NZ201683A - Pharmaceutical compositions comprising human proinsulin - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £01 683 201683 Priority Datc(s): Pil^P.: & Complete Specification Filed: Class: M.'KAY/j^-k.............

Publication Date: . '1.6 AUG J985 P.O. Journal, No: .... kg B3 No.: Date: NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION HUMAN PROINSULIN PHARMACEUTICAL FORMULATIONS M/We, ELI LILLY AND COMPANY, a corporation of the State of Indiana, U.S.A., having a principal place of business at 307 East McCarty Street, City of Indianapolis, State of Indiana, United States of America hereby declare the invention for which / we pray that a patent may be granted to j&&/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 - (followed by page la) 2016 —ice — HUMAN ■PROINGULIM PIIARMACEUTIC AL FORMULATIONC- Diabetes raellitus 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 frcan diabetes have been greatly aided by receiving controlled amounts of insulin. To the present time, the insulin used by diabetics has been isolated frcm animal pancreases, generally bovine and porcine. Both bovine and porcine insulin differ structurally frcm insulin generated by the human pancreas. Recently, it has become possible, by recombinant DNA methodology, to produce insulin identical 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 20 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 25 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 30 changes resulting in lesions in the retina and the 261C &J 201683 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 5 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 10 neuropathy; (4) large-vessel 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, 15 arteriolar nephrosclerosis, and pyelonephritis; and (8) problems during pregnancy, including increased incidence of large babies, stillbirths, miscarriages, neonatal deaths, and congenital defects.

Many, and perhaps all, of the diabetic com-20 plications are the result of the failure of insulin alone to restore the body to its natural hormonal balance.

This invention is directed to pharmaceutical compositions that more nearly achieve and maintain 25 natural hormonal homeostasis in a diabetic state than can be achieved by administration of insulin.

Hius, this invention concerns a pharmaceutical composition being in a form suitable for use as an antidiabetic agent which cctnprises human proinsulin in association with a pharmaceutical^ acceptable carrier. 20 T^e essential constituent of the pharma ceutical compositions of this invention is human pro-insulin. //V 201 The administration of human proinsulin using a composition in accordance with this invention will produce a more natural utilization of glucose and better blood glucose control, thereby diminishing hereinbefore described adverse diabetic complications. 5 Human proinsulin is available via a variety of routes, including organic synthesis, isolation from human pancreas by conventional methodology, and, more recently, recombinant DNA methodology.

In broad outline, the production of pro-10 insulin using recombinant DNA methodology 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 15 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 gene-containing 2o 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 25 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.

X^5Bi8A_ -4- 20168 The proinsulin amino acid sequence is cleaved frcm 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. 5 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.

As noted, the compositions of this invention are useful in promoting the attainment of natural hormonal homeostasis and thereby preventing or substantially diminishing or retarding those recognized diabetic complications. It is recognized that certain 15 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. 2o 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 2g 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. 2Q It also has been noted from recent studies [Podlecki et al., Diabetes, 31, Suppl. 2, 126A (1982)] 2016 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 5 role in and is necessary for the attainment of natural hormonal hcmostasis.

Schluter ^t al. , Diabetes 31, Suppl. 2, 135A (1982), describe studies that demonostrate that human insulin receptor binding is enhanced by the presence of 10 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 15 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 2o 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, on the basis that 1 mg. of human proinsulin affords 25 approximately 3.5 Units of human insulin activity, the dosage of human proinsulin 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 2o insulin activity per kilogram body weight per day. 2016 The ccxnposition is administered parenterally, including subcutaneous, intramuscular, and intravenous. The compositions of this invention comprise the active ingredient, human proinsulin, together with a pharma-ceutically acceptable carrier therefor and, optionally, ® other therapeutic ingredients. The amount of active ingredient 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 dele-terious to the recipient thereof.

Compositions of this invention suitable for parenteral administration conveniently comprise sterile aqueous solutions and/or suspensions of the pharma-ceutically active ingredients, which solutions or 15 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, 20 preservatives, dispersing agents, agents that promote rapid onset of action, agents that promote prolonged duration of action, and other such agents. Typical preservatives are, for example, phenol, m-cresol, methyl £-hydroxybenzoate, and others. Typical buffers 25 are, for example, sodium phosphate, sodium acetate, sodium citrate, and other known buffers.

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 ccmpo-30 sition ranges from about 2 to about 8, and, preferably, frcm about 6.8 to about 8.0. 2016 8 Other suitable additives are, for example, divalent zinc ion, 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 proinsulin, and protamine salt (for example, in the form of its sulfate), which, 5 if present at all, is generally present in an amount from about 0.5 mg. to about 20 mg. per 100 units of human proinsulin.

Examples of particular pharmaceutical compositions of this invention are provided in the examples 10 appearing hereinbelow.

Example 1 — Neutral Regular Human Proinsulin Formulation [40 Units (U) human proinsulin per cubic centimeter (cc.)] To prepare 10 cc. of the ccmposition, mix Human Proinsulin (3.5 U/mg.) 114 mg. (400 U) Phenol, distilled 20 mg.

Glycerin 160 mg.

Water and either 10% hydrochloric acid or 10% 20 sodium hydroxide sufficient to make a ccmposition volume of 10 cc. and a final pH of 7.0-7.8.

Example 2 — Protamine, Zinc Human Proinsulin Formulation £40 U human proinsulin per cc.] To prepare 10 cc. of the composition, mix Human Proinsulin (3.5 U/mg.) 114 mg. (400 U) Phenol, distilled 25 mg.

Zinc Oxide 0.95 -3.8 mg.

Glycerin 160 mg.

Protamine Sulfate 32 - 64 mg.

Sodium Phosphate, crystals 38 mg. 2016 "i4-5318A. -8- 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 3 — Isophane Protamine, Human Proinsulin Formulation [40 U human proinsulin per i cc. J To prepare 10 cc. of the composition, mix Human Proinsulin (3.5 U/mg.) 114 mg. (400 U) m-Cresol, distilled 16 mg.

Phenol, distilled 6.5 mg.

Glycerin 160 mg.

Protamine Sulfate 9.6 - 19.2 mg.

Sodium Phosphate, crystals 38 mg.

Water and either 10% hydrochloric acid or 10% 15 sodium hydroxide sufficient to make a composition volume of 10 cc. and a final pH of 7.1-7.4.

Example 4 — Zinc Human Proinsulin Formulation [40 U human proinsulin per cc.] To prepare 10 cc. of the ccmposition, mix Human Proinsulin (3.5 u/mg.) 114 mg. (400 U) Sodium Acetate, Anhydrous 16 mg.

Sodium Chloride, Granular 70 mg.

Methyl p-Hydroxybenzoate 10 mg.

Zinc Oxide 1-8 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. x-"53TB7r=tT4-- 201683 I (■'<:'£

Claims (4)

WHAT WE CLAIM IS:

1. A pharmaceutical ccmposition being in a form suitable for use as an antidiabetic agent which comprises human proinsulin in association with a pharmaceutically acceptable carrier.

2. A composition of claim 1, which contains

3. A composition of claim 1, which contains protamine salt.

4. A pharmaceutical ccmposition as claimed in any one of claims 1 to 3, substantially as hereinbefore described with reference to any one of the examples. 5 divalent zinc ion. PER ag;?A!" a ? 15 20 25 30