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

PATTY FAN-HAVARD, PHARM.D.
Associate Professor, Pharmacy Practice
The Ohio State University College of Pharmacy

FINAL REPORT

AFPE Award: MERCK-AFPE Clinical Pharmacy Post-Pharm.D. Fellowship in the Biomedical Research Sciences

AIMS: The goal of this AFPE research program is to acquire biomolecular and advanced analytical training to establish a translational research program focusing on placental and fetal/newborn outcomes associated with the use of HIV-1 protease inhibitors during pregnancy. The aim of this project is to establish an in vitro and in vivo model to study the effects of PIs on the de novo biosynthesis of 17?-estradiol (E2), assess placental and fetal outcomes following in-utero exposure to nelfinavir (NFV) and to characterize the disposition of NFV in fetoplacental unit.

STUDIES AND RESULTS: We observed a significant reduction in the biosynthesis of E2 following treatment with NFV in human JEG-3 choriocarcinoma cells. We examined the effect of NFV on E2 production during pregnancy in an in-vivo rodent model. A total of 27 female Sprague-Dawley rats were randomly assigned to control (C), low dose (LD) NFV (100 mg/kg/day) or high dose (HD) NFV-treated (400 mg/kg/day) group. Rats were mated during an overnight 12-hr period. Sperm-positive vaginal smears denoting day 0 of pregnancy. Dams were sacrificed and exsanguinized on day 20 of gestation. Plasma levels of E2 were measured by EIA. Data were analyzed by ANOVA, post hoc testing by the method of Tukey and with significance set at p<0.05. No differences were observed in the plasma levels of E2 among the treatment groups. The mean (+ SD) of plasma E2 levels were 122.9+ 45.9 pg/ml, 177.1+ 71.4 pg/ml and 133.5+ 49.4 pg/ml in C, LD-NFV and HD NFV-treated groups.

Clinical complications of glucose intolerance and hepatoxicities have been reported with the use of NFV in nonpregnant adults. In the placental and fetal outcomes study in rats, the findings of increased placental surface area and weight ratio, decreased fetal liver weight ratio, and a trend for higher maternal nonfasting plasma glucose in the LD and HD NFV-treated dams, are consistent with clinical and experimental models of gestational diabetes. The findings of a mild to moderate diffuse congestion, multifocal telangiectasia and myeloid and erythroid hyperplasia in the fetal livers were unexpected. It remains unknown if the abnormal fetal liver finding is associated with NFV exposure or alterations in the carbohydrate homeostasis during pregnancy. Nelfinavir undergoes extensive metabolism to the 3’-methoxy-4’-hydroxynelfinavir (M1) metabolite in rats. Our hypothesis is that NFV distributes across the placenta and is metabolized, albeit low, by the fetal liver. The progress in completing aim 3 of the study has been hampered by the lack of specificity in assessing NFV metabolite formation in the fetal liver using autoradiographic imaging and our unsuccessful development in separating the NFV and the M1 metabolite by high performance liquid chromatography. An on-line column switch chromatography tendem mass spectrometric method was recently developed and validated to separate NFV and the M1 metabolite peaks. The chromatographic conditions were performed using an extraction C8 column (MetaSil Basic 30?m (100Å) 10 x 2 mm ID), analytical C8 column
(5?m, 100Å BDS, 20 x 2 mm ID) at ambient temperature using an isocratic mobile phase containing 70:30 acetocetonitrile/0.1% ethyl acetate at a flow rate of 0.2 ml/min. The analytes were extracted for 3 minutes before switching to the analytical column. Reserpine (RSP) was used as the internal standard. The ESI consition produced transition of ions at m/z 568.2 ® 330 (NFV +H+) for NFV; m/z 598.2 ® 360 (M1 +H+) for M1; and, m/z 609.2 ® 397.4 (RSP + H+) for reserpine. The retention time was 7.94, 7.94 and 7.69 for NFV, M1 and RSP, respectively. The assay was validated in human plasma for NFV and M1 each over the concentration ranges of 0.5 to 20 ng/ml. The lowest limit of detection was at 0.5 ng/ml for NFV (% CV 9.51, accuracy 82%) and M1 (%CV 9.72, accuracy 81.67). The %CV and accuracy for NFV and M1 were 2.99% and 107.33 at 20 ng/ml. Study is on going to analyze NFV and M1 metabolite from fetal liver tissues collected from the LD and HD NFV-treated dams.

The OSU Department of Obstetrics and Gynecology recently established an HIV-High Risk Pregnancy Clinic (HHRPC), in collaboration with the ID Services, and Columbus Children’s Hospital. Pregnant women diagnosed with HIV infection are referred to the HHRPC for comprehensive obstetrical and medical care. An outcomes database was established to assess for maternal and newborn outcomes following antiretroviral chemoprophylaxis. Thirty patients were referred to the HHRPC, of which 21 delivered (refusal of ART in 1, AZT monotherapy in 1, 2 NRTIs in 1, 2 NRTIs with NVP in 13, and 2 NRTIs with PIs in 5). Four patients (19%) had active drug use history. Abnormal triple screen result was observed in 3 of 7 patients (42.9%) taking a protease inhibitor-containing regimen, and all fetuses were karyotypically normal. Maternal glucose intolerance, hyperlipidimia and abnormal LFTS were not observed among patients taking a PI-containing regimen. Rate of maternal anemia (1-3 gm/dl drop in Hgb) was 67% with ART, and rate of severe maternal hepatotoxicity (LFTs >4-5 times UL) was 15.4% with nevirapine-containing ART. Three cases of unusual maternal adverse events occurred, including drug rash, eosinophilia and systemic syndrome (DRESS), fatal postpartum cardiomyopathy, and the development of new onset fetal bradyarrhythmia requiring delivery near term following an increase in ddI dose. The rate of premature delivery (<37 weeks gestation) was 28.6%, LBW (<2500g) was 19%, and VLBW (<1500g) was 4.8%. Contributing factors for premature deliveries, LBW, VLBW included serious maternal adverse events to ART, cervical incompetence, chronic hypertension with superimposed pre-eclampsia, and recent cocaine use. No infants are HIV-positive to date.

SIGNIFICANCE: The relationship between antiretroviral therapy (ART) and adverse maternal and newborn outcomes has not been established, since the full spectrum of possible complications associated with ART during pregnancy is still uncertain. The research training gained during the past two years was critical in fostering and establishing a translational research program with the overall goal to provide clinical data on the safety of ART during pregnancy. The skills gained in animal research and biomolecular techniques provided the necessary in-vitro and in-vivo models to evaluate pharmacologic toxicities associated with ART. The recently established HIV High Risk Pregnancy Clinic provides the critical milieu to recruit patients for clinical trials to investigate the effects of pregnancy on pharmacokinetics of antiretroviral drugs and to determine if therapeutic drug monitoring may assist clinicians to improve efficacy and minimize toxicities.

TRAINING ACTIVITIES: The LC/MS/MS analytical methodology described above is new. The use of LC-MS/MS to analyze drug levels is state of the art since the method provides improved sensitivity and specificity, a critical technique in detecting the parent compound and its metabolites from the cord blood. The LC-MS/MS instrument is available in Dr. Kenneth Chan’s Biomedical Mass Spectrometry laboratory at the Division of Pharmaceutics under a collaborative arrangement. The extraction of the drug and metabolites from the organ tissue samples has been developed. Aim 3 of the study is ongoing and will be completed with Dr. Chan’s laboratory. I attended a summer workshop in biostatics to update new approaches to the analysis of data of biological, laboratory data, and clinical trials. The computer database for outcome analysis was established in consultation with Dr. Stacey Woodard at the Centers for Biostatistics. Patient information and data were prospectively entered once weekly at the HIV-High Risk Pregnancy Clinic.

OTHER SUPPORT: Federal-funding submission is in preparation for competitive and noncompetitive applications. Two grant applications will be submitted in collaboration with Dr. Michael T. Brady, Director of the Pediatric Pharmacology Research Unit (PPRU) at the Columbus Children’s Hospital. The PPRU is a network of 13 sites that has been funded by National Institute of Child Health and Human Development (NICHD) and its goal is to support a multi-disciplinary environment that promotes basic, clinical and translational research programs to investigate the use of medications in children. The OSU HIV High-Risk Pregnancy Clinic is now recognized as a new research program entity to be included in the overall research mission of the PPRU. Both noncompetitive and noncompetitive grant applications are in progress. Federal and institutional funding applications are also underway to incorporate pharmacokinetics and placental distribution studies of antiretroiviral agents in pregnancy with correlation to clinical maternal and newborn outcomes.

PUBLICATIONS:
1) Knudtson E, Para M, Boswell H and Fan-Havard P. DRESS syndrome and renal toxicity with nevirapine-containing regimen in a pregnant
patient with HIV. Am College of Obstet & Gynecol): In press.

2) Fan-Havard PF, Lieb CA, Monahan CM, Cho E, Wymer SK and Richard VB. Nelfinavir (NFV)-Induced Glucose Intolerance in Pregnancy:
Placental and Fetal Outcomes in Rats.[Abstract No. 428]. Presentation at the 40th Annual Meeting of the Infectious Diseases of America,
Chicago, IL. Oct. 24th-27th, 2002.

3) EJ Knudtson, LS Vasist, M Williams, MF Para, MT Brady, and P Fan-Havard. Maternal and Newborn Outcomes Following Implementation of a
Multidisciplinary HIV-High Risk Pregnancy Clinic. Abstract submitted to the 2003 Society for Gynecologic Investigation Annual Scientific
Meeting, March 27th – 30th, 2003.

4) P Fan-Havard, EJ Knudtson, LS Vasist, MF Para, N Rahkmanina, and MT Brady. Maternal Tolerability and Fetal Complications with Antiretroviral
Therapy in Pregnant HIV-1 Infected Women. Abstract submitted to the 10th Conference on Retroviruses and Opportunistic infections, Hynes
Convention Center, Boston, MA February 10-14, 2003

SPONSOR’S REPORT: The use of potent ART have resulted in a dramatic decline in the incidence of morbidity, mortality and perinatal transmission associated with HIV-infection. Women with HIV infection of childbearing age are now considering reproductive choices. Combination therapy is now routinely prescribed to reduce perinatal transmission by achieving undetectable viral load, and yet, the gestational safety data remain sparse. The established translational research program in collaboration with the HIV-High Risk Pregnancy Clinic will provide the necessary basic and clinical research infrastructure to investigate the safe use of ART during pregnancy.