NL8203316A - PHARMACEUTICAL PREPARATIONS WITH HUMAN PRO-INSULIN. - Google Patents

Description

5 3684-356 Ned. P & c

Pharmaceutical preparations containing human proinsulin.

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 administering regular amounts of insulin; got. So far, the insulin used by diabetics has been isolated from animal pancreatic glands, generally from cattle and pigs.

Both bovine and porcine insulin differ in their structure from insulin, r r 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 is not in itself 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 vessel abnormalities 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 supplies. 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 pruritisf (7) kidney diseases, such as diabetic; glomerulosclerosis, arteriolar nephrosclerosis and pyelonephritis; and (8) difficulties during pregnancy,,., S20 3 3 1 6 t, - 2 - such as increased incidence of large babies, stillbirths, miscarriages, neonatal deaths and congenital abnormalities.

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

The invention relates to pharmaceutical compositions which achieve and maintain natural hormonal homeostasis in the diabetic state more accurately than can be the case by administration of insulin.

Thus, the invention relates to a pharmaceutical composition containing human proinsulin in combination with a pharmaceutically acceptable carrier.

The essential ingredient of the pharmaceutical compositions of the invention is human proinsulin.

The administration of human proinsulin using a composition according to the invention causes a more natural utilization of glucose and a better control of the blood glucose level, thereby reducing the unfavorable diabetic complications described above.

Human proinsulin is available by various routes, including organic synthesis, isolation from human pancreas by conventional methods and more recently by recombinant DNA method.

Generally, the preparation of proinsulin using recombinant DNA methodology is such that, by isolation, construction, or combination of both, a DNA sequence encoding the amino acid sequence of human proinsulin is obtained. The human proinsulin DNA is then input into a suitable clone carrier in expression phase and expressed. The carrier is used to transform a suitable microorganism, after which this transformed microorganism is subjected to fermentation conditions leading to (a) production of further copies of the proinsulin containing vector gene and ( b) expressing pro-insulin or a pre-insulin product.

In the case where the expressed product is a pre-insulin product, it generally contains the amino acid series 35 of human pro-insulin, which at its amino end is linked to a fragment of a protein, which is normally expressed in the gene order in which the proinsulin gene has been introduced. The amino acid sequence for proinsulin is bound to the protein fragment via a specific cleavable site, in a representative case, methionine. This product is commonly referred to as 8203316 * 3 - a fused gene product.

The amino acid series of proinsulin is cleaved from the fused gene product using cyanogen bromine, after which the cysteine sulfhydryl portions of the amino acid series of proinsulin are stabilized by conversion into the corresponding S sulfonates.

The obtained pro-insulin S-sulfonate is purified and the purified pro-insulin S-sulfonate is then converted to pro-insulin by forming the three appropriately positioned disulfide bonds.

The obtained proinsulin product is purified.

As already mentioned, the compositions of the invention are useful for promoting the attainment of natural hormonal homeostasis, thereby preventing or significantly reducing or delaying the recognized diabetic complications. It has been recognized that some diabetics are unable to efficiently receive insulin by subcutaneous injection, due to the presence of proteases at the injection site, which rapidly decompose the insulin before it is allowed to be absorbed into the bloodstream. the receptor sites to be transported. If insulin is already administered to these diabetics, this must be done by intravenous injection. The necessary repeated intravenous injections 20 are undesirable because of their deleterious effect on the recipient's veins and the associated infections. It has been found that human pro-insulin is not degraded by these insulin-degrading proteases, so that it can be administered by subcutaneous injection. Its stability and therefore its availability promote the attainment of a natural hormonal homeostasis,

It has also been observed in recent research [Podlecki et al., Diabetes, 31, Suppl. 2, 126¾ (1982)] that human proinsulin is introduced into target tissues, eg fat cells. Although its particular intracellular activity on a molecular scale has not yet been determined, these findings further support the teaching here presented that human proinsulin plays an active role in and is necessary to achieve natural hormonal homeostasis.

Schluter et al., Diabetes 31_, Suppl. 2, 135a (1982) describe studies demonstrating that the receptor binding of human insulin is enhanced by the presence of human proinsulin. These results further support the teaching here presented that the availability and presence of human proinsulin leads to the promotion or restoration of natural hormonal homeostasis.

The amount of the compositions of the invention needed is 8203316-4.

is to maintain natural hormonal homeostasis or to achieve a state that more closely approximates natural hormonal homeostasis in the diabetic patient, of course, depends on the severity of the diabetic condition. In addition, the amount varies depending on the administration route. Finally, the amount of the composition administered and the frequency of administration should be decided by the treating physician. Assuming 1 mg. human proinsulin provides about 3.5 units of human insulin activity, however, generally the dosage of human proinsulin is such that about 0.02 to about 5 units of human insulin activity per kg. body weight per day and preferably about 0.1 to about 1 unit human insulin activity per kg. body weight and per day.

The preparation is administered parenterally, which includes subcutaneous, intramuscular and intravenous. The compositions of the invention contain the active ingredient, human proinsulin, together with a pharmaceutically acceptable carrier therefor and, if desired, other therapeutic ingredients. The amount of active ingredient in the composition can range from about 99.99 to about 0.01% by weight. The carrier must be acceptable in the sense that it is compatible with other components of the composition and is not harmful to the recipient thereof,

Compositions of the invention suitable for parenteral administration suitably 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 known agents of sodium chloride, glycerol, glucose, mannitol, sorbitol, etc. In addition, the compositions may contain a wide variety of adjuvants, such as buffering agents, preservatives, dispersants, agents that promote rapid onset of action, agents that promote long duration of action, and other such agents. Representative preservatives are, for example, phenol, m.cresol, methyl p-hydroxybenzoate, etc. Representative buffers are, for example, sodium phosphate, sodium acetate, sodium citrate, and other known buffers.

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 can range 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, is generally present in an amount of about 0.1 mg. to about 3 mg. per 100 units of human proinsulin, 8203316/5 and prortagine salt (e.g., in the form of my sulfate), which, if present, is generally present in an amount of about 0.5 mg . to about 20 mg. per 100 units of human proinsulin.

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

Example I

Neutral regular human proinsulin preparation [40 units 3 (E) human proinsulin per cm (ml)].

To prepare 10 ml of the preparation, 10 Human pro-insulin (3.5 U / mg) 114 mg are mixed. (400 E)

Phenol, distilled 20 mg.

Glycerol 160 mg.

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

example n

Preparation with protamine, zinc, human pro-insulin [40 E human pro-insulin per ml].

20 Human pro-insulin (3.5 U / mg) 114 mg is mixed to prepare 10 ml of the preparation. (400 E)

Phenol, distilled 25 mg.

Zinc oxide 0.95-3.8 mg.

Glycerol 160 mg.

Protamine sulfate 32-64 mg.

25 Sodium phosphate, crystals 38 mg.

Water and 10-1 / 1 hydrochloric acid or 10% / 1 sodium hydroxide solution in an amount sufficient to obtain a volume of 10 ml Vein preparation and a final pH of 7.1-7.4.

Example hi 30 Isophane protamine and human proinsulin containing preparation [40E human proinsulin per ml].

Human pro-insulin (3.5 U / mg) 114 mg is mixed to prepare 10 ml of the preparation. (400 U) m. Cresol, distilled 16 mg.

Phenol, distilled 6.5 mg.

Glycerol 160 mg.

Protamine sulfate 9.6-19.2 mg.

Sodium phosphate, crystals 38 mg.

Water and 10% hydrochloric acid or 10% sodium hydroxide solution sufficient to obtain 8203316-6 of a volume of the preparation of 10 ml and a final pH of 7.1-7.4.

Example IV

1 Preparation with zinc and human pro-insulin [40 E human pro-5 insulin per ml].

To prepare 10 ml of a preparation, human pro-insulin (3.5 U / mg) is mixed 114 mg. (400 U)

Sodium acetate, anhydrous 16 mg.

Sodium chloride, granular 70 mg.

10 Methyl p-hydroxybenzoate 10 mg.

Zinc oxide 1-8 mg.

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

8203316

Claims (2)

Preparation according to claim 1, characterized in that it contains divalent zinc ion. Preparation according to claim 1 or 2, characterized in that it contains protamine salt. 8203316