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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 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 Objectives of the Study • To study the impact of canine ABCB1 gene mutation on drug safety (Pgp 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 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. Background A transgenic mouse model expressing canine ABCB1 gene • 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 (ABCB1WT) were developed previously at FDA. Orzechowski KL, Yancy HF et al, Am J Vet Res 2012;73:1477-84 • This mouse model has the potential to be used in lieu of ivermectinsensitive Collies to assess the safety of P-gp substrate drugs. Pharmacogenomics Study Design in Animals Administration of P-gp substrates in mice: Ivermectin, Doramectin, Moxidectin, Digoxin Canine ABCB1-WT Canine ABCB1-Mutant Mouse Gene 1.0 ST Array Whole-genome gene expression microarray Gene pathway analysis Biomarker discovery A numerical scoring system developed to evaluate clinical signs of neurotoxicity Scores of 0, 1, 2, and 3 were assigned for no, mild, moderate, and severe clinical signs, respectively. Swain MD, Yancy HF et al. Res Vet Sci 2013;94: 656-661. ABCB1-1∆ mutant mice displayed neurotoxic clinical signs following administration of P-gp substrates. Mutant ID KI090 KI091 KI092 KI094 KI095 KI096 KI100 KI101 Sex M M M F F F M M Body weight (g) 30.6 33.3 31.8 24 27.9 24.7 39.6 39.7 P-gp substrate Digoxin Digoxin Doramectin Doramectin Moxidectin Moxidectin Ivermectin Ivermectin Ataxia 3 3 NA** NA 1 1 2 1 Lethargy 2 3 NA NA 1 2 2 0 Tremors 1 0 NA NA 0 0 1 0 Mice were euthanized at 6 hr due to severe clinical signs and undue pain or distress. • 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. Hierarchical clustering Analysis of Gene expression of ABCB1-1∆ mutant and ABCB1 wild-type mice administered P-gp substrate drugs Mutant Wild-type Genotype Drug treatment 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) Ivermectin 272 Digoxin 372 Doramectin 363 Moxidectin 1612 *Gene expression cut-off: ≥ 2-fold gene expression fold change Gene Pathway Analysis Digoxin 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. 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. Potential biomarkers Top of the commonly altered genes in ABCB1-1∆ mutant mice from all four drug treatment groups Fold change (Mutant/Wild-type) Gene GO biological process Gene Symbol Accession No. Ivermectin Digoxin Doramectin Moxidectin -14.4 -35.6 -4.1 -132.6 Transport, neuropeptide Glra1 NM_020492 signaling pathway Multicellular organismal Mab21l2 NM_011839 development -5.7 -6.3 -2.1 -45.9 Ebf3 NM_010096 Transcription, DNA-templated -15.3 -11.1 -4.5 -29.0 -15.7 -8.4 -10.8 -22.7 Transport, ion transport, Slc10a4 NM_173403 sodium ion transport Transport, drug transport, Slc18a2 NM_172523 neurotransmitter transport -22.7 -8.0 -8.0 -10.1 Ttr NM_013697 Transport 2.4 -11.5 10.3 -48.1 Dao NM_010018 Dopamine biosynthetic process -3.4 -5.9 -8.5 -25.5 -4.8 -9.8 -4.5 -24.0 Regulation of neuron Klk6 NM_011177 projection development Transport, chloride transport, Gabrq NM_020488 ion transport -15.1 -3.3 -13.4 -10.7 Alb NM_009654 Albumin; drug transport 5.0 110.6 158.5 36.9 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. 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. 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 Muchas Gracias a todos!