JPS5846026A - Medicine containing human insulin, human c-peptide and human proinsulin - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Diabetes is a metabolic disorder characterized by a defect in the body's tissues to oxidize carbohydrates at a normal rate, the key cause of which is insulin deficiency. Over the past 60 years, diabetic patients have been able to receive much relief from prescribed doses of insulin, but to this day, the insulin used for diabetic patients is still administered to animals (usually is collected from the pancreas of cows and pigs. Both bovine and porcine insulin are structurally different from insulin derived from the human pancreas. In recent years, it has become possible to produce insulin identical to the insulin produced in the human pancreas using recombinant DNA techniques. Such insulin could bring diabetics closer to their natural system than ever before.

However, it has long been well known that simply administering insulin to diabetic patients is insufficient to restore or maintain a normal metabolic state. Although insulin has clear effects on carbohydrate metabolism, diabetes is associated with other disorders, most if not all of which are related to vascular structure and function. These disabling defects are rarely completely corrected by conventional insulin therapy.

Diabetes-related vascular abnormalities are often referred to as "diabetic complications"
It generally causes abnormal changes in capillaries and causes lesions in the retina and kidneys. neurological disease
athy) is yet another diabetic complication that may or may not be directly or indirectly related to the well-known abnormal capillary changes. Examples of characteristic findings of diabetic complications include (1) eye diseases (retinopathy, cataract formation);

(2) Oral diseases (gingivitis, increased incidence of caries, periodontitis, large-scale alveolar bone resorption, etc.), (3) Mobility, sensory and autonomy (4) large blood vessel diseases, (5) capillary abnormalities, (6) skin diseases (diabetic xanthomas, diabetic lipoid necrosis, II tumor disease, mycosis, pruritus, etc.) .

(7) Kidney diseases (diabetic glomerulosclerosis, arteriolar nephrosclerosis, pyelonephritis, etc.), (8) Problems during pregnancy (increased incidence of macrosomia, stillbirth, miscarriage, neonatal death).

congenital malformations, etc.).

Many, perhaps all, of the diabetic complications! This is a result of the fact that insulin alone is insufficient to restore the body to its natural hormonal balance.

The present invention is more effective than by administering insulin alone.

The diabetic state is reduced to a natural hormonal steady state (homeost).
asis).

Thus, the present invention provides human insulin, human C-peptide and human proinsulin, together with a pharmaceutically acceptable carrier, in a molar ratio of human insulin:human C-peptide in the range of about 1 to about 1:1. Also, the weight ratio of human insulin:human proinsulin is from approximately 100 to approximately loo:
1.

The three essential components of the pharmaceutical composition of the present invention are human insulin, human C-peptide and human proinsulinte.

Human insulin using the composition according to the invention.

Human C-peptide and human proinsulin, when administered together, can utilize glucose more naturally and provide better control of glucose levels than insulin alone, thereby reducing the deleterious diabetic complications described above.・Human proinsulin can be obtained in various ways.

For example, it can be obtained by organic synthesis, conventional extraction from human pancreas, and more recently by recombinant DNA techniques.

In general, to produce proinsulin using recombinant DNA techniques, one must isolate and/or construct a DNA sequence encoding the amino acid sequence of human proinsulin; The human proinsulin DNA thus prepared is inserted in a readable phase into a suitable medium that allows cloning and expression.

The medium is transferred to a suitable microorganism, and the transformed microorganism is then subjected to conditions that allow (b) to produce copies of the al proinsulin gene-containing vector, and (b) to express proinsulin or a proinsulin precursor. Cultivate.

What is the proinsulin precursor produced by phenotypic expression?
Generally, a fragment of a protein normally expressed in the gene sequence into which the proinsulin gene is inserted is bound to the amino acid terminus of the human proinsulin amino acid sequence. The proinsulin amino acid sequence is attached to this protein fragment through a specific cleavage site (usually methionine). This product is conventionally referred to as a fusion gene product.

The proinsulin amino acid sequence is then cleaved from the fusion gene product using cyanogen bromide.

The sulfhydryl moiety of cysteine in the proinsulin amino acid sequence is stabilized by conversion to its corresponding S-sulfonic acid ester.

The resulting proinsulin S-sulfonate is purified and the purified proinsulin S-sulfonate is converted to proinsulin by forming disulfide bonds at three precise locations. The product proinsulin obtained is purified.

Human insulin, another active ingredient of the compositions of the present invention, can also be obtained in a variety of ways, including organic synthesis, extraction from human pancreas by conventional methods, conversion from extracted animal insulin, and conversion from human proinsulin. Obtained by transformation and recombinant DNA techniques.

However, human insulin can also be produced by enzymatically cleaving the produced human proinsulin using, for example, trypsin or carboxypeptidase B.

Using recombinant DNA techniques similar to those previously described in the section on producing human proinsulin, human insulin is produced by separately expressing and isolating human insulin A1111 and human insulin B chain, followed by their production. can be prepared by properly forming a disulfide bond.

Human insulin can also be prepared from porcine insulin. Human insulin differs from porcine insulin in only one amino acid, the carboxyl terminal amino acid of the B chain. Porcine insulin (DB 30 amino acids, alanine is truncated and replaced by threonine. In this regard, reference may be made to, for example, United States Patent No. 3.271..9).

Human C-peptide, which is yet another active ingredient of the compositions of the invention, is the part of the peptide that is present in human proinsulin and is attached to the insulin A and B chains. This peptide, called the "linking peptide", is removed during the production of human insulin from proinsulin.

The linking peptide present in human proinsulin is represented by the following formula.

Arg-Arg-Glu-Ala-Glu-Asp-L
eu-Gin-Val-Gly-Gin-Val-Gl
u-Le u-Gly-Gly-Gly-Pro
-Gly-A la-, Gly-! 9 er-Le
u-Gl n-Pro-Le u-Ala-Leu
-Glu-Gly-3e r-Le u-Gin-Ly
s-ArgThe human C-peptide present in the composition of the present invention differs from the above-mentioned linking peptide in that a total of two amino acids are removed, two from each end. Thus, human C-peptide has the following structure.

Glu-Ala-Glu-Asp-Leu-Gin-V
a 1-Gly-Gin-Val-Glu-Leu-G
ly-Gly-Gly-Pro-Gly-Ala-Gl
y-8er-Leu-Gin-Pro-Leu-Ala
-Leu-Glu-Gly-5er-Leu-Gin The human C-peptide component of the composition of the present invention is.

Chemical synthesis [e.g. N Yanaihara,
C, Yanaihara.

M, Sakagami t N, 5akura,
T, Hashimoto, and T.

N15hidasDiabetes I, 2Z (5
upp1. / ) + /'19-/6θ(/971
) or can be obtained by cleaving human proinsulin to produce human insulin.

Human C-peptide is also available along with human insulin during the enzymatic cleavage of human proinsulin as described above.

The composition of the invention contains human insulin, human C-pegtide and human proinsulin. Human proinsulin has a human insulin:human proinsulin weight ratio of approximately l:lOO to approximately lOO:l.
It exists as if the ratio of Preferably, the weight ratio of human insulin:human proinsulin is approximately /:2
The most preferred intersection is from about 4t:/ to about 20:l, more preferably from about 1:l to about 20:l. Another suitable weight ratio range of human insulin:human proinsulin is - from approximately /:30 to approximately lOO:l.

approximately 1:13; to approximately lOO:l, approximately l:l
from a to about 100: /, from about l:3o to about 2θ:c from about 1his to about 2°:l and from about l:lo to about 2θ:l.

The human C-peptide is present in the composition of the present invention such that the molar ratio of human insulin:human C-peptide is from about 1:1 to about 1:1. Preferably, the molar ratio of human insulin:human C-peptide is from about 2 to about 2:1, most preferably from about 1:1 to about 2:1.

As already mentioned, 9 the compositions of the present invention may assist in achieving a natural hormonal steady state, thereby preventing or substantially reducing or halting the progression of recognized diabetic complications. It is valid. A diabetic patient has
Even when insulin is injected subcutaneously, it does not receive it effectively because proteases are present in the injected area, which rapidly destroys insulin before it can be absorbed into the bloodstream and transported to receptors. It is known that it cannot be obtained. If such diabetic patients must receive insulin, they must do so by intravenous injection. The need for repeated intravenous injections is undesirable because it can harm the recipient's veins and cause associated infections. It has been discovered that human proinsulin is not degraded by the insulinolytic proteases described above, and thus human proinsulin can be administered by subcutaneous injection. The stability and bioavailability of human proinsulin make it a natural hormone.
Science ＼U＼ promotes ＼凰It seems possible.

Also, recent papers [Podlecki et al.
Diabetes +31+5upp1.2, 12
6AC/9J'2)] revealed that human proinsulin is taken up into target tissues (ie, adipose tissue). Although this special intracellular effect on a 9-molecule scale has not yet been confirmed, the above findings are true.

This further supports the present invention that human proinsulin is active and essential in achieving a natural hormonal steady state.

In addition to assisting in achieving a natural hormonal steady state, the acid complexes of the present invention, in which the weight ratio of human insulin:human proinsulin is approximately 1:1 or less, have the added benefit of very long-lasting hypoglycemic action. have an effect. In preferred compositions that produce this additive effect, the weight ratio of human insulin:human proinsulin is approximately l', Io
o to approximately l:l. Other preferred human insulin:human proinsulin weight ratios are approximately 1:3.
0 to approximately 1:/, more specifically, approximately /:30 to approximately /:10 or approximately /:10 to approximately l:l. Such compositions require fewer commonly used insulin formulation additives, such as protamine in NPH insulin and excess zinc in lentiinsulin. Both of the above additives are artificial and non-physiological.

Also, another document [Chluter, et al, +
Di'abetes, 3/rsuppl, ,
It has been shown that the binding of human insulin to the receptor is promoted in the presence of human proinsulin. This result was disappointing. It further supports the present invention that a free hormonal steady state is assisted and restored due to the presence of human proinsulin and its bioavailability. The amount of the composition of the present invention is necessary to maintain or approximate natural hormonal steady state in a diabetic patient.

Of course, it depends on the severity of the diabetic symptoms and also on the route of administration. Ultimately, the amount and frequency of administration of the 9 compositions will be at the discretion of the individual physician.

However, in general, the daily dose will be within the range of about 0.02 to about 5 active units of human insulin per kg of body weight.
Preferably, it is within the range of about θ to about 1 units.

The composition is administered parenterally, such as subcutaneously, intramuscularly, or intravenously. The composition of the present invention.

It contains the active ingredients human insulin, human C-peptide and human proinsulin together with its pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The total amount of active ingredients in the composition ranges from about 99% to about 0%.
0.01 weight percent. Carriers are acceptable if they are compatible with the other ingredients of the composition and are not deleterious to the recipient.

Compositions of the invention suitable for parenteral administration contain sterile aqueous solutions and/or suspensions of the pharmacologically active ingredient. The solution or suspension is generally sodium chloride, glycerin, glucose.

It is preferred to use mannitol, sorbitol or the like to make it isotonic with the blood of the recipient.

Additionally, the composition may include a number of adjuvants, e.g.

Buffer, preservative 9 Dispersant 9 Early onset promoter of action.

It may also contain prolonging agents or other known adjuvants. Typical preservatives include, for example, phenol, m-cresol, methyl p-hydroxybenzoate, and the like. Typical buffering agents include 2, e.g.
Examples include sodium phosphate, sodium acetate, and sodium citrate.

Additionally, acids such as hydrochloric acid and bases such as sodium hydroxide can be used for voice conditioning. Typically.

The pH of the aqueous composition is within the range of about 2 to about t, preferably about 6. It is within the range of about rθ from l.

Other suitable additives include, for example, divalent zinc ions and salts of protamine (e.g., in the sulfate form), although the content of divalent zinc ions, if present, may be low in human insulin. Generally from about ooiIIIg to about 0.03 g per 100 units of activity, the content of protamine salts, if present, is limited by the amount of human insulin activity l.
Generally from about 07q to about 31119 per Oθ unit.

Examples relating to individual pharmaceutical compositions of the invention.

This is illustrated by the examples shown below.

(Space below) Example/ Neutral standard human insulin: Human C-peptide: Human proinsulin preparation [Human insulin: Human C-
Peptide = l:q molar ratio; human insulin; human insulin 10θ: /M amount ratio i / cubic -1 = insulin θ units (U
) Containing [] To prepare the title composition (10CC).

Human zinc insulin IJ: / (21'U/#)
l100U human C-peptide
30Q Human Proinsulin 0/ge-phenol (distilled) 201R
The amount of the 1glycerin/6θ da composition is 7 Qcc, and sufficient water and 70% hydrochloric acid or 10% sodium hydroxide are mixed to give a final pI of 7θ to 7.

Example 2 Neutral standard human insulin: human C-peftide: human proinsulin preparation [human insulin: human C-
Pebutide = /: 1 molar ratio; human insulin: human proinsulin = 2.0: / weight ratio; containing 10 IJ of insulin per /cc] To prepare the title composition (/θcc).

Human zinc insulin (21U/Wiy) / θθθU human C-peptide / 9” If human proinsulin 1flW phenol (distilled), 20II 117 glycerin / 60 da The amount of the composition is /θcc, the final pH is 7θ~7g Mix enough water and 10% hydrochloric acid or 70% sodium hydroxide to

Example 3 Protamine, human zinc insulin: human C-peptide: human proinsulin preparation [human insulin: human C-peptide=/21 molar ratio; human insulin:
Human proinsulin = 20:/weight ratio; /contains 4 tOU of insulin per cc] To prepare the title composition (lOCC).

Human zinc insulin (2 U/■) lθOU human C-peptide Zada human broinsulin θ7 Daphenol (distilled) 23; W zinc oxide
θ7zadubriserin
/60'l19 protamine sulfate
Hiro θ ~ Otsu O■ Sodium phosphate (crystal)
31r111f! Mix enough water and 10% hydrochloric acid or 70% sodium hydroxide to give a composition volume of /θcc and a final voice of 77-711.

Examples Hiroprotamine, human zinc insulin: human C-heftide preparation [human insulin: human C-peratide-2:
1 toproinsulin=/:/weight ratio;/cc equivalent') containing 1001 J of insulin] To prepare the title composition Cl0Cc).

Human zinc insulin (2zaU/I'P) 100
01J human C-peptide 91
11g human proinsulin 361
q Phenol (distilled) 2! ;1
119 Zinc oxide 2.0
■Glycerin /60 ■Protamine sulfate lO~/j Misaki Sodium Phosphate (crystal) 31r■ Let the amount of the composition be /Qcc, and add enough water and 10% hydrochloric acid to make the final pI (71-71) Mix with 10% sodium hydroxide.

Example 5 Iaophane/protamine, human zinc insulin: human C-peptide: human gloinsulin preparation [human insulin: human C-peptide l: 1 molar ratio; human insulin; human gloinsulin =≠; 1 weight ratio H7cc Contains insulin lθU per unit] To prepare the title composition (/θcc).

Human zinc insulin (21U/1111')
11θOU human C-peptide Zadahito proinsulin 36'qm
-Cresol (distilled)/6Isg phenol (
Distilled) 6.3"IQ Glycerin 760~Protamine sulfate 12~2.Sodium phosphate (crystal) 311119 The amount of the composition is /θcc, and the final pH is 1i-7/~7'1. Mix enough water and 70% hydrochloric acid or 10% sodium hydroxide.

Example B Isophane protamine, human zinc insulin: human C-hair” tide: human proinsulin preparation [human insulin: human C-peptide = lI: 1 molar ratio; human insulin: human proinsulin = / 72MN
Ratio i/Contains 10 OU of insulin per CC] To prepare the title composition (10 cc).

Human zinc insulin (21rU/Mf) 10O
OU human C-peptide! Human gloinsulin 77qm-cresol (distilled) /6qm-cresol (distilled) 6. ! ；■Glycerin
/ o1ng protamine sulfate
3o~6.0 Sodium daphosphate (crystals) 3 and a sufficient amount of water and 7Qcc to make the final pH 7/~7'1.
Mix 0% hydrochloric acid or 70% sodium hydroxide.

Example 7 Human zinc insulin suspension: Human C-peptide: Human gloinsulin preparation [Human insulin: Human C-
Peptide=/:2 molar ratio; Human insulin: Human proinsulin=/Near 0 weight ratio; Contains 'IOU of insulin per /cc] To prepare the title composition (/θcc).

Human zinc insulin (2,!rU/l1lf/')
'10θU human C-peptide
/3'l19 human proinsulin
/'13ml1 Sodium acetate (anhydrous) /6 Misaki Sodium chloride (granular) 70qp-Methyl hydroxybenzoate 10■ Zinc oxide
063 ■ Amount of composition /θc
c and sufficient water and 70% hydrochloric acid or 10% sodium hydroxide to bring the final pH to 72-7j.

Example! Human zinc insulin suspension: human C-peptide: human broinsulin [lJ[human insulin: human C
- Peptide = l: 1 molar ratio human insulin: human proinsulin = 2,0: / weight ratio; / cc of insulin/θθU content] To prepare the title composition (10 CC).

Human zinc insulin (21U/elf>100
0U human C-peptide /91q
Human proinsulin 11tlg Sodium acetate (anhydrous)/6q Sodium chloride (granular) 70Wp-゛Hydroxy 1. Methyl fragrant IO”9 Zinc oxide L■ The amount of the composition is 7
Qcc and mix sufficient water and 10% hydrochloric acid or 70% sodium hydroxide to bring the final pH to 72-Z3.

Example 9 Neutral standard human insulin: human C-peptide: human proinsulin preparation [human insulin: human C-
Peptide=/:lI molar ratio human insulin: human proinsulin f00:f weight ratio; insulin 'AOU content per cc] To prepare the title composition (10 tl:e).

Human sodium insulin (2, rU/#) l
100 U Human C-Peptide 30 Dahytoproinsulin 0/Gudaphenol (distilled) 20''f/Glycerin/6θq Sufficient amount to bring the composition amount to /Qcc and final concentration to 70-1' water and 70% hydrochloric acid or 10% sodium hydroxide.

Example/θ Neutral standard human insulin: Human C-peptide: Human proinsulin preparation [Human insulin: Human C-
Peptide 2/2 1 molar ratio; human insulin: human proinsulin = co θ: 1 weight ratio; containing 10011 insulin per /cc] To prepare the title composition (10 CC).

Human sodium insulin C21U/181) 1
000U human C-peptide 79
■Human proinsulin 1llllf
Phenol (distilled) 2ON glycerin /1. The amount of Oq composition is 7Qcc, and enough water and 10% hydrochloric acid or 70% sodium hydroxide are mixed to give a final basis of 70-71.

Claims (9)

[Claims] (1) Human insulin, human O-peptide, and human proinsulin together with a pharmaceutically acceptable carrier such that the molar ratio of human insulin:human C-peptide is in the range of approximately /:l to approximately 1/:l. ,
Further, a pharmaceutical composition containing human insulin:human proinsulin in a weight ratio of approximately ./: 100 to approximately 100''. (2) The composition of claim (1), wherein the molar ratio of human insulin:human C-peptide is from about 1:2 to about 2:1. (3) The composition of claim (1), wherein the molar ratio of human insulin:human C-peptide is from about 1:1 to about 2:1. (4) The composition according to claim (1), wherein the weight ratio of human insulin:human proinsulin is from approximately / = IO to approximately ioo:i. (5) The composition according to claim (4), wherein the weight ratio of human insulin:human proinsulin is from about 1:2 to about 100:/. (6) The composition according to claim (1) or (4), wherein the weight ratio of human insulin:human proinsulin is from about 1:1 to about 2θ:1. (7) The composition according to claim (1) or (4), wherein the weight ratio of human insulin:human proinsulin is from about 1:1 to about 20:/. (8) Claims containing divalent zinc ions (1)
). (9) Claims containing salts of protamine (
1) The composition described. 0Q The composition of claim (1), wherein the weight ratio of human insulin:human proinsulin is from approximately /:30 to approximately ioo',i. A composition according to claim 1, wherein the weight ratio of Qυ human insulin:human proinsulin is from approximately l:13 to approximately lθO:l. The composition of claim (1), wherein the weight ratio of αa human insulin:human proinsulin is from about 1:3θ to about 20:/. 03. The composition according to claim (1), wherein the weight ratio of human insulin:human proinsulin is approximately /:/3 to approximately θ:l. The composition of claim 1, wherein the weight ratio of α→human insilin; human proinsulin is approximately l:lθ to approximately 2θ:l. 0υ The composition of claim (1), wherein the weight ratio of human insulin:human proinsulin is from about 1:loo to about 1:1. 0. The composition of claim (1), wherein the weight ratio of human insulin:human proinsulin is from approximately /:30 to approximately 1:1. The composition of claim (1), wherein the weight ratio of human insulin:human proinsulin is from about 1:30 to about l:lθ. α Enoki The composition according to claim (1), which has a human insulin:human proinsulin ratio.