U.S. patent application number 13/668000 was filed with the patent office on 2013-09-05 for rapid acting insulin formulation comprising an oligosaccharide.
This patent application is currently assigned to ADOCIA. The applicant listed for this patent is ADOCIA. Invention is credited to Gerard SOULA, Olivier SOULA, Remi SOULA.
Application Number | 20130231281 13/668000 |
Document ID | / |
Family ID | 49043172 |
Filed Date | 2013-09-05 |
United States Patent
Application |
20130231281 |
Kind Code |
A1 |
SOULA; Olivier ; et
al. |
September 5, 2013 |
Rapid acting insulin formulation comprising an oligosaccharide
Abstract
A composition in aqueous solution, including an insulin and at
least one oligosaccharide whose average degree of polymerization is
between 3 and 13 and whose polydispersity index PDI is above 1.0,
the oligosaccharide having partially substituted carboxyl
functional groups, the unsubstituted carboxyl functional groups
being salifiable.
Inventors: |
SOULA; Olivier; (Meyzieu,
FR) ; SOULA; Remi; (Lyon, FR) ; SOULA;
Gerard; (Meyzieu, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ADOCIA |
Lyon |
|
FR |
|
|
Assignee: |
ADOCIA
Lyon
FR
|
Family ID: |
49043172 |
Appl. No.: |
13/668000 |
Filed: |
November 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13287793 |
Nov 2, 2011 |
|
|
|
13668000 |
|
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Current U.S.
Class: |
514/5.9 |
Current CPC
Class: |
A61K 38/28 20130101;
A61K 31/702 20130101; A61K 9/0019 20130101; A61K 47/26 20130101;
A61K 47/61 20170801 |
Class at
Publication: |
514/5.9 |
International
Class: |
A61K 47/36 20060101
A61K047/36 |
Claims
1. A composition in aqueous solution, comprising insulin and at
least one oligosaccharide whose average degree of polymerization is
between 3 and 13 and whose polydispersity index PDI is above 1.0,
said oligosaccharide having partially substituted carboxyl
functional groups, the unsubstituted carboxyl functional groups
being salifiable.
2. The composition as claimed in claim 1, wherein it further
comprises at least one polyanionic compound.
3. The composition as claimed in claim 1, wherein the insulin is a
human insulin.
4. The composition as claimed in claim 1, wherein the insulin is an
insulin analog.
5. The composition as claimed in claim 4, wherein the insulin
analog is selected from the group comprising consisting of insulin
lispro (Humalog.RTM.), insulin aspart (Novolog.RTM.,
Novorapid.RTM.) and insulin glulisine (Apidra.RTM.).
6. The composition as claimed in claim 4, wherein the insulin
analog is insulin lispro (Humalog.RTM.).
7. The composition as claimed in claim 1, wherein the
oligosaccharide/insulin weight ratio is between 0.4 and 10.
8. The composition as claimed in claim 1, wherein the concentration
of polyanionic compound is between 1.4 and 35 mg/mL
9. The composition as claimed in claim 1, wherein oligosaccharide
is selected from the oligosaccharides of the following general
formula I: ##STR00005## In which: the oligosaccharide is a dextran,
F results from the coupling between linker arm R and an --OH
function of the oligosaccharide and being either an ester,
carbamate or ether function, R is a chain comprising between 1 and
15 carbons, optionally branched and/or unsaturated, comprising one
or more heteroatoms, such as O, N and/or S, and having at least one
carboxyl function, Phe is a residue of a phenylalanine derivative,
of absolute configuration L or D, produced from coupling between
the amine of the phenylalanine derivative and at least one acid
carried by group R prior to attachment to Phe, n represents the
mole fraction of R substituted with Phe and is between 0.3 and 0.9,
i represents the average mole fraction of groups F-R-[Phe]n borne
per saccharide unit and is between 0.5 and 2.5; when R is not
substituted with Phe, the acid or acids of group R are carboxylates
with an alkaline cation.
10. The composition as claimed in claim 2, wherein the anionic
compound is selected from the group consisting of anionic
molecules, anionic polymers and of compounds consisting of a
skeleton formed from a discrete number p between 1 and 8
(1.ltoreq.p.ltoreq.8) of identical or different saccharide units,
bound by identical or different glycosidic bonds that are naturally
carriers of carboxyl groups or are substituted with carboxyl
groups.
11. A pharmaceutical formulation comprising a composition as
claimed in claim 1.
12. The pharmaceutical formulation as claimed in claim 11, wherein
the concentration of insulin is between 240 and 3000 .mu.M (40 to
500 IU/mL).
13. The pharmaceutical formulation as claimed in claim 11, wherein
the concentration of insulin is between 600 and 1200 .mu.M (100 and
200 IU/mL).
14. A method for preparing a pharmaceutical formulation of insulin,
comprising at least one oligosaccharide whose average degree of
polymerization is between 3 and 13 and whose polydispersity index
PIN is above 1.0, alone nixed with a polyanionic compound, that
makes it possible, after administration, to accelerate the passage
of insulin into the blood and reduce glycemia more rapidly relative
to an oligosaccharide-free formulation alone or mixed with a
polyanionic compound.
15. The method as claimed in claim 14, wherein the oligosaccharide
is selected from the oligosaccharides of the following general
formula I: ##STR00006## In which: the oligosaccharide is a dextran,
F results from the coupling between linkage R and an --OH function
of the oligosaccharide and being either an ester function,
carbamate or ether function, R is a chain comprising between 1 and
15 carbons, optionally branched and/or unsaturated, comprising one
or more heteroatoms, such as 0, N and/or S, and having at least one
carboxyl function, Phe is a residue of a phenylalanine derivative,
of absolute configuration L or D, produced from coupling between
the amine of the phenylalanine derivative and at least one acid
carried by group R prior to attachment to Phe, n represents the
mole fraction of R substituted with Phe and is between 0.3 and 0.9,
i represents the average mole fraction of groups F-R-[Phe]n borne
per saccharide unit and is between 0.5 and 2.5; when R is not
substituted with Phe, the acid or acids of group R are carboxylates
with an alkaline cation, preferably such as Na+ or K+.
16. The method as claimed in claim 14, wherein the anionic compound
is selected from the group consisting of anionic molecules, anionic
polymers and of compounds consisting of a skeleton formed from a
discrete number p between 1 and 8 (1.ltoreq.p.ltoreq.8) of
identical or different saccharide units, bound by identical or
different glycosidic bonds that are naturally carriers of carboxyl
groups or are substituted with carboxyl groups.
17. A method of preparing a formulation of human insulin having an
insulin concentration between 240 and 3000 .mu.M (40 and 500
IU/mL), whose onset of in action in humans is less than that of the
reference formulation at the same insulin concentration in the
absence of oligosaccharide, wherein it comprises a step of adding,
to said formulation, at least one oligosaccharide having partially
substituted carboxyl functional groups.
18. The method as claimed in claim 17, wherein it further comprises
a step of adding at least one polyanionic compound to said
formulation.
19. The method as claimed in claim 17, for preparing a formulation
of human insulin having an insulin concentration of 600 .mu.M (100
IU/mL), whose onset of action in humans is less than 60 minutes,
wherein it comprises a step of adding, to said formulation, at
least one oligosaccharide having partially substituted carboxyl
functional groups.
20. The method as claimed in claim 19, wherein it further comprises
a step of adding at least one polyanionic compound to said
formulation.
21. A method of preparing a formulation of insulin analog having an
insulin concentration between 240 and 3000 .mu.M (40 and 500
IU/mL), whose onset of action in humans is less than that of the
reference formulation at the same insulin concentration in the
absence of oligosaccharide, wherein it comprises a step of adding,
to said formulation, at least one oligosaccharide having partially
substituted carboxyl functional groups.
22. The method as claimed in claim 21, wherein it further comprises
a step of adding at least one polyanionic compound to said
formulation.
23. The method as claimed in claim 21, for preparing a formulation
of insulin analog, having an insulin concentration of 600 .mu.M
(100 IU/mL), whose onset of action in humans is less than 30
minutes, wherein it comprises a step of adding, to said
formulation, at least one oligosaccharide having partially
substituted carboxyl functional groups.
24. The method as claimed in claim 23, wherein it further comprises
a step of adding at least one polyanionic compound to said
formulation.
25. The method as claimed in claim 17, wherein the oligosaccharide
is selected from the oligosaccharides of the following general
formula I: ##STR00007## In which: the oligosaccharide is a dextran,
F results from the coupling between linkage R and an --OH function
of the oligosaccharide and being either an ester-carbamate or ether
function, R is a chain comprising between 1 and 15 carbons,
optionally branched and/or unsaturated, comprising one or more
heteroatoms, such as O, N and/or S, and having at least one
carboxyl function, Phe is a residue of a phenylalanine derivative,
of absolute configuration L or D, produced from coupling between
the amine of the phenylalanine derivative and at least one acid
carried by group R prior to attachment to Phe, n represents the
mole fraction of R substituted with Phe and is between 0.3 and 0.9,
i represents the average mole fraction of groups F-R-[Phe]n borne
per saccharide unit and is between 0.5 and 2.5; when R is not
substituted with Phe, the acid or acids of group R are carboxylates
with an alkaline cation.
26. The method as claimed in claim 18, wherein the anionic compound
is selected from the group consisting of anionic molecules, anionic
polymers and of compounds consisting of a skeleton formed from a
discrete number p between 1 and 8 (1.ltoreq.p.ltoreq.8) of
identical or different saccharide units, bound by identical or
different glycosidic bonds that are naturally carriers of carboxyl
groups or are substituted with carboxyl groups.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a Continuation-in-Part of application Ser. No.
13/287,793 filed Nov. 2, 2011. The disclosure of the prior
application is hereby incorporated by reference herein in its
entirety.
BACKGROUND
[0002] The present invention relates to a rapid-acting insulin
formulation.
[0003] Since the production of insulin by genetic engineering, at
the beginning of the 1980s, diabetic patients have had the benefit
of human insulin for their treatment. This product has greatly
improved this therapy since the immunological risks associated with
the use of nonhuman, in particular porcine, insulin are eliminated.
However, human insulin injected subcutaneously only has a
hypoglycemic effect after -60 minutes, which means that diabetic
patients treated with human insulin must carry out the injection 30
minutes before a meal.
[0004] One of the problems that need to be solved for improving the
health and comfort of diabetic patients is to make insulin
formulations available that provide a hypoglycemic response more
quickly than that of human insulin and if possible approaching the
physiological response of a healthy individual. The endogenous
insulin secretion in a healthy individual is triggered immediately
by an increase in glycemia. The objective is to reduce as much as
possible the delay between injection of insulin and the start of a
meal.
[0005] It is now accepted that making such formulations available
is useful so that management of the disease is optimal.
[0006] Genetic engineering has made it possible to provide a
response with the development of rapid-acting insulin analogs.
These insulins are modified on one or two amino acids so that they
are absorbed more rapidly in the blood compartment after a
subcutaneous injection. These insulins lispro (Humalog.RTM.,
Lilly), aspart (Novolog.RTM., Novo) and glulisine (Apidra.RTM.,
SanofiAventis) are stable insulin solutions with a more rapid
hypoglycemic response than that of human insulin. Therefore
patients treated with these rapid-acting insulin analogs can
proceed with insulin injection just 15 minutes before a meal.
[0007] The principle of the rapid-acting insulin analogs is to form
hexamers with a concentration of 100 IU/mL for ensuring stability
of the insulin in the commercial product while promoting very rapid
dissociation of these hexamers into monomers after subcutaneous
injection in order to obtain rapid action.
[0008] Human insulin as formulated in its commercial form does not
allow a hypoglycemic response to be obtained that is close in terms
of kinetics of the physiological response generated by the start of
a meal (increase in glycemia), because at the concentration of use
(100 IU/mL), in the presence of zinc and other excipients such as
phenol or m-cresol, it assembles in the hexamer form whereas it is
active in the form of monomer and of dimer. Human insulin is
prepared in the form of hexamers so that it is stable for nearly 2
years at 4.degree. C., since in the form of monomers it has a very
strong tendency to aggregate and then to form fibrils, which causes
it to lose its activity. Moreover, in this aggregated form, it
presents an immunological risk for the patient.
[0009] Dissociation of the hexamers into dimers and of the dimers
into monomers delays its action by nearly 20 minutes compared with
a rapid-acting insulin analog (Brange J., et al., Advanced Drug
Delivery Review, 35, 1999, 307-335).
[0010] The kinetics of passage of insulin analogs into the blood,
and their kinetics of reduction of glycemia, are not optimal and
there is a real need for a formulation having an even shorter time
of action in order to approach the kinetics of endogenous insulin
secretion in healthy persons.
[0011] The company Biodel has proposed a solution to this problem
with a formulation of human insulin comprising EDTA and citric acid
as described in patent application US200839365. EDTA, by its
capacity for complexing zinc atoms, and citric acid, by its
interactions with the cationic zones present on the surface of
insulin, are described as destabilizing the hexameric form of
insulin and thus reducing its time of action.
[0012] However, such a formulation notably has the drawback of
dissociating the hexameric form of insulin, which is the only
stable form able to meet the stability requirements of the
pharmaceutical regulations.
[0013] PCT application WO2010/122385, in the name of the applicant,
is also known; this describes formulations of human insulin or
insulin analog for solving the various problems mentioned above by
adding a substituted polysaccharide comprising carboxyl groups.
[0014] However, the requirements arising from chronic and intensive
use or even pediatric use of such formulations lead a person
skilled in the art to try to use excipients whose molecular weight
and size are as small as possible to facilitate their
elimination.
[0015] The aim of reducing the size of the polysaccharides has led
a person skilled in the art to consider oligosaccharides, but owing
to their reduced size these do not have the same properties as the
polysaccharides, since there is loss of the polymer effect, as is
demonstrated in the comparative examples in the experimental
section, see notably the tests for insulin dissolution at the
isoelectric point and the tests of interaction with a model protein
such as albumin.
[0016] Despite these discouraging results, the applicant has
succeeded in developing formulations capable of accelerating
insulin by using oligomers alone or in combination with a
polyanionic compound.
[0017] Surprisingly, besides the fact that addition of this
polyanionic compound makes it possible to improve the inadequate
performance of the oligosaccharides, it makes it possible to obtain
performance equal to that obtained with a polysaccharide, even when
the oligosaccharide alone or the polyanionic compound alone has no
effect.
[0018] Moreover, as in the case when polysaccharides are used, the
hexameric nature of the insulin is not affected, therefore the
stability of the formulations is not affected.
[0019] The present invention can solve the various problems
described above since it notably makes it possible to produce a
formulation of insulin, human or analog, capable after
administration of accelerating the passage of human insulin or of
insulin analogs into the blood and of reducing glycemia more
rapidly compared with the corresponding commercial insulin
products.
[0020] It also allows a significant reduction in the time for the
start of action of a formulation of rapid-acting insulin
analog.
[0021] The invention consists of a composition, in aqueous
solution, comprising insulin and at least one oligosaccharide whose
average degree of polymerization is between 3 and 13 and whose
polydispersity index PDI is above 1.0, said oligosaccharide having
partially substituted carboxyl functional groups, the unsubstituted
carboxyl functional groups being salifiable.
[0022] In one embodiment, the average degree of polymerization is
below 10.
[0023] In one embodiment, the polydispersity index PDI is between
1.1 and 2.0.
[0024] In one embodiment, the polydispersity index PDI is between
1.2 and 1.8.
[0025] The degree of polymerization DP means the average number of
repeating units (monomers) per polymer chain. It is calculated by
dividing the number-average molecular weight by the average
molecular weight of the repeating unit.
[0026] The number-average molecular weight (Mn) means the
arithmetic mean of the weights of each of the polymer chains. Thus,
for a number ni of chains i of molecular weight Mi, we have
Mn=(.SIGMA.iniMi)/(.SIGMA.ini).
[0027] The weight-average molecular weight (Mw) is obtained from
Mw=(.SIGMA.iniMi2)/(.SIGMA.iniMi), ni being the number of polymer
chains i of molecular weight Mi.
[0028] The polymers can also be characterized by the distribution
of chain lengths, also called polydispersity index (PDI), and is
equal to Mw divided by Mn.
[0029] In one embodiment, the composition further comprises a
polyanionic compound.
[0030] In one embodiment, the insulin is human insulin.
[0031] Human insulin means an insulin obtained by synthesis or
recombination whose peptide sequence is the sequence of human
insulin, including the allelic variations and the homologs.
[0032] In one embodiment, the insulin is an insulin analog.
[0033] Insulin analog means a recombinant insulin whose primary
sequence contains at least one modification relative to the primary
sequence of human insulin.
[0034] In one embodiment the insulin analog is selected from the
group comprising insulin lispro (Humalog.RTM.), insulin aspart
(Novolog.RTM., Novorapid.RTM.) and insulin glulisine
(Apidra.RTM.).
[0035] In one embodiment, the insulin analog is insulin lispro
(Humalog.RTM.).
[0036] In one embodiment, the insulin analog is insulin aspart
(Novolog.RTM., Novorapid.RTM.).
[0037] In one embodiment, the insulin analog is insulin glulisine
(Apidra.RTM.).
[0038] The invention also relates to a pharmaceutical formulation
of insulin comprising a composition according to the invention.
[0039] In one embodiment, it relates to a pharmaceutical
formulation characterized in that the concentration of insulin is
between 240 and 3000 .mu.M (40 to 500 IU/mL).
[0040] In one embodiment, it relates to a pharmaceutical
formulation characterized in that the concentration of insulin is
between 600 and 1200 .mu.M (100 to 200 IU/mL).
[0041] In one embodiment, it relates to a pharmaceutical
formulation characterized in that the concentration of insulin is
600 .mu.M (100 IU/mL).
[0042] In one embodiment, it relates to a pharmaceutical
formulation characterized in that the concentration of insulin is
600 .mu.M (100 IU/mL).
[0043] In one embodiment, it relates to a pharmaceutical
formulation characterized in that the concentration of insulin is
1200 .mu.M (200 IU/mL).
[0044] In one embodiment, the invention relates to a complex
between human insulin and an oligosaccharide whose average degree
of polymerization is between 3 and 13 and whose polydispersity
index PDI is above 1.0, said oligosaccharide having partially
substituted carboxyl functional groups, the unsubstituted carboxyl
functional groups being salifiable.
[0045] It also relates to the use of this complex for preparing
formulations of human insulin that make it possible, after
administration, to accelerate the passage of human insulin into the
blood and reduce glycemia more rapidly relative to an
oligosaccharide-free formulation alone or mixed with a polyanionic
compound.
[0046] In one embodiment, the invention relates to a complex
between an insulin analog and an oligosaccharide whose average
degree of polymerization is between 3 and 13 and whose
polydispersity index PDI is above 1.0, said oligosaccharide having
partially substituted carboxyl functional groups, the unsubstituted
carboxyl functional groups being salifiable.
[0047] It also relates to the use of this complex for preparing
formulations of insulin analog that make it possible, after
administration, to accelerate the passage of the insulin analog
into the blood and reduce glycemia more rapidly relative to an
oligosaccharide-free formulation alone or mixed with a polyanionic
compound.
[0048] In one embodiment, the invention relates to the use of at
least one oligosaccharide whose average degree of polymerization is
between 3 and 13 and whose polydispersity index PDI is above 1.0,
alone or mixed with a polyanionic compound, for preparing a
pharmaceutical formulation of insulin that makes it possible, after
administration, to accelerate the passage of insulin into the blood
and reduce glycemia more rapidly relative to an
oligosaccharide-free formulation alone or mixed with a polyanionic
compound.
[0049] In one embodiment, the invention relates to the use of at
least one oligosaccharide whose average degree of polymerization is
between 3 and 13 and whose polydispersity index PDI is above 1.0,
alone or mixed with a polyanionic compound, for preparing a
formulation of insulin analog that makes it possible, after
administration, to accelerate the passage of the insulin analog
into the blood and reduce glycemia more rapidly relative to an
oligosaccharide-free formulation alone or mixed with a polyanionic
compound.
[0050] It is known by a person skilled in the art that the onset of
action of insulins is dependent upon the concentration of insulin.
Only values for the onset of action of formulations at 100 IU/mL
are documented.
[0051] The "regular" formulations of human insulin on the market at
a concentration of 600 .mu.M (100 IU/mL) have a onset of action
between 50 and 90 minutes and an offset of action of about 360 to
420 minutes in humans. The time to reach the peak insulin
concentration in the blood is between 90 and 180 minutes in
humans.
[0052] The formulations of rapid-acting insulin analogs on the
market at a concentration of 600 .mu.M (100 IU/mL) have a onset of
action between 30 and 60 minutes and an offset of action of about
240-300 minutes in humans. The time to reach the peak insulin
concentration in the blood is between 50 and 90 minutes in
humans.
[0053] The invention also relates to a method of preparing a
formulation of human insulin having an insulin concentration
between 240 and 3000 .mu.M (40 and 500 IU/mL), whose onset of
action in humans is less than that of the reference formulation at
the same insulin concentration in the absence of oligosaccharide,
characterized in that it comprises a step of adding, to said
formulation, at least one oligosaccharide whose average degree of
polymerization is between 3 and 13 and whose polydispersity index
PDI is above 1.0, said oligosaccharide having partially substituted
carboxyl functional groups, the unsubstituted carboxyl functional
groups being salifiable.
[0054] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0055] The invention also relates to a method of preparing a
formulation of human insulin having an insulin concentration
between 600 and 1200 .mu.M (100 and 200 IU/mL), whose onset of
action in humans is less than 60 minutes, characterized in that it
comprises a step of adding, to said formulation, at least one
oligosaccharide whose average degree of polymerization is between 3
and 13 and whose polydispersity index PDI is above 1.0, said
oligosaccharide having partially substituted carboxyl functional
groups, the unsubstituted carboxyl functional groups being
salifiable.
[0056] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0057] The invention also relates to a method of preparing a
formulation of human insulin having an insulin concentration of 600
.mu.M (100 IU/mL), whose onset of action in humans is less than 60
minutes, characterized in that it comprises a step of adding, to
said formulation, at least one oligosaccharide whose average degree
of polymerization is between 3 and 13 and whose polydispersity
index PDI is above 1.0, said oligosaccharide having partially
substituted carboxyl functional groups, the unsubstituted carboxyl
functional groups being salifiable.
[0058] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0059] The invention also relates to a method of preparing a
formulation of human insulin having an insulin concentration of
1200 .mu.M (200 IU/mL), whose onset of action in humans is at least
10% lower than that of the formulation of human insulin in the
absence of oligosaccharide, characterized in that it comprises a
step of adding, to said formulation, at least one oligosaccharide
whose average degree of polymerization is between 3 and 13 and
whose polydispersity index PDI is above 1.0, said oligosaccharide
having partially substituted carboxyl functional groups, the
unsubstituted carboxyl functional groups being salifiable.
[0060] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0061] The invention consists of preparing a so-called rapid-acting
formulation of human insulin, characterized in that it comprises a
step of adding, to said formulation, at least one oligosaccharide
whose average degree of polymerization is between 3 and 13 and
whose polydispersity index PDI is above 1.0, having partially
substituted carboxyl functional groups.
[0062] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0063] The invention also relates to a method of preparing a
formulation of human insulin at a concentration of 600 .mu.M (100
IU/mL) whose onset of action in humans is less than 60 minutes,
preferably less than 45 minutes, and more preferably less than 30
minutes, characterized in that it comprises a step of adding, to
said formulation, at least one oligosaccharide whose average degree
of polymerization is between 3 and 13 and whose polydispersity
index PDI is above 1.0, said oligosaccharide having partially
substituted carboxyl functional groups, the unsubstituted carboxyl
functional groups being salifiable.
[0064] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0065] The invention also relates to a method of preparing a
formulation of insulin analog having an insulin concentration
between 240 and 3000 .mu.M (40 and 500 IU/mL), whose onset of
action in humans is less than that of the reference formulation at
the same insulin concentration in the absence of oligosaccharide,
characterized in that it comprises a step of adding, to said
formulation, at least one oligosaccharide whose average degree of
polymerization is between 3 and 13 and whose polydispersity index
PDI is above 1.0, said oligosaccharide having partially substituted
carboxyl functional groups, the unsubstituted carboxyl functional
groups being salifiable.
[0066] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation
[0067] The invention also relates to a method of preparing a
formulation of insulin analog having an insulin concentration
between 600 and 1200 .mu.M (100 and 200 IU/mL), whose onset of
action in humans is less than that of the reference formulation at
the same concentration of insulin analog in the absence of
oligosaccharide, characterized in that it comprises a step of
adding, to said formulation, at least one oligosaccharide whose
average degree of polymerization is between 3 and 13 and whose
polydispersity index PDI is above 1.0, said oligosaccharide having
partially substituted carboxyl functional groups, the unsubstituted
carboxyl functional groups being salifiable.
[0068] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0069] The invention also relates to a method of preparing a
formulation of insulin analog having an insulin concentration of
600 .mu.mol/L (100 IU/mL), whose onset of action in humans is less
than that of the reference formulation at the same concentration of
insulin analog in the absence of oligosaccharide, characterized in
that it comprises a step of adding, to said formulation, at least
one oligosaccharide whose average degree of polymerization is
between 3 and 13 and whose polydispersity index PDI is above 1.0,
said oligosaccharide having partially substituted carboxyl
functional groups, the unsubstituted carboxyl functional groups
being salifiable.
[0070] The invention also relates to a method of preparing a
formulation of insulin analog having an insulin concentration of
1200 .mu.M (200 IU/mL), whose onset of action in humans is at least
10% lower than that of the formulation of insulin analog in the
absence of oligosaccharide, characterized in that it comprises a
step of adding, to said formulation, at least one oligosaccharide
whose average degree of polymerization is between 3 and 13, said
oligosaccharide having partially substituted carboxyl functional
groups, the unsubstituted carboxyl functional groups being
salifiable.
[0071] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0072] The invention also relates to a method of preparing a
formulation of insulin analog having an insulin concentration of
1200 .mu.mol/L (200 IU/mL), whose delay in action in humans is less
than 30 minutes, characterized in that it comprises a step of
adding, to said formulation, at least one oligosaccharide whose
average degree of polymerization is between 3 and 13 and whose
polydispersity index PDI is above 1.0, said oligosaccharide having
partially substituted carboxyl functional groups, the unsubstituted
carboxyl functional groups being salifiable.
[0073] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0074] The invention consists of preparing a so-called
very-rapid-acting formulation of insulin analog, characterized in
that it comprises a step of adding, to said formulation, at least
one oligosaccharide whose average degree of polymerization is
between 3 and 13 and whose polydispersity index PDI is above 1.0,
said oligosaccharide having partially substituted carboxyl
functional groups, the unsubstituted carboxyl functional groups
being salifiable.
[0075] In one embodiment, said method further comprises a step of
adding at least one polyanionic compound to said formulation.
[0076] In one embodiment, the insulin analog is selected from the
group comprising insulin lispro (Humalog.RTM.), insulin aspart
(Novolog.RTM., Novorapid.RTM.) and insulin glulisine
(Apidra.RTM.).
[0077] In one embodiment, the insulin analog is insulin lispro
(Humalog.RTM.).
[0078] In one embodiment, the insulin analog is insulin aspart
(Novolog.RTM., Novorapid.RTM.).
[0079] In one embodiment, the insulin analog is insulin glulisine
(Apidra.RTM.).
[0080] In one embodiment, the oligosaccharide is a functionalized
oligosaccharide whose average degree of polymerization is between 3
and 13 and whose polydispersity index PDI is above 1.0 and which
has carboxyl functional groups.
[0081] Said oligosaccharide is selected from the functionalized
oligosaccharides whose degree of polymerization is between 3 and 13
and whose polydispersity index PDI is above 1.0, consisting for the
most part of glycosidic bonds of the (1,6) type.
[0082] In one embodiment, the oligosaccharide consisting for the
most part of glycosidic bonds of the (1,6) type is a functionalized
dextran having carboxyl functional groups.
[0083] In one embodiment, the average degree of polymerization is
below 10.
[0084] Said oligosaccharide is functionalized with at least one
phenylalanine derivative, designated Phe: [0085] said phenylalanine
derivative being grafted or bonded to the oligosaccharide by
coupling with an acid function, said acid function being an acid
function borne by a linkage R bonded to the oligosaccharide by a
function F, said function F resulting from coupling between the
linker arm R and an --OH function of the oligosaccharide, [0086] F
being either an ester function, carbamate or ether function, [0087]
R being a chain comprising between 1 and 15 carbons, which is
optionally branched and/or unsaturated, comprising one or more
heteroatoms, such as O, N and/or S, and having at least one
carboxyl function, [0088] Phe being a residue of a phenylalanine
derivative, of absolute configuration L or D, the product of
coupling between the amine function of phenylalanine and at least
one acid function carried by the group R and/or an acid function
carried by the oligosaccharide bearing carboxyl functional
groups.
[0089] In one embodiment, the functionalized oligosaccharide is
selected from the oligosaccharides whose average degree of
polymerization is between 3 and 13 and whose polydispersity index
PDI is above 1.0, of the following general formula I:
##STR00001## [0090] the oligosaccharide is a dextran, [0091] F
results from coupling between the linker arm R and an --OH function
of the oligosaccharide and being either an ester, carbamate or
ether function, [0092] R is a chain comprising between 1 and 15
carbons, optionally branched and/or unsaturated, comprising one or
more heteroatoms, such as 0, N and/or S, and having at least one
carboxyl function, [0093] Phe is a residue of a phenylalanine
derivative, of absolute configuration L or D, produced from
coupling between the amine function of the phenylalanine derivative
and at least one acid function carried by group R prior to
attachment to Phe, [0094] n represents the mole fraction of the R
substituted with Phe and is between 0.1 and 0.9, preferably between
0.2 and 0.8, more preferably between 0.3 and 0.7, more preferably
between 0.3 and 0.5; [0095] i represents the average mole fraction
of the groups F-R-[Phe].sub.n borne per saccharide unit and is
between 0.5 and 3.0, preferably between 1.0 and 2.5, preferably
between 1.2 and 2.2, preferably between 1.4 and 2.0; [0096] when R
is not substituted with Phe, the acid or acids of group R are
carboxylates with an alkaline cation, preferably such as Na.sup.+,
K.sup.+, Ca.sup.2+ or Mg.sup.2+.
[0097] In one embodiment, n, which represents the degree of
substitution of the R substituted with Phe, is between 0.1 and 0.9,
preferably between 0.2 and 0.8, preferably between 0.3 and 0.7,
more preferably between 0.3 and 0.5.
[0098] In one embodiment, n, which represents the mole fraction of
the R substituted with Phe, is between 0.2 and 0.9, preferably
between 0.3 and 0.8, preferably between 0.3 and 0.6, more
preferably between 0.3 and 0.5.
[0099] In one embodiment, F is an ether function.
[0100] In one embodiment, F is a carbamate function.
[0101] In one embodiment, F is an ester function.
[0102] In one embodiment R is a chain comprising 1 carbon.
[0103] In one embodiment, the oligosaccharide according to the
invention is characterized in that the group R before optional
attachment to Phe, is selected from the following groups:
##STR00002##
or their salts of alkaline cations selected from the group
consisting of Na.sup.+ or K.sup.+.
[0104] In one embodiment, the oligosaccharide according to the
invention is characterized in that the group R before optional
attachment to Phe, is derived from citric acid.
[0105] In one embodiment, the oligosaccharide according to the
invention is characterized in that the group R before optional
attachment to Phe, is derived from malic acid.
[0106] In one embodiment, the oligosaccharide according to the
invention is characterized in that the phenylalanine derivative is
selected from the group consisting of phenylalanine,
alpha-methyl-phenylalanine, 3,4-dihydroxyphenylalanine, tyrosine,
alpha-methyl-tyrosine, O-methyl-tyrosine, alpha-phenylglycine,
4-hydroxyphenylglycine, 3,5-dihydroxyphenylglycine and their salts
of alkaline cations and phenylalaninol, phenylalaninamide, ethyl
benzyl amine, said derivatives being of absolute configuration L or
D.
[0107] In one embodiment, the oligosaccharide according to the
invention is characterized in that the phenylalanine derivative is
selected from the group consisting of phenylalanine and its salts
of alkaline cations, phenylalaninol, phenylalaninamide, ethyl
benzyl amine, said derivatives being of absolute configuration L or
D.
[0108] In one embodiment, the oligosaccharide according to the
invention is characterized in that the phenylalanine derivative is
selected from the group consisting of phenylalanine,
alpha-methyl-phenylalanine, 3,4-dihydroxyphenylalanine tyrosine,
alpha-methyl-tyrosine, O-methyl-tyrosine, alpha-phenylglycine,
4-hydroxyphenylglycine, 3,5-dihydroxyphenylglycine and their salts
of alkaline cations, said derivatives being of absolute
configuration L or D.
[0109] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is phenylalanine and its salts of alkaline cations, said derivative
being of absolute configuration L or D.
[0110] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is alpha-methyl-phenylalanine and its salts of alkaline cations,
said derivative being of absolute configuration L or D.
[0111] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is 3,4-dihydroxyphenylalanine and its salts of alkaline cations,
said derivative being of absolute configuration L or D.
[0112] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is tyrosine and its salts of alkaline cations, said derivative
being of absolute configuration L or D.
[0113] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is alpha-methyl-tyrosine and its salts of alkaline cations, said
derivative being of absolute configuration L or D.
[0114] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is O-methyl-tyrosine and its salts of alkaline cations, said
derivative being of absolute configuration L or D.
[0115] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is alpha-phenylglycine and its salts of alkaline cations, said
derivative being of absolute configuration L or D.
[0116] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is 4-hydroxyphenylglycine and its salts of alkaline cations, said
derivative being of absolute configuration L or D.
[0117] In one embodiment, the oligosaccharide according to the
invention is characterized in that the derivative of phenylalanine
is 3,5-dihydroxyphenylglycine and its salts of alkaline cations,
said derivative being of absolute configuration L or D.
[0118] In one embodiment, the derivatives of phenylalanine are in
the form of a racemic mixture.
[0119] In one embodiment, the derivatives of phenylalanine are in
the form of isolated isomers of absolute configuration D.
[0120] In one embodiment, the derivatives of phenylalanine are in
the form of isolated isomers of absolute configuration L.
[0121] In one embodiment, the oligosaccharide is selected from
oligodextrans.
[0122] In one embodiment, the oligodextran has a number-average
molecular weight below 3500 g/mol.
[0123] In one embodiment, the oligodextran has a number-average
molecular weight below 2500 g/mol.
[0124] In one embodiment, the oligosaccharide has an average degree
of polymerization between 3 and 13.
[0125] In one embodiment at least 50% of the population of
oligosaccharide has an average degree of polymerization below
10.
[0126] In one embodiment, the oligosaccharide has an average degree
of polymerization between 3 and 10.
[0127] In one embodiment, the oligosaccharide has an average degree
of polymerization between 3 and 6.
[0128] In one embodiment, the polydispersity index PDI is between
1.1 and 2.0.
[0129] In one embodiment, the polydispersity index PDI is between
1.2 and 1.8.
[0130] In one embodiment, the polyanionic compound is an anionic
molecule.
[0131] According to the invention, the anionic molecules are
selected from the group consisting of citric acid, aspartic acid,
glutamic acid, malic acid, tartaric acid, succinic acid, adipic
acid, oxalic acid, triphosphate and their salts of Na.sup.+,
K.sup.+, Ca.sup.2+ or Mg.sup.2+.
[0132] In one embodiment, the anionic molecule is citric acid and
its salts of Na.sup.+, K.sup.+, Ca.sup.2+ or Mg.sup.2+.
[0133] In one embodiment, the polyanionic compound is an anionic
polymer.
[0134] According to the invention, the anionic polymers are
selected from the group consisting of dextranmethylcarboxylic acid,
polyglutamic acid, polyaspartic acid, PAA (polyacrylic acid),
alginate, hyaluronic acid, polymers based on glucuronic acid or
based on galacturonic acid and their salts of Na.sup.+, K.sup.+,
Ca.sup.2+ or Mg.sup.2+.
[0135] In one embodiment, the anionic polymer has a number-average
molecular weight between 1 kg/mol and 15 kg/mol.
[0136] In one embodiment, the anionic polymer has a number-average
molecular weight between 1 kg/mol and 10 kg/mol.
[0137] In one embodiment, the anionic polymer has a number-average
molecular weight between 1 kg/mol and 8 kg/mol.
[0138] In one embodiment, the anionic polymer is a synthetic
dextran bearing carboxyl functions.
[0139] In one embodiment, the synthetic dextran bearing carboxyl
functions is selected from the group consisting of
carboxymethyldextran, carboxyethyldextran, dextran succinic acid,
dextran 2-butanedioic acid, dextran propanedioic acid.
[0140] In one embodiment, the anionic polymer is a
carboxymethyldextran whose mole fraction with respect to
carboxymethyl is between 0.5 and 3.
[0141] In one embodiment, the anionic polymer is a
carboxymethyldextran whose mole fraction with respect to
carboxymethyl is between 0.5 and 2.5.
[0142] In one embodiment, the anionic polymer is a
carboxymethyldextran whose mole fraction with respect to
carboxymethyl is between 1.0 and 2.0.
[0143] In one embodiment, the anionic polymer is a dextran succinic
acid whose mole fraction of succinic acid is between 0.5 and 3.
[0144] In one embodiment, the anionic polymer is a dextran succinic
acid whose mole fraction of succinic acid is between 0.5 and
2.5.
[0145] In one embodiment, the anionic polymer is a dextran succinic
acid whose mole fraction of succinic acid is between 1.0 and
2.0.
[0146] In one embodiment, the anionic polymer is a dextran
2-butanedioic acid whose mole fraction of 2-butanedioic acid is
between 0.2 and 3.
[0147] In one embodiment, the anionic polymer is a dextran
2-butanedioic acid whose mole fraction of 2-butanedioic acid is
between 0.5 and 2.5.
[0148] In one embodiment, the anionic polymer is a dextran
2-butanedioic acid whose mole fraction of 2-butanedioic acid is
between 0.7 and 2.0.
[0149] In one embodiment, the anionic polymer is a dextran
propanedioic acid whose mole fraction of propanedioic acid is
between 0.2 and 3.
[0150] In one embodiment, the anionic polymer is a dextran
propanedioic acid whose mole fraction of propanedioic acid is
between 0.5 and 2.5.
[0151] In one embodiment, the anionic polymer is a dextran
propanedioic acid whose mole fraction of propanedioic acid is
between 0.7 and 2.0.
[0152] In one embodiment, the polyanionic compound is selected from
anionic compounds consisting of a skeleton formed from a discrete
number p between 1 and 8 (1.ltoreq.p.ltoreq.8) of identical or
different saccharide units, bonded by identical or different
glycosidic bonds and naturally bearing carboxyl groups or
substituted with carboxyl groups, and salts thereof.
[0153] In one embodiment, the polyanionic compound consisting of a
skeleton formed from a discrete number of saccharide units is
obtained from a disaccharide compound selected from the group
comprising trehalose, maltose, lactose, sucrose, cellobiose,
isomaltose, maltitol and isomaltitol.
[0154] In one embodiment, the polyanionic compound consisting of a
skeleton formed from a discrete number of saccharide units is
obtained from a compound consisting of a skeleton formed from a
discrete number of saccharide units selected from the group
comprising maltotriose, maltotetraose, maltopentaose, maltohexaose,
maltoheptaose, maltooactose and isomaltotriose.
[0155] In one embodiment, the polyanionic compound consisting of a
skeleton formed from a discrete number of saccharide units is
selected from the group comprising carboxymethylmaltotriose,
carboxymethylmaltotetraose, carboxymethylmaltopentaose,
carboxymethylmaltohexaose, carboxymethylmaltoheptaose,
carboxymethylmaltooctaose and carboxymethylisomaltotriose.
[0156] In one embodiment, the insulin is a recombinant human
insulin as described in the European Pharmacopoeia and the United
States Pharmacopeia.
[0157] In one embodiment, the insulin is an insulin analog selected
from the group comprising insulin lispro (Humalog.RTM.), insulin
aspart (Novolog.RTM., Novorapid.RTM.) and insulin glulisine
(Apidra.RTM.).
[0158] In one embodiment, the oligosaccharide/insulin molar ratios
are between 0.2 and 7.
[0159] In one embodiment, the molar ratios are between 0.3 and
5.
[0160] In one embodiment, the molar ratios are between 0.6 and
4.
[0161] In one embodiment, the molar ratios are between 1 and 3.
[0162] In one embodiment, the molar ratios are between 1.2 and
3.
[0163] In one embodiment, the molar ratio is equal to 1.
[0164] In one embodiment, the molar ratio is equal to 2.
[0165] In one embodiment, the oligosaccharide/insulin weight ratios
are between 0.4 and 10.
[0166] In one embodiment, the weight ratios are between 0.6 and
7.
[0167] In one embodiment, the weight ratios are between 1.2 and
5.
[0168] In one embodiment, the weight ratios are between 1.6 and
4.
[0169] In one embodiment, the weight ratios are between 2 and
4.
[0170] In one embodiment, the concentration of functionalized
oligosaccharides is between 1.4 and 35 mg/mL.
[0171] In one embodiment, the concentration of functionalized
oligosaccharides is between 2.1 and 25 mg/mL.
[0172] In one embodiment, the concentration of functionalized
oligosaccharides is between 4.2 and 18 mg/mL.
[0173] In one embodiment, the concentration of functionalized
oligosaccharides is between 5.6 and 14 mg/mL.
[0174] In one embodiment, the concentration of functionalized
oligosaccharides is between 7 and 14 mg/mL.
[0175] In one embodiment, the concentration of anionic compound is
between 5 and 150 mM. In one embodiment, the concentration of
polyanionic compound is between 5 and 100 mM.
[0176] In one embodiment, the concentration of polyanionic compound
is between 5 and 75 mM.
[0177] In one embodiment, the concentration of polyanionic compound
is between 5 and 50 mM.
[0178] In one embodiment, the concentration of polyanionic compound
is between 5 and 30 mM.
[0179] In one embodiment, the concentration of polyanionic compound
is between 5 and 20 mM.
[0180] In one embodiment, the concentration of polyanionic compound
is between 5 and 10 mM.
[0181] In one embodiment, the concentration of polyanionic compound
is between 1 and 30 mg/mL.
[0182] In one embodiment, the concentration of polyanionic compound
is between 1.5 and 25 mg/mL.
[0183] In one embodiment, the concentration of polyanionic compound
is between 2 and 25 mg/mL.
[0184] In one embodiment, the concentration of polyanionic compound
is between 2 and 10 mg/mL.
[0185] In one embodiment, the concentration of polyanionic compound
is between 2 and 8 mg/mL.
[0186] In one embodiment, the oligosaccharide is sodium
dextranmethylcarboxylate modified with sodium phenylalaninate, q=4,
i=1.1, n=0.45.
[0187] In one embodiment, the oligosaccharide is sodium
dextranmethylcarboxylate modified with sodium phenylalaninate, q=4,
i=1.65, n=0.39.
[0188] In one embodiment, the oligosaccharide is sodium
dextranmethylcarboxylate modified with sodium phenylalaninate, q=4,
i=2.0, n=0.5.
[0189] In one embodiment, the oligosaccharide is sodium
dextranmethylcarboxylate modified with sodium phenylalaninate, q=4,
i=0.7, n=0.57.
[0190] In one embodiment, the oligosaccharide is sodium
dextranmethylcarboxylate modified with sodium phenylalaninate, q=4,
i=1.72, n=0.42.
[0191] In one embodiment, the oligosaccharide is sodium
dextranmethylcarboxylate modified with sodium phenylalaninate, q=4,
i=2.1, n=0.6.
[0192] In one embodiment, the polyanionic compound is sodium
dextranmethylcarboxylate.
[0193] In one embodiment, the polyanionic compound is sodium
citrate.
[0194] The composition can moreover be produced by simple mixing of
an aqueous solution of human insulin or insulin analog and of an
aqueous solution of oligosaccharide.
[0195] In one embodiment, the composition can be produced by simple
mixing of an aqueous solution of human insulin or insulin analog,
of an aqueous solution of oligosaccharide and of polyanionic
compound in solution or in the form of lyophilizate.
[0196] In one embodiment, the composition can be produced by simple
mixing of an aqueous solution of human insulin or insulin analog
and of oligosaccharide in the form of lyophilizate.
[0197] In one embodiment, the composition can be produced by simple
mixing of an aqueous solution of human insulin or insulin analog,
of oligosaccharide in the form of lyophilizate and of polyanionic
compound in solution or in the form of lyophilizate.
[0198] Preferably this composition is in the form of an injectable
solution.
[0199] In one embodiment, the concentration of human insulin or
insulin analog is between 240 and 3000 .mu.M (40 to 500 IU/mL).
[0200] In one embodiment, the concentration of human insulin or
insulin analog is between 600 and 1200 .mu.M (100 to 200
IU/mL).
[0201] In one embodiment, the concentration of human insulin or
insulin analog is 600 .mu.M (100 IU/mL).
[0202] In one embodiment, the concentration of human insulin or
insulin analog is 1200 .mu.M (200 IU/mL).
[0203] In one embodiment, the insulin concentration of the
solutions is 600 .mu.M or 100 IU/mL.
[0204] In one embodiment, the concentration of human insulin or
insulin analog of 600 .mu.M can be reduced by simple dilution, in
particular for pediatric applications.
[0205] The invention also relates to a pharmaceutical formulation
according to the invention, characterized in that it is obtained by
drying and/or lyophilization.
[0206] In the case of local and systemic release, the methods of
administration envisaged are by the intravenous, subcutaneous,
intradermal or intramuscular route.
[0207] The transdermal, oral, nasal, vaginal, ocular, buccal, and
pulmonary routes of administration are also envisaged.
[0208] The invention also relates to the use of a composition
according to the invention for the formulation of a solution of
human insulin or insulin analog with a concentration of 100 IU/mL
intended for implantable or portable insulin pumps.
[0209] The invention also relates to the use of a composition
according to the invention for the formulation of a solution of
human insulin or insulin analog with a concentration of 200 IU/mL
intended for implantable or portable insulin pumps.
BRIEF DESCRIPTION OF THE DRAWINGS
[0210] FIG. 1: the curves show that the formulation of human
insulin (curve plotted with squares corresponding to example B3,
Tmin glucose=61.+-.31 min) has a slower action than the commercial
formulation of insulin aspart (curve plotted with triangles
corresponding to example B1, Tmin glucose=44.+-.13 min).
[0211] FIG. 2: the curves show that the formulation of human
insulin alone (curve plotted with squares corresponding to example
B3, Tmax insulin=36.+-.33 min) induces slower absorption than the
commercial formulation of insulin aspart (Novolog.RTM.) (curve
plotted with triangles corresponding to example B1, Tmax
insulin=28.+-.13 min).
[0212] FIG. 3: the curves show that the formulation based on human
insulin comprising oligosaccharide 2 and polyanionic compound 1 as
excipient at 7.3 mg/mL (curve plotted with squares corresponding to
example B6, Tmin glucose=39.+-.11 min) has an action as rapid as
that of the commercial formulation of insulin aspart (Novolog.RTM.)
(curve plotted with triangles corresponding to example B1, Tmin
glucose=41.+-.9 min).
[0213] FIG. 4: the curves show that the formulation based on human
insulin comprising oligosaccharide 2 and polyanionic compound 1 as
excipients at 7.3 mg/mL (curve plotted with squares corresponding
to example B6, Tmax insulin=14.+-.9 min) induces an absorption that
is more rapid than the commercial formulation of insulin aspart
(Novolog.RTM.) (curve plotted with triangles corresponding to
example B1, Tmax insulin=24.+-.13 min).
[0214] FIG. 5: the curves show that the formulation based on human
insulin comprising oligosaccharide 2 and citrate at 9.3 mM as
excipients (curve plotted with squares corresponding to example B7,
Tmin glucose=36.+-.14 min) has a more rapid action than that of the
commercial formulation of insulin aspart (Novolog.RTM.) (curve
plotted with triangles corresponding to example B1, Tmin
glucose=53.+-.24 min).
[0215] FIG. 6: the curves show that the formulation comprising
oligosaccharide 2 and citrate at 9.3 mM as excipients (curve
plotted with squares corresponding to example B7, Tmax
insulin=15.+-.10 min) induces an absorption that is more rapid than
the commercial formulation of insulin aspart (Novolog.RTM.) (curve
plotted with triangles corresponding to example B1, Tmax
insulin=22.+-.10 min).
[0216] FIG. 7: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 9.3 mM as excipients (curve plotted with squares
corresponding to example B9, Tmin glucose=32.+-.9 min) has a more
rapid action than that of the commercial formulation of insulin
lispro (Humalog.RTM.) (curve plotted with triangles corresponding
to example B2, Tmin glucose=45.+-.16 min).
[0217] FIG. 8: the curves show that the formulation based on
Humalog.RTM. comprising oligosaccharide 2 and citrate at 9.3 mM as
excipient (curve plotted with squares corresponding to example B9,
Tmax insulin=12.+-.7 min) induces an absorption that is more rapid
than the commercial formulation of insulin lispro (Humalog.RTM.)
(curve plotted with triangles corresponding to example B2, Tmax
insulin=19.+-.10 min).
[0218] FIG. 9: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 6 mM as excipients (curve plotted with squares
corresponding to example B10, Tmin glucose=34.+-.12 min) has a more
rapid action than that of the commercial formulation of insulin
lispro (Humalog.RTM.) (curve plotted with triangles corresponding
to example B2, Tmin glucose=44.+-.14 min).
[0219] FIG. 10: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 6 mM as excipients (curve plotted with squares
corresponding to example B10, Tmax insulin=13.+-.8 min) induces an
absorption that is more rapid than the commercial formulation of
insulin lispro (Humalog.RTM.) (curve plotted with triangles
corresponding to example B2, Tmax insulin=18.+-.8 min).
[0220] FIG. 11: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 9.3 mM as excipients (curve plotted with squares
corresponding to example B11, Tmin glucose=31.+-.14 min) has a more
rapid action than that of the commercial formulation of insulin
lispro (Humalog.RTM.) (curve plotted with triangles corresponding
to example B2, Tmin glucose=44.+-.14 min).
[0221] FIG. 12: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 9.3 mM as excipients (curve plotted with squares
corresponding to example B11, Tmax insulin=15.+-.7 min) induces an
absorption that is more rapid than the commercial formulation of
insulin lispro (Humalog (curve plotted with triangles corresponding
to example B2, Tmax insulin=18.+-.8 min).
[0222] FIG. 13: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 18.6 mM as excipients (curve plotted with squares
corresponding to example B12, Tmin glucose=30.+-.5 min) has a more
rapid action than that of the commercial formulation of insulin
lispro (Humalog.RTM.) (curve plotted with triangles corresponding
to example B2, Tmin glucose=40.+-.12 min).
[0223] FIG. 14: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 18.6 mM as excipients (curve plotted with squares
corresponding to example B11, Tmax insulin=10.+-.4 min) induces an
absorption that is more rapid than the commercial formulation of
insulin lispro (Humalog.RTM.) (curve plotted with triangles
corresponding to example B2, Tmax insulin=23.+-.12 min).
[0224] FIG. 15: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipients at 7.3 mg/mL (curve plotted
with squares corresponding to example B13, Tmin glucose=32.+-.12
min) has a more rapid action than that of the commercial
formulation of insulin lispro (Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmin glucose=44.+-.14
min).
[0225] FIG. 16: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipients at 7.3 mg/mL (curve plotted
with squares corresponding to example B13, Tmax insulin=14.+-.7
min) induces a more rapid absorption than the commercial
formulation of insulin lispro Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmax insulin=18.+-.8
min).
[0226] FIG. 17: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipient at 14.6 mg/mL (curve plotted
with squares corresponding to example B14, Tmin glucose=30.+-.7
min) has a more rapid action than that of the commercial
formulation of insulin lispro (Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmin glucose=44.+-.14
min).
[0227] FIG. 18: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipient at 14.6 mg/mL (curve plotted
with squares corresponding to example B14, Tmax insulin=12.+-.5
min) induces a more rapid absorption of Humalog.RTM. than the
commercial formulation of insulin lispro (Humalog.RTM.) (curve
plotted with triangles corresponding to example B2, Tmax
insulin=18.+-.8 min).
[0228] FIG. 19: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 6 (curve
plotted with squares corresponding to example B8, Tmin
glucose=45.+-.19 min) has not an action that is more rapid than
that of the commercial formulation of insulin lispro (Humalog.RTM.)
(curve plotted with triangles corresponding to example B2, Tmin
glucose=50.+-.14 min).
[0229] FIG. 20: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 6 (curve
plotted with squares corresponding to example B8, Tmax
insulin=18.+-.10 min) does not induce a more rapid absorption of
Humalog.RTM. than the commercial formulation of insulin lispro
(Humalog.RTM.) (curve plotted with triangles corresponding to
example B2, Tmax insulin=20.+-.9 min).
[0230] FIG. 21: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 9.3 mM as excipient (curve plotted with squares
corresponding to example B9, Tmin glucose=32.+-.9 min) has a more
rapid action than that of the formulation based on insulin lispro
(Humalog.RTM.) comprising oligosaccharide 6 (curve plotted with
triangles corresponding to example B8, Tmin glucose=45.+-.19
min).
[0231] FIG. 22: the curves show that the formulation based on
Humalog.RTM. comprising oligosaccharide 2 and citrate at 9.3 mM as
excipient (curve plotted with squares corresponding to example B9,
Tmax insulin=12.+-.7 min) induces a more rapid absorption than the
formulation based on insulin lispro (Humalog.RTM.) comprising
oligosaccharide 6 (curve plotted with triangles corresponding to
example B8, Tmax insulin=18.+-.10 min).
[0232] FIG. 23: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 at 14.6
mg/mL (curve plotted with squares corresponding to example B15,
Tmin glucose=35.+-.5 min) has a more rapid action than that of the
commercial formulation of insulin lispro (Humalog.RTM.) (curve
plotted with triangles corresponding to example B2, Tmin
glucose=47.+-.18 min).
[0233] FIG. 24: the curves show that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 at 14.6
mg/Ml (curve plotted with squares corresponding to example B15,
Tmax insulin=12.+-.4 min) induces a more rapid absorption of
Humalog.RTM. than the commercial formulation of insulin lispro
(Humalog.RTM.) (curve plotted with triangles corresponding to
example B2, Tmax insulin=20.+-.11 min).
[0234] FIG. 25: the curves show that the formulation based on human
insulin comprising oligosaccharide 3 as excipient at 7.3 mg/mL
(curve plotted with squares corresponding to example B96, Tmin
glucose=46.+-.20 min) has a more rapid action than that of the
commercial formulation of human insulin (curve plotted with
triangles corresponding to example B3, Tmin glucose=64.+-.33
min).
[0235] FIG. 26: the curves shows that the formulation based on
human insulin comprising oligosaccharide 3 as excipient at 7.3
mg/mL (curve plotted with squares corresponding to example B96,
Tmax insulin=12.+-.6 min) induces an absorption that is more rapid
than the commercial formulation of human insulin (curve plotted
with triangles corresponding to example B3, Tmax insulin=26.+-.20
min).
[0236] FIG. 27: Bar charts representing CD signals at 251 nm
(degcm2dmol-1) intensities of (from left to right) Humalog.RTM.,
Citrate/Humalog.RTM., EDTA/Humalog.RTM., EDTA/Citrate/Humalog.RTM.,
Oligosaccharide 2/Citrate/Humalog.RTM. and Oligosaccharide
2/Oligosaccharide 3/Humalog.RTM. formulations. The charts show that
EDTA and the EDTA/citrate mixture completely destructures the R6
form of insulin lispro (Humalog.RTM.). EDTA therefore has a marked
effect of destabilization of the hexamer. In contrast, the citrate
alone, oligosaccharide 2 alone as well as the oligosaccharide
2/citrate mixture have almost no effect on the CD signal at 251 nm.
These compounds therefore have hardly any impact on the R6
structure of the hexamer and especially on the hexameric structure
of insulin, in contrast to EDTA, which destabilizes the
hexamer.
[0237] FIG. 28: Bar charts representing CD signals at 276 nm
(degcm2dmol-1) intensities of (from left to right) rhINS,
rhINS/EDTA, rhINS/Citrate, rhINS/EDTA/Citrate and
rhINS/Oligosaccharide 1 formulations. The bar charts show that EDTA
and the EDTA/Citrate combination have a very marked effect on the
hexameric structure of human insulin (complete dissociation of the
hexamer to dimers). Conversely, oligosaccharide 1 does not have a
significant effect on the hexameric structure of human insulin. In
contrast to EDTA, the formulations based on oligosaccharide 1 do
not dissociate the hexamer of human insulin.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Examples
TABLE-US-00001 [0238] AA. Oligosaccharides according to the
invention SUBSTITUENTS OLIGO- --F--R USUAL SACCHARIDES --F--R-Phe
NAME Oligosaccharide 1 q: 4 i: 1.1 n: 0.45 Oligosaccharide 2 q: 4
i: 1.65 n: 0.39 Oligosaccharide 3 q: 4 i: 2.0 n: 0.5
Oligosaccharide 4 q: 4 i: 0.7 n: 0.57 Oligosaccharide 5 q: 4 i:
1.72 n: 0.42 Oligosaccharide 6 q: 4 i: 2.1 n: 0.6 ##STR00003##
##STR00004## sodium dextran- methyl- carboxylate modified with
sodium phenyl- alaninate
AA1. Oligosaccharide 1: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0239] 40 g (0.74 mol of hydroxyl functions) of dextran with
weight-average molecular weight 1 kg/mol (DP=4, Pharmacosmos) and
115 g (0.99 mol) of sodium chloroacetate are dissolved in water at
65.degree. C. 123 mL of 10 N NaOH (1.23 mol) is added dropwise to
this solution and then the mixture is heated to 65.degree. C. The
mixture is then diluted with water, neutralized with acetic acid
and then purified by ultrafiltration on PES membrane of 1 kDa
against water. The oligosaccharide concentration of the final
solution is determined by dry extraction, then an acid/base assay
in a water/acetone mixture 50/50 (v/v) is carried out to determine
the average mole fraction of sodium methylcarboxylates.
[0240] According to dry extraction: [oligosaccharide]=37.6 mg/g
[0241] According to the acid base assay, the average mole fraction
of sodium methylcarboxylates is 1.1.
[0242] The solution of sodium dextranmethylcarboxylate is acidified
on a Purolite resin (anionic) to obtain dextranmethylcarboxylic
acid, which is then lyophilized for 18 hours.
[0243] 12 g of dextranmethylcarboxylic acid (61 mmol of
methylcarboxylic acid functions) is dissolved in DMF and then
cooled to 0.degree. C. A mixture of ethyl phenylalaninate,
hydrochloride salt (Bachem) (6 g, 26 mmol) in DMF is prepared. 2.6
g of triethylamine (26 mmol) is added to this mixture. A solution
of NMM (6.1 g, 61 mmol) and of EtOCOCl (6.6 g, 61 mmol) is then
added to the mixture at 0.degree. C. The solution of ethyl
phenylalaninate is then added and the mixture is stirred at
10.degree. C. An aqueous solution of imidazole is added and then
the mixture is heated to 30.degree. C. The medium is diluted with
water and then the solution obtained is purified by ultrafiltration
on PES membrane of 1 kDa against 0.1N NaOH, 0.9% NaCl and water.
The oligosaccharide concentration of the final solution is
determined by dry extraction. A sample of solution is lyophilized
and analyzed by .sup.1H NMR in D.sub.2O to determine the average
mole fraction of sodium methylcarboxylates functionalized with
sodium L-phenylalaninate.
[0244] According to dry extraction: [Oligosaccharide 1]=20.8
mg/g
[0245] According to .sup.1H NMR: the average mole fraction of
sodium methylcarboxylates functionalized with sodium
L-phenylalaninate is 0.45.
AA2. Oligosaccharide 2: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0246] 40 g (0.74 mol of hydroxyl functions) of dextran with
weight-average molecular weight 1 kg/mol (DP=4, Pharmacosmos) and
144 g (1.23 mol) of sodium chloroacetate are dissolved in water at
65.degree. C. 123 mL of 10 N NaOH (1.23 mol) is added dropwise to
this solution and then the mixture is heated at 65.degree. C. for
90 minutes. 86.3 g (0.74 mol) of sodium chloroacetate is then added
to the reaction mixture as well as 74.1 mL of 10N NaOH (0.74 mol)
dropwise and heating is continued at 65.degree. C. The mixture is
then diluted with water, neutralized with acetic acid and then
purified by ultrafiltration on PES membrane of 1 kDa against water.
The oligosaccharide concentration of the final solution is
determined by dry extraction, then an acid/base assay in a
water/acetone mixture 50/50 (v/v) is carried out to determine the
average mole fraction of sodium methylcarboxylates.
[0247] According to dry extraction: [oligosaccharide]=34.4 mg/g
[0248] According to the acid/base assay, the average mole fraction
of sodium methylcarboxylates is 1.65.
[0249] By a method similar to that used for preparing
oligosaccharide 1, a sodium dextranmethylcarboxylate functionalized
with sodium L-phenylalaninate is obtained.
[0250] According to dry extraction: [Oligosaccharide 2]=20.4
mg/g
[0251] According to .sup.1H NMR: average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.39.
AA3. Oligosaccharide 3: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0252] Oligosaccharide 3 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran of weight-average molecular weight 1 kg/mol (DP=4,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates optionally functionalized with sodium
L-phenylalaninate is 2.0. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.5.
[0253] This oligosaccharide is referenced polysaccharide 13 in the
priority document.
AA4. Oligosaccharide 4: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0254] 120 g (2.22 mol of hydroxyl functions) of dextran with
weight-average molecular weight 1 kg/mol (DP=4, Pharmacosmos) and
151 g (1.3 mol) of sodium chloroacetate are dissolved in water at
65.degree. C. 370 mL of 10 N NaOH (3.7 mol) is added dropwise to
this solution and then the mixture is heated to 65.degree. C. The
mixture is then diluted with water, neutralized with acetic acid
and then purified by ultrafiltration on PES membrane of 1 kDa
against water. The oligosaccharide concentration of the final
solution is determined by dry extraction, then an acid/base assay
in a water/acetone mixture 50/50 (v/v) is carried out to determine
the average mole fraction of sodium methylcarboxylates.
[0255] According to dry extraction: [oligosaccharide]=22.1 mg/g
[0256] According to the acid/base assay, the average mole fraction
of sodium methylcarboxylates is 0.7.
[0257] By a method similar to that used for preparing
oligosaccharide 1, a sodium dextranmethylcarboxylate functionalized
with sodium L-phenylalaninate is obtained.
[0258] According to dry extraction: [Oligosaccharide 4]=20.4
mg/g
[0259] According to .sup.1H NMR: the average mole fraction of
sodium methylcarboxylates functionalized with sodium
L-phenylalaninate is 0.57.
AA5. Oligosaccharide 5: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0260] Oligosaccharide 5 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran of weight-average molecular weight 1 kg/mol (DP=4,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates optionally functionalized with sodium
L-phenylalaninate is 1.72. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.42.
[0261] This oligosaccharide is referenced polysaccharide 12 in the
priority document.
AA6. Oligosaccharide 6: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0262] Oligosaccharide 6 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran of weight-average molecular weight 1 kg/mol (DP=4,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates optionally and functionalized with sodium
L-phenylalaninate is 2.1. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.6.
AB Polysaccharides, Counterexamples
[0263] AB1. Polysaccharide 1: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0264] Polysaccharide 1 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran of weight-average molecular weight 10 kg/mol (DP=39,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates is 1.06. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.43.
[0265] This polysaccharide corresponds to polysaccharide 1 of
application FR0901478.
AB2. Polysaccharide 2: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0266] Polysaccharide 2 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran with weight-average molecular weight 5 kg/mol (DP=19,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates is 1.65. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.39.
AB3. Polysaccharide 3: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0267] Polysaccharide 3 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran with weight-average molecular weight 5 kg/mol (DP=19,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates is 1.10. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.41.
AB4. Polysaccharide 4: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0268] Polysaccharide 4 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran with weight-average molecular weight 10 kg/mol (DP=39,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates is 1.65. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.39.
AB5. Polysaccharide 5: Sodium Dextranmethylcarboxylate
Functionalized with Sodium L-Phenylalaninate
[0269] Polysaccharide 5 is a sodium dextranmethylcarboxylate
functionalized with sodium L-phenylalaninate obtained from a
dextran with weight-average molecular weight 5 kg/mol (DP=19,
Pharmacosmos) according to the method described in patent
application FR 07/02316. The average mole fraction of sodium
methylcarboxylates is 1.10. The average mole fraction of sodium
methylcarboxylates functionalized with sodium L-phenylalaninate is
0.59.
AC Polyanionic Compounds
AC1. Polyanionic Compound 1: Sodium Dextranmethylcarboxylate
[0270] 40 g (0.74 mol of hydroxyl functions) of dextran with
weight-average molecular weight 1 kg/mol (DP=4, Pharmacosmos) and
144 g (1.23 mol) of sodium chloroacetate are dissolved in water at
60.degree. C. 123 mL of 10 N NaOH (1.23 mol) is added dropwise to
this solution and then the mixture is heated at 60.degree. C. for
90 minutes. 86.3 g (0.74 mol) of sodium chloroacetate is then added
to the reaction mixture as well as 74.1 mL of 10 N NaOH (0.74 mol)
dropwise. After 1 h of heating, the mixture is diluted with water,
neutralized with acetic acid and then purified by ultrafiltration
on PES membrane of 1 kDa against water. The oligosaccharide
concentration of the final solution is determined by dry
extraction, then an acid/base assay in a water/acetone mixture
50/50 (v/v) is carried out to determine the average mole fraction
of sodium methylcarboxylates.
[0271] According to dry extraction: [Polyanionic compound 1]=34.4
mg/g
[0272] According to the acid/base assay, the average mole fraction
of sodium methylcarboxylates is 1.65.
AC2. Polyanionic Compound 2: Sodium
Maltotriosemethylcarboxylate
[0273] Polyanionic compound 2 is a sodium
maltotriosemethylcarboxylate obtained by a method similar to that
used for preparing polyanionic compound 1. The average mole
fraction of sodium methylcarboxylates is 1.65.
B Preparation of the Solutions
B1. Solution of Rapid-Acting Insulin Analog Novolog.RTM. at 100
IU/mL.
[0274] This solution is a commercial solution of insulin aspart
from Novo Nordisk sold under the name Novolog.RTM.. This product is
a rapid-acting insulin aspart analog.
B2. Solution of Rapid-Acting Insulin Analog Humalog.RTM. at 100
IU/mL.
[0275] This solution is a commercial solution of insulin lispro
from Eli Lilly sold under the name Humalog.RTM.. This product is a
rapid-acting insulin analog.
B3. Solution of Regular Human Insulin Actrapid.RTM. at 100
IU/mL.
[0276] This solution is a commercial solution of human insulin from
Novo Nordisk sold under the name Actrapid.RTM.. This product is a
regular human insulin.
B4. Preparation of the Solutions of Excipients
[0277] Preparation of a solution of sodium citrate at 1.188 M.
[0278] A solution of sodium citrate is obtained by dissolving
9.0811 g of sodium citrate (30.9 mmol) in 25 mL of water in a
graduated flask. The pH is adjusted to exactly 7.4 by adding 1 mL
of 1M HCl. The solution is filtered on 0.22 .mu.m.
[0279] Preparation of a solution of m-cresol 130 mM.
[0280] A solution of m-cresol is obtained by dissolving 14.114 g of
m-cresol (130 mmol) in 986.4 mL of water in a 1 L graduated
flask.
[0281] Preparation of a solution of m-cresol and glycerin (96.6 mM
m-cresol and 566 mM glycerin).
[0282] 73.3 g of the solution of m-cresol at 130 mM is added to
5.26 g of glycerin and then diluted by adding 22.25 g of water. The
resultant solution of m-cresol and glycerin is homogenized for 30
minutes and then filtered on a 0.22 .mu.m membrane.
[0283] Preparation of a solution of Tween 20 at 32.7 mM.
[0284] A solution of Tween 20 is obtained by dissolving 2.0079 g of
Tween 20 (1.636 mmol) in 50 mL of water in a graduated flask. The
solution is filtered on a 0.22 .mu.m membrane.
B5. Preparation of a Solution of Human Insulin at 500 IU/mL.
[0285] 15 g of water is added to 563.6 mg of human insulin, then
the pH is lowered to acid pH by adding 5.98 g of 0.1N HCl. After
complete dissolution of the insulin at acid pH, the solution is
neutralized to pH 7.2 by adding 8.3 mL of 0.1N NaOH. The
concentration is then adjusted to 500 IU/mL by adding 0.76 g of
water. The solution is finally filtered on a 0.22 .mu.m
membrane.
B6. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and Polyanionic Compound 1.
[0286] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[polyanionic compound 1]/[insulin] of
2/2/1, the various reagents are mixed in the quantities stated
below:
TABLE-US-00002 Human insulin at 500 IU/mL 20 mL Solution of
polyanionic compound 1 at 34.74 mg/mL 21.01 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.99 mL Lyophilizate of
oligosaccharide 2 730 mg
The final pH is 7.4.+-.0.4.
[0287] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B7. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 9.3 mM of Citrate.
[0288] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2, the various reagents are
mixed in the quantities stated below:
TABLE-US-00003 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Solution of sodium
citrate at 1.188M 785 .mu.L
The final pH is 7.4.+-.0.4.
[0289] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B8. Preparation of a Solution of Insulin Lispro at 100 IU/mL in the
Presence of Oligosaccharide 6.
[0290] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 6]/[insulin lispro] of 2.0, the various
reagents are added in the quantities specified below:
TABLE-US-00004 Oligosaccharide 6 in lyophilized form 730 mg
Commercial solution of Humalog at 100 IU/ml 100 mL
[0291] The final pH is adjusted to 7.4.+-.0.4.
[0292] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B9. Preparation of a Solution of Insulin Lispro at 100 IU/mL in the
Presence of Oligosaccharide 2 and 9.3 mM of Citrate.
[0293] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 9.3 mM of citrate, the various reagents are added
in the quantities specified below:
TABLE-US-00005 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution of Humalog at 100 IU/ml 100 mL Solution of
sodium citrate at 1.188M 785 .mu.L
[0294] The final pH is adjusted to 7.4.+-.0.4. Optionally, 25 .mu.L
of solution of Tween 20 at 32.7 mM can be added to this solution
(final concentration of Tween 20=8 .mu.M).
[0295] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B10. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 6 mM of Citrate.
[0296] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 6 mM of citrate, the various reagents are added in
the quantities specified below:
TABLE-US-00006 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Solution of
sodium citrate at 1.188M 506 .mu.L
[0297] The final pH is adjusted to 7.4.+-.0.4.
[0298] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B11. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 9.3 mM of
Citrate.
[0299] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[lispro] of 2.0 and a concentration of
9.3 mM of citrate, the various reagents are added in the quantities
specified below:
TABLE-US-00007 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Solution of
sodium citrate at 1.188M 785 .mu.L
[0300] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B12. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 18.6 mM of Citrate.
[0301] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[lispro] of 2.0 and a concentration of
18.6 mM of citrate, the various reagents are added in the
quantities specified below:
TABLE-US-00008 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Solution of
sodium citrate at 1.188M 1570 .mu.L
[0302] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B13. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 7.3 mg/mL of Polyanionic
Compound 1.
[0303] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polyanionic compound 1]/[lispro] of
2/2/1, the various reagents are added in the quantities specified
below:
TABLE-US-00009 Oligosaccharide 2 in lyophilized form 730 mg
Polyanionic compound 1 in lyophilized form 730 mg Commercial
solution Humalog .RTM. 100 IU/ml 100 mL
[0304] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B14. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 14.6 mg/mL of Polyanionic
Compound 1.
[0305] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polyanionic compound 1]/[lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00010 Oligosaccharide 2 in lyophilized form 730 mg
Polyanionic compound 1 in lyophilized form 1460 mg Commercial
solution Humalog .RTM. 100 IU/ml 100 mL
[0306] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.M membrane and stored at 4.degree. C.
B15. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 at 14.6 mg/mL.
[0307] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 4, the various
reagents are added in the specified quantities:
TABLE-US-00011 Oligosaccharide 2 in lyophilized form 1460 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL
[0308] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B16. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 80 mM of Sodium
Tartrate.
[0309] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 80 mM of sodium tartrate, the various reagents are
added in the quantities specified below:
TABLE-US-00012 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium tartrate
1.552 g
[0310] For the tartrate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0311] The final pH is adjusted to 7.4.+-.0.4.
[0312] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B17. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 60 mM of
Phosphate.
[0313] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 60 mM of phosphate, the various reagents are added
in the quantities specified below:
TABLE-US-00013 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL
Na.sub.2HPO.sub.4.cndot.12H.sub.2O 2.148 g
[0314] For the phosphate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0315] The final pH is adjusted to 7.4.+-.0.4.
[0316] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
[0317] B18. Preparation of a Solution of Insulin Analog Lispro at
100 IU/mL in the Presence of Oligosaccharide 2 and 80 mM of Sodium
Aspartate.
[0318] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 80 mM of sodium aspartate, the various reagents
are added in the quantities specified below:
TABLE-US-00014 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium aspartate
1.416 g
[0319] For the aspartate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0320] The final pH is adjusted to 7.4.+-.0.4.
[0321] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B19. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 100 mM of Sodium
Glutamate.
[0322] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 100 mM of sodium glutamate, the various reagents
are added in the quantities specified below:
TABLE-US-00015 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium glutamate
1.691 g
[0323] For the glutamate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0324] The final pH is adjusted to 7.4.+-.0.4.
[0325] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B20. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 60 mM of Malic
Acid.
[0326] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 60 mM of malic acid, the various reagents are
added in the quantities specified below:
TABLE-US-00016 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Malic acid 0.805
g
[0327] For the malic acid, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0328] The final pH is adjusted to 7.4.+-.0.4.
[0329] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B21. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 80 mM of Sodium
Succinate.
[0330] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 80 mM of sodium succinate, the various reagents
are added in the quantities specified below:
TABLE-US-00017 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium succinate
1.296 g
[0331] For the succinate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0332] The final pH is adjusted to 7.4.+-.0.4.
[0333] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B22. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 50 mM of Sodium
Adipate.
[0334] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 50 mM of sodium adipate, the various reagents are
added in the quantities specified below:
TABLE-US-00018 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium adipate
0.951 g
[0335] For the adipate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0336] The final pH is adjusted to 7.4.+-.0.4.
[0337] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B23. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 80 mM of Sodium
Ascorbate.
[0338] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 80 mM of sodium ascorbate, the various reagents
are added in the quantities specified below:
TABLE-US-00019 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium ascorbate
1.585 g
[0339] For the ascorbate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0340] The final pH is adjusted to 7.4.+-.0.4.
[0341] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B24. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 2 and 10 mM of Sodium
Oxalate.
[0342] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[insulin lispro] of 2.0 and a
concentration of 10 mM of sodium oxalate, the various reagents are
added in the quantities specified below:
TABLE-US-00020 Oligosaccharide 2 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium oxalate
134 mg
[0343] For the oxalate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0344] The final pH is adjusted to 7.4.+-.0.4.
[0345] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B25. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 14.6 mg/mL of Polyglutamic
Acid.
[0346] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polyglutamic acid]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00021 Oligosaccharide 2 in lyophilized form 730 mg
Polyglutamic acid in lyophilized form 1460 mg Commercial solution
Humalog .RTM. 100 IU/ml 100 mL
For the polyglutamic acid, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0347] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B26. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 14.6 mg/mL of Polyaspartic
Acid.
[0348] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polyaspartic acid]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00022 Oligosaccharide 2 in lyophilized form 730 mg
Polyaspartic acid in lyophilized form 1460 mg Commercial solution
Humalog .RTM. 100 IU/ml 100 mL
[0349] For the polyaspartic acid, it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0350] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B27. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 14.6 mg/mL of Polyanionic
Compound 2.
[0351] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polyanionic compound 2]/[insulin lispro]
of 2/4/1, the various reagents are added in the quantities
specified below:
TABLE-US-00023 Oligosaccharide 2 in lyophilized form 730 mg
Polyanionic compound 2 in lyophilized form 1460 mg Commercial
solution Humalog .RTM. 100 IU/ml 100 mL
[0352] The polyanionic compound 2 can be used in the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0353] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B28. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 7.3 mg/mL of Sodium
Triphosphate.
[0354] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[triphosphate]/[insulin lispro] of 2/2/1,
the various reagents are added in the quantities specified
below:
TABLE-US-00024 Oligosaccharide 2 in lyophilized form 730 mg Sodium
triphosphate or polyphosphate 730 mg Commercial solution Humalog
.RTM. 100 IU/ml 100 mL
[0355] For the triphosphate or polyphosphate, it is possible to use
the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0356] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B29. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 14.6 mg/mL of Poly(Acrylic
Acid).
[0357] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[poly(acrylic acid)]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00025 Oligosaccharide 2 in lyophilized form 730 mg
Poly(acrylic acid) in lyophilized form 1460 mg Commercial solution
Humalog .RTM. 100 IU/ml 100 mL
[0358] For the poly(acrylic acid), it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0359] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.M membrane and stored at 4.degree. C.
B30. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 14.6 mg/mL of (Low Molecular
Weight) Sodium Alginate.
[0360] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[sodium alginate]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00026 Oligosaccharide 2 in lyophilized form 730 mg Sodium
alginate 1460 mg Commercial solution Humalog .RTM. 100 IU/ml 100
mL
[0361] For the alginate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0362] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B31. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 21.9 mg/mL of Polymer Based
on Glucuronic Acid.
[0363] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polymer based on glucuronic
acid]/[insulin lispro] of 2/6/1, the various reagents are added in
the quantities specified below:
TABLE-US-00027 Oligosaccharide 2 in lyophilized form 730 mg Polymer
based on glucuronic acid 2190 mg Commercial solution Humalog .RTM.
100 IU/ml 100 mL
[0364] For the polymers based on glucuronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0365] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B32. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 21.9 mg/mL of Polymer Based
on Galacturonic Acid.
[0366] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polymer based on galacturonic
acid]/[insulin lispro] of 2/6/1, the various reagents are added in
the quantities specified below:
TABLE-US-00028 Oligosaccharide 2 in lyophilized form 730 mg Polymer
based on galacturonic acid 2190 mg Commercial solution Humalog
.RTM. 100 IU/ml 100 mL
[0367] For the polymers based on galacturonic acid, it is possible
to use the acid form or the basic form in the form of sodium salt,
of potassium salt or of some other salt compatible with an
injectable formulation.
[0368] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B33. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 2 and 21.9 mg/mL of Polymer Based
on Hyaluronic Acid.
[0369] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 2]/[polymer based on hyaluronic
acid]/[insulin lispro] of 2/6/1, the various reagents are added in
the quantities specified below:
TABLE-US-00029 Oligosaccharide 2 in lyophilized form 730 mg Polymer
based on hyaluronic acid 2190 mg Commercial solution Humalog .RTM.
100 IU/ml 100 mL
[0370] For the polymers based on hyaluronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0371] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.M membrane and stored at 4.degree. C.
B34. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 80 mM of
Tartrate.
[0372] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 80 mM of tartrate, the various reagents are added
in the quantities specified below:
TABLE-US-00030 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium tartrate
1.552 g
[0373] For the tartrate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0374] The final pH is adjusted to 7.4.+-.0.4.
[0375] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B35. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 60 mM of
Phosphate.
[0376] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 60 mM of phosphate, the various reagents are added
in the quantities specified below:
TABLE-US-00031 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL
Na.sub.2HPO.sub.4.cndot.12H.sub.2O 2.148 g
[0377] For the phosphate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0378] The final pH is adjusted to 7.4.+-.0.4.
[0379] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B36. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 80 mM of Sodium
Aspartate.
[0380] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 80 mM of sodium aspartate, the various reagents
are added in the quantities specified below:
TABLE-US-00032 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium aspartate
1.416 g
[0381] For the aspartate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0382] The final pH is adjusted to 7.4.+-.0.4.
[0383] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B37. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 100 mM of Sodium
Glutamate.
[0384] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 100 mM of sodium glutamate, the various reagents
are added in the quantities specified below:
TABLE-US-00033 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium glutamate
1.691 g
[0385] For the glutamate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0386] The final pH is adjusted to 7.4.+-.0.4.
[0387] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B38. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 60 mM of Malic
Acid.
[0388] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 60 mM of malic acid, the various reagents are
added in the quantities specified below:
TABLE-US-00034 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Malic acid 0.805
g
[0389] For the malic acid, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0390] The final pH is adjusted to 7.4.+-.0.4.
[0391] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B39. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 80 mM of Sodium
Succinate.
[0392] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 80 mM of sodium succinate, the various reagents
are added in the quantities specified below:
TABLE-US-00035 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium succinate
1.296 g
[0393] For the succinate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0394] The final pH is adjusted to 7.4.+-.0.4.
[0395] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B40. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 50 mM of Sodium
Adipate.
[0396] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 50 mM of sodium adipate, the various reagents are
added in the quantities specified below:
TABLE-US-00036 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium adipate
0.951 g
[0397] For the adipate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0398] The final pH is adjusted to 7.4.+-.0.4.
[0399] The clear solution is filtered on a 0.22 .mu.M membrane and
stored at 4.degree. C.
B41. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the presence of Oligosaccharide 1 and 80 mM of Sodium
Ascorbate.
[0400] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 80 mM of sodium ascorbate, the various reagents
are added in the quantities specified below:
TABLE-US-00037 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium ascorbate
1.585 g
[0401] For the ascorbate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0402] The final pH is adjusted to 7.4.+-.0.4.
[0403] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B42. Preparation of a Solution of Insulin Analog Lispro at 100
IU/mL in the Presence of Oligosaccharide 1 and 10 mM of Sodium
Oxalate.
[0404] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[insulin lispro] of 2.0 and a
concentration of 10 mM of sodium oxalate, the various reagents are
added in the quantities specified below:
TABLE-US-00038 Oligosaccharide 1 in lyophilized form 730 mg
Commercial solution Humalog .RTM. 100 IU/ml 100 mL Sodium oxalate
134 mg
[0405] For the oxalate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0406] The final pH is adjusted to 7.4.+-.0.4.
[0407] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B43. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 14.6 Mg/mL of Polyglutamic
Acid.
[0408] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[polyglutamic acid]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00039 Oligosaccharide 1 in lyophilized form 730 mg
Polyglutamic acid in lyophilized form 1460 mg Commercial solution
Humalog .RTM. 100 IU/ml 100 mL
[0409] For the polyglutamic acid, it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0410] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B44. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 14.6 mg/mL of Polyaspartic
Acid.
[0411] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[polyaspartic acid]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00040 Oligosaccharide 1 in lyophilized form 730 mg
Polyaspartic acid in lyophilized form 1460 mg Commercial solution
Humalog .RTM. 100 IU/ml 100 mL
[0412] For the polyaspartic acid, it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0413] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B45. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 14.6 mg/mL of Polyanionic
Compound 2.
[0414] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[polyanionic compound 2]/[insulin lispro]
of 2/4/1, the various reagents are added in the quantities
specified below:
TABLE-US-00041 Oligosaccharide 1 in lyophilized form 730 mg
Polyanionic compound 2 in lyophilized form 1460 mg Commercial
solution Humalog .RTM. 100 IU/ml 100 mL
[0415] The polyanionic compound 2 can be used in the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0416] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B46. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 7.3 mg/mL of Sodium
Triphosphate.
[0417] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[triphosphate]/[insulin lispro] of 2/2/1,
the various reagents are added in the quantities specified
below:
TABLE-US-00042 Oligosaccharide 1 in lyophilized form 730 mg Sodium
triphosphate or polyphosphate 730 mg Commercial solution Humalog
.RTM. 100 IU/ml 100 mL
[0418] For the triphosphate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0419] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B47. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 14.6 mg/mL of Poly(Acrylic
Acid).
[0420] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[poly(acrylic acid)]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00043 Oligosaccharide 1 in lyophilized form 730 mg
Poly(acrylic acid) in lyophilized form 1460 mg Commercial solution
Humalog .RTM. 100 IU/ml 100 mL
[0421] For the poly(acrylic acid), it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0422] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B48. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 14.6 mg/mL of (Low Molecular
Weight) Sodium Alginate.
[0423] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[sodium alginate]/[insulin lispro] of
2/4/1, the various reagents are added in the quantities specified
below:
TABLE-US-00044 Oligosaccharide 1 in lyophilized form 730 mg Sodium
alginate 1460 mg Commercial solution Humalog .RTM. 100 IU/ml 100
mL
[0424] For the alginate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0425] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B49. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 21.9 mg/mL of Polymer Based
on Glucuronic Acid.
[0426] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[polymer based on glucuronic
acid]/[insulin lispro] of 2/6/1, the various reagents are added in
the quantities specified below:
TABLE-US-00045 Oligosaccharide 1 in lyophilized form 730 mg Polymer
based on glucuronic acid 2190 mg Commercial solution Humalog .RTM.
100 IU/ml 100 mL
[0427] For the polymers based on glucuronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0428] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.M membrane and stored at 4.degree. C.
B50. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 21.9 mg/mL of Polymer Based
on Galacturonic Acid.
[0429] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[polymer based on galacturonic
acid]/[insulin lispro] of 2/6/1, the various reagents are added in
the quantities specified below:
TABLE-US-00046 Oligosaccharide 1 in lyophilized form 730 mg Polymer
based on galacturonic acid 2190 mg Commercial solution Humalog
.RTM. 100 IU/ml 100 mL
[0430] For the polymers based on galacturonic acid, it is possible
to use the acid form or the basic form in the form of sodium salt,
of potassium salt or of some other salt compatible with an
injectable formulation.
[0431] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.M membrane and stored at 4.degree. C.
B51. Preparation of a Solution of Insulin Lispro at 100 IU/mL in
the Presence of Oligosaccharide 1 and 21.9 mg/mL of Polymer Based
on Hyaluronic Acid.
[0432] For a final volume of 100 mL of formulation, with a weight
ratio [oligosaccharide 1]/[polymer based on hyaluronic
acid]/[insulin lispro] of 2/6/1, the various reagents are added in
the quantities specified below:
TABLE-US-00047 Oligosaccharide 1 in lyophilized form 730 mg Polymer
based on hyaluronic acid 2190 mg Commercial solution Humalog .RTM.
100 IU/ml 100 mL
[0433] For the polymers based on hyaluronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0434] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.m membrane and stored at 4.degree. C.
B52. Preparation of a Solution of Insulin Analog (Insulin Lispro)
at 200 IU/mL.
[0435] The commercial formulation of insulin lispro (Humalog.RTM.)
was concentrated using AMICON Ultra-15 centrifugation tubes with a
cutoff at 3 kDa. The Amicon tubes were first rinsed with 12 mL of
deionized water. 12 mL of the commercial formulation was
centrifuged for 35 minutes at 4000 g at 20.degree. C. The volume of
retentate was measured and the concentration was estimated from the
volume of retentate. All retentates were combined and the total
concentration was estimated (>200 IU/mL).
[0436] The concentration of this concentrated solution of lispro
was adjusted to 200 IU/mL by adding the commercial formulation of
insulin lispro (Humalog.RTM.). The concentrated formulation of
concentrated insulin lispro has the same concentrations of
excipients (m-cresol, glycerin, phosphate) as the commercial
formulation at 100 IU/mL.
[0437] The final pH is adjusted to 7.4.+-.0.4. The clear solution
is filtered on a 0.22 .mu.M membrane and stored at 4.degree. C.
B53. Preparation of a Solution of Insulin Lispro at 200 IU/mL in
the Presence of Oligosaccharide 2 at 14.6 mg/mL and 9.3 mM of
Citrate.
[0438] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2/Lispro] of 2, the various reagents are
mixed in the quantities stated below
TABLE-US-00048 Insulin lispro at 200 IU/mL 100 mL Lyophilizate of
oligosaccharide 2 1460 mg Solution of sodium citrate at 1.188M 1570
.mu.L
[0439] The final pH is adjusted to 7.4.+-.0.4.
[0440] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B54. Preparation of a Solution of Insulin Lispro at 200 IU/mL in
the Presence of Oligosaccharide 2 at 14.6 mg/mL and of Polyanionic
Compound 1 at 14.6 Mg/mL.
[0441] For a final volume of 100 mL of formulation with a weight
ratio [(oligosaccharide 2/polyanionic compound 1/lispro] of 2/2/1,
the various reagents are mixed in the quantities stated below.
TABLE-US-00049 Insulin lispro at 200 IU/mL 100 mL Lyophilizate of
oligosaccharide 2 1460 mg Lyophilizate of polyanionic compound 1
1460 mg
[0442] The final pH is adjusted to 7.4.+-.0.4.
[0443] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B55. Preparation of a Solution of Insulin Lispro at 200 IU/mL in
the Presence of Oligosaccharide 2 at 14.6 mg/mL and of Polyanionic
Compound 1 at 29.2 mg/mL.
[0444] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2/polyanionic compound 1/lispro] of 2/4/1,
the various reagents are mixed in the quantities stated below.
TABLE-US-00050 Insulin lispro at 200 IU/mL 100 mL Lyophilizate of
oligosaccharide 2 1460 mg Lyophilizate of polyanionic compound 1
2920 mg
[0445] The final pH is adjusted to 7.4.+-.0.4.
[0446] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B56: Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 80 mM of Tartrate.
[0447] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 80 mM of tartrate, the
various reagents are mixed in the quantities stated below:
TABLE-US-00051 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium tartrate 1.552
g
[0448] For the tartrate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0449] The final pH is 7.4.+-.0.4.
[0450] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B57. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 80 mM of Phosphate.
[0451] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 80 mM of phosphate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00052 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL
Na.sub.2HPO.sub.4.cndot.12H.sub.2O 2.864 g
[0452] For the phosphate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0453] The final pH is 7.4.+-.0.4.
[0454] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B58 Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 80 mM of Aspartate.
[0455] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 80 mM of aspartate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00053 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium aspartate
1.416 g
[0456] For the aspartate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0457] The final pH is 7.4.+-.0.4.
[0458] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B59. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 100 mM of Glutamate.
[0459] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 100 mM of glutamate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00054 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium glutamate
1.691 g
[0460] For the glutamate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0461] The final pH is 7.4.+-.0.4.
[0462] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B60. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 60 mM of Malic Acid.
[0463] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 60 mM of malic acid,
the various reagents are mixed in the quantities stated below:
TABLE-US-00055 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Malic acid 0.805
g
[0464] For the malic acid, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0465] The final pH is 7.4.+-.0.4.
[0466] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B61. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 80 mM of Succinate.
[0467] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 80 mM of succinate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00056 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium succinate
1.296 g
[0468] For the succinate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0469] The final pH is 7.4.+-.0.4.
[0470] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B62. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 50 mM of Adipate.
[0471] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 50 mM of adipate, the
various reagents are mixed in the quantities stated below:
TABLE-US-00057 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium adipate 0.951
g
[0472] For the adipate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0473] The final pH is 7.4.+-.0.4.
[0474] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B63: Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 80 mM of Ascorbate.
[0475] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 80 mM of ascorbate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00058 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium ascorbate
1.585 g
[0476] For the ascorbate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0477] The final pH is 7.4.+-.0.4.
[0478] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B64: Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 10 mM of Oxalate.
[0479] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 2 and 10 mM of oxalate, the
various reagents are mixed in the quantities stated below:
TABLE-US-00059 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium oxalate 134
mg
[0480] For the oxalate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0481] The final pH is 7.4.+-.0.4.
[0482] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B65: Preparation of a Solution of Human Insulin at 100 mg/mL in the
Presence of oligosaccharide 2 and 14.6 mg/mL of polyglutamic
acid.
[0483] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[polyglutamic acid]/[insulin] of 2/4/1,
the various reagents are mixed in the quantities stated below:
TABLE-US-00060 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polyglutamic acid
1460 mg
[0484] For the polyglutamic acid, it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0485] The final pH is 7.4.+-.0.4.
[0486] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B66: Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 14.6 mg/mL of Polyaspartic
Acid.
[0487] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[polyaspartic acid]/[insulin] of 2/4/1,
the various reagents are mixed in the quantities stated below:
TABLE-US-00061 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polyaspartic acid
1460 mg
[0488] For the polyaspartic acid, it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0489] The final pH is 7.4.+-.0.4.
[0490] This clear solution is filtered on a 0.22 .mu.M membrane and
is then stored at +4.degree. C.
B67: Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 14.6 mg/mL of Polyanionic
Compound 2.
[0491] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[polyanionic compound 2]/[insulin] of
2/4/1, the various reagents are mixed in the quantities stated
below:
TABLE-US-00062 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polyanionic compound
2 1460 mg
[0492] The polyanionic compound 2 can be used in the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0493] The final pH is 7.4.+-.0.4.
[0494] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B68. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 7.3 mg/mL of Triphosphate.
[0495] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[triphosphate]/[insulin] of 2/2/1, the
various reagents are mixed in the quantities stated below:
TABLE-US-00063 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium triphosphate
or polyphosphate 730 mg
[0496] For the triphosphate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0497] The final pH is 7.4.+-.0.4.
[0498] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B69. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 14.6 mg/mL of Poly(Acrylic
Acid).
[0499] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[poly(acrylic acid)]/[insulin] of 2/4/1,
the various reagents are mixed in the quantities stated below:
TABLE-US-00064 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Poly(acrylic acid)
1460 mg
[0500] For the poly(acrylic acid), it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0501] The final pH is 7.4.+-.0.4.
[0502] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B70. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 14.6 mg/mL of (Low Molecular
Weight) Sodium Alginate.
[0503] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[alginate]/[insulin] of 2/4/1, the
various reagents are mixed in the quantities stated below:
TABLE-US-00065 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Alginate 1460 mg
[0504] For the alginate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0505] The final pH is 7.4.+-.0.4.
[0506] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B71. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 21.9 mg/mL of Polymer Based on
Glucuronic Acid.
[0507] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[polymer based on glucuronic
acid]/[insulin] of 2/6/1, the various reagents are mixed in the
quantities stated below:
TABLE-US-00066 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polymer based on
glucuronic acid 2190 mg
[0508] For the polymer based on glucuronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0509] The final pH is 7.4.+-.0.4.
[0510] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B72. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 21.9 mg/mL of Polymer Based on
Galacturonic Acid.
[0511] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[polymer based on galacturonic
acid]/[insulin] of 2/6/1, the various reagents are mixed in the
quantities stated below:
TABLE-US-00067 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polymer based on
galacturonic acid 2190 mg
[0512] For the polymer based on galacturonic acid, it is possible
to use the acid form or the basic form in the form of sodium salt,
of potassium salt or of some other salt compatible with an
injectable formulation.
[0513] The final pH is 7.4.+-.0.4.
[0514] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B73. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 2 and 21.9 mg/mL of Polymer Based on
Hyaluronic Acid.
[0515] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[polymer based on hyaluronic
acid]/[insulin] of 2/6/1, the various reagents are mixed in the
quantities stated below:
TABLE-US-00068 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 2 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polymer based on
hyaluronic acid 2190 mg
[0516] For the polymer based on hyaluronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0517] The final pH is 7.4.+-.0.4.
[0518] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B74. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 80 mM of Tartrate.
[0519] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 80 mM of tartrate, the
various reagents are mixed in the quantities stated below:
TABLE-US-00069 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium tartrate 1.552
g
[0520] For the tartrate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0521] The final pH is 7.4.+-.0.4.
[0522] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B75. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 80 mM of Phosphate.
[0523] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 80 mM of phosphate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00070 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL
Na.sub.2HPO.sub.4.cndot.12H.sub.2O 2.864 g
[0524] For the phosphate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0525] The final pH is 7.4.+-.0.4.
[0526] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B76: Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 80 mM of Aspartate.
[0527] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 80 mM of aspartate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00071 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium aspartate
1.416 g
[0528] For the aspartate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0529] The final pH is 7.4.+-.0.4.
[0530] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B77. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 100 mM of Glutamate.
[0531] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 100 mM of glutamate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00072 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium glutamate
1.691 g
[0532] For the glutamate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0533] The final pH is 7.4.+-.0.4.
[0534] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B78. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 60 mM of Malic Acid.
[0535] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2]/[insulin] of 1 and 60 mM of malic acid,
the various reagents are mixed in the quantities stated below:
TABLE-US-00073 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Malic acid 0.805
g
[0536] For the malic acid, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0537] The final pH is 7.4.+-.0.4.
[0538] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B79. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 80 mM of Succinate.
[0539] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 80 mM of succinate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00074 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium succinate
1.296 g
[0540] For the succinate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0541] The final pH is 7.4.+-.0.4.
[0542] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B80. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 50 mM of Adipate.
[0543] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 50 mM of adipate, the
various reagents are mixed in the quantities stated below:
TABLE-US-00075 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium adipate 0.951
g
[0544] For the adipate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0545] The final pH is 7.4.+-.0.4.
[0546] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B81. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 80 mM of Ascorbate.
[0547] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 80 mM of ascorbate,
the various reagents are mixed in the quantities stated below:
TABLE-US-00076 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium ascorbate
1.585 g
[0548] For the ascorbate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0549] The final pH is 7.4.+-.0.4.
[0550] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B82. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 10 mM of Oxalate
[0551] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[insulin] of 2 and 10 mM of oxalate, the
various reagents are mixed in the quantities stated below:
TABLE-US-00077 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium oxalate 134
mg
[0552] For the oxalate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0553] The final pH is 7.4.+-.0.4.
[0554] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B83. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 14.6 mg/mL of Polyglutamic
Acid.
[0555] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[polyglutamic acid]/[insulin] of 2/4/1,
the various reagents are mixed in the quantities stated below:
TABLE-US-00078 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polyglutamic acid
1460 mg
[0556] For the polyglutamic acid, it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0557] The final pH is 7.4.+-.0.4.
[0558] This clear solution is filtered on a 0.22 .mu.M membrane and
is then stored at +4.degree. C.
B84. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 14.6 mg/mL of Polyaspartic
Acid.
[0559] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[polyaspartic acid]/[insulin] of 2/4/1,
the various reagents are mixed in the quantities stated below:
TABLE-US-00079 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polyaspartic acid
1460 mg
[0560] For the polyaspartic acid, it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0561] The final pH is 7.4.+-.0.4.
[0562] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B85. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 14.6 mg/mL of Polyanionic
Compound 2.
[0563] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[polyanionic compound 2]/[insulin] of
2/4/1, the various reagents are mixed in the quantities stated
below:
TABLE-US-00080 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polyanionic compound
2 1460 mg
[0564] The polyanionic compound 2 can be used in the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0565] The final pH is 7.4.+-.0.4.
[0566] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B86. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 7.3 mg/mL of Triphosphate.
[0567] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[triphosphate]/[insulin] of 2/2/1, the
various reagents are mixed in the quantities stated below:
TABLE-US-00081 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Sodium triphosphate
or polyphosphate 730 mg
[0568] For the triphosphate, it is possible to use the acid form or
the basic form in the form of sodium salt, of potassium salt or of
some other salt compatible with an injectable formulation.
[0569] The final pH is 7.4.+-.0.4.
[0570] This clear solution is filtered on a 0.22 .mu.M membrane and
is then stored at +4.degree. C.
B87. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 14.6 mg/mL of Poly(Acrylic
Acid).
[0571] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[poly(acrylic acid)]/[insulin] of 2/4/1,
the various reagents are mixed in the quantities stated below:
TABLE-US-00082 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Poly(acrylic acid)
1460 mg
[0572] For the poly(acrylic acid), it is possible to use the acid
form or the basic form in the form of sodium salt, of potassium
salt or of some other salt compatible with an injectable
formulation.
[0573] The final pH is 7.4.+-.0.4.
[0574] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B88. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 14.6 mg/mL of (Low Molecular
Weight) Sodium Alginate.
[0575] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[alginate]/[insulin] of 2/4/1, the
various reagents are mixed in the quantities stated below:
TABLE-US-00083 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Alginate 1460 mg
[0576] For the alginate, it is possible to use the acid form or the
basic form in the form of sodium salt, of potassium salt or of some
other salt compatible with an injectable formulation.
[0577] The final pH is 7.4.+-.0.4.
[0578] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B89. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 21.9 mg/mL of Polymer Based on
Glucuronic Acid.
[0579] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[polymer based on glucuronic
acid]/[insulin] of 2/6/1, the various reagents are mixed in the
quantities stated below:
TABLE-US-00084 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polymer based on
glucuronic acid 2190 mg
[0580] For the polymer based on glucuronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0581] The final pH is 7.4.+-.0.4.
[0582] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B90. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 21.9 mg/mL of Polymer Based on
Galacturonic Acid.
[0583] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[polymer based on galacturonic
acid]/[insulin] of 2/6/1, the various reagents are mixed in the
quantities stated below:
TABLE-US-00085 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polymer based on
galacturonic acid 2190 mg
[0584] For the polymer based on galacturonic acid, it is possible
to use the acid form or the basic form in the form of sodium salt,
of potassium salt or of some other salt compatible with an
injectable formulation.
[0585] The final pH is 7.4.+-.0.4.
[0586] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B91. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 1 and 21.9 mg/mL of Polymer Based on
Hyaluronic Acid.
[0587] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 1]/[polymer based on hyaluronic
acid]/[insulin] of 2/6/1, the various reagents are mixed in the
quantities stated below:
TABLE-US-00086 Human insulin at 500 IU/mL 20 mL Solution of
oligosaccharide 1 at 36.01 mg/mL 20.27 mL Solution 96.6 mM
m-cresol/566 mM glycerin 30 mL Water 28.95 mL Polymer based on
hyaluronic acid 2190 mg
[0588] For the polymer based on hyaluronic acid, it is possible to
use the acid form or the basic form in the form of sodium salt, of
potassium salt or of some other salt compatible with an injectable
formulation.
[0589] The final pH is 7.4.+-.0.4.
[0590] This clear solution is filtered on a 0.22 .mu.m membrane and
is then stored at +4.degree. C.
B92. Preparation of a Solution of Human Insulin at 200 IU/mL.
[0591] 60.4 g of water is added to 884.7 mg of human insulin
comprising 2 Zn.sup.2+ ions per hexamer, and the pH is then
adjusted from 5.7 to 3 by adding 8 mL of 0.1N solution of HCl. The
solution is neutralized to pH 7 by adding 10 mL of 0.1N solution of
NaOH. The concentration is then adjusted to 200 IU/mL with 43.08 mL
of water. The final pH of this solution is 7.02. The solution is
finally filtered on a 0.22 .mu.m membrane.
B93. Preparation of a Solution of Human Insulin at 200 IU/mL in the
Presence of Oligosaccharide 2 at 14.6 Mg/mL and 9.3 mM of
Citrate.
[0592] For a final volume of 100 mL of formulation with a weight
ratio (oligosaccharide 2/human insulin) of 2, the various reagents
are mixed in the quantities stated below
TABLE-US-00087 Human insulin at 200 IU/mL 100 mL Lyophilizate of
oligosaccharide 2 1460 mg Solution of sodium citrate at 1.188M 1570
.mu.L
[0593] The final pH is adjusted to 7.4.+-.0.4.
[0594] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B94. Preparation of a Solution of Human Insulin at 200 IU/mL in the
Presence of Oligosaccharide 2 at 14.6 mg/mL and of Polyanionic
Compound 1 at 14.6 mg/mL.
[0595] For a final volume of 100 mL of formulation with a weight
ratio (oligosaccharide 2/polyanionic compound 1/human insulin) of
2/2/1, the various reagents are mixed in the quantities stated
below
TABLE-US-00088 Human insulin at 200 IU/mL 100 mL Lyophilizate of
oligosaccharide 2 1460 mg Lyophilizate of polyanionic compound 1
1460 mg
[0596] The final pH is adjusted to 7.4.+-.0.4.
[0597] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B95. Preparation of a Solution of Human Insulin at 200 IU/mL in the
Presence of Oligosaccharide 2 at 14.6 mg/mL and of Polyanionic
Compound 1 at 29.2 mg/mL.
[0598] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 2/polyanionic compound 1/human insulin] of
2/4/1, the various reagents are mixed in the quantities stated
below
TABLE-US-00089 Human insulin at 200 IU/mL 100 mL Lyophilizate of
oligosaccharide 2 1460 mg Lyophilizate of polyanionic compound 1
2920 mg
[0599] The final pH is adjusted to 7.4.+-.0.4.
[0600] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
B96. Preparation of a Solution of Human Insulin at 100 IU/mL in the
Presence of Oligosaccharide 3 at 7.3 mg/mL.
[0601] For a final volume of 100 mL of formulation with a weight
ratio [oligosaccharide 3/human insulin] of 2/1, the various
reagents are mixed in the quantities stated below
TABLE-US-00090 Human insulin at 500 IU/mL 20 mL Oligosaccharide 3
at 27.71 mg/mL 28.4 mL 96.6 mM m-cresol/566 mM glycerol 30 mL Water
(volume for dilution - volume of sodium hydroxide) 21.6 mL
[0602] The final pH is adjusted to 7.0.+-.0.3.
[0603] The clear solution is filtered on a 0.22 .mu.m membrane and
stored at 4.degree. C.
[0604] This formulation is referenced example 18 in the priority
document.
C Pharmacodynamics and Pharmacokinetics
C1. Protocol for Measuring the Pharmacodynamics of the Insulin
Solutions.
[0605] 12 domestic pigs of about 50 kg, previously catheterized in
the jugular, are fasted for 2.5 hours before the start of the
experiment. In the hour preceding the injection of insulin, 3 blood
samples are taken for determining the baseline glucose level.
[0606] Human insulin at a dose of 0.125 IU/kg (or 0.09 IU/kg for
the insulin analog) is injected subcutaneously in the neck, under
the animal's ear using a Novopen insulin pen fitted with a 31 G
needle.
[0607] Blood samples are then taken every 4 minutes for 20 min and
then every 10 minutes up to 3 hours. After each sampling, the
catheter is rinsed with a dilute heparin solution.
[0608] A drop of blood is taken for determining glycemia using a
glucometer.
[0609] The curves of glucose pharmacodynamics are then plotted and
the time taken to reach the minimum blood glucose level for each
pig is determined and reported as Tmin glucose. The mean value of
the Tmin glucose values is then calculated.
[0610] The remaining blood is collected in a dry tube and
centrifuged to isolate the serum. The insulin levels in the serum
samples are measured by Elisa Sandwich immunoassay for each
pig.
[0611] The pharmacokinetic curves are then plotted. The time taken
to reach the peak insulin concentration in the serum for each pig
is determined and reported as Tmax insulin. The mean value of the
Tmax insulin values is then calculated.
C2. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B1 and B3.
TABLE-US-00091 Dose Number of Example Insulin Oligosaccharide
Excipient (IU/kg) pigs B1 Aspart -- -- 0.125 11 B3 Human -- --
0.125 11
[0612] described in examples B1 and B3 are presented in FIG. 1.
Analysis of these curves shows that the formulation of human
insulin (curve plotted with squares corresponding to example B3,
Tmin glucose=61.+-.31 min) does indeed have a slower action than
the commercial formulation of insulin aspart (curve plotted with
triangles corresponding to example B1, Tmin glucose=44.+-.13
min).
[0613] The results for pharmacokinetics obtained with the
formulations described in examples B1 and B3 are presented in FIG.
2. Analysis of these curves shows that the formulation of human
insulin alone (curve plotted with squares corresponding to example
B3, Tmax insulin=36.+-.33 min) does indeed induce slower absorption
than the commercial formulation of insulin aspart (Novolog.RTM.)
(curve plotted with triangles corresponding to example B1, Tmax
insulin=28.+-.13 min). These results are in agreement with those in
the literature with an acceleration of a rapid-acting insulin
analog relative to a human insulin and therefore validate the
suitability of the model for the problem of measuring the
acceleration of an insulin.
C3. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B1 and B6.
TABLE-US-00092 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B1 Aspart -- -- 0.125 16 B6 Human 2
Polyanionic 0.125 8 compound 1 7.3 mg/mL
[0614] The results for pharmacodynamics obtained with the
formulations described in examples B1 and B6 are presented in FIG.
3. Analysis of these curves shows that the formulation based on
human insulin comprising oligosaccharide 2 and polyanionic compound
1 as excipient at 7.3 mg/mL (curve plotted with squares
corresponding to example B6, Tmin glucose=39.+-.11 min) makes it
possible to obtain an action as rapid as that of the commercial
formulation of insulin aspart (Novolog.RTM.) (curve plotted with
triangles corresponding to example B1, Tmin glucose=41.+-.9
min).
[0615] The results for pharmacokinetics obtained with the
formulations described in examples B1 and B6 are presented in FIG.
4. Analysis of these curves shows that the formulation based on
human insulin comprising oligosaccharide 2 and polyanionic compound
1 as excipients at 7.3 mg/mL (curve plotted with squares
corresponding to example B6, Tmax insulin=14.+-.9 mM) induces an
absorption that is more rapid than the commercial formulation of
insulin aspart (Novolog.RTM.) (curve plotted with triangles
corresponding to example B1, Tmax insulin=24.+-.13 min). As the
time parameters of insulin aspart between examples C2 and C3 are
similar, it can be deduced from this by extrapolation that the
formulation of example B6 also induces an acceleration relative to
the human insulin (example B3).
C4. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B1 and B7
TABLE-US-00093 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B1 Aspart -- -- 0.125 14 B7 Human 2 Citrate
9.3 mM 0.125 12
[0616] The results for pharmacodynamics obtained with the
formulations described in examples B1 and B7 are presented in FIG.
5. Analysis of these curves shows that the formulation based on
human insulin comprising oligosaccharide 2 and citrate at 9.3 mM as
excipients (curve plotted with squares corresponding to example B7,
Tmin glucose=36.+-.14 min) makes it possible to obtain a more rapid
action than that of the commercial formulation of insulin aspart
(Novolog.RTM.) (curve plotted with triangles corresponding to
example B1, Tmin glucose=53.+-.24 min).
[0617] The results for pharmacokinetics obtained with the
formulations described in examples B1 and B7 are presented in FIG.
6. Analysis of these curves shows that the formulation comprising
oligosaccharide 2 and citrate at 9.3 mM as excipients (curve
plotted with squares corresponding to example B7, Tmax
insulin=15.+-.10 min) induces an absorption that is more rapid than
the commercial formulation of insulin aspart (Novolog.RTM.) (curve
plotted with triangles corresponding to example B1, Tmax
insulin=22.+-.10 min). As the time parameters of insulin aspart
(Novolog.RTM.) between examples C2 and C4 are similar, it can be
deduced from this by extrapolation that the formulation of example
B7 also induces an acceleration relative to the human insulin
(example B3).
C5. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B2 and B9
TABLE-US-00094 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 0.09 23 B9 Lispro 2 Citrate
9.3 mM 0.09 12
[0618] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B9 are presented in FIG.
7. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 9.3 mM as excipients (curve plotted with squares
corresponding to example B9, Tmin glucose=32.+-.9 min) makes it
possible to obtain a more rapid action than that of the commercial
formulation of insulin lispro (Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmin glucose=45.+-.16
min).
[0619] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B9 are presented in FIG.
8. According to the invention, analysis of these curves shows that
the formulation based on Humalog.RTM. comprising oligosaccharide 2
and citrate at 9.3 mM as excipient (curve plotted with squares
corresponding to example B9, Tmax insulin=12.+-.7 min) induces an
absorption that is more rapid than the commercial formulation of
insulin lispro (Humalog.RTM.) (curve plotted with triangles
corresponding to example B2, Tmax insulin=19.+-.10 min).
C6. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B2 and B10
TABLE-US-00095 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 0.09 11 B10 Lispro 2
Citrate 6 mM 0.09 10
[0620] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B10 are presented in FIG.
9. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 6 mM as excipients (curve plotted with squares
corresponding to example B10, Tmin glucose=34.+-.12 min) makes it
possible to obtain a more rapid action than that of the commercial
formulation of insulin lispro (Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmin glucose=44.+-.14
min).
[0621] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B10 are presented in FIG.
10. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 6 mM as excipients (curve plotted with squares
corresponding to example B10, Tmax insulin=13.+-.8 min) induces an
absorption that is more rapid than the commercial formulation of
insulin lispro (Humalog.RTM.) (curve plotted with triangles
corresponding to example B2, Tmax insulin=18.+-.8 min).
C7. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B2 and B11.
TABLE-US-00096 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 0.09 11 B11 Lispro 1
Citrate 9.3 mM 0.09 11
[0622] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B11 are presented in FIG.
11. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 9.3 mM as excipients (curve plotted with squares
corresponding to example B11, Tmin glucose=31.+-.14 min) makes it
possible to obtain a more rapid action than that of the commercial
formulation of insulin lispro (Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmin glucose=44.+-.14
min).
[0623] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B11 are presented in FIG.
12. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 9.3 mM as excipients (curve plotted with squares
corresponding to example B11, Tmax insulin=15.+-.7 min) induces an
absorption that is more rapid than the commercial formulation of
insulin lispro (Humalog.RTM.) (curve plotted with triangles
corresponding to example B2, Tmax insulin=18.+-.8 min).
C8. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B2 and B12.
TABLE-US-00097 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 9 B12 Lispro 1 Citrate 18.6
0.09 11 mM
[0624] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B12 are presented in FIG.
13. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 18.6 mM as excipients (curve plotted with squares
corresponding to example B12, Tmin glucose=30.+-.5 min) makes it
possible to obtain a more rapid action than that of the commercial
formulation of insulin lispro (Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmin glucose=40.+-.12
min).
[0625] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B12 are presented in FIG.
14. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 1 and
citrate at 18.6 mM as excipients (curve plotted with squares
corresponding to example B11, Tmax insulin=10.+-.4 mM) induces an
absorption that is more rapid than the commercial formulation of
insulin lispro (Humalog.RTM.) (curve plotted with triangles
corresponding to example B2, Tmax insulin=23.+-.12 min).
C9. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from examples B2 and B13.
TABLE-US-00098 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 0.09 9 B13 Lispro 2
Polyanionic 0.09 11 compound 1 7.3 mg/mL
[0626] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B13 are presented in FIG.
15. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipients at 7.3 mg/mL (curve plotted
with squares corresponding to example B13, Tmin glucose=32.+-.12
min) makes it possible to obtain a more rapid action than that of
the commercial formulation of insulin lispro (Humalog.RTM.) (curve
plotted with triangles corresponding to example B2, Tmin
glucose=44.+-.14 min).
[0627] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B13 are presented in FIG.
16. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipients at 7.3 mg/mL (curve plotted
with squares corresponding to example B13, Tmax insulin=14.+-.7
min) induces a more rapid absorption than the commercial
formulation of insulin lispro Humalog.RTM.) (curve plotted with
triangles corresponding to example B2, Tmax insulin=18.+-.8
min).
C10. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B2 and B14.
TABLE-US-00099 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 0.09 10 B14 Lispro 2
Polyanionic 0.09 11 compound 1 14.6 mg/mL
[0628] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B14 are presented in FIG.
17. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipient at 14.6 mg/mL (curve plotted
with squares corresponding to example B14, Tmin glucose=30.+-.7
min) makes it possible to obtain a more rapid action than that of
the commercial formulation of insulin lispro (Humalog.RTM.) (curve
plotted with triangles corresponding to example B2, Tmin
glucose=44.+-.14 min).
[0629] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B14 are presented in FIG.
18. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
polyanionic compound 1 as excipient at 14.6 mg/mL (curve plotted
with squares corresponding to example B14, Tmax insulin=12.+-.5
min) induces a more rapid absorption of Humalog.RTM. than the
commercial formulation of insulin lispro (Humalog.RTM.) (curve
plotted with triangles corresponding to example B2, Tmax
insulin=18.+-.8 min). C11. Results for pharmacodynamics and
pharmacokinetics of the insulin solutions from examples B2 and
B8.
TABLE-US-00100 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 0.09 12 B8 Lispro 6 -- 0.09
12
[0630] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B8 are presented in FIG.
19. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 6 (curve
plotted with squares corresponding to example B8, Tmin
glucose=45.+-.19 min) does not allow an action to be obtained that
is more rapid than that of the commercial formulation of insulin
lispro (Humalog.RTM.) (curve plotted with triangles corresponding
to example B2, Tmin glucose=50.+-.14 min).
[0631] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B8 are presented in FIG.
20. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 6 (curve
plotted with squares corresponding to example B8, Tmax
insulin=18.+-.10 min) does not induce a more rapid absorption of
Humalog.RTM. than the commercial formulation of insulin lispro
(Humalog.RTM.) (curve plotted with triangles corresponding to
example B2, Tmax insulin=20.+-.9 min).
C12. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B9 and B8.
TABLE-US-00101 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B9 Lispro 2 Citrate 9.3 mM 0.09 12 B8
Lispro 6 -- 0.09 12
[0632] The results for pharmacodynamics obtained with the
formulations described in examples B8 and B9 are presented in FIG.
21. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 and
citrate at 9.3 mM as excipient (curve plotted with squares
corresponding to example B9, Tmin glucose=32.+-.9 min) makes it
possible to obtain a more rapid action than that of the formulation
based on insulin lispro (Humalog.RTM.) comprising oligosaccharide 6
(curve plotted with triangles corresponding to example B8, Tmin
glucose=45.+-.19 min).
[0633] The results for pharmacokinetics obtained with the
formulations described in examples B8 and B9 are presented in FIG.
22. According to the invention, analysis of these curves shows that
the formulation based on Humalog.RTM. comprising oligosaccharide 2
and citrate at 9.3 mM as excipient (curve plotted with squares
corresponding to example B9, Tmax insulin=12.+-.7 min) induces a
more rapid absorption than the formulation based on insulin lispro
(Humalog.RTM.) comprising oligosaccharide 6 (curve plotted with
triangles corresponding to example B8, Tmax insulin=18.+-.10
min).
C13. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B2 and B15.
TABLE-US-00102 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B2 Lispro -- -- 0.09 12 B15 Lispro 2 at
14.3 mg/mL -- 0.09 12
[0634] The results for pharmacodynamics obtained with the
formulations described in examples B2 and B15 are presented in FIG.
23. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 at 14.6
mg/mL (curve plotted with squares corresponding to example B15,
Tmin glucose=35.+-.5 min) makes it possible to obtain a more rapid
action than that of the commercial formulation of insulin lispro
(Humalog.RTM.) (curve plotted with triangles corresponding to
example B2, Tmin glucose=47.+-.18 min).
[0635] The results for pharmacokinetics obtained with the
formulations described in examples B2 and B15 are presented in FIG.
24. Analysis of these curves shows that the formulation based on
insulin lispro (Humalog.RTM.) comprising oligosaccharide 2 at 14.6
mg/Ml (curve plotted with squares corresponding to example B15,
Tmax insulin=12.+-.4 min) induces a more rapid absorption of
Humalog.RTM. than the commercial formulation of insulin lispro
(Humalog.RTM.) (curve plotted with triangles corresponding to
example B2, Tmax insulin=20.+-.11 min).
C14. Results for Pharmacodynamics and Pharmacokinetics of the
Insulin Solutions from Examples B3 and B96
TABLE-US-00103 Dose Number Example Insulin Oligosaccharide
Excipient IU/kg of pigs B3 Human -- -- 0.125 10 B96 Human 3 --
0.125 9
[0636] The results for pharmacodynamics obtained with the
formulations described in examples B3 and B96 are presented in FIG.
25. Analysis of these curves shows that the formulation based on
human insulin comprising oligosaccharide 3 as excipient at 7.3
mg/mL (curve plotted with squares corresponding to example B96,
Tmin glucose=46.+-.20 min) makes it possible to obtain a more rapid
action than that of the commercial formulation of human insulin
(curve plotted with triangles corresponding to example B3, Tmin
glucose=64.+-.33 min).
[0637] The results for pharmacokinetics obtained with the
formulations described in examples B3 and B96 are presented in FIG.
26. Analysis of these curves shows that the formulation based on
human insulin comprising oligosaccharide 3 as excipient at 7.3
mg/mL (curve plotted with squares corresponding to example B96,
Tmax insulin=12.+-.6 min) induces an absorption that is more rapid
than the commercial formulation of human insulin (curve plotted
with triangles corresponding to example B3, Tmax insulin=26.+-.20
min).
D Circular Dichroism
D1. State of Association of Insulin Lispro (Humalog.RTM.) by
Circular Dichroism (CD) in the Presence of Oligosaccharides
[0638] Circular dichroism makes it possible to study the secondary
and quaternary structure of insulin. The insulin monomers organize
into dimers and hexamers. The hexamer is the form of insulin that
is the most stable physically and chemically. There are two
hexameric forms, form R6 and form T6. Insulin lispro has a strong
CD signal at 251 nm characteristic of the hexameric form R6 (the
most stable form). Loss of the CD signal at 251 nm is connected
with destabilization of the hexamer (and therefore the first sign
of transformation of the hexamer to dimer).
[0639] EDTA and the EDTA/citrate mixture completely destructures
the R6 form of insulin lispro (FIG. 27). EDTA therefore has a
marked effect of destabilization of the hexamer. In contrast, the
citrate alone, oligosaccharide 2 alone as well as the
oligosaccharide 2/citrate mixture have almost no effect on the CD
signal at 251 nm. These compounds therefore have hardly any impact
on the R6 structure of the hexamer and especially on the hexameric
structure of insulin, in contrast to EDTA, which destabilizes the
hexamer.
D2. State of Association of Human Insulin by Circular Dichroism
(CD) in the Presence of Oligosaccharides
[0640] The CD signal at 276 nm (in the absence of m-cresol) is
characteristic of the hexameric form of human insulin (signal of
the hexamer around -300 nm, signal of the dimer between -200 nm and
-250 nm and signal of the monomer below -200). Loss of the CD
signal at 276 nm is therefore characteristic of destabilization of
the hexamer to dimers or monomers.
[0641] EDTA and the EDTA/citrate combination have a very marked
effect on the hexameric structure of human insulin (complete
dissociation of the hexamer to dimers, FIG. 28). Conversely,
oligosaccharide 1 does not have a significant effect on the
hexameric structure of human insulin. In contrast to EDTA, the
formulations based on oligosaccharide 1 do not dissociate the
hexamer of human insulin.
E Dissolution of Human Insulin and Insulin Analog at the
Isoelectric Point
E1. Dissolution of Human Insulin at its Isoelectric Point
[0642] Human insulin has an isoelectric point at 5.3. At this pH,
human insulin is precipitated. A test demonstrating the formation
of a complex of human insulin with the various oligosaccharides or
polysaccharides is carried out at the isoelectric point. If
interaction exists, it is possible to dissolve the insulin at its
isoelectric point.
[0643] A solution of human insulin at 200 IU/mL is prepared.
Solutions of oligosaccharides or of polysaccharides at different
concentrations (8, 30 or 100 mg/mL) in water are prepared. An
equivolume mixture (50/50) between the solution of insulin and the
solution of oligosaccharide or of polysaccharide is effected to
give a solution containing 100 UI/ML of human insulin and the
desired concentration of polysaccharide (4, 15 or 50 mg/mL). The pH
of the various solutions is adjusted to pH 5.3 by adding 200 mM
acetic acid.
[0644] The appearance of the solution is documented. If the
solution is cloudy, the oligosaccharide or the polysaccharide at
the concentration tested does not allow dissolution of the insulin.
If the solution is translucent, the oligosaccharide or the
polysaccharide permits dissolution of the insulin at the
concentration tested. In this way it is possible to determine the
concentration of oligosaccharide or polysaccharide necessary for
dissolving the insulin at its isoelectric point. The lower this
concentration, the greater is the affinity of the oligosaccharide
or of the polysaccharide for the insulin.
TABLE-US-00104 Dissolution of Dissolution of Dissolution of human
insulin human insulin human insulin at at 100 IU/mL at 100 IU/mL
100 IU/mL by the by the by the polysaccharide/ polysaccharide/
polysaccharide/ Polysaccharide/ oligosaccharide at oligosaccharide
at oligosaccharide Oligosaccharide 4 mg/mL 15 mg/mL at 50 mg/mL
Counter- examples Polysaccharide 1 Yes Yes Yes Polysaccharide 4 Yes
Yes Yes Polysaccharide 3 Yes Yes Yes Polysaccharide 2 Yes Yes Yes
Polysaccharide 5 Yes Yes Yes Examples Oligosaccharide 3 No Yes Yes
Oligosaccharide 6 No Yes Yes Oligosaccharide 2 No Yes Yes
Oligosaccharide 1 No No No Oligosaccharide 4 No No No
E2. Dissolution of Insulin Lispro at its Isoelectric Point
[0645] Insulin lispro has an isoelectric point at 5.3. At this pH,
insulin lispro is precipitated. A test demonstrating the formation
of a complex of insulin lispro with various oligosaccharides or
polysaccharides is carried out at the isoelectric point. If
interaction exists, it is possible to dissolve the insulin at its
isoelectric point.
[0646] The commercial formulation of insulin lispro (Humalog.RTM.)
is dialyzed against buffer PO4 1 mM (pH 7). After dialysis, the
concentration of insulin lispro is about 90 IU/mL. The lyophilizate
of oligosaccharide or of polysaccharide is weighed and dissolved in
the solution of insulin lispro to give formulations containing
insulin lispro at 90 IU/mL and the oligosaccharide or the
polysaccharide at the desired concentrations (4, 15 or 50 mg/mL).
The pH of the various solutions is adjusted to pH 5.3 by adding 200
mM acetic acid.
[0647] The appearance of the solution is documented. If the
solution is cloudy, the oligosaccharide or the polysaccharide at
the tested concentration does not allow dissolution of the insulin.
If the solution is translucent, the oligosaccharide or the
polysaccharide permits dissolution of the insulin at the
concentration tested. In this way it is possible to determine the
concentration of oligosaccharide or of polysaccharide necessary for
dissolving the insulin at its isoelectric point. The lower this
concentration, the greater is the affinity of the oligosaccharide
or of the polysaccharide for the insulin.
TABLE-US-00105 Dissolution Dissolution Dissolution of insulin of
insulin of insulin lispro at 90 lispro at 90 lispro at 90 IU/mL by
the IU/mL by the IU/mL by the polysaccharide/ polysaccharide/
polysaccharide/ Polysaccharide/ oligosaccharide at oligosaccharide
at oligosaccharide Oligosaccharide 4 mg/mL 15 mg/mL at 50 mg/mL
Counter- examples Polysaccharide 1 Yes Yes Yes Polysaccharide 3 Yes
Yes Yes Polysaccharide 2 Yes Yes Yes Examples Oligosaccharide 3 No
Yes Yes Oligosaccharide 6 No Yes Yes Oligosaccharide 2 No No Yes
Oligosaccharide 1 No No No Oligosaccharide 4 No No No
F Interaction with Albumin F1: In order to determine the
interactions between the various polysaccharides or
oligosaccharides and a model protein such as albumin, a Centricon
test (membrane with cutoff of 501(D) was carried out. A solution of
polysaccharide or of oligosaccharide at 7.3 mg/mL was diluted to
one-third in a solution of BSA at 20 mg/mL in PBS (concentration in
the mixture: 2.43 mg/mL of polymer, 13.3 mg/mL of albumin and about
100 mM of salt).
[0648] This mixture was centrifuged on the Centricon to cause about
half of the volume to pass through the membrane. The albumin is
retained quantitatively on the membrane of the Centricon. The
polysaccharides and oligosaccharides analyzed largely pass through
the membrane (for the polysaccharides with the highest molecular
weights, about 20% of the polysaccharide is retained).
[0649] After centrifugation the polysaccharide or oligosaccharide
is determined by UV in the filtrate. The percentage of BC bound to
the albumin is calculated from the following equation:
[polysaccharide or oligosaccharide in the filtrate in the presence
of albumin]/[polysaccharide or oligosaccharide in the filtrate in
the absence of albumin]*100
[0650] It can be seen very clearly that the polysaccharides with
molecular weight 5-15 kD are strongly retained by the albumin in
this test. In contrast, the oligosaccharides of lower molecular
weight 1-2 kD are retained far less by the albumin in this
test.
TABLE-US-00106 Polysaccharides/ % Polysaccharide/Oligosaccharide
Oligosaccharides bound to the BSA Counterexamples Polysaccharide 4
97% Polysaccharide 1 95% Polysaccharide 3 77% Polysaccharide 5 86%
Polysaccharide 2 82% Examples Oligosaccharide 4 27% Oligosaccharide
1 34% Oligosaccharide 2 48% Oligosaccharide 3 45% Oligosaccharide 6
51%
* * * * *