1. Using a transgenic mouse model expressing the canine ABCB1-1∆ gene to study pharmacogenomics and to identify biomarkers for predicting drug safety in dogs Min Zhu, DVM, Ph.D. Principle Investigator of Research Center for Veterinary Medicine U. S. Food and Drug Administration February, 2010 - October, 2014

2. Overview Objectives of the study Background - P-glycoprotein and ABCB1 gene - Collie dogs carrying the ABCB1-1∆ mutant gene - A transgenic mouse model for dogs Pharmacogenomics study Gene pathway analysis and biomarker discovery

3. Objectives of the Study To study the impact of canine ABCB1 gene mutation on drug safety (P- gp substrate drug) at the genomic level using a transgenic mouse model. To identify potential biomarkers that might be used to predict the safety of P-gp substrate drugs in dogs with the ABCB1-1∆ gene mutation. Ultimately, to support target animal safety studies that are part of the animal drug approval process. Biomarkers To protect Animal health

4. Background P-glycoprotein and ABCB1 gene P-glycoprotein (P-gp) belongs to superfamily of the ATP-binding cassette (ABC) transporters and it is encoded by the ABCB1 gene. It is a transmembrane protein widely present at the apical surface of epithelial cells (liver, intestine and kidney) and capillary endothelial cells (blood-brain barrier). It acts as a barrier to protect the cells within these organs by extruding various xenobiotics (toxin and drug) and endogenous metabolites. It exhibits broad substrate specificity, and P-gp substrates include drugs such as ivermectin, doramectin, moxidectin, and digoxin.

5. Background A transgenic mouse model expressing canine ABCB1 gene Orzechowski KL, Yancy HF et al, Am J Vet Res 2012;73:1477-84 Collie dogs are known to exhibit neurotoxicity when treated with ivermectin, a P-gp substrate, due to an ABCB1 gene mutation. The mutation, known as ABCB1-1∆, is a 4 base pair deletion in the ABCB1 gene, which results in a truncated and nonfunctional P-glycoprotein. A transgenic mouse model expressing the mutant canine ABCB1 gene (ABCB1-1∆), and one expressing the wild-type canine ABCB1 gene (ABCB1- WT) were developed previously at FDA. This mouse model has the potential to be used in lieu of ivermectin- sensitive Collies to assess the safety of P-gp substrate drugs.

6. Canine ABCB1-WTCanine ABCB1-Mutant Biomarker discovery Administration of P-gp substrates in mice: Ivermectin, Doramectin, Moxidectin, Digoxin Gene pathway analysis Pharmacogenomics Study Design in Animals Mouse Gene 1.0 ST Array Whole-genome gene expression microarray

7. A numerical scoring system developed to evaluate clinical signs of neurotoxicity Swain MD, Yancy HF et al. Res Vet Sci 2013;94: 656-661. Scores of 0, 1, 2, and 3 were assigned for no, mild, moderate, and severe clinical signs, respectively.

8. Mutant IDSexBody weight (g)P-gp substrateAtaxiaLethargyTremors KI090M30.6Digoxin321 KI091M33.3Digoxin330 KI092M31.8DoramectinNA**NA KI094F24DoramectinNA KI095F27.9Moxidectin110 KI096F24.7Moxidectin120 KI100M39.6Ivermectin221 KI101M39.7Ivermectin100 Clinical signs were observed in mice at 7 hr after administration of P-gp substrate drugs. The neurotoxic clinical signs were similar to those reported in dogs with the ABCB1-1∆ mutation. Mice were euthanized at 6 hr due to severe clinical signs and undue pain or distress. ABCB1-1∆ mutant mice displayed neurotoxic clinical signs following administration of P-gp substrates.

9. Hierarchical clustering Analysis of Gene expression of ABCB1-1∆ mutant and ABCB1 wild-type mice administered P-gp substrate drugs Genotype Drug treatment Mutant Wild-type

10. Gene expression was altered in ABCB1-1∆ mutant mice administered P-gp substrates as compared to ABCB1-WT mice. P-gp substrate# Genes altered in mutant mice (compared to WT mice) Ivermectin272 Digoxin372 Doramectin363 Moxidectin1612 *Gene expression cut-off: ≥ 2-fold gene expression fold change

11. Gene Pathway Analysis Digoxin

12. Ingenuity pathway analysis of altered gene signaling pathways/networks in ABCB1-1∆ mutant mice Displayed is the top ranked gene network associated with behavior from digoxin- treated mice. Genes with a ≥2-fold gene expression change were used for pathway analysis.

13. Biomarker discovery Common genes involved in response to P-gp substrates *Genes that were altered in ABCB1-1∆ mutant mice following administration of each of the P-gp substrates ivermectin, doramectin, moxidectin, and digoxin were compared. **Genes with a ≥2-fold gene expression change were used for comparison.

14. Gene Symbol Gene Accession No. GO biological process Fold change (Mutant/Wild-type) IvermectinDigoxinDoramectinMoxidectin Glra1NM_020492 Transport, neuropeptide signaling pathway-14.4-35.6-4.1-132.6 Mab21l2NM_011839 Multicellular organismal development-5.7-6.3-2.1-45.9 Ebf3NM_010096Transcription, DNA-templated-15.3-11.1-4.5-29.0 Slc10a4NM_173403 Transport, ion transport, sodium ion transport-15.7-8.4-10.8-22.7 Slc18a2NM_172523 Transport, drug transport, neurotransmitter transport-22.7-8.0 -10.1 TtrNM_013697Transport2.4-11.510.3-48.1 DaoNM_010018Dopamine biosynthetic process-3.4-5.9-8.5-25.5 Klk6NM_011177 Regulation of neuron projection development-4.8-9.8-4.5-24.0 GabrqNM_020488 Transport, chloride transport, ion transport-15.1-3.3-13.4-10.7 AlbNM_009654Albumin; drug transport5.0110.6158.536.9 Potential biomarkers Top of the commonly altered genes in ABCB1-1∆ mutant mice from all four drug treatment groups

15. Summary: As compared to ABCB1-WT mice, ABCB1-1∆ mutant mice exhibited neurotoxicity signs of ataxia, lethargy, and tremor similar to those reported in dogs with the ABCB1-1∆ mutation. Microarray analysis showed gene expression was altered in ABCB1-1∆ mutant mice following administration of P-gp substrates as compared to ABCB1-WT mice. Gene pathway analysis revealed that the altered genes were associated with behavior and nervous system development and function. Genes such as Gabrq, Dao, and albumin are potential biomarkers of neurotoxicity that might be used to predict the safety of P-gp substrates in dogs with the ABCB1-1∆ mutation.

16. Publication Min Zhu, Yi Ming, Heidi Swaim, Marla Swain, Michael Myers, Christine Deaver, Yolanda Jones, Xiaolin Wu, Robert Stephens, and Haile Yancy. Identifying biomarkers of neurotoxicity to predict the safety of P-glycoprotein substrates in transgenic mice expressing the canine ABCB1-1∆ mutant gene. American Journal of Veterinary Research, 2014. In press now. Disclaimers The experimental protocol was approved by the Animal Care and Use Committee at the Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (2011) and the Animal Welfare Act of 1966 (P.L. 89-544), as amended.

17. Acknowledgements U.S. Food and Drug Administration (FDA)/Center for Veterinary Medicine Dr. Haile Yancy Dr. Marla Swain Dr. Mike Myers Ms. Heidi Swaim Ms. Christine Deaver Ms. Yolanda Jones U.S. National Institutes of Health (NIH) Dr. Yi Ming Dr. Xiaolin Wu FDA/NIH inter-agency agreements

18. Muchas Gracias a todos!