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Details for Patent: 5,264,372

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Details for Patent: 5,264,372

Title: Receptor-based screening methods for amylin agonists and antagonists
Abstract:Methods for identifying or screening or characterizing or assaying or isolating known or candidate agonists and antagonists of amylin, comprising binding assays utilizing preparations containing specific receptors for amylin. Membranes from the brain that contain high density receptors for amylin are particularly useful for the methods of this invention, and as a source of amylin receptors.
Inventor(s): Beaumont; Kevin (San Diego, CA), Rink; Timothy J. (San Diego, CA)
Assignee: Amylin Pharmaceuticals, Inc. (San Diego, CA)
Filing Date:Mar 15, 1991
Application Number:07/670,231
Claims:1. An assay method for use in identifying or screening for compounds which inhibit the binding of amylin to amylin receptors, which comprises the steps of:

(a) bringing together a test sample and an amylin receptor preparation, said test sample containing one or more test compounds, and said amylin receptor preparation comprising an isolated cell or membrane preparation containing amylin receptors capable of detectably binding amylin and having a relative ligand binding profile such that the binding potency of amylin and salmon calcitonin to said receptor preparation is greater than the binding potency of CGRP, the binding potency of CGRP to said receptor preparation is greater than the binding potency of a mammalian calcitonin, said binding potency of said ligands being measured by determining the ability of said ligands to compete with an amylin ligand for binding to said amylin receptor preparation;

(b) incubating said test sample and said amylin receptor preparation under conditions which would permit the binding of amylin to said amylin receptors; and,

(c) determining the presence or amount of binding between said test sample and said amylin receptor preparation in order to identify those test samples containing one or more test compounds which detectably bind to said amylin receptors.

2. The assay method of claim 1 which further comprises,

(d) screening said test samples which detectably bind to said amylin receptors for in vitro or in vivo stimulation or inhibition of amylin-mediated activity; and,

(e) determining the presence or amount of stimulation or inhibition of amylin-mediated activity in order to identify those test samples which act as agonists or antagonists of amylin.

3. The assay method of claim 2 wherein said test samples which detectably bind to said amylin receptor protein are identified by measuring the displacement of a labelled first ligand from said amylin receptor preparation by said test sample, and comparing the measured displacement of said first labelled ligand from said amylin receptor preparation by said test sample with the measured displacement of said first labelled ligand from said amylin receptor preparation by one or more known second ligands.

4. The assay method of claim 1 wherein said amylin receptor preparation comprises isolated cells.

5. The assay method of claim 3 wherein said cells bearing said amylin receptor are brain cells.

6. The assay method of claim 5 wherein said brain cells are from the rat basal forebrain.

7. The assay method of claim 1 wherein said amylin receptor preparation comprises a membrane preparation.

8. The assay method of claim 7 wherein said membrane preparation is produced from brain cells.

9. The assay method of claim 8 wherein said brain cells are from the rat basal forebrain.

10. The assay method of claim 1 wherein said test samples which detectably bind to said amylin receptor protein are identified by measuring the displacement of a labelled first ligand form said amylin receptor preparation by said test sample, and comparing the measured displacement of said first labelled ligand from said amylin receptor preparation by said test sample with the measured displacement of said first labelled ligand from said amylin receptor preparation by one or more known second ligands.

11. The assay method of claim 10 wherein said labelled first ligand is amylin.

12. The assay method of claim 11 wherein said amylin is rat amylin.

13. The assay method of claim 12 wherein said rat amylin is .sup.125 I rat amylin.

14. The assay method of claim 10 wherein said labelled first ligand is an amylin agonist.

15. The assay method of claim 14 wherein said amylin agonist is selected from the group consisting of CGRP, salmon calcitonin, and eel calcitonin.

16. The assay method of claim 10 wherein said labelled first ligand is an amylin antagonist.

17. The assay method of claim 16 wherein said amylin antagonist is CGRP.sub.8-37.

18. The assay method of claim 10 wherein said first ligand is labelled with a member selected from the group consisting of radioactive isotopes, nonradioactive isotopes, fluorescent molecules, chemiluminescent molecules, and biotinylated molecules.

19. The assay method of any of claims 10, 11, 14, 16 or 18 wherein said known second ligand or ligands are selected from the group consisting of amylin, calcitonin, .alpha.CGRP, and .beta.CGRP.

20. The assay method of any of claims 10, 11, 14, 16 or 18 wherein said known second ligand or ligands are selected from the group consisting of human amylin, dog amylin, rat amylin, human calcitonin, rat calcitonin, eel calcitonin, salmon calcitonin, human .alpha.-CGRP, human .beta.-CGRP, rat.alpha.-CGRP, and rat .beta.-CGRP.

21. The assay method of any of claims 1, 2 or 10 wherein said test sample comprises one or more known test compounds.

22. The assay method of any of claims 1, 2 or 10 wherein said test sample comprises one or more unknown compounds.

23. The assay method of claim 1 wherein said compounds which inhibit the binding of amylin to amylin receptors are the agonists or antagonists of amylin.

24. The assay method of any of claims 1, 26, 27, 28, 37 or 38 wherein said mammalian calcitonin is rat calcitonin.

25. The assay method of any of claims 1, 26, 27, 28, 37 or 38 wherein said mammalian calcitonin is human calcitonin.

26. An assay method for use in identifying or screening for compounds which inhibit the binding of amylin to amylin receptors, which comprises the steps of:

(a) bringing together a test sample and an amylin receptor preparation, said test sample containing one or more test compounds, and said amylin receptor preparation comprising an isolated cell or membrane preparation containing amylin receptors capable of detectably binding amylin and having a relative ligand binding profile such that the binding potency of amylin and salmon calcitonin to said receptor preparation is greater than the binding potency of CGRP, the binding potency of CGRP to said receptor preparation is greater than the binding potency of a mammalian calcitonin, said binding potency of said ligands being measured by determining the ability of said ligands to compete with an amylin ligand for binding to said amylin receptor preparation;

(b) incubating said test sample and said amylin receptor preparation under conditions which would permit the binding of amylin to said amylin receptors;

(c) determining the presence or amount of binding between said test sample and said amylin receptor preparation in order to identify those test samples containing one or more test compounds which detectably bind to said amylin receptors;

(d) preparing two or more additional test samples from said test sample or samples that are determined to contain one or more test compounds which detectably bind to said amylin receptors, said additional test samples being characterized in that they contain a lesser number of test compounds that said test sample from which they were prepared; and,

(e) repeating steps (a)-(d) as many times as desired or until the test compound or compounds which bind to said amylin receptors have been identified.

27. An assay method for use in identifying or screening for compounds which inhibit the binding of amylin to amylin receptors which comprises the steps of,

(a) bringing together a test sample and an amylin receptor preparation, said test sample containing one or more test compounds, and said amylin receptor preparation comprising an isolated cell or membrane preparation containing amylin receptors capable of detectably binding amylin and having a relative ligand binding profile such that the binding potency of amylin and salmon calcitonin to said receptor preparation is greater than the binding potency of CGRP, the binding potency of CGRP to said receptor preparation is greater than the binding potency of a mammalian calcitonin, said binding potency of said ligands being measured by determining the ability of said ligands to compete with an amylin ligand for binding to said amylin receptor preparation;

(b) incubating said test sample and said amylin receptor preparation under conditions which would permit the binding of amylin to said amylin receptors;

(c) determining the presence or amount of binding between said test sample and said amylin receptor preparation in order to identify those test samples containing one or more test compounds which detectably bind to said amylin receptors;

(d) screening said test samples which detectably bind to said amylin receptors for in vitro or in vivo stimulation or inhibition of amylin-mediated activity;

(e) determining the presence or amount of stimulation or inhibition of amylin-mediated activity in order to identify those test samples which act as agonists or antagonists of amylin,

(f) preparing two or more additional test samples from said test sample or samples that are determined to stimulate or inhibit amylin-mediated activity, said additional test samples being characterized in that they contain a lesser number of test compounds than said test sample from which they were prepared; and,

(g) repeating steps (a)-(f) as many times as desired or until the test compound or compounds which bind to said amylin receptors have been identified.

28. An assay method for evaluating a known or candidate amylin agonist or antagonist compound for receptor binding selectivity, which comprises the steps of:

(a) bringing togehter said compound, a labelled amylin receptor ligand, and an amylin receptor preparation comprising an isolated cell or membrane preparation containing amylin receptors capable of detectably binding amylin, and determining or measuring the ability of said compound to compete against said labelled amylin receptor ligand for binding to said amylin receptor preparation, said amylin receptor preparation having a relative ligand binding profile such that the binding potency of amylin and salmon calcitonin to said receptor preparation is greater than the binding potency of CGRP, the binding potency of CGRP to said receptor preparation is greater than the binding potency of a mammalian calcitonin, said binding potency of said ligands being measured by determining the ability of said ligands to compete with an amylin ligand for binding to said amylin receptor preparation;

(b) brining together said compound, a labelled CGRP receptor ligand, and a CGRP receptor preparation, and determining or measuring the ability of said compound to compete against said labelled CGRP receptor ligand for binding to said CGRP receptor preparation; and/or,

(c) bringing together said compound, a labelled calcitonin receptor ligand, and a calcitonin receptor preparation, and determining or measuring the ability of said compound to compete against said labelled calcitonin receptor ligand for binding to said calcitonin receptor preparation; and,

(d) comparing the results of steps (a) and (b) and/or (c) in order to determine the receptor binding selectivity of said compound.

29. The assay method of claim 1 wherein said CGRP receptor preparation comprises hepatocytes.

30. The assay method of claim 29 wherein said hepatocytes are comprised of a primary cell culture or an established cell line.

31. The assay method of claim 30 wherein said hepatocytes comprise an established cell line, and said established hepatocyte cell line is the Hep G2 cell line.

32. The assay method of claim 28 wherein said CGRP receptor preparation comprises Hepatocyte membranes.

33. The assay method of claim 28 wherein said CGRP receptor preparation comprises myocytes.

34. The assay method of claim 33 wherein said myocytes are comprised of a primary cell culture or an established cell line.

35. The assay method of claim 28 wherein said CGRP receptor preparation comprises myocyte membranes.

36. The assay method of claim 35 wherein said myocytes comprise an established cell line, and said established cell line is the L6 cell line.

37. An assay method for evaluating a known candidate amylin agonist or antagonist for amylin receptor binding affinity, which comprises bringing together said compound, a labelled amylin receptor ligand, and an amylin receptor preparation, said amylin preparation comprising an isolated cell or membrane preparation containing amylin receptors capable of detectably binding amylin and having a relative ligand binding profile such that the binding potency of amylin and salmon calcitonin to said receptor preparation is greater than the binding potency of CGRP, the binding potency of CGRP to said receptor preparation is greater than the binding potency of a mammalian calcitonin, said binding potency of said ligands being measured by determining the ability of said ligands to compete with any amylin ligand for binding to said amylin receptor preparation, and determining or measuring the ability of said compound to compete against said labelled amylin receptor ligand for binding to said amylin receptor preparation.

38. An assay method for determining the presence or amount of any amylin receptor binding compound in a test sample to be assayed for said compound, which comprises the steps of:

(a) bringing together said test sample to be assayed and an amylin receptor preparation, said amylin receptor preparation comprising an isolated cell or membrane preparation containing amylin receptors capable of detectably binding amylin and having a relative ligand binding profile such that the binding potency of amylin and salmon calcitonin to said receptor preparation is greater than the binding potency of CGRP, the binding potency of CGRP to said receptor preparation is greater than the binding potency of a mammalian calcitonin, said binding potency of said ligands being measured by determining the ability of said ligands to compete with an amylin ligand for binding to said amylin receptor preparation;

(b) measuring the binding of said test sample to said amylin receptor preparation in the presence of a labelled amylin receptor ligand in order to determine the presence or amount of amylin receptor binding compound in said test sample.

39. The assay method of claim 38 wherein said amylin receptor binding compound is amylin.

40. The assay method of claim 38 wherein said amylin receptor binding compound is an amylin agonist.

41. The assay method of claim 38 wherein said amylin receptor binding compound is an amylin antagonist.

42. The assay method of claim 38 wherein said labelled ligand is amylin.

43. The assay method of claim 38 wherein said labelled ligand is an amylin agonist.

44. The assay method of claim 38 wherein said labelled ligand is an amylin antagonist.

45. The assay method of claim 38 wherein said amylin receptor binding compound is amylin, and said labelled ligand is amylin.

46. The assay method of claim 38 wherein said amylin receptor binding compound is an amylin agonist and said labelled ligand is an amylin agonist.

47. The assay method of claim 38 wherein said amylin receptor binding compound is an amylin antagonist and the labelled ligand is an amylin antagonist.

48. The assay method of claim 38 wherein said test sample is a biological fluid.

49. The assay method of claim 48 wherein said biological fluid is a member selected from the group consisting of blood, plasma, urine, cerebrospinal fluid, and lymph fluid.

50. The assay method of claim 38 wherein said test sample is an amylin preparation.

51. A process which comprises the use of the assay method of claim 38 to evaluate the stability of an amylin preparation.

52. A process which comprises the use of the assay method of claim 38 to evaluate the potency of an amylin preparation.

53. A process which comprises the use of the assay method of claim 38 to evaluate the solubility of an amylin preparation.

54. The assay method of claim 38 which further comprises,

(c) relating the amount of amylin receptor binding compound in said test sample with the amount of amylin receptor binding compound measured for a control sample in accordance with steps (a) and (b), said control sample being known to be free of any amylin receptor binding compound, and/or relating the amount of amylin receptor binding compound in said test sample with the amounts of amylin receptor binding compound measured for control samples containing known amounts of amylin receptor binding compound in accord acne with steps (a) and (b), to determine the presence or amount of amylin receptor binding compound in said test sample.

55. An assay method for use in identifying or screening for compounds which inhibit the binding of amylin or amylin receptors which comprises the steps of:

(a) bringing together a test sample and a receptor preparation, said test sample containing one or more test compounds, and said receptor preparation having a relative ligand binding profile such that the binding potency of amylin and salmon calcitonin to said receptor preparation is greater than the binding potency of CGRP, the binding potency of CGRP to said receptor preparation is greater than the binding potency of a mammalian calcitonin, said binding potency of said ligands being measured by determining the ability of said ligands to compete with an amylin ligand for binding to said amylin receptor preparation;

(b) incubating said test sample and said amylin receptor preparation under conditions which would permit the binding of amylin to said amylin receptors; and,

(c) determining the presence or amount of binding between said test sample and said amylin receptor preparation in order to identify those test samples containing one or more test compounds which detectably bind to said amylin receptors.

56. The method of claim 55 wherein said mammalian calcitonin is rat calcitonin.

57. The method of claim 55 wherein said mammalian calcitonin is human calcitonin.

58. The method of claim 55 wherein said amylin receptor preparation comprises an isolated cell preparation.

59. The method of claim 55 wherein said amylin receptor preparation comprises a membrane preparation.

60. An assay method for use in identifying or screening for compounds which inhibit the binding of amylin to amylin receptors which comprises the steps of:

(a) bringing together a test sample and an amylin receptor preparation, said test sample containing one or more test compounds, and said amylin receptor preparation comprising an isolated receptor protein preparation containing amylin receptors capable of binding amylin;

(b) incubating said test sample and said amylin receptor preparation under conditions which would permit the binding of amylin to said amylin receptors; and,

(c) determining the presence or amount of binding between said test sample and said amylin receptor preparation in order to identify those test samples containing one or more test compounds which detectably bind to said amylin receptors.

61. The assay method of claim 60 wherein said receptor protein is isolated from brain cells.

62. The assay method of claim 61 wherein said brain cells are from the rat basal forebrain.

63. An assay method for use in identifying or screening for compounds which inhibit the binding of amylin to amylin receptors which comprises the steps of:

(a) bringing together a test sample and a receptor preparation, said test sample containing one or more test compounds, and said receptor preparation consisting essentially of amylin receptors capable of binding amylin;

(b) incubating said test sample and said receptor preparation under conditions which would permit the binding of amylin to said amylin receptors; and,

(c) determining the presence or amount of binding between said test sample and said receptor preparation in order to identify those test samples containing one or more test compounds which detectably bind to said amylin receptors.
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