WO1991001738A1

WO1991001738A1 - Dithiochrome, an insulin-binding molecule with glucose metabolism-related pharmaceutical utility

Info

Publication number: WO1991001738A1
Application number: PCT/US1990/004163
Authority: WO
Inventors: Scott R. King
Original assignee: Thymax Corporation
Priority date: 1989-07-28
Filing date: 1990-07-30
Publication date: 1991-02-21
Also published as: IL95220A0; EP0497771A1; CA2064036A1; AU638267B2; AU6067490A; EP0497771A4; JPH05506772A

Abstract

A material obtained by: (i) lysing a eukaryotic cell mass and then combining the lysate obtained with a mixture of an aqueous buffer and an organic solvent; (ii) separating the organic phase from the water phase; (iii) subjecting the delipidated water phase extract to an insulin affinity chromatography matrix, a sulfhydryl exchange chromatography matrix, a C4,8,18- or phenyl reverse phase chromatography matrix, or an anion exchange chromatography matrix; (iv) and eluting the chromatography matrix to obtain a material capable of binding with insulin. This material may be used to treat patients suffering from elevated blood glucose or suboptimal glucose kinetics. An adduct of this compound with insulin may be used to treat patients for type I diabetes.

Description

DITHI0CHR0ME, AN INSULIN-BINDING MOLECULE WITH GLUCOSE METABOLISM-RELATED PHARMACEUTICAL UTILITY

Technical Field

The present invention relates to the extraction of a highly pure material heretofore available in low purity levels and known as glucose tolerance factor (GTF) , and to the use of this highly pure material in pharmaceutical applications.

Background Art

The importance of dietary chromium and its function in biological systems is long-established. Chromium is an essential trace metal which has been suggested to have an important role in normal glucose ho eostasis. Deficiency of chromium, or of its biologically active form, has been implicated in the pathogenesiε of some forms of glucose intolerance and diabetes mellitus.

Beginning in 1957, various investigators have sought to purify from natural sources, or to synthesize, a chromium-containing material called GTF, believed to be the biologically active form of chromium. From natural sources at best only partially purified materials (e.g., 5 to 10% wt. pure as determined by analysis of sample chromium content) were obtained. Synthetic efforts failed to yield GTF.

GTF is reported to be a naturally occurring low molecular weight (400-1200 dalton) organic compound which is water soluble and is reported to be stable against wet heat, acid, and alkyl treatments. Various investigators have reported that GTF is a complex of nicotinic acid, amino acid components, and Cr⁺³. Glycine, cysteine, and glutamic acid have been reported as appearing to be the amino acid components. See, Mooradian et al. Am. J. Clin. Nutr.. (1987), 45:877-895.

Toepfer et al_r J. Acrric. Food Che . , (1977) 25(1) :162-166 report that when Brewer's yeast is extracted with dilute alcohol and purified by ion exchange chromatography a product is obtained which possesses GTF activity. This material is reported to contain chromium, nicotinic acid, glycine, glutamic acid, and cysteine.

Toepfer et al also report that when 1 equivalent of trivalent chromium, Cr(Ac)₃.H₂0, is reacted with 2 equivalents of nicotinic acid, 2 equivalents of glycine, 1 equivalent of glutamic acid and 1 equivalent of cysteine a mixture of chromium complexes exhibiting GTF properties are obtained. These materials are however reported to be unstable, precipitating near neutral pH with resulting loss of biological activity.

Although various attempts have been made to isolate a purified form of the GTF product from natural sources and various attempts have been made to produce this material using synthetic methods, obtaining a purified GTF product has so far eluded investigators. In light of the importance of dietary chromium and problems associated with chromium deficiency, such as, e.g., insulin resistant diabetes, there is a strongly felt need for a method for readily obtaining purified GTF, or a material possessing the biological activity of GTF, in substantial quantities.

Disclosure of the Invention

Accordingly, it is an object of this invention to provide a method for obtaining a purified material having a high level of glucose tolerance factor activity.

It is another object of this invention to provide a method for efficiently obtaining a purified material having a high level of glucose tolerance factor activity.

It is another object of this invention to provide a purified material having a high level of glucose tolerance factor activity.

It is another object of this invention to provide an activated form of insulin useful in the therapy of patients for type I (juvenile or insulin-dependent) diabetes.

It is another object of this invention to provide a material useful for the treatment of type II diabetes, insulin resistance, gestational diabetes, stress induced diabetes, obesity, hyperlipidemia, atheroma and atherosclerosis, and other conditions associated with elevated blood glucose, subopti al glucose kinetics, or elevated blood insulin.

The inventor has now discovered a process and a Product which satisfy all of the above objects of this invention and other objects which will become apparent from the description of the invention given hereinbelow.

The product of the present invention comprises a compound named dithiochrome (DTC) by the inventor and which possesses a very high level of glucose tolerance factor activity. It is readily obtained by lysing a eukaryotic cell mass, wherein the eukaryotic cell mass is of fungus (including mushroom) , yeast, plant or animal origin. The lysate is then delipidated through contact with a mixture of an aqueous buffer (pH = 2 to 8, preferably 5 to 7) and an organic solvent selected from the group consisting of C_λ_ ₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and dichloromethane to obtain a delipidated water phasfe extract. This water phase extract is then subjected to (i) insulin affinity chromatography matrix, (ii) sulfhydryl exchange chromatography matrix, (iii) C_{4 8 18}- or phenyl reverse phase chromatogr phy, or (iv) anion exchange chromatography.

Dithiochrome binds to the insulin affinity or sulfhydryl exchange matrix. The chromatography matrix is first charged with the water phase extract. After washing the charged matrix, the dithiochrome may be obtained by treating the matrix with a low concentration of a reducing agent, e.g., an active thiol or dithiol. For example, the dithiochrome can be obtained by treating the matrix with (i) a 0.05 mM to 250 mM solution of an active dithiol, (ii) a 0.1 mM to 500 mM solution of an active thiol, (iii) with a gradient of from 0 mM to 250 mM, preferably 0 mM to 10 mM, of the active dithiol, or (iv) with a gradient of from 0 mM to 500 mM, preferably 0 mM to 20 mM, of the active thiol. Using a gradient is preferred.

With reverse phase chromatography, the water phase extract is subjected to a 0 to 50 vol.% (B) into (A) linear gradient. Component (A) is a 0.02 to 0.2M buffer having a pH of from 2 to 8 in 5 vol.% C₁_₃ lower alcohol, acetone, ethoxyethoxyethanol or acetonitrile containing 0.2 vol.% triethylamine. Component (B) is the lower alcohol, acetone, ethoxyethoxyethanol, or acetonitrile. The elution is monitored at λ .= 214 nm or 260 nm to detect elution of the dithiochrome.

With anion exchange chromatography, any anion exchange column can be used, including amine columns. The water extract is loaded onto the anionic exchange column. The column is then washed with water until a base line at λ = 214 nm or 260 nm is observed. The dithiochrome is then eluted with a 0 to 1 M salt linear gradient.

Brief Description of the Drawings

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying figures.

Figure 1 is a UV-VTS spectrum scan of the material obtained from the elution of an insulin column loaded with a yeast water phase extract. The absorbance at 260 nm is characteristic of dithiochrome, GTF and nicotinic acid.

Figures 2 and 3 show the bioactivity of DTC.

Best Mode for Carrying Out the Invention

The present invention makes possible the facile production, from natural sources, of a highly pure material containing glucose tolerance factor activity; a compound that is an essential part of normal glucose metabolism and which has been named dithiochrome by the inventors.

Because before the invention it was believed that GTF was present in low concentrations in yeast and other materials and because of its perceived relative instability it was believed that it would be especially difficult, if not impossible, to obtain a sufficient amount of GTF from natural materials to permit elucidation of its structure (Toepfer et al - 1977). The present invention is in part due to the inventors' discovery of facile processes which readily yield substantial amounts of this material, renamed as DTC, in heretofore unavailable highly purified form.

This compound may be administered by any known method of administration including enteral, suppository, subcutaneous, intravenous (IV) injection, intramuscular (IM) injection, or transdermally, to diabetics to alleviate their diabetes.

Dithiochrome may be given to a patient (e.g., a human) orally or by injection. For example it may be given orally for the treatment of type II diabetes, insulin resistant diabetes, gestational diabetes, stress induced diabetes, obesity, hyperϊipidemia, and other conditions associated with elevated blood glucose, elevated blood insulin, or suboptimal glucose kinetics.

Broadly, any unicellular eukaryote using sugar as its primary carbon source, of which yeast is a primary example may be used as a source of dithiochrome. Dithiochrome may be found in all eukaryotic cells, including yeast, fungus (including mushrooms) and eukaryotic cells of both animal and plant origin.

Dithiochrome is found in large amounts in yeast, liver, kidney, mushrooms and black pepper. Because of its high concentration in yeast and the low cost of this material, yeast is a preferred source for dithiochrome. Although either live yeast and dried yeast flakes can be used, of the two, live yeast has been found to be greatly superior starting material and is thus a preferred starting material.

In all of the manipulations outlined below, a pH range of 2 to 8, preferably 3 to 7, most preferably 5 to 7, can be used. In preferred embodiments of this invention, dithiochrome is obtained by the following method.

(i) The eukaryotic cell mass is first subjected to lysis conditions followed by a delipidation step. This can be carried out by: (ia) cooling the mass with liquid nitrogen followed by powdering the frozen mass obtained (e.g., with a blender) with subsequent addition of an aqueous buffer (pH = 2 to 8) and an organic solvent to the powdered mass; or by (ib) passing the eukaryotic cell mass through a French press, a sonicator, or similar device, followed by addition of an aqueous buffer (pH = 2 to 8) and an organic solvent thereto; or by (ic) contacting the eukaryotic cell mass with a mixture of water and an organic solvent, followed by transfer of the lysed mass obtained into a mixture of an aqueous buffer (pH = 2 to 8) and organic solvent. The organic solvent used in (ia) , (ib) and (ic) is selected from the group consisting of C_λ_₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethane, chloroform, and dichloromethane.

(ii) The organic phase is separated from the water phase and a water extract isolated. The isolated water extract is then optionally filtered and/or freeze-dried.

(iii) The water phase extract obtained in (ii) is charged onto (iiia) an insulin affinity chromatography matrix, (iiib) a sulfhydryl exchange chromatography matrix, (iiic) a C , 8, 18- or phenyl reverse phase chromatography matrix, or (iiid) an anionic exchange chromatography matrix.

(iv) The chromatography matrix is washed with, e.g., water or a solution having a moderate ionic strength. 91/01738 PCT/US90/04163

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(v) The chromatography matrix is then eluted to obtain DTC as a material capable of readily binding with insulin.

Elution step (v) is carried out as follows. When either the insulin infinity chromatography matrix or the sulfhydryl exchange chromatography matrix is used, a low concentration of a reducing agent, e.g., an active thiol or dithiol, is used. When the C_{4 8 18}- or phenyl reverse phase chromatography matrix is used a linear elution system of 0 to 50 vol.% (B) into (A), described above is used. When the anionic exchange chromatography matrix is used, elution is carried out with a salt.

In another particularly preferred embodiment, since the aqueous yeast extract obtained by lysis of the yeast cell mass contains 1 to 5 mM glutathione (GSH) and glutathione can disrupt the chromatography step by reducing the insulin disulfide bonds or occupying a majority of the binding sites on the insulin affinity column or on the sulfhydryl exchange column, the glutathione is initially separated from the dithiochrome product using a reverse phase column. Dithiochrome appears to have a partially hydrophobic surface and glutathione has many charged groups and little hydrophobicity. The aqueous extract is therefore advantageously loaded onto an open reverse phase column with the glutathione coming off in the wash which is discarded. DTC comes off the column when washed with organic solvent (e.g., 50:50 aqueous buffer/methanol) . The reverse phase eluent obtained is then loaded onto the insulin or sulfhydryl chromatography matrix.

After eltition from the insulin or sulfhydryl matrix with glutathione (a preferred method includes using a gradient rather than a wash) , the eluate contains glutathione (or cysteine) in addition to DTC. In this form, DTC can be stored because glutathione is a good reducing agent which prevents oxidation of the DTC. To obtain pure DTC, one may then repeat the glutathione separation using an open reverse phase column. Thus in a particularly preferred embodiment of this invention, which provides a particularly good yield and a product free of thiol, dithiochrome is obtained by:

(i¹) lysing a eukaryotic cell mass as noted above;

(ii¹) separating the organic phase from the water phase, isolating the water phase, and optionally filtering the water phase;

(iii') optionally applying the isolated water phase to a reverse phase chromatography matrix;

(iv¹) washing the chromatography matrix;

(v*) treating the chromatography matrix with a mixture of an aqueous buffer (pH = 2 to 8) and an organic solvent;

(vi¹) removing the organic solvent from the eluate under a vacuum to obtain a water phase free of glutathione;

(vii¹) applying the water phase extract to (viia) an insulin affinity chromatography matrix or to (viib) a sulfhydryl exchange chromatography matrix;

(viii¹) washing the chromatography matrix; (ix¹) treating the chromatography matrix with a reducing agent;

(x¹) applying the eluent to a reverse phase chromatography matrix; (xi¹) washing the chromatography matrix;

(xii*) treating the chromatography matrix with a mixture of aqueous buffer (pH = 2 to 8) and an organic solvent; and

(xiii) removing the organic solvent under a vacuum to obtain DTC as a material capable of binding with insulin.

Thus, in another preferred embodiment, the water phase extract isolated in step (ii) is loaded/applied onto a reverse phase column matrix and the flow through is discarded. The matrix is washed with water until base line is achieved monitoring λ = 214 nm or 260 nm. The matrix is then eluted with 50% 0.1M, pH = 3 phosphate buffer, 5% methanol with 0.2% triethylamine/50% methanol. Methanol is removed under vacuum. The solution is diluted with degassed PBS, pH -= 7.2.

The insulin/sulfhydryl exchange matrix is incubated for one hour at room temperature, end-over-end tumbling. It is then washed with PBS, washed with 1 M NaCl (pH = 3) and eluted with 16 mM GSH.

Dithiochrome has a molecular weight of approximately 920. In another embodiment, an extract containing at least about 35% wt. dithiochrome may be obtained by subjecting the water extract obtained in step (ii) above to gel exclusion chromatography and eluting all material having a molecular weight of approximately 920 (i.e., 720 to 1120, preferably 820 to 1020) .

i) lysis and delipidation of the eukaryotic cell mass:

As noted above, the eukaryotic cell mass used may be of fungus (including mushrooms) , yeast, plant or animal origin. The eukaryotic cells first subjected to lysis conditions, e.g., by grinding the cells, subjecting the cells to shear forces (e.g., by treatment with a blender) or contacting the cells with a mixture of water and organic solvent to cause their lysis.

In a preferred embodiment, the cell mass may be lysed using either one of two mechanical procedures, or a chemical procedure. The first mechanical procedure is based on cooling the cell mass with liquid nitrogen followed by powdering the frozen mass obtained with a blender with subsequent addition of the powdered product to an aqueous buffer and organic solvent mixture. The second mechanical procedure includes passing the cell mass through a French press, a sonicator, or similar device, with subsequent addition of an aqueous buffer and organic solvent mixture to the lysed cell mass obtained. The chemical procedure is based on contacting the cell mass with a mixture of water and organic solvent followed by transferring the lysed cellular mass to a mixture of an aqueous buffer and organic solvent. The organic solvent component is used in the water in an amount sufficient to denature proteins and disrupt (lyse) cells in the mass, namely in an amount of 1% to 99% (v/v) , preferably 25% to 50% (v/v) .

In the procedures set forth above, one obtains a lysed cell mass dissolved in a mixture of an aqueous buffer and the organic solvent. The organic solvent here is used to cause the delipidation of the lysed cell mass. The solvent used is preferably n-butanol, most preferably diethyl ether.

Any lower ether may be used as the organic solvent, including linear and cyclic ethers containing 2 to 8 carbon atoms. Illustrative examples of these ethers include dimethyl ether, diethyl ether, furan, tetrahydrofuran, 1,4- dioxane, etcetera. The advantage of using a lower ether, particularly diethyl ether, is that its high vapor pressure makes it easy to remove even residual quantities of the ether from the water extract.

Any lower alcohol which is either miscible or immiscible with water may be used, and combinations of these alcohols may also be used. These alcohols may contain from 1 to 5 carbon atoms, and may be linear, cyclic or branched. Illustrative examples of these alcohols include methanol, ethinol, n-propanol, i-propanol, n-butanol, i-butanol, t-butanol, n-pentanol, etc. The requirement imposed on the alcohol phase, when it is used, is that, at 22°C, it must form a separate phase from water. Some of the alcohols which may be used, e.g., methanol and ethanol, do not satisfy this condition. Such alcohols are accordingly used in the delipidation either (i) with activated charcoal or (ii) with a higher molecular weight alcohol in an amount appropriate to obtain an alcohol phase which forms a phase separate from water at 22°C.

As noted above, acetonitrile, dichloroethanes (both 1,1- and 1,2-), chloroform and dichloromethane (methylene chloride) can also be used as the organic solvent.

The lysed eukaryotic cell mass is homogenized in the water and organic solvent mixture for from 1/2 hour, preferably 2 hours to 24 hours, or longer, if necessary to cause the delipidation of the cell mass. The organic phase and the water phase are then allowed to form and are separated, preferably by centrifugation.

The organic phase (containing lipids) is discarded and the water phase is subjected to filtration or centrifugation tσ eliminate material not dissolved in the aqueous phase. The precipitate obtained from the water phase is discarded and the water phase is then subjected to lyophilization or to any other known method suitable for concentrating an aqueous biological extract which does not inactivate the dithiochrome, e.g., reverse osmosis or ion exchange chromatography.

Although both dried yeast flakes and live yeast may be used as the starting material, live yeast has been discovered by the inventor to provide greatly superior results, and is thus a preferred starting material. In a preferred embodiment, hydrated yeast that has been pelletized is formed into a ball and frozen in liquid nitrogen. The yeast ball is then broken into smaller chunks and the yeast cells disrupted with a blender by blending the yeast ball with liquid nitrogen. The blending is carried on until a fine frozen yeast powder (no chunks) is obtained.

An equal amount of buffer having a pH of 2 to 8, e.g., a sodium acetate or potassium phosphate buffer (weight yeast/volume buffer) is added to the yeast powder. The yeast buffer mixture is stirred under N₂ then centrifuged to obtain pellets which are discarded, with the supernatent being saved. The supernatent may then be extracted with the organic solvent, preferably an ether, e.g., diethylether.

ii) chromatography:

All of these chromatography procedures may be carried out at any practical temperature. Thus, for example, a temperature of from -10°C to 35°C, preferably 10°C to 35°C, and more preferably 20°C to 30°C, may be used.

In one embodiment, the water extract is then applied to an insulin affinity column. The insulin chromatography column used in the present invention may be any insulin affinity chromatography column using, as a support, a polysaccharide, agarose, cellulose, dextran, silica or any other inert, polymeric particles. The particles may be of spherical or mlcrocrystalline structure with a particle size of 0.1 μm to 500 μro.

The insulin n.ay be immobilized onto these supports using any known methods, including the aldehyde, cyanogen bromide, N-hydroxy succinimide ester, FMP or carbonyl diimidazole coupling methods. For example, using Affi-Prep 10 from Bio-Rad, Richmond, California, U.S.A. and insulin, one can readily make such an insulin affinity column. Alternatively, an Insulin-Actigel column may be used Sterogene Bioseparations, Inc., Arcadia, California USA 91006.

Once the water phase extract has been subjected to the insulin affinity chromatography matrix, the insulin affinity chromatography matrix is then subjected to a washing step. In any washing operation, used in this and the other manipulations, any salt or mixture of salts with an ionic strength ranging from 0.1 to 5 M, preferably about 1 M, can be used, at any pH from 2 to 8.

Following the washing operation, the insulin affinity chromatography matrix is treated with an active thiol or dithiol. In a preferred embodiment, gradient of active thiol or dithiol may be used using (i) a thiol concentration-which increases from 0 mM to 40 mM, or (ii) a dithiol concentration.which increases from 0 mM to 20 mM. Elution of dithiochrome occurs at approximately 5 to 20 mM of active thiol.or at approximately 2.5 to 10 mM of active dithiol, at room temperature. Alternatively dithiochrome may be eluted from the column by simply treating the column with an active thiol or dithiol at a concentration of from 10 mM to 40 mM, or from 5 mM to 20 mM, respectively.

The active thiol or dithiol which may be used to elute the dithiochrome may be any water-soluble thiol containing at least one sulfhydryl group. These active thiols/dithiols may contain additional hydroxyl groups to improve their water solubility. The thiols and dithiols which may be used include, dithioerythritol (DTE) , dithiothreitol (DTT) , glutathione, thioglycerol, cysteine, 2-mercaptoethanol or lipoic acid.

The purification procedure may be carried out at any practical temperature. Thus, for example, it may be carried out at a temperature of from -10°C to 35°C, more preferably 20°C to 30°C.

Alternatively, the water extract is then subjected to sulfhydryl exchange chromatography. The sulfhydryl exchange chromatography column used in the present invention may be any sulfhydryl exchange chromatography substance supported on, e.g., a polysaccharide, agarose, cellulose, dextran, any other inert polymeric support or a silicon support, e.g., sulfhydryl exchange material available on agarose from Pharmacia, Bio-Rad and Sigma Chemical Co. of St. Louis, Missouri 63178 USA, called Activated Thiol-Sepharose™ or Affigel 501™.

Once the water phase extract has been subjected to the chromatography matrix, the chromatography matrix is then subjected to a washing step to obtain a steady base line.

In this washing operation any solution with moderate ionic strength may be used. In this washing operation, any salt or mixture of salts with an ionic strength ranging from 0.1 to 5 M, preferably about 1 M, at any pH of 2 to 8 may be^' used. For example, 1M NaCl at pH = 3. Once a steady base line is obtained, the chromatography matrix is eluted with a reducing agent, e.g., a gradient of active thiol or dithiol. The gradient of active thiol or dithiol is performed at a dithiol concentratioij going from 0 mM to 250 mM, preferably 10 mM, or at a thiol concentration of from 0 mM to 500 mM, preferably 20 mM. Dithiochrome can be obtained by treating the matrix with 0.05 mM to 250 mM, preferably 2.5 mM to 10 mM of active dithiol (DTE) or 0.1 mM to 500 mM, preferably 5 mM to 20 mM of active thiol, at room temperature.

The active thiol or dithiol which may be used to elute the dithiochrome may be any low molecular weight, water soluble, thiol or dithiol containing at least one sulfhydryl group. These include materials containing from 2-6 carbon atoms. These active thiols may contain additional hydroxyl groups to improve their water solubility. Exemplary thiols/dithiols include dithiothreitol (DTT) , dithioerythritol (DTE) , glutathione, thioglycerol, 2-mercaptoethanol, lipoic acid, and cysteine.

In still other embodiments, DTC may be isolated by subjecting the water extract obtained in step (ii) above to either anion exchange chromatography or C_{4 8 18}- or phenyl reverse phase chromatography.

Using anion exchange chromatography, the water extract, which may be optionally freeze-dried, is applied to a quaternary amine anionic exchange chromatography matrix,, e.g., a QAE column, an AE (amino ethyl) column, or a DEAE column. The anion exchange chromatography matrix is washed with water until a baseline at λ ^*= 214 nm or 260 nm is observed. A .linear salt gradient (e.g., a 0 to 1 M NaCl, NaBr, KC1, KBr linear gradient) is employed to elute the dithiochrome. Using a reverse phase chromatography matrix, the isolated water extract, which may also be optionally freeze-dried, is loaded onto a C_{4 8} is" ^or phenyl reverse phase chromatography column, e.g., an Axxiom C₁₈ column. The flow through is discarded. The matrix is then washed with water until a baseline is achieved monitoring at λ = 214 nm or 260 nm. Then a 0 to 50 vol.% (B) into (A) linear gradient is then employed. Component (A) is a 0.02 to 0.2 M (sodium acetate, sodium phosphate) buffer with a pH of 2 to 6, 5 vol.% C₁_₃ lower alcohol, acetonitrile, acetone, or ethoxyethoxyethanol, with 0.2% triethylamine (TEA). Component (B) is the lower alcohol, acetone, ethoxyethoxyethanol or acetonitrile. Elution is monitored at λ = 214 nm.

iii) characteristics of DTC:

The compound obtained by the above processes provides a positive Ninhydrin test for primary and secondary amines, with a ratio of secondary to primary amines of about 2:1. It has a peak in the UV spectrum at 260 nm (nicotinic acid) (see Figure 1). It elutes from a QAE column at a 0.2 to 0.5 M salt, indicating that it is weak anion. It has at least one reactive thiol based on its behavior in sulfhydryl exchange chromatography. (DEAE-type ion exchangers have diethylaminoethyl functional groups. Their support may be cellulose, agarose, dextran, silica or polymeric particles. The particles may be of spherical or microcrystalline structure with a particle size of 0.1 μ to 500 μm.)

Dithiochrome readily binds with insulin when the two materials are combined in the same solution. This provides an insulin-dithiochrome adduct in which the insulin and the dithiochrome are covalently liked to each other. For example, the insulin-dithiochrome adduct may be obtained by adding insulin and an excess of dithiochrome to a solution of 25 mM potassium phosphate buffer (pH = 7.2) and allowing the solution ^*feo tumble overnight at a temperature of 4°C. The insulin-dithiochrome adduct may be recovered from the solution by subjecting the solution to size exclusion chromatography.

iv) uses of DTC:

Dithiochrome and the adduct ("activated insulin") may be administered using any method for the administration of a pharmaceutically active material; that is enteral, suppository, subcutaneous, intravenous, intramuscular and transdermal methods may be used.

For example, dithiochrome may be administered to diabetics to alleviate their diabetes, where it may be administered in two forms. Dithiochrome may be given orally useful for the treatment of type II diabetes, insulin resistance., gestational diabetes, stress induced diabetes, obesity, hyperlipidemia, atheroma and atherosclerosis, and other conditions associated with elevated blood glucose, suboptimal glucose kinetics, or elevated blood insulin. The insulin-dithiochrome adduct ("activated insulin") , may be used to treat a patient, in a manner similar to conventional insulin injection, as a therapy for type I (juvenile or insulin-dependent) diabetes. Optional therapy can consist of a mixture of activated insulin and insulin. The ratio of insulin activation may be 0.1% to 90%, preferably 1% to 35%.

Salts of-.dithiochrome and of the insulin-dithiochrome adduct, and in particular physiologically acceptable salts of the materials, are within the scope of this invention. Such salts contain a physiologically acceptable cation, for example, the cation of an alkali metal such as sodium, quaternary ammonium ions or protonated amines. Salts may be advantageously used because they impart greater water solubility.

Dithiochrome and the insulin-dithiochrome adduct may be formulated for use as pharmaceuticals for veterinary, for example in a mammalian context, or particularly human use by a variety of methods. Thus the present invention further provides pharmaceutical compositions containing either dithiochrome or an insulin-dithiochrome adduct. The dithiochrome and the insulin-dithiochrome adduct may each be present in an amount of 1 wt.% to 99 wt.% in these compositions where they are present together with a physiologically acceptable carrier or diluent.

Dithiochrome may be administered orally. For oral administration, compositions incorporating a liquid diluent containing dithiochrome may be used. For example, a conventional solid carrier material such as starch, lactose, dextrin or magnesium stearate may be used which permits the oral composition to be provided in a formed shape, for example as tablets, capsules (including spansules) , etc. Dithiochrome for oral administration may optionally be combined with peptide(s) or protein(s) that aid in intestinal absorption and stabilize transports.

The insulin-dithiochrome adduct may be administered to a patient as an aqueous, oily or emulsified composition incorporating a liquid diluent which is usually employed for parenteral (both intravenous and intramuscular) administration and is therefore sterile and pyrogen free.

Other forms of administration than by injection or through the oral route are possible in both human and veterinary contexts for both dithiochrome and the insulin-dithiochrome adduct. For example other forms known in the art such as the use of suppositories or pessaries may be used, particularly for human administration. Thus all forms of administration, enteral, suppository, subcutaneous, IV, IM and transdermal, may be used.

Pharmaceutical compositions may be formulated in unit dosage form, i.e., in the form of discrete portions each comprising a unit, dose, or a multiple or sub-multiple of a unit dose. While the dosage of active compound (i.e., either dithiochrome or insulin dithiochrome adduct) given will depend on various factors, including the disease being treated, dithiochrome and to insulin-dithiochrome adduct may be administered to patients as follows.

For the treatment of conditions associated with elevated bloo .glucose or suboptimal glucose kinetics, including type II diabetes, insulin resistant diabetes, gestational diabetes, stress induced diabetes, obesity, and hyperlipidemia, dithiochrome may be administered to a human using a daily dosage of 0.1 microgram to 1 gram per day, preferably 1 to 200 micrograms per day, most preferably 10 to 50 micrograms.

The insulin-dithiochrome adduct may be orally administered or injected in a manner similar to that used in conventional insulin therapy as a therapy for type I (juvenile or insulin-dependent) diabetes. The insulin-dithiochrome adduct may thus be used at a daily dosage of about 1 to 10³ units of insulin, preferably 4 to 10² units. Optional therapy can consist of administering a mixture of activated insulin and insulin, using a ratio of 0.1% to 90%, preferably 1% to 35% respectively.

The insulin-dithiochrome adduct may be injected in a manner similar to that used in conventional insulin therapy as a therapy for type I (juvenile or insulin-dependent) diabetes. The insulin-dithiochrome adduct may thus be used at a daily dosage of about 1 to 10³ units of insulin, preferably to 4 to 10² units of insulin.

However it should be appreciated that it may be 5 appropriate under certain circumstances to give daily dosages of- either of these two materials either below or above these levels.

Other features of this invention will become apparent in the course of the following description of exemplary 10 embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES

Extraction of Yeast Insulin-Actigel Chromatography:

Hydrated yeast that have been pelleted at 3000 rpm for

15 15 minutes are formed into a ball and frozen in liquid LN₂. The yeast ball is then broken into smaller chunks and the yeast cells are disrupted with a stainless steel bar blender by blending the yeast ball with LN₂. The blending is stopped after a fine frozen yeast powder exists (no

20 chunks) . An equal amount of sodium acetate buffer (pH 4-7) weight yeast/volume buffer is added to the powder in an erlenmeyer flask. The yeast buffer mixture is then stirred on a magnetic stirrer for 1 hour at 4°C under an N₂ atmosphere. The mixture is then centrifuged at 10,000 rpm

25 for 30 minutes. The yeast pellet is discarded and the supernatent saved. The supernatent is then extracted at room temperature with 6-10 equal volumes of diethylether to remove lipids. The excessive ether in the aqueous phase is removed in vacuo and the entire extract degassed. The

30 dilapidated extract is then centrifuged at 10,000 rpm for 30 minutes and the pellet is discarded. Insulin Affinity Chromatography:

The aqueous yeast extract is loaded on to a C₁₈ reverse phase matrix and the flow through is discarded. The matrix is washed with water until baseline is achieved monitoring at 214 nm or 260 nm. ' The matrix is eluted with 50% o.lM pH=3 phosphate buffet 5% MeOH with 0.2% triethylamine / 50% MeOH. MeOH js removed under vacuum. The solution is diluted with degassed PBS pH = 7.2.

The degassed eluate is added to insulin affinity chromatography matrix. The insulin matrix is incubated for 1 hour at room temperature, end-over-end tumbling. It is then washed with phosphate buffer (2 mM, pH = 7.2), washed with 1M NaCl pH = 3, and eluted with 16 mM GSH.

The glutathione is separated from DTC by reverse phase chromatography. The eluate is loaded on to a C₁₈ reverse phase matrix^-and the flow through is discarded. The matrix is washed with water until baseline is achieved monitoring at 214 nm or*260 nm. The matrix is eluted with 50% 0.1M pH =3 phosphate buffer 5% MeOH with 0.2% triethylamine/ 50% MeOH. MeOH is removed under vacuum.

Sulfhydryl Exchange Chromatography:

The aqueous yeast extract is loaded on to a C₁₈ reverse phase matrix and the flow through is discarded. The matrix is washed with water until baseline is achieved monitoring a lambda = 254 nm. The matrix is eluted with 50% O.lM pH =3 phosphate buffer 5% MeOH with 0.2% triethylamine / 50% MeOH. MeOH is removed under vacuum. The solution is diluted with phophate buffer (25 mM, pH = 7.2).

The degassed eluate is added to sulfhydryl exchange chromatography matrix. The insulin matrix is incubated for 1 hour at room temperature, end-over-end tumbling. It is then washed with phosphate buffer, washed with 1M NaCl pH = 3, and eluted with 16 mM GSH.

The glutathione is separated from DTC by reverse phase chromatography. The eluate is loaded on to a C₁₈ reverse phase matrix and the flow through is discarded. The matrix is washed with water until baseline is achieved monitoring at 254 nm. The matrix is eluted with 50% O.lM pH = 3 phosphate buffer 5% MeOH with 0.2% triethylamine / 50% MeOH. MeOH is removed under vacuum.

C_1E-Reverse Phase Chromatography:

Standard 5 [mu]m, 25 cm Axxio C₁₈ column is used under gradient conditions to fractionate the crude delipidated yeast extract. 20 μl of the crude yeast extract is injected on the HPLC. A 0-50% B into A linear gradient is employed (A 0.1 M potassium phosphate buffer pH=3 5% MeOH w/0.2% Triethylamine (TEA) B MeOH). The elution is monitored at 214 nm and the column flow rate is 0.7 ml/ in. The bioactive fractions are collected from 15 to 40% in the gradient elution. The chromatogram is complex and consists of at least 30 peaks.

Anion exchange chromatography: OAE

A 3ml JT Baker SPE column is loaded with 3ml of the crude yeast extract. The column is washed with H₂0 until a baseline at 254 nm is observed. A 0 to 1 M NaCl linear gradient is employed. DTC elutes early in the gradient (indicating it is a weak anion) .

Chemical Analysis: Ninhvdrin: DTC is ninhydrin positive. Both primary and secondary amines are present, in a ratio of approximately 2:1.

Chromiu : Bioactive samples of DTC test positive for chromium in tests performed at a commercial laboratory.

Thiol: DTC is a thiol or dithiol based on its behavior on sulfhydryl exchange chromatography.

Biological Activity:

Dithiochrome increases the activity of insulin. The biological assay is based on this property. Authentic DTC will increase the ability of insulin to stimulate both glucose uptake and glucose oxidation. Insulin is required for this activity. Cells do not respond to DTC alone, but only in the presence of both DTC and insulin.

Cellular biological assays can be performed with any insulin responsive cell. These include yeast cells, and muscle, fat and liver cells (myocytes, adipocytes and hepatocytes) from any mammal. Cell lines that are insulin sensitive are also useful. For cellular assays, glucose uptake can be measured at least two ways. One is to monitor the concentration of glucose in test wells containing cells, insulin and various concentrations and DTC using a standard assay for glucose. A second method is to measure uptake of deoxyglucose labelled with tritium.

In vivo assays can be performed on any animal with insulin resistance or insulin resistance diabetes. Dithiochrome or activated insulin are administered and blood sugar or blood insulin are monitored.

(1) Preparation of 3T3-L1 cells. A mouse adipocyte line (American Type Culture Cell Line CCL 92.1) known at 3T3-L1 is grown to a monolayer and subjected to conditions that cause it to differentiate to cells with adipocyte characteristics.

(2) Preparation of Rat epididymal fat cells.

Fat cells from rats are prepared according to the method of Rodbell (Rodbell, Martin: Metabolism of isolated Fat Cells. Journal of Biological Chemistry. 239:375-380 (1964)).

(3) Glucose consumption assay.

Trypsinized 3T3-L1 cells or collagenase-treated fresh rat adipocytes are plated into 24 well plates. Cells are then cultured in the presence of insulin ranging from 0 to 50 mM, with and without dithiochrome, in the presence of 4 mM glucose. A sample of the medium is taken at time zero and at intervals up to 10 hours. The samples are analyzed for glucose and the glucose consumption rate is determined by the method of least squares.

Figure 2 shows the results a glucose consumption assay. The lower graphs show the raw data, that is, the UV absorbance from glucose hexokinase assay. The graph on top shows the glucose consumption rate as determined by least squares slope of the glucose measurements. Note that at zero insulin concentration DTC alone has no effect on glucose consumption.

(4) Glucose uptake assay.

Trypsinized 3T3-L1 cells or collagenase-treated fresh rat adipocytes are plated into 24 well plates. Cells are then cultured in the presence Of insulin ranging from 0 to 50 mM, with and without dithiochrome, in the absence of glucose. After a period ranging from 5 minutes to ten hours, deoxyglucose labelled with tritium is added. After 5 minutes the cells are quenched, and washed. The level of tritium remaining (and therefore in the cells) is measured with a liquid scintillation counter.

Figure 3 shows the results of a glucose uptake assay done 5 hours after the addition of insulin and DTC. Again note that at a zero insulin concentration DTC alone has no effect on glucose consumption.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

1. A material obtained by:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C_x_₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(ii) separating the organic phase from the water phase and isolating a water phase extract;

(iii) applying said water phase extract to (iiia) an insulin affinity chromatography matrix or to (iiib) a sulfhydryl chromatography matrix;

(iv) washing said chromatography matrix; and

(v) passing through said chromatography matrix a reducing agent to elute therefrom a material capable of binding with insulin.

2. The material of Claim 1, wherein the eluate obtained in step (v) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain said material capable of binding with insulin.

3. The material of Claim 2, wherein said water phase extract obtained in step (ii) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain an eluate from which the organic solvent is removed to provide a water phase extract which is then subjected to step (iii) .

4. The material of Claim 1, wherein said lysing in step (i) comprises, (ia) contacting said eukaryotic cell mass with liquid nitrogen followed by powdering the frozen cell mass obtained, or (ib) passing said eukaryotic cell mass through a French press or sonicator, or (ic) combining said eukaryotic cell mass with a mixture of water and said organic solvent.

5. The material of Claim 1, said material displaying a positive Ninhydrin test for primary amines.

6. The material of Claim 1, said material displaying a positive Ninhydrin test for secondary amines.

7. The material of Claim 1, said material displaying a positive Ninhydrin test for both primary and secondary amines.

8. The material of Claim 1, said material having a UV spectSrum peak at 260 nm.

9. The material of Claim 1, said material having a reactive sulfhydryl group.

10. A material obtained by:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of _s2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C-^_g alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(iii) applying said water phase extract to (iiia) an anion chromatography matrix or to (iiib) a C_{4 8 18}- or phenyl reverse phase chromatography matrix;

(iv)* washing said chromatography matrix; and

(v) eluting from said chromatography matrix a material capable of binding with insulin.

11. The material of Claim 10, wherein said lysing in step (i) comprises, (ia) contacting said eukaryotic cell mass with liquid nitrogen followed by powdering the frozen cell mass obtained, or (ib) passing said eukaryotic cell mass through a French press or sonicator, or (ic) combining said eukaryotic cell mass with water and said organic solvent.

12. A pharmaceutical composition comprising, in association with a pharmaceutically acceptable carrier or diluent, a material obtained by:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C^_g alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(iii) applying said water phase extract to (iiia) an insulin affinity chromatography matrix or to (iiib) a sulfhydryl exchange chromatography matrix; (iv) washing said chromatography matrix; and

13. The composition of Claim 12, wherein the eluate obtained in step (v) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain said material capable of binding with insulin.

14. The composition of Claim 13, wherein said water phase extract obtained in step (ii) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to. obtain an eluate from which the organic solvent is removed to provide a water phase extract which is then subjected to step (iii).

15. The composition material of Claim 12, wherein said lysing in step (i) comprises, (ia) contacting said eukaryotic cell mass with liquid nitrogen followed by powdering the frozen cell mass obtained, or (ib) passing said eukaryotic cell mass through a French press or sonicator, or (ic) combining said eukaryotic cell mass with a mixture of "water and said organic solvent.

16. The pharmaceutical composition of Claim 12, wherein said pharmaceutical carrier or diluent is suitable for oral administration.

17. A dithiochrome-insulin adduct, wherein dithiochrome and insulin are covalently linked together and dithiochrome -Is a material obtained by: (i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C₁_₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(iii) applying said water phase extract to (iiia) an insulin affinity chromatography matrix or to (iiib) a sulfhydryl exchange chromatography matrix;

(iv) washing said chromatography matrix; and

18. The adduct of Claim 17, wherein the eluate obtained in step (v) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain said material capable of binding with insulin.

19. The adduct of Claim 18, wherein said water phase extract obtained in step (ii) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain an eluate from which the organic solvent is removed to provide a water phase extract which is then subjected to step (iii) .

20. The adduct of Claim 17, wherein said lysing in^' step (i) comprises, (ia) contacting said eukaryotic cell mass with liquid nitrogen followed by powdering the frozen cell mass obtained, or (ib) passing said eukaryotic cell mass through a French press or sonicator, or (ic) combining said eukaryotic cell mass with water and said organic solvent.

21. A process for obtaining a purified material possessing glucose tolerance factor activity, comprising:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C_λ_₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(iv) washing said chromatography matrix; and

22. The process of Claim 21, wherein the eluate obtained in step (v) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain said material capable of binding with insulin.

23. The process of Claim 22, wherein said water phase extract obtained in step (ii) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain an eluate from which the organic solvent is removed to provide a water phase extract which is then subjected to step (iii) .

24. The process of Claim 21, wherein said lysing in step (i) comprises, (ia) contacting said eukaryotic cell mass with liquid nitrogen followed by powdering the frozen cell mass obtained, or (ib) passing said eukaryotic cell mass through a French press or sonicator, or (ic) combining said eukaryotic cell mass with a mixture of water and said organic solvent.

25. The process of Claim 24, comprising, in step (iϋ) applying said water phase to an insulin affinity chromatography matrix.

26. The process of Claim 24, comprising, in step (iii) , applying said water phase extract to a sulfhydryl chromatography matrix.

27. The process of Claim 24, wherein said organic solvent is diethylether.

28. The process of Claim 21, comprising passing through said chromatography matrix, as said reducing agent, a thiol or a dithiol at a concentration of up to 500 M for said thiol and up to 250 mM for said dithiol.

29. The process of Claim 26, wherein said sulfhydryl exchange chromatography matrix is Actigel-T™, Activated Thiol-Sepharose™ or Affigel 501™.

30. A process for obtaining a purified material possessing glucose tolerance factor activity, comprising:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C₁_₅ alcohols,* C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(iϋ) applying said water phase extract to an anion exchange chromatography matrix;

(iv) washing said chromatography matrix; and

(v) passing through said chromatography matrix a salt solution to elute therefrom a material capable of binding with insulin.

31. A process for obtaining a purified material possessing glucose tolerance factor activity, comprising:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C₁_₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(iii) applying said water phase extract to a C_{4 8 18}-, or phenyl reverse phase chromatography matrix; (iv) subjecting said chromatography matrix to a linear gradient of (B) into (A), wherein (A) is a 0.02 to 0.2 M solution of a buffer having a pH of 2 to 8, containing 5 vol.% of a C₁_₃ alcohol, acetone, ethoxyethoxyethanol or acetonitrile, and 0.2 vol.% of triethylamine, and wherein (B) is said C___₃ alcohol, acetone, ethoxyethoxyethanol or acetonitrile, to obtain a material capable of binding with insulin.

32. A method for treating a patient suffering from either elevated blood glucose or suboptimal glucose kinetics, comprising administering to said patient a material obtained by:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C _₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(iv) washing said chromatography matrix; and

(v) passing through said chromatography matrix a solution of a reducing agent to elute therefrom a material capable of binding with insulin.

33. The method of Claim 32, wherein the eluate obtained in step (v) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain said material capable of binding with insulin.

34. The method of Claim 35, wherein said water phase extract obtained in step (ii) is applied to a reverse phase chromatography matrix "which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain an eluate from which the organic solvent is removed to provide a water phase extract which is then subjected to step (iii) .

35. The^" method of Claim 32, wherein said patient is being treated for type II diabetes, insulin resistance, gestational diabetes, stress induced diabetes, obesity, hyperlipidemia atheroma or atherosderoris.

36. A method for treating a patient suffering from type I diabetes, comprising administering to said patient a dithiochrome-insulin adduct, wherein said dithiochrome is a material obtained by:

(i) , lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C___₅ alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

37. The method of Claim 36, wherein the eluate obtained in step (v) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain said material capable of binding with insulin.

38. The method of Claim 37, wherein said water phase extract obtained in step (ii) is applied to a reverse phase chromatography matrix which is washed and then treated with a mixture of said aqueous buffer and said organic solvent to obtain an eluate from which the organic solvent is removed to provide a water phase extract which is then subjected to step (iii) .

39. The method of Claim 36, wherein said patient is being treated for juvenile or insulin-dependent diabetes.

40. A process for obtaining a material possessing glucose tolerance factor activity, comprising:

(i) lysing a eukaryotic cell mass and combining the lysate obtained with a mixture of an aqueous buffer having a pH of 2 to 8 and an organic solvent, wherein said organic solvent is selected from the group consisting of C_1-5 alcohols, C₂_₈ ethers, acetonitrile, dichloroethanes, chloroform, and methylene chloride;

(ii) separating the organic phase from the water phase and isolating a water phase extract; (iii) subjecting said water phase extract to gel exclusion chromatography to isolate all material having a molecular weight of approximately 720 to 1120 to obtain a material capable of binding with insulin.

41. The process of Claim 40, comprising isolating all material having a molecular weight of approximately 820 to approximately 1020.