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 ResearchCenter 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 (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

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

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.

Canine ABCB1-WT Canine 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 ArrayWhole-genome gene expression microarray

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.

     Mutant ID Sex Body weight (g) P-gp substrate Ataxia Lethargy TremorsKI090 M 30.6 Digoxin 3 2 1KI091 M 33.3 Digoxin 3 3 0KI092 M 31.8 Doramectin NA** NA NAKI094 F 24 Doramectin NA NA NAKI095 F 27.9 Moxidectin 1 1 0KI096 F 24.7 Moxidectin 1 2 0KI100 M 39.6 Ivermectin 2 2 1KI101 M 39.7 Ivermectin 1 0 0

• 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.

Hierarchical clustering Analysis of Gene expression of ABCB1-1∆ mutant and ABCB1 wild-type mice administered P-gp substrate drugs

GenotypeDrug treatment

Mutant Wild-type

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.

Gene Symbol

Gene

Accession No.

GO biological process

Fold change (Mutant/Wild-type)

Ivermectin Digoxin Doramectin Moxidectin

Glra1 NM_020492

Transport, neuropeptide

signaling pathway -14.4 -35.6 -4.1 -132.6

Mab21l2 NM_011839

Multicellular organismal

development -5.7 -6.3 -2.1 -45.9

Ebf3 NM_010096 Transcription, DNA-templated -15.3 -11.1 -4.5 -29.0

Slc10a4 NM_173403

Transport, ion transport,

sodium ion transport -15.7 -8.4 -10.8 -22.7

Slc18a2 NM_172523

Transport, drug transport,

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

Klk6 NM_011177

Regulation of neuron

projection development -4.8 -9.8 -4.5 -24.0

Gabrq NM_020488

Transport, chloride transport,

ion transport -15.1 -3.3 -13.4 -10.7

Alb NM_009654 Albumin; drug transport 5.0 110.6 158.5 36.9

Potential biomarkers Top of the commonly altered genes in ABCB1-1∆ mutant mice from all four drug treatment groups

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 YancyDr. Marla SwainDr. Mike MyersMs. Heidi SwaimMs. Christine DeaverMs. Yolanda Jones

•U.S. National Institutes of Health (NIH)Dr. Yi MingDr. Xiaolin Wu

•FDA/NIH inter-agency agreements

Muchas Gracias a todos!