TAMI R. ARGO, Pharm.D.
Clinical Psychopharmacology Fellow
University of Iowa College of Pharmacy

FIRST YEAR-PROPOSED RESEARCH

TITLE OF RESEARCH: Infection risk among patients receiving long-term clozapine treatment.

AIMS: Clozapine, an atypical antipsychotic, is efficacious in the treatment of resistant psychotic illness, but problematic side effects, including agranulocytosis, limit its use as first line therapy. Clozapine has been determined to have immunomodulatory properties, with the ability to alter plasma levels of cytokine and cytokine receptors. Complex networking of these endogenous substances occurs during infectious disease, helping to mediate the primary host response. While patients who develop significant neutropenia are clearly at high risk for infection, it is unknown whether patients who fail to reach the hematological monitoring thresholds for clozapine discontinuation are also at some increased risk for infection.

STUDIES AND RESULTS: De-identified data from Iowa Medicaid claims will be used to identify drug exposure. Each Medicaid pharmacy claim identifies the drug product dispensed by National Drug Code (NDC) and drug name, and each claim gives the date that the fill was provided. Demographic information including sex and date of birth will be obtained from a separate Medicaid enrollment file. Patient identifiers in the datasets have been encrypted to protect patient confidentiality.
Patients will be deemed eligible if they meet all of the following criteria:
1.) are continuously Medicaid eligible in the two years prior to and two years
following clozapine initiation;
2.) have continuous clozapine use in the two years following initiation; and
3.) have no evidence of concurrent immunosuppressive disease state or
medication.
The occurrence of infection will be defined as a fill for an antibiotic prescription during the four year period surrounding clozapine initiation, including antibacterials, antifungals, and non-HIV antivirals.

Specific Aim 1: To compare the number of antibiotic prescription claims for patients in 24 consecutive months pre-clozapine treatment versus a period of 24 consecutive months while being treated with clozapine, and determine if patients were at a significantly increased risk of infection after the initiation of clozapine.

Specific Aim 2: To characterize the time course of infection risk based on the claim frequency within different time intervals following clozapine initiation, including 1 month, 3 months, 6 months, 12 months and 12+ months. An adjustment may be necessary in this analysis to correct for the time of year when clozapine was initiated (e.g. winter vs. summer).
Specific Aim 3: To characterize risk factors for clozapine-related infection. Analysis variables may include, but not be limited to, age, gender, length of treatment with clozapine, prior antipsychotic use, time of year when clozapine was initiated, and time of year antibiotic was prescribed.

Specific Aim 4: To identify a cohort of patients initiating risperidone treatment using the same procedure described for clozapine, and compare infection rates in the pre-initiation and post-initiation periods between clozapine and risperidone groups. Adjustments will be made for any identified confounding variables.

SIGNIFICANCE: Immunological mechanisms have been etiologically implicated in the occurrence of clozapine-induced agranulocytosis; however, a full understanding of the hematological complications that may accompany clozapine therapy is yet to be elucidated. Clozapine has been shown to alter plasma cytokine and cytokine receptor levels, which may have activating, proliferative, and differentiating effects on lymphoid cells. Previous study in hospitalized patients suggests antibiotics are more frequently prescribed in patients treated with clozapine. Future study will help determine if patients taking clozapine are at increased risk for infection, and will shed additional light on possible mechanisms by which this may occur.

TRAINING ACTIVITIES: As part of the next year of my fellowship, I will be responsible for developing the scientific hypothesis and experimental methods for the research project included in this application. While the focus of this fellowship is primarily clinical research, laboratory training will also be included. I will work with Dr. Vicki Ellingrod, Pharm.D. to learn laboratory techniques in pharmacogenetic research, including polymerase chain reaction and restriction fragment length polymorphisms. Our clinical psychopharmacology laboratory is equipped for various HPLC drug assays, which I can receive training in to aid in my research. I expect to develop expertise in data analysis both by experiential training and didactic course work in biostatistics and epidemiology. I will continue my coursework and complete the Master’s program in Epidemiology at the University of Iowa College of Public Health in May 2005. As a crucial part of my training, I anticipate developing proficiency with multiple statistical software packages, including SAS. I plan to maintain some degree of clinical practice by continuing to consult on the general and medical psychiatry wards and in the outpatient clinics, as well as providing drug information, including literature searches and clinical reviews, to physicians, nurses, and other members of the medical team. I will assist in the preceptoring of Doctor of Pharmacy students as part of their psychopharmacology clinical rotation, as well as develop and present lectures in the psychopharmacology lecture series for the psychiatry medical residents. I plan on attending several seminars and meetings, including the Sharing our Skills VA Psychiatric Specialty Training Workshop, the annual meeting of the College of Psychiatric and Neurologic Pharmacists, and the annual meeting of the American College of Clinical Pharmacy, and will prepare and present results from this and other research studies. Continuing education in psychiatry is available through multiple seminars and lectures I will be attending and participating in, including weekly Grand Rounds and Research Seminar in the Department of Psychiatry, weekly Psychopharmacology Seminars presented to the resident physicians in the Department of Psychiatry, and weekly Division of Clinical and Administrative Pharmacy Faculty Seminars. Paul J. Perry, Ph.D. will serve as my mentor and will have primary responsibility for all aspects of the fellowship program. Statistical support, assistance in study design and implementation, and additional mentoring will be also be provided by Elizabeth A. Chrischilles, Ph.D., Brian C. Lund, Pharm.D., M.S., and Ryan M. Carnahan, Pharm.D., M.S..

JODI L. GRABINSKI, Pharm.D.
FIRST YEAR-PROPOSED RESEARCH

TITLE: Pharmacogenomic Analysis of Drug Metabolizing Enzymes Associated with Tamoxifen in Breast Cancer Patients

AIMS: The objectives of this study are to examine the extent to which genetic polymorphisms within the cytochrome P450 enzymes influence the formation of 4-hydroxytamoxifen, evaluate the impact of polymorphisms in the SULT1A1 gene on the conjugation of the 4-hydroxytamoxifen, and investigate the functional significance of these genes on clinical outcome.

1. Determine the CYP2C9, CYP2D6, and SULT1A1 genotypes in breast cancer patients receiving adjuvant tamoxifen therapy
2. Determine the extent to which tamoxifen is metabolized to its metabolites
3. Prospectively evaluate the correlation between genotype, tamoxifen metabolism, and patient outcome

STUDIES AND RESULTS: Three hundred women with early stage breast cancer who have been receiving adjuvant tamoxifen therapy for at least 8 weeks will be recruited from the Cancer Therapy and Research Center. After obtaining informed consent, two 7 mL blood samples will be collected from the patient. One sample will be used for DNA extraction using the QIAamp Midi Kit (Qiagen, Inc. ®) and quantified by spectrophotometry and the other will be used for high-performance liquid chromatography (HPLC) analysis of tamoxifen and its metabolites. Extracted DNA will be aliquoted for the study and the remainder will be stored in our anonymized Pharmacogenomic DNA Bank. Patient specific information including age, ethnicity, and menopausal status will be combined with disease characteristics (tumor size, histological grade, lymph node involvement) as well as molecular characteristics (ER/PR status, HER2 overexpression, BRCA1/BRCA2) and will be collected at the time of enrollment.

Aim 1: Genotype data for the CYP2C9 gene will be obtained using an assay we developed on the ABI®7000 Sequence Detection System. Validation of this assay was accomplished using restriction fragment length polymorphism (RFLP) and sequencing. Real-time PCR followed by allelic discrimination using fluorescently labeled probes identify the CYP2C9*2 (Arg144Cys) and the CYP2C9*3 (Ile359Leu) alleles. Genotyping for the SULT1A1 gene (Arg213His) will be accomplished using RFLP analysis. CYP2D6 genotypes will be determined by sequencing or pyrosequencing.

Aim 2: Quantification of tamoxifen and its metabolites will be evaluated using HPLC analysis. Metabolites that will be quantitated include N-desmethyltamoxifen, 4-hydroxytamoxifen, and a newly identified metabolite 4-hydroxy-N-desmethyltamoxifen.

Aim 3: Determination of the functional significance of these polymorphisms on progression free and overall survival will be evaluated by patient follow-up every 6 months. Cox proportional hazards regression will be used to determine the independent effects of genotype on survival.

SIGNIFICANCE: Retrospective data has demonstrated that women who are homozygous for the SULT1A1*2 allele receiving adjuvant tamoxifen therapy have three times the risk of death compared to those who are homozygous wild-type or heterozygous (Nowell S. J Natl Cancer Inst, 2002). However, there is currently no published data prospectively evaluating how polymorphisms within the SULT1A1 gene and other highly polymorphic genes involved in the metabolism of tamoxifen impact response to therapy. This study will provide a more comprehensive understanding of how polymorphisms within a number of genes involved in the metabolism of tamoxifen affect progression free and overall survival.

TRAINING ACTIVITIES: I am currently enrolled in an NIH K30-sponsored Master of Science in Clinical Investigation program in which I have taken classes in research ethics, clinical research methods, biostatistics and the integration of molecular biology into clinical research. I have also completed a molecular biology class through the graduate school. During my second year of the program I will be involved with classes in data management, scientific writing, clinical research methods and biostatistics. I have had the opportunity this year to gain experience in PCR, DNA extraction and purification, real-time PCR, restriction enzyme digestion, electrophoresis, HPLC and tissue culture techniques. Over the next two years, I will continue to gain experience in sequencing, LC/MS and statistical analysis. I will also be further expanding our pharmacogenomic analysis of tamoxifen by evaluating polymorphisms within tamoxifen’s target, the estrogen receptor using DNA from our Pharmacogenomic DNA Bank. John G. Kuhn, Pharm.D. will continue to serve as my mentor and Brad Pollock, Ph.D. will assist in statistical support. Clinical support is being provided by South Texas Oncology and Hematology, a community oncology group directed by Lon Smith, M.D.