trager, W.F., Motley, C.H. & Howald, W. Interaction of
secobarbital with warfarin pseudoracemates. Clin Pharmacol Ther 28,
187-195 (1980).
37. Indiana Univerisity Department of Medicine Web Site. Cytochrome P450 drug
interaction table. Avaiable at http://medicine.iupui.edu/flockhart/table.htm.
Accessed Nov 26, 2005.
38. Thijssen, H.H., Soute, B.A., Vervoort, L.M. & Claessens, J.G. Paracetamol
(acetaminophen) warfarin interaction: NAPQI, the toxic metabolite of
paracetamol, is an inhibitor of enzymes in the vitamin K cycle. Thromb
Haemost 92, 797-802 (2004).
39. Booth, S.L. & Centurelli, M.A. Vitamin K: a practical guide to the dietary
management of patients on warfarin. Nutr Rev 57, 288-296 (1999).
40. Blickstein, D., Shaklai, M. & Inbal, A. Warfarin antagonism by avocado.
Lancet 337, 914-915 (1991).
41. Cambria-Kiely, J.A. Effect of soy milk on warfarin efficacy. Ann
Pharmacother 36, 1893-1896 (2002).
42. Bartle, W.R., Madorin, P. & Ferland, G. Seaweed, vitamin K, and warfarin. Am
J Health Syst Pharm 58, 2300 (2001).
43. Monterrey-Rodriguez, J. Interaction between warfarin and mango fruit. Ann
Pharmacother 36, 940-941 (2002).
44. Bartle, W.R. Grapefruit juice might still be factor in warfarin response. Am J
62
Health Syst Pharm 56, 676 (1999).
45. Suvarna, R., Pirmohamed, M. & Henderson, L. Possible interaction between
warfarin and cranberry juice. Bmj 327, 1454 (2003).
46. O''Reilly, R.A. Lack of effect of fortified wine ingested during fasting and
anticoagulant therapy. Arch Intern Med 141, 458-459 (1981).
47. Lambert, J.P. & Cormier, A. Potential interaction between warfarin and
boldo-fenugreek. Pharmacotherapy 21, 509-512 (2001).
48. Wong, A.L. & Chan, T.Y. Interaction between warfarin and the herbal product
quilinggao. Ann Pharmacother 37, 836-838 (2003).
49. Izzat, M.B., Yim, A.P. & El-Zufari, M.H. A taste of Chinese medicine! Ann
Thorac Surg 66, 941-942 (1998).
50. Page, R.L., 2nd & Lawrence, J.D. Potentiation of warfarin by dong quai.
Pharmacotherapy 19, 870-876 (1999).
51. Lam, A.Y., Elmer, G.W. & Mohutsky, M.A. Possible interaction between
warfarin and Lycium barbarum L. Ann Pharmacother 35, 1199-1201 (2001).
52. Davis, N.B., Nahlik, L. & Vogelzang, N.J. Does PC-SPEs interact with
warfarin? J Urol 167, 1793 (2002).
53. Matthews, M.K., Jr. Association of Ginkgo biloba with intracerebral
hemorrhage. Neurology 50, 1933-1934 (1998).
54. Rosado, M.F. Thrombosis of a prosthetic aortic valve disclosing a hazardous
interaction between warfarin and a commercial ginseng product. Cardiology
99, 111 (2003).
55. Taylor, J.R. & Wilt, V.M. Probable antagonism of warfarin by green tea. Ann
Pharmacother 33, 426-428 (1999).
56. Heck, A.M., DeWitt, B.A. & Lukes, A.L. Potential interactions between
alternative therapies and warfarin. Am J Health Syst Pharm 57, 1221-1227;
quiz 1228-1230 (2000).
57. Vesell, E.S. & Page, J.G. Genetic control of dicumarol levels in man. J Clin
Invest 47, 2657-2663 (1968).
58. JP von Wartburg, P.S. Atypical human liver alcohol dehydrogenase. Ann NY
Acad Sci 151, 936-946 (1968).
59. Marez, D. et al. Polymorphism of the cytochrome P450 CYP2D6 gene in a
European population: characterization of 48 mutations and 53 alleles, their
frequencies and evolution. Pharmacogenetics 7, 193-202 (1997).
60. Meyer, U.A. & Zanger, U.M. Molecular mechanisms of genetic
polymorphisms of drug metabolism. Annu Rev Pharmacol Toxicol 37, 269-296
(1997).
61. Breakthrough of the year. Areas to watch in 2005. Science 306, 2014 (2004).
63
62. http://www.ncbi.nlm.nih.gov/SNP/. (2005).
63. Girelli, D. et al. Polymorphisms in the factor VII gene and the risk of
myocardial infarction in patients with coronary artery disease. N Engl J Med
343, 774-780 (2000).
64. Shikata, E. et al. Association of pharmacokinetic (CYP2C9) and
pharmacodynamic (factors II, VII, IX, and X; proteins S and C; and
gamma-glutamyl carboxylase) gene variants with warfarin sensitivity. Blood
103, 2630-2635 (2004).
65. Taube, J., Halsall, D. & Baglin, T. Influence of cytochrome P-450 CYP2C9
polymorphisms on warfarin sensitivity and risk of over-anticoagulation in
patients on long-term treatment. Blood 96, 1816-1819 (2000).
66. Aithal, G.P., Day, C.P., Kesteven, P.J. & Daly, A.K. Association of
polymorphisms in the cytochrome P450 CYP2C9 with warfarin dose
requirement and risk of bleeding complications. Lancet 353, 717-719 (1999).
67. Higashi, M.K. et al. Association between CYP2C9 genetic variants and
anticoagulation-related outcomes during warfarin therapy. Jama 287,
1690-1698 (2002).
68. Loebstein, R. et al. Interindividual variability in sensitivity to warfarin--Nature
or nurture? Clin Pharmacol Ther 70, 159-164 (2001).
69. Margaglione, M. et al. Genetic modulation of oral anticoagulation with
warfarin. Thromb Haemost 84, 775-778 (2000).
70. Sullivan-Klose, T.H. et al. The role of the CYP2C9-Leu359 allelic variant in
the tolbutamide polymorphism. Pharmacogenetics 6, 341-349 (1996).
71. Phillips, K.A., Veenstra, D.L., Oren, E., Lee, J.K. & Sadee, W. Potential role
of pharmacogenomics in reducing adverse drug reactions: a systematic review.
Jama 286, 2270-2279 (2001).
72. Leung, A.Y. et al. Genetic polymorphism in exon 4 of cytochrome P450
CYP2C9 may be associated with warfarin sensitivity in Chinese patients.
Blood 98, 2584-2587 (2001).
73. Zarza J, H.J., Montes R, Alberca I, Lopez ML, Rocha E Leu208Val and
Ile181Leu variants of cytochrome P450 CYP2C9 are not related to the
acenocoumarol dose requirement in a Spanish population. Blood 100, 734
(2002).
74. Rettie, A.E. et al. CYP2C9 exon 4 mutations and warfarin dose phenotype in
Asians. Blood 101, 2896-2897 (2003).
75. Chang, J.-G. No exon 4 polymorphism of cytochrome P450 CYP2C9 in
Taiwanese. Blood 101, 5086-5087 (2003).
76. Sconce, E.A. et al. The impact of CYP2C9 and VKORC1 genetic
64
polymorphism and patient characteristics upon warfarin dose requirements:
proposal for a new dosing regimen. Blood 106, 2329-2333 (2005).
77. Yuan, H.Y. et al. A novel functional VKORC1 promoter polymorphism is
associated with inter-individual and inter-ethnic differences in warfarin
sensitivity. Hum Mol Genet 14, 1745-1751 (2005).
78. Bodin, L. et al. Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide
reductase (VKORC1) genotypes as determinants of acenocoumarol sensitivity.
Blood 106, 135-140 (2005).
79. Harrington, D.J. et al. Pharmacodynamic resistance to warfarin associated
with a Val66Met substitution in vitamin K epoxide reductase complex subunit
1. Thromb Haemost 93, 23-26 (2005).
80. Rieder, M.J. et al. Effect of VKORC1 haplotypes on transcriptional regulation
and warfarin dose. N Engl J Med 352, 2285-2293 (2005).
81. Wadelius, M. et al. Common VKORC1 and GGCX polymorphisms associated
with warfarin dose. Pharmacogenomics J 5, 262-270 (2005).
82. Loebstein, R. et al. Common genetic variants of microsomal epoxide
hydrolase affect warfarin dose requirements beyond the effect of cytochrome
P450 2C9. Clin Pharmacol Ther 77, 365-372 (2005).
83. Yu, H.C., Chan, T.Y., Critchley, J.A. & Woo, K.S. Factors determining the
maintenance dose of warfarin in Chinese patients. Qjm 89, 127-135 (1996).
84. Weibert RT, P.L. Differences in warfarin dose requirements between Asian and
Caucasian patients [abstract]. Clin Pharmacol Ther 49, 151 (1991).
85. Zhao, F. et al. Novel CYP2C9 genetic variants in Asian subjects and their
influence on maintenance warfarin dose. Clin Pharmacol Ther 76, 210-219
(2004).
86. Li, T. et al. Identification of the gene for vitamin K epoxide reductase. Nature
427, 541-544 (2004).
87. Rost, S. et al. Mutations in VKORC1 cause warfarin resistance and multiple
coagulation factor deficiency type 2. Nature 427, 537-541 (2004).
88. Takahashi, H. et al. Determination of unbound warfarin enantiomers in human
plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase
liquid chromatography with ultraviolet or fluorescence and on-line circular
dichroism detection. J Chromatogr B Biomed Sci Appl 701, 71-80 (1997).
89. Takahashi, H. et al. Population differences in S-warfarin metabolism between
CYP2C9 genotype-matched Caucasian and Japanese patients. Clin Pharmacol
Ther 73, 253-263 (2003).