| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 27.10.98 |
| PATENT TITLE |
Sulfated glycolipids and antibodies thereto for prophylaxis or therapy of diabetes |
| PATENT ABSTRACT |
Methods for the treatment of prediabetes or diabetes in an individual comprise administering to the individual a therapeutically active agent comprising a glycolipid having a galactose-3-O-sulfate moiety capable of binding to islet cell antibodies. Methods of detecting or quantifying islet cell antibodies in a sample, methods for the detection of Langerhans islet cells, methods for monitoring the development of prediabetes, diabetes or symptoms thereof in an individual and methods for prophylactic treatment against the development of prediabetes or diabetes also involve the use of a glycolipid having a galactose-3-O-sulfate moiety. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | February 28, 1995 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Berkow et al., eds., "The Merck Manual" sixteenth edition, Merck Research Laboratories (Rahway, NJ) 1992, pp. 955-970. Goodman et al., eds., "Goodman and Gilman's The Pharmaceutical Basis of Therapeutics", Pergamon Press, Inc. (Elmsford, NY) 1990, pp. 1471-1475. Zubay "Biochemistry", The Benjamin/Cummings Publishing Company, Inc. (Menlo Park, CA) 1983, pp. 20-22. Stevenson et al., Chem. Abs. No. 78882x, STN International, vol. 116, No. 9 (1992). Fredman et al. Biochem. J. 1988, 251, 17-22. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
I claim: 1. A method for treatment of prediabetes or diabetes in an individual by inducing in said individual a tolerance to a galactosylceramide-3-sulfate active agent of the formula ##STR1## a lactosylceramide-3-sulfate active agent of the formula ##STR2## or a seminolipid active agent of the formula ##STR3## the method comprising administering the galactosylceramide-3-sulfate active agent, the lactosylceramide-3-sulfate active agent, or the seminolipid active agent to the individual. 2. A method of treating prediabetes or diabetes in an individual by raising in said individual suppressor or regulator cells or antibodies directed against lymphocytes recognizing a galactosylceramide-3-sulfate active agent of the formula ##STR4## a lactosylceramide-3-sulfate active agent of the formula ##STR5## or a seminolipid active agent of the formula ##STR6## the method comprising administering lymphocytes having inhibited cytotoxicity and recognizing the galactosylceramide-3-sulfate active agent, the lactosylceramide-3-sulfate active agent or the seminolipid active agent. 3. A method for therapy of prediabetes or diabetes in an individual, comprising contacting the blood stream of the individual with an immobilized galactosylceramide-3-sulfate active agent of the formula ##STR7## immobilized lactosylceramide-3-sulfate active agent of the formula ##STR8## or immobilized seminolipid active agent of the formula ##STR9## and removing from the blood stream of the individual antibodies or lymphocytes which recognize the galactosylceramide-3-sulfate active agent, the lactosylceramide-3-sulfate active agent, or the seminolipid active agent. 4. A method of detecting or quantifying islet cell antibodies in a sample, comprising contacting the sample with an antigen comprising a glycolipid having a galactose-3-O-sulfate moiety capable of binding to islet cell antibodies, and detecting or quantifying as islet cell antibodies those antibodies that bind to the glycolipid. 5. The method according to claim 4, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR10## a lactosylceramide-3-sulfate of the formula ##STR11## or a seminolipid of the formula ##STR12## 6. The method according to claim 4, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR13## 7. The method according to claim 4, wherein the glycolipid comprises a lactosylceramide-3-sulfate of the formula ##STR14## 8. The method according to claim 4, wherein the glycolipid comprises a seminolipid of the formula ##STR15## 9. A method for detection of Langerhans islet cells, comprising histologically or cytologically staining a pancreatic preparation using an antibody or lectin directed against a sulfated glycolipid antigen comprising a galactosylceramide-3-sulfate of the formula ##STR16## a lactosylceramide-3-sulfate of the formula ##STR17## or a seminolipid of the formula ##STR18## 10. The method according to claim 9, wherein the antibody is Sulph I monoclonal antibody. 11. A method for treatment of prediabetes or diabetes in an individual, comprising administering to the individual a glycolipid therapeutic active agent having a galactose-3-O-sulfate moiety capable of binding to islet cell antibodies. 12. A method for treatment of prediabetes or diabetes in an individual, comprising administering to the individual a galactosylceramide-3-sulfate therapeutic active agent of the formula ##STR19## a lactosylceramide-3-sulfate therapeutic active agent of the formula ##STR20## or a seminolipid therapeutic active agent of the formula ##STR21## 13. The method according to claim 12, wherein the galactosylceramide-3-sulfate therapeutic active agent of the formula ##STR22## is administered. 14. A method for treatment of prediabetes or diabetes in an individual, comprising administering to the individual a lactosylceramide-3-sulfate of the formula ##STR23## 15. A method for treatment of prediabetes or diabetes in an individual, comprising administering to the individual a seminolipid of the formula ##STR24## 16. A method of detecting and optionally quantifying islet cell antibodies sin an individual, comprising contacting a sample of body fluid from the individual with an antigen comprising a glycolipid having a galactose-3-O-sulfate moiety capable of bonding to islet cell antibodies, and detecting and optionally quantifying as islet cell antibodies those antibodies that bind to the glycolipid. 17. The method according to claim 16, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR25## a lactosylceramide-3-sulfate of the formula ##STR26## or a seminolipid of the formula ##STR27## 18. The method according to claim 16, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR28## 19. The method according to claim 16, wherein the glycolipid comprises a lactosylceramide-3-sulfate of the formula ##STR29## 20. The method according to claim 16, wherein the glycolipid comprises a seminolipid of the formula ##STR30## 21. A method for monitoring development of prediabetes or diabetes in an individual, comprising contacting a sample of body fluid from the individual with an antigen comprising a glycolipid having a galactose-3-O-sulfate moiety capable of binding to islet cell antibodies, and detecting and optionally quantifying as islet cell antibodies those antibodies that bind to the glycolipid. 22. The method according to claim 21, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR31## a lactosylceramide-3-sulfate of the formula ##STR32## or a seminolipid of the formula ##STR33## 23. The method according to claim 21, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR34## 24. The method according to claim 21, wherein the glycolipid comprises a lactosylceramide-3-sulfate of the formula ##STR35## 25. The method according to claim 21, wherein the glycolipid comprises a seminolipid of the formula ##STR36## 26. A method for prophylactic treatment against the development of prediabetes or diabetes in an individual being at risk of developing prediabetes or diabetes, comprising administering to the individual a therapeutically active agent comprising a glycolipid having galactose-3-O-sulfate moiety capable of binding to islet cell antibodies. 27. The method according to claim 26, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR37## a lactosylceramide-3-sulfate of the formula ##STR38## or a seminolipid of the formula ##STR39## 28. The method according to claim 26, wherein the glycolipid comprises a galactosylceramide-3-sulfate of the formula ##STR40## 29. The method according to claim 26, wherein the glycolipid comprises a lactosylceramide-3-sulfate of the formula ##STR41## 30. The method according to claim 26, wherein the glycolipid comprises a seminolipid of the formula ##STR42## -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Insulin-dependent (type 1) diabetes is a disease in which both humoral and cellular immunological mechanisms seem to play a role in the pathogenesis. For the study of type 1 diabetes there are several animal models, of which the BB rat is among the most important. The BB rat spontaneously develops diabetes at the age 60-120 days, and the diabetes is dependent on a functional thymus-dependent immune system. Lymphocyte infiltration (insulitis) occurs in the islets of Langerhans, and autoantibodies against islet cells can be detected. Some groups have been able to raise islet-cell-specific monoclonal autoantibodies from the BB rat or the composite NOD mouse. The recognition of relevant beta cell antigen(s) seems crucial for an understanding of the disease mechanisms behind diabetes mellitus, for a possible prevention of the disease, and for a detection of relevant marker antibodies. Quantitatively changed antigen expression, dependent on the degree of function, might be a mechanism relevant to several study findings which indicate a diabetogenetic importance of the degree of insulin production. Among the most important studies, the following should be mentioned. Increased incidence of diabetes is seen after increased insulin-production. In the low-dose streptozotocin diabetic mouse model there is, after ventromedial lesion of the hypothalamic region, a higher incidence and a more severe diabetes. In humans there is an increased incidence of true insulin-dependent diabetes in the last trimester of pregnancy, a period characterized by high insulin output from the beta-cells. Decreased incidence of diabetes is seen after decreased insulin-production. In the BB rat there is a lower incidence of diabetes, if prophylactic insulin treatment is given during the diabetes-risk period, a procedure which reduces the in situ production of insulin. In the low-dose streptozotocin model there is a lower incidence of diabetes after diet containing low amounts of carbohydrate. Finally, hyperinsulinsemia is observed in relation to the diabetogenesis. In the EMC-M virus mouse model there are high insulin concentrations in peripheral blood before the onset of clinical diabetes. In parallel, human first-degree relatives of diabetic patients show increased insulin-response to glycaemic stimulus. Recently, several groups have suggested different proteins as antigen-candidates; these include glutamic acid decarboxylase (64 kD autoantigen), heat-shock protein 65, and a 38 kD protein in the membrane of the insulin secretory granule. However, the antigen could as well be of non-protein structure and, in fact, islet cell antibodies (ICA)--present in the majority of the diabetic patients at diagnosis and labelling both beta and alpha cells--are believed to be directed against a glycolipid. Furthermore, several monoclonal antibodies directed against beta cells have a ganglioside epitope (3G5, A285, R2D6). Sulfatide (3'-sulfogalactosylceramide) is an acidic glycosphingolipid located at the cell membrane. It is an early marker for the differentiation of oligodendrocytes and Schwann cells (Zalc, B. and Baumann, N., Adv. Exp. Med. Biol. 152: 439-443, 1982) and becomes highly enriched in the myelin. With the aim to produce an antibody to sulfatide that was specific enough to be used for the quantitative assay of sulfatide in tissues and body fluids in demyelinating disorders, Fredman et al. (Biochem. J. 251: 17-22, 1988) produced the monoclonal antibody Sulph I. Balb/c mice were immunized with sulfatide coated on Salmonella minnesota membrane. Spleen cells from the mice were fused with mouse myeloma cells, the resulting hybridomas were screened against sulfatide by an ELISA method, and positive hybrids were cloned by limiting dilution. The subclass of the antibody was found to be IgG1. Fredman et al. found that the monoclonal antibody Sulph I have affinity to three glycolipids: galactosylceramide-3-sulfate (sulfatide), lactosylceramide-3-sulfate and seminolipid. These three glycolipids all have the same terminal group: galactose-3-O-sulfate. The non-sulfated glycolipids, galactosylceramide and lactosylceramide, and also some bis-sulfated mucopolysaccharides did not bind the antibody. Removal of the fatty acid from sulfatide and seminolipid resulting in their corresponding lyso compounds gave a remarkable diminution in their binding to the antibody. Sulfated glycolipids are somewhat unusual molecules. Galactosyl sulfatide is relatively abundant in brain tissue, particularly myelin. It is synthesized by oligodendroglial cells and is a differentiation marker for these cells. In vitro, the biosynthesis and cell surface expression of galactosyl sulfatide on oligodendrocytes is regulated, and culture of these cells in the presence of sulfatide-specific monoclonal antibodies significantly affects cell growth and differentiation. Although islet cells are believed to be of endodermal origin, recent findings have shown sharing of antigen determinants between islets of Langerhans and neural tissue. Glutamic acid decarboxylase is present in both tissues like different kinds of gangliosides. Furthermore, the statistical coincidence of Type 1 (insulin-dependent) diabetes and the neurological disorder "Stiff man syndrome", and possibly also "Guillian-Barre syndrome" (inflammatory demyelinating polyradiculoneuropathy), strengthens the interest in possible joint antigens. In Guillian-Barre syndrome, patients display anti-sulfatide antibodies and by using the monoclonal anti-sulfatide antibody, Sulph I, Fredman et al. (above) have shown structural changes involving the corresponding antigen in the form of demyelinization. FINDINGS ON WHICH THE INVENTION IS BASED The following Example 1 examines the presence of sulfatide (or very similar sulfated glycolipids) in islet cells and especially in beta cells; the possible labelling by Sulph I was investigated on pancreatic histological sections as well as on isolated islet cells and on the beta cell fraction and the non-beta cell fraction thereof, separated by means of a fluorescence activated cell sorter. It was shown that an epitope on the glycolipid sulfatide or on closely related structures are present in islet cells--both beta and non-beta cells--but seems not to be detectable in other examined cells apart from tubular and glomerular cells in the kidney and neural tissue. Furthermore, the used antisulfatide monoclonal antibody gives a very bright staining of islets of Langerhans which may be advantageous for a convenient and reliable detection of islet cells. Islet cell antibodies (ICA) from Type 1 diabetic patients label--like Sulph I--both beta cells and alpha cells, and increasing evidence indicates that ICA are directed against a glycolipid. On frozen sections, the islet cell antigen has been found to have properties of a sialic-acid-containing glycolipid. ICA reactivity of sera could be blocked by preincubation with monoanglioside-glycolipid extracts from human pancreas. Furthermore, human islets contain G.sub.M1 -G.sub.M2 ganglioside and, in studies using rat islets, the expression of the gangliosides was metabolically regulable similar to what has been found for the antigens corresponding to the monoclonal antibodies IC2 and A285. Type 1 diabetic sera displayed reactivity against another ganglioside (G.sub.T3) from RIN tumors. Finally, autoantibodies to human pancreatic fucogangliosides in the sera of ICA-positive Type 1 diabetic patients have been described using thin layer chromatography. The following Example 2 investigates islets of Langerhans for sulfatide structures, as demonstrated by staining with the monoclonal anti-sulfatide antibody, Sulph I. Since an (auto)immune etiopathogenesis to diabetic late complications has been suggested, also kidney tissue as well as neurological structures were investigated. Furthermore, patients with newly diagnosed Type 1 diabetes were examined for the presence of anti-sulfatide antibodies. A distinct staining by Sulph I of islets of Langerhans and of tubular structures and glomeruli in the diabetic kidney was found, and in peripheral blood anti-sulfatide antibodies related to Type 1 diabetes were demonstrated. This investigation showed labelling of islets of Langerhans, kidney structures and neural tissue by the same monoclonal antibody, Sulph I, which is directed against sulfatide. The tissues mentioned all suffer during the natural course of Type 1 diabetes. By immunoelectronmicroscopy the Sulph I labelling of alpha and beta cells was found to be related to the membrane and the content of the secretory granules. Also ICA seems to label the membranes of insulin-secretory granules. Likewise, the Type 1 diabetic T-cell reactive 38 kD protein involves the insulin-secretory-granule membrane. Carboxypeptidase H, a possible beta cell autoantigen identified as a target of ICA in a lambda GT11 cDNA library, is an enzyme within the beta cell secretory granule and exists as a membrane and (?) soluble form. On the other hand is GAD described to be localized around synaptic-like microvesicles in beta cells. Staining of the glomeruli with Sulph I was seen both in BB rat and in man, but only when Type 1 diabetes was developed. Labelling of mesangial cells and capillary loops was seen, whereas basal membranes remained unstained. The reason for the diabetic labelling is unknown. It is unlikely that the staining is caused by substantial deposits since diabetes does not induce morphological changes in the BB rat glomeruli, except for thickening of the basement membrane. Thus, no changes in the diabetic BB rat glomeruli are seen in the cells associated with the peripheral capillary wall, in the fractional volumes of the mesangial cells or of the mesangial matrix. Between membranous glomerulopathy and Guillian-Barre syndrome, a link has been suggested, but proof of an association between these diseases does not exist. Furthermore, the Guillian-Barre nephropathy does not necessarily depend on antigen share but might solely be due to immune complexes having their primary origin in the demyelinization process. Staining of the distal tubuli with Sulph I is in good agreement with earlier demonstration of sulfatide in these structures. It has been suggested that sulfatide in the tubuli is implicated in the passive diffusion of sodium chloride from the lumen into the interstitial space. In Guillian-Barre syndrome anti-sulfatide antibodies are seen. Since the present study by using Sulph I has demonstrated sulfatide in islet cells (as in neural tissue) we looked for antibodies against sulfatide in newly developed Type 1 diabetic patients and found 88% of them (at the cut off level mentioned in the Result section) to have a titre of up to 1:3200 at diagnosis. Furthermore, in the remission period 6 months later, at which time the disease is stabilizing 59% were positive. Our findings suggest that anti-sulfatide antibodies could be useful as markers for Type 1 diabetes. Today, by far the most commonly used marker is ICA, which is positive in about 70% of newly diagnosed Type 1 diabetics in titres up to 1:128. GAD antibodies have been found to be present in 81% of early stage patients, whereas insulin autoantibodies are detectable in about 40% of newly diagnosed Type 1 diabetics. In monitoring the disease process it seems advantageous to use more than one marker. Clinical co-incidence between diabetes and neurological disorders is also seen in a diabetes animal model. The strain of encephalomyocarditis virus (EMC-M) used in our laboratory produces diabetes in approximately one third of BALB/c/BOM mice and paresis in about 90 per cent. Attention should be paid to sulfatide as a common antigen but the question of its pathogenetic importance has yet been addressed. SUMMARY OF THE INVENTION The foregoing evidence suggests that sulfated glycolipids, in particular such containing galactose-3-O-sulfate, and more particularly galactosylceramide-3-sulfate, lactosylceramide-3-sulfate and seminolipid, are antigens against which Islet Cell Antibodies (ICA) responsible for the development of diabetes and associated complications are raised, and that Sulph I is representative for such ICA. Accordingly, the invention comprises said sulfated glycolipids and specific catchers therefore (antibodies or lectins) for use in the prophylaxis or thereby of prediabetes, diabetes and/or associated complications in an individual. By therapy in this context should not be understood the normal substitution therapy (insulin or any substances that stimulate the production of insulin), but any steps taken to stop the destruction or improve the regeneration of beta cells or to prevent the development of complications often associated with diabetes. The sulfated glycolipids may be used for prophylaxis of prediabetes, diabetes and/or associated complications in an individual by inducing tolerance to such antigens in the individual, for example by perinatal administration of a sulfated glycolipid to said individual. The theory is that by presenting the antigen to the individual at around birth the immune system of the individual will recognize the antigen as belonging to the organism self and will develop tolerance to it. The sulfated glycolipids may also be used for prophylaxis or therapy of prediabetes, diabetes and/or associated complications in an individual by raising suppressor or regulator cells or antibodies against lymphocytes, recognizing the antigenic sulfated glycolipids, in said individual. This can for example be done by removing lymphocytes from the individual, contacting them with a sulfated glycolipid in vitro to make them recognize this antigen, irradiating the lymphocytes to inhibit their cytotoxicity, and (a) returning them to the individual to raise suppressor or regulator cells or antibodies against lymphocytes reactive with this antigen, or (b) administering them parenterally to another mammal in order to raise antibodies against lymphocytes reactive with this antigen in said mammal and then isolating serum containing the antibodies from said mammal and administering it to the individual. The sulfated glycolipids may further be used for prophylaxis or therapy of prediabetes, diabetes and/or associated complications in an individual by removing antibodies and/or lymphocytes recognizing the antigenic sulfated glycolipids form the blood stream of the individual. This can for example be done by contacting the blood stream of the individual with an immobilized sulfated glycolipid to remove antibodies and/or lymphocytes recognizing the antigenic sulfated glycolipids from the individual. The catchers for said glycolipids, and in particular the antibodies directed against them, may also be used for prophylaxis or therapy of prediabetes, diabetes and/or associated complications in an individual by raising anti-antibodies in said individual. This can for example be done by parenterally administering an antibody against sulfated glycolipids, (a) to said individual in a sufficient amount to raise anti-antibodies in said individual, or (b) to another mammal in order to raise anti-antibodies in said mammal and then isolating serum containing the anti-antibodies from said mammal and administering it to the individual. The invention further comprises the use of sulfated glycolipids in particular such containing a galactose-3-O-sulfate moiety as the antigen in antigen-antibody assays for the detection and, optionally, quantitation of islet cell antibodies (ICA) or other antibodies relevant to prediabetes, diabetes and associated complications in an individual. The antigen-antibody assay is carried out on a sample of body fluid taken from the individual, and as examples of antigen-antibody assays in common use may be mentioned ELISA, radioimmunoassay, countercurrent electrophoresis and immunofluorescence. The detection and quantitation of ICA or other antibodies relevant to prediabetes, diabetes and associated complications in an individual may serve to assess the risk of said individual for developing diabetes, and repeated quantitations of ICA or other relevant antibodies in the individual may be used to monitor the development of such risk. Likewise, repeated quantitations of ICA or other relevant antibodies in an individual already suffering from prediabetes or diabetes and, possibly, associated complications may be used to monitor the development of the disease and/or associated complications or to monitor the results of a treatment of the disease and/or associated complications. Finally the invention comprises the use of catchers for sulfated glycolipids, in particular such containing a galactose-3-O-sulfate moiety, for the detection of Langerhans islet cells by histologic or cytologic staining of pancreatic preparations. I have found that this is a very convenient and reliable method of detecting Langerhans islets in pancreatic tissue preparations as well as isolated islet cells in pancreatic cell fractions. Practical embodiments of histologic staining of pancreatic tissue sections and cytologic staining of isolated cell fractions are illustrated in the following Example 1. BRIEF DESCRIPTION OF THE DRAWINGS The figures are reproductions of photographs showing: FIG. 1. Labelling by anti-sulfatide monoclonal antibody, Sulph I, of islets of Langerhans in pancreas from a Lewis rat using APAAP technique as described in Example 1, Materials and Methods. FIG. 2. Labelling of the beta-cell fraction of isolated islet cells from a Lewis rat with Sulph I and FITC conjugate using immunofluoresence technique as described in Example 1, Materials and Methods. A spackled, bright surface fluorescence of the cells is seen. FIG. 3. The same labelling as in FIG. 2 but of the non-beta-cell fraction of islet cells. The intensity of fluorescence after Sulph I staining seems to be equal for beta and non-beta cells. FIG. 4. Sulfatide, chromatographed on thin-layer plate, immunostained with plasma from a newly diagnosed Type 1 diabetic patient. The plasma dilutions are 1:100, 1:400, 1:800, 1:1600, 1:3200 and 1:6400, respectively. The amount of sulfatide applied on the plate was 500 pmol. A detailed description of the chromatography and the immunostaining is given in materials and methods. FIG. 5. Sulph I-stained pancreatic sections. Original magnification .times.66. FIG. 5a is from a Lewis rat; FIG. 5b from a pig; and FIG. 5c from a Macaca facicularis monkey. FIG. 6. Electronimicrograph illustrating .beta.-granules (arrows) labelled with 15 nm colloidal-gold. Original magnification .times.25.000. FIG. 7. Sulph I-stained kidney sections. FIG. 7a is from a Macaca facicularis monkey (original magnification .times.80); arrows indicate the tubular basement membrane which is seen as a distinct uncolored structure. FIG. 7b is from a non-diabetic BB rat (.times.132). FIG. 7c is from a diabetic BB rat (.times.132); mesangial staining is indicated by arrow. FIG. 7d is from a non-diabetic human (.times.50); glomeruli indicated by arrows. FIG. 7e is from a NIDDM human (.times.50). FIG. 7f is from an IDDM human (.times.66); mesangial staining indicated by arrow. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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