The Pharmacogenomics Journal (2004) 4, 40–48 & 2004 Nature Publishing Group All rights reserved 1470-269X/04 $25.00 www.nature.com/tpj ORIGINAL ARTICLE Warfarin sensitivity related to CYP2C9, CYP3A5, ABCB1 (MDR1) and other factors M Wadelius1,4 ABSTRACT 1 The required dose of the oral anticoagulant warfarin varies greatly, and KSo¨rlin overdosing often leads to bleeding. Warfarin is metabolised by cytochrome 2 O Wallerman P450 enzymes CYP2C9, CYP1A2 and CYP3A. The target cell level of warfarin J Karlsson1 may be dependent on the efflux pump P-glycoprotein, encoded by the Q-Y Yue3 adenosine triphosphate-binding cassette gene ABCB1 (multidrug resistance PKE Magnusson2 gene 1). Genetic variability in CYP2C9, CYP3A5 and ABCB1 was analysed in 2 201 stable warfarin-treated patients using solid-phase minisequencing, C Wadelius pyrosequencing and SNaPshot. CYP2C9 variants, age, weight, concurrent H Melhus1 drug treatment and indication for treatment significantly influenced warfarin dosing in these patients, explaining 29% of the variation in dose. CYP3A5 did 1Department of Medical Sciences, Clinical not affect warfarin dosing. An ABCB1 haplotype containing the exon 26 Pharmacology, University Hospital, Uppsala, Sweden; 2Department of Genetics and Pathology, 3435T variant was over-represented among low-dose patients. Thirty-six Medical Genetics, Rudbeck Laboratory, Uppsala, patients with serious bleeding complications had higher prothrombin time Sweden; 3Medical Products Agency, Uppsala, international normalised ratios than 189 warfarin-treated patients without Sweden serious bleeding, but there were no significant differences in CYP2C9, CYP3A5 or ABCB1 genotypes and allelic variants. Correspondence: The Pharmacogenomics Journal (2004) 4, 40–48. doi:10.1038/sj.tpj.6500220 M Wadelius, Department of Medical Published online 16 December 2003 Sciences, Clinical Pharmacology, University Hospital, SE-751 85 Uppsala, Sweden. Keywords: warfarin; bleeding; CYP2C9; CYP3A5; ABCB1; MDR1 Tel: þ 46 18 611 49 45 Fax: þ 46 18 51 92 37 E-mail: [email protected] 4Temporary address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome INTRODUCTION Campus, Hinxton, Cambridge CB10 1SA, UK. Nearly 1% of the Swedish population is treated with the oral anticoagulant Tel: þ 44 1223 49 47 09. warfarin. The indications for treatment include atrial fibrillation, heart valve prosthesis, recurrent stroke, deep vein thrombosis and pulmonary embolism.1 Interindividual variation in the response to warfarin and a narrow therapeutic range are two of the factors that make warfarin therapy difficult to handle. In all, bleeding complication during treatment with warfarin is the most common adverse drug reaction with a fatal or disabling outcome.2,3 Warfarin is a coumarin derivative that interferes with the recycling of vitamin K in the liver.2 Vitamin K is involved in the carboxylation of the precursor proteins for the coagulation factors II, VII, IX and X. In the presence of warfarin, the activity of these components in the blood is lowered by around 10–40% and coagulation is inhibited.3,4 The effect is delayed until dysfunctional coagulation factors have been synthesised, which takes 4–5 days.4 Patients treated with warfarin are monitored by prothrombin time (PT) expressed as an international normalised ratio (INR). PT INR measures the sum of activity of the vitamin K- Received: 09 July 2003 dependent coagulation factors II, VII and X. For most indications, the ideal PT Revised: 03 October 2003 4–6 Accepted: 14 October 2003 INR value lies between 2.0 and 3.0. One exception is heart valve prosthesis, Published online 16 December 2003 which requires a higher PT INR. Warfarin related to CYP2C9, CYP3A5 and ABCB1 (MDR1) M Wadelius et al 41 The dose requirement of warfarin varies more than 10-fold hepatocytes, which indicates that warfarin is a P-GP between patients.7 In general, women need lower doses than substrate in the liver.27 In theory, low P-GP activity in men.4 A high dietary intake of vitamin K leads to a higher hepatocytes may lead to a high level of warfarin in the target dose requirement, while old age is associated with lower cell, causing warfarin sensitivity in some people. doses.8–11 Diseases that increase the sensitivity to warfarin The ABCB1 gene, also called the multidrug resistance gene include liver dysfunction impairing the synthesis of coagu- 1 (MDR1), encodes P-GP. There are many known poly- lation proteins, hypermetabolic states that increase the morphisms in ABCB1: for example, À12T4C in exon 1 in clearance of coagulation factors and congestive heart failure the 50 untranslated region, À1G4A at the initiation of that alters the distribution of warfarin.3,6,11 Warfarin is translation in exon 2, 1199G4A in exon 11 leading to highly bound to serum albumin, which in combination amino-acid change S400N, 1236C4T in exon 12 at a wobble with its extensive cytochrome P450 (CYP) metabolism and position G412G, 2677G4TorG4A in exon 21 leading to narrow therapeutic range makes warfarin susceptible to drug A893S or T, and in exon 26 two polymorphisms at wobble interactions.12 positions, 3396C4T A1132A and 3435C4T I1145I.28,30 An asymmetric carbon in the warfarin molecule gives rise Polymorphisms of ABCB1 are suggested to be important to the enantiomeric forms S- and R-warfarin.13 Both for variability in drug bioavailability, but the pharmacolo- enantiomers are eliminated extensively via hepatic metabo- gical implication of these polymorphisms has not been fully lism with a low extraction ratio. The S-form is a 3–5 times established.30 more potent vitamin K antagonist than the R-form.6,7 Haemorrhage is the most common adverse reaction to CYP2C9 is the main enzyme responsible for metabolism of coumarin anticoagulants, and a great under-reporting of the active S-warfarin,7,13 which renders the genetic variation these events is believed to exist.3,31 Swedish studies have of this metabolic pathway extremely important.14 In shown that 4.5% of warfarin-treated patients experience addition to the CYP2C9*1 wild-type allele, at least 11 major bleeding and 0.5% suffer fatal complications.31 The variant alleles are known to exist (http://www.imm.ki.se/ risk of bleeding is 10 times higher during the first month cypalleles/ in September 2003). The most common variants compared to after the first year.5 There is a clear relationship CYP2C9*2 (430C4T, R144C) and CYP2C9*3 (1075A4C, between haemorrhage and the intensity of treatment, with I359L) generate enzymes with impaired hydroxylation of PT INR elevation being a strong predictor.3,5,10,32 Age, S-warfarin due to amino-acid changes, and several studies concurrent medication, specific comorbid conditions, espe- have shown that these variants have an effect on warfarin cially cerebrovascular, kidney, heart and liver disease as well dose requirement.7,11,15–24 as prosthetic heart valves, are independent risk factors.5,32 R-warfarin is mainly metabolised by the CYP enzymes Several studies suggest that patients with CYP2C9 variant CYP1A2 and CYP3A, with CYP3A likely to be the most alleles have a higher incidence of bleeding complications important.13 CYP3A activity is derived from CYP3A4 and than carriers of the wild-type genotype.15,16,20,22 The risk of CYP3A5 that have similar substrate specificities. CYP3A5 is haemorrhage must always be weighed against the preven- considered to be 17–50% of the total CYP3A content in the tion of thromboembolism, and in most patients the livers of people that express it.25,26 There is considerable preventive effect outweighs the risk of bleeding.4,32 intra- and interindividual variation in CYP3A4 enzyme The aim of the study was to identify factors that influence activity, and even though numerous polymorphisms have the effect of warfarin and the required dose. Warfarin dose been found, the molecular basis of this is not yet under- requirement was compared to genetic variation of CYP2C9, stood. On the other hand, CYP3A5 exists in distinct fast and CYP3A5 and ABCB1, age, gender, weight, smoking, indica- slow variants,25 and is therefore worth studying even if it is tion for treatment, and concurrent medication. In the case– less abundant than CYP3A4. CYP3A5*1 encodes an active control part, we studied these factors in relation to warfarin enzyme, whereas CYP3A5*3 (6986A4G) and CYP3A5*6 bleeding. (14690G4A) are inactive due to splicing defects. The CYP3A5*3 allele is the most common cause of low CYP3A5 functionality in Caucasians, while CYP3A5*6 is rarely found. RESULTS There is preliminary evidence that P-glycoprotein (P-GP) Dose Requirement contributes to warfarin disposition.27 P-GP belongs to a class The first aim was to identify factors that influence the of adenosine triphosphate-binding cassette (ABC) transport required dose of warfarin. The patients were recruited in proteins.28 It serves as a potent efflux pump for a wide 2000, when they were 28–88 years old (Table 1). They had variety of lipophilic compounds that are frequently CYP3A stable PT values, and had been treated with warfarin for substrates.29 P-GP is expressed in tissues such as the between 2.4 months and 26 years (median 2 years). intestine, liver, kidney, blood–brain barrier and placenta.28 Individual warfarin dose requirement varied between 4.5 Warfarin is well absorbed from the intestine with over 90% and 77.25 mg per week. The most frequent indications for bioavailability, making it unlikely that intestinal P-GP anticoagulation were atrial fibrillation, heart valve prosthe- affects the absorption of warfarin