U.S. patent application number 14/223408 was filed with the patent office on 2014-07-24 for preparation comprising insulin, nicotinamide and an amino acid.
This patent application is currently assigned to Novo Nordisk A/S. The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Svend Havelund, Svend Ludvigsen, Helle Naver, Helle Birk Olsen, Ulla Ribel-Madsen, Morten Schlein, Jeppe Sturis.
Application Number | 20140206610 14/223408 |
Document ID | / |
Family ID | 41351568 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140206610 |
Kind Code |
A1 |
Olsen; Helle Birk ; et
al. |
July 24, 2014 |
Preparation Comprising Insulin, Nicotinamide and an Amino Acid
Abstract
Insulin preparations comprising an insulin compound or a mixture
of two or more insulin compounds, a nicotinic compound and an amino
acid.
Inventors: |
Olsen; Helle Birk;
(Alleroed, DK) ; Havelund; Svend; (Bagsvaerd,
DK) ; Ribel-Madsen; Ulla; (Virum, DK) ;
Sturis; Jeppe; (Vaerloese, DK) ; Naver; Helle;
(Alleroed, DK) ; Schlein; Morten; (Vaerloese,
DK) ; Ludvigsen; Svend; (Lynge, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
41351568 |
Appl. No.: |
14/223408 |
Filed: |
March 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13671034 |
Nov 7, 2012 |
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14223408 |
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13309134 |
Dec 1, 2011 |
8324157 |
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13671034 |
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PCT/EP2010/059069 |
Jun 25, 2010 |
|
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13309134 |
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61222168 |
Jul 1, 2009 |
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Current U.S.
Class: |
514/6.3 |
Current CPC
Class: |
A61P 3/10 20180101; A61K
31/198 20130101; A61P 3/08 20180101; A61K 47/02 20130101; A61K
31/455 20130101; A61P 9/00 20180101; A61K 9/0019 20130101; A61K
31/465 20130101; A61P 9/10 20180101; A61K 45/06 20130101; A61K
47/183 20130101; A61K 38/28 20130101; A61K 47/22 20130101; A61K
31/198 20130101; A61K 2300/00 20130101; A61K 31/455 20130101; A61K
2300/00 20130101; A61K 38/28 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/6.3 |
International
Class: |
A61K 47/22 20060101
A61K047/22; A61K 47/18 20060101 A61K047/18; A61K 47/02 20060101
A61K047/02; A61K 38/28 20060101 A61K038/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 2009 |
EP |
09163940.1 |
Claims
1. An insulin preparation comprising: an insulin compound; a
nicotinic compound; arginine; and a buffer.
2. The insulin preparation of claim 1, wherein the buffer is a
phosphate buffer.
3. The insulin preparation of claim 2, wherein the insulin compound
comprises B28Asp human insulin.
4. The insulin preparation of claim 1, wherein the insulin compound
is selected from the group consisting of B28Asp human insulin,
B28LysB29Pro human insulin, and B3LysB29Glu human insulin.
5. The insulin preparation of claim 4, wherein the buffer is a
phosphate buffer.
6. An insulin preparation comprising: B28Asp human insulin;
nicotinamide; zinc; arginine; and a phosphate buffer.
7. The insulin preparation of claim 6, wherein the nicotinamide is
present at a concentration ranging from about 1 mM to about 300
mM.
8. The insulin preparation of claim 7, wherein less than about 4
zinc ions are present per six B28Asp human insulin molecules.
9. The insulin preparation of claim 8, wherein the arginine is
present in a concentration from about 10 mM to about 60 mM.
10. The insulin preparation of claim 9, wherein the arginine is
present in a concentration ranging from about 10 mM to about 40
mM.
11. The insulin preparation of claim 10, wherein the preparation
has a pH of about 7.4 or less.
12. The insulin preparation of claim 11, wherein the preparation
has a pH of about 7.1.
13. The insulin preparation of claim 6 wherein the nicotinamide is
present at a concentration ranging from about 80 mM to about 260
mM.
14. The insulin preparation of claim 13, wherein the arginine is
present in a concentration ranging from about 10 mM to about 40
mM.
15. The insulin preparation of claim 14, wherein the preparation
has a pH of about 7.4 or less.
16. The insulin preparation of claim 15, wherein less than about 4
zinc ions are present per six B28Asp human insulin molecules.
17. The insulin preparation of claim 14, wherein the preparation
has a pH of about 7.1.
18. The insulin preparation of claim 17, wherein less than about 4
zinc ions are present per six B28Asp human insulin molecules.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/671,034,filed Nov. 7, 2012, which is a continuation of U.S.
application Ser. No. 13/309,134, filed Dec. 1, 2011, now U.S. Pat.
No. 8,324,157, which is a continuation of PCT/EP2010/059069, filed
Jun. 25, 2010, which claimed priority of European Patent
Application 09163940.1, filed Jun. 26, 2009; this application
further claims priority under 35 U.S.C. .sctn.119 of U.S.
Provisional 61/222,168, filed Jul. 1, 2009; the contents of all
above-named applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to pharmaceutical preparations
comprising an insulin compound, a nicotinic compound and an amino
acid.
BACKGROUND OF THE INVENTION
[0003] Diabetes mellitus is a metabolic disorder in which the
ability to utilize glucose is partly or completely lost. About 5%
of all people suffer from diabetes and the disorder approaches
epidemic proportions.
[0004] Since the introduction of insulin in the 1920's, continuous
improvements have been made in the treatment of diabetes. To help
avoid high glycaemia levels, diabetic patients often practice
multiple injection therapy, whereby insulin is administered with
each meal. As diabetic patients have been treated with insulin for
several decades, there is a major need for safe and life-quality
improving insulin preparations. Among the commercially available
insulin preparations, rapid-acting, intermediate-acting and
prolonged-acting preparations can be mentioned.
[0005] In the treatment of diabetes mellitus, many varieties of
pharmaceutical preparations of insulin have been suggested and
used, such as regular insulin (such as Actrapid.RTM.), isophane
insulin (designated NPH), insulin zinc suspensions (such as
Semilente.RTM., Lente.RTM., and Ultralente.RTM.), and biphasic
isophane insulin (such as NovoMix.RTM.). Human insulin analogues
and derivatives have also been developed, designed for particular
profiles of action, i.e. fast action or prolonged action. Some of
the commercially available insulin preparations comprising such
rapid acting insulin analogues include NovoRapid.RTM. (preparation
of B28Asp human insulin), Humalog.RTM. (preparation of B28LysB29Pro
human insulin) and Apidra.RTM. (preparation of B3LysB29Glu human
insulin).
[0006] International applications WO 91/09617 and WO/9610417 (Novo
Nordisk NS) disclose insulin preparations containing nicotinamide
or nicotinic acid or a salt thereof.
[0007] Most often pharmaceutical preparations of insulins are
administered by subcutaneous injection. Important for the patient
is the action profile of the insulin, meaning the action of insulin
on glucose metabolism as a function of time from injection. In this
profile, inter alia, the time of the onset, the maximum value and
the total duration of action are important. In the case of bolus
insulins, a variety of insulin preparations with different action
profiles are desired and requested by the patients. One patient
may, on the same day, use insulin preparations with very different
action profiles. The action profile desired for example, depends on
the time of the day and the amount and composition of the meal
eaten by the patient.
[0008] Equally important for the patient is the chemical stability
of the insulin preparations, for example, due to the abundant use
of pen-like injection devices such as devices which contain
Penfill.RTM. cartridges, in which an insulin preparation is stored
until the entire cartridge is empty which may be at least 1 to 2
weeks for devices containing 1.5-3.0 ml cartridges. During storage,
covalent chemical changes in the insulin structure occur. This may
lead to formation of molecules which may be less active and/or
potentially immunogenic such as deamidation products and higher
molecular weight transformation products (dimers, polymers).
Furthermore, also important is the physical stability of the
insulin preparations, since long term storage may eventually lead
to formation of insoluble fibrils, which are biologically inactive
and potentially immunogenic.
SUMMARY OF THE INVENTION
[0009] The invention relates to insulin preparations with
favourable absorption rate and favourable chemical and physical
stability. The present invention relates to insulin preparations
comprising human insulin and/or analogues thereof, nicotinamide or
nicotinic acid and/or salts thereof, and arginine.
[0010] In one embodiment, the present invention relates to an
insulin preparation comprising: [0011] an insulin compound, [0012]
a nicotinic compound, and [0013] arginine. In another embodiment
the insulin preparation may further comprise glutamic acid.
[0014] In another embodiment, the present invention also
contemplates a method for the treatment of diabetes mellitus in a
subject or for reducing the blood glucose level in a subject
comprising administering to a subject or mammal an insulin
preparation according to the invention.
DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the development in percentage of total insulin
content of degradation products during 2 weeks of storage at
37.degree. C. of preparations according to the present invention.
The letter A refers to a NovoRapid.RTM. reference and remaining
letters correspond to insulin aspart preparations as described in
Table 1 of Example 1. Compared to the NovoRapid.RTM. preparation
(preparation A), addition of nicotinamide (preparations B and D)
leads to an increased formation of degradation products, whereas
the combined addition of nicotinamide, glutamic acid and arginine
(preparations C and E), has a mostly similar degradation pattern,
with lower formation of HMWP.
[0016] FIG. 2 shows the development in percentage of total insulin
content of degradation products during 2 weeks of storage at
37.degree. C. of preparations according to this invention. The
letter A refers to a NovoRapid.RTM. reference and remaining letters
correspond to insulin aspart preparations as described in Table 1
of Example 1. The combined addition of nicotinamide, glutamic acid
and arginine, preparations F, G, H, and I, differing in buffer
system, phosphate or tris buffer, and concentration of insulin and
Zn, 0.6 mM and 0.3 mM or 1.2 mM and 0.6 mM, has a degradation
pattern similar to the NovoRapid.RTM. preparation, preparation
A.
[0017] FIG. 3 shows the glucose concentration (mean+/-SEM, N=8) in
plasma after subcutaneous injection in pigs of a 1 nmol/kg dose at
0 minutes of preparations according to this invention. The letter A
refers to a NovoRapid.RTM. reference and remaining letters
correspond to insulin aspart preparations as described in Table 1
of Example 1. Compared to the NovoRapid.RTM. preparation
(preparation A) the initial rate of plasma glucose lowering is
faster for the preparation with addition of nicotinamide
(preparation N) and even faster for a combination of nicotinamide
and arginine (preparation M).
[0018] FIG. 4 shows the glucose concentration in plasma
(mean+/-SEM, N=7) after subcutaneous injection in pigs of a 1
nmol/kg dose at 0 minutes of preparations according to this
invention. The letter A refers to a NovoRapid.RTM. reference and
remaining letters correspond to insulin aspart preparations as
described in Table 1 of Example 1. Compared to the NovoRapid.RTM.
preparation (preparation A), the initial rate of plasma glucose
lowering is faster for a preparation with a combination of
nicotinamide, arginine and glutamic acid (preparation L) and for a
preparation with a combination of nicotinamide and arginine
(preparation K).
[0019] FIG. 5 shows the insulin aspart concentration in plasma
(mean+/-SEM, N=7) after subcutaneous injection in pigs of a 1
nmol/kg dose at 0 minutes of preparations according to this
invention. The letter A refers to a NovoRapid.RTM. reference and
remaining letters correspond to insulin aspart preparations as
described in Table 1 of Example 1. Compared to the NovoRapid.RTM.
preparation, (preparation A), the initial absorption rate of the
insulin component of the preparations with nicotinamide
(preparation J), the combination of nicotinamide and arginine
(preparation K), and the combination of nicotinamide, arginine and
glutamic acid (preparation L) is markedly faster.
[0020] FIG. 6 shows the time course for fibril formation can be
described by a sigmoidal curve using Equation 1.
DESCRIPTION OF THE INVENTION
[0021] The absorption after subcutaneous injection of the insulin
compound in the insulin preparations of the present invention was
surprisingly found to be faster than that of the reference insulin
preparations. This property is useful for rapid-acting insulins, in
particular in connection with a multiple injection regimen where
insulin is given before each meal. With faster onset of action, the
insulin can conveniently be taken closer to the meal than with
conventional rapid acting insulin solutions. Furthermore, a faster
disappearance of insulin probably diminishes the risk of post-meal
hypoglycaemia.
[0022] The insulin preparations of the present invention are
rapid-acting insulin preparations comprising an insulin compound
such as insulin aspart, a nicotinic compound, such as nicotinamide
and the amino acid arginine. Optionally, the insulin preparations
of the present invention may comprise further amino acids such as
glutamic acid. These insulin preparations have a rapid absorption
profile that mimics normal physiology more closely than existing
therapies. Furthermore, the insulin preparations of the present
invention have chemical and physical stability suitable for
commercial pharmaceutical preparations.
[0023] The insulin preparations of the present invention provide an
even faster onset of action compared with existing insulin
therapies. Such ultra-fast insulin preparations have the advantage
of restoring first phase insulin release, injection convenience and
shutting down hepatic glucose production. The insulin preparations
of the present invention have a favourable absorption rate from
subcutis into plasma with an increase in initial absorption rate
ranging from 1.5 to 5 times, when compared to conventional
preparations such as NovoRapid.RTM., as suggested by several PK/PD
experiments in pigs. This faster absorption rate may improve
glycaemic control and convenience and may allow for a shift from
pre-meal to post-meal dosing. The present invention is based in
part, on the surprising discovery that although, the addition of
nicotinamide allows the increase in absorption rate, it also has a
negative effect on chemical stability by significantly increasing
the amount of HMWP. The insulin preparations of the present
invention have an improved chemical stability by addition of
arginine, which is reflected in e.g. a reduction in the formation
of dimers and polymers and desamido insulins after storage. The
insulin preparations of the present invention may furthermore also
have improved physical stability, which may be useful for use in
pumps.
[0024] The present invention provides an insulin preparation
comprising an insulin compound according to the present invention
which is present in a concentration from about 0.1 mM to about 10.0
mM, and wherein said preparation has a pH from 3 to 8.5. The
preparation also comprises a nicotinic compound and arginine. The
preparation may further comprise protease inhibitor(s), metal ions,
a buffer system, preservative(s), tonicity agent(s), chelating
agent(s), stabilizers and surfactants.
[0025] In one embodiment the insulin preparations comprise a human
insulin, an analogue or combinations thereof, nicotinamide and/or
nicotinic acid and/or salts thereof and arginine and/or salts
thereof.
[0026] In one embodiment, the insulin preparations according to the
present invention comprise an aqueous solution of B28Asp human
insulin, nicotinamide and arginine.
[0027] The content of B28Asp human insulin in the solutions of this
invention may be in the range of 15 to 500 international units
(IU)/ml, preferably in the range of 50 to 333 IU/ml, in
preparations for injection. However, for other purposes of
parenteral administration, the content of insulin compound may be
higher.
[0028] There is also described herein an insulin preparation
comprising an insulin compound, a nicotinic compound and glutamic
acid.
[0029] In the present context the unit "IU" corresponds to 6
nmol.
[0030] The term "insulin aspart" refers to the human insulin
analogue B28Asp human insulin.
[0031] The term "onset" refers to the time from injection until the
PK curve shifts to an increase.
[0032] The term "absorption rate" refers to the slope of the PK
curve.
[0033] An "insulin compound" according to the invention is herein
to be understood as human insulin, an insulin analogue and/or any
combination thereof.
[0034] The term "human insulin" as used herein means the human
hormone whose structure and properties are well-known. Human
insulin has two polypeptide chains that are connected by disulphide
bridges between cysteine residues, namely the A-chain and the
B-chain. The A-chain is a 21 amino acid peptide and the B-chain is
a 30 amino acid peptide, the two chains being connected by three
disulphide bridges: one between the cysteines in position 6 and 11
of the A-chain, the second between the cysteine in position 7 of
the A-chain and the cysteine in position 7 of the B-chain, and the
third between the cysteine in position 20 of the A-chain and the
cysteine in position 19 of the B-chain.
[0035] The hormone is synthesized as a single-chain precursor
proinsulin (preproinsulin) consisting of a prepeptide of 24 amino
acids followed by proinsulin containing 86 amino acids in the
configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a
connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are
cleavage sites for cleavage of the connecting peptide from the A
and B chains.
[0036] By "insulin analogue" as used herein is meant a polypeptide
derived from the primary structure of a naturally occurring
insulin, for example that of human insulin, by mutation. One or
more mutations are made by deleting and/or substituting at least
one amino acid residue occurring in the naturally occurring insulin
and/or by adding at least one amino acid residue. The added and/or
substituted amino acid residues can either be codable amino acid
residues or other naturally occurring amino acid residues.
[0037] In one embodiment an insulin analogue comprises less than 8
modifications (substitutions, deletions, additions and any
combination thereof) relative to the parent insulin, alternatively
less than 7 modifications relative to the parent insulin,
alternatively less than 6 modifications relative to the parent
insulin, alternatively less than 5 modifications relative to the
parent insulin, alternatively less than 4 modifications relative to
the parent insulin, alternatively less than 3 modifications
relative to the parent insulin, alternatively less than 2
modifications relative to the parent insulin.
[0038] Mutations in the insulin molecule are denoted stating the
chain (A or B), the position, and the three letter code for the
amino acid substituting the native amino acid. By "desB30" or
"B(1-29)" is meant a natural insulin B chain or analogue thereof
lacking the B30 amino acid residue, and by B28Asp human insulin is
meant human insulin wherein the amino acid residue in position 28
of the B chain has been substituted with Asp.
[0039] Examples of insulin analogues are such wherein Pro in
position 28 of the B chain is mutated with Asp, Lys, Leu, Val, or
Ala and/or Lys at position B29 is mutated with Pro, Glu or Asp.
Furthermore, Asn at position B3 may be mutated with Thr, Lys, Gln,
Glu or Asp. The amino acid residue in position A21 may be mutated
with Gly. The amino acid in position B1 may be mutated with Glu.
The amino acid in position B16 may be mutated with Glu or His.
Further examples of insulin analogues are the deletion analogues
e.g. analogues where the B30 amino acid in human insulin has been
deleted (des(B30) human insulin), insulin analogues wherein the B1
amino acid in human insulin has been deleted (des(B1) human
insulin), des(B28-B30) human insulin and des(B27) human insulin.
Insulin analogues wherein the A-chain and/or the B-chain have an
N-terminal extension and insulin analogues wherein the A-chain
and/or the B-chain have a C-terminal extension such as with two
arginine residues added to the C-terminal of the B-chain are also
examples of insulin analogues. Further examples are insulin
analogues comprising combinations of the mentioned mutations.
Insulin analogues wherein the amino acid in position A14 is Asn,
Gln, Glu, Arg, Asp, Gly or His, the amino acid in position B25 is
His and which optionally further comprises one or more additional
mutations are further examples of insulin analogues. Insulin
analogues of human insulin wherein the amino acid residue in
position A21 is Gly and wherein the insulin analogue is further
extended in the C-terminal with two arginine residues are also
examples of insulin analogues.
[0040] Further examples of insulin analogues include, but are not
limited to: DesB30 human insulin; AspB28 human insulin;
AspB28,desB30 human insulin; LysB3,GluB29 human insulin;
LysB28,ProB29 human insulin; GlyA21,ArgB31,ArgB32 human insulin;
GluA14,HisB25 human insulin; HisA14,HisB25 human insulin;
GluA14,HisB25,desB30 human insulin; HisA14, HisB25,desB30 human
insulin; GluA14,HisB25,desB27,desB28,desB29,desB30 human insulin;
GluA14,HisB25,GluB27,desB30 human insulin;
GluA14,HisB16,HisB25,desB30 human insulin;
HisA14,HisB16,HisB25,desB30 human insulin;
HisA8,GluA14,HisB25,GluB27,desB30 human insulin;
HisA8,GluA14,GluB1,GluB16,HisB25,GluB27,desB30 human insulin; and
HisA8,GluA14,GluB16,HisB25,desB30 human insulin.
[0041] The term "nicotinic compound" includes nicotinamide,
nicotinic acid, niacin, niacin amide and vitamin B3 and/or salts
thereof and/or any combination thereof.
[0042] According to the present invention, the concentration of the
nicotinic compound and/or salts thereof is in the range from about
1 mM to about 300 mM or from about 5 mM to about 200 mM.
[0043] The term "arginine" or "Arg" includes the amino acid
arginine and/or a salt thereof.
[0044] In one embodiment, the insulin preparation comprises 1 to
100 mM of arginine.
[0045] In one embodiment, the insulin preparation comprises 1 to 20
mM of arginine.
[0046] In one embodiment, the insulin preparation comprises 20 to
90 mM of arginine.
[0047] In one embodiment, the insulin preparation comprises 30 to
85 mM of arginine.
[0048] The term "glutamic acid" or "Glu" includes the aminoacid
glutamic acid and/or a salt thereof.
[0049] In one embodiment, the insulin preparation comprises 1 to
100 mM of glutamic acid.
[0050] In one embodiment, the insulin preparation comprises 20 to
90 mM of glutamic acid.
[0051] In one embodiment, the insulin preparation comprises 30 to
85 mM of glutamic acid.
[0052] The term "pharmaceutical preparation" or "insulin
preparation" as used herein means a product comprising an insulin
compound, i.e., a human insulin, an analogue thereof and/or
combinations thereof and a nicotinic compound and an amino acid,
optionally together with other excipients such as preservatives,
chelating agents, tonicity modifiers, bulking agents, stabilizers,
antioxidants, polymers and surfactants, metal ions, oleaginous
vehicles and proteins (e.g., human serum albumin, gelatine or
proteins), said insulin preparation being useful for treating,
preventing or reducing the severity of a disease or disorder by
administration of said insulin preparation to a person. Thus, an
insulin preparation is also known in the art as a pharmaceutical
preparation or pharmaceutical composition.
[0053] The buffer may be selected from the group consisting of, but
not limited to, sodium acetate, sodium carbonate, citrate, sodium
dihydrogen phosphate, disodium hydrogen phosphate, sodium
phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine,
malic acid, succinate, maleic acid, fumaric acid, tartaric acid,
aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of the invention.
[0054] The insulin preparation of the present invention may further
comprise other ingredients common to insulin preparations, for
example zinc complexing agents such as citrate, and phosphate
buffers.
[0055] Glycerol and/or mannitol and/or sodium chloride may be
present in an amount corresponding to a concentration of 0 to 250
mM, 0 to 200 mM or 0 to 100 mM.
[0056] Stabilizers, surfactants and preservatives may also be
present in the insulin preparations of this invention.
[0057] The insulin preparations of the present invention may
further comprise a pharmaceutically acceptable preservative. The
preservative may be present in an amount sufficient to obtain a
preserving effect. The amount of preservative in an insulin
preparation may be determined from e.g. literature in the field
and/or the known amount(s) of preservative in e.g. commercial
products. Each one of these specific preservatives constitutes an
alternative embodiment of the invention. The use of a preservative
in pharmaceutical preparations is described, for example in
Remington: The Science and Practice of Pharmacy, 19.sup.th edition,
1995.
[0058] The preservative present in the insulin preparation of this
invention may be as in the heretofore conventional insulin
preparations, for example phenol, m-cresol and methylparaben.
[0059] The insulin preparation of the present invention may further
comprise a chelating agent. The use of a chelating agent in
pharmaceutical preparations is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0060] The insulin preparation of the present invention may further
comprise a stabilizer. The term "stabilizer" as used herein refers
to chemicals added to polypeptide containing pharmaceutical
preparations in order to stabilize the peptide, i.e. to increase
the shelf life and/or in-use time of such preparations. For
convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0061] The insulin preparation of the present invention may further
comprise a surfactant. The term "surfactant" as used herein refers
to any molecules or ions that are comprised of a water-soluble
(hydrophilic) part, the head, and a fat-soluble (lipophilic)
segment. Surfactants accumulate preferably at interfaces, which the
hydrophilic part is orientated towards the water (hydrophilic
phase) and the lipophilic part towards the oil- or hydrophobic
phase (i.e. glass, air, oil etc.). The concentration at which
surfactants begin to form micelles is known as the critical micelle
concentration or CMC. Furthermore, surfactants lower the surface
tension of a liquid. Surfactants are also known as amphipathic
compounds. The term "detergent" is a synonym used for surfactants
in general. The use of a surfactant in pharmaceutical preparations
is well-known to the skilled person. For convenience reference is
made to Remington: The Science and Practice of Pharmacy, 19.sup.th
edition, 1995.
[0062] In a further embodiment the invention relates to an insulin
preparation comprising an aqueous solution of an insulin compound
of the present invention, and a buffer, wherein said insulin
compound is present in a concentration from 0.1 mM or above, and
wherein said preparation has a pH from about 3.0 to about 8.5 at
room temperature (.about.25.degree. C.).
[0063] The present invention also relates to methods for producing
the insulin preparations of the invention.
[0064] In one embodiment, the method for making insulin
preparations of the invention comprises:
[0065] a) preparing a solution by dissolving the insulin compound
or a mixture of insulin compounds in water or buffer;
[0066] b) preparing a solution by dissolving a divalent metal ion
in water or buffer;
[0067] c) preparing a solution by dissolving a preservative in
water or buffer;
[0068] d) preparing a solution by dissolving an isotonicity agent
in water or buffer;
[0069] e) preparing a solution by dissolving a surfactant and/or a
stabilizer in water or buffer;
[0070] f) mixing solution a) and one or more of solutions b), c),
d), and e);
[0071] Finally adjusting the pH of the mixture in f) to the desired
pH followed by a sterile filtration.
[0072] The insulin preparations of the present invention can be
used in the treatment of diabetes by parenteral administration. It
is recommended that the dosage of the insulin preparations of this
invention which is to be administered to the patient be selected by
a physician.
[0073] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of
a syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. As a
further option, the insulin preparations containing the insulin
compound of the invention can also be adapted to transdermal
administration, e.g. by needle-free injection or from a patch,
optionally an iontophoretic patch, or transmucosal, e.g. buccal,
administration.
[0074] Insulin preparations according to the present invention may
be administered to a patient in need of such treatment at several
sites, for example, at topical sites, for example, skin and mucosal
sites, at sites which bypass absorption, for example,
administration in an artery, in a vein, in the heart, and at sites
which involve absorption, for example, administration in the skin,
under the skin, in a muscle or in the abdomen.
[0075] In one embodiment of the invention the insulin preparation
is an aqueous preparation, i.e. preparation comprising water. Such
preparation is typically a solution or a suspension. In a further
embodiment of the invention the insulin preparation is an aqueous
solution.
[0076] The term "aqueous preparation" is defined as a preparation
comprising at least 50% w/w water. Likewise, the term "aqueous
solution" is defined as a solution comprising at least 50% w/w
water, and the term "aqueous suspension" is defined as a suspension
comprising at least 50% w/w water.
[0077] Aqueous suspensions may contain the active compounds in
admixture with excipients suitable for the manufacture of aqueous
suspensions.
[0078] In one embodiment, the insulin preparations of this
invention are well-suited for application in pen-like devices used
for insulin therapy by injection.
[0079] In one embodiment the insulin preparations of the present
invention can be used in pumps for insulin administration.
[0080] The term "physical stability" of the insulin preparation as
used herein refers to the tendency of the protein to form
biologically inactive and/or insoluble aggregates of the protein as
a result of exposure of the protein to thermo-mechanical stresses
and/or interaction with interfaces and surfaces that are
destabilizing, such as hydrophobic surfaces and interfaces.
Physical stability of the aqueous protein preparations is evaluated
by means of visual inspection and/or turbidity measurements after
exposing the preparation filled in suitable containers (e.g.
cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual
inspection of the preparations is performed in a sharp focused
light with a dark background. The turbidity of the preparation is
characterized by a visual score ranking the degree of turbidity for
instance on a scale from 0 to 3 (a preparation showing no turbidity
corresponds to a visual score 0, and a preparation showing visual
turbidity in daylight corresponds to visual score 3). A preparation
is classified physically unstable with respect to protein
aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the preparation can be evaluated by
simple turbidity measurements well-known to the skilled person.
Physical stability of the aqueous protein preparations can also be
evaluated by using a spectroscopic agent or probe of the
conformational status of the protein. The probe is preferably a
small molecule that preferentially binds to a non-native conformer
of the protein. One example of a small molecular spectroscopic
probe of protein structure is Thioflavin T. Thioflavin T is a
fluorescent dye that has been widely used for the detection of
amyloid fibrils. In the presence of fibrils, and perhaps other
protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about
482 nm when bound to a fibril protein form. Unbound Thioflavin T is
essentially non-fluorescent at the wavelengths.
[0081] The term "chemical stability" of the protein preparation as
used herein refers to changes in the covalent protein structure
leading to formation of chemical degradation products with
potential less biological potency and/or potential increased
immunogenic properties compared to the native protein structure.
Various chemical degradation products can be formed depending on
the type and nature of the native protein and the environment to
which the protein is exposed. Increasing amounts of chemical
degradation products is often seen during storage and use of the
protein preparation. Most proteins are prone to deamidation, a
process in which the side chain amide group in glutaminyl or
asparaginyl residues is hydrolysed to form a free carboxylic acid
or asparaginyl residues to form an IsoAsp derivative. Other
degradations pathways involves formation of high molecular weight
products where two or more protein molecules are covalently bound
to each other through transamidation and/or disulfide interactions
leading to formation of covalently bound dimer, oligomer and
polymer degradation products (Stability of Protein Pharmaceuticals,
Ahem. T. J. & Manning M. C., Plenum Press, New York 1992).
Oxidation (of for instance methionine residues) can be mentioned as
another variant of chemical degradation. The chemical stability of
the protein preparation can be evaluated by measuring the amount of
the chemical degradation products at various time-points after
exposure to different environmental conditions (the formation of
degradation products can often be accelerated by for instance
increasing temperature). The amount of each individual degradation
product is often determined by separation of the degradation
products depending on molecule size and/or charge using various
chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC). Since
HMWP products are potentially immunogenic and not biologically
active, low levels of HMWP are advantageous.
[0082] The term "stabilized preparation" refers to a preparation
with increased physical stability, increased chemical stability or
increased physical and chemical stability. In general, a
preparation must be stable during use and storage (in compliance
with recommended use and storage conditions) until the expiration
date is reached.
[0083] The term "diabetes" or "diabetes mellitus" includes type 1
diabetes, type 2 diabetes, gestational diabetes (during pregnancy)
and other states that cause hyperglycaemia. The term is used for a
metabolic disorder in which the pancreas produces insufficient
amounts of insulin, or in which the cells of the body fail to
respond appropriately to insulin thus preventing cells from
absorbing glucose. As a result, glucose builds up in the blood.
[0084] Type 1 diabetes, also called insulin-dependent diabetes
mellitus (IDDM) and juvenile-onset diabetes, is caused by B-cell
destruction, usually leading to absolute insulin deficiency.
[0085] Type 2 diabetes, also known as non-insulin-dependent
diabetes mellitus (NIDDM) and adult-onset diabetes, is associated
with predominant insulin resistance and thus relative insulin
deficiency and/or a predominantly insulin secretory defect with
insulin resistance.
[0086] The term "pharmaceutically acceptable" as used herein means
suited for normal pharmaceutical applications, i.e., not giving
rise to any serious adverse events in patients.
[0087] The term "treatment of a disease" as used herein means the
management and care of a patient having developed the disease,
condition or disorder and includes treatment, prevention or
alleviation of the disease. The purpose of treatment is to combat
the disease, condition or disorder. Treatment includes the
administration of the active compounds to eliminate or control the
disease, condition or disorder as well as to alleviate the symptoms
or complications associated with the disease, condition or
disorder, and prevention of the disease, condition or disorder.
[0088] In another embodiment, an insulin analogue according to the
invention is used as a medicament for delaying or preventing
disease progression in type 2 diabetes.
[0089] In one embodiment of the present invention, the insulin
preparation according to the invention is for use as a medicament
for the treatment or prevention of hyperglycemia including stress
induced hyperglycemia, type 2 diabetes, impaired glucose tolerance,
type 1 diabetes, and burns, operation wounds and other diseases or
injuries where an anabolic effect is needed in the treatment,
myocardial infarction, stroke, coronary heart disease and other
cardiovascular disorders is provided.
[0090] In a further embodiment of the present invention, a method
for the treatment or prevention of hyperglycemia including stress
induced hyperglycemia, type 2 diabetes, impaired glucose tolerance,
type 1 diabetes, and burns, operation wounds and other diseases or
injuries where an anabolic effect is needed in the treatment,
myocardial infarction, coronary heart disease and other
cardiovascular disorders, stroke, the method comprising
administering to a patient in need of such treatment an effective
amount for such treatment of an insulin preparation according to
the invention, is provided.
[0091] The treatment with an insulin preparation according to the
present invention may also be combined with a second or more
pharmacologically active substances, e.g. selected from
antidiabetic agents, antiobesity agents, appetite regulating
agents, antihypertensive agents, agents for the treatment and/or
prevention of complications resulting from or associated with
diabetes and agents for the treatment and/or prevention of
complications and disorders resulting from or associated with
obesity.
[0092] The treatment with an insulin preparation according to the
present invention may also be combined with bariatric surgery--a
surgery that influences the glucose levels and/or lipid homeostasis
such as gastric banding or gastric bypass.
[0093] The production of polypeptides, e.g., insulins, is well
known in the art. An insulin analogue according to the invention
may for instance be produced by classical peptide synthesis, e.g.
solid phase peptide synthesis using t-Boc or Fmoc chemistry or
other well established techniques, see e.g. Greene and Wuts,
"Protective Groups in Organic Synthesis", John Wiley & Sons,
1999. The insulin analogue may also be produced by a method which
comprises culturing a host cell containing a DNA sequence encoding
the analogue and capable of expressing the insulin analogue in a
suitable nutrient medium under conditions permitting the expression
of the insulin analogue. For insulin analogues comprising
non-natural amino acid residues, the recombinant cell should be
modified such that the non-natural amino acids are incorporated
into the analogue, for instance by use of tRNA mutants. Hence,
briefly, the insulin analogues according to the invention are
prepared analogously to the preparation of known insulin
analogues.
[0094] Several methods may be used for the production of human
insulin and human insulin analogues. For example three major
methods which are used in the production of insulin in
microorganisms are disclosed in WO2008034881. Two of these involve
Escherichia coli, with either the expression of a large fusion
protein in the cytoplasm (Frank et al. (1981) in Peptides:
Proceedings of the 7.sup.th American Peptide Chemistry Symposium
(Rich & Gross, eds.), Pierce Chemical Co., Rockford, Ill. pp
729-739), or use of a signal peptide to enable secretion into the
periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third
method utilizes Saccharomyces cerevisiae to secrete an insulin
precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770).
The prior art discloses a number of insulin precursors which are
expressed in either E. coli or Saccharomyces cerevisiae, vide U.S.
Pat. No. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845
and EP 0741188.
[0095] The insulin analogues are produced by expressing a DNA
sequence encoding the insulin analogue in question in a suitable
host cell by well known technique as disclosed in e.g. U.S. Pat.
No. 6,500,645. The insulin analogue is either expressed directly or
as a precursor molecule which has an N-terminal extension on the
B-chain or a C-terminal extension on the B-chain. The N-terminal
extension may have the function of increasing the yield of the
directly expressed product and may be of up to 15 amino acid
residues long. The N-terminal extension is to be cleaved of in
vitro after isolation from the culture broth and will therefore
have a cleavage site next to B1. N-terminal extensions of the type
suitable in the present invention are disclosed in U.S. Pat. No.
5,395,922, and EP 765,395. The C-terminal extension may have the
function of protecting the mature insulin or insulin analogue
molecule against intracellular proteolytic processing by host cell
exoproteases. The C-terminal extension is to be cleaved of either
extra-cellularly in the culture broth by secreted, active
carboxypeptidase or in vitro after isolation from the culture
broth. A method for producing mature insulin and insulin analogs
with C-terminal extensions on the B-chain that are removed by
carboxypetidase are disclosed in WO 08037735. The target insulin
product of the process may either be a two-chain human insulin or a
two-chain human insulin analogue which may or may not have a short
C-terminal extension of the B-chain. If the target insulin product
will have no C-terminal extension of the B-chain, then said
C-terminal extension should be capable of subsequently being
cleaved off from the B-chain before further purification steps.
[0096] The present invention also contemplates the following
non-limiting list of embodiments, which are further described
elsewhere herein: [0097] 1. An insulin preparation comprising:
[0098] an insulin compound, [0099] a nicotinic compound, and [0100]
arginine. [0101] 2. The insulin preparation according to embodiment
1, wherein the insulin compound is human insulin or an insulin
analog. [0102] 3. The insulin preparation according to any of the
preceding embodiments, wherein the insulin compound is B28Asp human
insulin. [0103] 4. The insulin preparation according to any of the
preceding embodiments, wherein the insulin compound is B28LysB29Pro
human insulin. [0104] 5. The insulin preparation according to any
of the preceding embodiments, wherein the insulin compound is
B3LysB29Glu human insulin. [0105] 6. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in a range selected from the following:
0.1-10.0 mM; 0.1-3.0 mM; 0.1-2.5 mM; 0.1-2.0 mM; 0.1-1.5 mM;
0.2-2.5 mM; 0.2-2.0 mM; 0.2-1.5 mM; 0.3-3.0 mM; 0.3-2.5 mM; 0.3-2.0
mM; 0.3-1.5 mM; 0.5-1.3 mM and 0.6-1.2 mM. [0106] 7. The insulin
preparation according to any of the preceding embodiments, wherein
the insulin compound is present in the amount from about 0.1 mM to
about 10.0 mM. [0107] 8. The insulin preparation according to any
of the preceding embodiments, wherein the insulin compound is
present in the amount from about 0.1 mM to about 3.0 mM. [0108] 9.
The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount
from about 0.1 mM to about 2.5 mM. [0109] 10. The insulin
preparation according to any of the preceding embodiments, wherein
the insulin compound is present in the amount from about 0.1 mM to
about 2.0 mM. [0110] 11. The insulin preparation according to any
of the preceding embodiments, wherein the insulin compound is
present in the amount from about 0.1 mM to about 1.5 mM. [0111] 12.
The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount
from about 0.2 mM to about 2.5 mM. [0112] 13. The insulin
preparation according to any of the preceding embodiments, wherein
the insulin compound is present in the amount from about 0.2 mM to
about 2.0 mM. [0113] 14. The insulin preparation according to any
of the preceding embodiments, wherein the insulin compound is
present in the amount from about 0.2 mM to about 1.5 mM. [0114] 15.
The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount
from about 0.3 mM to about 3.0 mM. [0115] 16. The insulin
preparation according to any of the preceding embodiments, wherein
the insulin compound is present in the amount from about 0.3 mM to
about 2.5 mM. [0116] 17. The insulin preparation according to any
of the preceding embodiments, wherein the insulin compound is
present in the amount from about 0.3 mM to about 2.0 mM. [0117] 18.
The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount
from about 0.3 mM to about 1.5 mM. [0118] 19. The insulin
preparation according to any of the preceding embodiments, wherein
the insulin compound is present in the amount from about 0.5 mM to
about 1.3 mM. [0119] 20. The insulin preparation according to any
of the preceding embodiments, wherein the insulin compound is
present in the amount from about 0.3 mM to about 1.2 mM. [0120] 21.
The insulin preparation according to any of the preceding
embodiments, wherein the insulin compound is present in the amount
from about 0.6 mM to about 1.2 mM. [0121] 22. The insulin
preparation according to any of the preceding embodiments, wherein
the insulin compound is present in the amount of about 0.6 or about
1.2 mM. [0122] 23. The insulin preparation according to any of the
preceding embodiments, wherein the insulin compound is present in
the amount of about 0.3 mM. [0123] 24. The insulin preparation
according to any of the preceding embodiments, wherein the insulin
compound is present in the amount of about 0.6 mM. [0124] 25. The
insulin preparation according to any of the preceding embodiments,
wherein the insulin compound is present in the amount of about 1.2
mM. [0125] 26. The insulin preparation according to any of the
preceding embodiments, wherein the nicotinic compound is selected
from the group consisting of nicotinamide, nicotinic acid, niacin,
niacin amide and vitamin B3 and/or salts thereof and/or any
combination thereof. [0126] 27. The insulin preparation according
to any of the preceding embodiments, wherein the nicotinic compound
is selected from nicotinamide and nicotinic acid and/or salts
thereof and/or any combination thereof. [0127] 28. The insulin
preparation according to any of the preceding embodiments, wherein
the nicotinic compound is present in a range selected from the
following: 1-300 mM; 5-200 mM; 40-120 mM, 70-140 mM or 80-130 mM.
[0128] 29. The insulin preparation according to any of the
preceding embodiments, comprising from about 1 mM to about 300 mM
of the nicotinic compound. [0129] 30. The insulin preparation
according to any of the preceding embodiments, comprising from
about 8 mM to about 260 mM of the nicotinic compound. [0130] 31.
The insulin preparation according to any of the preceding
embodiments, comprising from about 5 mM to about 200 mM of the
nicotinic compound. [0131] 32. The insulin preparation according to
any of the preceding embodiments, comprising from about 1 mM to
about 150 mM of the nicotinic compound. [0132] 33. The insulin
preparation according to any of the preceding embodiments,
comprising from about 5 mM to about 20 mM of the nicotinic
compound. [0133] 34. The insulin preparation according to any of
the preceding embodiments, comprising from about 20 mM to about 120
mM of the nicotinic compound. [0134] 35. The insulin preparation
according to any of the preceding embodiments, comprising from
about 40 mM to about 120 mM of the nicotinic compound. [0135] 36.
The insulin preparation according to any of the preceding
embodiments, comprising from about 20 mM to about 40 mM of the
nicotinic compound. [0136] 37. The insulin preparation according to
any of the preceding embodiments, comprising from about 60 mM to
about 80 mM of the nicotinic compound. [0137] 38. The insulin
preparation according to any of the preceding embodiments,
comprising from about 70 mM to about 140 mM of the nicotinic
compound. [0138] 39. The insulin preparation according to any of
the preceding embodiments, comprising from about 80 mM to about 130
mM of the nicotinic compound. [0139] 40. The insulin preparation
according to any of the preceding embodiments, comprising about 8
mM, 30 mM, 100 mM or 130 mM of the nicotinic compound. [0140] 41.
The insulin preparation according to any of the preceding
embodiments, comprising about 8 mM of the nicotinic compound.
[0141] 42. The insulin preparation according to any of the
preceding embodiments, comprising about 30 mM, 100 mM or 130 mM of
the nicotinic compound. [0142] 43. The insulin preparation
according to any of the preceding embodiments, comprising about 30
mM of the nicotinic compound. [0143] 44. The insulin preparation
according to any of the preceding embodiments, comprising about 100
mM of the nicotinic compound. [0144] 45. The insulin preparation
according to any of the preceding embodiments, comprising about 130
mM of the nicotinic compound. [0145] 46. The insulin preparation
according to any of the preceding embodiments, comprising about 150
mM of the nicotinic compound. [0146] 47. The insulin preparation
according to any of the preceding embodiments, comprising the
following ranges of arginine compound: 1-100 mM, 5-120 mM, 8-85 mM,
20-90 mM, 30-90 mM, 30-85 mM, 30-60 mM or 10-40 mM. [0147] 48. The
insulin preparation according to any of the preceding embodiments,
comprising the following ranges of arginine compound: 1-120 mM,
8-85 mM or 1-40 mM. [0148] 49. The insulin preparation according to
any of the preceding embodiments, comprising from about 1 mM to
about 120 mM of arginine. [0149] 50. The insulin preparation
according to any of the preceding embodiments, comprising from
about 1 mM to about 100 mM of arginine. [0150] 51. The insulin
preparation according to any of the preceding embodiments,
comprising from about 5 mM to about 120 mM of arginine. [0151] 52.
The insulin preparation according to any of the preceding
embodiments, comprising from about 20 mM to about 90 mM of
arginine. [0152] 53. The insulin preparation according to any of
the preceding embodiments, comprising from about 30 mM to about 85
mM of arginine. [0153] 54. The insulin preparation according to any
of the preceding embodiments, comprising from about 8 mM to about
85 mM of arginine. [0154] 55. The insulin preparation according to
any of the preceding embodiments, comprising from about 30 mM to
about 60 mM of arginine. [0155] 56. The insulin preparation
according to any of the preceding embodiments, comprising from
about 10 mM to about 40 mM of arginine. [0156] 57. The insulin
preparation according to any of the preceding embodiments,
comprising from about 1 mM to about 40 mM of arginine. [0157] 58.
The insulin preparation according to any of the preceding
embodiments, wherein arginine is present in a range selected from
the following: 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9
mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mM, 45 mM, 50
mM, 55 mM or 60 mM. [0158] 59. The insulin preparation according to
any of the preceding embodiments, comprising about 1 mM of
arginine. [0159] 60. The insulin preparation according to any of
the preceding embodiments, comprising about 2 mM of arginine.
[0160] 61. The insulin preparation according to any of the
preceding embodiments, comprising about 3 mM of arginine. [0161]
62. The insulin preparation according to any of the preceding
embodiments, comprising about 4 mM of arginine. [0162] 63. The
insulin preparation according to any of the preceding embodiments,
comprising about 5 mM of arginine. [0163] 64. The insulin
preparation according to any of the preceding embodiments,
comprising about 6 mM of arginine. [0164] 65. The insulin
preparation according to any of the preceding embodiments,
comprising about 7 mM of arginine. [0165] 66. The insulin
preparation according to any of the preceding embodiments,
comprising about 8 mM of arginine. [0166] 67. The insulin
preparation according to any of the preceding embodiments,
comprising about 9 mM of arginine. [0167] 68. The insulin
preparation according to any of the preceding embodiments,
comprising about 10 mM of arginine. [0168] 69. The insulin
preparation according to any of the preceding embodiments,
comprising about 15 mM of arginine. [0169] 70. The insulin
preparation according to any of the preceding embodiments,
comprising about 20 mM of arginine. [0170] 71. The insulin
preparation according to any of the preceding embodiments,
comprising about 25 mM of arginine. [0171] 72. The insulin
preparation according to any of the preceding embodiments,
comprising about 30 mM of arginine. [0172] 73. The insulin
preparation according to any of the preceding embodiments,
comprising about 35 mM of arginine. [0173] 74. The insulin
preparation according to any of the preceding embodiments,
comprising about 40 mM of arginine. [0174] 75. The insulin
preparation according to any of the preceding embodiments,
comprising about 45 mM of arginine. [0175] 76. The insulin
preparation according to any of the preceding embodiments,
comprising about 50 mM of arginine. [0176] 77. The insulin
preparation according to any of the preceding embodiments,
comprising about 55 mM of arginine. [0177] 78. The insulin
preparation according to any of the preceding embodiments,
comprising about 60 mM of arginine. [0178] 79. The insulin
preparation according to any of the preceding embodiments, further
comprising glutamic acid. [0179] 80. The insulin preparation
according to embodiment 79, wherein glutamic acid is present in a
range selected from the following: 1-100 mM, 20-90 mM, 30-90 mM,
30-85 mM or 30-50 mM. [0180] 81. The insulin preparation according
to embodiment 79, comprising from about 1 mM to about 100 mM of
glutamic acid. [0181] 82. The insulin preparation according to
embodiment 79, comprising from about 20 mM to about 90 mM of
glutamic acid. [0182] 83. The insulin preparation according to
embodiment 79, comprising from about 30 mM to about 85 mM of
glutamic acid. [0183] 84. The insulin preparation according to
embodiment 79, comprising from about 30 mM to about 50 mM of
glutamic acid. [0184] 85. The insulin preparation according to
embodiment 79, comprising about 30 mM or 50 mM of glutamic acid.
[0185] 86. The insulin preparation according to embodiment 79,
comprising about 30 mM of glutamic acid. [0186] 87. The insulin
preparation according to embodiment 79, comprising about 50 mM of
glutamic acid. [0187] 88. The insulin preparation according to any
of the preceding embodiments, which further comprises a metal ion,
preservative agent(s), isotonicity agent(s) and stabilizer(s),
detergent(s), and buffer(s). [0188] 89. The insulin preparation
according to embodiment 88, wherein said buffer is Tris. [0189] 90.
The insulin preparation according to embodiment 89, comprising from
about 2 mM to about 50 mM of Tris. [0190] 91. The insulin
preparation according to embodiment 89, comprising from about 10 mM
to about 40 mM of Tris. [0191] 92. The insulin preparation
according to embodiment 89, comprising from about 20 mM to about 30
mM of Tris. [0192] 93. The insulin preparation according to
embodiment 89, comprising about 10 mM, 20 mM, 30 mM or 40 mM of
Tris. [0193] 94. The insulin preparation according to embodiment
89, comprising about 10 mM of Tris. [0194] 95. The insulin
preparation according to embodiment 89, comprising about 20 mM of
Tris. [0195] 96. The insulin preparation according to embodiment
89, comprising about 30 mM of Tris. [0196] 97. The insulin
preparation according to embodiment 89, comprising about 40 mM of
Tris. [0197] 98. The insulin preparation according to embodiment
89, wherein the metal ion is zinc. [0198] 99. The insulin
preparation according to embodiment 98, wherein less than about 6
zinc ions are present per hexamer of insulin compound. [0199] 100.
The insulin preparation according to embodiment 98, wherein less
than about 4 zinc ions are present per hexamer of insulin compound.
[0200] 101. The insulin preparation according to embodiment 98,
wherein less than about 3 zinc ions are present per hexamer of
insulin compound. [0201] 102. The insulin preparation according to
embodiment 98, wherein the zinc:insulin molar ratio is from about
2:6 to about 5:6. [0202] 103. The insulin preparation according to
embodiment 98, wherein the zinc:insulin molar ratio is from about
2.5:6 to about 4.5:6.
[0203] 104. The insulin preparation according to embodiment 98,
wherein the zinc:insulin molar ratio is from about 3:6 to about
4:6. [0204] 105. The insulin preparation according to embodiment
98, wherein the zinc:insulin molar ratio is about 2:6. [0205] 106.
The insulin preparation according to embodiment 98, wherein the
zinc:insulin molar ratio is about 2.5:6. [0206] 107. The insulin
preparation according to embodiment 98, wherein the zinc:insulin
molar ratio is about 3:6. [0207] 108. The insulin preparation
according to embodiment 98, wherein the zinc:insulin molar ratio is
about 3.5:6. [0208] 109. The insulin preparation according to
embodiment 98, wherein the zinc:insulin molar ratio is about 4:6.
[0209] 110. The insulin preparation according to embodiment 98,
wherein the zinc:insulin molar ratio is about 4.5:6. [0210] 111.
The insulin preparation according to embodiment 98, wherein the
zinc:insulin molar ratio is about 5:6. [0211] 112. The insulin
preparation according to embodiment 88, wherein the stabilizer is a
non-ionic detergent. [0212] 113. The insulin preparation according
to embodiment 112, wherein the detergent is polysorbate 20 (Tween
20) or polysorbate 80 (Tween 80). [0213] 114. The insulin
preparation according to embodiment 112, wherein the detergent is
polysorbate 20 (Tween 20). [0214] 115. The insulin preparation
according to embodiment 112, wherein the detergent is polysorbate
80 (Tween 80). [0215] 116. The insulin preparation according to any
of embodiments 112-115, comprising from about 5 to 100 ppm, from
about 10 to about 50 ppm or from about 10 to about 20 ppm of
polysorbate. [0216] 117. The insulin preparation according to
embodiment 88, further comprising a phenolic compound. [0217] 118.
The insulin preparation according to embodiment 117, wherein said
phenolic compound is present in the amount from about 0 to about 6
mg/ml or from about 0 to about 4 mg/ml. [0218] 119. The insulin
preparation according to embodiment 88, further comprising
m-cresol. [0219] 120. The insulin preparation according to
embodiment 119, wherein m-cresol is present in the amount from
about 0.5 to about 4.0 mg/ml or from about 0.6 to about 4.0 mg/ml.
[0220] 121. An insulin preparation according to any of the previous
embodiments, wherein the pH is neutral to weakly basic. [0221] 122.
An insulin preparation according to any of the previous
embodiments, wherein the pH is from about 7.0 to about 8.0. [0222]
123. An insulin preparation according to any of the previous
embodiments, wherein the pH is about 7.0. [0223] 124. An insulin
preparation according to any of the previous embodiments, wherein
the pH is about 7.1. [0224] 125. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 7.2.
[0225] 126. An insulin preparation according to any of the previous
embodiments, wherein the pH is about 7.3. [0226] 127. An insulin
preparation according to any of the previous embodiments, wherein
the pH is about 7.4. [0227] 128. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 7.5.
[0228] 129. An insulin preparation according to any of the previous
embodiments, wherein the pH is about 7.6. [0229] 130. An insulin
preparation according to any of the previous embodiments, wherein
the pH is about 7.7. [0230] 131. An insulin preparation according
to any of the previous embodiments, wherein the pH is about 7.8.
[0231] 132. An insulin preparation according to any of the previous
embodiments, wherein the pH is about 7.9. [0232] 133. An insulin
preparation according to any of the previous embodiments, wherein
the pH is about 8.0. [0233] 134. A method of reducing the blood
glucose level in mammals by administering to a patient in need of
such treatment a therapeutically active dose of an insulin
preparation according to any of the preceding embodiments. [0234]
135. A method for the treatment of diabetes mellitus in a subject
comprising administering to a subject an insulin preparation
according to any of the preceding embodiments. [0235] 136. A method
according to any of the preceding embodiments, for parenteral
administration. [0236] 137. An insulin preparation according to any
of the preceding embodiments, for use in the treatment or
prevention of hyperglycemia including stress induced hyperglycemia,
type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and
burns, operation wounds and other diseases or injuries where an
anabolic effect is needed in the treatment, myocardial infarction,
stroke, coronary heart disease and other cardiovascular disorders
and treatment of critically ill diabetic and non-diabetic
patients.
[0237] The invention is further illustrated by the following
examples which are not to be construed as limiting.
[0238] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law).
[0239] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way.
[0240] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0241] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability, and/or enforceability of such patent
documents.
[0242] This invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as
permitted by applicable law.
EXAMPLES
Example 1
Preparation of Pharmaceutical Preparations
[0243] The pharmaceutical preparations of the present invention may
be formulated as an aqueous solution. The aqueous medium is made
isotonic, for example, with sodium chloride or glycerol.
Furthermore, the aqueous medium may contain zinc ions, for example
added as zinc acetate or zinc chloride, buffers and preservatives.
Arginine may be added as Arg, HCl. The pH value of the preparation
is adjusted to the desired value and may be between about 3 to
about 8.5, between about 3 and about 5 or about 6.5 and about 7.5
depending on the isoelectric point, pI, of the insulin in
question.
TABLE-US-00001 TABLE 1 Composition of insulin preparations
according to this invention Insulin m- Phos- Arginine, Nicotin-
Glutamic aspart Zn Phenol cresol NaCl phate Tris Glycerol HCl amide
acid (mM) (mM) (mM) (mM) (mM) (mM) (mM) (% w/v) (mM) (mM) (mM) pH
A* 0.6 0.3 16 16 10 7 1.6 7.4 B 0.6 0.3 16 16 2 7 130 7.4 C 0.6 0.3
16 16 2 7 50 80 50 7.4 D 0.6 0.3 16 16 2 7 130 7.4 E 0.6 0.3 16 16
2 7 50 80 50 7.4 F 0.6 0.3 16 16 20 7 30 80 30 7.4 G 0.6 0.3 16 16
20 7 30 80 30 7.4 H 1.2 0.6 16 16 20 7 30 80 30 7.4 I 1.2 0.6 16 16
20 7 30 80 30 7.4 J 0.6 0.3 16 16 10 7 1.3 80 7.4 K 0.6 0.3 16 16
10 7 0.77 30 80 7.4 L 0.6 0.3 16 16 10 7 0.24 30 80 30 7.4 M 0.6
0.3 16 16 10 7 60 100 7.4 N 0.6 0.3 16 16 10 7 1.13 100 7.4
*Commercially available NovoRapid .RTM.
TABLE-US-00002 TABLE 2 Composition of further insulin preparations
according to this invention Prep- ara- [Insulin [phe- [Nicotin-
tion aspart] [Zn2+] nol] [Arg] [Gly] [Glu] [His] amide] nr. mM mM
mM mM mM mM mM mM 1 0.6 0.3 32 260 2 0.6 0.3 32 10 260 3 0.6 0.3 32
20 260 4 0.6 0.3 32 30 260 5 0.6 0.3 32 40 260 6 0.6 0.3 32 50 260
7 0.6 0.3 32 50 260 8 0.6 0.3 32 50 260 9 0.6 0.3 32 50 260
Example 2
Analysis of Insulin Chemical Stability
Size Exclusion Chromatography
[0244] Quantitative determination of high molecular weight protein
(HMWP) and monomer insulin aspart was performed on Waters insulin
(300.times.7.8mm, part nr wat 201549) with an eluent containing
2.5M acetic acid, 4 mM L-arginine and 20% (V/V) acetonitrile at a
flow rate of 1 ml/min. and 40.degree. C. Detection was performed
with a tuneable absorbance detector (Waters 486) at 276 nm.
Injection volume was 40 .mu.l and a 600 .mu.M human insulin
standard. HMWP and concentration of the preparations were measured
at each sampling point.
Reverse Phase Chromatography (UPLC)
[0245] Determination of the insulin aspart related impurities were
performed on a UPLC system using a BEH RP C8 2.1.times.100 mm
column, particle size of 1.7 .mu.m. Waters part no 186002878. with
a flow rate of 0.5 ml/min., at 40.degree. C. detection at 220 nm.
Elution was performed with a mobile phase consisting of the
following:
[0246] A. 10% (w/V) acetonititrile, 2.8% (w/w) sodium sulphate,
0.3% (w/w) o-phosphoric acid, pH 3.5.
[0247] B. 70% (w/V) acetonitrile. Gradient: 0-11 min isocratic with
73%/27% of A/B, 11-12 linear change to 52%/48% A/B, 13-15 min.
linear change to 73%/27% of A/B, 15-20 min. isocratic gradient at
73%/27% of A/B.
[0248] The amount of B28iso-aspartate, desamido and other related
impurities were determined as absorbance area measured in percent
of total absorbance area determined after elution of the
preservatives. The RP-UPLC method is equivalent to the analytical
method used for quality control of Novo Nordisk marketed insulin
aspart pharmaceuticals.
[0249] Addition of arginine reduces the amount of degradation
products formed, especially HMWP and des-amido forms, increasing
the concentration of arginine in the range 10 to 50 mM leads to
further reduction of degradation. The physical stability measured
as lag time in the ThT assay is reduced upon addition of arginine
and is increasingly reduced when the arginine concentration is
increased. The overall performance of 50 mM arginine is superior to
50 mM glycine, 50 mM glutamic acid, or 50 mM histidine regarding
reduction of the formation of degradation products, as is shown in
Table 3 below.
TABLE-US-00003 TABLE 3 Physical and chemical stability data for
insulin preparations 1-9 of Table 2 (Example 1). Chemical stability
Content of degradation product (%) measured as difference Physical
stability, between content after incubation for 2 weeks at
37.degree. C. and at 4.degree. C. Preparation lag time (min) in B28
des-amido Other related nr. ThT assay IsoAsp forms impurities HMWP
1 160 1.17 3.67 1.73 1.36 2 80 1.30 3.05 0.82 0.65 3 80 1.30 2.49
0.64 0.34 4 60 1.31 2.26 0.79 0.20 5 60 1.27 2.27 0.37 0.19 6 40
1.36 1.99 0.47 0.16 7 100 1.26 4.72 2.21 1.11 8 50 1.39 3.41 1.07
0.70 9 0 1.75 6.99 2.22 1.01
Example 3
Pharmacokinetic (PK)/Pharmacodynamic (PD) Studies in LYD Pig Model
and Plasma Analysis Assay
PK/PD Studies in LYD Pigs
[0250] The PK/PD studies were performed on domestic female pigs,
LYD cross-breed, weighing between 55 and 110 kg. The pigs were
catheterised into the jugular vein through an ear vein at least 2
days before start of the study. The last meal before the start of
the study was served to the animals approx. 18 hours prior to the
injection of the test preparation, and the animals had free access
to water at all time during the fasting period and the test
period.
[0251] At time 0 hours the test preparation was given subcutaneous
on the lateral side of the neck. A blood sample was drawn prior
dosing and at regular time intervals after dosing samples were
drawn from the catheter and sampled into 1.5 ml glass tubes
pre-coated with heparin. The blood samples were kept in ice water
until separation of plasma by centrifugation for 10 min. 3000 rpm
at 4.degree. C., which was done within the first 30 minutes. Plasma
samples were stored at 4.degree. C. for short time (2-3 hours) or
at -18.degree. C. for long term storage and were analysed for
glucose on YSI or Konelab 30i and for insulin Aspart concentration
by LOCI.
Luminescent Oxygen Channeling Immunoassay (LOCI) for Insulin Aspart
Quantification
[0252] The insulin Aspart LOCI is a monoclonal antibody-based
sandwich immunoassay and applies the proximity of two beads, the
europium-coated acceptor beads and the streptavidin coated
donor-beads. The acceptor beads were coated with a specific
antibody against human insulin and recognize insulin Aspart in
plasma samples. A second biotinylated antibody bind specific to
insulin Aspart and together with the streptavidin coated beads,
they make up the sandwich. Illumination of the
beads-aggregate-immunocomplex releases singlet oxygen from the
donor beads which channels into the acceptor beads and triggers
chemiluminescence. The chemiluminescence was measured and the
amount of light generated is proportional to the concentration of
insulin Aspart.
[0253] Compared to the marketed product NovoRapid.RTM., the initial
rate of plasma glucose lowering is faster for the preparations of
the present invention (FIGS. 3 and 4). Likewise, when compared to
NovoRapid.RTM., the initial absorption rate of the insulin
component of the preparations of the present invention, is markedly
faster (FIG. 5).
Example 4
General Introduction to ThT Fibrillation Assays for the Assessment
of Physical Stability of Protein Formulations
[0254] Low physical stability of a peptide may lead to amyloid
fibril formation, which is observed as well-ordered, thread-like
macromolecular structures in the sample eventually resulting in gel
formation. This has traditionally been measured by visual
inspection of the sample. However, that kind of measurement is very
subjective and depending on the observer. Therefore, the
application of a small molecule indicator probe is much more
advantageous. Thioflavin T (ThT) is such a probe and has a distinct
fluorescence signature when binding to fibrils [Naiki et al. (1989)
Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309,
274-284].
[0255] The time course for fibril formation can be described by a
sigmoidal curve (FIG. 6) with the following expression [Nielsen et
al. (2001) Biochemistry 40, 6036-6046]:
F = f i + m i t + f f + m f t 1 + - [ ( t - t 0 ) / .tau. ] Eq . (
1 ) ##EQU00001##
[0256] Here, F is the ThT fluorescence at the time t. The constant
t.sub.0 is the time needed to reach 50% of maximum fluorescence.
The two important parameters describing fibril formation are the
lag-time calculated by t.sub.0-Z.tau. and the apparent rate
constant k.sub.app=1/.tau..
[0257] Formation of a partially folded intermediate of the peptide
is suggested as a general initiating mechanism for fibrillation.
Few of those intermediates nucleate to form a template onto which
further intermediates may assembly and the fibrillation proceeds.
The lag-time corresponds to the interval in which the critical mass
of nucleus is built up and the apparent rate constant is the rate
with which the fibril itself is formed.
Sample Preparation
[0258] Samples were prepared freshly before each assay. Each sample
composition is described in each example. The pH of the sample was
adjusted to the desired value using appropriate amounts of
concentrated NaOH and HClO.sub.4or HCl. Thioflavin T was added to
the samples from a stock solution in H.sub.2O to a final
concentration of 1 .mu.M.
[0259] Sample aliquots of 200 .mu.l were placed in a 96 well
microtiter plate (Packard OptiPlate.TM.-96, white polystyrene).
Usually, four or eight replica of each sample (corresponding to one
test condition) were placed in one column of wells. The plate was
sealed with Scotch Pad (Qiagen).
Incubation and Fluorescence Measurement
[0260] Incubation at given temperature, shaking and measurement of
the ThT fluorescence emission were done in a Fluoroskan Ascent FL
fluorescence platereader or Varioskan platereader (Thermo
Labsystems). The temperature was adjusted to 37.degree. C. The
orbital shaking was adjusted to 960 rpm with an amplitude of 1 mm
in all the presented data. Fluorescence measurement was done using
excitation through a 444 nm filter and measurement of emission
through a 485 nm filter.
[0261] Each run was initiated by incubating the plate at the assay
temperature for 10 min. The plate was measured every 20 minutes for
a desired period of time. Between each measurement, the plate was
shaken and heated as described.
Data Handling
[0262] The measurement points were saved in Microsoft Excel format
for further processing and curve drawing and fitting was performed
using GraphPad Prism. The background emission from ThT in the
absence of fibrils was negligible. The data points are typically a
mean of four or eight samples and shown with standard deviation
error bars. Only data obtained in the same experiment (i.e. samples
on the same plate) are presented in the same graph ensuring a
relative measure of fibrillation between experiments.
[0263] The data set may be fitted to Eq. (1). However, since full
sigmodial curves are not always achieved during the measurement
time, lag times were here visually determined from the ThT
fluorescence curve as the time point at which the ThT fluorescence
is different than the background level.
Measurement of Initial and Final Concentrations
[0264] The peptide concentration in each of the tested formulations
were measured both before application in the ThT fibrillation assay
("Initial") and after completion of the ThT fibrillation ("After
ThT assay"). Concentrations were determined by reverse HPLC methods
using a pramlintide standard as a reference. Before measurement
after completion 150 .mu.l was collected from each of the replica
and transferred to an Eppendorf tube. These were centrifuged at
30000 G for 40 mins. The supernatants were filtered through a 0.22
.mu.m filter before application on the HPLC system.
* * * * *