NL192192C - Pharmaceutical preparations containing human insulin and human C peptide. - Google Patents

Description

1 192192

Pharmaceutical preparations containing human insulin and human C peptide

The invention relates to a pharmaceutical composition comprising, in combination with a pharmaceutically acceptable carrier, human insulin and an additional polypeptide.

Such compositions are known from CH-A-330,318, in which insulin is combined with a synthetic basic polypeptide (eg polylysine) to increase the stability of insulin at physiological pH in the presence of protamine. The problem is that synthetic polypeptides are exogenous substances and therefore do not play a role in natural hormonal homeostasis.

The present invention solves the above-mentioned problem by combining the human insulin with human C-peptide, and surprisingly thereby enhances the action of the human insulin in reducing the effects of diabetes when and this is characteristic of the in the preamble mentioned method, the preparation is to be administered parenterally and that the additional polypeptide is human C-peptide, wherein the molar ratio of human insulin to human C-peptide is 1: 4 to 4: 1, wherein a natural mixture containing human insulin and human C peptide is excluded.

Diabetes mellitus is a metabolic disease, characterized by the failure of body tissues to oxidize carbohydrates at the normal rate. Its main factor is a lack of insulin. During the last 60 years, people suffering from diabetes have been greatly helped by receiving regular amounts of insulin. Thus far, insulin 20 used by diabetics has been isolated from animal pancreatic glands, generally from cattle and pigs. Bovine and porcine insulin both differ in their structure from insulin, which is made up of the human pancreas. Recently, recombinant DNA methodology has made it possible to produce insulin identical to that produced by the human pancreas {Goeddel, D.V. et al., Proc. Natl. Acad. Sci. USA, 76, 106-110, (1979) and Chance, R.E. et al., Diabetes Care, 4, 147-154, (1981)}. The use of such insulin allows the diabetic to mimic the natural system more accurately than has hitherto been possible.

Nevertheless, it has long been recognized that administration of insulin to the diabetic alone is not sufficient to restore and / or maintain normal metabolic status. Although insulin has a pronounced effect on carbohydrate metabolism, diabetes mellitus carries with it further abnormalities, most or all of which are related to the structural function of blood vessels. The deficiencies leading to these abnormalities are rarely fully corrected by conventional insulin therapy.

The abnormalities in the vessels related to diabetes are called "complications of diabetes". They generally consist of microangiopathic changes leading to damage to the retina and kidney. Neuropathy is another diabetic complication, which may or may not be directly or indirectly related to the reported microangiopathic changes. Examples of specific manifestations of complications of diabetes are (1) diseases of the eye, such as retinopathy, cataract formation, glaucoma and extraocular muscle deformities; (2) oral diseases, such as gingivitis, an increased incidence of dental caries, periodontal disease and strong resorption of the dental bone; (3) 40 motor, sensory and autonomic neuropathy; (4) disease of the great vessels; (5) microangiopathy; (6) skin diseases, such as xanthoma diabeticorum, necrobiosis lipoidica diabeticorum, furunculosis, mycosis and pruritis; (7) kidney diseases, such as diabetic glomerulosclerosis, arteriolar nephrosclerosis and pyelonephritis; and (8) difficulties during pregnancy, including an increased incidence of large babies, stillbirths, miscarriages, neonatal deaths and congenital abnormalities.

45 Many and perhaps all diabetic complications result from the inadequacy of insulin alone to restore the body to its natural hormonal balance; the present and above-described invention improves in this situation.

US-A-2,789,080 discloses a composition of insulin and an esterified albumin of animal or human origin for the same stability reasons previously mentioned. However, this publication does not mention or suggest the use of human C peptide.

Administration of human insulin, combined with human C peptide, mimics the natural situation. For each insulin molecule produced, a C peptide molecule is formed; therefore, the molar ratio of insulin to human C-peptide should preferably be between 1: 2 and 2: 1, and most preferably, since there is evidence that the C protein in the peripheral circulation is more stable than 55 insulin (Gonen et al, in Proinsulin, Insulin, C-peptide, Eds Baba, Kaneko and Yanaihara, Excepta Medica, Amsterdam-Oxford, 1979, p. 246) between 1: 1 and 2: 1.

Human insulin is available through various routes, such as organic synthesis, isolation from 192192 human pancreas, conversion of human proinsulin, conversion of isolated animal insulin, and more recently recombinant DNA methodology.

Human insulin can be prepared from viral insulin by cleaving off the carboxyl-terminal amino acid of the B chain: Alanine, the B-30 amino acid of porcine insulin, and replacing it with threonine. See, for example, U.S. Pat. No. 3,279,961 in this regard.

The other active ingredient of the composition of the invention, human C-peptide, is part of a peptide contained in human proinsulin to which the insulin A and B chains are bound. This peptide, also known as the "compound peptide", is removed from proinsulin during the formation of human insulin. The compound peptide 10 present in human proinsulin has the formula:

Arg-Arg-Glu-Ala-Glu-Asp-Leu-GIn-Val-Gly-GIn-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-GIn-Pro-

Leu-Ala-Leu-Glu-Gly-Ser-Leu-GIn-Lys-Arg.

The human C peptide present in the composition of the invention differs from the compound peptide by removing 4 amino acids, 2 at each end. Thus, the human C peptide 15 has the structure:

Glu-Ala-Glu-Asp-Leu-GIn-Val-Gly-GIn-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-GIn-Pro-Leu-Ala-

Leu-Glu-Gly-Ser-Leu-GIn.

The human C peptide, which is a constituent of the composition of the invention, can be prepared 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 it may be prepared from human proinsulin by cleavage in the preparation of human insulin.

As already mentioned, the compositions of the invention are suitable for promoting the attainment of natural hormonal homeostasis and thus preventing or significantly reducing or delaying the recognized diabetic complications. The amount of the compositions of the invention necessary to maintain or achieve natural hormonal homeostasis that more closely approximates natural hormonal homeostasis in the diabetic depends, of course, on the severity of the diabetic condition and on the route of administration. Generally, however, the dosage is in a range providing 0.02 to 5 units of human insulin per kg of body weight and per day, and preferably 0.1 to 1 unit of human insulin per kg of body weight and per day.

The preparation is administered parenterally, such as subcutaneously, intramuscularly and intravenously. The compositions of the invention contain the active ingredients, human insulin and human C-peptide, together with a pharmaceutically acceptable carrier therefor and, optionally, other therapeutic ingredients. The total amount of active ingredients present in the composition is 99.99 to 0.01% by weight. The carrier must be acceptable in that it is compatible with the other ingredients of the composition and is not harmful to its recipient.

Compositions of the invention suitable for parenteral administration usually include sterile aqueous solutions and / or suspensions of the pharmaceutically active ingredients, which solutions or suspensions are preferably made isotonic with the blood of the recipient, generally using sodium chloride , glycerol, glucose, mannitol and sorbitol. In addition, the compositions 40 may contain any number of further adjuvants, such as buffers, preservatives, dispersants, agents that promote rapid onset of action, agents that promote extended duration of action, and other known agents. Examples of representative preservatives are phenol, m-cresol and methyl p-hydroxybenzoate. Examples of representative buffers are sodium phosphate, sodium acetate and sodium citrate.

In addition, an acid, e.g., hydrochloric acid, or a base, e.g., sodium hydroxide, can be used to control the pH. Generally, the pH of the aqueous preparation ranges from 2 (solution) to 8 (suspension) and preferably from 6.8 to 8.0.

Other suitable additives are e.g. divalent zinc ion, which, if present, is generally present in an amount of 0.01 to 0.5 mg per 100 units of human insulin and protamine salt 50 (e.g. in the form of the sulfate ), which, if present, is generally present in an amount of 0.1 to 2 mg per 100 units of human insulin.

Example I

Neutral regular human insulin: human C peptide preparation [1: 4 human insulin: human 55 C peptide on a molar basis at 40 units (E) insulin per cm3 (ml)].

To prepare 10 ml of the preparation, mix: 3 192192

Human zinc insulin (28 U / mg) 400 U

Human C peptide 30 mg

Phenol, distilled 20 mg

Glycerol 160 mg 5 Water and 10% hydrochloric acid or 10% sodium hydroxide solution, sufficient to obtain a preparation with a volume of 10 ml and a final pH of 7.0-7.8.

Example II

Neutral regular human insulin: human C peptide preparation [1: 1 human insulin: human 10 C peptide on a molar basis at 100 U insulin per ml].

Human zinc insulin (28 U / mg) 1000 U is mixed to prepare 10 ml of the preparation

Human C peptide 19 mg

Phenol, distilled 20 mg 15 Glycerol 160 mg

Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.0-7.8.

Example III

Protamine, zinc human insulin: human C peptide preparation [1: 1 human insulin: human C peptide on a molar basis at 40 U insulin per ml].

Human zinc insulin (28 U / mg) 400 U is mixed to prepare 10 ml of the preparation

Human C peptide 8 mg 25 Phenol, distilled 25 mg

Zinc oxide 0.78 mg

Glycerol 160 mg

Protamine sulfate 4.0-6.0 mg

Sodium phosphate, crystals 38 mg 30 Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.1-7.4.

Example IV

Protamine, zinc human insulin: human C peptide preparation [2: 1 human insulin: human C peptide 35 on a molar basis at 100 U insulin per ml].

Human zinc insulin (28 U / mg) 1000 U is mixed to prepare 10 ml of the preparation

Human C peptide 9 mg

Phenol, distilled 25 mg 40 Zinc oxide 2.0 mg

Glycerol 160 mg

Protam in sulfate 10-15 mg

Sodium phosphate, crystals 38 mg

Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a 45 composition having a volume of 10 ml and a final pH of 7.1-7.4.

Example V

Isophane protamine, zinc human insulin: human C peptide preparation [1: 1 human insulin: human C peptide on a molar basis at 40 U insulin per ml].

50 To prepare 10 ml of the preparation is mixed

Human zinc insulin (28 U / mg) 400 U

Human C peptide 8 mg m-Cresol, distilled 16 mg

Phenol, distilled 6.5 mg 55 Glycerol 160 mg

Protamine sulfate 1.2-2.4 mg

Sodium phosphate, crystals 38 mg 192194 4

Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.1-74.

Example VI

5 Isophane protamine, zinc human insulin: human C peptide preparation [4: 1 human insulin: human C peptide on a molar basis at 100 U insulin per ml].

Human zinc insulin (28 U / mg) 1000 U is mixed to prepare 10 ml of the preparation

Human C peptide 5 mg 10 m-Cresof, distilled 16 mg

Phenol, distilled 6.5 mg

Glycerol 160 mg

Protamine sulfate 3.0-6.0 mg

Sodium phosphate, crystals 38 mg 15 Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.1-7.4.

Example VII

Zinc formulation human insulin suspension: human C peptide [1: 2 human insulin: human 20 C peptide on a molar basis at 40 U insulin per ml].

Human zinc insulin (28 U / mg) 400 U is mixed to prepare 10 ml of the preparation

Human C peptide 15 mg

Sodium acetate, anhydrous 16 mg 25 Sodium chloride, granular 70 mg

Methyl p-hydroxybenzoate 10 mg

Zinc oxide 0.63 mg

Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.2-7.5.

30

Example VIII

Zinc formulation human insulin suspension: human C peptide [1: 1 human insulin: human C peptide on a molar basis at 100 U insulin per ml].

To prepare 10 ml of the preparation, 35 Human zinc insulin (28 U / mg) 1000 U are mixed

Human C peptide 19 mg

Sodium acetate, anhydrous 16 mg

Sodium chloride, granular 70 mg

Methyl p-hydroxybenzoate 10 mg 40 Zinc oxide 1.6 mg

Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.2-7.5.

Example IX

45 Neutral regular human insulin: human C peptide preparation [1: 4 human insulin: human C peptide on a molar basis at 40 U insulin per ml].

To prepare 10 ml of the preparation, human sodium insulin (28 U / mg) 400 U is mixed

Human C peptide 30 mg 50 Phenol, distilled 20 mg

Glycerol 160 mg

Water and 10% hydrochloric acid or 10% sodium hydroxide solution in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.0-7.8.

55 Example X

Neutral regular human insulin: human C peptide preparation [1: 1 human insulin: human C peptide on a molar basis at 100 U insulin per ml].

Claims (4)

5 192192 To prepare 10 ml of the preparation, human sodium insulin (28 U / mg) 1000 E human C peptide 19 mg Phenol, distilled 20 mg 5 Glycerol 160 mg Water and 10% hydrochloric acid or 10%% is mixed s caustic soda in sufficient quantity to obtain a preparation with a volume of 10 ml and a final pH of 7.0-7.8. 10 Conclusions Pharmaceutical composition comprising, in combination with a pharmaceutically acceptable carrier, human insulin and an additional polypeptide, characterized in that the composition can be administered parenterally and that the additional polypeptide is human C-peptide, in which the molar ratio of 15 human insulin to human C peptide is from about 1: 4 to about 4: 1, excluding a natural mixture containing human insulin and human C peptide. Preparation according to claim 1, characterized in that the molar ratio between human insulin and human C-peptide is 1: 1 to 2: 1. Preparation according to claims 1-2, characterized in that it contains divalent zinc ion. Preparation according to claims 1-3, characterized in that it contains protamine salt.