Note: Descriptions are shown in the official language in which they were submitted.
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PREPARATION COMPRISING INSULIN, NICOTINAMIDE AND AN AMINO ACID
FIELD OF THE INVENTION
The present invention relates to pharmaceutical preparations comprising an
insulin com-
BACKGROUND OF THE INVENTION
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 ep-
idemic proportions.
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 of-
ten 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
In the treatment of diabetes mellitus, many varieties of pharmaceutical
preparations of
insulin have been suggested and used, such as regular insulin (such as
Actrapide), isophane
insulin (designated NPH), insulin zinc suspensions (such as Semilente , Lente
, and Ul-
International applications WO 91/09617 and WO/9610417 (Novo Nordisk NS)
disclose insu-
lin preparations containing nicotinamide or nicotinic acid or a salt thereof.
European applica-
tion EP1283051 allegedly discloses insulin preparation containing arginine,
where arginine is
used as buffering agent. EP1283051 allegedly discloses improvement of physical
stability of
Most often pharmaceutical preparations of insulins are administered by
subcutane-
ous 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,
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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 prepa-
rations 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.
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 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-
3.0mIcartridges. 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). Further-
more, also important is the physical stability of the insulin preparations,
since long term stor-
age may eventually lead to formation of insoluble fibrils, which are
biologically inactive and
potentially immunogenic.
SUMMARY OF THE INVENTION
The invention relates to insulin preparations with favourable absorption rate
and favourable
chemical and physical stability. The present invention relates to insulin
preparations compris-
ing human insulin and/or analogues thereof, nicotinamide or nicotinic acid
and/or salts there-
of and arginine.
In one embodiment, the present invention relates to an insulin preparation
comprising:
= an insulin compound,
= a nicotinic compound, and
= arginine.
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 compris-
ing administering to a subject or mammal an insulin preparation according to
the invention.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the development in percentage of total insulin content of
degradation prod-
ucts during 2 weeks of storage at 37 C of preparations according to the
present invention.
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The letter A refers to a NovoRapid reference and remaining letters correspond
to insulin as-
part preparations as described in Table 1 of Example 1. Compared to the
NovoRapid prep-
aration (preparation A), addition of nicotinamide (preparations B and D) leads
to an in-
creased 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.
Figure 2 shows the development in percentage of total insulin content of
degradation prod-
ucts during 2 weeks of storage at 37 C of preparations according to this
invention. The letter
A refers to a NovoRapid reference and remaining letters correspond to insulin
aspart prepa-
rations as described in Table 1 of Example 1. The combined addition of
nicotinamide, glu-
tamic acid and arginine, preparations F, G, H, and I, differing in buffer
system, phosphate or
tris buffer, and concentration of insulin and Zn, 0.6mM and 0.3mM or 1.2mM and
0.6mM, has
a degradation pattern similar to the NovoRapid preparation, preparation A.
Figure 3 shows the glucose concentration (mean +/- SEM, N=8) in plasma after
subcutane-
ous injection in pigs of a lnmol/kg dose at 0 minutes of preparations
according to this inven-
tion. The letter A refers to a NovoRapid reference and remaining letters
correspond to insu-
lin aspart preparations as described in Table 1 of Example 1. Compared to the
NovoRapid
preparation (preparation A) the initial rate of plasma glucose lowering is
faster for the prepa-
ration with addition of nicotinamide (preparation N) and even faster for a
combination of nico-
tinamide and arginine (preparation M).
Figure 4 shows the glucose concentration in plasma (mean +/- SEM, N=7) after
subcutane-
ous injection in pigs of a lnmol/kg dose at 0 minutes of preparations
according to this inven-
tion. The letter A refers to a NovoRapid reference and remaining letters
correspond to insu-
lin aspart preparations as described in Table 1 of Example 1. Compared to the
NovoRapid
preparation (preparation A), the initial rate of plasma glucose lowering is
faster for a prepara-
tion with a combination of nicotinamide, arginine and glutamic acid
(preparation L) and for a
preparation with a combination of nicotinamide and arginine (preparation K).
Figure 5 shows the insulin aspart concentration in plasma (mean +/- SEM, N=7)
after subcu-
taneous 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 reference and remaining letters
correspond to
insulin aspart preparations as described in Table 1 of Example 1. Compared to
the No-
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voRapid preparation, (preparation A) , the initial absorption rate of the
insulin component of
the preparations with nicotinamide (preparation J), the combination of
nicotinamide and ar-
ginine (preparation K), and the combination of nicotinamide, arginine and
glutamic acid
(preparation L) is markedly faster.
Figure 6 shows the glucose concentration in plasma (mean +/- SEM, N=8, each
pig dosed
twice) after subcutaneous injection in pigs of a lnmol/kg dose at 0 minutes of
preparations
according to this invention. The letter A refers to a NovoRapid reference and
number 11
corresponds to a insulin aspart preparation as described in Table 3 of Example
1. Compared
to the NovoRapid preparation (preparation A), the initial rate of plasma
glucose lowering is
faster for a preparation with a combination of nicotinamide and arginine
(preparation 11).
Figure 7 shows the insulin aspart concentration in plasma (mean +/- SEM, N=8,
each pig
dosed twice) after subcutaneous injection in pigs of a 1 nmol/kg dose at 0
minutes of prepa-
rations according to this invention. The letter A refers to a NovoRapid
reference and number
11 corresponds to a insulin aspart preparation as described in Table 3 of
Example 1. Com-
pared to the NovoRapid preparation (preparation A), the initial absorption
rate of the insulin
component of the preparations with nicotinamide and arginine (preparation 11)
is markedly
faster.
DESCRIPTION OF THE INVENTION
The absorption after subcutaneous injection of the insulin compound in the
insulin prepara-
tions 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 connec-
tion with a multiple injection regimen where insulin is given before each
meal. With faster on-
set 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 dimin-
ishes the risk of post-meal hypoglycaemia.
The insulin preparations of the present invention are rapid-acting insulin
prepara-
tions 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. These insulin preparations have a
rapid absorp-
tion 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.
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The insulin preparations of the present invention provide fast-acting insulin
prepara-
tions which are not only physically stable, but surprisingly also chemically
stable. 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
5 first phase insulin release, injection convenience and shutting down
hepatic glucose produc-
tion. 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 , 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 inven-
tion 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 inven-
tion 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.
The present invention provides an insulin preparation comprising an insulin
com-
pound according to the present invention which is present in a concentration
from about 0.1
mM to about 10.0mM, 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.
In one embodiment the metal ion is zinc, wherein zinc is added as zinc acetate
or
zinc chloride.
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.
In one embodiment, the insulin preparations according to the present invention
comprise an aqueous solution of B28Asp human insulin, nicotinamide and
arginine.
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 con-
tent of insulin compound may be higher.
In the present context the unit "IU" corresponds to 6 nmol.
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The term "insulin aspart" refers to the human insulin analogue B28Asp human
insu-
lin.
The term "onset" refers to the time from injection until the PK curve shifts
to an in-
crease.
The term "absorption rate" refers to the slope of the PK curve.
An "insulin compound" according to the invention is herein to be understood as
hu-
man insulin, an insulin analogue and/or any combination thereof.
The term "human insulin" as used herein means the human hormone whose struc-
ture and properties are well-known. Human insulin has two polypeptide chains
that are con-
nected 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 po-
sition 20 of the A-chain and the cysteine in position 19 of the B-chain.
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 pep-
tide from the A and B chains.
By "insulin analogue" as used herein is meant a polypeptide derived from the
pri-
mary 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 res-
idue occurring in the naturally occurring insulin and/or by adding at least
one amino acid res-
idue. The added and/or substituted amino acid residues can either be codable
amino acid
residues or other naturally occurring amino acid residues.
In one embodiment an insulin analogue comprises less than 8 modifications
(substi-
tutions, deletions, additions and any combination thereof) relative to the
parent insulin, alter-
natively less than 7 modifications relative to the parent insulin,
alternatively less than 6 mod i-
fications 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, alterna-
tively less than 3 modifications relative to the parent insulin, alternatively
less than 2 modifi-
cations relative to the parent insulin.
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"
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or "B(1-29)" is meant a natural insulin B chain or analogue thereof lacking
the B30 amino ac-
id residue, and by B28Asp human insulin is meant human insulin wherein the
amino acid resi-
due in position 28 of the B chain has been substituted with Asp.
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 ana-
logues wherein the B1 amino acid in human insulin has been deleted (des(B1)
human insu-
lin), des(B28-630) 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 ex-
amples 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 addi-
tional mutations are further examples of insulin analogues. Insulin analogues
of human insu-
lin 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.
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
hu-
man insulin; HisA14,HisB25 human insulin; GluA14,HisB25,desB30 human insulin;
HisA14,
HisB25,desB30 human insulin; GluA14,HisB25,desB27,desB28,desB29,desB30 human
insu-
lin; 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.
The term "nicotinic compound" includes nicotinamide, nicotinic acid, niacin,
niacin
amide and vitamin B3 and/or salts thereof and/or any combination thereof.
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According to the present invention, the concentration of the nicotinic
compound
and/or salts thereof is in the range from about 1mM to about 300mM or from
about 5mM to
about 200mM.
The term "arginine" or "Arg" includes the amino acid arginine and/or a salt
thereof,
e.g. arginine hydrochloride or arginine glutamate.
In one embodiment, the insulin preparation comprises 1 to 100mM of arginine.
In one embodiment, the insulin preparation comprises 1 to 20mM of arginine.
In one embodiment, the insulin preparation comprises 20 to 90mM of arginine.
In one embodiment, the insulin preparation comprises 30 to 85mM of arginine.
The term "glutamic acid" or "Glu" includes the aminoacid glutamic acid and/or
a salt
thereof.
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 aminoacid,
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.
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 (tris),
bicine, tricine,
malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic
acid, ethylendiamine or
mixtures thereof. Each one of these specific buffers constitutes an
alternative embodiment of
the invention.
The insulin preparation of the present invention may further comprise other
ingredi-
ents common to insulin preparations, for example zinc complexing agents such
as citrate,
and phosphate buffers.
Glycerol and/or mannitol and/or sodium chloride may be present in an amount
cor-
responding to a concentration of 0 to 250mM, 0 to 200mM or 0 to 100mM.
Stabilizers, surfactants and preservatives may also be present in the insulin
preparations of this invention.
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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, 19th edition, 1995.
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
methylpara-
ben.
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, 19th edition, 1995.
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, 19th edition, 1995.
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 wa-
ter (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 prepare-
tions is well-known to the skilled person. For convenience reference is made
to Remington:
The Science and Practice of Pharmacy, 19th edition, 1995.
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 0.1mM or above, and
wherein said
preparation has a pH from about 3.0 to about 8.5 at room temperature (-25 C).
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The present invention also relates to methods for producing the insulin
preparations
of the invention.
In one embodiment, the method for making insulin preparations of the invention
comprises:
5 a) preparing a solution by dissolving the insulin compound or a
mixture of insulin
compounds in water or buffer;
b) preparing a solution by dissolving a divalent metal ion in water or buffer;
c) preparing a solution by dissolving a preservative in water or buffer;
d) preparing a solution by dissolving a tonicity agent in water or buffer;
10 e) preparing a solution by dissolving a surfactant and/or a stabilizer
in water or buff-
er;
f) mixing solution a) and one or more of solutions b), c), d), and e);
Finally adjusting the pH of the mixture in f) to the desired pH followed by a
sterile fil-
tration.
In one embodiment, the method for making insulin preparations of the invention
comprises:
a) preparing separate stock solutions of preservative agents, tonicity agent,
metal
ion salt, arginine or arginine salt and nicotinic compound;
b) preparing a solution by adding stock solution(s) of preservative or a
mixture of
preservatives to water or buffer;
c) adding stock solution of tonicity agent to solution b);
d) preparing a solution by dissolving the insulin compound or a mixture of
insulin
compounds in water or buffer; adding HCI to acidify this solution;
e) adding stock solution of metal ion salt to d);
f) adding e) to c) and stirring;
g) adding stock solution of arginine or arginine salt to f) and stirring;
h) adding stock solution of nicotinic compound to g) and adjusting pH with
Na0H/HCI to desired pH.
In one embodiment, the method for making insulin preparations of the invention
comprises:
a) preparing separate stock solutions of preservative agents, tonicity agent,
metal
ion salt, and a mixed stock solution of Arginine or Arginine salt and
nicotinic compound
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b) preparing a solution by adding stock solution(s) of preservative or a
mixture of
preservatives to water or buffer;
c) adding stock solution of tonicity agent to solution b);
d) preparing a solution by dissolving the insulin compound or a mixture of
insulin
compounds in water or buffer; adding HCI to acidify this solution;
e) adding stock solution of metal ion salt to d);
f) adding e) to c) and stirring;
g) adding mixed stock solution of Arginine or Arginine salt and nicotinic
compound to
f) and stirring;
h) adjusting pH of g) with Na0H/HCI to desired pH.
The insulin preparations of the present invention can be used in the treatment
of di-
abetes by parenteral administration. It is recommended that the dosage of the
insulin prepa-
rations of this invention which is to be administered to the patient be
selected by a physician.
Parenteral administration may be performed by subcutaneous, intramuscular, in-
traperitoneal 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.
Insulin preparations according to the present invention may be administered to
a pa-
tient 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.
In one embodiment of the invention the insulin preparation is an aqueous
prepara-
tion, i.e. preparation comprising water. Such preparation is typically a
solution or a suspen-
sion. In a further embodiment of the invention the insulin preparation is an
aqueous solution.
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 com-
prising at least 50 %w/w water.
Aqueous suspensions may contain the active compounds in admixture with
excipients
suitable for the manufacture of aqueous suspensions.
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In one embodiment, the insulin preparations of this invention are well-suited
for ap-
plication in pen-like devices used for insulin therapy by injection.
In one embodiment the insulin preparations of the present invention can be
used in
pumps for insulin administration.
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 inspec-
tion 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 tempera-
tures 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 tur-
bidity in daylight corresponds to visual score 3). A preparation is classified
physically unsta-
ble with respect to protein aggregation, when it shows visual turbidity in
daylight. Alterna-
tively, 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 essen-
tially non-fluorescent at the wavelengths.
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 prod-
ucts 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 hydro-
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13
lysed to form a free carboxylic acid or asparaginyl residues to form an !soAsp
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, Ahern.
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 deg-
radation 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
immu-
nogenic and not biologically active, low levels of HMWP are advantageous.
The term "stabilized preparation" refers to a preparation with increased
physical sta-
bility, 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.
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 prevent-
ing cells from absorbing glucose. As a result, glucose builds up in the blood.
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.
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.
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.
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, pre-
vention or alleviation of the disease. The purpose of treatment is to combat
the disease, con-
dition 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 complica-
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14
tions associated with the disease, condition or disorder, and prevention of
the disease, condi-
tion or disorder.
In its broadest sense, the term a "critically ill patient", as used herein
refers to a pa-
tient who has sustained or are at risk of sustaining acutely life-threatening
single or multiple
organ system failure due to disease or injury, a patient who is being operated
and where
complications supervene, and a patient who has been operated in a vital organ
within the last
week or has been subject to major surgery within the last week. In a more
restricted sense,
the term a "critically ill patient", as used herein refers to a patient who
has sustained or are at
risk of sustaining acutely life-threatening single or multiple organ system
failure due to dis-
ease or injury, or a patient who is being operated and where complications
supervene. In an
even more restricted sense, the term a "critically ill patient", as used
herein refers to a patient
who has sustained or are at risk of sustaining acutely life-threatening single
or multiple organ
system failure due to disease or injury. Similarly, these definitions apply to
similar expres-
sions such as "critical illness in a patient" and a "patient is critically
ill". Examples of a criti-
cally ill patient is a patient in need of cardiac surgery, cerebral surgery,
thoracic surgery, ab-
dominal surgery, vascular surgery, or transplantation, or a patient suffering
from neurological
diseases, cerebral trauma, respiratory insufficiency, abdominal peritonitis,
multiple trauma or
severe burns, or critical illness polyneuropathy.
The term "anabolism" as used herein, means the set of metabolic pathways that
construct molecules from smaller units. These reactions require energy. One
way of
categorizing metabolic processes, whether at the cellular, organ or organism
level is as
'anabolic' or as 'catabolic', which is the opposite. Anabolism is powered by
catabolism, where
large molecules are broken down into smaller parts and then used up in
respiration. Many
anabolic processes are powered by adenosine triphosphate (ATP). Anabolic
processes tend
toward "building up" organs and tissues. These processes produce growth and
differentiation
of cells and increase in body size, a process that involves synthesis of
complex molecules.
Examples of anabolic processes include the growth and mineralization of bone
and
increases in muscle mass. Endocrinologists have traditionally classified
hormones as
anabolic or catabolic, depending on which part of metabolism they stimulate.
The balance
between anabolism and catabolism is also regulated by circadian rhythms, with
processes
such as glucose metabolism fluctuating to match an animal's normal periods of
activity
throughout the day. Some examples of the "anabolic effects" of these hormones
are
increased protein synthesis from amino acids, increased appetite, increased
bone
remodeling and growth, and stimulation of bone marrow, which increases the
production of
red blood cells. Through a number of mechanisms anabolic hormones stimulate
the
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formation of muscle cells and hence cause an increase in the size of skeletal
muscles,
leading to increased strength.
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.
5 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 includ-
ing stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance,
type 1 diabe-
tes, 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 car-
10 diovascular disorders is provided.
In a further embodiment of the present invention, a method for the treatment
or pre-
vention of hyperglycemia including stress induced hyperglycemia, type 2
diabetes, impaired
glucose tolerance, type 1 diabetes, and burns, operation wounds and other
diseases or inju-
ries where an anabolic effect is needed in the treatment, myocardial
infarction, coronary
15 heart disease and other cardiovascular disorders, stroke, the method
comprising administer-
ing to a patient in need of such treatment an effective amount for such
treatment of an insulin
preparation according to the invention, is provided.
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 associ-
ated with diabetes and agents for the treatment and/or prevention of
complications and dis-
orders resulting from or associated with obesity.
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.
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 syn-
thesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or
other well estab-
lished 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 ca-
pable of expressing the insulin analogue in a suitable nutrient medium under
conditions per-
mitting 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
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16
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.
Several methods may be used for the production of human insulin and human insu-
lin analogues. For example three major methods which are used in the
production of insulin
in microorganisms are disclosed in W02008034881. 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 7th American Peptide Chemistry Symposium (Rich &
Gross,
eds.), Pierce Chemical Co., Rockford, III. 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 meth-
od 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.55,962,267, WO
95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.
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. US 6500645. The insulin analogue is either expressed directly or as a
precursor mole-
cule 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 exten-
sion 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 US 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 ana-
logs with C-terminal extensions on the B-chain that are removed by
carboxypetidase are dis-
closed 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 exten-
sion 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.
The present invention also contemplates the following non-limiting list of
embodi-
ments, which are further described elsewhere herein:
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17
1. An insulin preparation comprising:
= an insulin compound,
= a nicotinic compound, and
= arginine.
2. The insulin preparation according to embodiment 1, wherein the insulin
compound is hu-
man insulin or an insulin analog.
3. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is B28Asp human insulin, B3Ly5B29Glu human insulin or
B28LysB29Pro
human insulin.
4. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is B28Asp human insulin.
5. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is B3Ly5B29Glu human insulin or B28LysB29Pro human insulin.
6. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is B28LysB29Pro human insulin.
7. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is B3Ly5B29Glu human insulin.
8. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in a range selected from the following: 0.1-10.0mM;
0.1-
3.0mM; 0.1-2.5mM; 0.1-2.0mM; 0.1-1.5mM; 0.2-2.5mM; 0.2-2.0mM; 0.2-1.5mM; 0.3-
3.0mM; 0.3-2.5mM; 0.3-2.0mM; 0.3-1.5mM; 0.5-1.3mM and 0.6-1.2mM.
9. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.1mM to about 10.0mM.
10. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.1mM to about 3.0mM.
11. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.1mM to about 2.5mM.
12. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.1mM to about 2.0mM.
13. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.1mM to about 1.5mM.
14. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.2mM to about 2.5mM.
15. The insulin preparation according to any of the preceding embodiments,
wherein the in-
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18
sulin compound is present in the amount from about 0.2mM to about 2.0mM.
16. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.2mM to about 1.5mM.
17. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.3mM to about 3.0mM.
18. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.3mM to about 2.5mM.
19. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.3mM to about 2.0mM.
20. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.3mM to about 1.5mM.
21. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.5mM to about 1.3mM.
22. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.3mM to about 1.2mM.
23. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount from about 0.6mM to about 1.2mM.
24. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount of about 0.6 or about 1.2mM.
25. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount of about 0.3mM.
26. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount of about 0.6mM.
27. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount of about 0.9mM.
28. The insulin preparation according to any of the preceding embodiments,
wherein the in-
sulin compound is present in the amount of about 1.2mM.
29. The insulin preparation according to any of the preceding embodiments,
wherein the nic-
otinic compound is selected from the group consisting of nicotinamide,
nicotinic acid, nia-
cm, niacin amide and vitamin B3 and/or salts thereof and/or any combination
thereof.
30. The insulin preparation according to any of the preceding embodiments,
wherein the nic-
otinic compound is selected from nicotinamide and nicotinic acid and/or salts
thereof
and/or any combination thereof.
31. The insulin preparation according to any of the preceding embodiments,
wherein the nic-
otinic compound is nicotinamide and/or salts thereof.
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32. The insulin preparation according to any of the preceding embodiments,
wherein the nic-
otinic compound is present in a range selected from the following: 1-300mM; 5-
200mM;
40-120mM, 70-140mM or 80-130mM.
33. The insulin preparation according to any of the preceding embodiments,
comprising from
about 1mM to about 300mM of the nicotinic compound.
34. The insulin preparation according to any of the preceding embodiments,
comprising from
about 8mM to about 260mM of the nicotinic compound.
35. The insulin preparation according to any of the preceding embodiments,
comprising from
about 50mM to about 250mM of the nicotinic compound.
36. The insulin preparation according to any of the preceding embodiments,
comprising from
about 80mM to about 250mM of the nicotinic compound.
37. The insulin preparation according to any of the preceding embodiments,
comprising from
about 80mM to about 180mM of the nicotinic compound.
38. The insulin preparation according to any of the preceding embodiments,
comprising from
about 5mM to about 200mM of the nicotinic compound.
39. The insulin preparation according to any of the preceding embodiments,
comprising from
about 1mM to about 150mM of the nicotinic compound.
40. The insulin preparation according to any of the preceding embodiments,
comprising from
about 5mM to about 20mM of the nicotinic compound.
41. The insulin preparation according to any of the preceding embodiments,
comprising from
about 20mM to about 120mM of the nicotinic compound.
42. The insulin preparation according to any of the preceding embodiments,
comprising from
about 40mM to about 120mM of the nicotinic compound.
43. The insulin preparation according to any of the preceding embodiments,
comprising from
about 20mM to about 40mM of the nicotinic compound.
44. The insulin preparation according to any of the preceding embodiments,
comprising from
about 60mM to about 80mM of the nicotinic compound.
45. The insulin preparation according to any of the preceding embodiments,
comprising from
about 70mM to about 140mM of the nicotinic compound.
46. The insulin preparation according to any of the preceding embodiments,
comprising from
about 80mM to about 130mM of the nicotinic compound.
47. The insulin preparation according to any of the preceding embodiments,
comprising
about 8mM, 30mM, 100mM or 130mM of the nicotinic compound.
48. The insulin preparation according to any of the preceding embodiments,
comprising
about 8mM of the nicotinic compound.
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49. The insulin preparation according to any of the preceding embodiments,
comprising
about 30mM, 100mM or 130mM of the nicotinic compound.
50. The insulin preparation according to any of the preceding embodiments,
comprising
about 30mM of the nicotinic compound.
5 51. The insulin preparation according to any of the preceding
embodiments, comprising
about 80mM of the nicotinic compound.
52. The insulin preparation according to any of the preceding embodiments,
comprising
about 100mM of the nicotinic compound.
53. The insulin preparation according to any of the preceding embodiments,
comprising
10 about 120mM of the nicotinic compound.
54. The insulin preparation according to any of the preceding embodiments,
comprising
about 130mM of the nicotinic compound.
55. The insulin preparation according to any of the preceding embodiments,
comprising
about 150mM of the nicotinic compound.
15 56. The insulin preparation according to any of the preceding
embodiments, comprising
about 155mM of the nicotinic compound.
57. The insulin preparation according to any of the preceding embodiments,
comprising
about 180mM of the nicotinic compound.
58. The insulin preparation according to any of the preceding embodiments,
comprising
20 about 230mM of the nicotinic compound.
59. The insulin preparation according to any of the preceding embodiments,
comprising the
following ranges of arginine compound: 1-100mM, 5-120mM, 8-85mM, 20-90mM, 30-
90mM, 30-85mM, 30-60mM or 10-40mM.
60. The insulin preparation according to any of the preceding embodiments,
comprising the
following ranges of arginine compound: 1-120mM, 8-85mM or 1-40mM.
61. The insulin preparation according to any of the preceding embodiments,
comprising from
about 1mM to about 120mM of arginine.
62. The insulin preparation according to any of the preceding embodiments,
comprising from
about 1mM to about 100mM of arginine.
63. The insulin preparation according to any of the preceding embodiments,
comprising from
about 5mM to about 120mM of arginine.
64. The insulin preparation according to any of the preceding embodiments,
comprising from
about 20mM to about 90mM of arginine.
65. The insulin preparation according to any of the preceding embodiments,
comprising from
about 30mM to about 85mM of arginine.
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66. The insulin preparation according to any of the preceding embodiments,
comprising from
about 8mM to about 85mM of arginine.
67. The insulin preparation according to any of the preceding embodiments,
comprising from
about 30mM to about 60mM of arginine.
68. The insulin preparation according to any of the preceding embodiments,
comprising from
about 10mM to about 40mM of arginine.
69. The insulin preparation according to any of the preceding embodiments,
comprising from
about 10mM to about 30mM of arginine.
70. The insulin preparation according to any of the preceding embodiments,
comprising from
about 10mM to about 60mM of arginine.
71. The insulin preparation according to any of the preceding embodiments,
comprising from
about 1mM to about 40mM of arginine.
72. The insulin preparation according to any of the preceding embodiments,
wherein arginine
is present in a range selected from the following: 1mM, 2mM, 3mM, 4mM, 5mM,
6mM,
7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM, 45mM, 50mM,
55mM or 60mM.
73. The insulin preparation according to any of the preceding embodiments,
comprising
about 1mM of arginine.
74. The insulin preparation according to any of the preceding embodiments,
comprising
about 2mM of arginine.
75. The insulin preparation according to any of the preceding embodiments,
comprising
about 3mM of arginine.
76. The insulin preparation according to any of the preceding embodiments,
comprising
about 4mM of arginine.
77. The insulin preparation according to any of the preceding embodiments,
comprising
about 5mM of arginine.
78. The insulin preparation according to any of the preceding embodiments,
comprising
about 6mM of arginine.
79. The insulin preparation according to any of the preceding embodiments,
comprising
about 7mM of arginine.
80. The insulin preparation according to any of the preceding embodiments,
comprising
about 8mM of arginine.
81. The insulin preparation according to any of the preceding embodiments,
comprising
about 9mM of arginine.
82. The insulin preparation according to any of the preceding embodiments,
comprising
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about 10mM of arginine.
83. The insulin preparation according to any of the preceding embodiments,
comprising
about 11mM of arginine.
84. The insulin preparation according to any of the preceding embodiments,
comprising
about 12mM of arginine.
85. The insulin preparation according to any of the preceding embodiments,
comprising
about 13mM of arginine.
86. The insulin preparation according to any of the preceding embodiments,
comprising
about 14mM of arginine.
87. The insulin preparation according to any of the preceding embodiments,
comprising
about 15mM of arginine.
88. The insulin preparation according to any of the preceding embodiments,
comprising
about 17mM of arginine.
89. The insulin preparation according to any of the preceding embodiments,
comprising
about 20mM of arginine.
90. The insulin preparation according to any of the preceding embodiments,
comprising
about 22mM of arginine.
91. The insulin preparation according to any of the preceding embodiments,
comprising
about 25mM of arginine.
92. The insulin preparation according to any of the preceding embodiments,
comprising
about 30mM of arginine.
93. The insulin preparation according to any of the preceding embodiments,
comprising
about 35mM of arginine.
94. The insulin preparation according to any of the preceding embodiments,
comprising
about 40mM of arginine.
95. The insulin preparation according to any of the preceding embodiments,
comprising
about 45mM of arginine.
96. The insulin preparation according to any of the preceding embodiments,
comprising
about 50mM of arginine.
97. The insulin preparation according to any of the preceding embodiments,
comprising
about 55mM of arginine.
98. The insulin preparation according to any of the preceding embodiments,
comprising
about 60mM of arginine.
99. The insulin preparation according to any of the preceding embodiments,
which further
comprises a buffer(s).
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23
100. The insulin preparation according to embodiment 99, wherein said
buffer is a phos-
phate buffer.
101. The insulin preparation according to embodiment 100, comprising from
about
lmg/mL to about 20mg/mL of phosphate buffer.
102. The insulin preparation according to embodiment 100, comprising from
about
lmg/mL to about 15mg/mL of phosphate buffer.
103. The insulin preparation according to embodiment 100, comprising from
about
1mg/mL to about 10mg/mL of phosphate buffer.
104. The insulin preparation according to embodiment 100, comprising about
3mg/mL of
phosphate buffer.
105. The insulin preparation according to embodiment 99, wherein said
buffer is Tris.
106. The insulin preparation according to embodiment 105, comprising from
about 2mM
to about 50mM of Tris.
107. The insulin preparation according to embodiment 105, comprising from
about 10mM
to about 40mM of Tris.
108. The insulin preparation according to embodiment 105, comprising from
about 20mM
to about 30mM of Tris.
109. The insulin preparation according to embodiment 105, comprising about
10mM,
20mM, 30mM or 40mM of Tris.
110. The insulin preparation according to embodiment 105, comprising about
7mM of
Tris.
111. The insulin preparation according to embodiment 105, comprising about
10mM of
Tris.
112. The insulin preparation according to embodiment 105, comprising about
20mM of
Tris.
113. The insulin preparation according to embodiment 105, comprising about
30mM of
Tris.
114. The insulin preparation according to embodiment 105, comprising about
40mM of
Tris.
115. The insulin preparation according to any of the preceding embodiments,
which fur-
ther comprises a metal ion.
116. The insulin preparation according to embodiment 115, wherein the metal
ion is zinc.
117. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is from about 0:6 to about 6:6.
118. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
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24
ratio is from about 0:6 to about 5:6.
119. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is from about 1:6 to about 6:6.
120. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is from about 2:6 to about 6:6.
121. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is from about 2:6 to about 5:6.
122. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is from about 2.5:6 to about 4.5:6.
123. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is from about 3:6 to about 4:6.
124. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 2:6.
125. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 2.5:6.
126. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3:6.
127. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.1:6.
128. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.2:6.
129. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.3:6.
130. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.4:6.
131. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.5:6.
132. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.6:6.
133. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.7:6.
134. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.8:6.
135. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 3.9:6.
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136. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 4:6.
137. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 4.5:6.
5 138. The insulin preparation according to embodiment 116, wherein the
zinc:insulin molar
ratio is about 5:6.
139. The insulin preparation according to any one of the preceding
embodiments, which
further comprises a stabilizer(s).
140. The insulin preparation according to embodiment 139, wherein the
stabilizer is a
10 non-ionic detergent.
141. The insulin preparation according to embodiment 140, wherein the
detergent is pol-
ysorbate 20 (Tween 20) or polysorbate 80 (Tween 80).
142. The insulin preparation according to embodiment 140, wherein the
detergent is pol-
ysorbate 20 (Tween 20).
15 143. The insulin preparation according to embodiment 140, wherein the
detergent is pol-
ysorbate 80 (Tween 80).
144. The insulin preparation according to embodiment 140, comprising from
about 5 to
100ppm, from about 10 to about 50ppm or from about 10 to about 20ppm of
polysorbate.
145. The insulin preparation according to any one of the preceding
embodiments, which
20 further comprises a preservative agent(s).
146. The insulin preparation according to embodiment 145, wherein the
preservative is a
phenolic compound.
147. The insulin preparation according to embodiment 146, wherein said
phenolic com-
pound is present in the amount from about 0 to about 6mg/m1 or from about 0 to
about
25 4mg/ml.
148. The insulin preparation according to embodiment 146, wherein said
phenolic com-
pound is present in the amount of from about 5 to about 100mM.
149. The insulin preparation according to embodiment 146, wherein said
phenolic com-
pound is present in the amount of from about 5 to about 50mM.
150. The insulin preparation according to embodiment 146, wherein said
phenolic com-
pound is present in the amount of from about 5 to about 30mM.
151. The insulin preparation according to embodiment 146, wherein said
phenolic com-
pound is present in the amount of about 16mM.
152. The insulin preparation according to embodiment 146, wherein said
phenolic com-
pound is present in the amount of about 14.4mM.
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153. The insulin preparation according to embodiment 145, wherein said
preservative is
m-cresol.
154. The insulin preparation according to embodiment 153, wherein m-cresol
is present
in the amount from about 0.5 to about 4.0mg/m1 or from about 0.6 to about
4.0mg/ml.
155. The insulin preparation according to embodiment 153, wherein m-cresol
is present
in the amount of from about 5 to about 100mM.
156. The insulin preparation according to embodiment 153, wherein m-cresol
is present
in the amount of from about 5 to about 50mM.
157. The insulin preparation according to embodiment 153, wherein m-cresol
is present
in the amount of from about 5 to about 30mM.
158. The insulin preparation according to embodiment 153, wherein m-cresol
is present
in the amount of about 16mM.
159. The insulin preparation according to embodiment 153, wherein m-cresol
is present
in the amount of about 14.4mM.
160. The insulin preparation according to any of the preceding embodiments,
further
comprising glycerol in the amount from about 0.5 to about 2.5%.
161. The insulin preparation according to any of the preceding embodiments,
further
comprising glycerol in the amount from about 0.7 to about 2.0%.
162. The insulin preparation according to any of the preceding embodiments,
further
comprising glycerol in the amount from about 1.0 to about 1.5%.
163. The insulin preparation according to any of the preceding embodiments,
further
comprising glycerol in the amount of about 1.25%.
164. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is neutral to weakly basic.
165. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is from about 6.8 to about 8Ø
166. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 6.8.
167. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 6.9.
168. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7Ø
169. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.1.
170. The insulin preparation according to any of the preceding embodiments,
wherein the
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pH is about 7.2.
171. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.3.
172. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.4.
173. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.5.
174. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.6.
175. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.7.
176. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.8.
177. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 7.9.
178. The insulin preparation according to any of the preceding embodiments,
wherein the
pH is about 8Ø
179. A method of reducing the blood glucose level in mammals by
administering to a pa-
tient in need of such treatment a therapeutically active dose of an insulin
preparation ac-
cording to any of the preceding embodiments.
180. A method for the treatment of diabetes mellitus in a subject
comprising administer-
ing to a subject an insulin preparation according to any of embodiments 1-178.
181. A method according to any of embodiments 179-180, for parenteral
administration.
182. An insulin preparation according to any of embodiments 1-178, for use
in the treat-
ment or prevention of hyperglycemia including stress induced hyperglycemia,
type 2 dia-
betes, 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 treat-
ment of critically ill diabetic and non-diabetic patients.
183. The insulin preparation according to embodiment 182, for use in the
treatment or
prevention of hyperglycemia including stress induced hyperglycemia, type 2
diabetes,
impaired glucose tolerance, type 1 diabetes, and burns and operation wounds,
myocar-
dial infarction, stroke, coronary heart disease and other cardiovascular
disorders.
184. The insulin preparation according to embodiment 182-183, for use in
the treatment
of hyperglycemia type 2 diabetes and type 1 diabetes.
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185. An insulin preparation comprising:
= an insulin compound,
=a nicotinic compound
=arginine, and
=a buffer.
186. The insulin preparation according to embodiment 185, wherein the
insulin com-
pound is human insulin or an insulin analog.
187. The insulin preparation according to any one of embodiments 185 or
186, wherein
the insulin compound is selected from the group consisting of B28Asp human
insulin,
B3Ly5B29Glu human insulin and B28LysB29Pro.
188. The insulin preparation according to embodiment 186, wherein the
insulin com-
pound is B28Asp human insulin.
189. The insulin preparation according to any one of embodiments 185-188,
wherein the
insulin compound is present in the amount from about 0.2mM to about 2.0mM.
190. The insulin preparation according to any one of embodiments 185-189,
wherein the
insulin compound is present in the amount from about 0.6mM to about 1.2mM.
191. The insulin preparation according to any one of embodiments 185-190,
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.
192. The insulin preparation according to any one of embodiments 185-191,
wherein the
nicotinic compound is nicotinamide.
193. The insulin preparation according to embodiment 192, comprising from
about 1mM
to about 250mM of the nicotinic compound.
194. The insulin preparation according to embodiment 192, comprising from
about 1mM
to about 250mM of the nicotinic compound.
195. The insulin preparation according to embodiment 192, comprising from
about 80mM
to about 230mM of the nicotinic compound.
196. The insulin preparation according to any one of embodiments 185-195,
comprising
from about 10mM to about 60mM of arginine.
197. The insulin preparation according to any one of embodiments 185-196,
comprising
from about 10mM to about 30mM of arginine.
198. The insulin preparation according to any one of embodiments 185-197,
comprising
about 20mM arginine.
199. The insulin preparation according to any one of embodiments 185-198,
wherein said
buffer is a phosphate buffer.
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200. The insulin preparation according to any one of embodiments 185-199,
wherein said
buffer is Tris.
201. The insulin preparation according to any one of embodiments 185-200,
which may
further comprises preservative agent(s), isotonicity agent(s) and/or
stabilizer(s).
202. The insulin preparation according to any one of embodiments 185-201,
which further
comprises a metal ion.
203. The insulin preparation according to embodiment 202, wherein the metal
ion is zinc.
204. The insulin preparation according to embodiment 203, wherein the
zinc:insulin molar
ratio is from about 0:6 to about 3.5:6.
205. The insulin preparation according to embodiment 203, wherein the
zinc:insulin molar
ratio is from about 2:6 to about 3:6.
206. The insulin preparation according to any one of embodiments 185-205,
wherein the
preparation has a pH of less than 7.4.
207. The insulin preparation according to any one of embodiments 185-205,
wherein the
preparation has a pH of about 7.4.
208. The insulin preparation according to any one of embodiments 185-205,
wherein the
preparation has a pH of about 7.1
209. An insulin preparation comprising: B28Asp human insulin; nicotinamide;
zinc; argin-
ine; and a phosphate buffer.
210. The insulin preparation of embodiment 209, wherein the B28Asp human
insulin is
present in a concentration ranging from about 0.6 mM to about 1.2 mM, and
wherein the
nicotinamide is present at a concentration ranging from about 80 mM to about
260 mM,
and wherein the arginine is present in a concentration ranging from about 10
mM to
about 40 mM, and wherein less than about 4 zinc ions are present per six
B28Asp human
insulin molecules, and wherein the preparation has a pH of about 7.4 or less.
211. An insulin preparation consisting essentially of:
a. B28Asp human insulin, wherein the B28Asp human insulin is present in a
concentration ranging from about 0.6 mM to about 1.2 mM;
b. Nicotinamide, wherein the nicotinamide is present in a concentration
ranging
from about 80 mM to abour 260 mM;
c. Zinc, wherein less than about 4 zinc ions are present per six B28Asp human
insulin molecules;
d. Arginine, wherein the the arginine is present in a concentration ranging
from
about 10 mM to about 30 mM; and
e. a phosphate buffer;
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wherein the preparation has a pH of about 7.1.
212. A method of reducing the blood glucose level in mammals by
administering to a
mammal in need of such treatment a therapeutically active dose of an insulin
preparation
according to any one of the preceding embodiments.
5 213. A method for the treatment of diabetes mellitus in a subject
comprising administer-
ing to a subject an insulin preparation according to any one of embodiments 1-
211.
214. An insulin preparation according to any one of embodiments 1-211, 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
10 other diseases or injuries where an anabolic effect is needed in the
treatment, myocardial
infarction, stroke, coronary heart disease and other cardiovascular disorders
and treat-
ment of critically ill diabetic and non-diabetic patients.
215. An insulin preparation according to any one of embodiments 1-211, for
use in the
treatment of hyperglycemia type 2 diabetes and type 1 diabetes.
Further embodiments of the invention relate to the following:
216. An insulin preparation comprising:
= an insulin compound,
= a nicotinic compound, and
= arginine.
217. The insulin preparation according to embodiment 216, wherein the
insulin com-
pound is human insulin or an insulin analog.
218. The insulin preparation according to embodiments 216-217, wherein the
insulin
compound is B28Asp human insulin.
219. The insulin preparation according to any one of embodiments 216-218,
wherein the
insulin compound is B28LysB29Pro human insulin.
220. The insulin preparation according to any one of embodiments 216-219,
wherein the
insulin compound is B3Ly5B29Glu human insulin.
221. The insulin preparation according to any one of embodiments 216-220,
wherein the
insulin compound is present in the amount from about 0.2mM to about 2.0mM.
222. The insulin preparation according to any one of embodiments 216-221,
wherein the
insulin compound is present in the amount from about 0.3mM to about 1.2mM.
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223. The insulin preparation according to any one of embodiments 216-222,
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.
224. The insulin preparation according to any one of embodiments 216-223,
comprising
from about 1mM to about 150mM of the nicotinic compound.
225. The insulin preparation according to any one of embodiments 216-224,
comprising
from about 1mM to about 85mM of arginine.
226. The insulin preparation according to any one of 216-225, which further
comprises a
metal ion, preservative agent(s), isotonicity agent(s) and stabilizer(s) and
buffer(s).
227. A method of reducing the blood glucose level in mammals by
administering to a pa-
tient in need of such treatment a therapeutically active dose of an insulin
preparation ac-
cording to any one of embodiments 216-226.
228. A method for the treatment of diabetes mellitus in a subject
comprising administer-
ing to a subject an insulin preparation according to any one of embodiments
216-226.
229. An insulin preparation according to any one of embodiments 216-226,
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 treat-
ment of critically ill diabetic and non-diabetic patients.
The invention is further illustrated by the following examples which are not
to be construed as
limiting.
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 refer-
ence 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).
All headings and sub-headings are used herein for convenience only and should
not
be construed as limiting the invention in any way.
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
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be construed as indicating any non-claimed element as essential to the
practice of the inven-
tion.
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.
This invention includes all modifications and equivalents of the subject
matter re-
cited in the claims appended hereto as permitted by applicable law.
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EXAMPLES
Example 1
Preparation of pharmaceutical preparations
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 glyc-
erol. 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, HCI. 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 isoe-
lectric point, pl, of the insulin in question.
Table 1. Composition of insulin preparations according to this invention
Insulin Zn Phenol m- NaCI Phos- Tris Glyce- Arginine, Nicotin-
Glutamic pH
aspart (mM) (mM) cresol (mM) phate (mM) rol HCI amide acid
(mM) (mM) (mM) (%w/v) (mM) (mM) (mM)
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
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Table 2. Composition of further insulin preparations according to this
invention
Preparation [Insulin [Zn2+] [phenol] [Arg] [Gly] [Glu] [His] [Nicotin pH
nr. aspart] mM mM mM mM mM mM amide]
mM mM
1 0.6 0.3 32 260 7.4
2 0.6 0.3 32 10 260 7.4
3 0.6 0.3 32 20 260 7.4
4 0.6 0.3 32 30 260 7.4
0.6 0.3 32 40 260 7.4
6 0.6 0.3 32 50 260 7.4
7 0.6 0.3 32 50 260 7.4
8 0.6 0.3 32 50 260 7.4
9 0.6 0.3 32 50 260 7.4
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Table 3. Composition of further insulin preparations according to this
invention
Preparation [Insulin [Zn2+] [phenol] [m- [Arg] [Glycerol] [tris] [Nicotin
pH
nr. aspart] mM mM cresol] mM %w/vol mM amide]
mM mM mM
10 0.6 0.35 16 16 1.3 7 80
7.4
11 0.6 0.35 16 16 10 1.3 7 80
7.4
12 0.6 0.35 16 16 30 1.3 7 80
7.4
13 0.6 0.35 16 16 50 1.3 7 80
7.4
Table 4. Composition of further insulin preparations according to this
invention
Preparation [Insulin [Zn2+] [phenol] [m- [Arg] [Glycerol]
[Phos- [Nicotin pH
nr. aspart] mM mM mM cresol] mM %w/vol phate]
amide] mM
mM mg/mL
14 0.6 0.30 16 16 20 1.08 3 80
7.1
15 0.6 0.30 16 16 20 - 3 230
7.1
16 0.6 0.20 16 16 20 - 3 230
7.1
17 0.6 0.24 16 16 20 0.24 3 180
7.1
18 0.6 0.28 16 16 20 0.45 3 155
7.1
19 0.6 0.25 16 16 20 0.75 3 120
7.1
20 0.6 0.20 16 16 20 1.08 3 80
7.1
5
10 Example 2
Analysis of insulin chemical stability
Size Exclusion Chromatography
Quantitative determination of high molecular weight protein (HMWP) and monomer
insulin
15 aspart was performed on Waters insulin (300 x 7.8mm, part nr wat 201549)
with an eluent
containing 2.5M acetic acid, 4mM L-arginine and 20 %(VN) acetonitrile at a
flow rate of
1m1/min. and 40 C. Detection was performed with a tuneable absorbance detector
(Waters
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486) at 276nm. Injection volume was 40p1 and a 600pM human insulin standard.
HMWP and
concentration of the preparations were measured at each sampling point.
Reverse phase chromatography (U PLC)
Determination of the insulin aspart related impurities were performed on a
UPLC system us-
ing a BEH RP 082.1 x 100mm column, particle size of 1,7pm. Waters part no
186002878.
with a flow rate of 0,5m1/min., at 40 C detection at 220nm. Elution was
performed with a
mobile phase consisting of the following:
A. 10 % (w/V) acetonititrile, 2.8% (w/w) sodium sulphate, 0.3 % (w/w) o-
phosphoric
acid, pH 3.5.
B. 70 % (w/V) acetonitrile. Gradient: 0-11 min isocratic with 73%/27% of NB,
11-12
linear change to 52%148% NB, 13-15 min. linear change to 73%/27% of NB, 15-20
min. iso-
cratic gradient at 73%/27% of NB.
The amount of B28iso-aspartate, desamido and other related impurities were de-
termined 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.
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
50mM 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 50mM arginine
is superior to
50 mM glycine, 50mM glutamic acid, or 50mM histidine regarding reduction of
the formation
of degradation products, as is shown in Table 4 below.
The insulin preparations of the present invention provide fast-acting insulin
prepara-
tions which are not only physically stable, but surprisingly also chemically
stable.
Table 5. Physical and chemical stability data for insulin preparations 1-9 of
Table 2
Preparation nr. Physical stabil- Chemical stability
ity, lag time Content of degradation product (%) measured
as differ-
(min) in ThT ence between content after incubation for 2
weeks at
assay 37 C and at 4 C
B28 !soAsp des-amido Other re- HMWP
forms lated impuri-
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ties
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
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
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Table 6. Chemical stability data for insulin preparations 10-13 of Table 3
Preparation nr. Chemical stability
Content of degradation product (%) measured as differ-
ence between content after incubation for 2 weeks at
37 C and at 4 C
B28 !soAsp des-amido Other re- HMWP
forms lated impuri-
ties
1.35 1.90 1.71 0.91
11 1.45 1.44 0.98 0.39
12 1.43 1.07 0.75 0.21
13 1.46 0.99 0.84 0.16
Table 7. Physical and chemical stability data for insulin preparations 14-20
of Table 4
Preparation Physical stabil- Chemical stability 5
nr. ity, lag time Content of degradation product ( /0) measured
after incu-
(min) in ThT bation for 4 weeks at 37 C
assay B28 !soAsp desamido Other related HMWP
forms impurities
14 133 2.7 2.9 2.1 0.4
15 87 2.7 3.6 2.3 0.4
16 50 2.8 4.4 2.7 0.4
17 65 2.7 3.5 2.3 0.4
18 107 2.7 3.3 2.2 0.5
19 85 2.6 3.1 2.3 0.5
20 59 2.6 3.3 2.3 0.5
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Example 3
Pharmacokinetic (PK)/ Pharmacodynamic (PD) studies in LYD pig model and plasma
analysis assay
PK/PD studies in LYD pigs
The PK/PD studies were performed on domestic female pigs, LYD cross-breed,
weighing
between 55 and 110kg. 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.
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 1.5m1 glass tubes pre-coated
with heparin.
The blood samples were kept in ice water until separation of plasma by
centrifugation for
10min. 3000rpm at 4 C, which was done within the first 30 minutes. Plasma
samples were
stored at 4 C for short time (2-3 hours) or at -18 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
The insulin Aspart LOCI is a monoclonal antibody-based sandwich immunoassay
and ap-
plies 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 hu-
man 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
chemilumines-
cence. The chemiluminescence was measured and the amount of light generated is
propor-
tional to the concentration of insulin Aspart.
Compared to the marketed product NovoRapid , the initial rate of plasma
glucose lowering is
faster for the preparations of the present invention (Figures 3 and 4).
Likewise, when com-
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pared to NovoRapid , the initial absorption rate of the insulin component of
the preparations
of the present invention, is markedly faster (Figure 5).
5 Example 4
General introduction to ThT fibrillation assays for the assessment of physical
stability
of protein formulations
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
10 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].
15 The time course for fibril formation can be described by a sigmoidal
curve with the following
expression [Nielsen et al. (2001) Biochemistry 40, 6036-6046];
f +m ft
F = f, + m,t + ________________ ),
1+e t" Eq.(i)
20 Here, F is the ThT fluorescence at the time t. The constant to is the
time needed to
reach 50% of maximum fluorescence. The two important parameters describing
fibril forma-
tion are the lag-time calculated by to ¨ 2T and the apparent rate constant
kapp =
= = = ==
==
.= = ff + mft
c.)
c.) - =
2 -
o kapp = lit
f,
H
- f, + mIt
=
-111111111111p111111111111
to Ti me
Lag-time = to -
CA 02821613 2013 06 13
WO 2012/080362 PCT/EP2011/072809
41
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
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 HCIator HCI. Thioflavin T was added to the
samples
from a stock solution in H20 to a final concentration of 1 M.
Sample aliquots of 2000 were placed in a 96 well microtiter plate (Packard
Opti-
PlateTm-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
Incubation at given temperature, shaking and measurement of the ThT
fluorescence emis-
sion were done in a Fluoroskan Ascent FL fluorescence platereader or Varioskan
platereader
(Thermo Labsystems). The temperature was adjusted to 37 C. The orbital
shaking was ad-
justed to 960rpm with an amplitude of 1mm in all the presented data.
Fluorescence meas-
urement was done using excitation through a 444nm filter and measurement of
emission
through a 485nm filter.
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 meas-
urement, the plate was shaken and heated as described.
Data handling
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.
CA 02821613 2013 06 13
WO 2012/080362 PCT/EP2011/072809
42
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
The peptide concentration in each of the tested formulations were measured
both before ap-
plication in the ThT fibrillation assay ("Initial") and after completion of
the ThT fibrillation ("Af-
ter ThT assay"). Concentrations were determined by reverse HPLC methods using
a pramlin-
tide standard as a reference. Before measurement after completion 150p1 was
collected from
each of the replica and transferred to an Eppendorf tube. These were
centrifuged at 30000 G
for 40mins. The supernatants were filtered through a 0.22pm filter before
application on the
HPLC system.
