JASON J. EVERLY, PHARM.D.
University of Arizona, College of Pharmacy & Arizona Cancer Center

PROPOSED RESEARCH REPORT

TITLE(S) OF RESEARCH: Characterization of expression patterns for 5-, 12-, and 15-lipoxygenase enzymes in human non-small cell lung adenocarcinoma cells lines with both smoking (NCI-H1395 and NCI-H1437) and nonsmoking (NCI-H2126) histories.

AIMS: To define the expression patterns for 5-, 12-, and 15-lipoxygenase enzymes in human non-small cell lung adenocarcinoma cells lines with both smoking (NCI-H1395 and NCI-H1437) and nonsmoking (NCI-H2126) histories using the known chemopreventative agents zileuton, caffeic acid, and NDGA as controls.

STUDIES AND RESULTS: Characterization of Selected Cell Lines: Individual cell lines will be characterized by measuring lipoxygenase metabolites (LTB4; Cysteinyl leukotrienes (LTC4, LTD4, and LTE4); 5(S)-HETE; 12(S)-HETE; 15(S)-HETE; 13(S)-HODE). Measurements will be collected at 10% percent cell confluency intervals. Indicators of cell culture health will be tested and confirmed. Lipoxygenase metabolite formation will be measured after cell harvesting techniques. Results will establish baseline lipoxygenase metabolite patterns prior to further study.

Quantification of Lipoxygenase Metabolites by ELISA: Our highly sensitive methodology (Assay Designs, USA) will allow for quantification of LTB4, cysteinyl leukotrienes, 12(S)HETE, and 15(S)HETE in cell culture lysates. ELISA data analysis will be conducted utilizing AssayZap 3.0, Biosoft U.K.
Western Blotting of Lipoxygenase Enzymes and Downstream Metabolites: Western blotting or immunoblotting is a well established technique for protein analysis. The 5-, 12-, and 15-LO enzymes as well as 5(S)-HETE cannot be quantified by ELISA, and will need to be quantified using Western blotting instead. Data will be analyzed via NIH ImageJ (NIH Image Processing and Analysis in Java). Western blotting will also be used to validate the results observed in ELISA methodologies.

Known Lipoxygenase Inhibitors:

DRUG TREATMENT GROUPS
DRUG CLASSIFICATION DOSE RANGE (µM) TREATMENT DURATION
ZILEUTON 5-LO inhibitor 2.0 to 60.0 2 min to 48 hrs
CAFFEIC ACID 5- & 12-LO inhibitor 0.1 to 20.0 2 min to 48 hrs
NDGA 5-, 12-, 15-LO inhibitor 1.0 to 10.0 2 min to 48 hrs

Dose Determination and Drug Response Utilizing MTT Assay: We will utilize the MTT CPA as a cell proliferation indicator after treatment with the compounds listed above to determine the IC10 and IC50 of these compounds. These results will be used to establish a dose correlating baseline lipoxygenase expression and metabolite formation to growth inhibition.

Data Analysis

Preliminary data from the ELISA experiments will be collected by the Bio-Rad Benchmark Microplate Reader and processed by the Bio-Rad Benchmark Microplate 5.0 Software. We will convert the ELISA data to a format compatible with two validated ELISA software programs: Assay Zap and ELISA for Windows (NIH). The expression data produced from Western blotting will be quantified by the ImageJ software. The Emax Model will be utilized for evaluation of the MTT CPA data to determine IC10 and IC50 drug concentrations for dose determination and for correlating drug dose to cellular response. Differences in lipoxygenase expression and lipoxygenase metabolite formation and cellular response (e.g., growth inhibition) will be determined by the Fisher’s exact test.

SIGNIFICANCE: Lipoxygenase expression and metabolite formation data is available for only two non-small cell lung adenocarcinoma cells lines (A549 and NCI-H1264), and focuses only on 5-Lipoxygenase. This research would provide data on (1) the differences and similarities of lipoxygenase expression and metabolite formation between smokers and nonsmokers (2) the sum effect that lipoxygenase enzymes and metabolites have in terms of tumor growth and apoptosis. Expression patterns will be measured by using the known chemopreventative agents zileuton, caffeic acid, and NDGA as controls.

TRAINING ACTIVITIES: I will gain extensive experience in a variety of laboratory skills over the next 2 years. Such skills include (but are not limited to) work with cancer cell lines, a mouse lung adenocarcinoma model, protein identification and quantification, and various aspects of drug treatment. Familiarity with these procedures will enable me to transition into a career in chemopreventative drug development research with ease. To augment my fellowship experience, I will receive formal clinical research training through the Arizona Clinical Research Training Program (AzCRTP) offered through the Mel and Enid Zuckerman Arizona College of Public Health. The AzCRTP objectives are to both educate program scholars in the principles, methods, and techniques used in clinical research and to provide the tools necessary to become independently funded clinical researchers. Individuals are awarded an AzCRTP Certificate of Completion at the end of the two year program. The program is comprised of classes in biostatistics, epidemiology, bioethics, and grant/scientific writing and federal regulatory guidelines. I will also have the opportunity to participate in ongoing Phase I trials as a sub-Principal Investigator. Timothy L. Brenner, Pharm.D. will serve as my mentor and will have primary responsibility for all aspects of the fellowship program. Dr. Robert T. Dorr, Ph.D., R.Ph., will serve as a co-mentor, providing additional leadership and guidance with regard to drug development, research abilities, and professional development. Laboratory training and support will be provided by Senior Research Specialist, Jennifer Oas and Research Technician, Katherine Larson.

OTHER SUPPORT: In compliance with AFPE, Dr. Jason Everly’s stipend will be paid for by the University of Arizona College of Pharmacy. His fellowship is not and will not be supplemented with any external funding such as other fellowships, scholarships, grant awards, etc. from NIH, AHA, ACS, ACCP, or any other organization or government agency during the term of this AFPE award.

TAMI ARGO, PHARM.D.
GlaxoSmithKline-AFPE Post-Pharm.D. Fellow in the Biomedical Research Sciences
University of Iowa College of Pharmacy

- FINAL RESEARCH REPORT -

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. The primary objective of this study was to replicate findings of a small, retrospective chart review in a higher functioning non-institutionalized population using a pharmacoepidemiological design.

STUDIES AND RESULTS: De-identified data from Iowa Medicaid claims were used to characterize drug exposure. Each Medicaid pharmacy claim identified the drug product dispensed by National Drug Code (NDC) and drug name, and each claim gave the date that the fill was provided. Demographic information including sex and date of birth were obtained from a separate Medicaid enrollment file. Patient identifiers in the datasets were encrypted to protect patient confidentiality.
Patients were 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;
3.) have no evidence of concurrent health maintenance organization (HMO) eligibility, and;
3.) have no evidence of concurrent immunosuppressive disease state or medication.
The occurrence of infection was defined as a fill for an antibiotic prescription during the four year period surrounding clozapine initiation, including antibacterials, antifungals, and non-HIV antivirals.

For the statistical analysis, the categorical risk of any antibiotic claim during the pre- vs. post-clozapine period was compared using McNemar’s test for correlated proportions. The total number of antibiotic claims before and during clozapine use was compared using a one-sample student t-test for matched pairs. For each analysis, an alpha less than 0.05 was considered statistically significant. For the student t-test, two tails of the sampling distribution were considered.
A total of 321 patients met all selection criteria and were included in the analysis. The risk of any antibiotic use during clozapine treatment compared to the period before clozapine initiation was not statistically different. Furthermore, the total number of antibiotics used to treat infections did not change significantly from the pre-clozapine period to the period during clozapine treatment.

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 suggested antibiotics are more frequently prescribed in patients treated with clozapine. This work did not replicate these previous findings.

PUBLICATIONS, POSTERS, AND LECTURES: Poster of work presented at the College of Psychiatric and Neurologic Pharmacists 2005 Annual Meeting, March 10-13, San Diego, CA. Submitting work for publication in International Clinical Psychopharmacology.

BENEFITS: This study was specifically performed in an attempt to replicate previous study findings using similar methodologies to previously published work. Those methodologies maintain several important limitations that came to be noticed as I worked through the project and contemplated my results. Future work is being designed to account and control for the bias and confounding that I found to be important when looking for this association between clozapine use and infection risk. These factors may include age, gender, as well as seasonality of clozapine initiation. Further work will include multivariate modeling to determine risk of antibiotic use with clozapine therapy. The current project failed to replicate previous findings. However, additional study using appropriate design and methods is warranted to further probe this relationship.

This award has provided me the opportunity to pursue research and academic coursework in the area of pharmacoepidemiology. Because of the support provided to me from this award, I had the opportunity to work on this project, my first original research project. This award was invaluable in helping me, a young researcher, gain valuable time and resources in which to pursue the areas of learning and research that most interested me. I am convinced that the skills and background that I have acquired through my coursework and through working on this research project will contribute significantly to future research endeavors as a faculty member. With the provisions of this award, I was also able to attend the 2005 College of Psychiatric and Neurologic Pharmacists Annual Meeting, and present my research results for this project, and gain valuable feedback from other researchers and clinicians in my field of expertise.

CURRENT STATUS: Clinical Assistant Professor, University of Texas at Austin, College of Pharmacy.

JODI GRABINSKI, PHARM.D.
PFIZER-AFPE Clinical Pharmacy Post-Pharm.D. Fellow in the Biomedical Research Sciences
University of Texas College of Pharmacy

- FINAL RESEARCH REPORT -

TITLE OF RESEARCH: “Pharmacogenomic Analysis of Drug Metabolizing Enzymes Associated with Tamoxifen in Breast Cancer Patients.”

AIMS: The objectives of this study was to examine the extent to which genetic polymorphisms within the cytochrome P450 enzymes influence the conversion of tamoxifen to 4-hydroxytamoxifen and other metabolites, 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.

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

STUDIES AND RESULTS: Three hundred and one women and two males with early stage breast cancer who were receiving adjuvant tamoxifen therapy for at least 8 weeks were enrolled on the study. Patients were recruited from the Cancer Therapy and Research Center from January 2004 to January 2005. After obtaining informed consent, two 7 mL blood samples were collected from the patient. One sample was used for DNA extraction using the QIAamp Midi Kit (Qiagen, Inc. ®) and quantified by spectrophotometry and the other was used for high-performance liquid chromatography (HPLC) analysis of tamoxifen and its metabolites. Extracted DNA was aliquoted for the study and the remainder was stored in our anonymized Pharmacogenomic DNA Bank. Patient specific information including age, ethnicity, and menopausal status combined with disease characteristics (tumor size, histological grade, lymph node involvement) and molecular characteristics (ER/PR status, HER2 overexpression, BRCA1/BRCA2) was collected at the time of enrollment.

Samples were genotyped for the SULT1A1, CYP2C9 and CYP2D6 genes by Pyrosequencing. Our lab developed a novel Pyrosequencing assay for the SULT1A1 gene that was validated by genotyping samples by both restriction fragment length polymorphism (RFLP) and Pyrosequencing to determine the percent concordance. As a result of the development of this assay we were able to efficiently genotype over 300 samples and determine the genotypic and allelic frequency of the SULT1A1 gene in our population. Our cohort was comprised of approximately one-third Hispanics in which there is no genotypic or allelic frequency data previously reported. From our study we identified that Hispanics have a higher frequency of homozygous variant genotypes (SULT1A1 *2/*2) compared with Caucasians (0.18 versus 0.13, p=0.26) although not statistically different. However, when we controlled for age <60 or = 60 there was a significant difference between Hispanics and Caucasians in the older age group (p=0.01). Whether or not this finding will influence progression free or overall survival cannot be determined until our cohort matures. We also evaluated the correlation between the SULT1A1 gene individually on a number of clinical and biological markers including age, tumor size, histological grade, stage of disease, menopausal status, HER2 overexpression and ER/PR status and found no significant associations. We also evaluated the influence of this particular polymorphism on tamoxifen and metabolite levels and found no association (p=0.99).

The impact of CYP2C9 polymorphisms was also evaluated in relation to tamoxifen and metabolite levels and individually did not influence levels. We recently completed genotyping for the CYP2D6 and will also evaluate how variant genotypes influence the conversion of tamoxifen to its metabolites as previous studies have suggested that this particular gene influences the levels of the intermediate metabolites.

From the Pharmacogenomic DNA bank that we initiated from this protocol, we expanded our analysis of tamoxifen to include the evaluation of polymorphisms within the estrogen receptor gene. We are evaluating four polymorphisms within the ERa gene. Genotyping for the Put II and Be I polymorphisms was conducted using Taxman genotyping assays. Polymorphisms within exon 4 and exon 8 will be genotyped using Pyrosequencing assays. As very few polymorphisms within the ERß gene have been identified, we are currently sequencing 8 axons within the gene in an effort to identify polymorphisms that predict response to tamoxifen therapy.

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 (Newell S. J Natal 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 influence response to therapy. This study provides a more comprehensive understanding of how polymorphisms within a number of genes involved in the metabolism of tamoxifen impact progression free and overall survival.

The data we have obtained to date from our study has provided important information regarding genotypic and allelic frequencies within the SULT1A1 gene within the Hispanic population. We also identified that SULT1A1 genotype is not associated with other clinical and biologic markers in breast cancer or tamoxifen and metabolite levels. As we continue to analyze our data, we will have a better understanding of how these genes individually as well as collaboratively influence the metabolism of tamoxifen and their impact on response to therapy.

TRAINING ACTIVITIES: During my fellowship I completed a NIH K30-sponsored Master of Science in Clinical Investigation program in which I took classes in research ethics, clinical research methods, biostatistics, data management, scientific writing and the integration of molecular biology into clinical research. I also completed a molecular biology class through the graduate school. I had the opportunity to gain experience in PCR, DNA extraction and purification, real-time PCR, restriction enzyme digestion, electrophoresis, Pyrosequencing, HPLC and tissue culture techniques. I was responsible for the protocol development and consent forms associated with the study. John G. Kuhn, Pharm.D. served as my mentor and Brad Pollock, Ph.D. assisted in statistical support. South Texas Oncology and Hematology provided M.D clinical support a community oncology group directed by Lon Smith.

OTHER SUPPORT: Dr. Kuhn currently receives funding from the NCI U01 CA62426-10.

PUBLICATIONS:
Grabinski JL. Development of a SULT1A1 pyrosequencing assay. (In submission)
Grabinski JL, Smith LS, Chisholm GB, Drengler R, Rodriguez GI, Lang AS, Kalter SP, Garner AM, Fichtel LM, Hollsten J, Pollock BH, Kuhn JG. Genotypic and allelic frequencies of SULT1A1 polymorphisms in women receiving adjuvant tamoxifen therapy. Breast Cancer Res Treat. (In press)