Kathryn M. Momary

Regulatory polymorphism in vitamin K epoxide reductase complex subunit 1 (VKORC1) affects gene expression and warfarin dose requirement

Warfarin dose requirements have been associated with 2 main haplotypes in VKORC1, but the responsible polymorphisms remain unknown. To search for regulatory polymorphisms, we measured allelic mRNA expression of VKORC1 in human liver, heart, and B lymphocytes. The observed 2-fold allelic mRNA expression imbalance narrowed possible candidate SNPs to -1639G>A and 1173C<T. This genotype effect was observed selectively in the liver but not in heart or lymphocytes. In vitro expression of VKORC1 gene constructs, including coding and promoter regions, failed to reveal any genotype effect on transcription and mRNA processing. Chromatin immunoprecipitation with antibodies against acetyl-histone3 and K4-trimethyl-histone3 revealed preferential association of the promoter -1639 G allele with active chromatin, consistent with enhanced mRNA expression. The minor -1639 A allele generates a suppressor E-box binding site, apparently regulating gene expression by a mechanism undetectable with reporter gene assays. A clinical association study demonstrated that promoter SNP -1639G>A, and the tightly linked intron1 SNP 1173C>T, predict warfarin dose more accurately than intron 2 SNP 1542G>C in blacks. Increased warfarin dose requirement in blacks was accounted for by lower frequency of the -1639 A allele. Therefore, -1639G>A is a suitable biomarker for warfarin dosing across ethnic populations.

Genetic and Clinical Predictors of Warfarin Dose Requirements in African Americans

The objective of this study was to determine whether, in African‐American patients, additional vitamin K oxidoreductase complex subunit 1 (VKORC1), cytochrome P450 2C9 (CYP2C9), CYP4F2, or apolipoprotein E (APOE) polymorphisms contribute to variability in the warfarin maintenance dose beyond what is attributable to the CYP2C9*2 and *3 alleles and the VKORC1 −1639G>A genotype. In a cohort of 226 African‐American patients, weekly warfarin dose requirements were lower in those with the CYP2C9*8 allele (34 (30–47) mg; P = 0.023) and the CYP2C9 *2, *3, *5, *6, or *11 allele (33(28–40 mg); P < 0.001) as compared with those with the CYP2C9*1/*1 genotype (43 (35–56) mg). The combination of CYP2C9 alleles, VKORC1 −1639G>A genotype, and clinical variables explained 36% of the interpatient variability in warfarin dose requirements. By comparison, a model without the CYP2C9*5, *6, *8, and *11 alleles explained 30% of the variability in dose. No other VKORC1, CYP4F2, or APOE polymorphism contributed to the variance. The inclusion of additional CYP2C9 variants may improve the predictive ability of warfarin dosing algorithms for African Americans.

Factors influencing warfarin dose requirements in African–Americans

INTRODUCTION African-Americans are under-represented in studies assessing contributors to warfarin response. Our primary objective was to determine whether the genes for cytochrome P450 (CYP) 2C9, nicotinamide adenine dinucleotide phosphate, reduced, quinone oxidoreductase (NQO1) and vitamin K epoxide reductase complex subunit 1 (VKORC1) are associated with warfarin dose requirements in African-Americans. PATIENTS AND METHODS The following factors were assessed: demographics; clinical data; the CYP2C9 Arg144Cys (*2), Ile358Leu (*3) and Asp360Glu (*5); NQO1 Pro187Ser (*1/*2); and VKORC1 G6853C genotypes were analyzed in 115 African-Americans on stable warfarin doses. RESULTS Allele frequencies were 0.05 for the CYP2C9 *2, *3 or *5 alleles; 0.20 for NQO1 *2; and 0.25 for VKORC1 6853C. Possession of a CYP2C9*2, *3 or *5 allele was associated with a 38% lower warfarin dose compared with the *1/*1 genotype (30 +/- 13 vs 48 +/- 18 mg/week; p = 0.003). Neither the NQO1 *1/*2 nor VKORC1 G6853C genotype was associated with warfarin dose requirements in the population as a whole or in CYP2C9*1 allele homozygotes. Multiple regression analysis revealed that CYP2C9 genotype (p = 0.015), age (p < 0.001) and body surface area (p < 0.001) were jointly associated with warfarin dose requirements, and together explained 33% of the variability in warfarin dose requirements among African-Americans. DISCUSSION Our data suggest that CYP2C9 genotype, age and body size are important determinants of warfarin dose requirements in African-Americans. Our data further suggest that the VKORC1 G6853C polymorphism alone may not be useful for predicting warfarin dose requirements in this racial group.

Genetic Causes of Clopidogrel Nonresponsiveness: Which Ones Really Count?

Clopidogrel decreases the morbidity and mortality associated with several cardiovascular diseases. However, clopidogrel is a prodrug that needs to be metabolized to the active thiol metabolite by the cytochrome P450 (CYP) system. This activation is a source of significant interindividual variability in clopidogrel responsiveness. Drug interactions with and genetic variation in CYP3A4, CYP3A5, and CYP2C19 enzymes have been implicated in decreasing active metabolite production. In addition, polymorphisms in the genes encoding P‐glycoprotein (an efflux transporter) and purinergic receptor P2Y12 (the active site for clopidogrel) have been studied for their role in clopidogrel responsiveness. Several large studies have recently assessed the role of genetic variation in clopidogrel responsiveness as characterized by clinical outcomes. In this review, we summarize the genetic causes of clopidogrel nonresponsiveness, with a focus on larger outcomes‐based studies. A MEDLINE search of the English‐language literature (1990–2008) was conducted to identify studies that examined these relationships; additional citations were obtained from the articles retrieved from the literature search. Polymorphisms in CYP2C19 and, to a lesser extent, the adenosine 5`‐triphosphate‐binding cassette gene, ABCB1, contribute to variability in clopidogrel responsiveness. Specifically patients possessing at least one variant CYP2C19 allele (CYP2C19* 2,* 3) have impaired clopidogrel responsiveness due to decreased formation of the active metabolite. In addition, one study found that considering ABCB1 genotype in addition to CYP2C19 allowed better prediction of clopidogrel nonresponsiveness. However, routine genotyping for CYP2C19 or ABCB1 polymorphisms in order to predict clopidogrel responsiveness cannot be recommended at this time because of logistic and cost considerations.

Pharmacogenomics of Warfarin dose requirements in Hispanics

While Hispanics are the largest and most rapidly growing minority population in the United States, they are underrepresented in pharmacogenomic studies with warfarin. We sought to determine the combination of clinical and genetic influences of warfarin dose requirements in Hispanics. In addition, we tested the performance of published warfarin dosing algorithms derived from largely non-Hispanic cohorts in an inner-city U.S. Hispanic population. Genetic samples and clinical data were obtained from 50 Hispanics on a stable dose of warfarin. The contribution of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex-1 (VKORC1) genotypes and clinical factors to warfarin dose requirements was determined. The correlation between the predicted dose using published algorithms and therapeutic dose was also assessed. Compared to the VKORC1-1639 GG genotype, warfarin dose requirements were 30% and 62% lower with the GA and AA genotypes, respectively (p=0.001). The combination of the VKORC1-1639G>A and CYP2C9 genotypes and clinical factors explained 56% of the inter-patient variability in warfarin dose. Warfarin dose predicted using algorithms derived from mostly non-Hispanic cohorts was significantly correlated with the therapeutic dose in our Hispanic cohort (r(2)=0.43 to 0.49; p<0.001); the predicted dose was within 1.0 mg/day of the therapeutic dose for 40% to 50% of patients. Our data suggest that factors influencing warfarin dose requirements in Hispanic Caucasians are similar to those previously described in European Caucasians and that dosing algorithms derived from non-Hispanic Caucasian cohorts are applicable to Hispanics living in the U.S.

A comparison of students' self-assessments with faculty evaluations of their communication skills.

Objective. To compare students’ self-assessment of their communication skills with faculty members’ formal evaluation of their skills in a therapeutics course. Methods. Over a 3-year period, faculty members evaluated second-year pharmacy students’ communication skills as part of a requirement in a therapeutics course. Immediately following an individual oral assessment and again following a group oral assessment, students self-assessed their communication skills using the same rubric the faculty members had used. Students’ self-assessments were then compared with faculty members’ evaluation of students’ communication skills. Results. Four hundred one (97.3%) students consented to participate in this study. Faculty evaluation scores of students for both the individual and group oral assessments were significantly higher than students’ self-assessment scores. Students’ self-assessment scores of their communication skills increased from the individual to the group oral assessment. Conclusion. Students’ self-assessments of communication skills were consistently lower than faculty members’ evaluations. Greater use of oral assessments throughout the pharmacy curriculum may help to improve students’ confidence in and self-assessment of their communication skills.

Effect of Combination Nicotinic Acid and Gemfibrozil Treatment on Intermediate Density Lipoprotein, and Subclasses of Low Density Lipoprotein and High Density Lipoprotein in Patients With Combined Hyperlipidemia

The goal of this study was to determine, using analytic ultracentrifugation, the effect of nicotinic acid alone or nicotinic acid added to gemfibrozil on lipoprotein subclass distribution, including intermediate-density lipoprotein (IDL; low-density to very low density flotation rate [S(f)] 12 to 20); low-density lipoprotein (LDL) subfractions LDL-I (S(f) 7 to 12), LDL-II (S(f) 5 to 7), LDL-III (S(f) 3 to 5), and LDL-IV (S(f) 0 to 3); and high-density lipoprotein (HDL) subfractions HDL(2) (high-density flotation rate 3.5 to 9.0) and HDL(3) (high-density flotation rate 0 to 3.5). Patients with combined hyperlipidemia were randomized to nicotinic acid (1,500 mg/day) plus placebo or nicotinic acid plus gemfibrozil (1,200 mg/d) for 12 weeks. Baseline characteristics were similar between the 2 groups, and mean LDL cholesterol (180 +/- 33 mg/dl) and triglycerides (310 +/- 126 mg/dl) were typical for patients with combined hyperlipidemia. Treatment with nicotinic acid resulted in a reduction in dense LDL (S(f) 5 to 7; p = 0.02), which was counterbalanced by an increase in buoyant LDL (S(f) 7 to 12; p = 0.03) that resulted in no significant LDL mass or LDL cholesterol change. IDL was reduced (p = 0.005) and HDL(2) increased by 143% (p = 0.004). The combination of nicotinic acid and gemfibrozil resulted in a further 17.8% reduction in apolipoprotein B (p = 0.06), a further 33.8% reduction in IDL (p = 0.06), and a greater reduction in the apolipoprotein B/apolipoprotein A-I ratio (p = 0.02). The combination of nicotinic acid and gemfibrozil reduced atherogenic by IDL 71%, dense LDL-III by 52%, and apolipoprotein B by 37% and increased protective HDL(2) by 90%. In conclusion, this investigation revealed that a combination of a fibric acid derivative and nicotinic acid offers greater improvement in detailed lipoprotein subclass distribution and apolipoprotein ratios than monotherapy.

Factors associated with clopidogrel nonresponsiveness

Clopiodgrel therapy is the standard of care in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention. However, there is a significant amount of interindividual variability in clopidogrel responsiveness. Clopidogrel is a prodrug and requires metabolism via several CYP450 enzymes in order to exert its antiplatelet effects. Interference in these activation steps is the primary cause of clopidogrel nonresponsiveness. This review focuses on genetic polymorphisms in, and drug interactions with, the CYP450 enzymes that are associated with clopidogrel nonresponsiveness. In addition, clinical factors that effect clopidogrel responsiveness are also reviewed. Particular emphasis is placed on those factors that are not only associated with a change in clopidogrel pharmacokinetics or pharmacodynamics, but are also associated with an increased risk of adverse cardiovascular events. Currently, the majority of data assessing clopidogrel nonresponsiveness focus on genetic variation in CYP2C19 and drug interactions with proton pump inhibitors. However, genetic variation in other CYP450 enzymes, other drug interactions and clinical causes have been studied and are also reviewed here. It is important for healthcare practitioners to recognize all of the causes of clopidogrel nonresponsiveness, especially as novel antiplatelet alternatives become available.

Cytochrome P450 2C9 mediated metabolism in people with and without cancer.

OBJECTIVES To compare cytochrome P450 activity in people with and without cancer and examine the relationship between CYP2C9 activity and serum cytokine levels. PATIENTS AND METHODS 10 subjects with cancer who were currently receiving treatment and 10 additional subjects without cancer who were matched to the subjects with cancer based on gender and race were enrolled into the study. Serial blood samples were drawn to measure tolbutamide in the plasma before and after oral tolbutamide 500 mg. Total urine excreted was collected from 0 to 12 h following the dose. Tolbutamide and its metabolites were measured in plasma and urine by HPLC. CYP2C9 genotype was determined by PCR and pyrosequencing and cytokine values were determined by ELISA. RESULTS The mean apparent oral clearance (cancer, 19.5 +/- 10.5 vs. non-cancer, 15.8 +/- 5.0 ml/min) and the mean urinary metabolic ratio from 0 to 12 h were similar (838 +/- 693 vs. 775 +/- 390). Neither age nor genotype statistically affected the outcomes. Mean interleukin-6 (7.2 +/- 9.4 vs. 1.5 +/- 1.3 pg/ml) and tissue necrosis factor-a (26.2 +/- 71.2 vs. 1.5 +/- 1.3 pg/ml) were 5- to 7-fold higher, respectively, in subjects with cancer. No statistically significant correlation between cytokine values and oral clearance or urinary metabolic ratio was found. CONCLUSIONS CYP2C9 activity as measured by apparent oral clearance and urinary metabolic ratio following oral tolbutamide appear similar in people with and without cancer. Serum cytokine values appear higher in patients with cancer, although the differences did not reach statistical significance.

Genetic Testing for Early Detection of Individuals at Risk of Coronary Heart Disease and Monitoring Response to Therapy: Challenges and Promises

Coronary heart disease (CHD) often presents suddenly with little warning. Traditional risk factors are inadequate to identify the asymptomatic high-risk individuals. Early identification of patients with subclinical coronary artery disease using noninvasive imaging modalities would allow the early adoption of aggressive preventative interventions. Currently, it is impractical to screen the entire population with noninvasive coronary imaging tools. The use of relatively simple and inexpensive genetic markers of increased CHD risk can identify a population subgroup in which benefit of atherosclerotic imaging modalities would be increased despite nominal cost and radiation exposure. Additionally, genetic markers are fixed and need only be measured once in a patient’s lifetime, can help guide therapy selection, and may be of utility in family counseling.