FR2513126A1 - PHARMACEUTICAL FORMULATIONS COMPRISING HUMAN INSULIN AND HUMAN C-PEPTIDE - Google Patents

Abstract

THE INVENTION RELATES TO A PHARMACEUTICAL COMPOSITION COMPRISING, IN ASSOCIATION WITH A PHARMACEUTICALLY ACCEPTABLE SUPPORT, HUMAN INSULIN AND HUMAN C-PEPTIDE IN A MOLAR RATIO OF BETWEEN ABOUT 1: 4 AND ABOUT 4: 1, THIS COMPOSITION BEING USEFUL FOR THE TREATMENT OF DIABETES, AS WELL AS TO PROMOTE THE OBTAINING OF NATURAL HORMONAL HOMEOSTASE TO PREVENT, MITIGATE OR SENSITIVELY STOP DIABETIC COMPLICATIONS.

Description

PHARMACEUTICAL FORMULATIONS COMPRISING INSULIN

HUMAN AND HUMAN PEPTIDE.

  Diabetes mellitus is a metabolic disorder characterized by the fact that body tissues fail to oxidize carbohydrates on a diet

  normal Its most important factor is a defi-

  insulin science In the past 60 years,

  people with diabetes have been considered

  aided by receiving regulated amounts of

  sulirne Currently, the insulin used by diabetics has been isolated from the pancreas of animals, generally bovines and pigs. The insulin of bovids and pigs has a different structure.

  that of 1 iinsulin produced by the human pancreas.

  Recently, by the 1 l DNA methodology (DNA = acid

  deoxyribonucleic) recombinant, it became pos-

  sible (prepare it (L insulin identical to that produced by the human pancreas The use of this insulin will allow: diabetics, to imitate the

  natural system more tightly than it was pos-

sible so far.

  o; Nevertheless, it has been recognized since the past that the mere administration of insulin to a diabetic is insufficient to restore and / or maintain the normal metabolic state. Although insulin manifests its effect on the metabolism of carbohydrates, diabetes mellitus causes additional disorders, most, if not all, of which relate to the structure and function of blood vessels

  leading to these disorders are rarely corrected com-

  completely by conventional insulin therapy.

  The vascular anomalies associated with the dia-

  animals are often called "diabetes complications".

  They generally consist of micro- changes

  angiopathics giving rise to damage to the retina

  and kidney Neuropathy is a complica-

  an additional diabetic which may be related

13126

  tion or not, directly or indirectly, with the aforementioned microangiopathic changes Among the

  specific manifestations of complications of the di-

  beast, there are, for example: (1) eye diseases, including retinopathy, cataract formation,

  glaucoma and extraocular muscle paralysis

  res; (2) diseases of the mouth, particularly gingivitis, increased occurrence of dental caries, periodontitis and stronger resorption of the alveolar bone; (3) motor, sensory and autonomous neuropathies; (4) large vessel disease; (5) micro-angiopathy; (6) diseases of the

  skin, especially the xanthoma of diabetes mellitus, necro-

  lipoid biosis of diabetes mellitus, furunculosis,

  yeast and pruritus; (7) kidney disease,

  including diabetic glomerulosclerosis, nephro-

  arteriolar sclerosis and pyelonephritis; and (8) problems during pregnancy, including an increase in the birth of large babies, childbirth, stillbirth, miscarriages,

  neonatal deaths and birth defects.

  Many and perhaps all of the diabetic complications result from the fact that insulin alone cannot bring the body back

to its natural hormonal balance.

  The present invention relates to composites

  Pharmaceutical measures enabling a diabetic subject to achieve and maintain a state closer to natural hormonal homeostasis than that which can be obtained by the administration of insulin alone. Therefore, the present invention relates to a pharmaceutical composition comprising, in combination

  with a pharmaceutically acceptable carrier,

  human sulin and human C-peptide in a molar ratio (human insulin / human C-peptide) lying

between about 1: 4 and about 4: 1.

  The two essential constituents of the composi-

  The pharmaceutical compositions of the present invention are human insulin and human C-peptide. Human insulin can be obtained in various ways, in particular by organic synthesis, by isolation of the human pancreas, by transformation of pro-insulin, by transformation of animal insulin

  isolated and, more recently, by the methodology

  of recombinant deoxyribonucleic acid.

  By adopting the deoxy acid methodology

  recombinant ribonucleic, human insulin can be prepared by separate expression and isolation of the A chain and the B chain of human insulin by

  then forming their correct disulfide bonds.

  Alternatively, the expression product of deoxy acid

  recombinant ribonucleic acid can be human proinsulin itself or a proinsulin precursor

  which is transformed into human pro-insulin.

  The pro-insulin is then cleaved enzymatically,

  for example, using trypsin and carboxy-

  peptidase B to obtain human insulin.

  Human insulin can also be pre-

  adorned from pig insulin Human insulin differs from pig insulin by a single amino acid, that is to say the terminal carboxy-amino acid of the B chain Alanine, c that is to say amino acid B-30 from insulin from pigs, is cleaved and replaced by threonine In this respect, we

  refer, for example, to the patent of the United States of America

Risk No. 3,276,961.

  The other active constituent of the composition of the present invention, namely the human C-peptide,

  is a portion of a peptide present in the pro-insu-

  Human iin and to which the A and B chains of insulin are joined This peptide called "binding peptide"

  is eliminated during the production of insulin hu-

  main from proinsulin The binding peptide present in human proinsulin corresponds to the following formula:

  Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Va 1-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 compound

  tion of the present invention differs from the binding peptide by the elimination of four amino acids,

  namely two at each end Therefore, the C-pep-

  human body has the following 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 present invention can be obtained by chemical synthesis see, for example, N Yanaihara and C. Yanaihara, M Sakagami, N Sakura, T Hashimoto and T. Nishida, "Diabetes" 27 (supplement 1), 149-160 (1978) l

  or from human pro-insulin following its cleavage

ge to get human insulin.

  Consequently, as indicated, the active constituents of the composition of the present invention can be obtained in various ways,

  including human proinsulin In its large

  lines, insulin production by adopting

  the methodology with recombinant deoxyribonucleic acid

  nant consists in obtaining, by insulation and / or by-

  truction, a deoxyribo- acid coding sequence

  nucleic acid for the amino acid sequence of the pro-

  human insulin The deoxy acid is then introduced

  human proinsulin ribonucleic in the read phase

  ture in a suitable cloning and expression vehicle

  The vehicle is used to transform a

  appropriate microorganism, after which the microorganism-

  transformedism is subject to firm conditions

  leading to: (a) the production of additional copies

  of the vector containing the pro- gene

  insulin and (b) expression of pro-insulin or a

  pro-insulin precursor.

  If the expression product is a precursor

  of pro-insulin, it will usually include the sequence

  this amino acid of human proinsulin joined, at its amino terminus, to a fragment of a protein normally expressed in the gene sequence into which the proinsulin gene has been introduced The amino acid sequence of pro-insulin is joined to the protein fragment via a specifically cleavable site, in particular methionine This product is usually called "fused genetic product".

  g O The amino acid sequence of the pro-

  insulin is cleaved from the fused gene product using cyanogen bromide, after which the sulfhydryl fractions of cysteine from the amino acid sequence of proinsulin are stabilized

  by transformation into their corresponding S-sulfonates

  dants. We purify pro-insulin S-sulfonate

  obtained, then we transform the S-sulfonate from pro-

  insulin purified into pro-insulin by formation of

  three correctly located disulfide bonds.

  When purifying pro-insulin,

  this is cleaved enzymatically, specifically-

  deny using trypsin and carboxypeptidase B,

  thus giving rise to the formation of human insulin

  ne and human C-peptide, The compositions of the present invention contain human insulin and human C-peptide in a molar ratio of between about 1: 4

  and about 4: 1 Preferably, the ratio between the

  human sulin and human C-peptide is between about 1: 2 and about 2: 1, better still, between about

1: 1 and approximately 2: 1.

  As noted, the compositions of the

  present invention are useful in promoting the

  natural hormonal homeostasis and thus em-

  catch or attenuate or significantly slow down the well-known diabetic complications which have been mentioned above Of course, the quantity

  in which the compositions of the present invention

  tion should be used to maintain homeosta-

  natural hormone or to achieve a state closer to hormonal homeostasis

  natural in a diabetic subject, will depend on the

  vity of the diabetic state In addition, this amount will vary depending on the mode of administration Finally, the amount of composition administered and the frequency of this administration will be left to the choice of the individual doctor However, as a general rule, the dosage will be within the interval giving about 0.02 to about 5 units of human insulin activity per kg of body weight per day, preferably,

  between about 0.1 and about 1 unit of activity dtin-

  human sulin per kg of body weight per day.

  The composition is administered by the

  rentérale, in particular, by subcutaneous route, by

  intramuscular and intravenous

  positions of the present invention include the

  active ingredients, namely insulin and C-pep-

  tide humans together with pharmaceutical support-

  acceptable for the latter and possibly with other therapeutic ingredients The quantity

  total of the active ingredients present in the composition

  sition is between about 99.99 and about 0.01% by weight The support must be acceptable in the sense that it must be compatible with other components of the composition and it must not be harmful to the

recipient of it.

  The compositions of the present invention which are suitable for administration by the

  parenteral, advantageously include suspen-

  sterile aqueous solutions and / or solutions

  pharmaceutically active ingredients, these solutions or suspensions being preferably made isotonic with the recipient's blood, generally using sodium chloride, glycerin, glucose,

  mannitol, sorbitol and known analogues.

  In addition, the compositions may contain either adjuvant such as buffering agents, preservatives, dispersing agents, agents promoting rapid onset of activity, agents promoting prolonged activity and other known agents As specific preservatives, mention will be made, for example, of phenol,

  m-cresol, methyl p-hydroxybenzoate and others.

  As specific buffering agents, there will be mentioned, for example, sodium phosphate, sodium acetate,

sodium citrate and others.

  In addition, an acid such as

  hydrochloric acid, or a base such as hydro-

  Sodium xyde to adjust the p H As a general rule,

  the p H of the aqueous composition will be between

  about 2 and about 8, preferably between about

6.8 and around 8.

  Among other suitable additives are

  For example, the divalent zinc ion, the amount of which, if present, is generally between about 0.01 mg and about 0.5 mg per 100 units of human insulin, as well as a protamine salt

  (for example, in the form of its sulfate) of which the quan-

  tity, if present, is generally between

  approximately 0.1 mg and approximately 2 mg per 100 active units

vity of human insulin.

  Examples of compounds will be given below.

  particular pharmaceutical according to the pre-

invention.

Example 1

  Regular regular human insulin / C-pep- formulation

  human tide molar ratio -between human insulin and human C-peptide = 1: 4 to 40 units of insulin

per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) 400 units Human C-peptide 30 mg Phenol (distilled) 20 mg Glycerin 160 mg Water and either 10% hydrochloric acid, or 10% sodium hydroxide: sufficient quantity to obtain a composition with a volume of 10 cm 3 and a

final p of 7-7.8.

Example 2

  Formulation of regular regular human insulin / C-pep-

  human tide molar ratio between human insulin and human C-peptide = 1: 1 to 100 units of insulin

per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) 1000 units Human C-peptide 19 mg Phenol (distilled) 20 mg Glycerin 160 mg Water and either 10% hydrochloric acid, or 10% sodium hydroxide: sufficient quantity to obtain a composition with a volume of 10 cm 3 and a final p H of 7-7.8.

Example 3

  Formulation of human insulin protamine-zinc / human C-peptide The molar relationship between 11 human insulin and the

  Human C-peptide = 1: 1 to 40 units of insulin per cm 3 lJ.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) 400 units Human C-peptide 8 mg Phenol (distilled) 25 mg Zinc oxide 0578 mg Glycerin 160 mg Protamine sulfate 4.0- 6.0 mg sodium phosphate (crystals) 38 mug Water and either 10% hydrochloric acid or 1% sodium hydroxide 0%: sufficient quantity to obtain a composition with a volume of 10 rcm 3 and deun

final p of 7.1-7.4.

Example 4

  Formulation of human insulin pr ,, tamine-zinc / human C-peptide lrappor L molar between human insulin and

  Human C-peptide = 2: 1 to 100 units of insulin per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) Human C-peptide Phenol (distilled) Zinc oxide Glycerin Protamine sulphate Sodirum phosphate (crystals) 1,000 units 9 mg mg 2 mg mg -15 mg 38 mg Water and either 10% hydrochloric acid or 10% sodium hydroxide: sufficient quantity to obtain a composition with a volume of 10 cm 3 and a

final p of 7.1-7.4.

Example 5

  Formulation of human insulin isophane-protamine-zinc / human C-peptide molar relationship between insulin

  human and human C-peptide = 1: 1 to 40 units of in-

sulin per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) 400 units Human C-peptide 8 mg m-cresol (distilled) 16 mg Phenol (distilled) 6.5 mg Glycerin 160 mg Sulfate protamine 1.2-2.4 mg Sodium phosphate (crystals) 38 mg Water and either 10% hydrochloric acid or 10% sodium hydroxide: sufficient quantity to obtain a volume composition of 10 cm 3 and dlun

final p of 7.1-7.4.

Example 6

  Formulation of human insulin isophane-protamine-zinc / human C-peptide molar ratio between human insulin and human C-peptide = 4: 1 to 100 units

insulin per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) 1,000 units Human C-peptide 5 mg m-cresol (distilled) 16 mg Phenol (distilled) 6.5 mg Glycerin 160 mg Sulfate of protamine 3, O 0-6.0 mg Sodium phosphate (crystals) 38 mg Water and either 10% hydrochloric acid or 10% sodium hydroxide: sufficient quantity to obtain a composition of a volume of 10 cm 3 and a final p H of 7.1-7.4.

Example 7

  Formulation of a human zinc insulin / human C-peptide suspension molar relationship between insulin

  human and human C-peptide = 1: 2 to 40 units of in-

sulin per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) 400 units 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 Water and either 10% hydrochloric acid or 10% sodium hydroxide: sufficient quantity to obtain a composition with a volume of 10 cm 3 and a

final p of 7.2-7.5.

Example 8

  Formulation of a human zinc insulin / human C-peptide suspension molar ratio between human insulin and human C-peptide = 1: 1 to 100 units

insulin per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human zinc insulin (28 units / mg) Human C-peptide Sodium acetate (anhydrous)

Sodium chloride (granular).

  methyl p-hydroxybenzoate Zinc oxide 1.00 O units 19 mg 16 mg mg mg 1.6 mg Water and either 10% hydrochloric acid or 10% sodium hydroxide: sufficient quantity to obtain a composition with a volume of 10 cm 3 and a

final p of 7.2-7.5.

Example 9

  Formulation of regular human insulin neutral / C-pep-

  human tide molar ratio between human insulin and human C-peptide = 1: 4 to 40 units of insulin

per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human sodium insulin (28 units / mg) 400 units Human C-peptide 30 mg Phenol (distilled) 20 mg Glycerin 160 mg Water and either 10% hydrochloric acid, or 10% sodium hydroxide: sufficient quantity to obtain a composition with a volume of 10 cm 3 - and a

final p of 7.0-7.8.

Example 10

  Formulation of regular regular human insulin / C-pep-

  human tide molar ratio between human insulin and human C-peptide = 1: 1 to 100 units of insulin

per cm 3 l.

  To prepare 10 cm 3 of the composition, the following are mixed: Human sodium insulin (28 units / mg) 1,000 units Human C-peptide 19 mg Phenol (distilled) 20 mg Glycerin 160 mg Water and either 10% hydrochloric acid , or 10% sodium hydroxide: sufficient quantity to obtain a composition with a volume of 10 cm 3 and a

final p of 7.0-7.8.

M 3

Claims (4)

  1 Pharmaceutical composition comprising, in association with a pharmaceutically acceptable carrier, human insulin and human C-peptide in a molar ratio (between human insulin and human Cpeptide) of between approximately 1: 4 and approximately 4: 1.   2, Composition according to claim 1,   characterized in that the molar ratio between insulin   human line and the human C-peptide is between about 1: 2 and about 2: 1.   3 Composition according to claim 1,   characterized in that the molar rapporb between the in-   human sulin and the human C-peptide is between about 1: 1 and about 2: 1.   4 Composition according to claim 1,   characterized in that it contains the zinc ion biva-   slow. Composition according to claim 1,   characterized in that it contains a prota salt mine.