AIMS: Warfarin anticoagulant drug therapy poses a challenge to clinicians due to its narrow therapeutic range and interpatient variability. This study represents the first pharmacogenomic (multiple gene) approach to investigate the genetic causes of variable warfarin sensitivity. The aims of this study are to:
1. Determine if genetic polymorphisms in the genes encoding the coagulation factors VII and X are associated with variable warfarin maintenance dose requirements.
2. Determine if CYP2C9 metabolizing enzyme polymorphisms, along with coagulation polymorphisms provides a better predictor of warfarin dose requirements than CYP2C9 polymorphisms alone.
3. Determine the contribution of non-genetic factors to warfarin dose requirements.

STUDIES AND RESULTS: Patients on a stable weekly maintenance dose of warfarin will be recruited from the Shands at the University of Florida and Gainesville VA Medical Center Anticoagulation Clinics. Patients must be on a stable weekly maintenance dose of warfarin for a minimum of three consecutive clinic visits over a time period of at least eight weeks. Maintenance dose requirements (expressed as mg/week) cannot vary by more than 10% and INR values associated with each visit must be within goal range. Patients will be excluded from the study if they have liver cirrhosis, advanced malignancy, hospitalization within the previous four weeks of the index visit, or a febrile/diarrheal illness within two weeks of their visit. A total of four hundred patients will be enrolled in the study. After signing informed consent, each patient will be asked to provide a genetic sample by swishing and then expectorating mouthwash to provide a buccal sample. Following collection of the genetic sample, a brief interview will be done to assess for non-genetic factors that may impact warfarin dose requirements. Genomic DNA will be isolated from each mouthwash sample using a commercially available kit (Gentra PuregeneÒ). Resulting DNA concentrations will be quantitated by a standard spectrophotometric method.

Specific Aim 1: Genotypes will be determined for two polymorphisms in the Factor VII gene. These include a single nucleotide polymorphism resulting in a substitution of glutamine for arginine at position 353 in the catalytic domain (R353Q), and a 10-bp insertion in the promoter region of the Factor VII gene. Genotypes will also be determined for two polymorphisms between nucleotides –343A and –342G. Single nucleotide polymorphisms in the Factor VII and Factor X genes will be determined by PCR with a single primer extension method (96-well plate kit from Orchid BioSciencesÒ). Insertion/deletion polymorphisms will be determined with PCR and electrophoretic separation on an 8% polyacrylamide (PAGE) gel.

Specific Aim 2: Genotypes will be determined for two single nucleotide polymorphisms in the CYP2C9 metabolizing enzyme. These include the CYP2C9*2 variant in which cysteine substitutes arginine at position 144, and the CYP2C9*3 variant in which leucine substitutes isoleucine at position 359 of the gene. Genotypes for CYP2C9*2 and CYP2C9*3 will be determined by PCR with a single primer extension method.

Specific Aim 3: Patients will be asked about concomitant medications and herbal products that they have taken during the past three visits. They will also be asked for information regarding the number of servings of vitamin K foods they eat each week, with specific emphasis on foods that are high in vitamin K. Concomitant drug therapy will be given a rank score on the relative impact each drug has on warfarin’s therapeutic effect. Dietary intake of vitamin K will also be given a rank score based on published nutritional information on the vitamin K content in various foods in the Factor X gene. These include a single nucleotide polymorphism at position –40 in the promoter region along with a hexanucleotide insertion.

SIGNIFICANCE: A pharmacogenomic approach is necessary to better understand the impact of genetic variation on warfarin sensitivity. Investigations of multiple gene polymorphisms allow us to take into account the complex interaction of proteins and enzymes involved in warfarin’s pharmacologic action. This study addresses the genetic variability in both drug targets and drug metabolizing enzymes, while at the same time incorporating the contribution of non-genetic factors to the overall variability in response. This study will provide the necessary foundation for future prospective studies in which a patient’s genetic makeup may be one factor utilized in the selection of an appropriate warfarin dose.

TRAINING ACTIVITIES: Over the course of the next year, I will gain extensive experience in a variety of different molecular biology techniques. These include isolation of genomic DNA, amplification of DNA by the polymerase chain reaction (PCR), vertical and horizontal gel electrophoresis, and determination of genotype by both single primer extension methods and restriction fragment length polymorphism (RFLP). I will also have active involvement in biweekly cardiovascular and clinical pharmacology journal clubs that serve to provide continual insight into hypothesis generation and study design. I will also have the opportunity to participate in ongoing molecular biology seminars given by the Interdisciplinary Center for Biotechnology Research at the University of Florida, along with seminars hosted by the UF Center for Pharmacogenomics. I have recently completed courses in the fundamentals of research design, analysis of biomedical research data, and pharmacokinetics/biopharmaceutics. Julie A. Johnson, Pharm.D. will serve as my mentor and will have primary responsibility for all aspects of the fellowship program. Taimour Langaee, Ph.D. will be responsible for teaching and assisting me on different molecular biology techniques. Statistical support will be provided by Susan P. McGorray, Ph.D.

PUBLICATIONS, POSTERS, AND LECTURES: N/A

BRIAN OVERHOLSER
Pharmacodynamics Fellow
Purdue University College of Pharmacy

PROPOSED RESEARCH REPORT

AIMS: This proposal details a clinical study, specifically, designed to investigate potential sex-based pharmacokinetic differences and responses to digoxin. The study design will further provide insight into mechanisms, which may be responsible for any such pharmacokinetic or pharmacodynamic differences. This study aims to:
1. Characterize and compare digoxin pharmacokinetic parameters following intravenous (IV) and oral (PO) administration in healthy males and females.
2. Characterize sex-based response, determined by measures of inotropy, chronotropy, and dromotropy following IV and PO digoxin administration in males and females.
3. Characterize oral digoxin pharmacokinetics and pharmacodynamics in males and females when P-glycoprotein is inhibited with ketoconazole.

STUDIES AND RESULTS: A prospective, randomized, three-way crossover design will be employed to evaluate and compare sex-based differences in digoxin pharmacokinetics between healthy males and females and the potential impact on three measures of response (positive inotropy, negative chronotropy, and negative dromotropy) to digoxin. Twenty healthy, normal volunteers (10 M, 10 F) between 18 to 45 years of age will be recruited for study participation. Patients will be eligible if they are healthy, non-smokers, and are not taking chronic medications. An initial interview will be conducted to ascertain information regarding the subject’s past medical history. Females will be excluded if they are breast-feeding or pregnant. White blood cells (WBCs) will be isolated from venous blood samples (10 ml), collected during initial screening. MDR1 genotype will be determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays to detect the 3435C>T polymorphism on exon 26 and the 2677G>T polymorphism on exon 21. On the morning of the study days, after a minimum eight-hour fast, subjects will report to the Indiana University, General Clinical Research Center and be randomly assigned to the following treatment protocols.
Arm A IV Digoxin 0.5 mg infused into a forearm vein over 10 minutes
Arm B PO Digoxin 0.5 mg administered
Arm C PO Ketoconazole 200 mg administered daily for 7 consecutive days
PO Digoxin 0.5 mg administered 2 hours after ketoconazole dose on day 5
Serial blood and urine samples will be collected up to 48 hours after digoxin administration in each study arm. Serum and urine digoxin concentrations will be determined by a fluorescence polarization immunoassay (TDxFLx; Abbott Laboratories, Abbott Park, Ill). Lead II electrocardiogram rhythm strips will be obtained immediately following each blood sample collection. Continuous wave Doppler echocardiography, a non-invasive approach to characterize left ventricular systolic function, will be performed prior to and 1, 4, 8, 12, and 24 hours after digoxin administration in each arm. Measures of left ventricular systolic function will be used as a measure of positive inotropic response for comparison between males and females in all three study arms.
Specific Aim 1: Initial pharmacokinetic parameter estimates will be obtained by noncompartmental methods. The appropriate pharmacokinetic and pharmacodynamic models will be fitted to the data. Estimates of pharmacokinetic parameters will be obtained. Standard pharmacokinetic equations will be used to calculate secondary parameters of interest.
Specific Aim 2: The digoxin concentration-response relationships (inotropic measures, heart rate, PR-interval response measures) will, most likely, exhibit counterclockwise hysteresis. Each subject's concentration-response data, therefore, will be plotted and if hysteresis is observed as expected; the peripheral compartment concentrations will be modeled. The Emax, Sigmoid Emax and linear pharmacodynamic models will be fitted to the digoxin concentration- response data. If the Emax or Sigmoid Emax model is selected as the appropriate model, EC50 will be used to compare digoxin sensitivity between males and females.
Specific Aim 3: Pharmacokinetic and pharmacodynamic analyses following oral digoxin administration described above will be repeated for subjects on day 5 of a 7 day ketoconazole treatment regimen (Arm C).

SIGNIFICANCE: The proposed study will characterize potential sex-based pharmacokinetic and response differences to digoxin. It will also provide insightful information on the mechanistic explanation of any such difference. Females may have less functional P-glycoprotein leading to increased digoxin exposure, potentially increasing response. If females with left ventricular dysfunction have increased inotropic action, it may explain an observed increased mortality rate when receiving digoxin. Numerous available drugs that are substrates for P-glycoprotein will be influenced if sex-based differences exist in the function of the efflux transporter.

TRAINING ACTIVITIES: Throughout pre and post-doctoral training thus far, the fellow has been exposed to several clinical, analytical, and laboratory-based techniques that will complement the research project to be conducted over the next year. A majority of coursework, focused on statistical and pharmacokinetic/pharmacodynamic analyses along with trial design and grant writing techniques have been completed. The fellow will continue to participate in weekly departmental journal clubs and will attend and present at seminars and journal clubs in the Division of Clinical Pharmacology at the Indiana University School of Medicine. In addition, ongoing laboratory work investigating the inhibitory actions of b-lactams on the PEPT1 transporter in cytokine treated Caco-2 cell monolayers, along with the validation of a fluoroquinolone assay with high-performance liquid chromatography and ultraviolet detection will be completed over the next year. These activities combined with the research project detailed above will comprise the final year of training. The study described herein will contribute significantly to the fellowship training by including exposure to pharmacogenomic techniques and pharmacodynamic analyses. Specifically, the fellow will be trained in the isolation of genomic deoxyribonucleic acid (DNA) from WBC’s and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. In addition to the genetic component, the study described in this fellowship application will present a clinical question to be, partly, answered through response measures following digoxin administration. This will require a pharmacodynamic analysis that will utilize three measures of digoxin response to determine drug concentration-response relationships. This will, therefore, complement current pharmacokinetic and laboratory-based training skills that the fellow has achieved. In combination with the analytical tools already acquired, exposure to the investigations in the final year of this fellowship program will enable the fellow to achieve sound translational-based research skills.