Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-02
2003-11-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S458000, C435S459000, C435S461000, C435S366000
Reexamination Certificate
active
06642003
ABSTRACT:
FIELD OF THE INVENTION
Embodiments of the present invention are directed to a human pancreatic cell line transfected so as to express &bgr;-cell differentiation factor IDX-1. Cells so transfected respond to glucagon-like peptide-1 by differentiating into insulin-secreting &bgr; cells.
Embodiments of the present invention are directed to a human pancreatic cell line transfected so as to express &bgr;-cell differentiation factor IDX-1. Cells so transfected respond to glucogon-like/peptide-1 by differentiating into insulin-secreting &bgr;cells.
BACKGROUND OF THE INVENTION
Insulin is essential for proper metabolism in humans: in addition to its familiar role as the chief regulator of blood sugar levels in humans, it is essential for carbohydrate, lipid, and protein metabolism, as well. Pancreatic beta (&bgr;) cells of the islets of Langerhans, epithelial cells dispersed throughout the pancreas, secrete insulin. When &bgr; cells are destroyed or their function impaired, insulin production declines, and diabetes results.
The most common form of diabetes, presenting in nearly a million new cases every year in the United States, is type II diabetes. Type II refers to a group of disorders characterized by high blood levels of glucose (hyperglycemia) and a resistance to insulin. Administering insulin to such patients tends not to produce its usual effect: in healthy individuals, insulin increases glucose uptake by skeletal muscle and decreases glucose production in the liver; in individuals with type II diabetes, insulin tends not to do so. Many patients with type II diabetes, therefore, do not respond well to insulin therapy, even when it is administered at high doses.
Drugs that promote insulin secretion or that lower glucose levels by other means are commonly prescribed to treat patients with type II diabetes. Sulfonylureas are the principal drugs prescribed to such patients. They stimulate insulin production by directly stimulating &bgr; cells; the effectiveness of such drugs therefore depends on the number of functioning &bgr; cells remaining in the pancreas. Repaglinide also stimulates insulin production by stimulating &bgr; cells, but differs structurally from the sulfonylureas. Other drugs, such as troglitazone (known better by its brand name, REZULIN®) and metformin, lower glucose levels by reducing glucose production in the liver and by promoting insulin sensitivity. Another drug, acarbose, inhibits digestive enzyme secretion and thereby delays digestion of carbohydrates (which when broken down in the body ultimately yield glucose). The efficacy of these drugs is tested first in vitro using existing cell lines that seek to model insulin-secreting &bgr; cells. None of these cell lines provides a satisfactory model, however, because they lose their responsiveness to glucose. As a result, in vitro studies of insulin-secreting drugs currently provide only limited information regarding their efficacy.
Understanding the function and development of insulin-secreting &bgr; cells is a critical step in developing better drugs to treat—and ultimately cure—diabetes. Pancreatic endocrine and exocrine cells (the cells that secrete insulin and other hormones) originate from a precursor epithelial cell during the development of the pancreas. G. Teitelman and J. K. Lee, “Cell lineage analysis of pancreatic islet cell development: glucagon and insulin cells arise from catecholarninergic precursor present in the pancreatic duct.”
Dev. Biol
. 121:454-466, 1987; R. L. Pictet, W. R. Clark, R. H. Williams, and W. J. Rutter, “An ultrastructual analysis of the developing embryonic pancreas.”
Dev. Biol
. 29:436-467, 1972 (the foregoing publications, and all other publications cited herein, are incorporated by reference in their entirety). Various differentiation factors are required to achieve the mature phenotype characteristic of islet beta &bgr;-cells.
New &bgr;-cells are formed from existing islets and from ductal epithelial cells. The latter source has greater intrinsic biological relevance. Indeed, the possibility of differentiating insulin-secreting cells from non-endocrine cells supports the hypothesis that the biological source (pancreatic ductal epithelium) for this compensatory mechanism may be present even in the setting of a generalized destruction of the entire population of islet &bgr;-cells. This is strongly supported by recent studies demonstrating that primary cultures of epithelial ductal cells (from human and mouse pancreas) are susceptible to undergo differentiation into endocrine cells. V. K. Ramiya, M. Maraist, K. E. Arfors, D. A. Schatz, A. E. Peck, J. G. Conmelius, “Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.”
Nature Medicine
, 6(3):278-82, 2000; S. Bonner-Weir, M. Taneja, G. C. Weir, K. Tatarkiewicz, K. H. Song, A. Sharma, J. J. O'Neil, “In vitro cultivation of human islets from expanded ductal tissue.
Proc. Natl. Aca. Sci. USA
, 14:7999-8004, 1997.
Growth and differentiation of islet &bgr;-cells is not limited to the embryological state. A constant remodeling of size and function of the islets of Langerhans occurs during the entire life of individuals and is likely to play an essential role in the prevention of diabetes. In adult rats, two independent pathways are utilized for the proliferation of pancreatic endocrine cells: in the first pathway of proliferation, new endocrine cells arise from the division and differentiation of cells within the islets; in the second pathway, the islets cells originate from precursor cells located in the pancreatic ductal epithelium. S. Bonner-Weir, L. A. Baxter, G. T. Schuppin, F. E. Smith, “A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development.”
Diabetes
42:1715-1720, 1993.
It has yet to be determined whether in the normal ductal epithelium there are different populations of cells, some of which are capable of differentiating into endocrine cells, while others have merely a structural role in defining the epithelial wall It is also possible that all pancreatic ductal epithelial cells could represent a not-fully-differentiated population of cells capable of acquiring a new phenotype Under specific stimuli, but this, too, has yet to be determined; at present, this possibility is a matter of speculation. It is likely that a coordinated activation of multiple differentiation factors, in a fashion similar to the sequence of events occurring during fetal development, is required for the cellular growth of the endocrine pancreas of adults. The mechanism (or mechanisms) for the activation of such a complex regulatory network in adulthood is poorly understood.
An incretin hormone, glucagon-like-peptide-1 (GLP-1), is believed to play a role in the development of the pancreas, though researchers have disagreed as to precisely what this role is. A decade ago, for example, U.S. Pat. No. 5,120,712, the entirety of which is incorporated by reference, stated that “The failure to identify any physiological role for GLP-1 caused some investigators to question whether GLP-1 was in fact a hormone and whether the relatedness between glucagon and GLP-1 might be artifactual.” Researchers have more recently learned that GLP-1 has a function in rats. Bonner-Weir et al., for example, demonstrated that an analog of the incretin hormone glucagon-like-peptide-1 (GLP-1), termed exendin4, was able to increase islet mass in adult animals previously subjected to subtotal pancreatectomy. G. Xu, D. A. Stoffers, J. F. Habener, S. Bonner-Weir, “Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.”
Diabetes
48:2270-2276, 1999. Similarly, the inventor has demonstrated that treating glucose-intolerant aging Wistar rats with GLP-1 restored normal glucose tolerance and induced islet cell proliferation. Y. Wang, R. Perfetti, N. H. Greig, H. W. Holloway, K. A. DeOre, C. Montrose-Rafizadeh, D. Elahi, J. M. Egan, “Glucagon-like peptide-1 can reverse
Cedars-Sinai Medical Center
Ketter James
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