*13 genotype

Clinica Chimica Acta 412 (2011) 2343–2345

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Case report

Low dose requirement for warfarin treatment in a patient with CYP2C9*3/*13 genotype Min-Jung Kwon a, Young-Keun On b, Wooseong Huh b, d, Jae-Wook Ko d, Duk-Kyung Kim b, June Soo Kim b,⁎, Soo-Youn Lee c, d,⁎⁎ a

Departments of Laboratory Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Departments of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Departments of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea d Department of Clinical Pharmacology and Therapeutics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 16 December 2010 Received in revised form 31 May 2011 Accepted 27 June 2011 Available online 19 July 2011 Keywords: Warfarin PT INR CYP2C9 VKORC1 Genotype

a b s t r a c t Background: Cytochrome P450 2C9 (CYP2C9) is the major isoform of the CYP2C subfamily, and is involved in the metabolism of many clinically important therapeutic agents. Here we describe a patient who was intolerant of warfarin treatment because of impaired drug metabolism related to the CYP2C9*3/*13 genotype and high warfarin sensitivity associated with the VKORC1 1173TT genotype. Case: A 64-year-old Korean man had taken warfarin for treatment of an occluded left subclavian artery and atrial fibrillation. Although the warfarin doses prescribed were not high (14 mg/week) during the induction of anticoagulation, the prothrombin time (PT) was elevated to over 120 s after two weeks of warfarin therapy. For this patient, a very low dose of warfarin (4 mg/week) was required in order to achieve target INR values. Conclusions: To the best of our knowledge, this is the first report of a patient with CYP2C9*3/*13 and VKORC1 1173TT genotypes who had slower than normal warfarin metabolism, resulting in the need to administer an extremely low dose of warfarin in order to achieve the target INR value. Our report reinforces the relevance of pharmacogenetic testing to explain warfarin dose variability and to enable individualized dosage adjustments for improved warfarin treatment outcomes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Warfarin is an oral anticoagulant commonly used for the treatment and prevention of arterial and venous thrombosis. Warfarin has a narrow therapeutic range, as its dose requirement has wide intra- and inter-individual variability, which makes prescribing the correct dosage problematic [1]. This variability is influenced by gender, age, body mass index, smoking, diet, comorbid conditions, drug interactions and genetic factors. Pharmacogenetics refers to the study of genetic background to predict inter-individual variability related to drug responsiveness and effectiveness, and to predict the risk of adverse effects from medications [2]. Polymorphisms in genes encoding CYP2C9, a member of the drug metabolizing cytochrome P450 subfamily, and the vitamin K epoxide

⁎ Correspondence to: J.S. Kim, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, Republic of Korea, 135-710. Tel.: + 82 2 3410 3414; fax: + 82 2 3410 0031. ⁎⁎ Correspondence to: S.Y. Lee, Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, Republic of Korea, 135-710. Tel.: +82 2 3410 1834; fax: +82 2 3410 2719. E-mail addresses: [email protected] (J.S. Kim), [email protected] (S.-Y. Lee). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.06.040

reductase complex subunit 1 (VKORC1) account for the variable therapeutic effects of warfarin [3]. To date, at least 34 allelic variants of CYP2C9 have been reported [4]. Two allelic variants, CYP2C9*2 (p.Arg144Cys) and CYP2C9*3 (p.Ile359Leu), are associated with enhanced sensitivity to warfarin therapy, and patients carrying these mutations often require reduced doses of warfarin during induction because of impaired drug metabolism [5]. We herein describe a patient with the CYP2C9*3/*13 and VKORC1 1173TT genotype who demonstrated an intolerance to a usual warfarin dosing pattern. 2. Clinical case The patient was a 64-year-old Korean man, who was referred to our hospital because of cyanotic color changes to his left second and fifth fingertips, accompanied by tingling in his left arm. The patient's past medical history was significant and included a cerebral infarct, controlled diabetes mellitus, and hypercholesterolemia. His medication regimen during the previous six months included daily ginkgo biloba leaf extract (80 mg), nicergoline (10 mg), metformin (500 mg), fenofibate (100 mg), and celecoxib (200 mg), in addition to twice daily acetaminophen (650 mg). Physical examination revealed a blood pressure of 159/98 mmHg in the left arm and 125/98 mmHg in the

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right arm and a heart rate of 96 beats/min. His body weight was 71.6 kg, and his height was 169.6 cm. The initial laboratory results were unremarkable, with a normal prothrombin time (PT) of 12.8 s (s) and a baseline international normalized ratio (INR) of 0.97. Results of routine chemical studies, hematological studies and thyroid function tests indicated normal values. An electrocardiogram verified atrial fibrillation and left atrial enlargement (45 mm). Computed tomographic (CT) angiography and duplex scans detected a segmental occlusion in the left subclavian artery area of approximately 35 mm and a mild atherosclerotic change in the proximal internal carotid artery. Based on these findings, we started the patient on oral anticoagulant therapy at 4 mg/day. Seven days later, the patient exhibited PT prolongation to 45.7 s, with an INR of 4.45. The warfarin dose was then reduced to 2 mg/day. Two weeks later, the patient was seen again at an outpatient clinic, where his PT was over the upper limit of the clinical reportable range (N120 s) in our laboratory and his INR was expected to be higher than 16.0. Warfarin was discontinued at that time, and 10 mg of phytonadione (vitamin K activity) was administered. Three days later, his PT had stabilized and the patient was admitted for a left common carotid artery to left axillary artery bypass, using an ipsilateral and reversed greater saphenous vein. The patient was heparinized with an initial bolus of 2500 IU heparin followed by a continuous heparin infusion during the operation. On the third postoperative day, the patient was restarted on warfarin at 2 mg/day, at which point his PT was 22.3 s, with an INR of 1.91. On the seventh postoperative day, his INR increased to 2.64, and we then determined the plasma concentrations of warfarin and 7-hydroxywarfarin to be 0.645 μg/mL (reference range, 0.420–3.310 μg/mL) [6] and 0.015 μg/mL, respectively, with a ratio of warfarin to 7-hydroxywarfarin of 43. The patient's warfarin treatment was then reduced even further to an alternating dose of 0.5 mg/day to 1 mg/day, which resulted in a PT range of 21.7–27.4 s (INR 1.84–2.45). After ten postoperative days, the patient's vitamin K level in the serum was close to its upper limit at 2.45 ng/mL (reference range, 0.2–2.7 ng/mL) on warfarin treatment of 1 mg/day. At this point, his PT was 26.6 s with an INR of 2.36. The difficulty in finding the optimal treatment dose during the induction period of anticoagulation continued for more than three weeks. The patient has continued to receive warfarin therapy at an outpatient clinic, and his PT has remained within the recommended target therapeutic range (INR 2–3), despite a total warfarin dose of only 4.0 mg/week. Direct sequencing analysis of the patient's CYP2C9 gene revealed two different genetic variations. The first was a T-to-C substitution at nucleotide position 269 in exon 2, which replaces leucine with proline at codon 90 (c.269 T N C, p.Leu90Pro). The second was an A-to-C substitution at nucleotide position 1075 in exon 7, which replaced isoleucine with leucine at codon 359 (c.1075A N C, p.Ile359Leu). No other sequence variations were observed. The patient was a poor warfarin metabolizer, with a genotype of CYP2C9*3/*13 (Fig. 1). In addition, a direct sequence analysis of the VKORC1 gene revealed a 1173 TT polymorphism. This is the first report of a patient with the

CYP2C9*3/*13 and VKORC1 1173 TT genotypes who demonstrated warfarin intolerance. 3. Discussion This is the first report of a patient with the CYP2C9*3/*13 and VKORC1 1173TT genotype who demonstrated warfarin intolerance. The patient did not experience any bleeding events, and his over anticoagulation was discovered in the analysis of his pharmacodynamic effects (PT N120 s; INR N 16.0) during the induction period, while the patient was taking not high warfarin dose (14 mg/week) for treatment induction. In addition, this patient did not present with any comorbid diseases such as heart failure, liver disease, renal insufficiency, hypothyroidism, or hyperthyroidism. When this patient's chart was thoroughly reviewed, it was noted that his treatment regimen included some medications that could potentially increase the oral anticoagulant effect of warfarin when taken concurrently, including acetaminophen, fenofibrate, and celecoxib [7]. We could not identify a change of the warfarin requirement when these drugs were stopped in our patient, because the clinicians did not discontinue these drugs. However, we could infer that the effects of these drugs were not a major cause for the difficultly in metabolizing warfarin because the usual increase in anticoagulation in the presence of these drugs is about 13%–29% [8–10]. In this patient, the daily maintenance dose of warfarin required to achieve a target INR range was 0.56 mg (4.0 mg/week), which is an approximately a 70% reduction in the usual dose. The patient's ratio of warfarin to 7-hydroxywarfarin was 43, which is close to the upper limit when compared with other wafarinized patients' ratios of 6.8 to 57.3 [6], despite the fact that the patient was not taking a high warfarin dose (2 mg/day). We suspect that the warfarin metabolism was impaired in this patient. In order to determine if the patient's over anticoagulation might be related to genetic alteration involved in drug metabolism, we performed sequencing analysis of CYP2C9 and VKORC1. At present, clinicians typically adjust warfarin doses based on PT INR and clinical response. Recently, pharmacogenetic research has explored the relationship between genotypes and the appropriate doses of oral anticoagulants in hopes that we might be able to optimize therapy by reducing the amount of time needed to achieve effective anticoagulation [11]. Moreover, in August 2007, the US Food and Drug Administration updated the label information for warfarin to encourage the use of genetic information in initiating warfarin therapy. Although the CYP2C9 allele designated CYP2C9*13 has been identified in Chinese subjects who were poor metabolizers of lornoxicam [12] and losartan [13], there have been no reports about the effects of this variant in warfarin therapy. Results of kinetic experiments showed that CYP2C9*13 had a lower intrinsic rate of clearance of lornoxicam 5′-hydroxylation than CYP2C9*3 and CYP2C9*1, because of an increase in the Michaelis–Menten constant (Km) (2.79 vs.

Fig. 1. Direct sequencing analysis detects nucleotide changes in the CYP2C9 gene in a patient with CYP2C9*3/*13 showing p.Leu90Pro and p.Ile359Leu substitutions (indicated by arrows).

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1.61 and 1.24 μM, respectively), and a decrease in its maximal reaction velocity (Vmax) (0.22 vs. 0.28 and 0.83 pmol/min/pmol, respectively) [14]. In this way, the CYP2C9*13 allele, which has lower activity than the wild type allele, is associated with an increased risk of adverse drug events. Allele frequencies of CYP2C9 and VKORC1 genotypes have been shown to be markedly different among global populations [15]. Only five CYP2C9 genotypes, including CYP2C9*1/*1, *1/*3, *1/*13, *1/*14, and *3/*4, have been observed to date in the Korean population [16–18]. The allele frequency of CYP2C9*1/*13 was reported to be 0.6% in 358 healthy Korean subjects [16] and 0.4% in 265 Korean patients with mechanical heart valve replacement [18]. However, there have been no previous reports of subjects identified as compound heterozygotes for the CYP2C9*3/*13 allele with the associated clinical implications. Our patient was a compound heterozygote for the mutant allele, which likely resulted in a substantially reduced catalytic efficiency of the translated product. In addition, patients with the 1173 TT genotype for the VKORC1 gene require lower daily doses of warfarin than those carrying the CT or CC genotypes. To the best of our knowledge, this is the first report of a patient with the CYP2C9*3/*13 and VKORC1 1173TT genotypes who had a slower than normal warfarin metabolism, resulting in the need for an extremely low dose of warfarin in order to achieve the target INR value. Our study reveals the functional and clinical significance of these CYP2C9 and VKORC1 genotypes in warfarin therapy. In addition, our report confirms the relevance of pharmacogenetic testing to explain warfarin dose variability among different patients and to enable individualized dosage adjustments for improved of warfarin treatment outcomes. Acknowledgements This study was supported by grants from the Korean Ministry of Education, Science and Technology, FPR08A2-130 of the 21C Frontier Functional Proteomics Program, the Korea Healthcare technology R&D Project, Ministry of Health, Welfare & Family Affairs, Republic of Korea (A070001) and the Samsung Medical Center Research Development program CRS110-54-2.

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