Perspectives in Pharmacology Building Individualized Medicine: Prevention of Adverse Reactions to Warfarin Therapy

Warfarin is the most widely used oral anticoagulant in the world for patients with venous thrombosis, pulmonary embolism, chronic atrial fibrillation, and prosthetic heart valves. Approximately 30 genes contribute to therapeutic effects of warfarin, and genetic polymorphisms in these genes may modulate its anticoagulant activity. In contrast to monogenic pharmacogenetic traits, warfarin drug response is a polygenic trait, and development of diagnostic tools predictive of adverse reactions to warfarin requires a novel approach. A combination of two strategies, biochemical isolation of allelic variants and linkage disequilibrium association studies, was used to find an association between genetic polymorphisms in the candidate genes and warfarin response. A strong association was found between genetic polymorphisms in six genes, including VKORC1, CYP2C9, PROC, EPHX1, GGCX, and ORM1, and interindividual variability in the anticoagulant effect of warfarin; the strongest predictors were VKORC1 and CYP2C9. Generation of single nucleotide polymorphism (SNP)-based dense genetic maps made it possible to identify haplotypes associated with drugresponse phenotypes. Discrimination between haplotypes associated with warfarin dose phenotypes can be achieved by a limited set of informative polymorphisms (tag SNPs). The use of tag SNPs in pharmacogenomic analysis provides a promising tool for dissecting polygenic traits of drug response. The human genome is variable both within generations (more than 7 million SNPs with a minor allele frequency at least 5%) and between generations (175 mutations per diploid genome per generation) (Kruglyak and Nickerson, 2001). Genetic variations make us unique in many senses, including our response to drug therapy. Pharmacogenomics uses the tools of human genetics to tailor medicinal treatment to an individual’s genetic makeup. To this end, phenotypic manifestations (a therapeutic outcome or an adverse drug event, ADE) are considered in relation to the underlying genetic background of a patient. In a pregenome era, these studies were based on biochemical isolation and characterization of drug-metabolizing enzymes and their genes, followed by sequence and functional analysis of possible mutant variants. Characterization of mutations led to development of a genotyping assay that was used to perform genetic analysis of patient DNA. Initial achievements of pharmacogenomics were related to detection of simple monogenic traits, e.g., abrogation of drug metabolism due to inactivation of a single enzyme. Characterization of inactivating mutations in arylamine-N-acetyltransferase 2, cytochrome P450 2D6, and thiopurine S-methyltransferase provided molecular mechanisms of adverse drug events caused by izoniazid, debrisoquine, and mercaptopurine (Gonzalez et al., 1988; Vatsis et al., 1991; Krynetski et al., 1995). Biochemical analysis does not rely on statistical evaluation of genetic markers and therefore works equally well for common and rare genotypes. Such functional studies require extensive knowledge of the biotransformation, transport, and physiological activity of the drug in question. In contrast, linkage disequilibrium (LD) association studies rely on dense maps of human genome where nearly every human gene and genomic region is marked by a sequence variation. With development of massive parallel methods for SNP detection, linkage analysis and association studies are increasingly used to effectively characterize complex polyArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.117952. ABBREVIATIONS: SNP, single nucleotide polymorphism; LD, linkage disequilibrium; ADE, adverse drug event(s); INR, international normalized ratio; VKOR, vitamin K epoxide reductase; ORM, orosomucoid. 0022-3565/07/3222-427–434$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 322, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 117952/3231400 JPET 322:427–434, 2007 Printed in U.S.A. 427 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from genic traits of drug response. The association studies make use of statistical analysis of genetic markers (e.g., SNPs) in large groups of patients stratified according to their dose requirements. This approach uses genetic markers rather than functional polymorphisms and therefore does not provide information about possible mechanisms behind alterations in drug response. Instead, it relies on the assumption that a mutant allele is to be correlated with an allele of an assayed SNP. Ideally, both approaches should result in identification of the same set of functionally important polymorphisms, which in reality is rarely achieved. Whereas functional studies proved to be efficient in analysis of single genes, association studies hold great promise in identifying multiple alleles contributing to polygenic traits. A clinically important example for these two strategies has been provided by recent dissection of warfarin-pharmacogenomic determinants. Metabolism, transport, and pharmacological effects of warfarin are determined by interplay of approximately 30 genes, and genetic analysis of this pharmacological pathway is still in progress (Wadelius and Pirmohamed, 2006). Warfarin is the most widely used oral anticoagulant in the world for patients with venous thrombosis, pulmonary embolism, chronic atrial fibrillation, and prosthetic heart valves (Wadelius and Pirmohamed, 2006). The therapeutic index for individual patients is narrow; therefore, patients are closely monitored by international normalized ratio (INR) for prothrombin time. Clinical use of warfarin is complicated due to variations in individual response, and prescribed doses may vary 20-fold or more because dose requirements, the stability of anticoagulation, and risk of bleeding are determined by a complex combination of environmental and genetic factors. Whereas nongenetic factors, such as intake of vitamin K, disease, age, gender, concurrent medications, and body surface area, have been taken into consideration by clinicians, the genetic analysis still remains an underused tool, and clinicians must be further educated on how their patients can benefit from timely recognition of pharmacogenetic variations (Krynetskiy and Evans, 2004). Recent progress in elucidating pharmacogenetic variables contributing to variance in warfarin dose made it practical to implement genotyping of patients before beginning warfarin therapy (Geisen et al., 2005; Voora et al., 2005). A rational selection of genetic markers for genotyping poses a problem for applied pharmacogenomics, and the choice of informative polymorphisms predictive of altered warfarin pharmacokinetics or pharmacodynamics still remains to be a matter of discussion. The current article aims to describe available genotyping assays for evaluating patients’ genotype as a risk factor in warfarin treatment-related complications. Warfarin Therapy and Clinical Complications An ADE is any undesired and harmful effect of a drug that usually falls in one of two categories: 1) adverse drug reactions (ADR) defined as “any response to a drug that is noxious and unintended and occurs at doses normally used in man for the prophylaxis, diagnosis, or therapy of diseases” (World Health Organization, 1969); or 2) therapeutic failure, i.e., the failure to achieve the desired therapeutic outcome, with an event that can harm the patient. The goal of warfarin therapy is to administer the lowest possible dose of anticoagulant to prevent clot formation or expansion while trying to avoid unintended ADE from overanticoagulation. Warfarin-related ADR result in unintended hypoprothrombinemia, with or without frank hemorrhage. ADE are common with warfarin, thus being a leading cause of drug-induced hospital admissions (McDonnell and Jacobs, 2002). The incidence of these ADR ranges from 6 to 39% of warfarin patients annually and is directly related to the intensity of anticoagulation (Levine et al., 1995). By contrast, therapeutic failure of warfarin treatment is an event that produces unintended thromboembolism due to suboptimal anticoagulation in these patients. Warfarin has both anticoagulant and antithrombotic activity. The anticoagulant activity of warfarin depends on the clearance of functional clotting factors from the systemic circulation. The clearance is determined by half-lives of the clotting factors, with earliest changes in prothrombin time (24 to 26 h after warfarin administration) due to the clearance of factor VII (half-life of approximately 6 h). Early changes in prothrombin times do not reflect the full antithrombotic effects of warfarin, which are achieved by the 5th day in most patients after the functional clearance of prothrombin (half-life of approximately 50 h) (Hirsh et al., 1995). Therefore, the maximal anticoagulant effect is achieved after 72 to 96 of exposure. Pharmacogenomic interpatient differences in warfarin response are an important group of factors that need to be considered for warfarin dosing. Mutations in genes involved in the therapeutic effect of warfarin cause two distinct phenotypes—increased risk of hypoprothrombinemia and warfarin resistance. In many cases, genotype can be a critical variable in determining the optimal dosing to achieve anticoagulant and antithrombotic effects (Voora et al., 2005). Molecular Basis for Warfarin-Induced Adverse Drug Reactions The biochemical basis of warfarin action is reasonably well understood (Fig. 1). The anticoagulant effect of warfarin is achieved through inhibition of vitamin K epoxide reductase (VKOR) activity, resulting in a decrease of the reduced form of vitamin K. The deficiency of the reduced form of vitamin K results in abrogation of post-translational modification of the blood coagulation components, including clotting factors II, VII, IX, and X; proteins C, S, and Z; and several other proteins, thus reducing their activity (Rost et al., 2005). Unintended hypoprothrombinemia in a genetically predisposed subpopulation of patients most often originates from reduced metabolic inactivation of warfarin by CYP2C9 or decreased expression of the drug target (VKOR). CYP2C9 is the major isoform of human liver cytochromes P450 that modulates the physiological effect of warfarin. Low rate of oxidative metabolism catalyzed by CYP2C9 hypomorphic alleles *2 and *3 results in slow metabolic inactivation of warfarin, which in turn leads to undesired hypoprothrombinemia (Daly and King, 2003; Palkimas et al., 2003). The decrease in CYP2C9 activity is most often caused by the nonsynonymous SNPs present in CYP2C9*2 (rs1799853: T; R144C) and CYP2C9*3 (rs1057910: C; I359L). The reduced activity of CYP2C9*2 mutant (R144C) may reflect its diminished affinity for cytochrome P450 reductase, which interacts with the positively charged residues on the surface of 428 Krynetskiy and McDonnell at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from