NL8203315A - PHARMACEUTICAL PREPARATIONS CONTAINING HUMAN INSULIN AND HUMAN C-PEPTIDE. - Google Patents

Description

P · "-: —— S 3684-357 Eng. * - *

P T & C

Pharmaceutical preparations containing human insulin and human C peptide.

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. So far it is through diabetic! used insulin isolated% from animal pancreas glands, generally from cattle and pigs. Bovine and pig insulin both differ in their structure from insulin, which is formed by the human pancreas. Recently, recombinant DNA methodology has made it possible to produce insulin identical to that produced by the human pancreas. 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 the normal metabolic state. Although insulin has a pronounced effect on carbohydrate metabolism, diabetes mellitus involves further abnormalities, which are largely or all related to the function of blood vessels. The deficiencies leading to these abnormalities are rarely completely corrected by usual insulin therapy.

The abnormalities in the vessels associated with diabetes are often referred to as "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 paralysis; (2) oral diseases, such as gingivitis, an increased incidence of dental caries, periodontal disease and strong resorption of the dental bone; (3) motor, sensory and autonomic neuropathy; (4) disease of the great vessels; (5) microangiopathy; (6) skin diseases, such as xanthoma dia-35 beticorum, necrobiosis lipoidica diabeticorum, furunculosis, mycosis and pruritis; (7) kidney diseases, such as diabetic glomerulosclerosis, arteriolar nephrosclerosis and pyelonephritis; and (8) difficulties during pregnancy, 8203315 i - 2 - including an increased incidence of large babies, stillbirths, miscarriages, neonatal deaths and congenital abnormalities.

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

The invention relates to pharmaceutical compositions which approach and maintain hormonal homeostasis in a diabetic state more closely than can be achieved by administration of insulin alone.

Thus, the invention relates to the pharmaceutical composition which, in combination with a pharmaceutically acceptable carrier, contains human insulin and human C peptide in a molar ratio of human insulin to human C peptide from about 1: 4 to about 4: 1.

The two essential ingredients of the pharmaceutical compositions of the invention are human insulin and human C peptide.

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

When using recombinant DNA methodology, human insulin can be prepared by separately expressing and isolating the human insulin A chain and the human insulin B chain, followed by formation of their proper disulfide bond. Alternatively, the product expressed by recombinant DNA method may be human proinsulin itself or a precursor of human proinsulin converted to human proinsulin. The proinsulin is then enzymatically cleaved, e.g., using trypsin and carboxypeptidase B, to form human insulin.

Human insulin can also be prepared from pig insulin. Human insulin differs from porcine insulin in a single amino acid, i.e., the B chain carboxyl-terminal amino acid. Alanine, the B-30 amino acid from pig insulin is split off and replaced by threonine. See, for example, U.S. Pat. No. 3,276,961 in this regard.

The other active ingredient of the composition of the invention, human C-peptide, is part of a peptide, which is present in human proinsulin, to which the insulin A and B chains are bound. Also referred to as a "compound peptide", this peptide is removed from proinsulin during human insulin formation.

The compound peptide contained in human proinsulin has the formula: 8203315 - 3 -

Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-

Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg.

The human C peptide present in the composition of the invention differs from the compound peptide by removing 54 amino acids, 2 at each end. Thus, the human C peptide has the structure:

Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-

Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln.

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 can be prepared from human proinsulin by cleavage in the preparation of human insulin.

As already mentioned, the active ingredients of the composition of the invention are available by various routes involving human proinsulin. Generally outlined, the production of insulin using recombinant DNA methodology involves obtaining a DNA-20 sequence encoding the amino acid sequence of human pro-insulin by isolation, construction, or combination of both.

The human proinsulin DNA is then introduced into a suitable clone carrier in expression phase and expressed. The carrier is used to convert a suitable microorganism, after which the converted microorganism is subjected to fermentation conditions, leading to (a) the formation of further copies of the vector containing the proinsulin gene, and ( b) expressing pro-insulin or a pre-insulin product.

In case the expressed product is a pre-product of proinsulin, it generally contains the amino acid series 30 of human proinsulin, which is bound at its amino terminal group to a fragment of a protein, which is normally expressed is placed in the gene series into which the pro-insulin gene has been introduced. The amino acid sequence of proinsulin is bound to the protein fragment via a specifically cleavable plant, in representative cases methionine. This product is commonly referred to as a combined gene product.

The amino acid sequence of proinsulin is cleaved from the combined gene product using cyanogen bromine, after which the sulfhydryl groups of cysteine of the amino acid series of proinsulin are stabilized by putting them in the corresponding S sulfonates.

8203315> - 4 -

The obtained pro-insulin S-sulfonate is purified and the purified pro-insulin S-sulfonate is then converted to pro-insulin by forming the 3 properly disulfide bonds.

After purification of the proinsulin, it is enzymatically cleaved, typically using trypsin and carboxypeptidase B, leading to the formation of human insulin and human C peptide.

The compositions of the invention contain human insulin and human C peptide in a molar ratio of from about 1: 4 to about 4: 1. Preferably, the ratio of human insulin to human C peptide is about 1: 2 to about 2: 1 and most preferably about 1: 1 to about 2: 1.

As already mentioned, the compositions of the invention are useful in 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. In addition, the amount varies depending on the route of administration. Ultimately, the amount of preparation administered and the frequency of administrations should be determined by the treating physician. Generally, however, the dosage is in a range providing about 0.02 to about 5 units of human insulin per kg of body weight and per day, and preferably about 0.1 to about 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 from about 99.99 to about 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 decomposed 8203315-5. - catcher, generally using sodium chloride, glycerol, glucose, mannitol, sorbitol and the like known agents. In addition, the compositions 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, n-cresol, methyl p-hydroxybenzoate and others. Examples of representative buffers are sodium phosphate, sodium acetate, sodium citrate, etc.

In addition, an acid, eg hydrochloric acid, or a base, eg.

use sodium hydroxide to control the pH. Generally, the pH of the aqueous preparation ranges from about 2 to about 8 and preferably from about 6.8 to about 8.0.

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

Examples of specific pharmaceutical preparations of the invention are provided in the embodiments below.

example i

Neutral regular human insulin: human C-peptide preparation [1: 4 human insulin: human C-peptide on a molar basis at 40 3 units (E) insulin per cm (ml)].

To prepare 10 ml of the preparation, mix:

Human zinc insulin (28 U / mg) 400 U

Human C peptide 30 mg.

Phenol, distilled 20 mg.

Glycerol 160 mg.

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 C peptide on a molar basis at 100 U insulin per ml].

For the preparation of 10 ml of Vein the preparation is mixed 8203315 * - 6 -

Human zinc insulin (28 U / mg) 1000 E.

Human C peptide 19 mg.

Phenol, distilled 20 mg.

Glycerol 160 mg.

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

. Example III

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Protamine, zinc human insulin: human C peptide preparation 10 [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.

Phenol, distilled 25 mg.

Zinc oxide 0.78 mg.

Glycerol 160 mg. .

Protamine sulfate 4.0-6.0 mg.

Sodium phosphate, crystals 38 mg.

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

example iv

Protamine, zinc human insulin: human C peptide preparation 25 [2: 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 9 mg.

Phenol, distilled 25 mg.

Zinc oxide 2.0 mg.

Glycerol 160 mg.

Protamine 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 preparation with a volume of 10 ml and a final pH of 7.1-7.4.

Example V

Isophane protamine, zinc human insulin: human C-peptide- 8203315 * - 7 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.

m. Cresol, distilled 16 mg.

Phenol, distilled 6.5 mg.

Glycerol 160 mg.

Protamine sulfate 1.2-2.4 mg.

10 Sodium phosphate, crystals 38 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 7.1-7.4.

Example VI

15 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.]

Mix to prepare 10 ml of the preparation

Human zinc insulin (28 U / mg) 1000 E.

Human C peptide 5 mg.

m. Cresol, distilled 16 mg.

Phenol, distilled 6.5 mg.

Glycerol 160 mg.

Protamine sulfate 3.0-6.0 mg.

25 Sodium phosphate, crystals 38 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 7.1-7.4.

Pre-free VII

30 Zinc human insulin suspension human C-peptide preparation. [1: 2 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 15 mg.

Sodium acetate, anhydrous 16 mg.

Sodium chloride, granular 70 mg.

Methyl p-hydroxybenzoate 10 mg.

Zinc oxide 0.63 mg.

8203315 W 'S-v - 8 -

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 VIII

5 Zinc 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, human zinc insulin (28 U / mg) 1000 U is mixed

Human C peptide 19 mg.

Sodium acetate, anhydrous 16 mg.

Sodium chloride, granular 70 mg.

Methyl p-hydroxybenzoate 10 mg.

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

Neutral regular human insulin: human C peptide preparation 20 [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.

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.

30 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.]

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

Human C peptide 19 mg.

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

from 7.0-7.8. 8 2 0 3 3 1 5

Claims (5)

A facsimile pharmaceutical composition, characterized in that it contains human insulin and human C peptide in combination with a pharmaceutically acceptable carrier in a molar ratio of human insulin to human C peptide from about 1: 4 to about 4: 1. Preparation according to claim 1, characterized in that the molar ratio between human insulin and human C-peptide is about 1: 2 to about 2: 1. Preparation according to claim 1 or 2, characterized in that the molar ratio between human insulin and human C-peptide is about 1: 1 to 10 about 2: 1. Preparation according to claims 1-3, characterized in that it contains divalent zinc ion. Preparation according to claims 1-4, characterized in that it contains the protamine salt. 8203315