Generation and Characterization of a Human Bradykinin Receptor B1 Transgenic Rat as a Pharmacodynamic Model
- J. Fred Hess,
- Richard W. Ransom,
- Zhizhen Zeng,
- Raymond S. L. Chang,
- Patricia J. Hey,
- Lee Warren,
- Charles M. Harrell,
- Kathryn L. Murphy,
- Tsing-Bau Chen,
- Patricia J. Miller,
- Edward Lis,
- Duane Reiss,
- Raymond E. Gibson,
- M. Kristine Markowitz,
- Robert M. DiPardo,
- Dai-Shi Su,
- Mark G. Bock,
- Robert J. Gould and
- Douglas J. Pettibone
- Departments of Molecular Neurology (J.F.H, R.W.R., P.J.H., L.W., C.M.H., K.L.M., T.-B.C., P.J.M., E.L., D.R., D.J.P), Imaging (Z.Z., R.S.L.C., R.E.G), Medicinal Chemistry (M.K.M., R.M.D., D.-S.S., M.G.B), and Biomedical Research (R.J.G.) Merck Research Laboratories, West Point, Pennsylvania
- Address correspondence to:
Dr. J. Fred Hess, Merck Research Lab, WP26A-3000, P.O. Box 4, West Point PA 19486. E-mail: fred_hess{at}merck.com
Abstract
Antagonists of the B1 bradykinin receptor (B1R) offer the promise of novel therapeutic agents for the treatment of inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the human B1R. To circumvent these issues, we generated a transgenic rat expressing the human B1R under the control of the neuron-specific enolase promoter. Membranes prepared from whole brain homogenates of heterozygous transgenic rats indicate a B1R expression level of 30 to 40 fmol/mg; there is no detectable B1R expression in control nontransgenic rats. The pharmacological profile of the B1R expressed in the transgenic rat matches that expected of the human, but not the rat receptor. The mapping of the transgene insertion site to rat chromosome 1 permitted the development of a reliable assay for the identification of homozygous transgenic rats. Significantly, homozygous transgenic rats express 2-fold more B1R than heterozygous animals. Autoradiographic analyses of tissue sections from transgenic rats reveal that the B1R is broadly expressed in both the brain and spinal cord. The human B1R expressed in the transgenic rat functions in an in vitro contractile assay and thus has the potential to elicit a functional response in vivo. Using the humanized B1R transgenic rat, an assay was developed that is suitable for the routine evaluation of a test compound's ability to occupy the human B1R in the central nervous system.
Footnotes
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This work was presented in abstract form at the 2003 Annual Meeting of the Society for Neuroscience in New Orleans, LA.
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DOI: 10.1124/jpet.104.066886.
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ABBREVIATIONS: B1R, bradykinin receptor B1; B2R, bradykinin receptor B2; CNS, central nervous system; NSE, neuron-specific enolase; PCR, polymerase chain reaction; CMV, cytomegalovirus; kb, kilobase; CHO, Chinese hamster ovary; RT, room temperature; DALK, des-Arg10, Leu9kallidin; DAK, des-Arg10 kallidin; DABK, des-Arg9 bradykinin; LPS, lipopolysaccharide; compound A, N-{2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl}-2-[(2R)-1-(2-naphthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide; compound B, (3R)-3-(3,4-dichlorophenyl)-N-{(1R)-1-[4-(4,5-dihydro-1H-imidazol-2-yl)benzyl]-2-oxo-2-pyrrolidin-1-ylethyl}-3-[(2-naphthylsulfonyl)amino]propanamide; compound C, 2-{(2R)-1-[(3,4-dichlorophenyl)sulfonyl]-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl}-N-{2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl}acetamide; compound D, 3-{[(2,2-difluorocyclopropyl)carbonyl]amino}-N-{(1R)-1-[2′-(methoxycarbonyl)-5′-methylbiphenyl-4-yl]ethyl}-4-methylpyridin-2-aminium chloride; compound E, N-[4-chloro-2-({(1R)-1-[3′-fluoro-2′-(3-methyl-1,2,4-oxadiazol-5-yl)biphenyl-4-yl]ethyl}amino)pyridin-3-yl]-3,3,3-trifluoropropanamide.
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- Accepted March 29, 2004.
- Received February 12, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
