Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-10-31
2004-08-10
Kunz, Gary (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600
Reexamination Certificate
active
06774109
ABSTRACT:
BACKGROUND OF THE INVENTION
Amylin, a 37-amino acid polypeptide structurally related to calcitonin gene-related protein [Cooper, Endocrine Review, 15:163 (1994)], is primarily synthesized, packaged, and secreted from the &bgr;-cells of pancreatic islets. Apart from the islet of Langerhans, amylin-like immunoreactivity activity has also been detected in lung, gastrointestinal tract, and the nervous system. See Miyazato, M., et. al., Bioch. Bioph. Res. Comm., 181:293 (1991); Chance, et al., Brain Res., 539:352 (1991); Mulder, et al., Gastroenterology, 107:712 (1994).
The presence of an abnormally high concentration of amylin in the blood, i.e., hyperamylinemia, has been found in patients with pancreatic cancer [Permert, et al., N. Engl. J. Med, 330:313 (1994)], obese patients [Huang, et al., Hypertension, 19 (Supp. I):101 (1992)], and in prediabetic patients [Erickson, J., Diabetologia, 35:291 (1992)]. The hyperamylinemic state has been associated with both diabetes as well as amyloid formations. DeKoning, et al., Proc. Natl. Acad. Sci. USA, 91:8467 (1994). Amyloid formation causes destruction of &bgr;-islet cells and eventually pancreatic dysfunction. Johnson, et al., Lab. Invest. 66:522 (1992); Lorenzo, et al., Nature, 368:756 (1994).
Native somatostatin is comprised of both a 14-amino acid isoform (somatostatin-14) and a 18-amino acid isoform (somatostatin-28). Reichlin, New Eng. J. Medicine, 309(24):1495 (1983). Five distinct somatostatin receptors have been identified and characterized. Hoyer, et al., Naunyn-Schmiedeberg's Arch. Pharmacol., 350:441 (1994). Somatostatin produces a variety of effects, including modulation of hormone release, e.g., growth hormone, glucagon, insulin, amylin, and neurotransmitter release. Some of these effects have been associated with its binding to a specific somatostatin receptor. For example, the inhibition of growth hormone has been attributed to somatostatin type-2 receptor (“SSTR-2”). Raynor, et al., Molecular Pharmacol. 43:838 (1993); Lloyd, et al. Am. J. Physiol. 268:G102 (1995). Because of the short half-life of the native somatostatin, various somatostatin analogs have been developed, e.g., for the treatment of acromegaly. Raynor, et al., Molecular Pharmacol. 43:838 (1993).
SUMMARY OF THE INVENTION
The present invention is based on the discovery that ligands selective for somatostatin type-5 receptor (“SSTR-5”) are effective in inhibiting release of amylin from pancreas cells.
An aspect of this invention relates to a method of determining the ability of a compound to both bind to somatostatin type-5 receptor and inhibit amylin release from amylin-secreting pancreas cells. The method includes the steps of (i) obtaining a preparation, either a cell preparation or a membrane preparation, which contains SSTR-5; (ii) incubating the preparation, the compound, and a SSTR-5 ligand, at least one of the ligand and the compound being detectably labeled; (iii) determining the ability of the compound to compete against the ligand for binding to SSTR-5; (iv) if and only if the compound is determined to be able to bind to SSTR-5, obtaining amylin-secreting pancreatic cells (e.g., cells in an intact pancreas from a rodent such as a rat or a mouse, pancreatic islet cells such as &bgr; cells, or amylinoma cells); (v) incubating the compound, the pancreatic cells, and an amylin release stimulator (e.g., glucose or D-glyceraldehyde) under conditions in which the amylin release stimulator would induce release of amylin from the pancreatic cells; and (vi) determining the ability of the compound to inhibit amylin release.
By “SSTR-5 ligand” is meant a compound which binds to SSTR-5, e.g., somatostatin-14, somatostatin-28, an analog of somatostatin-14 or somatostatin-28 (such as [Tyr
11
]-somatostatin-14), or an antibody raised against of SSTR-5. Either the ligand or the test compound can be labeled with a radioactive isotope, or a nonradioactive (e.g., fluorescent, chemiluminescent, or biotinylated) molecule. Preferably, [
125
I-Tyr
11
]-somatostatin-14 is used as a labeled SSTR-5 ligand to practice the above-described method. Other examples of labeled ligands include
125
I-LTT-somatostatin-28 [Patel, et al., Endocrinol., 135(6):2814 (1994)] and
125
I-CGP 23996 [Raynor, et al., Mol. Pharm. 44:385-392 (1993)].
Examples of the pancreatic cells which are used to practice the above method include both rodent and human islet cells (e.g., &bgr; and &dgr; cells) and pancreatic tumor cells (e.g., amylinoma cells). The pancreatic cells can be incubated either in vitro or in vivo. Examples of an in vitro system include an isolated rat pancreas, the rat &bgr;-cell line RINm5f, and the hamster &bgr;-cell line H1T-T15. As an example of an in vivo system, Sprague-Dawley or Zucker fatty rats can be used as animal models to test the amylin inhibition activity of test somatostatin analogs. An amylin release stimulator, e.g. 16.7 mm of glucose, can be injected into the animal. The test somatostatin analog is then injected into the animals at various concentrations. Blood samples can be taken from the animal, and the amount of amylin present before and after the injection of the test somatostatin analog can be determined by radioimmunoassay.
By “amylin release stimulator” is meant a compound which stimulates the release of amylin stored in pancreatic cells. Examples of amylin release stimulators include glucose, D-glyceraldehyde, or L-arginine.
The method described above can be used to screen for new compounds capable of inhibiting amylin release from the pancreas in a patient. To promote efficiency, when the method is used in a screening project, two or more test compounds can be linked together as a single sample, and, if necessary, subsequently divided and retested.
Another aspect of this invention relates to a method of treating (i.e., either preventing or ameliorating) hyperamylinemia by inhibiting release of amylin from the pancreas in a subject (i.e., a mammal, such as a human being) who suffers from, or is predisposed to suffer from, hyperamylinemia. The method includes administering (e.g., parenterally, intravenously, subcutaneously, transdermally, intramucously, or via implantation of a sustained release formulation), to the subject an amount of a SSTR-5 agonist, the amount being effective in treating hyperamylinemia, i.e., in lowering amylin levels in the bloodstream by inhibiting the release of amylin from pancreas cells.
What is meant by “SSTR-5 agonist” is a compound which (i) is more selective for SSTR-5 than for SSTR-2, i.e., its K
i
for SSTR-5 is lower than that for SSTR-2 (as determined by one of the receptor binding assays described in the working examples below); and (ii) inhibits the release of amylin from pancreas cells induced by an amylin release stimulator (as determined by one of the functional assays described in the working examples below). Both examples of SSTR-5 agonists and the procedures of selecting them appear in the “Description of the Invention” section below.
The above-described therapeutic method can be used to treat a subject suffering from pancreatic cancer, prediabetic symptoms, or non-insulin dependent diabetics.
An effective amount depends upon the conditions being treated, the route of administration chosen, and the specific activity of the compound used, and ultimately will be decided by the attending physician or veterinarian. While it is possible for a SSTR-5 agonist to be administered as the pure or substantially pure compound, it is preferable to present it as a pharmaceutical formulation or preparation. The formulations to be used in the present invention, for both humans and animals, comprise any of the SSTR-5 agonists to be described below, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier must be “acceptable” in the sense of being compatible with the active ingredient(s) of the formulation (and preferably, capable of stabilizing peptides) and not deleterious to the s
Cawthorne Michael Anthony
Davenport Michelle
Dunmore Simon Jon
Feeney Alan F.
Fish & Richardson
Hayes Robert C.
Kunz Gary
Morrill Brian R.
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