Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-22
2003-03-04
Baker, Anne-Marie (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C530S350000
Reexamination Certificate
active
06528271
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns transgenic mice useful for screening compounds for their ability to control pain, methods of controlling pain in subjects in need thereof, methods of screening a compound for activity in controlling pain, and/or screening compounds for opioid receptor agonist activity.
BACKGROUND OF THE INVENTION
G protein coupled receptors (GPCRs) have important roles in mediating fundamental physiological processes such as vision, olfaction, cardiovascular function, and pain perception. Cellular communication through GPCRs requires the coordination of processes governing receptor activation, desensitization, and resensitization. However, the relative contribution of desensitization mechanisms to the overall homeostatic process still remains largely unexplored in vivo. GPCR kinases (GRKs) act to phosphorylate activated receptors and promote their interaction with &bgr;arrestins. This, in turn, prevents further coupling with G proteins and disrupts normal activation of the second messenger signaling cascade. By this mechanism, GRKs and &bgr;arrestins can act to dampen GPCR signaling, thereby leading to desensitization of the receptor (S. Ferguson, et al.,
Annu Rev Biochem
67, 653 (1998)). At least six GRKs (GRK1-6) and four arrestins (visual and cone arrestin, &bgr;arrestin-1 and -2) have been discovered; however, the functional significance of such redundancy is unclear.
Overexpression or inactivation of certain GRKs leads to modulation of receptor responsiveness (W. Koch, et al.,
Science
268, 1350 (1995); H. Rockman et al.,
Proc Natl Acad Sci USA
93, 9954 (1996); D. Choi et al.
J Biol Chem
272, 17223 (1997); G. Iaccarino et al.,
Am J Physiol
275, H1298 (1998); K. Peppel, et al.,
J Biol Chem
272, 25425 (1997); H. Rockman, et al.,
J Biol Chem
273, 18180 (1998). J. Walker et al.,
Am J Physiol
276, R 1214 (1999)). In addition, mice that are deficient in &bgr;arrestin-1 display increased cardiac contractility in response to &bgr;-adrenergic receptor agonists (D. Conner et al.,
Circ Res
81, 1021 (1997)).
SUMMARY OF THE INVENTION
Pain perception (nociception) is mediated by a cascade of events from the point of the stimulus to integrative circuits in the brain. Nociception involves signals that are mediated by several classes of receptors and signal transduction mechanisms such as GPCRs for substance P, opioid peptides, etc. and ion channels such as NMDA receptors. Antinociception has been known for more than 1000 years to be induced by the alkaloid compound, morphine, which functions as an agonist at the &mgr; opioid receptor. The activity of agonists for signaling through GPCRs is usually limited by cellular mechanisms that dampen the signal of the agonist, a process referred to as desensitization. These mechanisms include phosphorylation of agonist-activated receptors by specific receptor kinases called GRKs followed by the interaction of the phosphorylated GPCR with any of the members of the arrestin family of proteins. Morphine-mediated antinociception is known to wane with time, however the contribution of the desensitization is controversial and for all practical purposes is unknown. With the &bgr;arrestin knockout mice disclosed herein, it is shown that interfering with (eliminating) one of the key protein components of the desensitization mechanism greatly enhances the potency and efficacy of the antinociceptive properties of morphine.
Accordingly, a first aspect of the present invention is a knockout mouse useful for testing the efficacy of potential analgesic agents, the cells of said mouse containing at least one inactive endogenous &bgr;arrestin gene (preferably the &bgr;arrestin-2 gene), the mouse exhibiting a phenotype of decreased sensitivity to pain after administration of a &mgr; opioid receptor agonist such as morphine as compared to the corresponding wild type mouse. The mouse may be heterozygous or homozygous for the inactive endogenous &bgr;arrestin gene. The mouse is useful for evaluating potential analgesic drugs, and particularly for evaluating the contribution of the desensitization mechanisms to the antinociceptive effects of endogenous opioids.
A second aspect of the invention is a method of controlling pain in a subject. The method comprises inhibiting &bgr;arrestin binding to the phosphorylated &mgr; opioid receptor in said subject in an amount effective to induce or enhance analgesia in the subject. The method may be carried out with or without concurrently administering a &mgr; opioid receptor agonist (typically an opiate such as morphine) to said subject.
A third aspect of the present invention is a method of screening a compound for activity in potentiating &mgr; opioid receptor agonist activity (e.g., morphine activity). The method comprises determining whether or not the compound inhibits &bgr;arrestin binding to a phosphorylated &mgr; opioid receptor. The inhibition of such binding by the compound indicates the compound is active in potentiating &mgr; opioid receptor agonist activity.
A particular aspect of the present invention is a method of screening a compound for activity in controlling pain. The method comprises determining whether or not the compound inhibits &bgr;arrestin binding to phosphorylated &mgr; opioid receptor. The inhibition of such binding by the compound indicates the compound is active in controlling pain (i.e., is a candidate compound for controlling pain, and should be subjected to further screening and testing for pain control). Any degree of inhibition may be examined, with greater inhibition of binding indicating potentially greater activity of the compound being tested.
Further aspects of the present invention include compounds produced or identified by the methods described hereinabove and pharmaceutical formulations of the same, along with the use of such compounds for the preparation of a medicament for the potentiation of the activity of &mgr; opioid receptor agonists such as morphine, and/or for the control of pain, in a subject in need thereof, either alone or in combination with a &mgr; opioid receptor agonist such as morphine.
The foregoing and other objects and aspects of the present invention are explained in detail in the drawings herein and the specification set forth below.
REFERENCES:
patent: 5591618 (1997-01-01), Chantry et al.
patent: 5597699 (1997-01-01), Lanzara
patent: 5658783 (1997-08-01), Grandy et al.
patent: 5661184 (1997-08-01), Helton et al.
patent: 5821067 (1998-10-01), Grandy et al.
patent: 5882944 (1999-03-01), Sadee
patent: 5891646 (1999-04-01), Barak et al.
patent: 6007986 (1999-12-01), Sadée
patent: RE36547 (2000-02-01), Crain et al.
patent: 6028175 (2000-02-01), Grandy et al.
patent: 6087115 (2000-07-01), Gershengorn et al.
patent: 6096756 (2000-08-01), Crain et al.
patent: 6103492 (2000-08-01), Yu
patent: 6110693 (2000-08-01), Barak et al.
patent: WO98/55635 (1998-12-01), None
Bohn, Laura M.,Enchanced Morphine Analgesia in Mice Lacking &bgr;-Arrestin 2, Science, (Washington D.C.), vol. 286, No. 5449, Dec. 24, 1999, pp. 2495-2498.
Chen, Jeannie, et al.,Increased Susceptibility to Light Damage in an Arrestin Knockout Mouse Mode of Oguchi Disease(Stationary Night Blindness),Investigative Ophthalmology&Visual Science, vol. 40, No. 12, Nov. 1999, pp. 2978-2982.
Kovoor, Abraham, et al.,&mgr; and &sgr; Opioid Receptors Are Differentially Desensitized by the Coexpression of &bgr;-Adrenergic Receptor Kinase 2 and &bgr;-Arrestin 2 in Xenopus Oocytes, The Journal of Biological Chemistry, (U.S.A.), vol. 272, No. 44, Oct. 31, 1997, pp. 27605-27611.
Mathier, Michael, A. et al.,Enhanced Left Ventricular Contractile Responses to Acute &bgr;-Adrenergic Stimulation in a &bgr;-Arrestin 1 Knockout Mouse, Scientific Sessions of the American Heart Assoiciation, Orlando, Florida, US, vol. 96, No. 8, Suppl, 1997, XP-000983952, p. I-445.
Schulz, Rüdiger, et al.,Phosducin, &bgr;-arrestin and Opioid receptor migration, European Journal of Pharmacology, vol., 375, No. 1-3, Elsevier Science B.V., Jun. 30, 1999, pp. 349-357.
Whistler, Jennifer, L., et
Bohn Laura M.
Caron Marc G.
Lefkowitz Robert J.
Lin Fang-Tsyr
Baker Anne-Marie
Burns Doane Swecker & Mathis L.L.P.
Duke University
LandOfFree
Inhibition of &bgr;arrestin mediated effects prolongs and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Inhibition of &bgr;arrestin mediated effects prolongs and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Inhibition of &bgr;arrestin mediated effects prolongs and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3059435