Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2001-04-12
2004-02-03
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S366000, C435S371000
Reexamination Certificate
active
06686197
ABSTRACT:
The present invention relates to a method for the large scale production of preparation of mature and immature pancreatic endocrine cells and their use for treatment of diabetes mellitus.
BACKGROUND TO THE INVENTION
Diabetes mellitus is defined as a chronic state of hyperglycaemia. This metabolic disturbance appears when insulin release has become insufficient, either as a result of a primary defect at the level of the insulin-producing beta cells or, secondary, when the beta cells fail to compensate for an increased peripheral resistance to insulin. The shift to elevated glucose levels can be counteracted by sustained adjustments in life style and by daily administration of hypoglycemic agents, under form of insulin injections or sulphonylurea tablets. Current treatment does however not succeed in a complete normalization of glucose homeostasis. Diabetic patients thus face the risk of developing chronic complications as a consequence of recurrent episodes of hyperglycemia. They are known to exhibit, as a group, a higher incidence of retinopathy and blindness, of nephropathy and renal failure, of neuropathy and amputations, of vasculopathy and cardiovascular disease. Diabetes is therefore considered as a major health problem. The disease is diagnosed in more than 5 percent of the Western population. Its impact on each patient's quality of life is variable but life-long (Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, Diabetes Care 20,1183-1197, 1997).
A variety of stategies are currently explored in an attempt to find ways that stop the progression of the disease at any of its preclinical or clinical stages. Several are directed towards the pancreatic beta cells with the purpose of) reinstalling a functional beta cell mass that is sufficient to restore, at least in part, an endogenous control circuit in which insulin is released as a function of the metabolic needs. There are essentially two ways to achieve this goal. The first involves an implantation of foreign beta cells in order to replace the endogenous beta cell population or supplement it. It has been shown to correct the diabetic state in patients who had completely lost their endogenous beta cell mass (Warnock et al, Diabetologia 35:89-95, 1992; Ricordi et al, Transplantation 53;407-414, 1992; Gores et al, Lancet 341:19-21, 1993; Scharp et al, Transplantation 51:76-85, 1991). The second consists in administering drugs which increase the endogenous functional beta cell mass, either by inducing neoformation of beta cells, prolonging their survival or correcting their homeostatic function. Both strategies require the availability of large numbers of beta cells, either as cell grafts for implantation or as test model for screening and developing new drugs in the laboratory.
The number of patients who could benefit from a beta cell graft is, conservatively, estimated at 0.5 percent of the total population, which largely exceeds the number of candidates for other types of grafts. It is also clear that development of drugs acting on the beta cells involves extensive preclinical screening and testing for which large numbers of normal cells will be necessary. There is not yet a source of beta cells which can adequately fulfil both needs. Human pancreata have been used to produce beta cell preparations for transplantations as well as for in vitro studies but the number of donor organs is largely insufficient; moreover, criteria on human organ donation impede their use for drug development. These restrictions raise the need for producing beta cells from other species.
Among the larger mammals, pigs are considered as a potentially useful source of beta cell preparations since their use for medical applications faces fewer ethical obstacles than primates or other domestic animals, since pigs are relatively easy to breed and since porcine insulin is very similar to human insulin. Methods have been developed to isolate islet and tissue preparations from fetal, neonatal and adult pig pancreata. These preparations can normalize a diabetic state in immune-incompetent and in immune-competent mice (Korsgren et al, Surgery 113, 205-214, 1993; Korbutt et al, J Clin Invest 97, 2119-2129, 1996; Thomas et al, Transplantation 67:846-854, 1999; Lu et al, Xenotransplantation 5, 154-163, 1998). Fetal pig islet preparations have already been transplanted in diabetic patients, however without success (Groth et al, Lancet 344, 1402-1404, 1994). It is still unknown whether and if so, how successful xenotransplantation can be carried out in man. Use of reaggregated beta cell preparations with selected size and cellular composition might help a search for such conditions. Our studies in rodents have shown that purified islet endocrine cell aggregates exhibit a lower immunogenicity as allograft than intact islet tissue (Pipeleers et al, Diabetes 40, 908-919, 1991; Pipeleers et al, Diabetes 40, 920-930, 1991; Pipeleers-Marichal et al, Diabetes 40, 931-938, 1991, Pipeleers et al, Diabetologia 34,390-396, 1991). They also illustrate how variations in cellular composition influence the metabolic capacity of the grafts (Keymeulen et al, Diabetologia 40:1152-1158, 1997; Keymeulen et al, Diabetes 45,1814-1821, 1996). While these experiments demonstrated the usefulness of composing beta cell grafts in the laboratory, they did not offer an adequate methodology for clinical implantation. The methods that we used for composing the rat beta cell grafts do not allow large scale preparations of pancreatic endocrine cells. They involve prior isolation of the islets of Langerhans (herein defined as micro-organs with a diameter >100 &mgr;m containing a mixture of endocrine cell types including the insulin producing beta cells) and thus discard beta cells that are present as single cells or as small cell aggregates; the single beta cells are assumed to be adjacent to immature endocrine cells, i.e. cells which can differentiate into beta cells. As a result of this removal, little is known about these beta cells and the immature endocrine cells. Their relative proportion (with respect to the numbers incorporated in islets) is however not insignificant during early phases of life (In't Veld et al, Diabetologia 35:272-276,1992); in the human pancreas, they remain numerous throughout adult life (Bouwens and Pipeleers, Diabetologia 41:629-633, 1993). Since the migration and association of pancreatic endocrine cells into typical islet structures is considered as a step in maturation (Pictet and Rutter, development of the embryonic pancreas. In: Steiner D F, Freinkel N (eds) Handbook of Physiology, Section 7 Endocrinology Vol I: Endocrine Pancreas, Baltimore; Williams & Wilkins, 1972 pp25-66), the endocrine cells which do not occur in these micro-organs can be defined as “immature”. Although the properties of immature beta cells and immature endocrine cells have not been well characterized, they are likely different from those of the “islets” which have matured under influence of their typical microanatomy and neighbouring endocrine cells (Orci and Unger, The Lancet 2;1243-1244,1975; Pipeleers, Experientia 440:1114-1126,1984; Pipeleers, Diabetologia 30:277-291, 1987). The islet functions are considered as typical for mature beta cells; mature beta cells are larger than their immature counterparts (Pipeleers, unpublished observations). There is indirect evidence that the “immature” beta cells can achieve a growth of the beta cell mass. The loss of these cells during the isolation procedure is thus expected to result in a purified endocrine cell preparation which is only representative for the mature cell population, which contains only a subpopulation of the beta cells and which exhibits a low capacity for growth, three consequences that are disadvantageous when the isolated cells are to be used for the above-mentioned strategies, namely the construction of beta cell grafts and the development of drugs which aim to increase the functional beta cell mass.
In order to overcome said problems the invention provides a method accor
Beta-Cell, N.V.
Lankford , Jr. Leon B.
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
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