GB2104381A - Pharmaceutical formulations comprising human insulin, human c-peptide, and human proinsulin - Google Patents

Abstract

A pharmaceutical composition comprises, in association with a pharmaceutically acceptable carrier, human insulin, human C-peptide, and human proinsulin, said human C-peptide being present in a molar ratio, human insulin to human C- peptide, of from about 1:4 to about 4:1, and said human proinsulin being present in a weight ratio, human insulin to human proinsulin, of from about 1:100 to about 100:1.

Description

SPECIFICATION Pharmaceutical formulations comprising human insulin, human c-peptide, and human proinsulin Diabetes mellitus is a metabolic disorder characterized by the failure of body tissues to oxidize carbohydrates at the normal rate. Its most important factor is a deficiency of insulin. During the last 60 years people suffering from diabetes have been greatly aided by receiving controlled amounts of insulin. To the present time, the insulin used by diabetics has been isolated from animal pancreases, generally bovine and porcine. Both bovine and porcine insulin differ structurally from insulin generated by the human pancreas. Recently, it has become possible, by recombinant DNA methodology, to produce insulin identicai to that produced by the human pancreas.The use of such insulin will enable the diabetic to more closely mimic the natural system than heretofore has been possible.

Nevertheless, it long has been recognized that administration of insulin to the diabetic is alone insufficient to restore and/or maintain the normal metabolic state. Although insulin has its manifested effect on carbohydrate metabolism, diabetes mellitus carries additional disorders, most if not all of which are related to the structure and function of blood vessels. The deficiencies leading to these disorders rarely are completely corrected by conventional insulin therapy.

Those vascular abnormalities associated with diabetes often are referred to as "complications of diabetes". They consist generally of microangiopathic changes resulting in lesions in the retina and the kidney. Neuropathy represents an additional diabetic complication which may or may not be related directly or indirectly to the noted microangiopathic changes.Examples of specific manifestations of diabetes complications are (1) diseases of the eye, including retinopathy, cataract formation, glaucoma, and extraocular muscle palsies; (2) diseases of the mouth, including gingivitis, increased incidence of dental caries, periodontal disease, and greater resorption of the alveolar bone; (3) motor, sensory, and autonomic neuropathy; (4) largevessel disease; (5) microangiopathy; (6) diseases of the skin, including xanthoma diabeticorum, necrobiosis lipoidica diabeticorum, furunculosis, mycosis, and pruritis; (7) diseases of the kidneys, including diabetic glomerulosclerosis, arteriolar nephrosclerosis, and pyelonephritis; and (8) problems during pregnancy, including increased incidence of large babies, stilibirths, miscarriages, neonatal deaths, and congenitai defects.

Many, and perhaps all, of the diabetic complications are the result of the failure of insulin alone to restore the body to its natural hormonal balance.

This invention concerns pharmaceutical compositions that more nearly achieve and maintain natural hormonal homeostasis in a diabetic state than can be achieved by administration of insulin alone.

Thus, this invention concerns a pharmaceutical composition which comprises, in association with a pharmaceutically acceptable carrier, human insulin, human C-peptide, and human proinsulin, said human C-peptide being present in a ratio on a molar basis, human insulin to human C-peptide, of from about 1:4 to about 4: 1, and said human proinsulin being present in a ratio on a weight basis, human insulin to human proinsulin, of from about 1:100 to about 100:1.

The three essential constituents of the pharmaceutical compositions of this invention are human insulin, human C-peptide, and human proinsulin.

The administration of a combination of human insulin, human C-peptide, and human proinsulin using a composition in accordance with this invention will produce a more natural utilization of glucose and better glucose control than is achieved by insulin alone, thereby diminishiny hereinbefore described adverse diabetic complications.

Human proinsulin is available via a variety of routes, including organic synthesis, isolation from human pancreas by conventional methodoiogy, and, more recently, recombinant DNA methodology.

In broad outline, the production of proinsulin using recombinant DNA methodoloty involves obtaining, whether by isolation, construction, or a combination of both, a sequence of DNA coding for the amino acid sequence of human proinsulin. The human proinsulin DNA then is inserted in reading phase into a suitable cloning and expression vehicle. The vehicle is used to transform a suitable microorganism after which the transformed microorganism is subjected to fermentation conditions leading to (a) the production of additional copies of the proinsulin genecontaining vector and (b) the expression of proinsulin or a proinsulin precursor product.

In the event the expression product is a proinsulin precursor, it generally will comprise the human proinsulin amino acid sequence joined at its amino terminal end to a fragment of a protein normally expressed in the gene sequence into which the proinsulin gene has been inserted. The proinsulin amino acid sequence is joined to the protein fragment through a specifically cleavable site, typically methionine. This product is customarily referred to as a fused gene product.

The proinsulin amino acid sequence is cleaved from the fused gene product using cyanogen bromide after which the cysteine sulfhydryl moieties of the proinsulin amino acid sequence are stabilized by conversion to their corresponding S-sulfonates.

The resulting proinsulin S-sulfonate is purified, and the purified proinsulin S-sulfonate then is converted to proinsulin by formation of the three properly located disulfide bonds. The resulting proinsulin product is purified.

A second active constituent of the compositions of this invention, human insulin, also is available via a variety of routes, including organic synthesis, isolation from human pancreas by conventional techniques, conversion of isolated animal insulin, conversion of human proinsulin, and recombinant DNA methodology.

Human proinsulin, however produced, can be enzymatically cleaved, for example, using trypsin and carboxypeptidase B, to produce human Insulin.

Using recombinant DNA methodology in a manner analogous to that hereinbefore described for the production of human proinsulin, human insulin can be prepared by the separate expression and isolation of human insulin A-chain and human insulin B-chain followed by their proper disulfide bond formation.

Human insulin can also be prepared from porcine insulin. Human insulin differs from porcine insulin by a single amino acid, i.e., the B-chain carboxyl terminal amino acid. Alanine, the B-30 amino acid of porcine insulin, is cleaved and replaced by threonine. In this regard, see, for example, U.S. Patent No. 3,276,961.

A third active constituent of the composition of this invention, human C-peptide, is a portion of a peptide present in human proinsulin and to which the insulin A- and B-chains are joined.

The latter peptide, termed a "connecting peptide' is removed during production of human insulin from proinsulin. The connecting peptide present in human proinsulin has the formula Arg-Arg-Gíu-Ala-Gíu-Asp-Leu-Gín-Vaí-Gíy-Gín-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu- Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-GIn-Lys-Arg.

The human C-peptide present in the composition of this invention differs from the connecting peptide by elimination of four amino acids, two at each end. Thus, the human C-peptide has the structure Gíu-Aía-Glu-Asp-Leu-Gln-Val-Gly-Gín-Val-Gíu-Leu-Gly-Gly-Gly-Pro-Gly-Aía-Gly-Ser-Leu-Gln-Pro- Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln.

The human C-peptide constituent of the composition of this invention can be produced by chemical synthesis, see, e.g., N. Yanaihara, C. Yanaihara, M. Sakagami, N. Sakura, T.

Hashimoto, and T. Nishida, Diabetes 27(suppl. 1) 149-160 (1978), or from human proinsulin as a result of its cleavage to produce human insulin, Human C-peptide also is available in conjunction with human insulin via the aforedescribed enzymatic cleavage of human proinsulin.

The compositions of this invention contain human insulin, human C-peptide. and human proinsulin. Human proinsulin is present in a weight ratio, human insulin to human proinsulin, of from about 1:100 to about 1 00:1. Preferably, the weight ratio of human insulin to human proinsulin is from about 1:2 to about 1 00:1, more preferably, from about 1:1 to about 20:1, and, most preferably, from about 4:1 to about 20:1.Additional preferred weight ratio ranges, human insulin to human proinsulin, are from about 1:30 to about 100:1; from about 1:15 to about 100:1; from about 1:10 to about 100:1; from about 1:30 to about 20:1; from about 1:15 to about 20:1; and from about 1:10 to about 20:1, Human C-peptide is present in the compostions of this invention in a molar ratio, human insulin to human C-peptide. of from about 1:4 to about 4:1. Preferably, the molar ratio of human insulin to human C-peptide is from about 1:2 to about 2:1, and, most preferably, from about 1:1 to about 2:1.

As noted, the compositions of this invention are useful in promoting the attainment of natural hormonal homeostatis and thereby preventing or substantially diminishing or retarding those recognized diabetic complications. It is recognized that certain diabetics are unable to effectively receive insulin by subcutaneous injection due to the presence of proteases at the injection site that rapidly destroy the insulin before it has an opportunity to be absorbed into the bloodstream and transported to the receptor sites. These diabetics, if they are to receive insulin at all, must receive it by intravenous injection. The necessary repeated intravenous injections are undesirable due to their deleterious effect on the veins of the recipient and infections associated therewith. It has been discovered that human proinsulin is not degraded by these insulin-degrading proteases and, thus, it can be administered by subcutaneous injection. Its stability and thus availability promote attainment of natural hormonal homeostasis. Moreover, since insulin and proinsulin together form complexes, it can be expected that proinsulin will afford protection for the otherwise degradable insulin.

It also has been noted from recent studies [Podlecki et al., Diabetes, 31, Suppl. 2, 126A (1 982)] that human proinsulin is internalized into target tissues, e.g. fat cells. Although its particular intracellular action on a molecular scale is as yet undetermined, these findings further support the disclosure herein that human proinsulin plays an active role in and is necessary for the attainment of natural hormonal homeostasis.

In addition to promoting the attainment of natural hormonal homeostasis, those compositions of this invention in which the weight ratio, human insulin to human proinsulin, is equal to or less than about 1:1, afford the added benefit of an extra long hypoglycemic effect. Preferred compositions which carry this added benefit have a weight ratio, human insulin to human proinsulin, of from about 1:100 to about 1:1. Other preferred weight ratios, human insulin to human proinsulin, are from about 1:1, and, more particularly, from about 1:30 to about 1:1 0, or.from about 1:10 to about 1:1. Such compositions will minimize the need for customary insulin formulation additives such as protamine which is present in NPH insulin or excess zinc which is present in lente insulin. Both such additives are artificial and unphysiologic.

Schluter et al., Diabetes 31, Suppl. 2, 1 35A (1982), describe studies that demonstrate that human insulin receptor binding is enhanced by the presence of human proinsulin. These results again further support the disclosure herein that the availability and presence of human proinsulin results in the promotion or restoration of natural hormonal homeostasis. The amount of the compositions of this invention necessary to maintain natural hormonal homeostasis or to achieve a state that more nearly approaches natural hormonal homeostasis in the diabetic, of course, will depend upon the severity of the diabetic condition. Moreover, the amount will vary depending upon the route of administration. Ultimately, the amount of composition administered and the frequency of such administration will be at the discretion of the particular physician.In general, however, the dosage will be in the range affording from about 0.02 to about 5 units of human insulin activity per kilogram body weight per day, and, preferably, from about 0.1 to about 1 unit of human insulin activity per kilogram body weight per day.

The composition is administered parenterally, including subcutaneous, intramuscular, and intravenous. The compositions of this invention comprise the active ingredients, human insulin, human C-peptide, and human proinsulin, together with a pharmaceutically acceptable carrier therefor and, optionally, other therapeutic ingredients. The total amount of active ingredients present in the composition ranges from about 99.99 to about 0.01 percent by weight. The carrier must be acceptable in the sense that it is compatible with other components of the composition and is not deleterious to the recipient thereof.

Compositions of this invention suitable for parenteral administration conveniently comprise sterile aqueous solutions and/or suspensions of the pharmaceutically active ingredients, which solutions 7r suspensions preferably are made isotonic with the blood of the recipient, generally using sodium chloride, glycerin, glucose, mannitol, sorbitol, and similar known agents. In addition, the compositions may contain any of a number of adjuvants, such as buffers, preservatives, dispersing agents, agents that promote rapid onset of action, agents that promote prolonged duration of action, and other known agents. Typical preservatives are, for example, phenol, necresol, methyl phydroxybenzoate, and others. Typical buffers are, for example, sodium phosphate, sodium acetate, sodium citrate, and others.

Moreover, an acid, such as hydrochloric acid, or a base, such as sodium hydroxide, can be used for pH adjustment. In general, the pH of the aqueous composition ranges from about 2 to about 8, and, preferably, from about 6.8 to about 8.0.

Other suitable additives are, for example, divalent zinc ion, which, if present at all, is generally present in an amount from about 0.01 mg. to about 0.5 mg per 100 units of human insulin, and protamine salt (for example, in the form of its sulfate), which, if present at all, is generally present in an amount from about 0.1 mg. to about 3 mg. per 100 units of human insulin activity.

Examples of particular pharmaceutical compositions of this invention are provided in the examples appearing hereinbelow.

Example 1 Neutral Regular Human Insulin: Human C-Peptide: Human Proinsulin Formulation [1:4 human insulin: human C-peptide on molar basis and 100:1 human insulin: human proinsulin on weight basis at 40 Units (U) insulin per cubic centimeter (cc.)] To prepare 10 cc. of the composition, mix Human Zinc Insulin (28 U/mg.) 400 U Human C-Peptide 30 mg.

Human Proinsulin 0.14 mg.

Phenol, distilled 20 mg.

Glycerin 160 mg.

Water and either 10% hydrochloric acid or 10% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7 0-7.8.

Example 2 Neutral Regular Human Insulin: Human C-Peptide: Human Proinsulin Formulation [1:1 human insulin: human C-peptide on molar basis and 20:1 human insulin: human proinsulin on weight basis at 100 U insulin per cc.] To prepare 10 cc. of the composition. mix Human Zinc Insulin (28 U/mg ) 1000 U Human C-Peptide 1 9 mg Human Proinsulin 1.8 mg.

Phenol, distilled 20 mg Glycerin 160 mg.

Water and either 10% hydrochloric acid or 10% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7 0-7.8.

Example 3 Protamine, Zinc Human Insulin: Human C-Peptide: Human Proinsulin Formulation [1:1 human insulin: human C-peptide on molar basis and 20:1 human insulin: human proinsulin on weight basis at 40 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Zinc Insulin (28 U/mg.) 400 U Human C-Peptide 8 mg.

Human Proinsulin 0.7 mg Phenol, distilled 25 mg.

Zinc Oxide 0.78 mg.

Glycerin 160 mg.

Protamine Sulfate 4.0-6.0 mg.

Sodium Phosphate, crystals 38 mg.

Water and either 10% hydrochloric acid or 10% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.1-7.4.

Example 4 Protamine, Zinc Human Insulin: Human C-Peptide: Human Proinsulin Formulation [2:1 human insulin: human C-peptide on molar basis and 1:1 human insulin: human proinsulin on weight basis at 100 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Zinc Insulin (28 U/mg.) 1000 U Human C-Peptide 9 mg.

Human Proinsulin 36 mg.

Phenol, distilled 25 mg.

Zinc oxide 2.0 mg.

Glycerin 160 mg.

Protamine Sulfate 10-15 mg.

Sodium Phosphate, crystals 38 mg.

Water and either 10% hydrochloric acid or 1OU/o sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.1-7.4.

Example 5 Isophane Protamine, Zinc Human Insulin: Human C-Peptide: Human Proinsulin Formulation [1:1 human insulin: human C-peptide on molar basis and 4:1 human insulin: human proinsulin on weight basis at 40 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Zinc Insulin (28 U/mg.) 400 U Human C-Peptide 8 mg.

Human Proinsulin 3.6 mg.

m-Cresol, distilled 1 6 mg.

Phenol, distilled 6.5 mg.

Glycerin 160 mg.

Protamine Sulfate 1.2-2.4 mg.

Sodium Phosphate, crystals 38 mg.

Water and either 10% hydrochloric acid or 10% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.1-7.4.

Example 6 Isophane Protamine, Zinc Human Insulin: Human C-Peptide: Human Proinsulin Formulation [4:1 human insulin: human C-peptide on molar basis and 1:2 human insulin: human proinsulin on weight basis at 100 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Zinc Insulin (28 U/mg.) 1000 U Human C-Peptide 5 mg.

Human Proinsulin 71 mg.

m-Cresol, distilled 1 6 mg.

Phenol, distilled 6.5 mg.

Glycerin 160 mg.

Protamine Sulfate 3.0-6.0 mg.

Sodium Phosphate, Crystals 38 mg.

Water and either 10% hydrochloric acid or 10% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.1-7.4.

Example 7Zinc Human Insulin Suspension: Human C-Peptide: Human Proinsulin Formulation [1:2 human insulin: human C-peptide on molar basis and 1:10 human insulin: human proinsulin on weight basis at 40 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Zinc Insulin (28 U/mg.) 400 U Human C-Peptide 1 5 mg.

Human Proinsulin 1 43 mg.

Sodium Acetate, Anhydrous 1 6 mg.

Sodium Chloride, Granular 70 mg.

Methyl p-Hydroxybenzoate 10 mg.

Zinc Oxide 0.63 mg.

Water and either 10% hydrochloric acid or 10% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.2-7.5.

Example 8Zinc Human Insulin Suspension: Human C-Peptide: Human Proinsulin Formulation [1:1 human insulin: human C-peptide on molar basis and 20:1 human insulin: human proinsulin on weight basis at 100 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Zinc Insulin (28 U/mg.) 1000 U Human C-Peptide 1 9 mg.

Human Proinsulin 1.8 mg.

Sodium Acetate, Anhydrous 16 mg.

Sodium Chloride, Granular 70 mg.

Methyl pHydroxybenzoate 10 mg.

Zinc Oxide 1.6 mg.

Water and either 10% hydrochloric acid or 10% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.2-7.5.

Example 9 Neutral Regular Human Insulin: Human C-Peptide: Human Proinsulin Formulation [1:4 human insulin: human C-peptide on molar basis and 100:1 human insulin: human proinsulin on weight basis at 40 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Sodium Insulin (28 U/mg.) 400 U Human C-Peptide 30 mg.

Human Proinsulin 0.14 mg.

Phenol, distilled 20 mg.

Glycerin 160 mg.

Water and either 10% hydrochloric acid or 1 0% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.0-7.8.

Example 10 Neutral Regular Human Insulin: Human C-Peptide: Human Proinsulin Formulation [1:1 human Insulin: human C-peptide on molar basis and 20:1 human insulin: human proinsulin on weight basis at 100 U insulin per cc.] To prepare 10 cc. of the composition, mix Human Sodium Insulin (28 U/mg.) 1000 U Human C-Peptide 1 9 mg.

Human Proinsulin 1.8 mg.

Phenol, distilled 20 mg.

Glycerin 160 mg.

Water and either 10% hydrochloric acid or 1 0% sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.0-7.8.

Claims (12)

1. A pharmaceutical composition which comprises. in association with a pharmaceutically acceptable carrier, human insulin. human C-peptide. and human proinsulin, said human Cpeptide being present in a ratio on a molar basis, human insulin to human C-peptide, of from 1:4 to about 4:1, and said human proinsulin being present in a ratio on a weight basis, human insulin to human proinsulin, of from about 1:100 to about 100:1.

2. Composition of claim 1. in which the molar ratio of human insulin to human C-peptide is from about 1:2 to about 2:1.

3. Composition of claim 1, in which the molar ratio of human insulin to human C-peptide is from about 1:1 to about 2:1.

4. Composition of claim 1, in which the weight ratio of human insulin to human proinsulin is from about 1:10 to about 100:1.

5. Composition of claim 4, in which the weight ratio of human insulin to human proinsulin is from about 1:2 to about 100:1.

6. Composition of claim 1 to 4, in which the weight ratio of human insulin to human proinsulin is from about 1:1 to about 20: 1.

7. Composition of claim 1 or 4, in which the weight ratio of human insulin to human proinsulin is from about 4:1 to about 20:1.

8. Composition of claim 1. which contains divalent zinc ion.

9. Composition of claim 1. which contains protamine salt.

10. Composition of claim 1, in which the weight ratio of human insulin to human proinsulin is from about 1:30 to about 100:1.

11. Composition of claim 1, in which the weight ratio of human insulin to human proinsulin is from about 1:1 5 to about 100:1,

1 2. Composition of claim 1, in which the weight ratio of human insulin to human proinsulin is from about 1:30 to about 20:1.

1 3. Composition of claim 1, in which the weight ratio of human insulin to human proinsulin is from about 1:1 5 to about 20:1.

1 4. Composition of claim 1. in which the weight ratio of human insulin to human proinsulin is from about 1:10 to about 20:1.

1 5. Composition of claim 1, in which the weight ratio of human insulin to human proinsulin is from about 1:100 to about 1:1.

1 6. Composition of claim 1. in which the weight ratio of human insulin to human proinsulin is from about 1:30 to about 1:1.

1 7. Composition of claim 1. in which the weight ratio of human insulin to human proinsulin is from about 1:30 to about 1:10.

1 8. Composition of claim 1. in which the weight ratio of human insulin to human proinsulin is from about 1:10 to about 1:1.

1 9. A pharmaceutical composition as claimed in claims 1 to 18, substantially as hereinbe fore described with reference to any one of the examples.