A major impediment to clinical implantation of pharmacogenetic information is the availability of cost-effective clinical tests at the time of prescribing. A major initiative of our program has been to develop a pharmacogenetic assay that is

• comprehensive in covering all of the genes with prescribing guidance available (currently 22 genes),
• inexpensive to perform ($100),
• preemptively tested in order to provide the information in advance of prescribing drugs and
• guided with a report to educate clinicians and patients regarding the outcome of the assay.

To develop this project, we have used a methodology called MIPs, which stands for molecular inversion probes. This approach is essentially a custom pull-down or capture of the 22 genes for which we wish to determine genetic information. We can capture those genes for each patient and fully sequence them to identify genetic variation. The genetic variations are interpreted according to already published guidelines as to the patient’s responses to various drugs. These data allow us to produce a report for each patient which details their responses to over 100 drugs that can then be used to provide the guidance for “the right drug or the right dose of drug” on an individualized basis.

Pharmacogenes that we target in PGx1:

ABCB1, BCHE, CYP2B6, CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5, DPYD, G6PD, HLA-A, HLA-B, IL28B, ITPA, NAT2, NUDT15, RYR1, SLCO1B1, TPMT, TYMS, UGT1A1, and VKORC1

This PGx1 assay is currently undergoing clinical laboratory improvement amendments, or CLIA, validation. These are U.S. federal regulatory standards that apply to all clinical laboratory testing performed on humans. This assay is innovative and unique in the approach we have taken to develop it. It uses next gen sequencing to provide the data for a low cost. This is an important breakthrough to pharmacogenetic testing. Now that we have championed this approach, we are using it in a number of different projects.

Our ability to develop cost-effective assays for several different sets of genes that have pharmacological and pharmacogenetic relevance has enabled the development of multiple different projects using this approach. All of these projects are now in various stages of development; hey have been developed by my lab in collaboration with other faculty in the UNC School of Medicine and the Gillings School of Global Public Health.

• Treatments for Incontinence. Fesoterodine is a commonly used agent for treatment of incontinence that demonstrates variable efficacy. After oral administration, fesoterodine is rapidly and extensively hydrolyzed to its active metabolite. The active metabolite is further metabolized in the liver via two major pathways involving CYP2D6 and CYP3A4. In a pilot study, we are genotyping 75 patients that have been treated with fesoterodine to look for genetic markers of efficacy.Collaborator: Jennifer Wu, M.D., associate professor, UNC Department of Obstetrics and Gynecology, School of Medicine.

• Pharmacogenetics in kidney transplant. Tacrolimus is the standard treatment for immunosuppression in solid transplant. It is metabolized largely by CYP3A5. This gene has a large variability in activity with Caucasians often having a nonfunctional version of the gene and African American patients rarely having that same allele. Kidney transplant patients are on a wait list usually for months before a kidney becomes available. This pilot project will genotype patients on the wait list so the appropriate dose of tacrolimus is initiated after transplant. We will monitor tacrolimus levels and clinical outcomes of genotype-guided dosing.Collaborators: Tomas Kozlowski, M.D., chief of transplant, School of Medicine, and Bob Dupuis, Pharm.D., UNC Eshelman School of Pharmacy.

• Pharmacogenetics in bone-marrow transplant. Tacrolimus is also used as a standard of care immunosuppressive agent in allogeneic hematopoietic stem cell transplant. Patients with the genotype associated with CYP3A5 function experience lower trough concentrations putting these patients at higher risk for developing acute graft versus host disease (aGVHD). And aGVHD is one of the major life-threatening sequelae associated with allogeneic HSCT.DNA will be collected from consenting patients who have undergone allogeneic HSCT at UNC. Associations between genotype and time to target tacrolimus trough level, time to aGVHD and severity of aGVHD will be assessed. Information from this retrospective pharmacogenetics study will help inform the development of a population PK/PD model that will identify the most precise starting dose for patients with the alternate genotype.Collaborators: Dan Crona, Ph.D., UNC Eshelman School of Pharmacy. Paul Armistead, Marcie Riches, and Thomas Shea (UNC Bone Marrow Transplant, School of Medicine), Kamakshi Rao, Pharm.D., and Maurice Alexander, Pharm.D., (UNC Bone Marrow Transplant, Department of Pharmacy, UNC Eshelman School of Pharmacy), and Eric Weimer (UNC McLendon Labs, School of Medicine).

• Pharmacogenetics in the chronic pain clinic. Multiple drugs that are prescribed for chronic pain management have pharmacogenetic information available, CYP2D6 being the most prominent among numerous other genes. In the drug classes used, the opioids are of particular concern because of addictive properties. We have developed a panel of 16 genes that we can assay to provide guidance for prescribing in a pain setting. We have initiated a pilot project with the UNC Chronic Pain Clinic to implement pharmacogenetic testing.Collaborators: Tim Ives, Pharm.D., M.P.H., UNC Eshelman School of Pharmacy and physicians from the UNC Chronic Pain Clinic.

• Implementation of PGx in a multiclinic setting. We have proposed that the analytical assay we have developed for assessment of the pharmacogenetics of pain can be implemented in multiple different pain clinics. A proposal will be submitted (R01 – July submission) for the implementation of this work. This is specifically a call for implementation science and we have engaged the National Implementation Research Network at UNC to be active participants for this collaborative effort.Collaborators, Dean Fixsen, Ph.D., and Caryn Ward, Ph.D., National Implementation Research Network. The groups that have contracted to participate in this five-clinic network are:
• UNC Chronic Pain Clinic (Tim Ives, Pharm.D., MPH, UNC Eshelman School of Pharmacy),
• Mission Hospital Chronic Pain Clinic (Lynn Dressler, Dr.P.H., Mission Hospital, Asheville),
• UNC Chronic Pain Clinic – Anesthesia (Dr. Wunnova, School of Medicine) and
• Novant Hospital System, Greenville

• Pharmacogenetics in the Catheterization Clinic. A project using clinical factors and CYP2C19 genotype to provide genotype-guided therapy selection in high-risk patients undergoing percutaneous coronary intervention in the Catheterization Lab has been initiated by George A. Stouffer, MD, UNC Chief of Cardiology, School of Medicine and Craig Lee, Pharm.D., Ph.D., UNC Eshelman School of Pharmacy. We are assessing a full panel of pharmacogenetic genes in these same patients for a retrospective analysis of cost-effectiveness of comprehensive pharmacogenetic preemptive testing.Collaborators: Stephanie Wheeler, Ph.D., UNC Gillings School of Global Public Health

• Pharmacogenetics of salt sensitivity and blood pressure. The salt sensitivity phenotype has been indicated as a significant risk factor for mortality independent of blood pressure. Identifying this phenotype is important in the management of hypertension and can help tailor nutrition and pharmaceutical interventions for patients in an individualized way. This has now developed into two separate but linked projects, one using a Caucasian cohort.Collaborators: Martin Kohlmeier, M.D., Ph.D., UNC Nutrition Research Institute School of Medicine, UNC Gillings School of Global Public Health; Robin Felder, Ph.D., University of Virginia; Scott Williams, Ph.D., Case Western Reserve University; and Jose Pedro, M.D., Ph.D., George Washington University.

• Pharmacogenetics of salt sensitivity and blood pressure. With the recognition that there are strong ethnic differences in phenotype susceptibility and allele frequencies for SNPs and genes, this second project aims to develop a multilocus genetic algorithm to predict salt sensitivity in an African American cohort.Collaborators: Mildred Pointer, Ph.D., FAHA director, Cardio-metabolic Program and NC Central University, Martin Kohlmeier, M.D., Ph.D., UNC Nutrition Research Institute School of Medicine, UNC Gillings School of Global Public Health. R01 grant proposal being developed.

• Assessment of epigenetic factors in dioxin response. Establishing how environmental chemical exposures may impact human health is extraordinarily difficult, particularly in inaccessible tissues like the brain. Moreover, existing strategies fail to account for genetic variability among individuals, and determining who is most susceptible remains a significant obstacle in the field. This study will measure the specific molecular effects of a highly toxic chemical, TCDD (dioxin). It is often not feasible to sample brain tissue in humans, but other tissues like blood and skin can be readily sampled. This raises the question of whether epigenomic profiles associated with EDC exposure in blood and skin are good surrogates for exposure in brain.Collaborators: David Aylor Ph.D., NCSU; Greg Crawford, Ph.D., Duke University; and Heather Patisaul Ph.D., NCSU Joint U01 with Duke, NCSU, UNC.

• Genetic etiology of cancer drug response. Numerous genes potentially influence anti-cancer agent drug response, but current candidate-gene approaches are severely limited. In response to these limitations, this project undertakes pharmacogenomic assessment of cytotoxic effect of the majority of FDA approved anti-cancer compounds using an ex vivo model system to determine the heritability of drug-induced cell killing to prioritize drugs for pharmacogenomic mapping.Collaborators Alison Motsinger-Reif, Ph.D., NCSU and Howard McLeod, Pharm.D., Moffitt Cancer Center.

• Personalization of therapeutic efficacy and risk. Nonsteroidal anti-inflammatory drugs (NSAIDs) are consumed by tens of millions worldwide. Although they relieve pain and inflammation, we understand poorly their mechanism of action. They also cause serious gastrointestinal and cardiovascular adverse effects and despite enrolling more than 100,000 patients in randomized trials, we still do not know the NSAID of choice for patients with arthritis and heart disease or if NSAIDs differ in clinical efficacy.Collaborators: Garret Fitzgerald, Ph.D., PENN lead investigator. Multiple other sites including UNC.

Pharmacogenomics Educational Initiatives

Pharmacists, with extensive training in pharmacology and pharmacotherapy and accessible to patients are ideally suited to champion clinical pharmacogenomics. The public will also drive clinical pharmacogenomics as consumers of pharmacogenomics tests. Therefore, Tim Wiltshire has developed educational initiatives for four groups: graduate students, student pharmacists, current practicing pharmacists and the public.

1. Graduate Students. Currently we have two graduate courses in our Pharm.D. curriculum. DPET 832, and 838. Federico Innocenti, M.D., Ph.D., is the course coordinator for 832, and I have developed and been the course coordinator for DPET 838, Methods in Pharmacogenetics. Our graduate program is undergoing a revision and will likely undergo a transformation over the next few years. As part of the changes in our curricula we are looking to reduce the amount of didactic teaching for the graduate program.

2. Pharm.D. students. Ideally, student pharmacists may benefit from pharmacogenomics educational interventions early in the curriculum to build a foundation for advanced discussion. Student pharmacists beginning their second year were surveyed regarding their attitudes and perceived competencies on pharmacogenomics prior to and following an educational intervention focusing on the practical applications of pharmacogenomics in the clinical setting. Also to reflect the current landscape of direct-to-consumer genomic testing, student pharmacists were offered the opportunity to obtain genotyping using 23andMe kits. This educational intervention, including personal genotyping, was feasible and positively enhanced students’ reflections and attitudes toward pharmacogenomics in a professional pharmacy program.

3. Continuing Education of Pharmacists. Members of CPIT have presented numerous continuing education sessions on pharmacogenomics that I have developed, including opportunities through Kroger Pharmacy, the North Carolina Association of Pharmacists and the North Carolina Area Health Education Center system. In May 2017, the UNC Eshelman School of Pharmacy Division of Pharmacotherapy and Experimental Therapeutics and CPIT will be hosting the annual Chapel Hill Pharmaceutical Sciences Conference, which will focus on personalized medicine. Danny Gonzalez, Ph.D., and Tim Wiltshire, Ph.D., are the coordinators of this meeting. The effectiveness of PGx1 will also be evaluated as a teaching tool to current practicing pharmacists.

4. Educating pharmacists in the community pharmacy. CPIT is partnering with Kroger Pharmacy to offer pharmacogenomics education to members of the public in the spring of 2017. This educational intervention will include virtual reality and videos on pharmacogenomics produced in collaboration with the UNC Eshelman School of Pharmacy Center of Innovation in Pharmacy Simulation. PGx1 will be offered to members of the public in order to better educate them on clinical pharmacogenomics.

Protocols

• Sequenom
  • Genotyping protocoling Protocol
  • Chip Tracker
  • Plate Layout Template (MTP)
• Protein Assay
  • MSD Sandwich ELISA Assay
• Cell Culture
  • MEF Transfection
• Reverse Transcriptase and Q-PCR
  • Combined RT & Q-PCR Protocol
• Oxaliplatin PP

Posters

• “In-Vitro Drug Toxicity Screening”
• “Modeling Depression in Mice to Identify Genetic Mechanisms of Mood Disorder”