Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-12-21
2001-11-27
Kemmerer, Elizabeth (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C514S008100, C514S013800, C514S014800, C514S015800
Reexamination Certificate
active
06323178
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the pharmaceutical and medical arts. In particular the invention pertains to beta-lipotropin, fragments and analogs thereof, pharmaceutical formulations, and methods for using same in treating diabetes and other associated conditions in mammals.
Proopiomelanocortin (POMC) is a neuropeptide precursor molecule which is translocated to secretory pathways within neuroendocrine cells. POMC is cleaved by the action of specific endopeptidases to yield peptides such as adrenocorticotrophic hormone (ACTH), Beta-lipotropin (BLT), Beta-endorphin, and Melanocyte Stimulating Hormone (MSH). The processing of POMC into one or more specific peptides occurs in a tissue and cell specific manner (See generally, M. Castro and E. Morrison,
Crit. Rev. Neurobiol.,
11, 35-57, 1997; Roberts, J. L. and Herbert, E.,
Proc Nat Acad Sci,
74, 4826 (1977); Roberts, J. L. and Herbert, E.,
Proc.Nat.Acad.Sci
74, 5300 (1977); Mains, et al.,
Proc.Nat.Acad.Sci
74, 3014 (1977)). POMC is produced mainly in the pituitary gland and hypothalamus. Post-translational processing of POMC in the anterior pituitary produces ACTH and BLT. On the other hand, the major products of the intermediate pituitary are &agr;-MSH, CLIP, &ggr;-lipotropin, &bgr;-endorphin, and &bgr;-MSH, while in the hypothalamus, POMC is processed primarily into &ggr;-MSH and &bgr;-endorphin.
POMC-derived peptides perform a variety of important roles in metabolic and physiological regulation. For example, ACTH, a 39 amino acid peptide, stimulates secretion of glucocorticoids from the adrenal cortex. The MSH's, on the other hand, stimulate melanin synthesis by melanocytes in the skin, and also appear to be involved in fat metabolism. &bgr;-endorphin derives from the carboxyl end of BLT (viz. Residues 59 to 89 of the human sequence), and possesses analgesic activity that is antagonized by naloxone, a known antagonist for morphine. Thus, POMC-derived peptide hormones have diverse roles in physiologic and metabolic regulation.
Proper glucose and fuel metabolism depend on the non-POMC related peptide, insulin. Specifically, insulin stimulates glycogen, fatty acid, and protein synthesis, and also stimulates glycolysis. Insulin is critical in promoting entry of glucose into muscle and fat cells.
Defective insulin metabolism may lead to diabetes. Type 1 diabetics require exogenous insulin administration for proper control of fuel and glucose metabolism. On the other hand, Type 2 diabetics typically do not require exogenous insulin until the later stages of the disease. Proper control of glucose and fuel metabolism is essential for effective management of diabetes. Without this, there can be serious, perhaps even fatal, consequences including ketoacidosis, coma, retinopathy, diabetic microangiopathy, atherosclerosis, myocardial infarction, stroke, gangrene, hypertriglyceridemia, hypercholesterolemia, cardiomyopathy, dermopathy, diabetic foot syndrome, nephropathy, urinary tract infection, papillary necrosis, cataracts, diabetic gastroenteropathy, constipation, peripheral vascular disease, and even death. In many instances, a delicate balance must be struck between administration of too much insulin and too little insulin. Therefore, an ideal therapy for diabetes would be one that controls blood glucose levels by improving sensitivity to insulin.
Described herein is a method for treatment and pharmaceutical composition that is effective in treating or preventing type 1 and type 2 diabetes, and associated complications thereof, comprising the administration of a pharmaceutically effective amount of beta-lipotropin and/or fragments and/or analogs thereof.
BRIEF SUMMARY OF THE INVENTION
The present invention provides isolated proteins comprising beta-lipotropin (BLT), analogs thereof, fragments thereof, nucleic acids encoding same, and methods for producing and using BLT in the treatment of diabetes and complications associated therewith.
Disclosed herein are methods for treating diabetes and complications thereof in mammals, including humans, by administering pharmaceutically effective amounts of BLT, analogs thereof, or functional fragments thereof. The invention relates further to methods of treatment for type 1 and type 2 diabetes, retinopathy, diabetic microangiopathy, atherosclerosis, myocardial infarction, stroke, gangrene, hypertriglyceridemia, hypercholesterolemia, cardiomyopathy, dermopathy, diabetic foot syndrome, nephropathy, urinary tract infection, papillary necrosis, cataracts, diabetic gastroenteropathy, constipation, and peripheral vascular disease.
Disclosed herein are methods for producing BLT in
E. coli
and yeast. In
E. coli
BLT is produced as a fusion protein which can be recovered from cell lysates in the presence of high salt by conventional purification methods. The BLT fusion protein contains a recognition site for specific proteases which are used to separate the fusion partner from BLT. In the yeast
Pichia pastoris
, the fusion protein is cleaved upon secretion of the protein from the cell such that native BLT can be recovered intact from the culture medium.
In one embodiment the present invention relates to a substantially pure protein having the amino acid sequence which is SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 7.
In still another embodiment the present invention relates to an isolated nucleic acid compound encoding a fusion protein or recombinant protein or peptide comprising beta lipotropin.
In another embodiment the present invention relates to at least one isolated nucleic acid compound encoding a protein or peptide of the present invention.
In still another embodiment the present invention relates to a vector comprising an isolated nucleic acid compound of the invention.
In yet another embodiment the present invention relates to a vector comprising an isolated nucleic acid of the invention, wherein said isolated nucleic acid compound is operably-linked to a promoter sequence.
In another embodiment the present invention relates to a host cell containing a vector of the present invention.
In yet another embodiment the present invention relates to a method for constructing a recombinant host cell having the potential to express beta-lipotropin, said method comprising introducing into said host cell by any suitable means a vector of the invention.
In still another embodiment the present invention relates to a method for expressing beta-lipotropin in a recombinant host cell, said method comprising culturing said recombinant host cell under conditions suitable for gene expression.
In another embodiment the present invention relates to a pharmaceutical formulation comprising as an active ingredient beta-lipotropin, analog, or functional fragment thereof, associated with one or more pharmaceutically acceptable carriers, excipients, or diluents therefor.
In still another embodiment the present invention relates to a pharmaceutical formulation wherein said beta-lipotropin, analog, or functional fragment thereof, is human beta-lipotropin.
In yet another embodiment the present invention relates to beta-lipotropin, analog, or functional fragment thereof, for use in treating diabetes or complications thereof.
In still another embodiment the present invention relates to fragments of BLT having insulinotropic activity.
In still another embodiment the present invention relates to a peptide having insulinotropic activity selected from the group consisting of SEQ ID NO:9 through SEQ ID NO:13.
In still another embodiment the present invention relates to a peptide having insulinotropic activity selected from the group consisting of SEQ ID NO:14 through SEQ ID NO:25.
In still another embodiment the present invention relates to a functional analog of a beta-lipotropin peptide disclosed herein.
In still another embodiment the present invention relates to a method for treating diabetes comprising administration of a therapeutically effective amount of at least one peptide selected from the group consisting of SEQ ID NO:9 through SEQ ID NO:25.
In still another embodiment the present
Hale John Edward
Heath, Jr. William Francis
Heiman Mark Louis
Schoner Brigitte Elisabeth
Eli Lilly and Company
Kemmerer Elizabeth
Webster Thomas D.
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