Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL FORMULATIONS COMPRISING
HUMAN INSULIN, HUMAN C-PEPTIDE,
AND HUMAN PROINSULIN
Diabetes mellitus 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 from diabetes have been greatly
aided by receiving controlled amounts of insulin. To
the present time, the insulin used by diabetics has
10 been isolated from 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 re-
combinant DNA methodology, to produce insulin identical
15 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
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
25 vessels. The deficiencies leading to these disorders
rarely are completely corrected by conventional insulin
therapy.
Those vascular abnormalities associated with
diabetes often are referred to as "complications of
30 diabetes". They consist generally of microangiopathic
3~
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changes resulting in lesions in the retina and the
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 com-
plications are (1) diseases of the eye, includingretinopathy, cataract formation, glaucoma, and extra-
ocular muscle palsies; (2) diseases of the mouth,
including gingivitis, increased incidence of dental
caries, periodontal disease, and greater resorption of
10 the alveolar bone; (3) tor, sensory, and autoncmic
neuropathy; (4) large-vessel disease; (5) microangio-
pathy, (6) diseases of the skin, including xantho~a
diabeticorum, necrobiosis lipoidica diabeticorum,
furunculosis, mycosis, and pruritis; (7) diseases of
15 the kidneys, including diabetic glomerulosclerosis,
arteriolar nephrosclerosis, and pyelonephritis, and
(8) problems during pregnancy, including increased
incidence of large babies, ~tillbirths, miscarriages,
neonatal deaths, and congenital defects.
Many, and perhaps all, of the diabetic com-
plications are the result of the failure of insulin
alone to restore the body to its natural hormonal
balance.
This invention concerns pharmaceutical
25 compositions that more nearly achieve and maintain
natural hormonal homeosta~is in a diabetic state than
can be achieved by administration of insulin alone.
Thus, this invention concerns a pharmaceu-
tical composition which compri~es, in association with
30 a pharmaceutically acceptable carrier, human insulin,
human C-peptide, and human proinsulin, said human C-
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peptide being present in a ratio on a molar basis,
human insulin to human C-peptide, of from about 1:4 to
about 4:1, and said human proinsulin being present in a
ratio on a weight basis, human insulin to human pro-
insulin, of from about 1:100 to about 100:1.
The three essential constituents of the
pharmaceutical cpositions of this invention are human
insulin, human C-peptide, and human proinsulin.
The administration of a combination of human
insulin, human C-peptide, and human proinsulin using a
10 composition in accordance with this invention will
produce a more natural utilization of glucose and
better glucose control than is achieved by insulin
alone, thereby diminishing hereinbefore described
adverse diabetic complications.
lS 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-
20 insulin using recombinant DNA methodology involvesobtaining, 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
2~ 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
30 vector and (b) the expression of proinsulin or a
proinsulin precursor product.
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In the event the expression product is a
proinsulin precursor, it generally will comprise the
human proinsulin amino acid sequence joined at its
amino tenminal end to a fragment of a protein normaLly
expressed in the gene sequence into which the pro-
insulin 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.
The proinsulin amino acid ~equence is cleaved
from the fused gene product using cyanogen bromide
after which the cysteine sulfhydryl moieties of the
proinsulin amino acid sequence are stabilized by con-
version to their corresponding S-sulfonates.
The resulting proinsulin S-sulfonate is
puriied, and the purified proinsulin S-sulfonate then
is converted to proinsulin by formation of the three
properly located disulfide bonds. The resulting pro-
insulin product i5 purified.
A second active constituent of the composi-
tions of this invention, human insulin, also is avail-
able via a variety of routes, including organic syn-
thesis, isolation from human pancreas by conventional
techniques, conversion of isolated animal insulin,
25 conversion of human proinsulin, and recombinant DNA
methodology.
Human proin~ulin, however produced, can be
enzymatically cleaved, for example, using trypsin and
carboxypeptida~e B, to produce human insulin.
Using recombinant DNA methodology in a manner
analogous to that hereinbefore described for the pro-
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duction of human proinsulin, human insulin can be
prepared by the separate expression and isolation of
human insulin A-chain and human insulin B-chain fol-
lowed by their proper disulfide bond formation.
Human insulin can also be prepared from
porcine insulin. Human insulin differs from porcine
insulin by a ~ingle amino acid, i.e., the B-chain
carboxyl terminal amino acid. Alanine, the B-30 amino
acid of porcine insulin, is cleaved and replaced by
threonine. In this regard, see, for example, U.S.
lO Patent No. 3,276,961.
A third active constituent of the composition
of this invention, human C-peptide, is a portion of a
peptide pre~ent in human proinsulin and to which the
insulin A- and B-chains are joined. The latter p~ptide,
15 termed a "connecting peptide", is removed during produc-
tion of human insulin from proinsulin. The connecting
peptide present in human proinsulin has the formula
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-
20 Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg.
The human C-peptide present in the composi-
tion of this invention differ~ from the connecting
peptide by elimination of four amino acids, two 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 constituent of the com-
30 position of this invention can be produced by chemicalsynthesis, see, e.g., N. Yanaihara, C. Yanaihara, M.
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Sakagami, N. Sakura, T. Hashimoto, and T. ~ishida,
Diabetes 27 (suppl. 1), 149-160 (1978), or from human
_
proinsulin as a result of its cleavage to produce human
insulin.
Human C-peptide also is available in conjunc-
tion with human insulin via the aforedescribed enzymaticcleavage of human proinsulin.
The compositions of this invention contain
human insulin, human C-peptide, and human proinsulin.
Human proinsulin i8 present in a weight ratio, human
10 insulin to human proinsulin, of from about 1:100 to
about 100:1. Preferably, the weight ratio of human
insulin to human proinsulin is from about 1:2 to about
100:1, more preferably, from about 1:1 to about 2Q:l,
and, most preferably, from about 4:1 to about 20:1.
15 Additional preferred weight ratio ranges, human insulin
to human proinsulin, are from about 1:30 to about 100:1;
from about 1:15 to about 100:1, from about 1:10 to
about 100:1, from about 1:30 to about 20:1; from about
1:15 to about 20:1; and from about 1:10 to about 20:1.
Human C-peptide is present in the composi-
tions of this invention in a molar ratio, human insulin
to human C-peptide, of from about 1:4 to about 4:1.
Preferably, the molar ratio of human insulin to human
C-peptide i8 from about 1:2 to about 2:1, and, most
25 preferably, from about 1:1 to about 2:1.
As noted, the cpositions of this invention
are useful in promoting the attainment of natural
hormonal homeostasis and thereby preventing or sub-
stantially diminishing or retarding those recognized
30 diabetic complications. It is recognized that certain
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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.
These diabetics, if they are to receive insulin at all,
must receive it by intravenous injection. The neces-
sary repeated intravenous injections are undesirable
due to their deleterious effect on the veins of the
recipient and infections associated therewith. It has
lO been discovered that human proinsulin is not degraded
by these insulin-degrading proteases and, thus, it can
be administered by subcutaneous injection. Its sta-
bility and thus availability promote attainment of
natural honmonal homeosta~is Moreover, since insulin
15 and proinsulin together form camplexes, it can be
expected that proinsulin will afford protection for the
otherwise degradable insulin.
It also has been noted from recent studies
[Podlecki et al., Diabetes, 31, Suppl. 2, 126A (1982)]
20 that human proinsulin is internalized into target
tissues, e.g. fat cells. Although its particular
intracellular action on a molecular scale i9 as yet
undetermined, these findings further support the
disclosure herein that human proinsulin plays an active
2S role in and is necessary for the attainment of natural
hormonal homeostasis.
In addition to promoting the attainment of
natural hormonal homeostasis, those compositoins of
this invention in which the weight ratio, human
30 insulin to human proinsulin, is equal to or less than
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about 1:1, afford the added benefit of an extra long
hypoglycemic effect. Preferred compo~itions which
carry this added benefit have a weight ratio, human
insulin to human proinsulin, of from about 1:100 to about
1:1. Other preferred weight ratios, human insulin to
human proinsulin, are from about 1:30 to about 1:1,
and, more particularly, from about 1:30 to about l:l0,
or from about 1:10 to about 1:1. Such compositions
will minimize the need for customary insulin formulation
additives such as protamine which is present in ~PH
10 insulin or excess zinc which is present in lente insulin.
Both such additives are artificial and unphysiologic.
Schluter et al., Diabetes 31, Suppl. 2, 135A
(1982), de~cribe studies that demonstrate that human
insulin receptor binding is enhanced by the presence of
15 human proinsulin. These results again further support
the disclosure herein that the availability and presence
of human proinsulin results in the pro~otion or restora-
tion of natural hormonal homeostasis. The amount of
the compositions of this invention necessary to main-
20 tain natural hormonal homeostasis or to achieve a statethat more nearly approaches natural hormonal hcmeo-
stasis in the diabetic, of course, will depend upon the
severity of the diabetic condition. Moreover, the
amount will vary depending upon the route of admin-
25 istration. Ultimately, the amount of compositionadministered and the frequency of such administration
will be at the discretion of the particular physician.
In general, however, the dosage will be in the range
affording from about 0.02 to about 5 units of human
30 insulin activity per kilogram body weight per day, and,
preferably, from about 0.1 to about l unit of human
insulin activity per kilogram body weight per day.
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The composition i8 administered parenterally,
including subcutaneous, intramuscular, and intravenous.
The compositions of this invention ccmprise the active
ingredients, human insulin, human C-peptide, and human
proinsulin, together with a pharmaceutically acceptable
carrier therefor and, optionally, other therapeutic
ingredients. The total amount of active ingredients
present 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
10 other components of the ccmposition 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-
15 ceutically active ingredients, which solutions orsuspensions preferably are made isotonic with the blood
of the recipient, generally u~ing sodium chloride,
glycerin, glucose, mannitol, sorbitol, and similar
known agents. In addition, the compositions may
20 contain any of a number of adjuvants, such as buffers,
preservatives, dispersing agents, agents that prc~ote
rapid onset of action, agents that promote prolonged
duration of action, and other known agents. Typical
preservatives are, for example, phenol, _-cresol,
25 methyl ~-hydroxybenzoate, and others. Typical buffers
are, for example, sodium phosphate, sodium acetate,
sodium citrate, and others.
Moreover, an acid, such as hydrochloric acid,
or a base, such as sodium hydroxide, can be used ~or pH
3~ adjustment. In g~neral, the pH of the aqueous composi-
tion ranges frcm about 2 to about 8, and, preferably,
from about 6.8 to about 8Ø
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Other suitable additives are, for example,
divalent zinc ion, which, if present at all, is gener-
ally present in an amount from about 0.01 mg. to about
0.5 mg. per 100 units of human insulin, and protamine
salt (for example, in the form of its sulfate), which,
if present at all, is generally present in an amount
frcm about 0.1 mg. to about 3 mg. per 100 units of
human insulin activity.
Examples of particular pharmaceutical com-
positions of this invention are provided in the exam-
10 pLes appearing hereinbelow.
ExamPle 1 -- ~eutral Regular Human Insulin:Human
C-Peptide:Human Proinsulin Formulation
[1:4 human insulin:human C-peptide on
molar ba~is and 100:1 human insulin:
human proinsulin on weight basis at
40 Units (U) insulin per cubic centi-
- meter (cc.)]
To prepare 10 cc. of the composition, mix
Human Zinc Insulin (28 U/mg.)400 U
Human C-Peptide 30 mg.
Human Proinsulin 0.14 mg.
Phenol, distilled 20 mg.
Glycerin 160 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 7.0-7.8.
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Example 2 -- Neutral Regular Human Insulin:Human
C-Peptide:Human Proinsulin Formulation
[1:1 human insulin:human C-peptide on
molar basi-~ and 20:1 human insulin:human
proinsulin on weight basis at 100 U
insulin per cc.]
To prepare 10 cc. of the composition, mix
Human Zinc Insulin (28 U/mg.) 1000 U
Human C-Peptide 19 mg.
10 Human Proinsulin L.8 mg.
Phenol, distilled 20 mg.
Glycerin 160 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 7.0-7.8.
Example 3 -- Protamine, Zinc Human Insulin:Human
C-Peptide:Human Proinsulin Formulation
[1:1 human insulin:human C-peptide on
molar basis and 20:1 human insulin:human
proinsulin on weight basis at 40 U
insulin per cc.]
To prepare 10 cc. of the composition, mix
Human Zinc Insulin (28 U/mg.) 400 U
25 Human C-Peptide 8 mg.
Human Proinsulin O.7 mg.
Phenol, distilled 25 mg.
Zinc Oxide 0.78 mg.
Glycerin 160 mg.
30 Protamine Sulfate 4.0 - 6.0 mg.
Sodium Phosphate, crystals38 mg.
Water and either 10~ hydrochloric acid or 10~
sodium hydroxide sufficient to make a camposition
volume of 10 cc. and a final pH of 7.1-7.4.
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Example 4 -- Protamine, Zinc Human Insulin:Human
C-Peptide:Human Proinsulin Fonnulation
[2:1 human insulin:human C-peptide on
molar basis and 1:1 human insulin:human
proinsulin on weight basis at lO0 U
insulin per cc.]
To prepare 10 cc. of the ccmposition, mix
Human Zinc Insulin (28 U/mg.) 1000 IJ
Human C-Peptide 9 mg.
Human Proinsulin 36 mg.
Phenol, distilled 25 mg.
Zinc oxide 2.0 mg.
Glycerin 160 mg.
Protamine Sulfate 10 - 15 mg.
Sodium Phosphate, crystals38 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 7.1-7.4.
Example 5 -- Isophane Protamine, Zinc Human Insulin:
Human C-Peptide:Human Proinsulin Formu-
lation ~l:l human insulin:human C-peptide
on molar basis and 4:1 human insulin:
human proinsulin on weight basis at 40 U
insulin per cc.]
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To prepare 10 cc. of the composition, mix
Human Zinc Insulin (28 U/mg.) 400 U
Human C-Peptide 8 mg.
Human Proinsulin 3.6 mg.
_-Cre~ol, distilled 16 mg.
Phenol, distilled 6.5 mg.
Glycerin 160 mg.
Protamine Sulfate 1.2 - 2.4 mg.
Sodium Phosphate, crystals38 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 7.1-7.4.
Example 6 -- Isophane Protamine, Zinc Human Insulin:
Human C-Peptide:Human Proinsulin Formu-
lation [4:1 human insulin:human C-peptide
on molar basis and 1:2 human in~ulin:
human proinsulin on weight basis at
100 U insulin per cc.]
To prepare 10 cc. of the composition, mix
Human Zinc Insulin (28 U/mg.) 1000 U
Human C-Peptide 5 mg.
Human Proinsulin 71 mg.
m-Cresol, distilled 16 mg.
Phenol, distilled Z.5 mg.
Glycerin 160 mg.
Protamine Sulfate 3.0 - 6.0 mg.
Sodium Phosphate, Crystals38 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 7.1-7.4.
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Example 7 -- Zinc Human Insulin Suspension:Human
C-Peptide:Human Proinsulin Formulation
[1:2 human insulin:human C-peptide on
molar basis and 1:10 human insulin:human
proinsulin on weight basis at 40 U
insulin per cc.]
To prepare 10 cc. of the composition, mix
Human Zinc Insulin (28 U/mg.) 400 U
Human C-Peptide 15 mg.
10 Human Proinsulin 143 mg.
Sodium Acetate, Anhydrous16 mg.
Sodium Chloride, Granular70 mg.
Methyl _-Hydroxybenzoate10 mg.
Zinc Oxide 0.63 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.
Example 8 -- Zinc Human Insulin Suspension:Human
C-Peptide:Human Proinsulin Formulation
tl:l human insulin:human C-peptide on
molar basis and 20:1 human insulin:human
proinsulin on weight basis at 100 U
insulin per cc.
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To prepare 10 cc. of the composition, mix
Human Zinc Insulin (28 Utmg.) 1000 U
Human C-Peptide 19 mg.
Human Proinsulin 1.8 mg.
Sodium Acetate, Anhydrous16 mg.
Sodium Chloride, Granular70 mg.
Methyl ~-Hydroxybenzoate10 mg.
Zinc Oxide 1.6 mg.
Water and either 10% hydrochloric acid or 10%
sodium hydroxide sufficient to make a ccmposition
volume of 10 cc. and a final pH of 7.2-7.5.
Example 9 -- Neutral Regular Human Insulin:Human
C-Peptide:Human Proinsulin Formulation
[1:4 human insulin:human C-peptide on
molar basis and 100:1 human insulin:
human proinsulin on weight basis at
40 U insulin per cc.]
To prepare 10 cc. of the composition, mix
Human Sodium Insulin (28 U/mg.) 400 U
Human C-Peptide 30 mg.
Human Proinsulin 0.14 mg.
Phenol, distilled 20 mg.
Glycerin 160 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 7.0-7.8.
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Example 10 -- ~eutral Regular Human Insulin:Human
C-Peptide:Human Proinsulin Formulation
[1:1 human insulin:human C-peptide on
molar basis and 20:1 human insulin:human
proinsulin on weight basis at 100 U
insulin per cc.]
To prepare 10 cc. of the composition, mix
Human Sodium Insulin (28 U/mg.) 1000 U
Human C-Peptide 19 mg.
Human Proinsulin 1.8 mg.
Phenol, distilled 20 mg.
Glycerin 160 mg.
Water and either 10% hydrochloric acid or 10%
sodium hydroxide sufficient to make a composition
lS volume of lO cc. and a final pH of 7.0-7.8.
