Transcript Document

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!