U.S. patent application number 10/338101 was filed with the patent office on 2003-07-31 for insoluble compositions for controlling blood glucose.
Invention is credited to Brader, Mark Laurence.
Application Number | 20030144181 10/338101 |
Document ID | / |
Family ID | 27371371 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030144181 |
Kind Code |
A1 |
Brader, Mark Laurence |
July 31, 2003 |
Insoluble compositions for controlling blood glucose
Abstract
The present invention relates to insoluble compositions
comprising a protein selected from the group consisting of insulin,
insulin analogs, and proinsulins; a derivatized protein selected
from the group consisting of derivatized insulin, derivatized
insulin analog, and derivatized proinsulin; a complexing compound;
a hexamer-stabilizing compound; and a divalent metal cation.
Formulations of the insoluble composition are suitable for both
parenteral and non-parenteral delivery for treating hyperglycemia
and diabetes. Microcrystal forms of the insoluble precipitate are
pharmaceutically analogous to the neutral protamine Hagedorn (NPH)
insulin crystal form. Surprisingly, it has been discovered that
suspension formulations of such insoluble compositions possess
unique and controllable dissolution properties that provide
therapeutically advantageous glucodynamics compared with insulin
NPH formulations.
Inventors: |
Brader, Mark Laurence;
(Indianapolis, IN) |
Correspondence
Address: |
ELI LILLY AND COMPANY
PATENT DIVISION
P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Family ID: |
27371371 |
Appl. No.: |
10/338101 |
Filed: |
January 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10338101 |
Jan 7, 2003 |
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09217275 |
Dec 21, 1998 |
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6531448 |
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60068601 |
Dec 23, 1997 |
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60088859 |
Jun 11, 1998 |
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60109940 |
Nov 25, 1998 |
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Current U.S.
Class: |
514/6.3 ;
514/5.9; 514/6.4; 514/6.6; 514/6.9 |
Current CPC
Class: |
A61K 38/28 20130101;
A61P 3/10 20180101; A61P 5/50 20180101 |
Class at
Publication: |
514/3 ;
514/4 |
International
Class: |
A61K 038/28 |
Claims
I claim:
1. An insoluble composition, comprising: a) a protein selected from
the group consisting of insulin, insulin analog, and proinsulin; b)
a derivatized protein selected from the group consisting of
derivatized insulin, derivatized insulin analog, and derivatized
proinsulin; c) a complexing compound; d) a hexamer-stabilizing
compound; and e) a divalent metal cation.
2. The composition of claim 1 which is an amorphous
precipitate.
3. The composition of claim 1 which is a microcrystal.
4. The composition of claim 3, wherein the microcrystal has
rod-like morphology.
5. The composition of claim 3, wherein the microcrystal has
irregular morphology.
6. The composition of claim 1, wherein the derivatized protein is
selected from the group consisting of fatty acid-acylated insulin,
fatty acid-acylated insulin analogs, and fatty acid acylated
proinsulins.
7. The composition of claim 6, wherein the completing compound is
protamine, the hexamer-stabilizing compound is a phenolic
preservative, and the divalent metal cation is zinc.
8. The composition of claim 1, wherein the derivatized protein is
selected from the group consisting of fatty acid-acylated insulin
and fatty acid-acylated insulin analogs.
9. The composition of claim 8, wherein the complexing compound is
protamine, the hexamer-stabilizing compound is a phenolic
preservative, and the divalent metal cation is zinc.
10. The composition of claim 1, wherein the derivatized protein
selected from the group consisting of acylated insulin, acylated
insulin analogs, and acylated proinsulins.
11. The composition of claim 1, wherein the derivatized protein is
selected from the group consisting of acylated insulin and acylated
insulin analogs.
12. The composition of claim 11, wherein the derivatized protein is
mono-acylated at its Lys-N.epsilon.-amino group.
13. The composition of claim 12, wherein the complexing compound is
protamine, which is present at about 0.15 mg to about 0.5 mg per
3.5 mg of total protein.
14. The composition of claim 13, wherein the divalent metal cation
is zinc, which is present at about 0.3 mole to about 0.7 mole per
mole of total protein.
15. The composition of claim 14, wherein the hexamer-stabilizing
compound is a phenolic preservative selected from the group
consisting of phenol, m-cresol, o-cresol, p-cresol, chlorocresol,
methylparaben, and mixtures thereof, and is present at a ratio of
at least 3 moles of phenolic preservative to 6 moles of total
protein.
16. The composition of claim 15, wherein the protein is selected
from the group consisting of insulin and insulin analogs.
17. The composition of claim 16, wherein the protein is
insulin.
18. The composition of claim 17, wherein the derivatized protein is
insulin that is mono-acylated at the LysB29-N.epsilon.-amino
group.
19. The composition of claim 18, wherein the derivatized protein is
insulin that is acylated with a straight-chain, saturated fatty
acid.
20. The composition of claim 19, wherein the straight-chain,
saturated fatty acid is selected from the group consisting of
n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic
acid, and n-decanoic acid.
21. The composition of claim 20, wherein the mole ratio between the
protein and the derivatized protein is from about 1.9 to about
9:1.
22. The composition of claim 21, wherein the straight-chain,
saturated fatty acid is selected from the group consisting of
n-hexanoic acid, n-octanoic acid, and n-decanoic acid.
23. The composition of claim 22, wherein the straight-chain,
saturated fatty acid is selected from the group consisting of
n-octanoic acid and n-decanoic acid
24. The composition of claim 23, wherein the straight-chain,
saturated fatty acid is n-octanoic acid.
25. The composition of claim 24, wherein the mole ratio between the
protein and the derivatized protein is from about 1:3 to about
3:1.
26. The composition of claim 22, wherein the mole ratio between the
protein and the derivatized protein is from about 1:9 to about
1:1.
27. The composition of claim 16, wherein the protein is an insulin
analog.
28. The composition of claim 27, wherein the protein is LysB28,
ProB29-human insulin analog.
29. The composition of claim 27, wherein the protein is
AspB28-human insulin analog.
30. The composition of claim 1, wherein the protein is insulin or
an insulin analog, and the derivatized protein is an acylated
protein selected from the group consisting of acylated insulin and
acylated insulin analogs.
31. The composition of claim 1, wherein the derivatized protein is
insulin that is mono-acylated at its LysB29-N.epsilon. amino group
with a straight-chain, saturated fatty acid.
32. The composition of claim 31, wherein the straight-chain,
saturated fatty acid is selected from the group consisting of
n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic
acid, and n-decanoic acid.
33. The composition of claim 32, wherein the derivatized protein is
selected from the group consisting of B29-N.epsilon.-hexanoyl-human
insulin, B29-N.epsilon.-octanoyl-human insulin, and
B29-N.epsilon.-decanoyl-human insulin.
34. The composition of claim 31, wherein the straight-chain,
saturated fatty acid selected from the group consisting of
n-dodecanoic acid, n-tetradecanoic acid, and n-hexadecanoic
acid.
35. The composition of claim 1, wherein the derivatized protein is
di-acylated at the Lys-N.epsilon.-amino group and is also acylated
at one N-terminal N.alpha.-amino group, and wherein the fatty acid
is selected from the group consisting of n-hexanoic acid,
n-heptanoic acid, n-octanoic acid, n-nonanoic acid, and n-decanoic
acid.
36. The composition of claim 1, wherein the derivatized protein is
acylated with a branched-chain, saturated fatty acid.
37. The composition of claim 36, wherein the derivatized protein is
acylated with a branched-chain, saturated fatty acid having from
three to ten carbon atoms in its longest branch.
38. The composition of claim 1, wherein the derivatized protein is
an insulin analog that is mono-acylated at its Lys-N.epsilon.-amino
group with a straight-chain, saturated fatty acid.
39. The composition of claim 38, wherein the fatty acid is selected
from the group consisting of n-hexanoic acid, n-heptanoic acid,
n-octanoic acid, n-nonanoic acid, and n-decanoic acid.
40. The composition of claim 38, wherein the derivatized protein is
an insulin analog that is mono-acylated at the N.epsilon.-amino
group with a fatty acid selected from the group consisting of
n-dodecanoic acid, n-tetradecanoic acid, and n-hexadecanoic
acid.
41. The composition of claim 38, wherein the derivatized protein is
selected from the group consisting of fatty acid-acylated animal
insulins, fatty acid-acylated monomeric insulin analogs, fatty
acid-acylated deletion analogs, and fatty acid-acylated pI-shifted
insulin analogs.
42. The composition of claim 41, wherein the derivatized protein is
fatty acid-acylated des(B30)-human insulin analog, fatty
acid-acylated LysB28, ProB29-human insulin analog, or fatty
acid-acylated AspB28-human insulin analog.
43. The composition of claim 42, wherein the derivatized protein is
fatty acid-acylated des(B30)-human insulin analog.
44. The composition of claim 43, wherein the derivatized protein is
B29-N.epsilon.-myristoyl-des(B30)-human insulin analog.
45. The composition of claim 42, wherein the derivatized protein is
fatty acid-acylated LysB28, ProB29-human insulin analog.
46. The composition of claim 45, wherein the derivatized protein is
B28-N.epsilon.-myristoyl-LysB28, ProB29-human insulin analog.
47. The composition of claim 42, wherein the derivatized protein is
fatty acid-acylated AspB28-human insulin analog.
48. The composition of claim 1, wherein the mole ratio between the
protein and the derivatized protein is from about 1:9 to about
9:1.
49. The composition of claim 48, wherein the ratio is from about
1:3 to about 3:1.
50. The composition of claim 48, wherein the ratio is from about
1:9 to about 1:1.
51. The composition of claim 1, wherein the protein is insulin.
52. The composition of claim 1, wherein the protein is an insulin
analog.
53. The composition of claim 52, wherein the protein is a monomeric
insulin analog.
54. A suspension formulation, comprising an insoluble phase and a
solution phase, wherein the insoluble phase comprises the insoluble
composition of claim 1, and the solution phase comprises an aqueous
solvent.
55. The suspension formulation of claim 54, wherein the solution
phase further comprises a phenolic preservative at a concentration
of about 0.5 mg per mL to about 6 mg per mL of solution, a
pharmaceutically acceptable buffer, and an isotonicity agent.
56. The suspension formulation of claim 54, wherein the solution
phase further comprises insulin, an insulin analog, a derivatized
insulin, or a derivatized insulin analog.
57. The suspension formulation of claim 56, wherein the solution
phase comprises insulin.
58. The suspension formulation of claim 56, wherein the solution
phase comprises derivatized insulin.
59. The suspension formulation of claim 56, wherein the solution
phase comprises an insulin analog.
60. The suspension formulation of claim 56, wherein the insulin
analog is a monomeric insulin analog.
61. The suspension formulation of claim 56, wherein the insulin
analog is LysB28, ProB29-human insulin analog.
62. The suspension formulation of claim 56, wherein the insulin
analog is AspB28-human insulin analog.
63. The suspension formulation of claim 54, wherein the solution
phase further comprises a protein selected from insulin and insulin
analogs and a derivatized protein selected from derivatized insulin
and derivatized insulin analogs.
64. The suspension formulation of claim 63 wherein the protein in
the solution phase is the same protein that is in the insoluble
phase, and wherein the derivatized protein in the solution phase is
the same derivatized protein that is in the insoluble phase.
65. The suspension formulation of claim 54, wherein the insoluble
phase consists essentially of an amorphous precipitate.
66. The suspension formulation of claim 54, wherein the insoluble
phase consists essentially of a microcrystal.
67. The suspension formulation of claim 54, wherein the insoluble
phase consists of a mixture of amorphous precipitate and
microcrystal.
68. The suspension formulation of claim 54, wherein the solution
phase further comprises zinc and protamine, wherein the ratio of
zinc to total protein in the suspension formulation is from about 5
to about 7 mole of zinc atoms per mole of total protein, and the
ratio of protamine to total protein in the suspension formulation
is from about 0.25 mg to about 0.5 mg per mg of total protein.
69. A method of treating diabetes comprising administering the
composition of claim 1 to a patient in need thereof in a quantity
sufficient to regulate blood glucose levels in the patient.
70. A method of treating hyperglycemia comprising administering the
composition of claim 54 to a patient in need thereof in a quantity
sufficient to regulate blood glucose levels in the patient.
71. A hybrid hexamer composition, comprising six monomers and zinc,
wherein at least one monomer is selected from the group consisting
of insulin, insulin analogs, and proinsulins, and at least one
monomer is selected from the group consisting of derivatized
insulin, derivatized insulin analogs, and derivatized
proinsulins.
72. A mixed hexamer composition, comprising zinc protein hexamers
and zinc derivatized protein hexamers, wherein the zinc protein
hexamers comprise zinc and a protein selected from the group
consisting of insulin, insulin analogs, and proinsulins, and
wherein the zinc derivatized protein hexamers comprise zinc and a
derivatized protein selected from the group consisting of
derivatized insulin, derivatized insulin analogs, and derivatized
proinsulins.
73. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation in an
aqueous solvent having a pH that will permit the formation of
hexamers, and b) adding a complexing compound.
74. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation in an
aqueous solvent having a pH that will not permit the formation of
hexamers, and b) adjusting the pH to between about 6.8 and about
7.8; and c) adding a complexing compound.
75. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a hexamer-stabilizing
compound, and a divalent metal cation in an aqueous solvent having
a pH that will permit the formation of hexamers; b) separately,
dissolving a derivatized protein, a hexamer-stabilizing compound,
and a divalent metal cation in an aqueous solvent having a pH that
will permit the formation of hexamers; c) thoroughly mixing
together the solutions from steps a) and b); and d) adding a
complexing compound to the solution produced in step c).
76. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a hexamer-stabilizing
compound, a divalent metal cation, and a complexing compound in an
aqueous solvent, wherein the resulting solution has a pH at which
precipitation does not occur; b) separately, dissolving a
-derivatized protein, a hexamer-stabilizing compound, a divalent
metal cation, and a complexing compound in an aqueous solvent,
wherein the resulting solution has a pH at which precipitation does
not occur; c) thoroughly mixing together the solutions from steps
a) and b); and d) adjusting the pH of the solution of step c) to a
value at which precipitation occurs.
77. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, a divalent metal cation, and a
complexing compound in an aqueous solvent, wherein the resulting
solution has a pH at which precipitation does not occur; and b)
adjusting the pH of the solution of step a) to a value at which
precipitation occurs.
78. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation, in an
aqueous solvent, wherein the resulting solution has a pH at which
precipitation will not occur when a complexing agent is added; b)
adding a complexing compound; and c) adjusting the pH of the
solution of step b) to a value at which precipitation occurs.
79. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a hexamer-stabilizing
compound, and a divalent metal cation in an aqueous solvent,
wherein the resulting solution has a pH at which precipitation will
not occur when a complexing compound is added; b) separately,
dissolving a derivatized protein, a hexamer-stabilizing compound,
and a divalent metal cation in an aqueous solvent, wherein the
resulting solution has a pH at which precipitation will not occur
when a complexing compound is added; c) thoroughly mixing together
the solutions from steps a) and b); d) adding complexing compound
to the solution of step c); and e) adjusting the pH of the solution
of step d) to a value at which precipitation occurs.
80. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a protein derivative, a
hexamer-stabilizing compound, and a divalent metal cation in an
aqueous solvent, wherein the resulting solution has a pH at which
precipitation will not occur when a complexing compound is added;
b) adjusting the pH of the solution of step a) to a value at which
precipitation will occur when a complexing compound is added; and
c) adding a complexing compound to the solution of step b).
81. A process for preparing the insoluble composition of claim 1
comprising: a) dissolving a protein, a hexamer-stabilizing
compound, and a divalent metal cation in an aqueous solvent,
wherein the resulting solution has a pH at which precipitation will
not occur when a complexing compound is added; b) separately,
dissolving a derivatized protein, a hexamer-stabilizing compound,
and a divalent metal cation in an aqueous solvent, wherein the
resulting solution has a pH at which precipitation will not occur
when a complexing compound is added; c) thoroughly mixing together
the solutions from steps a) and b); d) adjusting the pH of the
solution of step c) to a value at which precipitation will occur
when a complexing compound is added; and e) adding a complexing
compound to the solution of step d).
82. A process for preparing hybrid hexamers, comprising dissolving
a protein, a derivatized protein, a hexamer-stabilizing compound,
and a divalent metal cation in an aqueous solvent having a pH that
will permit the formation of hexamers.
83. A process for preparing hybrid hexamers, comprising: a)
dissolving a protein, a derivatized protein, a hexamer-stabilizing
compound, and a divalent metal cation in an aqueous solvent having
a pH that will not permit the formation of hexamers, and b)
adjusting the pH to between about 6.8 and about 7.8.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention is in the field of human medicine. More
particularly, this invention is in the field of pharmaceutical
treatment of the diseases of diabetes and hyperglycemia.
[0003] 2. Description of Related Art
[0004] It has long been a goal of insulin therapy to mimic the
pattern of endogenous insulin secretion in normal individuals. The
daily physiological demand for insulin fluctuates and can be
separated into two phases: (a) the absorptive phase requiring a
pulse of insulin to dispose of the meal-related blood glucose
surge, and (b) the post-absorptive phase requiring a sustained
delivery of insulin to regulate hepatic glucose output for
maintaining optimal fasting blood glucose.
[0005] Accordingly, effective therapy for people with diabetes
generally involves the combined use of two types of exogenous
insulin formulations: a rapid acting meal time insulin provided by
bolus injections and a long-acting, so-called, basal insulin,
administered by injection once or twice daily to control blood
glucose levels between meals. An ideal basal insulin will provide
an extended and "flat" time action--that is, it will control blood
glucose levels for at least 12 hours, and preferably for 24 hours
or more, without significant risk of hypoglycemia. Furthermore, an
ideal basal insulin should be mixable with a soluble meal-time
insulin, and should not cause irritation or reaction at the site of
administration. Finally, basal insulin preparations that are
suspension formulations should be able to be readily, and uniformly
resuspended by the patient prior to administration.
[0006] As is well understood by those skilled in this art,
long-acting insulin formulations have been obtained by formulating
normal insulin as microcrystalline suspensions for subcutaneous
injection. Examples of commercial basal insulin preparations
include NPH (Neutral Protamine Hagedorn) insulin, protamine zinc
insulin (PZI), and ultralente (UL).
[0007] Early versions of present-day commercial NPH insulin that
contained a surplus of protamine were developed in the 1930's by
Scott, et al. [J. Pharmacol. Exp. Ther. 58:78, et seq. (1936)] and
Hagedorn, et al. [J. Am. Med. Assoc. 106:177-180 (1936)]. In 1946,
NPH insulin having isophane proportions of insulin and protamine,
together with zinc, were developed by Krayenbuhl, et al. [Rep.
Steno Mem. Hosp. Nord. Insulinlab. 1:60, et seq. (1946)]. These
workers found that when insulin and protamine were combined in
so-called isophane proportions at a neutral pH, in the presence of
zinc and a phenolic compound, that an amorphous precipitate formed,
and that upon standing the amorphous precipitate was transformed
into oblong, tetragonal crystals having pyramidal shaped ends.
These crystals have been described as rod-like. The isophane ratio
of insulin and protamine sulfate is observed to be about 0.09 mg of
protamine sulfate per mg of insulin. Zinc is needed in an amount of
at least about 3.5 .mu.g per mg of insulin, and a phenolic compound
at a concentration higher than about 0.1%.
[0008] Insulin NPH is the most widely-used insulin preparation,
constituting from 50 to 70 per cent of the insulin used worldwide.
It is a suspension of a microcrystalline complex of insulin, zinc,
protamine, and one or more phenolic preservatives. NPH insulin
preparations are commercially available incorporating human
insulin, pork insulin, beef insulin, or mixtures thereof. Also,
NPH-like preparations of a monomeric insulin analog, LysB298,
ProB29-human insulin analog, are known in the art [abbreviated
herein as "NPL": De Felippis, M. R., U.S. Pat. No. 5,461,031,
issued Oct. 24, 1995; De Felippis, M. R., U.S. Pat. No. 5,650,486,
issued Jul. 22, 1997; and De Felippis, M. R., U.S. Pat. No.
5,747,642, issued May 5, 1998]. It is widely accepted that insulin
NPH provides extended control of blood glucose compared with
regular insulin because insulin must first dissolve from the
insulin NPH microcrystals before it can be absorbed. With regular
insulin, there is no dissolution needed prior to absorption. For
insulin NPH, dissolution is the rate-controlling step in
determining the pharmacodynamics and pharmacokinetics.
[0009] NPH insulin microcrystals possess a distinctive rod-shaped
morphology of typical dimensions about 5 microns long by 1 micron
thick and 1 micron wide. The extended duration of action of NPH
insulin microcrystals results from their slow absorption from the
subcutaneous injection site.
[0010] Therapy using currently-available NPH insulin preparations
fails to provide the ideal "flat" pharmacokinetics necessary to
maintain optimal fasting blood glucose for an extended period of
time between meals. Consequently, treatment with NPH insulin can
result in undesirably high levels of insulin in the blood, which
may cause life-threatening hypoglycemia.
[0011] In addition to failing to provide an ideal flat
pharmacokinetic profile, the duration of action of NPH insulin also
is not ideal. In particular, a major problem with NPH therapy is
the "dawn phenomenon" which is hyperglycemia that results from the
loss of effective glucose control overnight while the patient is
sleeping. These deficiencies in glycemic control contribute to
serious long-term medical complications of diabetes and impose
considerable inconvenience and quality-of-life disadvantages to the
patient.
[0012] Protamine zinc insulin (PZI) has a composition similar to
NPH, but contains higher levels of protamine and zinc than NPH. PZI
preparations may be made as intermediate-acting amorphous
precipitates or long-acting crystalline material. PZI, however, is
not an ideal basal insulin pharmaceutical because it is not mixable
with a soluble meal-time insulin, and the high zinc and protamine
can cause irritation or reaction at the site of administration.
[0013] Human insulin ultralente is a microcrystalline preparation
of insulin having higher levels of zinc than NPH, and not having
either protamine or a phenolic preservative incorporated into the
microcrystal. Human ultralente preparations provide moderate time
action that is not suitably flat, and they do not form stable
mixtures with insulin. Furthermore, they are difficult to
resuspend.
[0014] There have been attempts to address the perceived
inadequacies of known insulin suspensions. Fatty acid-acylated
insulins have been investigated for basal control of blood glucose
[Havelund, S., et al., WIPO publication WO95/07931, Mar. 23, 1995].
Their extended time action is caused by binding of the fatty acyl
portion of these molecules to serum albumin. The fatty acyl chain
lengths of these molecules is such as to take advantage of the
fatty acid binding capability of serum albumin. The fatty acid
chains used in fatty acid-acylated insulins are typically longer
than about ten carbon atoms, and chain lengths of fourteen and
sixteen carbon atoms are optimal for binding to serum albumin and
extending time action.
[0015] Unlike NPH insulin, which is insoluble, the aforementioned
fatty acid-acylated insulins are soluble at the usual therapeutic
concentrations of insulin. However, the time action of these
preparations may not be sufficiently long enough, or flat enough,
to provide ideal basal control, and they are less potent than
insulin, thereby requiring administration of greater amounts of the
drug agent [Radziuk, J., et al., Diabetologia 41:116-120, 489-490
(1998)].
[0016] Whittingham, J. L., et al. [Biochemistry 36:2826-2831
(1997)1 crystallized B29-N.epsilon.-tetradecanoyl-des(B30)-human
insulin analog as a hexamer complex with zinc and phenol for the
purpose of structural studies by X-ray crystallography. The hexamer
was found to be in the R6 conformation, and to have certain
properties different from hexamers of human insulin. Whittingham,
et al. do not disclose any pharmaceutical or pharmacological
properties of the crystal that was formed, nor do they suggest that
such a crystal would have any advantageous properties for treating
diabetes or hyperglycemia. It is not possible to predict from
Whittingham, et al. whether protamine-containing crystals of the
NPH type could be formed with derivatized insulins and insulin
analogs, or what the pharmacokinetics or pharmacodynamic response
of such crystals would be.
[0017] Thus, there remains a need to identify insulin preparations
that have flatter and longer time action than NPH insulin, that are
mixable with soluble, meal-time insulins, that can be readily
resuspended, and that do not pose risk of irritation or reaction at
the site of administration. I discovered quite surprisingly that
these properties are provided by insoluble compositions that
include a derivatized protein, an un-derivatized protein, zinc,
protamine, and a phenolic preservative. In addition to the
properties mentioned above, the insoluble compositions provide
flexibility of control over the duration and shape of the
glucodynamic response profile. They are thought to function as
controlled release compositions, wherein, the release rate is
controlled by the proportion and nature of the derivatized protein.
Thus, one aspect of the present invention is an insoluble
composition comprising an un-derivatized protein, a derivatized
protein, a complexing compound, a hexamer-stabilizing compound, and
a divalent metal cation. Other aspects of this invention that
relate to the preparation, formulation, and use of such
compositions will be discussed herein.
[0018] There are no examples known to me of mixtures of derivatized
and un-derivatized insulins, as those terms are to be understood in
the context of the present disclosure. Crystals comprised of
proinsulin and insulin (Steiner, D. F., Nature 243:528-530 (1973);
Low, B. W., et al. Nature 248:339-340 (1974)] and crystals
comprised of a insulin or an insulin analog having approximately
the same isoelectric point as insulin and an insulin analog having
additional basic amino acids [Dorschug, M., et al., U.S. Pat. No.
5,028,587, issued Jul. 2, 1991] are known.
[0019] Steiner produced crystals comprised of proinsulin and
insulin with mole ratios of about 1:11, 1:5, 1:2, and 1:1,
respectively (i.e., 0.5, 1, 2, and 3 moles of proinsulin per 6
moles total insulin and proinsulin) in 0.095 M sodium citrate, pH
6.0, 0.03 M NaCl, 0.012 M ZnC12, and 16% acetone. The proportion of
proinsulin greatly affected the rate of crystallization. The
crystals differed greatly from those of pure insulin under the same
conditions, and were characterized as rhombohedral crystals with
rounded borders. There was great variability within and between
preparations. The utility ascribed to crystallizing proinsulin and
insulin was that it facilitated isolating small amounts of
proinsulin and related structures from pancreatic extracts. The
author speculated that crystallization may occur between precursor
and product peptides, and among other closely related proteins.
[0020] Low, B. W., et al. produced very large crystals comprised of
equimolar proportions of beef or pork insulin and their respective
proinsulins, wherein the proinsulin and insulin were formed into
homogenous hexamers prior to crystallization. Analysis by X-ray
crystallography and quantitative electrophoresis supported a
conclusion that the unit cell in the crystals was formed of twelve
insulin hexamers and twelve proinsulin hexamers. It was
specifically stated that no studies were known to suggest that
insulin and proinsulin form mixed dimers and hexamers in
solution.
[0021] Dorschug, M., et al. disclosed crystals comprised of
insulin, des(PheB1) insulin, des(ThrB30) human insulin, or
des(AlaB30) beef insulin, and at least one insulin having a basic
modification at the C-terminal end of the B chain ("modified
insulins"). Such modified insulins are disclosed, for example, in
European Patent Application No. 132,769. Globin or protamine
sulfate were stated to be auxiliary compounds that could be used in
the crystal preparations. There are no examples of the use of
protamine, nor any suggestion that the inventors appreciated the
effect of adding such compounds. Furthermore, the modified insulins
used in Dorschug, et al. are different than the derivatives used in
the present invention.
[0022] As mentioned above, I have unexpectedly observed that when a
protein selected from insulin, an insulin analog, and proinsulin is
made less soluble in an aqueous solvent or more lipophilic by
derivatizing one or more of its reactive side groups, the
derivatized protein and an un-derivatized protein selected from
insulin, an insulin analog, and proinsulin can be incorporated into
insoluble precipitates and into NPH-like crystals with protamine.
When such proteins are jointly precipitated or crystallized to form
insoluble compositions, the rate at which the proteins dissolve
from the insoluble composition is greatly reduced compared with the
rate at which physically similar insoluble compositions comprised
of un-derivatized protein dissolve.
[0023] I have furthermore discovered that both amorphous
precipitates and microcrystals comprised of derivatized protein,
protein, a complexing compound, a divalent metal cation, and a
hexamer-stabilizing compound provide flatter and longer time action
than do physically similar microcrystals comprised solely of
un-derivatized protein. Additionally, I have surprisingly
discovered that the benefits of flatter and longer time action can
be obtained even from amorphous precipitates comprised of one of
the proteins and a derivatized protein.
SUMMARY OF THE INVENTION
[0024] Accordingly, in its broadest aspect, the present invention
provides insoluble compositions comprising a derivatized protein
selected from the group consisting of insulin derivatives, insulin
analog derivatives, and proinsulin derivatives, a protein selected
from the group consisting of insulin, insulin analogs, and
proinsulins, a complexing compound, a hexamer-stabilizing
-compound, and a divalent metal cation. The derivatized protein is
either less soluble in an aqueous solvent than is the
un-derivatized protein, is more lipophilic than un-derivatized
insulin, or produces a complex with zinc and protamine that is less
soluble than the corresponding complex with the un-derivatized
protein. The insoluble compositions of the present invention may be
in the form of amorphous precipitates, or more preferably, in the
form of microcrystals. The microcrystals may be either rod-shaped
or irregular in morphology. These insoluble compositions are useful
for treating diabetes and hyperglycemia, and provide the advantages
of having flatter and longer time action than NPH insulin. The
insoluble compositions are mixable in a formulation with soluble
protein or with soluble derivatized protein, or both. Furthermore,
by varying the ratio between protein and derivatized protein, the
extent of protraction of the time action can be finely controlled
over a very great range of time-action, from that nearly the same
as NPH insulin to much greater than that of NPH insulin.
[0025] More specifically, the present invention provides insoluble
compositions of proteins and fatty acid-acylated proteins that are
useful for treating diabetes and hyperglycemia. These compositions
are comprised of fatty acid-acylated protein selected from the
group consisting of fatty acid-acylated insulin, fatty
acid-acylated insulin analog, and fatty acid-acylated proinsulin,
protein selected from the group consisting of insulin, insulin
analogs, and proinsulin, protamine, a phenolic preservative, and
zinc. The present invention is distinct from previous fatty
acid-acylated insulin technology in that the extension of time
action of the present invention does not rely necessarily on
albumin-binding, though albumin binding may further protract the
time action of certain of the compositions of the present
invention. The invention provides a microcrystal comprising a
protein selected from the group consisting of insulin, insulin
analog, and proinsulin, a derivatized protein selected from the
group consisting of derivatized insulin, derivatized insulin
analog, and derivatized proinsulin, a complexing compound a
divalent metal cation, and a hexamer-stabilizing compound. The
microcrystals of the present invention are useful for treating
diabetes and for controlling blood glucose in a patient in need
thereof.
[0026] The invention provides an amorphous precipitate comprising a
protein selected from the group consisting of insulin, insulin
analog, and proinsulin; a derivatized protein selected from the
group consisting of derivatized insulin, derivatized insulin
analog, and derivatized proinsulin, a completing compound a
divalent metal cation, and a hexamer-stabilizing compound. The
amorphous precipitates of the present invention are useful for
treating diabetes and for controlling blood glucose in a patient in
need thereof. They are also useful as intermediates in the
formation of the microcrystals of the present invention.
[0027] The invention provides aqueous suspension formulations
comprising an insoluble composition and an aqueous solvent. One
such aqueous suspension formulation is comprised of a
microcrystalline composition of the present invention and an
aqueous solvent. Another such aqueous suspension formulation
comprises an amorphous precipitate of the present invention and an
aqueous solvent. The soluble, aqueous phase of the present
suspension formulations may optionally be comprised of a protein,
such as human insulin, or a soluble analog of human insulin, such
as a monomeric insulin analog, that control blood glucose
immediately following a meal, and may additionally or alternatively
be comprised of a derivatized protein. The formulations of the
present invention have superior pharmacodynamics compared with
human insulin NPH, and their time-action can be purposefully
selected over a wide range, from just slightly extended compared
with human insulin NPH to very greatly extended compared with human
insulin NPH.
[0028] The invention also provides processes for preparing hybrid
hexamers, mixed hexamers, the amorphous precipitates, and the
co-crystals of the present invention,
[0029] The invention provides a method of treating diabetes or
hyperglycemia comprising, administering to a patient in need
thereof a sufficient quantity of an insoluble composition of the
present invention to regulate blood glucose levels in the
patient.
[0030] The invention includes hybrid hexamer compositions
comprising a protein selected from the group consisting of insulin,
insulin analog, and proinsulin; a derivatized protein selected from
the group consisting of derivatized insulin, derivatized insulin
analogs, and derivatized proinsulins, and zinc. The hybrid hexamers
of the present invention are useful for treating diabetes and for
controlling blood glucose in a patient in need thereof. They are
also useful as intermediates in the formation of the insoluble
compositions of the present invention, which are themselves useful
for treating diabetes and for controlling blood glucose in a
patient in need thereof. Hybrid hexamers are believed to be formed
when a protein and a derivatized protein are first mixed together
under conditions that strongly favor dissolution into lower states
of aggregation than the hexameric state, and second, the conditions
are changed to strongly favor the hexameric aggregation state.
[0031] The invention includes mixed hexamer compositions, comprised
zinc hexamers of a protein selected from the group consisting of
insulin, an insulin analog, or proinsulin and zinc hexamers of a
derivatized protein selected from the group consisting of a
derivatized insulin, derivatized insulin analog, or a derivatized
proinsulin. The mixed hexamers of the present invention are useful
for treating diabetes and for controlling blood glucose in a
patient in need thereof. They are also useful as intermediates in
the formation of the insoluble compositions of the present
invention, which are themselves useful for treating diabetes and
for controlling blood glucose in a patient in need thereof. Mixed
hexamers are believed to be formed when a protein and a derivatized
protein are first separately dissolved under conditions that favor
the hexameric aggregation state, and then are mixed together under
conditions that continue to strongly favor the hexameric
aggregation state.
[0032] The invention includes the use of an insoluble composition
of the present invention to prepare a medicament for the treatment
of diabetes or hyperglycemia.
BRIEF DESCRIPTION OF THE DRAWING
[0033] FIG. 1 depicts the dissolution over a five-hour period of
co-crystals of the present invention having ratios of human insulin
to B29-N.epsilon.-octanoyl-human insulin of 3:1 (- - -), 1:1 (--),
and 1:3 (), compared with the dissolution of a preparation of human
insulin-protamine crystals (. . .).
[0034] FIG. 2 depicts dissolution data from the same experiments as
depicted in FIG. 1, but with the time axis extended to show data
for the 1:3 co-crystal up to about 13.5 hours. Dissolution of
co-crystals of the present invention having ratios of human insulin
to B29-N.epsilon.-octanoyl-human insulin of 3:1 (- - -), 1:1 (--),
and 1:3 () are compared with the dissolution of a preparation of
human insulin-protamine crystals (. . .).
[0035] FIG. 3 depicts the dissolution of co-crystals of the present
invention having ratios of human insulin to
B29-N.epsilon.-octanoyl-human insulin of 1:1 (-) and 1:3 (--),
compared with the dissolution of with microcrystals comprised only
of B29-N.epsilon.-octanoyl-human insulin (), or a preparation of
human insulin-protamine crystals (. . .).
[0036] FIG. 4 depicts the dissolution of co-crystals of the present
invention having a 1:3 ratio of human insulin to
B29-N.epsilon.-octanoyl-- human insulin (), compared with
preparations of human insulin-protamine crystals (. . .), human
insulin ultralente (-) and beef ultralente (--)
DESCRIPTION OF THE INVENTION
[0037] The term "mixed hexamers" refers to a mixture of protein
hexamers and derivatized protein hexamers, wherein the protein
hexamers are comprised of zinc and a protein selected from the
group consisting of insulin, insulin analogs, and proinsulins, and
wherein the derivatized protein hexamers are comprised of zinc and
a derivatized protein selected from the group consisting of
derivatized insulin, derivatized insulin analogs, and derivatized
proinsulins. The level of zinc actually incorporated into a hexamer
is between about 2 and about 4 atoms of zinc per hexamer.
[0038] The term "hybrid hexamer" refers to a hexamer comprised of
six monomers and zinc, wherein at least one monomer is selected
from the group consisting of insulin, insulin analogs, and
proinsulins, and at least one monomer is selected from the group
consisting of derivatized insulin, derivatized insulin analogs, and
derivatized proinsulins. The level of zinc actually incorporated
into a hexamer is commonly known to be between about 2 and about 4
atoms of zinc per hexamer.
[0039] As used herein, the term "co-crystal" means a microcrystal
of the present invention.
[0040] The term "insoluble composition" refers to matter in either
a microcrystalline state or in an amorphous precipitate state. The
presence of microcrystals or amorphous precipitate can be
ascertained by visual and microscopic examination. Solubility
depends on solvent, and a particular composition may be insoluble
in one solvent, but soluble in another.
[0041] The term "microcrystal" means a solid that is comprised
primarily of matter in a crystalline state, wherein the individual
crystals are predominantly of a single crystallographic composition
and are of a microscopic size, typically of longest dimension
within the range 1 micron to 100 microns. The term
"microcrystalline" refers to the state of being a microcrystal.
[0042] The term "rod-like" means the distinctive microcrystal
morphology that is also described as pyramidal-tipped tetragonal
rods. The morphology of microcrystals of the present invention are
easily determined by microscopic examination.
[0043] The term "irregular morphology" is a characterization of
microcrystals whose morphology, as determined by microscopic
examination, is not readily classified into any of the well-known
crystal types, is not a single type of crystal morphology, or is
not readily determinable because the size of the crystals is too
small for certain classification.
[0044] The term "amorphous precipitate" refers to insoluble
material that is not crystalline in form. The person of ordinary
skill can distinguish crystals from amorphous precipitate. The
amorphous precipitates of the present invention have advantageous
pharmacological properties in their own right, and also are
intermediates in the formation of the microcrystals of the present
invention.
[0045] The term "protein" may have its common meaning, that is, a
polymer of amino acids. The term "protein," as used herein, also
has a narrower meaning, that is, a protein selected from the group
consisting of insulin, insulin analogs, and proinsulins. The term
"un-derivatized proteins" also refers to a protein selected from
the group consisting of insulin, insulin analogs, and
proinsulins.
[0046] As used in the claims, and elsewhere as the context
dictates, the term "total protein" refers to the combined amount of
protein (insulin, an insulin analog, or a proinsulin) and
derivatized protein (derivatized insulin, a derivatized insulin
analog, or a derivatized proinsulin). Although protamine and other
known complexing compounds are also proteins in the broadest sense
of that term, the term "total protein" does not include them.
[0047] The term "derivatized protein" refers to a protein selected
from the group consisting of derivatized insulin, derivatized
insulin analogs, and derivatized proinsulin that is derivatized by
a functional group such that the derivatized protein is either less
soluble in an aqueous solvent than is the un-derivatized protein,
is more lipophilic than un-derivatized insulin, or produces a
complex with zinc and protamine that are less soluble than the
corresponding complex with the un-derivatized protein. The
determination of either the solubility or lipophilicity of proteins
and derivatized proteins is well-known to the skilled person. The
solubility of derivatized proteins and protein in complexes with
zinc and protamine can be readily determined by well-known
procedures [Graham and Pomeroy, J. Pharm. Pharmacol. 36:427-430
(1983), as modified in DeFelippis, M. R. and Frank, B., EP
735,048], or the procedure used herein.
[0048] Many examples of such derivatized proteins are known in the
art, including benzoyl, p-tolyl-sulfonamide carbonyl, and indolyl
derivatives of insulin and insulin analogs [Havelund, S., et al.,
WO95/07931, published Mar. 23, 1995]; alkyloxycarbonyl derivatives
of insulin (Geiger, R., et al., U.S. Pat. No. 3,684,791, issued
Aug. 15, 1972; Brandenberg, D., at al., U.S. Pat. No. 3,907,763
issued Sep. 23, 1975); aryloxycarbonyl derivatives of insulin
[Brandenberg, D., et al., U.S. Pat. No. 3,907,763, issued Sep. 23,
1975]; alkylcarbamyl derivatives [Smyth, D. G., U.S. Pat. No.
3,864,325, issued Feb. 4, 1975; Lindsay, D. G., et al., U.S. Pat.
No. 3,950,517, issued Apr. 13, 1976); carbamyl, O-acetyl
derivatives of insulin [Smyth, D. G., U.S. Pat. No. 3,864,325
issued Feb. 4, 1975]; cross-linked, alkyl dicarboxyl derivatives
[Brandenberg, D., et al., U.S. Pat. No. 3,907,763, issued Sep. 3,
1975]; N-carbamyl, 0-acetylated insulin derivatives [Smyth, D. G.,
U.S. Pat. No. 3,868,356, issued Feb. 25, 1975]; various O-alkyl
esters [Markussen, J., U.S. Pat. No. 4,343,898, issued Aug. 10,
1982; Morihara, K., et al., U.S. Pat. No. 4,400,465, issued Aug.
23, 1983; Morihara, K., et al., U.S. Pat. No. 4,401,757, issued
Aug. 30, 1983; Markussen, J., U.S. Pat. No. 4,489,159, issued Dec.
18, 1984; Obermeier, R., et al., U.S. Pat. No. 4,601,852, issued
Jul. 22, 1986; and Andresen, F. H., et al., U.S. Pat. No.
4,601,979, issued Jul. 22, 1986]; alkylamide derivatives of insulin
[Balschmidt, P., et al., U.S. Pat. No. 5,430,016, issued Jul. 4,
1995]; various other derivatives of insulin (Lindsay, D. G., U.S.
Pat. No. 3,869,437, issued Mar. 4, 1975]; and the fatty
acid-acylated proteins that are described herein.
[0049] The term "acylated protein" as used herein refers to a
derivatized protein selected from the group consisting of insulin,
insulin analogs, and proinsulin that is acylated with an organic
acid moiety that is bonded to the protein through an amide bond
formed between the acid group of an organic acid compound and an
amino group of the protein. In general, the amino group may be the
al-amino group of an N-terminal amino acid of the protein, or may
be the .epsilon.-amino group of a Lys residue of the protein. An
acylated protein may be acylated at one or more of the three amino
groups that are present in insulin and in most insulin analogs.
Mono-acylated proteins are acylated at a single amino group.
Di-acylated proteins are acylated at two amino groups. Tri-acylated
proteins are acylated at three amino groups. The organic acid
compound may be, for example, a fatty acid, an aromatic acid, or
any other organic compound having a carboxylic acid group that will
form an amide bond with an amino group of a protein, and that will
lower the aqueous solubility, raise the lipophilicity, or decrease
the solubility of zinc/protamine complexes of the derivatized
protein compared with the un-derivatized protein.
[0050] The term "fatty acid-acylated protein" refers to a an
acylated protein selected from the group consisting of insulin,
insulin analogs, and proinsulins that is acylated with a fatty acid
that is bonded to the protein through an amide bond formed between
the acid group of the fatty acid and an amino group of the protein.
In general, the amino group may be the .alpha.-amino group of an
N-terminal amino acid of the protein, or may be the e-amino group
of a Lys residue of the protein. A fatty acid-acylated protein may
be acylated at one or more of the three amino groups that are
present in insulin and in most insulin analogs. Mono-acylated
proteins are acylated at a single amino group. Di-acylated proteins
are acylated at two amino groups. Tri-acylated proteins are
acylated at three amino groups. Fatty acid-acylated insulin is
disclosed in a Japanese patent application 1-254,699. See also,
Hashimoto, M., et al., Pharmaceutical Research, 6:171-176 (1989),
and Lindsay, D. G., et al., Biochemical J. 121:737-745 (1971).
Further disclosure of fatty acid-acylated insulins and fatty
acylated insulin analogs, and of methods for their synthesis, is
found in Baker, J. C., et al, U.S. Pat. No. 08/342,931, filed Nov.
17, 1994 and issued as U.S. Pat. No. 5,693,609, Dec. 2, 1997;
Havelund, S., et al., WO95/07931, published Mar. 23, 1995, and a
corresponding U.S. Pat. No. 5,750,497, May 12, 1998; and Jonassen,
I., et al., WO96/29342, published Sep. 26, 1996. These disclosures
are expressly incorporated herein by reference for describing fatty
acid-acylated insulins and fatty acid-acylated insulin analogs and
for enabling preparation of the same.
[0051] The term "fatty acid-acylated protein" includes
pharmaceutically acceptable salts and complexes of fatty
acid-acylated proteins. The term "fatty acid-acylated protein" also
includes preparations of acylated proteins wherein the population
of acylated protein molecules is homogeneous with respect to the
site or sites of acylation. For example, N.epsilon.-mono-acylated
protein, B1-N.alpha.-mono-acylated protein,
A1-N.alpha.-mono-acylated protein, A1,B1-N.alpha.-di-acylated
protein, N.epsilon.,A1-N.alpha.,di-acylated protein,
N.epsilon.,B1-N.alpha.,di-acy- lated protein, and
N.epsilon.,A1,B1-N.alpha.,tri-acylated protein are all encompassed
within the term "fatty acid-acylated protein" for the purpose of
the present invention. The term also refers to preparations wherein
the population of acylated protein molecules has heterogeneous
acylation. In the latter case, the term "fatty acid-acylated
protein" includes mixtures of mono-acylated and di-acylated
proteins, mixtures of mono-acylated and tri-acylated proteins,
mixtures of di-acylated and tri-acylated proteins, and mixtures of
mono-acylated, di-acylated, and tri-acylated proteins.
[0052] The term "insulin" as used herein, refers to human insulin,
whose amino acid sequence and special structure are well-known.
Human insulin is comprised of a twenty-one amino acid A-chain and a
thirty-amino acid B-chain which are cross-linked by disulfide
bonds. A properly cross-linked insulin contains three disulfide
bridges: one between position 7 of the A-chain and position 7 of
the B-chain, a second between position 20 of the A-chain and
position 19 of the B-chain, and a third between positions 6 and 11
of the A-chain.
[0053] The term "insulin analog" means proteins that have an
A-chain and a B-chain that have substantially the same amino acid
sequences as the A-chain and B-chain of human insulin,
respectively, but differ from the A-chain and B-chain of human
insulin by having one or more amino acid deletions, one or more
amino acid replacements, and/or one or more amino acid additions
that do not destroy the insulin activity of the insulin analog.
[0054] "Animal insulins" are analogs of human insulin, and
therefore, are insulin analogs, as defined herein. Four such animal
insulins are rabbis, pork, beef, and sheep insulin. The amino acid
substitutions that distinguish these animal insulins from human
insulin are presented below for the reader's convenience.
1 Amino Acid Position A8 A9 A10 B30 human insulin Thr Ser Ile Thr
rabbit insulin Thr Ser Ile Ser pork insulin Thr Ser Ile Ala beef
insulin Ala Ser Val Ala sheep insulin Ala Gly Val Ala
[0055] Another type of insulin analog, "monomeric insulin analog"
is well-known in the art. Monomeric insulin analogs are
structurally very similar to human insulin, and have activity
similar or equal to human insulin, but have one or more amino acid
deletions, replacements or additions that tend to disrupt the
contacts involved in dimerization and hexamerization which results
in their having less tendency to associate to higher aggregation
states. Monomeric insulin analogs are rapid-acting analogs of human
insulin, and are disclosed, for example, in Chance, R. E., et al.,
U.S. Pat. No. 5,514,646, May 7, 1996; Brems, D. N., et al. Protein
Engineering, 5:527-533 (1992); Brange, J. J. V., et al., EPO
publication No. 214,826, published March 18, 1987; Brange, J. J.
V., et al., U.S. Pat. No. 5,618,913, Apr. 8, 1997; and Brange, J.,
et al., Current Opinion in Structural Biology 1:934-940 (1991). An
example of monomeric insulin analogs is described as human insulin
wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val,
or Ala, and wherein Lys at position B29 is Lys or is substituted
with Pro, and also, AlaB26-human insulin, des(B28-B30)-human
insulin, and des(B27)-human insulin. The monomeric insulin analogs
employed as derivatives in the present crystals, or employed
un-derivatized in the solution phase of suspension formulations,
are properly cross-linked at the same positions as is human
insulin.
[0056] Another group of insulin analogs for use in the present
invention are those wherein the isoelectric point of the insulin
analog is between about 7.0 and about 8.0. These analogs are
referred to as "pI-shifted insulin analogs." Examples of such
insulin analogs include ArgB31, ArgB32-human insulin, GlyA21,
ArgB31, ArgB32-human insulin, ArgA0,ArgB31, ArgB32-human insulin,
and ArgA0,GlyA21, ArgB31, ArgB32-human insulin.
[0057] Another group of insulin analogs consists of insulin analogs
that have one or more amino acid deletions that do not
significantly disrupt the activity of the molecule. This group of
insulin analogs is designated herein as "deletion analogs." For
example, insulin analogs with deletion of one or more amino acids
at positions B1-B3 are active. Likewise, insulin analogs with
deletion of one or more amino acids at positions B28-B30 are
active. Examples of "deletion analogs" include des(B30)-human
insulin, desPhe(B1)-human insulin, des(B27)-human insulin,
des(B28-B30)-human insulin, and des(B1-B3)-human insulin. The
deletion analogs employed as derivatives in the present crystals,
or employed un-derivatized in the solution phase of suspension
formulations, are properly cross-linked at the same positions as is
human insulin.
[0058] Amidated amino acids, and particularly asparagine residues
in insulin, are known to be chemically unstable [Jorgensen, K. H.,
et al. U.S. Pat. No. 5,008,241, issued Apr., 16, 1991; Dorschug,
M., U.S. Pat. No. 5,656,722, issued Aug., 12, 1997]. Particularly,
they are prone to deamidation and various rearrangement reactions
under certain conditions that are well-known. Therefore,
optionally, an insulin analog may be insulin or an insulin analog
that has one or more of its amidated residues replaced with other
amino acids for the sake of chemical stability. For example, Asn or
Gln may be replaced with a non-amidated amino acid. Preferred amino
acid replacements for Asn or Gln are Gly, Ser, Thr, Asp or Glu. It
is preferred to replace one or more Asn residues. In particular,
AsnA18, AsnA21, or AsnB3, or any combination of those residues may
be replaced by Gly, Asp, or Glu, for example. Also, GlnA15 or
GlnB4, or both, may be replaced by either Asp or Glu. Preferred
replacements are Asp at B21, and Asp at B3. Also preferred are
replacements that do not change the charge on the protein molecule,
so that replacement of Asn or Gln with neutral amino acids is also
preferred.
[0059] The term "proinsulin" means a single-chain peptide molecule
that is a precursor of insulin. Proinsulin may be converted to
insulin or to an insulin analog by chemical or, preferably,
enzyme-catalyzed reactions. In proinsulin, proper disulfide bonds
are formed as described herein. Proinsulin comprises insulin or an
insulin analog and a connecting bond or a connecting peptide. A
connecting peptide has between 1 and about 35 amino acids. The
connecting bond or connecting peptide connects to a terminal amino
acid of the A-chain and to a terminal amino acid of the B-chain by
an .alpha.-amide bond or by two .alpha.-amide bonds, respectively.
Preferably, none of the amino acids in the connecting peptide is
cysteine. Preferably, the C-terminal amino acid of the connecting
peptide is Lys or Arg. Proinsulin may have the formula X-B-C-A-Y or
may have the formula X-A-C-B-Y, wherein X is hydrogen or is a
peptide of from 1 to about 100 amino acids that has either Lys or
Arg at its C-terminal amino acid, Y is hydroxy, or is a peptide of
from 1 to about 100 amino acids that has either Lys or Arg at its
N-terminal amino acid, A is the A-chain of insulin or the A-chain
of an insulin analog, C is a peptide of from 1 to about 35 amino
acids, none of which is cysteine, wherein the c-terminal amino acid
is Lys or Arg, and B is the B-chain of insulin or the B-chain of an
insulin analog.
[0060] A "pharmaceutically acceptable salt" means a salt formed
between any one or more of the charged groups in a protein and any
one or more pharmaceutically acceptable, non-toxic cations or
anions. Organic and inorganic salts include, for example, those
prepared from acids such as hydrochloric, sulfuric, sulfonic,
tartaric, fumaric, hydrobromic, glycolic, citric, maleic,
phosphoric, succinic, acetic, nitric, benzoic, ascorbic,
p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, propionic,
carbonic, and the like, or for example, ammonium, sodium,
potassium, calcium, or magnesium.
[0061] The verb "acylate" means to form the amide bond between a
fatty acid and an amino group of a protein. A protein is "acylated"
when one or more of its amino groups is combined in an amide bond
with the acid group of a fatty acid.
[0062] The term "fatty acid" means a saturated or unsaturated,
straight chain or branched chain fatty acid, having from one to
eighteen carbon atoms.
[0063] The term "C1 to C18 fatty acid" refers to a saturated,
straight chain or branched chain fatty acid having from one to
eighteen carbon atoms.
[0064] The term "divalent metal cation" refers to the ion or ions
that participate to form a complex with a multiplicity of protein
molecules. The transition metals, the alkaline metals, and the
alkaline earth metals are examples of metals that are known to form
complexes with insulin. The transitional metals are preferred. Zinc
is particularly preferred. Other transition metals that may be
pharmaceutically acceptable for complexing with insulin proteins
include copper, cobalt, and iron.
[0065] The term "complex" has two meanings in the present
invention. In the first, the term refers to a complex formed
between one or more atoms in the proteins that form the complex and
one or more divalent metal cations. The atoms in the proteins serve
as electron-donating ligands. The proteins typically form a hexamer
complex with divalent transition metal cations. The second meaning
of "complex" in the present invention is the association between
the complexing compound and hexamers. The "complexing compound" is
an organic molecule that typically has a multiplicity of positive
charges that binds to, or complexes with hexamers in the insoluble
composition, thereby stabilizing them against dissolution. Examples
of complexing compounds suitable in the present invention include
protamine, surfen, various globin proteins [Brange, J. , Galenics
of Insulin, Springer-Verlag, Berlin Heidelberg (1987)], and various
polycationic polymer compounds known to complex with insulin.
[0066] The term "protamine" refers to a mixture of strongly basic
proteins obtained from fish sperm. The average molecular weight of
the proteins in protamine is about 4,200 [Hoffmann, J. A., et al.,
Protein Expression and Purification, 1:127-133 (1990)]. "Protamine"
can refer to a relatively salt-free preparation of the proteins,
often called "protamine base." Protamine also refers to
preparations comprised of salts of the proteins. Commercial
preparations vary widely in their salt content.
[0067] Protamines are well-known to those skilled in the insulin
art and are currently incorporated into NPH insulin products. A
pure fraction of protamine is operable in the present invention, as
well as mixtures of protamines. Commercial preparations of
protamine, however, are typically not homogeneous with respect to
the proteins present. These are nevertheless operative in the
present invention. Protamine comprised of protamine base is
operative in the present invention, as are protamine preparations
comprised of salts of protamine, and those that are mixtures of
protamine base and protamine salts. Protamine sulfate is a
frequently used protamine salt. All mass ratios referring to
protamine are given with respect to protamine free base. The person
of ordinary skill can determine the amount of other protamine
preparations that would meet a particular mass ratio referring to
protamine.
[0068] The term "suspension" refers to a mixture of a liquid phase
and a solid phase that consists of insoluble or sparingly soluble
particles that are larger than colloidal size. Mixtures of NPH
microcrystals and an aqueous solvent form suspensions. Mixtures of
amorphous precipitate and an aqueous solvent also forms a
suspension. The term "suspension formulation" means a
pharmaceutical composition wherein an active agent is present in a
solid phase, for example, a microcrystalline solid, an amorphous
precipitate, or both, which is finely dispersed in an aqueous
solvent. The finely dispersed solid is such that it may be
suspended in a fairly uniform manner throughout the aqueous solvent
by the action of gently agitating the mixture, thus providing a
reasonably uniform suspension from which a dosage volume may be
extracted. Examples of commercially available insulin suspension
formulations include, for example, NPH, PZI, and ultralente. A
small proportion of the solid matter in a microcrystalline
suspension formulation may be amorphous. Preferably, the proportion
of amorphous material is less than 10%, and most preferably, less
than 1% of the solid matter in a microcrystalline suspension.
Likewise, a small proportion of the solid matter in an amorphous
precipitate suspension may be microcrystalline.
[0069] "NPH insulin" refers to the "Neutral Protamine Hagedorn"
preparation of insulin. Synonyms include human insulin NPH and
insulin NPH, among many others. Humulin.RTM. N is a commercial
preparation of NPH insulin. A related term is "NPL" which refers to
an NPH-like preparation of LysB28, ProB29-human insulin analog. The
meaning of these terms, and the methods for preparing them will be
familiar to the person of ordinary skill in the insulin formulation
art.
[0070] The term "aqueous solvent" refers to a liquid solvent that
contains water. An aqueous solvent system may be comprised solely
of water, may be comprised of water plus one or more miscible
solvents, and may contain solutes. The more commonly-used miscible
solvents are the short-chain organic alcohols, such as, methanol,
ethanol, propanol, short-chain ketones, such as acetone, and
polyalcohols, such as glycerol.
[0071] An "isotonicity agent" is a compound that is physiologically
tolerated and imparts a suitable tonicity to a formulation to
prevent the net flow of water across cell membranes that are in
contact with an administered formulation. Glycerol, which is also
known as glycerin, is commonly used as an isotonicity agent. Other
isotonicity agents include salts, e.g., sodium chloride, and
monosaccharides, e.g., dextrose and lactose.
[0072] The insoluble compositions of the present invention contain
a hexamer-stabilizing compound. The term "hexamer-stabilizing
compound" refers to a non-proteinaceous, small molecular weight
compound that stabilizes the protein or derivatized protein in a
hexameric aggregation state. Phenolic compounds, particularly
phenolic preservatives, are the best known stabilizing compounds
for insulin and insulin derivatives. Hexamer-stabilizing compounds
stabilize the hexamer by binding to it through specific
inter-molecular contacts. Examples of hexamer-stabilizing agents
include: various phenolic compounds, phenolic preservatives,
resorcinol, 4'-hydroxyacetanilide, 4-hydroxybenzamide, and
2,7-dihyroxynaphthalene. Multi-use formulations of the insoluble
compositions of the present invention will contain a preservative,
in addition to a hexamer-stabilizing compound. The preservative
used in formulations of the present invention may be a phenolic
preservative, and may be the same as, or different from the
hexamer-stabilizing compound.
[0073] The term "preservative" refers to a compound added to a
pharmaceutical formulation to act as an anti-microbial agent. A
parenteral formulation must meet guidelines for preservative
effectiveness to be a commercially viable multi-use product. Among
preservatives known in the art as being effective and acceptable in
parenteral formulations are benzalkonium chloride, benzethonium,
chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben,
chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric
nitrate, thimerosal, benzoic acid, and various mixtures thereof.
See, e.g., Wallhusser, K. -H., Develop. Biol. Standard, 24:9-28
(1974) (S. Krager, Basel).
[0074] The term "phenolic preservative" includes the compounds
phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben,
and mixtures thereof. Certain phenolic preservatives, such as
phenol and m-cresol, are known to bind to insulin-like molecules
and thereby to induce conformational changes that increase either
physical or chemical stability, or both [Birnbaum, D. T., et al.,
Pharmaceutical. Res. 14:25-36 (1997); Rahuel-Clermont, S., et al.,
Biochemistry 36:5837-5845 (1997)].
[0075] The term "buffer" or "pharmaceutically acceptable buffer"
refers to a compound that is known to be safe for use in insulin
formulations and that has the effect of controlling the pH of the
formulation at the pH desired for the formulation. The pH of the
formulations of the present invention is from about 6.0 to about
8.0. Preferably the formulations of the present invention have a pH
between about 6.8 and about 7.8. Pharmaceutically acceptable
buffers for controlling pH at a moderately acidic pH to a
moderately basic pH include such compounds as phosphate, acetate,
citrate, arginine, TRIS, and histidine. "TRIS" refers to
2-amino-2-hydroxymethyl-1,3, -propanediol, and to any
pharmacologically acceptable salt thereof. The free base and the
hydrochloride form are two common forms of TRIS. TRIS is also known
in the art as trimethylol aminomethane, tromethamine, and
tris(hydroxymethyl)aminomethane. Other buffers that are
pharmaceutically acceptable, and that are suitable for controlling
pH at the desired level are known to the chemist of ordinary
skill.
[0076] The term "administer" means to introduce a formulation of
the present invention into the body of a patient in need thereof to
treat a disease or condition.
[0077] The term "treating" refers to the management and care of a
patient having diabetes or hyperglycemia, or other condition for
which insulin administration is indicated for the purpose of
combating or alleviating symptoms and complications of those
conditions. Treating includes administering a formulation of
present invention to prevent the onset of the symptoms or
complications, alleviating the symptoms or complications, or
eliminating the disease, condition, or disorder.
[0078] As mentioned above, the present invention provides insoluble
compositions that have properties similar to NPH insulin in certain
respects, and superior to NPH insulin in other respects. They are
similar to NPH insulin in respect to their physical properties, as
described hereafter. A light microscope equipped with an oil
immersion objective and a crossed polarizer was utilized to examine
microcrystals comprised of B29-N.epsilon.-octanoyl-human insulin,
insulin, zinc, protamine, and phenol prepared according to the
present invention. Examination at 1000.times.magnification showed
that these microcrystals were single and rod-like, exhibiting a
uniform crystal morphology. The sizes of these microcrystals fell
generally within the range of approximately 2 microns long to 8
microns long. A direct comparison using this microscope showed that
the morphology of these microcrystals appeared to be similar to
that of commercially manufactured pork NPH microcrystals, which has
elsewhere been described as rod-like. The size range of these
microcrystals was also similar to that of commercially manufactured
NPH microcrystals, which generally have an average length of about
5 microns. The commercial manufacturing specification for the mean
length of NPH microcrystals is from 1 micron to 40 microns.
[0079] The microcrystals of the present invention are, however,
unexpectedly and unpredictably different from NPH insulin crystals
in their dissolution properties, and in their time action. In
particular, the microcrystals of the present invention dissolve
much more slowly under conditions that simulate physiologic
conditions than do NPH insulin crystals, and provide a longer and
flatter profile of blood glucose control than does NPH insulin.
This was demonstrated by the following experiments.
[0080] Certain derivatized proteins, in soluble form, were found to
have time actions not significantly different from regular human
insulin. Three groups of animals were used. Each animal in the
first group received a dose (0.75 nmol/kg) of Humulin.RTM. R
(soluble human insulin), each animal in the second group received a
dose (0.75 nmol/kg) of soluble B29-N.epsilon.-octanoyl-human
insulin ("C8-hI"), and each animal in the third group received a
dose (0.75 nmol/kg) of soluble B29-N.epsilon.-decanoyl-human
insulin ("C10-hI"). The experiments were carried out essentially as
described in Example 5, with five dogs per group. The proteins were
administered subcutaneously. Blood glucose concentrations were
determined, and are presented in the table below.
[0081] Table 1. Blood glucose concentrations before and after
administration of Humulin.RTM. R, soluble
B29-N.epsilon.-octanoyl-human insulin ("C8-hI"), or soluble
B29-N.epsilon.-decanoyl-human insulin ("C10-hI") in normal dogs
simultaneously administered 20 somatostatin to create a transient
diabetic state. Values are mean.+-.standard error.
2 Blood Glucose Concentration (mg/dL) Soluble Soluble Time (h)
Humulin .RTM. R C8-hI C10-hI -0.5 110 .+-. 2 115 .+-. 4 108 .+-. 2
0 101 .+-. 2 101 .+-. 7 96 .+-. 4 0.5 83 .+-. 5 80 .+-. 5 85 .+-. 6
1 54 .+-. 6 52 .+-. 4 70 .+-. 5 1.5 49 .+-. 4 51 .+-. 2 57 .+-. 4 2
48 .+-. 4 51 .+-. 2 52 .+-. 3 2.5 55 .+-. 4 60 .+-. 3 56 .+-. 4 3
59 .+-. 2 65 .+-. 4 58 .+-. 4 3.5 65 .+-. 2 73 .+-. 5 63 .+-. 4 4
71 .+-. 2 85 .+-. 6 68 .+-. 4 5 87 .+-. 2 110 .+-. 8 79 .+-. 3 6
104 .+-. 3 124 .+-. 4 91 .+-. 7 7 119 .+-. 8 145 .+-. 14 106 .+-. 8
8 144 .+-. 5 153 .+-. 16 119 .+-. 11
[0082] These data clearly show that soluble
B29-N.epsilon.-octanoyl-human insulin and
B29-N.epsilon.-decanoyl-human insulin, administered subcutaneously
to normal dogs in a transient diabetic state, provide glucose
lowering roughly comparable to that obtained with soluble human
insulin. Most notably, soluble B29-N.epsilon.-octanoyl-human
insulin shows a quicker onset, and shorter time action than does
human insulin.
[0083] In a second experiment, the dissolution rate of co-crystals
of insulin and B29-N.epsilon.-octanoyl-human insulin prepared in
accordance with the present invention was found to be markedly
longer than that of a commercially manufactured NPH-pork insulin.
This was most unexpected in view of the data above. The rate of
dissolution may be measured using well-known procedures (Graham and
Pomeroy, J. Pharm. Pharmacol. 36:427-430 (1983), as modified in De
Felippis, M. R. and Frank, B., EP 735,048], or the procedure used
herein.
[0084] The dissolution rate of pork insulin NPH microcrystals was
measured by placing 5 microliters of U100 pork insulin NPH into 3
mL of Dulbecco's phosphate buffered saline (without calcium or
magnesium) in a 1 cm path length square quartz cuvette at a
temperature of 22.degree. C. This solution was stirred at a
constant rate using a magnetic cuvette stirrer. Absorbance
measurements at 320 nm were taken at 1 minute intervals. The
absorbance at 320 nm corresponds to the light scattered by the
insoluble particles present in the aqueous suspension.
Consequently, as the microcrystals dissolve, the absorbance
approaches zero. Pork insulin NPH microcrystals were completely
dissolved after about 20 minutes.
[0085] The dissolution rate of protamine-zinc crystals of human
insulin containing no co-crystallized acylated human insulin was
also found to be about 20 minutes by the procedure used above.
These crystals of NPH-human insulin were prepared essentially by
the procedure of Preparation #1, below, except that no acylated
protein was used, and 7 parts of human insulin were used. The data
generated from this experiment are presented in FIG. 1 as the
dashed line.
[0086] Co-crystals of the present invention comprised of human
insulin and B29-N.epsilon.-octanoyl-LysB29 human insulin were
prepared as described in Preparations #2, 4, and 5, below. In these
preparations, human insulin and the acylated insulin were used in
pre-crystallization mass ratios of 3:1, 1:1, and 1:3,
respectively.
[0087] A procedure as described above was followed to measure the
dissolution rate of these co-crystals. In summary, a volume of 12
microliters of each protamine-zinc-B29-N.epsilon.-octanoyl-LysB29
human insulin-human insulin co-crystalline suspension (containing
no more than 50 U/mL) was placed into 3 mL of Dulbecco's phosphate
buffered saline (without calcium or magnesium) in a 1 cm path
length square quartz cuvette. This solution was stirred at the same
constant rate and at the same temperature of 22.degree. C. The data
generated from this experiment are presented in FIG. 1, and show
that the 3:1 co-crystals required more than 100 minutes to
dissolve, that the 1:1 co-crystals required more than 150 minutes
to dissolve, and that the 1:3 co-crystals required did not
completely dissolve even after 400 minutes.
[0088] The time required for the absorbance during dissolution to
reach half way from the starting absorbance value to the final
absorbance value is defined as the t/.sub.1/2 value. The
t.sub.1/2values for these preparations are presented below in Table
2.
3TABLE 2 Dissolution t.sub.1/2 values for Iletin NPH, Human Insulin
NPH, and co-crystals of B29-octanoyl-human insulin and human
insulin % B29- octanoyl- human % human % porcine t.sub.1/2 value
Identity insulin insulin insulin (minutes) Iletin NPH 0 0 100 6
Human NPH 0 100 0 7 3:1 co-crystal 25 75 0 38 1:1 co-crystal 50 50
0 117 1:3 co-crystal 75 25 0 >400
[0089] These experiments establish that, in Dulbecco's phosphate
buffered saline (without calcium and magnesium), a solution that
mimics the interstitial fluid in certain aspects, the rate of
dissolution of the 3:1, 1:1, and 1:3 co-crystals is significantly
slower than that of pork NPH microcrystals. These experiments also
establish that the rate of dissolution of protamine-zinc-human
insulin microcrystals is very similar to that of pork NPH insulin
(Iletin NPH). These results further establish that the rate of
dissolution of protamine-zinc-B29-N.epsilon.-octanoyl-Ly- sB29
human insulin-human insulin co-crystals depends on the ratio of
human insulin to B29-N.epsilon.-octanoyl-LysB29 human insulin
present in the co-crystals, and, in particular, the rate of
dissolution of protamine-zinc-B29-N.epsilon.-octanoyl-LysB29 human
insulin-human insulin co-crystals decreases as the proportion of
human insulin decreases.
[0090] Another dissolution method, based on HPLC determination of
dissolved protein and derivatized protein, was used to compare the
dissolution rate of the 1:3 microcrystals described above with
commercial preparations of human insulin NPH, human insulin
ultralente, and beef ultralente, and also to study the effects on
dissolution rate of varying the ratio of protein to derivatized
protein.
[0091] A volume (O.5 mL) of a U100 formulation was suspended in 200
mL of Dulbecco's phosphate buffered saline adjusted to pH 7.4 in a
water-jacketed dissolution apparatus maintained at 25.degree. C.
The dissolution buffer also contained 1 mg/mL human serum albumin
to prevent adsorption losses of dissolved insulins. The dissolution
medium was stirred at a constant rate of 180 rpm. At regular
intervals, 3.5 mL of this solution was taken out and filtered
through a 0.22 .mu.m, low protein-binding filter. The first 0.5 mL
of the filtrate was discarded and the next 1.5 mL of the filtrate
was acidified with 4 mL of 5 N HCl and subjected to HPLC analysis.
The concentrations of the dissolved insulins were determined based
on HPLC peak areas and the results were expressed as the percent
dissolved as a function of time, with 100% being the total area of
insulins in unfiltered samples. If the total area in unfiltered
samples decreased slightly as a function of time, a linearly
corrected value was used as 100%, for computation of percent
dissolved at each time point. The results of these studies are
presented in FIGS. 3 and 4.
[0092] The data of FIG. 3 show that the greater the fraction of
derivatized protein incorporated into the microcrystal, the slower
the dissolution rate. The data of FIG. 4 show that the 1:3
microcrystal of human insulin and B29-octanoyl human insulin
dissolves significantly more slowly than both human insulin NPH and
human insulin ultralente. Most significantly, the 1:3 microcrystal
has a dissolution rate very similar to that of beef ultralente.
Beef ultralente has long been considered a nearly ideal long-acting
insulin preparations, both because of its very long protraction of
biological activity, and because of the flatness of the
pharmacodynamic response after its administration. It is thus
predicted that these microcrystals may approach or exceed beef
ultralente in their ability to provide control of basal glucose
output for very long time periods.
[0093] Because the time action profile of NPH preparations is
related strongly to the rate of dissolution of the microcrystals in
the subcutaneous interstitial fluid, it is concluded from these
experiments that the microcrystalline compositions of the present
invention possess a more protracted duration of action when
administered subcutaneously to diabetic patients than existing
commercial NHP insulin preparations. Importantly, these results
also establish that the present invention makes it possible to
control, and even to optimize, the duration of action in patients
by manipulating the ratio of protein to derivatized protein.
[0094] This ability to manipulate the duration of action by simply
varying the ratios of protein and derivatized protein is most
significant when viewed against the historical backdrop.
Historically, a major obstacle to the development of insulin
formulations for controlling basal glucose output has been that
their time-action has been inflexibly linked to the inherent
molecular properties of the protein, for example, albumin-binding
affinity, isoelectric point, or solubility. The consequence was
that only a single time-action was possible for each molecule or
formulation, and the only recourse for improving the
pharmacokinetics was a further modification of the molecule.
[0095] A long sought goal has been to develop controlled-release
delivery systems where the pharmacokinetics can be precisely and
conveniently adjusted by manipulating the formulation matrix. Much
academic and industrial research in this area has been directed
towards insulin over the past 15 years, however, the goal of
controlled release has proven particularly elusive because of the
extremely narrow therapeutic index of insulin, its chronic-use
requirement, as well as economic considerations which bias against
sophisticated and expensive processes and formulations.
[0096] A major advantage of the present invention is that it is a
controlled release system where the pharmacokinetics of insulin
release can be more conveniently controlled than other proposed
controlled release technologies. That is, an insoluble composition
comprised of a protein insulin with a derivatized protein provides
a convenient means to adjust the dissolution rate and hence make
controlled-release possible. While not intended to be limiting, it
is believed that the pharmacological efficacy of the insoluble
compositions of the present invention is based on the slow release
of a consistent proportion of protein and derivatized protein from
the composition. It is further believed, without limiting the
invention, that a significant underlying feature of this invention
is the complete or nearly complete homogeneity of the insoluble
composition. For microcrystals, it is believed that every
individual microcrystal in the suspension is comprised of very
nearly the same ratio of protein and derivatized protein. This
ratio closely reflects the ratio of protein to derivatized protein
combined in solution prior to crystallization. It is also believed
that, as the microcrystals dissolve, a consistent and predetermined
proportion of protein and derivatized protein is released
throughout the entire duration of dissolution. The significance of
this behavior is considerable because it results in a constant, but
reduced rate of release of the two active molecules from the site
of injection to the bloodstream. In order to achieve this object,
particular attention must be given to the process for preparing the
compositions.
[0097] The present discovery that it is possible to crystallize
protein with derivatized protein, and thereby obtain a homogeneous
co-crystal, was a surprising finding in view of the complexity of
the parameters affecting protein crystallization: specific and
non-specific inter-molecular interactions, such as,
hydrogen-bonding, electrostatic interactions, hydrophobic
interactions, van der Waals forces, excluded volume effects,
solubility, and steric bulk. Although the fundamental understanding
of small molecule crystallization is relatively advanced, for
complex macromolecules it is still largely a qualitative science
and current practice consists of empirically applying large arrays
of crystallization recipes.
[0098] The hydrophobic effect has been proposed as a major driving
force for protein association [Chothia, C. et al., Nature
256:705-708 (1975)]. Furthermore, strong correlation between
surface hydrophobicity and protein-protein contacts have been noted
(Young, L., et al., Protein Science 3:717-729 (1994)]. Yet, in view
of the complexity of protein crystallization, the finding that
derivatized insulins can co-crystallize with un-derivatized protein
in a manner that is isomorphous, or nearly isomorphous to normal
insulin, is surprising.
[0099] The co-crystallization of two related therapeutic proteins
for the purpose of modifying therapeutic behavior is a new concept.
To a large extent this is because the vast majority of protein
crystallization work is motivated by the goal of obtaining single,
large, homogeneous crystals suitable for x-ray diffraction
structural analysis. Although protein co-crystals are known, such
co-crystal systems are characterized as host-substrate complexes
e.g. a protein and its receptor, protein-DNA complexes, and protein
receptor-drug complexes. These host-substrate complex co-crystals
are fundamentally different than the co-crystals of the present
invention because there is no complex formed between the protein
and derivatized protein.
[0100] The predictability of the proportions of protein and
derivatized protein and the homogeneity of the insoluble
compositions of the present invention were demonstrated by
measuring (by HPLC) the concentrations of protein and derivatized
protein during dissolution studies using two preparations of
microcrystals that were prepared with known quantities of human
insulin (protein) and B29-octanoyl-human insulin (derivatized
protein). In the first preparation, the mass proportion of protein
added to derivatized protein added was 1:3. In the second
preparation, the proportion was 55:45. For the 1:3 microcrystals,
B29-octanoyl-human insulin constituted between about 73% and 76% of
the total protein for 14 measurements over 10 hours during
dissolution under conditions as described for the dissolution test
above. No trend in the data was observed. For the 55:45
microcrystals, B29-octanoyl-human insulin constituted between about
43% and 47% of the total protein for 9 measurements taken over a
period of about 3.75 hours during dissolution under conditions as
described for the dissolution test above. Again, no trend in the
data was observed.
[0101] The insoluble compositions of the present invention may be
crystals with rod-like morphology or with an irregular morphology,
or they may be amorphous precipitates. Preferred insoluble
compositions are comprised of acylated insulin or acylated insulin
analog, zinc ions, which are present at about 0.3 to about 0.7 mole
per mole of total protein, a phenolic preservative selected from
the group consisting of phenol, m-cresol, o-cresol, p-cresol,
chlorocresol, methylparaben, and mixtures thereof and is present in
sufficient proportions with respect to total protein to stabilize
the T3R3 or R6 hexamer conformation, and protamine, which is
present at about 0.15 to about 0.7 mole per mole of total
protein.
[0102] A preferred group of insulin analogs for preparing
derivatized insulin analogs used to form the present insoluble
compositions consists of animal insulins, deletion analogs, and
pI-shifted analogs. A more preferred group consists of animal
insulins and deletion analogs. Deletion analogs are yet more
preferred.
[0103] Another preferred group of insulin analogs for use in the
microcrystals of the present invention consists of the monomeric
insulin analogs. Particularly preferred are those monomeric insulin
analogs wherein the amino acid residue at position B28 is Asp, Lys,
Leu, Val, or Ala, the amino acid residue at position B29 is Lys or
Pro, the amino acid residue at position B10 is His or Asp, the
amino acid residue at position B1 is Phe, Asp or deleted alone or
in combination with a deletion of the residue at position B2, the
amino acid residue at position B30 is Thr, Ala, Ser, or deleted,
and the amino acid residue at position B9 is Ser or Asp; provided
that either position B28 or B29 is Lys.
[0104] Another preferred group of insulin analogs for use in the
present invention consists of those wherein the isoelectric point
of the insulin analog is between about 7.0 and about 8.0. These
analogs are referred to as "pI-shifted insulin analogs." Examples
of pI-shifted insulin analogs include, for example, ArgB31,
ArgB32-human insulin, GlyA21, ArgB31, ArgB32-human insulin,
ArgA0,ArgB31, Arg32-human insulin, and ArgA0,GlyA21, ArgB31,
ArgB32-human insulin.
[0105] Another preferred group of insulin analogs consists of
LysB28, ProB29-human insulin (B28 is Lys; B29 is Pro); AspB28-human
insulin (B28 is Asp), AspB1-human insulin, ArgB31, ArgB32-human
insulin, ArgA0-human insulin, AspB1,GluB13-human insulin,
AlaB26-human insulin, GlyA21-human insulin, des(ThrB30)-human
insulin, and GlyA21, ArgB31, ArgB32-human insulin.
[0106] Especially preferred insulin analogs include LysB28,
ProB29-human insulin, des(ThrB30)-human insulin, AspB28-human
insulin, and AlaB26-human insulin. Another especially preferred
insulin analog is GlyA21, ArgB31, ArgB32-human insulin [Dorschug,
M., U.S. Pat. No. 5,656,722, Aug. 12, 1997]. The most preferred
insulin analog is LysB28, ProB29-human insulin.
[0107] The preferred derivatized proteins are acylated proteins,
and the preferred acylated proteins for the microcrystals and
formulations of the present invention are fatty acid-acylated
insulin, and fatty acid-acylated insulin analogs. Fatty
acid-acylated human insulin is highly preferred. Fatty
acid-acylated insulin analogs are equally highly preferred.
[0108] The particular group used to derivatize insulin, an insulin
analog, or a proinsulin (collectively, protein) may be any chemical
moiety that does not significantly reduce the biological activity
of the protein, is not toxic when bonded to the protein, and most
importantly, reduces the aqueous solubility, raises the
lipophilicity, or decreases the solubility of zinc/protamine
complexes of the derivatized protein.
[0109] One preferred group of acylating moieties consists of fatty
acids that are straight chain and saturated. This group consists of
methanoic acid (C1), ethanoic acid (C2), propanoic acid (C3),
n-butanoic acid (C4), n-pentanoic acid (C5), n-hexanoic acid (C6),
n-heptanoic acid (C7), n-octanoic acid (C8), n-nonanoic acid (C9),
n-decanoic acid (C10), n-undecanoic acid (C11), n-dodecanoic acid
(C12), n-tridecanoic acid (C13), n-tetradecanoic acid (C14),
n-pentadecanoic acid (C15), n-hexadecanoic acid (C16),
n-heptadecanoic acid (C17), and n-octadecanoic acid (C18).
Adjectival forms are formyl (C1), acetyl (C2), propionyl (C3),
butyryl (C4), pentanoyl (C5), hexanoyl (C6), heptanoyl (C7),
octanoyl (C8), nonanoyl (C9), decanoyl (C10), undecanoyl (C11),
dodecanoyl (C12), tridecanoyl (C13), tetradecanoyl (C14) or
myristoyl, pentadecanoyl (C15), hexadecanoyl (C16) or palmitic,
heptadecanoyl (C17), and octadecanoyl (C18) or stearic.
[0110] A preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having an even number of carbon
atoms--that is, C2, C4, C6, C8, C10, C12, C14, C16, and C18
saturated fatty acids.
[0111] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having an odd number of carbon
atoms--that is, C1, C3, C5, C7, C9, C11, C13, C15, and C17
saturated fatty acids.
[0112] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having more than 5 carbon
atoms--that is, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16,
C17, and C18 saturated fatty acids.
[0113] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having less than 9 carbon
atoms--that is, C1, C2, C3, C4, C5, C6, C7, and C8 saturated fatty
acids.
[0114] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having between 6 and 8 carbon
atoms--that is, C6, C7, and C8, saturated fatty acids.
[0115] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having more than between 4 and 6
carbon atoms--that is, C4, C5, and C6, saturated fatty acids.
[0116] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having more than between 2 and 4
carbon atoms--that is, C2, C3, and C4, saturated fatty acids.
[0117] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having less than 6 carbon
atoms--that is, C1, C2, C3, C4, and C5 saturated fatty acids.
[0118] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having less than 4 carbon
atoms--that is, C1, C2, and C3 saturated fatty acids.
[0119] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having more than 9 carbon
atoms--that is, C10, C11, C12, C13, C14, C15, C16, C17, and C18
saturated fatty acids.
[0120] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having an even number of carbon
atoms and more than 9 carbon atoms--that is, C10, C12, C14, C16,
and C18 saturated fatty acids.
[0121] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having 12, 14, or 16 carbon
atoms, that is, C12, C14, and C16 saturated fatty acids.
[0122] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having 14 or 16 carbon atoms,
that is, C14 and C16 saturated fatty acids. Fatty acids with 14
carbons are particularly preferred. Fatty acids with 16 carbons are
also particularly preferred.
[0123] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of saturated fatty acids having between 4 and 10
carbon atoms, that is C4, C5, C6, C7, C8, C9, and C10 saturated
fatty acids.
[0124] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of saturated fatty acids having an even number
of carbon atoms between 4 and 10 carbon atoms, that is C4, C6, C8,
and C10 saturated fatty acids.
[0125] Another preferred group of fatty acids for forming the fatty
acid-acylated proteins used in the microcrystals of the present
invention consists of fatty acids having between 6, 8, or 10 carbon
atoms. Fatty acids with 6 carbons are particularly preferred. Fatty
acids with 8 carbons are also particularly preferred. Fatty acids
with 10 carbons are particularly preferred.
[0126] The skilled person will appreciate that narrower preferred
groups are made by combining the preferred groups of fatty acids
described above.
[0127] Another preferred group of acylating moieties consists of
saturated fatty acids that are branched. A branched fatty acid has
at least two branches. The length of a "branch" of a branched fatty
acid may be described by the number of carbon atoms in the branch,
beginning with the acid carbon. For example, the branched fatty
acid 3-ethyl-5-methylhexanoi- c acid has three branches that are
five, six, and six carbons in length. In this case, the "longest"
branch is six carbons. As another example,
2,3,4,5-tetraethyloctanoic acid has five branches that are 4, 5, 6,
7, and 8 carbons long. The "longest" branch is eight carbons. A
preferred group of branched fatty acids are those having from three
to ten carbon atoms in the longest branch.
[0128] A representative number of such branched, saturated fatty
acids will be mentioned to assure the reader's comprehension of the
range of such fatty acids that may be used as acylating moieties of
the proteins in the present invention: 2-methyl-propioinic acid,
2-methyl-butyric acid, 3-methyl-butyric acid,
2,2-dimethyl-propionic acid, 2-methyl-pentanoic acid,
3-methyl-pentanoic acid, 4-methyl-pentanoic acid,
2,2-dimethyl-butyric acid, 2,3-dimethyl-butyric acid,
3,3-dimethyl-butyric acid, 2-ethyl-butyric acid, 2-methyl-hexanoic
acid, 5-methyl-hexanoic acid, 2,2-dimethyl-pentanoic acid,
2,4-dimethyl-pentanoic acid, 2-ethyl-3-methyl-butyric acid,
2-ethyl-pentanoic acid, 3-ethyl-pentanoic acid,
2,2-dimethyl-3-methyl-but- yric acid, 2-methyl-heptanoic acid,
3-methyl-heptanoic acid, 4-methyl-heptanoic acid,
5-methyl-heptanoic acid, 6-methyl-heptanoic acid,
2,2-dimethyl-hexanoic acid, 2,3-dimethyl-hexanoic acid,
2,4-dimethyl-hexanoic acid, 2,5-dimethyl-hexanoic acid,
3,3,-dimethyl-hexanoic acid, 3,4-dimethyl-hexanoic acid,
3,5-dimethyl-hexanoic acid, 4,4-dimethyl-hexanoic acid,
2-ethyl-hexanoic acid, 3-ethyl-hexanoic acid, 4-ethyl-hexanoic
acid, 2-propyl-pentanoic acid, 2-ethyl-hexanoic acid,
3-ethyl-hexanoic acid, 4-ethyl-hexanoic acid, 2-(1-propyl)pentanoic
acid, 2-(2-propyl)pentanoic acid, 2,2-diethyl-butyric acid,
2,3,4-trimethyl-pentanoic acid, 2-methyl-octanoic acid,
4-methyl-octanoic acid, 7-methyl-octanoic acid,
2,2-dimethyl-heptanoic acid, 2,6-dimethyl-heptanoic acid,
2-ethyl-2-methyl-hexanoic acid, 3-ethyl-5-methyl-hexanoic acid,
3-(1-propyl)-hexanoic acid, 2-(2-butyl)-pentanoic acid,
2-(2-(2-methylpropyl))pentanoic acid, 2-methyl-nonanoic acid,
8-methyl-nonanoic acid, 6-ethyl-octanoic acid,
4-(1-propyl)-heptanoic acid, 5-(2-propyl)-heptanoic acid,
3-methyl-undecanoic acid,2-pentyl-heptanoic acid,
2,3,4,5,6-pentamethyl-heptanoic acid, 2,6-diethyl-octanoic acid,
2-hexyl-octanoic acid, 2,3,4,5,6,7-hexamethyl-- octanoic acid,
3,3-diethyl-4,4-diethyl-hexanoic acid, 2-heptyl-nonanoic acid,
2,3,4,5-tetraethyl-octanoic acid, 2-octyl-decanoic acid, and
2-(1-propyl)-3-(1-propyl)-4,5-diethyl-6-methyl-heptanoic acid.
[0129] Yet another preferred group of acylating moieties consists
of cyclic alkyl acids having from 5 to 24 carbon atoms, wherein the
cyclic alkyl moiety, or moieties, have 5 to 7 carbon atoms. A
representative number of such cyclic alkyl acids will be mentioned
to assure the reader's comprehension of the range of such acids
that may be used as acylating moieties of the proteins in the
present invention:
[0130] cyclopentyl-formic acid, cyclohexyl-formic acid,
1-cyclopentyl-acetic acid, 2-cyclohexyl-acetic acid,
1,2-dicyclopentyl-acetic acid, and the like.
[0131] A preferred group of derivatized proteins for use in the
insoluble compositions of the present invention consists of
mono-acylated proteins. Mono-acylation at the .epsilon.-amino group
is most preferred. For insulin, mono-acylation at LysB29 is
preferred. Similarly, for certain insulin analogs, such as, LysB28,
ProB29-human insulin analog, mono-acylation at the .epsilon.-amino
group of LysB28 is most preferred. Mono-acylation at the
.alpha.-amino group of the B-chain (B1) is also preferred.
Mono-acylation at the .alpha.-amino group of the A-chain (A1) is
also preferred.
[0132] Another preferred group of acylated proteins for use in the
insoluble compositions of the present invention consists of
di-acylated proteins. The di-acylation may be, for example, at the
.epsilon.-amino group of Lys and at the .alpha.-amino group of the
B-chain, or may be at the .epsilon.-amino group of Lys and at the
.alpha.-amino group of the A-chain, or may be at the .alpha.-amino
group the A-chain and at the .alpha.-amino group of the
B-chain.
[0133] Another preferred group of acylated proteins for use in the
insoluble compositions of the present invention consists of
tri-acylated proteins. Tri-acylated proteins are those that are
acylated at the .epsilon.-amino group of Lys, at the .alpha.-amino
group of the B-chain, and at the .alpha.-amino group of the
A-chain.
[0134] It is also preferred to use acylated proteins that are a
mixture of mono-acylated and di-acylated proteins.
[0135] It is likewise preferred to use acylated proteins that are a
mixture of mono-acylated and tri-acylated proteins.
[0136] Another preferred group of acylated proteins consists of a
mixture of di-acylated and tri-acylated proteins.
[0137] Also preferred is to use acylated proteins that are a
mixture of mono-acylated, di-acylated, and tri-acylated
proteins.
[0138] Certain fatty acid-acylated proteins used in the present
microcrystals will be mentioned to assure the reader's
comprehension of the scope of the present invention. The list is
illustrative, and the fact that a particular fatty acid-acylated
protein is not mentioned does not mean that a microcrystal
containing it is not within the scope of the present invention.
[0139] B29-N.epsilon.-Formyl-human insulin.
[0140] B1-N.alpha.-Formyl-human insulin.
[0141] A1-N.alpha.-Formyl-human insulin.
[0142] B29-N.epsilon.-Formyl-, B1-N.alpha.-formyl-human
insulin.
[0143] B29-N.epsilon.-Formyl-, A1-N.alpha.-formyl-human
insulin.
[0144] A1-N.alpha.-Formyl-, B1-N.alpha.-formyl-human insulin.
[0145] B29-N.epsilon.-Formyl-, A1-N.alpha.-formyl-,
B1-N.alpha.-formyl-human insulin.
[0146] B29 -N.epsilon.-Acetyl -human insulin.
[0147] B1-N.alpha.-Acetyl-human insulin.
[0148] A1-N.alpha.-Acetyl-human insulin.
[0149] B29-N.epsilon.-Acetyl-, B1-N.alpha.-acetyl-human
insulin.
[0150] B29-N.epsilon.-Acetyl-, A1-N.alpha.-acetyl-human
insulin.
[0151] A1-N.alpha.-Acetyl-, B1-N.alpha.-acetyl-human insulin.
[0152] B29-N.epsilon.-Acetyl-, A1-N.alpha.-acetyl-,
B1-N.alpha.-acetyl-human insulin.
[0153] B29-N.epsilon.-Propionyl-human insulin.
[0154] B1-N.alpha.-Propionyl-human insulin.
[0155] A1-N.alpha.-Propionyl-human insulin.
[0156] B29-N.epsilon.-Propionyl-, B1-N.alpha.-propionyl-human
insulin.
[0157] B29-N.epsilon.-Propionyl-, A1-N.alpha.-propionyl-human
insulin.
[0158] A1-N.alpha.-Propionyl-, B1-N.alpha.-propionyl-human
insulin.
[0159] B29-N.epsilon.-Propionyl-, A1-N.alpha.-propionyl-,
B1-N.alpha.-propionyl-human insulin.
[0160] B29-N.epsilon.-Butyryl-human insulin.
[0161] B1-N.alpha.-Butyryl-human insulin.
[0162] A1-N.alpha.-Butyryl-human insulin.
[0163] B29-N.epsilon.-Butyryl-, B1-N.alpha.-butyryl-human
insulin.
[0164] B29-N.epsilon.-Butyryl-, A1-N.alpha.-butyryl-human
insulin.
[0165] A1-N.alpha.-Butyryl-, B1-N.alpha.-butyryl-human insulin.
[0166] B29-N.epsilon.-Butyryl-, A1-N.alpha.-butyryl-,
B1-N.alpha.-butyryl-human insulin.
[0167] B29-N.epsilon.-Pentanoyl-human insulin.
[0168] B1-N.alpha.-Pentanoyl-human insulin.
[0169] A1-N.alpha.-Pentanoyl-human insulin.
[0170] B29-N.epsilon.-Pentanoyl-, B1-N.alpha.-pentanoyl-human
insulin.
[0171] B29-N.epsilon.-Pentanoyl-, A1-N.alpha.-pentanoyl-human
insulin.
[0172] A1-N.alpha.-Pentanoyl-, B1-N.alpha.-pentanoyl-human
insulin.
[0173] B29-N.epsilon.-Pentanoyl-, A1-N.alpha.-pentanoyl-,
B1-N.alpha.-pentanoyl-human insulin.
[0174] B29-N.epsilon.-Hexanoyl-human insulin.
[0175] B1-N.alpha.-Hexanoyl-human insulin.
[0176] A1-N.alpha.-Hexanoyl-human insulin.
[0177] B29-N.epsilon.-Hexanoyl-, B1-N.alpha.-hexanoyl-human
insulin.
[0178] B29-N.epsilon.-Hexanoyl-, A1-N.alpha.-hexanoyl-human
insulin.
[0179] A1-N.alpha.-Hexanoyl-, B1-N.alpha.-hexanoyl-human
insulin.
[0180] B29-N.epsilon.-Hexanoyl-, A1-N.alpha.-hexanoyl-,
B1-N.alpha.-hexanoyl-human insulin.
[0181] B29-N.epsilon.-Heptanoyl-human insulin.
[0182] B1-N.alpha.-Heptanoyl-human insulin.
[0183] A1-N.alpha.-Heptanoyl-human insulin.
[0184] B29-N.epsilon.-Heptanoyl-, B1-N.alpha.-heptanoyl-human
insulin.
[0185] B29-N.epsilon.-Heptanoyl-, A1-N.alpha.-heptanoyl-human
insulin.
[0186] A1-N.alpha.-Heptanoyl-, B1-N.alpha.-heptanoyl-human
insulin.
[0187] B29-N.epsilon.-Heptanoyl-, A1-N.alpha.-heptanoyl-,
B1-N.alpha.-heptanoyl-human insulin.
[0188] B29-N.epsilon.-Octanoyl-human insulin.
[0189] B1-N.alpha.-Octanoyl-human insulin.
[0190] A1-N.alpha.-Octanoyl-human insulin.
[0191] B29-N.epsilon.-Octanoyl-, B1-N.alpha.-octanoyl-human
insulin.
[0192] B29-N.epsilon.-Octanoyl-, A1-N.alpha.-octanoyl-human
insulin.
[0193] A1-N.alpha.-Octanoyl-, B1-N.alpha.-octanoyl-human
insulin.
[0194] B29-N.epsilon.-Octanoyl-, A1-N.alpha.-octanoyl-,
B1-N.alpha.-octanoyl-human insulin.
[0195] B29-N.epsilon.-Nonanoyl-human insulin.
[0196] B1-N.alpha.-Nonanoyl-human insulin.
[0197] A1-N.alpha.-Nonanoyl-human insulin.
[0198] B29-N.epsilon.-Nonanoyl-, B1-N.alpha.-nonanoyl-human
insulin.
[0199] B29-N.epsilon.-Nonanoyl-, A1-N.alpha.-nonanoyl-human
insulin.
[0200] A1-N.alpha.-Nonanoyl-, B1-N.alpha.-nonanoyl-human
insulin.
[0201] B29-N.epsilon.-Nonanoyl-, A1-N.alpha.-nonanoyl-,
B1-N.alpha.-nonanoyl-human insulin.
[0202] B29-N.epsilon.-Decanoyl-human insulin.
[0203] B1-N.alpha.-Decanoyl-human insulin.
[0204] A1-N.alpha.-Decanoyl-human insulin.
[0205] B29-N.epsilon.-Decanoyl-, B1-N.alpha.-decanoyl-human
insulin.
[0206] B29-N.epsilon.-Decanoyl-, A1-N.alpha.-decanoyl-human
insulin.
[0207] A1-N.alpha.-Decanoyl-, B1-N.alpha.-decanoyl-human
insulin.
[0208] B29-N.epsilon.-Decanoyl-, A1-N.alpha.-decanoyl-,
B1-N.alpha.-decanoyl-human insulin.
[0209] B29-N.alpha.-Undecanoyl-human insulin.
[0210] B1-N.alpha.-undecanoyl-human insulin.
[0211] A1-N.alpha.-Undecanoyl-human insulin.
[0212] B29-N.epsilon.-Dodecanoyl-human insulin.
[0213] B1-N.alpha.-Dodecanoyl-human insulin.
[0214] A1-N.alpha.-Dodecanoyl-human insulin.
[0215] B29-N.epsilon.-Tridecanoyl-human insulin.
[0216] B1-N.alpha.-Tridecanoyl-human insulin.
[0217] A1-N.alpha.-Tridecanoyl-human insulin.
[0218] B29-N.epsilon.-Tetradecanoyl-human insulin.
[0219] B1-N.alpha.-Tetradecanoyl-human insulin.
[0220] A1-N.alpha.-Tetradecanoyl-human insulin.
[0221] B29-N.epsilon.-Pentadecanoyl-human insulin.
[0222] B1-N.alpha.-Pentadecanoyl-human insulin.
[0223] A1-N.alpha.-Pentadecanoyl-human insulin.
[0224] B29-N.epsilon.-Hexadecanoyl-human insulin.
[0225] B1-N.alpha.-Hexadecanoyl-human insulin.
[0226] A1-N.alpha.-Hexadecanoyl-human insulin.
[0227] B29-N.epsilon.-Heptadecanoyl-human insulin.
[0228] B1-N.alpha.-Heptadecanoyl-human insulin.
[0229] A1-N.alpha.-Heptadecanoyl-human insulin.
[0230] B29-N.epsilon.-Octadecanoyl-human insulin.
[0231] B1-N.alpha.-Octadecanoyl-human insulin.
[0232] A1-N.alpha.-Octadecanoyl-human insulin.
[0233] B28-N.epsilon.-Formyl-LysB28, ProB29-human insulin
analog.
[0234] B1-N.alpha.-Formyl-LysB28, ProB29-human insulin analog.
[0235] A1-N.alpha.-Formyl-LysB28, ProB29-human insulin analog.
[0236] B28-N.epsilon.-Formyl-, B1-N.alpha.-formyl-LysB28,
ProB29-human insulin analog.
[0237] B28-N.epsilon.-Formyl-, A1-N.alpha.-formyl-LysB28,
ProB29-human insulin analog.
[0238] A1-N.alpha.-Formyl-, B1-N.alpha.-formyl-LysB28, ProB29-human
insulin analog.
[0239] B28-N.epsilon.-Formyl-, A1-N.alpha.-formyl-,
B1-N.alpha.-formyl-LysB28, ProB29-human insulin analog.
[0240] B28-N.epsilon.-Acetyl-LysB28, ProB29-human insulin
analog.
[0241] B1-N.alpha.-Acetyl-LysB28, ProB29-human insulin analog.
[0242] A1-N.alpha.-Acetyl-LysB28, ProB29-human insulin analog.
[0243] B28-N.epsilon.-Acetyl-, B1-N.alpha.-acetyl-LysB28,
ProB29-human insulin analog.
[0244] B28-N.epsilon.-Acetyl-, A1-N.alpha.-acetyl-LysB28,
ProB29-human insulin analog.
[0245] A1-N.alpha.-Acetyl-, B1-N.alpha.-acetyl-LysB28, ProB29-human
insulin analog.
[0246] B28-N.epsilon.-Acetyl-, A1-N.alpha.-acetyl-,
B1-N.alpha.-acetyl-LysB28, ProB29-human insulin analog.
[0247] B28-N.epsilon.-Propionyl-LysB28, ProB29-human insulin
analog.
[0248] B1-N.alpha.-Propionyl-LysB28, ProB29-human insulin
analog.
[0249] A1-N.alpha.-Propionyl-LysB28, ProB29-human insulin
analog.
[0250] B28-N.epsilon.-Propionyl-, B1-N.alpha.-propionyl-LysB28,
ProB29-human insulin analog.
[0251] B28-N.epsilon.-Propionyl-, A1-N.alpha.-propionyl-LysB28,
ProB29-human insulin analog.
[0252] A1-N.alpha.-Propionyl-, B1-N.alpha.-propionyl-LysB28,
ProB29-human insulin analog.
[0253] B28-N.epsilon.-Propionyl-, A1-N.alpha.-propionyl-,
B1-N.alpha.-propionyl-LysB28, ProB29-human insulin analog.
[0254] B28-N.epsilon.-Butyryl-LysB28, ProB29-human insulin
analog.
[0255] B1-N.alpha.-Butyryl-LysB28, ProB29-human insulin analog.
[0256] A1-N.alpha.-Butyryl-LysB28, ProB29-human insulin analog.
[0257] B28-N.epsilon.-Butyryl-, B1-N.alpha.-butyryl-LysB28,
ProB29-human insulin analog.
[0258] B28-N.epsilon.-Butyryl-, A1-N.alpha.-butyryl-LysB28,
ProB29-human insulin analog.
[0259] A1-N.alpha.-Butyryl-, B1-N.alpha.-butyryl-LysB28,
ProB29-human insulin analog.
[0260] B28-N.epsilon.-Butyryl-, A1-N.alpha.-butyryl-,
B1-N.alpha.-butyryl-LysB28, ProB29-human insulin analog.
[0261] B28-N.epsilon.-Pentanoyl-LysB28, ProB29-human insulin
analog.
[0262] B1-N.alpha.-Pentanoyl-LysB28, ProB29-human insulin
analog.
[0263] A1-N.alpha.-Pentanoyl-LysB28, ProB29-human insulin
analog.
[0264] B28-N.epsilon.-Pentanoyl-, B1-N.alpha.-pentanoyl-LysB28,
ProB29-human insulin analog.
[0265] B28-N.epsilon.-Pentanoyl-, A1-N.alpha.-pentanoyl-LysB28,
ProB29-human insulin analog.
[0266] A1-N.alpha.-Pentanoyl-, B1-N.alpha.-pentanoyl-LysB28,
ProB29-human insulin analog. B28-N.epsilon.-Pentanoyl-,
A1-N.alpha.-pentanoyl-, B1-N.alpha.-pentanoyl-LysB28, ProB29-human
insulin analog.
[0267] B28-N.epsilon.-Hexanoyl-LysB28, ProB29-human insulin
analog.
[0268] B1-N.alpha.-Hexanoyl-LysB28, ProB29-human insulin
analog.
[0269] A1-N.alpha.-Hexanoyl-LysB28, ProB29-human insulin
analog.
[0270] B28-N.epsilon.-Hexanoyl-, B1-N.alpha.-hexanoyl-LysB28,
ProB29-human insulin analog.
[0271] B28-N.epsilon.-Hexanoyl-, A1-N.alpha.-hexanoyl-LysB28,
ProB29-human insulin analog.
[0272] A1-N.alpha.-Hexanoyl-, B1-N.alpha.-hexanoyl-LysB28,
ProB29-human insulin analog.
[0273] B28-N.epsilon.-Hexanoyl-, A1-N.alpha.-hexanoyl-,
B1-N.alpha.-hexanoyl-LysB28, ProB29-human insulin analog.
[0274] B28-N.epsilon.-Heptanoyl-LysB28, ProB29-human insulin
analog.
[0275] B1-N.alpha.-Heptanoyl-LysB28, ProB29-human insulin
analog.
[0276] A1-N.alpha.-Heptanoyl-LysB28, ProB29-human insulin
analog.
[0277] B28-N.epsilon.-Heptanoyl-, B1-N.alpha.-heptanoyl-LysB28,
ProB29-human insulin analog.
[0278] B28-N.epsilon.-Heptanoyl-, A1-N.alpha.-heptanoyl-LysB28,
ProB29-human insulin analog.
[0279] A1-N.alpha.-Heptanoyl-, B1-N.alpha.-heptanoyl-LysB28,
ProB29-human insulin analog.
[0280] B28-N.epsilon.-Heptanoyl-, A1-N.alpha.-heptanoyl-,
B1-N.alpha.-heptanoyl-LysB28, ProB29-human insulin analog.
[0281] B28-N.epsilon.-octanoyl-LysB28, ProB29-human insulin
analog.
[0282] B1-N.alpha.-Octanoyl-LysB28, ProB29-human insulin
analog.
[0283] A1-N.alpha.-Octanoyl-LysB28, ProB29-human insulin
analog.
[0284] B28-N.epsilon.-Octanoyl-, B1-N.alpha.-octanoyl-LysB28,
ProB29-human insulin analog.
[0285] B28-N.epsilon.-Octanoyl-, A1-N.alpha.-octanoyl-LysB28,
ProB29-human insulin analog.
[0286] A1-N.alpha.-Octanoyl-, B1-N.alpha.-octanoyl-LysB28,
ProB29-human insulin analog.
[0287] B28-N.epsilon.-Octanoyl-, A1-N.alpha.-octanoyl-,
B1-N.alpha.-octanoyl-LysB28, ProB29-human insulin analog.
[0288] B28-N.epsilon.-Nonanoyl-LysB28, ProB29-human insulin
analog.
[0289] B1-N.alpha.-Nonanoyl-LysB28, proB29-human insulin
analog.
[0290] A1-N.alpha.-Nonanoyl-LysB28, ProB29-human insulin
analog.
[0291] B28-N.epsilon.-Nonanoyl-, B1-N.alpha.-nonanoyl-LysB28,
ProB29-human insulin analog.
[0292] B28-N.epsilon.-Nonanoyl-, A1-N.alpha.-nonanoyl-LysB28,
ProB29-human insulin analog.
[0293] A1-N.alpha.-Nonanoyl-, B1-N.alpha.-nonanoyl-LysB28,
ProB29-human insulin analog.
[0294] B28-N.epsilon.-Nonanoyl-, A1-N.alpha.-nonanoyl-,
B1-N.alpha.-nonanoyl-LysB28, ProB29-human insulin analog.
[0295] B28-N.epsilon.-Decanoyl-LysB28, ProB29-human insulin
analog.
[0296] B1-N.alpha.-Decanoyl-LysB28, ProB29-human insulin
analog.
[0297] A1-N.alpha.-Decanoyl-LysB28, ProB29-human insulin
analog.
[0298] B28-N.epsilon.-Decanoyl-, B1-N.alpha.-decanoyl-LysB28,
ProB29-human insulin analog.
[0299] B28-N.epsilon.-Decanoyl-, A1-N.alpha.-decanoyl-LysB28,
ProB29-human insulin analog.
[0300] A1-N.alpha.-Decanoyl-, B1-N.alpha.-decanoyl-LysB28,
ProB29-human insulin analog.
[0301] B28-N.epsilon.-Decanoyl-, A1-N.alpha.-decanoyl-,
B1-N.alpha.-decanoyl-LysB28, ProB29-human insulin analog.
[0302] B28-N.epsilon.-Undecanoyl-LysB28, ProB29-human insulin
analog.
[0303] B1-N.alpha.-Undecanoyl-LysB28, ProB29-human insulin
analog.
[0304] A1-N.alpha.-Undecanoyl-LysB28, ProB29-human insulin
analog.
[0305] B28-N.epsilon.-Dodecanoyl-LysB28, ProB29-human insulin
analog.
[0306] B1-N.alpha.-Dodecanoyl-LysB28, ProB29-human insulin
analog.
[0307] A1-N.alpha.-Dodecanoyl-LysB28, ProB29-human insulin
analog.
[0308] B28-N.epsilon.-Tridecanoyl-LysB28, ProB29-human insulin
analog.
[0309] B1-N.alpha.-Tridecanoyl-LysB28, ProB29-human insulin
analog.
[0310] A1-N.alpha.-Tridecanoyl-LysB28, ProB29-human insulin
analog.
[0311] B28-N.epsilon.-Tetradecanoyl-LysB28, ProB29-human insulin
analog.
[0312] B1-N.alpha.-Tetradecanoyl-LysB28, ProB29-human insulin
analog.
[0313] A1-N.alpha.-Tetradecanoyl-LysB28, ProB29-human insulin
analog.
[0314] B28-N.epsilon.-Pentadecanoyl-LysB28, ProB29-human insulin
analog.
[0315] B1-N.alpha.-Pentadecanoyl-LysB28, ProB29-human insulin
analog.
[0316] A1-N.alpha.-Pentadecanoyl-LysB28, ProB29-human insulin
analog.
[0317] B28-N.epsilon.-Hexadecanoyl-LysB28, ProB29-human insulin
analog.
[0318] B1-N.alpha.-Hexadecanoyl-LysB28, ProB29-human insulin
analog.
[0319] A1-N.alpha.-Hexadecanoyl-LysB28, ProB29-human insulin
analog.
[0320] B28-N.epsilon.-Heptadecanoyl-LysB28, ProB29-human insulin
analog.
[0321] B1-N.alpha.-Heptadecanoyl-LysB28, ProB29-human insulin
analog.
[0322] A1-N.alpha.-Heptadecanoyl-LysB28, ProB29-human insulin
analog.
[0323] B28-N.epsilon.-Octadecanoyl-LysB28, ProB29-human insulin
analog.
[0324] B1-N.alpha.-octadecanoyl-LysB28, ProB29-human insulin
analog.
[0325] A1-N.alpha.-Octadecanoyl-LysB28, ProB29-human insulin
analog.
[0326] B29-N.epsilon.-Pentanoyl-GlyA21, ArgB31, ArgB32-human
insulin.
[0327] B1-N.alpha.-Hexanoyl-GlyA21, ArgB31, ArgB32-human
insulin.
[0328] A1-N.alpha.-Heptanoyl-GlyA21, ArgB31, ArgB32-human
insulin.
[0329] B29-N.epsilon.-Octanoyl-, B1-N.alpha.-octanoyl-GlyA21,
ArgB31, ArgB32-human insulin.
[0330] B29-N.epsilon.-Propionyl-A1-N.alpha.-propionyl-GlyA21,
ArgB31, ArgB32-human insulin.
[0331] A1-N.alpha.-Acetyl, B1-N.alpha.-acetyl-GlyA21, ArgB31,
ArgB32-human insulin.
[0332] B29-N.epsilon.-Formyl-, A1-N.alpha.-formyl-,
B1-N.alpha.-formyl-GlyA21, ArgB31, ArgB32-human insulin.
[0333] B29-N.epsilon.-Formyl-des(TyrB26)-human insulin.
[0334] B1-N.alpha.-Acetyl-AspB28 -human insulin.
[0335] B29-N.epsilon.-Propionyl-, A1-N.alpha.-propionyl-,
B1-N.alpha.-propionyl-AspB1, AspB3, AspB21-human insulin.
[0336] A1-N.alpha.-Butyryl-AspB10-human insulin.
[0337] B29-N.epsilon.-Pentanoyl-GlyA21-human insulin.
[0338] B1-N.alpha.-Hexanoyl-GlyA21-human insulin.
[0339] A1-N.alpha.-Heptanoyl-GlyA21-human insulin.
[0340] B29-N.epsilon.-Octanoyl-, B1-N.alpha.-octanoyl-GlyA21-human
insulin.
[0341] B29-N.epsilon.-Propionyl-,
A1-N.alpha.-propionyl-GlyA21-human insulin.
[0342] A1-N.alpha.-Acetyl, B1-N.alpha.-acetyl-GlyA21-human
insulin.
[0343] B29-N.epsilon.-Formyl-, A1-N.alpha.-formyl-,
B1-N.alpha.-formyl-GlyA21-human insulin.
[0344] B29-N.epsilon.-Butyryl-des(ThrB30)-human insulin.
[0345] B1-N.alpha.-Butyryl-des(ThrB30)-human insulin.
[0346] A1-N.alpha.-Butyryl-des(ThrB30)-human insulin.
[0347] B29-N.epsilon.-Butyryl-,
B1-N.alpha.-butyryl-des(ThrB30)-human insulin.
[0348] B29-N.epsilon.-Butyryl-,
A1-N.alpha.-butyryl-des(ThrB30)-human insulin.
[0349] A1-N.alpha.-Butyryl-, B1-N.alpha.-butyryl-des(ThrB30)-human
insulin.
[0350] B29-N.epsilon.-Butyryl-, A1-N.alpha.-butyryl-,
B1-N.alpha.-butyryl-des (ThrB30)-human insulin.
[0351] Aqueous compositions containing water as the major solvent
are preferred. Aqueous suspensions wherein water is the solvent are
highly preferred.
[0352] The compositions of the present invention are used to treat
patients who have diabetes or hyperglycemia. The formulations of
the present invention will typically provide derivatized protein at
concentrations of from about 1 mg/mL to about 10 mg/mL. Present
formulations of insulin products are typically characterized in
terms of the concentration of units of insulin activity (units/mL),
such as U40, U50, U100, and so on, which correspond roughly to
about 1.4, 1.75, and 3.5 mg/mL preparations, respectively. The
dose, route of administration, and the number of administrations
per day will be determined by a physician considering such factors
as the therapeutic objectives, the nature and cause of the
patient's disease, the patient's gender and weight, level of
exercise, eating habits, the method of administration, and other
factors known to the skilled physician. In broad range, a daily
dose would be in the range of from about 1 nmol/kg body weight to
about 6 nmol/kg body weight (6 nmol is considered equivalent to
about 1 unit of insulin activity). A dose of between about 2 and
about 3 nmol/kg is typical of present insulin therapy.
[0353] The physician of ordinary skill in treating diabetes will be
able to select the therapeutically most advantageous means to
administer the formulations of the present invention. Parenteral
routes of administration are preferred. Typical routes of
parenteral administration of suspension formulations of insulin are
the subcutaneous and intramuscular routes. The compositions and
formulations of the present invention may also be administered by
nasal, buccal, pulmonary, or occular routes.
[0354] Glycerol at a concentration of 12 mg/mL to 25 mg/mL is
preferred as an isotonicity agent. Yet more highly preferred for
isotonicity is to use glycerol at a concentration of from about 15
mg/mL to about 17 mg/mL.
[0355] M-cresol and phenol, or mixtures thereof, are preferred
preservatives in formulations of the present invention.
[0356] Insulin, insulin analogs, or proinsulins used to prepare
derivatized proteins can be prepared by any of a variety of
recognized peptide synthesis techniques including classical
(solution) methods, solid phase methods, semi-synthetic methods,
and more recent recombinant DNA methods. For example, see Chance,
R. E., et al., U.S. Pat. No. 5,514,646, May 7, 1996; EPO
publication number 383,472, Feb. 7, 1996; Brange, J. J. V., et al.
EPO publication number 214,826, Mar. 18, 1987; and Belagaje, R. M.,
et al., U.S. Pat. No. 5,304,473, Apr. 19, 1994, which disclose the
preparation of various proinsulin and insulin analogs. These
references are expressly incorporated herein by reference.
[0357] Generally, derivatized proteins are prepared using methods
known in the art. The publications listed above to describe
derivatized proteins contain suitable methods to prepare
derivatized proteins. Those publications are expressly incorporated
by reference for methods of preparing derivatized proteins. To
prepare acylated proteins, the protein is reacted with an activated
organic acid, such as an activated fatty acid. Activated fatty
acids are derivatives of commonly employed acylating agents, and
include activated esters of fatty acids, fatty acid halides,
activated amides of fatty acids, such as, activated azolide
derivatives [Hansen, L. B., WIPO Publication No. 98/02460, Jan. 22,
1998], and fatty acid anhydrides. The use of activated esters,
especially N-hydroxysuccinimide esters of fatty acids, is a
particularly advantageous means of acylating a free amino acid with
a fatty acid. Lapidot, et al. describe the preparation of
N-hydroxysuccinimide esters and their use in the preparation of
N-lauroyl-glycine, N-lauroyl-L-serine, and N-lauroyl-L-glutamic
acid. The term "activated fatty acid ester" means a fatty acid
which has been activated using general techniques known in the art
[Riordan, J. F. and Vallee, B. L., Methods in Enzymology,
XXV:494-499 (1972); Lapidot, Y., et al., J. Lipid Res. 8:142-145
(1967)]. Hydroxybenzotriazide (HOBT), N-hydroxysuccinimide and
derivatives thereof are particularly well known for forming
activated acids for peptide synthesis.
[0358] To selectively acylate the .epsilon.-amino group, various
protecting groups may be used to block the .alpha.-amino groups
during the coupling. The selection of a suitable protecting group
is known to one skilled in the art and includes
p-methoxybenzoxycarbonyl (pmZ). Preferably, the .epsilon.-amino
group is acylated in a one-step synthesis without the use of
amino-protecting groups. A process for selective acylation at the
N.epsilon.-amino group of Lys is disclosed and claimed by Baker, J.
C., et al., U.S. Pat. No. 5,646,242, Jul. 8, 1997, the entire
disclosure of which is incorporated expressly by reference. A
process for preparing a dry powder of an acylated protein is
disclosed and claimed by Baker, J. C., et al., U.S. Pat. No.
5,700,904, Dec. 23, 1997, the entire disclosure of which is
incorporated herein expressly by reference.
[0359] The primary role of zinc in the present invention is to
facilitate formation of Zn(II) hexamers of the protein and
derivatized protein, either separately as mixed hexamers, or
together as hybrid hexamers. Zinc facilitates the formation of
hexamers of insulin, and of insulin analogs. Zinc likewise promotes
the formation of hexamers of derivatized insulin and insulin
analogs. Hexamer formation is conveniently achieved by bringing the
pH of a solution comprising protein, or derivatized protein, or
both into the neutral region in the presence of Zn(II) ions, or by
adding Zn(II) after the pH has been adjusted to the neutral
region.
[0360] For efficient yield of microcrystals or amorphous
precipitate, the molar ratio of zinc to total protein in the
microcrystal and amorphous precipitate of the present invention is
bounded at the lower limit by about 0.33, that is, the
approximately two zinc atoms per hexamer which are needed for
efficient hexamerization. The microcrystal and amorphous
precipitate compositions will form suitably with about 2 to about
4-6 zinc atoms present when no compound that competes with insulin
for zinc binding is present. Even more zinc may be used during the
process if a compound that competes with the protein for zinc
binding, such as one containing citrate or phosphate, is present.
Excess zinc above the minimum amount needed for efficient
hexamerization may be desirable to more strongly drive
hexamerization. Also, excess zinc above the minimum amount can be
present in a formulation of the present invention, and may be
desirable to improve chemical and physical stability, to improve
suspendability, and possibly to further extend time-action.
Consequently, there is a fairly wide range of zinc:protein ratios
allowable in the insoluble compositions, processes, and
formulations of the present invention.
[0361] In accordance with the present invention, zinc is present in
the formulation in an amount of from about 0.3 mole to about 7
moles per mole of total protein and more preferably about from 0.3
mole to about 1.0 mole of total protein. Yet more highly preferred
is a ratio of zinc to derivatized protein from about 0.3 to about
0.7 mole of zinc atoms per mole of total protein. Most highly
preferred is a ratio of zinc to total protein from about 0.30 to
about 0.55 mole of zinc atoms per mole of total protein. For higher
zinc formulations that are similar to PZI preparations, the zinc
ratio is from about 5 to about 7 moles of zinc per mole of total
protein.
[0362] The zinc compound that provides zinc for the present
invention may be any pharmaceutically acceptable zinc compound. The
addition of zinc to insulin preparations is known in the art, as
are pharmaceutically acceptable sources of zinc. Preferred zinc
compounds to supply zinc for the present invention include zinc
chloride, zinc acetate, zinc citrate, zinc oxide, and zinc
nitrate.
[0363] A complexing compound is required for the microcrystals and
precipitates of the present invention. The complexing compound must
be present in sufficient quantities to cause substantial
precipitation and crystallization of the hexamers. Such quantities
can be, readily determined for a particular preparation of a
particular complexing compound by simple titration experiments.
Ideally, the complexing compound concentration is adjusted so that
there is negligible complexing compound remaining in the soluble
phase after completion of precipitation and crystallization. This
requires combining the complexing compound based on an
experimentally determined "isophane" ratio. This ratio is expected
to be very similar to that of NPH and NPL. However, it may be
slightly different because derivatization may affect the nature of
the protein-protamine interaction.
[0364] When protamine is the complexing compound, it is present in
the microcrystal in an amount of from about 0.15 mg to about 0.5 mg
per 3.5 mg of the total protein. The ratio of protamine to total
protein is preferably from about 0.25 to about 0.40 (mg/mg). More
preferably the ratio is from about 0.25 to about 0.38 (mg/mg).
Preferably, protamine is in an amount of 0.05 mg to about 0.2 mg
per mg of the total protein, and more preferably, from about 0.05
to about 0.15 milligram of protamine per milligram of total
protein. Protamine sulfate is the preferred salt form of protamine
for use in the present invention. When protamine sulfate, or other
salt form of protamine is used, the mass of it to be used would
have to be adjusted with respect to the mass of protamine free base
that would be used for the same application by a factor equal to
the ratio of the molecular weights of the salt form and
protamine.
[0365] To further extend the time action of the compositions of the
present invention or to improve their suspendability, additional
protamine and zinc may be added after crystallization. Thus, also
within the present invention are formulations having protamine, at
higher than isophane ratios. For these formulations, the protamine
ratio is from 0.25 mg to about 0.5 mg of protamine per mg of total
protein.
[0366] A required component of the microcrystals and precipitates
of the present invention is a hexamer stabilizing compound. The
structures of three hexameric conformations have been characterized
in the literature, and are designated T6, T3R3, and R6. In the
presence of hexamer stabilizing compound, such as various phenolic
compounds, the R6 conformation is stabilized. Therefore, it is
highly likely that hexamers are in the R6 conformation, or the T3R3
conformation in the crystals and precipitates produced in the
presence of a hexamer stabilizing compound, such as phenol or
m-cresol, among others. A wide range of hexamer stabilizing
compounds are suitable. They must be present in sufficient
proportions with respect to total protein to stabilize the R6
hexamer conformation. To accomplish this, at least 2 or at least 3
moles of hexamer stabilizing compound per hexamer are required for
effective hexamer stabilization. It is preferred that at least 3
moles of hexamer stabilizing compound per hexamer be present in the
microcrystals and precipitates of the present invention. The
presence of higher ratios of hexamer stabilizing compound, at least
up to 25 to 50-fold higher, in the solution from which the
microcrystals and precipitates are prepared will not adversely
affect hexamer stabilization.
[0367] In formulations of the present invention, a preservative may
be present, especially if the formulation is intended to be sampled
multiple times. As mentioned above, a wide range of suitable
preservatives are known. Preferably, the preservative is present in
the solution in an amount suitable to provide an antimicrobial
effect sufficient to meet pharmacopoeial requirements.
[0368] Preferred preservatives are the phenolic preservatives,
which are enumerated above. Preferred concentrations for the
phenolic preservative are from about 2 mg to about 5 mg per
milliliter of the aqueous suspension formulation. These
concentrations refer to the total mass of phenolic preservatives
because mixtures of individual phenolic preservatives are
contemplated. Suitable phenolic preservatives include, for example,
phenol, m-cresol, and methylparaben. Preferred phenolic compounds
are phenol and m-cresol. Mixtures of phenolic compounds, such as
phenol and m-cresol, are also contemplated and highly preferred.
Examples of mixtures of phenolic compounds are 0.6 mg/mL phenol and
1.6 mg/mL m-cresol, and 0.7 mg/mL phenol and 1.8 mg/mL
m-cresol.
[0369] The microcrystals of the present invention are preferably
oblong-shaped, also known as "rod-like", single crystals that are
comprised of a protein, a derivatized protein, a divalent cation,
and including a complexing compound and a hexamer-stabilizing
compound. The mean length of the microcrystals of the present
invention preferably is within the range of 1 micron to 40 microns,
and more preferably is within the size range of 3 microns to 15
microns.
[0370] A preferred composition comprises from about 3 mg to about 6
mg of protamine sulfate per 35 mg of total protein, and from about
0.1 to about 0.4 mg zinc per 35 mg of total protein. Another
preferred composition comprises from about 10 mg to about 17 mg of
protamine sulfate per 35 mg of total protein, and from about 2.0 to
about 2.5 mg zinc per 35 mg of total protein. Another preferred
composition comprises, per mL, protamine sulfate, 0.34-0.38 mg;
zinc, 0.01-0.04 mg; and total protein, 3.2-3.8 mg.
[0371] Both an un-derivatized protein and a derivatized protein are
required for the present co-crystals and amorphous precipitates.
The ratio between the masses of these proteins determines the
degree of time extension of the preparations. A preferred ratio of
the number of moles of the protein to the number of moles of the
derivatized protein is between about 1:100 and about 100:1. A
further preferred ratio of the number of moles of the protein to
the number of moles of the derivatized protein is between about 1:1
and about 100:1. Another preferred ratio of the number of moles-of
the protein to the number of moles of the derivatized protein is
between about 1:1 and about 20:1. Yet other preferred ratios of the
number of moles of the protein to the number of moles of the
derivatized protein are: between about 2:1 and about 20:1; between
about 2:1 and 10:1; between about 2:1 and 5:1; between about 3:1
and 5:1; between 1:1 and 1:20; between 1:1 and 1:10; between about
1:2 and about 1:20; between about 1:2 and 1:10; between about 1:2
and 1:5; between about 1:3 and 1:5; between about 10:1 and about
1:10; between about 9:1 and about 1:9; between about 5:1 and about
1:5; and between about 3:1 and about 1:3.
[0372] The present invention provides processes for preparing the
compositions. Also, the use of the present insoluble compositions
to prepare medicaments for controlling blood glucose, and for
treating diabetes or hyperglycemia is contemplated. The amorphous
precipitates and microcrystals of the present invention can be
prepared for use in medicaments, or other uses, by many different
processes.
[0373] In summary, suitable processes are comprised generally of
the steps in one of the following sequences:
[0374] solubilization (if starting with dry material),
[0375] hexamerization, homogenization, complexation,
precipitation,
[0376] crystallization, and optionally formulation; or
[0377] solubilization (if starting with dry material),
[0378] homogenization, hexamerization, complexation,
precipitation,
[0379] crystallization, and optionally formulation.
[0380] Solubilization means the dissolution of derivatized protein
and protein sufficiently to allow them to form hexamers.
Hexamerization refers to the process wherein molecules of protein
and derivatized protein bind with zinc(II) atoms to form hexamers.
Complexation denotes the formation of insoluble complexes between
the hexamers and protamine. Precipitation results typically from
the formation of insoluble complexes. Crystallization involves the
conversion of precipitated hexamer/protamine complexes into
crystals, typically, rod-like crystals.
[0381] Solubilization is carried out by dissolving the derivatized
protein and protein in an aqueous solvent. The aqueous solvent may
be, for example, an acidic solution, a neutral solution, or a basic
solution. The aqueous solvent may be comprised partially of a
miscible organic solvent, such as ethanol, acetonitrile,
dimethylsulfoxide, and the like. Acidic solutions may be, for
example, solutions of HCl, advantageously from about 0.01 N HCl to
about 1.0 N HCl. Other acids that are pharmaceutically acceptable
may be employed as well. Basic solutions may be, for example,
solutions of NaOH, advantageously from about 0.01 N NaOH to about
1.0 N NaOH, or higher. Other bases that are pharmaceutically
acceptable may be employed as well. For the sake of protein
stability, the concentration of acid or base is preferably as low
as possible while still being effective to adequately dissolve the
protein and derivatized protein.
[0382] Most proteins (insulin, insulin analogs, and proinsulins)
and many derivatized proteins may be dissolved to suitable
concentrations at neutral pH. Solutions to dissolve derivatized
proteins at neutral pH may contain a buffer and optionally, one or
more additional solutes such as salts, phenolic compounds, zinc,
and isotonicity agents.
[0383] When hexamerization occurs before homogenization, two
populations of homogenous hexamers are formed first, and then the
populations are mixed, thereby forming mixed hexamers. When
homogenization occurs first, hexamerization yields hybrid hexamers.
As mentioned above, to prepare insoluble compositions comprised of
hybrid hexamers, protein and derivatized protein are homogenized
under conditions favoring dissociation to monomer or dimer
aggregation states prior to hexamerization with a divalent metal
cation. To achieve the necessary dissociation, the protein and
derivatized protein may be mixed under strongly acidic or strongly
basic conditions. The degree of dissociation, and therefore,
homogenization is influenced by the solution conditions chosen for
this step. Insulin and related proteins readily self-associate in a
series of reactions producing dimers, hexamers, and other
associated forms. The distribution of these association forms at
equilibrium is dependent on many parameters, including pH. These
association reactions are commonly thought to involve primarily
monomer-dimer-hexamer assembly. Consequently, depending on the
solution conditions chosen, homogenization should accomplish the
mixing of monomers, dimers, or a mixture thereof. Homogenization in
1 N HCl, for example, could involve a higher fraction of monomer
mixing than in 0.1 N HCl, which would probably involve more dimer
mixing. For the preparation of compositions comprised of hybrid
hexamers, the homogenization process will be effective provided
that only a very small or negligible fraction of homogeneous
hexamers of the protein or derivatized protein exist under the
homogenization conditions employed.
[0384] Compositions comprised of mixed hexamers incorporate
predominantly two types of hexamers, namely hexamers of the
protein, and hexamers of the derivatized protein. In this case, the
homogenization step occurs after the hexamerization step, and
achieves the homogenization of the hexamers prior to complexation
with the complexing compound. Consequently, the homogenization step
is performed under solution conditions that stabilize the
Zn(II)-insulin hexamer. Solution conditions that stabilize insulin
hexamers are well known in the literature.
[0385] The solution conditions required for hexamerization are
those that allow the formation of the hybrid hexamers or mixed
hexamers in solution. These conditions will be identical or very
similar to the conditions under which insulin or insulin analogs
are made to hexamerize. Typically, hexamerization requires zinc and
a neutral to slightly basic pH, which is taken to be from about pH
6.8 to about pH 8.4. The presence of a hexamer-stabilizing compound
advantageously influences hexamerization by promoting the R6 or the
T3R3 conformations of the derivatized protein, and in certain
instances, of the protein also. For certain monomeric insulin
analogs, a hexamer-stabilizing compound is required to form
hexamers.
[0386] For compositions comprised of hybrid hexamers, seven
hexameric species are expected: P.sub.6, P.sub.5D.sub.1,
P.sub.4D.sub.2, P.sub.3D.sub.3, P.sub.2D.sub.4, P.sub.1D.sub.5, and
D.sub.6, where P represents the protein monomer, and D represents
the derivatized protein monomer. The statistical distribution of
hexamers is expected to conform to a Poisson distribution, and will
be influenced by the relative proportion of protein and derivatized
protein, and by the degree of dissociation prior to hexamerization.
For example, from a homogenized solution constituted predominantly
of dimers, four major hybrid hexamer species are expected: P.sub.6,
P.sub.4D.sub.2, P.sub.2D.sub.4, and D.sub.6. For compositions
comprised of mixed hexamers, only two hexameric species are
expected to predominate: P6 and D.sub.6.
[0387] The complexation step must involve the combination a
complexing compound with hexamer under solution conditions where
each is initially soluble. This could be accomplished by combining
separate solutions of hexamers and of protamine, or by first
forming a solution of protein, derivatized protein, and protamine
at acidic or basic pH, and then shifting the pH to the neutral
range.
[0388] During crystallization, the solution conditions must
stabilize the crystallizing species, and promote the conversion of
precipitate to solute to crystal. Thus, the solution conditions
will determine the rate and outcome of crystallization.
Crystallization likely involves a complex equilibrium involving
non-crystalline precipitate, dissolved hexamer-protamine complexes,
and crystal. To obtain microcrystals, the conditions chosen for
crystallization must drive the equilibrium toward crystal
formation. Also, in light of the hypothesized equilibrium, the
solubility of the derivatized protein is expected to profoundly
affect crystallization rate and size because lower solubility will
likely slow the net conversion from precipitate to solution to
crystal. Furthermore, it is well-recognized that slowing the rate
of crystallization often results in larger crystals. Thus, the
crystallization rate and crystal size are thought to depend on the
size and nature of the derivatizing moiety on the derivatized
protein.
[0389] Crystallization parameters that influence the
crystallization rate and the size of crystals of the present
invention are: acyl group size and nature; temperature; the
presence and concentration of compounds that compete with the
protein and derivatized protein for zinc, such as citrate,
phosphate, and the like; the nature and concentration of phenolic
compound(s); zinc concentration; the presence and concentration of
a miscible organic solvent; the time permitted for crystallization;
the pH and ionic strength; buffer identity and concentration; the
concentration of precipitants; the presence of seeding materials;
the shape and material of the container; the stirring rate; and the
total protein concentration. Temperature and the concentration of
competing compounds are thought to be of particular importance.
[0390] Competing compounds, such as citrate, may affect the rate at
which crystals form, and indirectly, crystal size and quality.
These compounds may exert their effect by forming coordination
complexes with zinc in solution, thus competing with the relatively
weak zinc binding sites on the surface of the hexamer for zinc.
Occupation of these weak surface binding sites probably impedes
crystallization. Additionally, many derivatized proteins are
partially insoluble in the presence of little more than 0.333 zinc
per mole of derivatized protein, and the presence of competing
compounds restores solubility, and permits crystallization. The
optimum concentration of competing compound can be determined using
routine techniques for any combination of protein and derivatized
protein. As an upper limit, of course, is the concentration at
which zinc is precipitated by the competing compound, or the
concentration at which residual competing compound would be
pharmaceutically unacceptable, such as, when it would cause pain or
irritation at the site of administration.
[0391] An example of a process for preparing the precipitates and
crystals of the present invention follows. A measured amount of the
derivatized protein and a measured amount of the protein are
dissolved in, or are combined to form a solution in an aqueous
solvent containing a hexamer-stabilizing compound, such as a
phenolic compound. To this solution is added a solution of zinc as
one of its soluble salts, for example Zn(II)Cl.sub.2, to provide
from about 0.3 moles of zinc per mole of derivatized insulin to
about 0.7 moles, or to as much as 1.0 moles, of zinc per mole of
total protein (protein+derivatized protein). Absolute ethanol, or
another miscible organic solvent, may optionally be added to this
solution in an amount to make the solution from about 5% to about
10% by volume organic solvent. This solution may then be filtered
through a 0.22 micron, low-protein binding filter. A protamine
solution is prepared by dissolving a measured amount of protamine
in an aqueous solvent. This solution may be filtered through a 0.22
micron, low-protein binding filter. The solution of protein and
derivatized protein and the protamine solution are combined,
whereupon a precipitate forms initially. The resulting suspension
is stirred slowly at room temperature (typically about
20-25.degree. C.), whereupon microcrystals are formed within a
period from about 4 hours to about 10 days.
[0392] The microcrystals may then be separated from the mother
liquor and introduced into a different solvent, for storage and
administration to a patient. Examples of appropriate aqueous
solvents are as follows: water for injection containing 25 mM TRIS,
5 mg/mL phenol and 16 mg/mL glycerol; water for injection
containing 2 mg/mL sodium phosphate dibasic, 1.6 mg/mL m-cresol,
0.65 mg/mL phenol, and 16 mg/mL glycerol; and water for injection
containing 25 mM TRIS, 5 mg/mL phenol, 0.1 M trisodium citrate, and
16 mg/mL glycerol.
[0393] In another process for preparing the insoluble compositions
of the present invention, for example, a measured mass of dry
derivatized protein and a measured mass of dry protein are
dissolved together in an acidic aqueous solvent, such as 0.1 N -1.0
N HCl. This solution is stirred to insure thorough mixing of
derivatized protein and protein. The ratio of derivatized protein
powder to protein powder in this mixture is predefined to achieve a
similar ratio of derivatized protein to protein in the insoluble
composition to be produced. A separately prepared aqueous solution
comprised of a phenolic preservative and, optionally, a
pharmaceutically acceptable buffer, is combined with the acidic
solution of the proteins. The pH of the resulting solution is then
adjusted to about 6.8 to about 8.4, preferably from about 6.8 to
about 8.0, or preferably to a pH of from about 7.2 to about 7.8,
and most preferably from about 7.4 to about 7.8. To this solution
is added a solution of zinc as one of its soluble salts, for
example Zn(II)Cl.sub.2, to provide from about 0.3 moles of zinc per
mole of total insulin to about 4 moles of zinc per mole of total
insulin. This solution is adjusted to a pH as given above, and
preferably to about 7.4-7.6, and may then be filtered through a
0.22 micron, low-protein binding filter. A solution of protamine is
prepared by dissolving a measured mass of protamine in an aqueous
solvent. The protamine solution may be filtered through a 0.22
micron, low-protein binding filter. The solution of protein and
derivatized protein and the protamine solution are combined,
whereupon a precipitate forms initially. The resulting suspension
is stirred slowly at room temperature (typically about
20-25.degree. C.), whereupon microcrystals are formed within a
period from about 4 hours to about 10 days.
[0394] In another process for preparing the insoluble compositions
of the present invention, a measured amount of a derivatized
protein is first dissolved in an aqueous solvent containing a
phenolic preservative. To this solution is added a solution of zinc
as one of its soluble salts, for example Zn(II)Cl.sub.2, to provide
from about 0.3 moles of zinc per mole of derivatized protein to
about 4 moles of zinc per mole of derivatized protein. The pH of
the resulting solution is then adjusted to about 6.8 to about 8.4,
preferably from about 6.8 to about 8.0, or preferably to a pH of
from about 7.2 to about 7.8, and most preferably from about 7.4 to
about 7.8. A second solution is prepared separately wherein a
measured amount of a protein selected from the group consisting of
insulin, insulin analogs, and proinsulin is dissolved in an aqueous
solvent containing a phenolic preservative. To this solution is
added a solution of zinc as one of its soluble salts, for example
Zn(II)Cl.sub.2, to provide from about 0.3 moles of zinc per mole of
protein to about 4 moles of zinc per mole of protein. The pH of the
resulting solution is then adjusted to about 6.8 to about 8.4,
preferably from about 6.8 to about 8.0, or preferably to a pH of
from about 7.2 to about 7.8, and most preferably from about 7.4 to
about 7.8, or 7.4-7.6. Portions of the derivatized protein solution
and the protein solution are combined in a ratio that is predefined
in order to achieve a similar ratio of derivatized protein to
protein in the insoluble composition. This solution is stirred to
insure thorough mixing of derivatized protein and protein. This
solution is then adjusted to a pH of about 7.6, and may then be
filtered through a 0.22 micron, low-protein binding filter. A
protamine solution is prepared separately by dissolving a measured
amount of protamine in an aqueous solvent. This protamine solution
may be filtered through a 0.22 micron, low-protein binding filter.
The solution of protein and derivatized protein and the protamine
solution are combined, whereupon a precipitate forms initially. The
resulting suspension is stirred slowly at room temperature
(typically about 20-25.degree. C.), whereupon microcrystals are
formed within a period from about 4 hours to about 10 days.
[0395] While not describing all of the very many types of processes
that will produce the insoluble compositions of the present
invention in any way, the following are yet further processes of
the present invention:
[0396] dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation in an
aqueous solvent having a pH that will permit the formation of
hexamers, and adding a complexing compound;
[0397] dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation in an
aqueous solvent having a pH that will not permit the formation of
hexamers, adjusting the pH to between about 6.8 and about 7.8, and
adding a complexing compound;
[0398] dissolving a protein, a hexamer-stabilizing compound, and a
divalent metal cation in an aqueous solvent having a pH that will
permit the formation of hexamers, separately, dissolving a
derivatized protein, a hexamer-stabilizing compound, and a divalent
metal cation in an aqueous solvent having a pH that will permit the
formation of hexamers, thoroughly mixing together these two
solution, and then adding a complexing compound;
[0399] dissolving a protein, a hexamer-stabilizing compound, a
divalent metal cation, and a complexing compound in an aqueous
solvent, wherein the resulting solution has a pH at which
precipitation does not occur, separately, dissolving a derivatized
protein, a hexamer-stabilizing compound, a divalent metal cation,
and a complexing compound in an aqueous solvent, wherein the
resulting solution has a pH at which precipitation does not occur,
thoroughly mixing together these two solutions, and adjusting the
pH of the solution of to a value at which precipitation occurs;
[0400] dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, a divalent metal cation, and a
complexing compound in an aqueous solvent, wherein the resulting
solution has a pH at which precipitation does not occur and
adjusting the pH of the solution to a value at which precipitation
occurs;
[0401] dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation, in an
aqueous solvent, wherein the resulting solution has a pH at which
precipitation will not occur when a complexing agent is added,
adding a complexing compound, and adjusting the pH of the solution
of step b) to a value at which precipitation occurs;
[0402] dissolving a protein, a hexamer-stabilizing compound, and a
divalent metal cation in an aqueous solvent, wherein the resulting
solution has a pH at which precipitation will not occur when a
completing compound is added, separately, dissolving a derivatized
protein, a hexamer-stabilizing compound, and a divalent metal
cation in an aqueous solvent, wherein the resulting solution has a
pH at which precipitation will not occur when a complexing compound
is added, thoroughly mixing together these two solutions, adding
complexing compound to the solution, and adjusting the pH to a
value at which precipitation occurs;
[0403] dissolving a protein, a protein derivative, a
hexamer-stabilizing compound, and a divalent metal cation in an
aqueous solvent, wherein the resulting solution has a pH at which
precipitation will not occur when a complexing compound is added,
adjusting the pH of the solution to a value at which precipitation
will occur when a complexing compound is added, and adding a
complexing compound to the solution;
[0404] dissolving a protein, a hexamer-stabilizing compound, and a
divalent metal cation in an aqueous solvent, wherein the resulting
solution has a pH at which precipitation will not occur when a
complexing compound is added, separately, dissolving a derivatized
protein, a hexamer-stabilizing compound, and a divalent metal
cation in an aqueous solvent, wherein the resulting solution has a
pH at which precipitation will not occur when a complexing compound
is added; thoroughly mixing together these two solutions, adjusting
the pH of the solution of step c) to a value at which precipitation
will occur when a complexing compound is added, and adding a
complexing compound to the solution;
[0405] In a preferred embodiment, the microcrystals are prepared in
a manner that obviates the need to separate the microcrystals from
the mother liquor. Thus, it is preferred that the mother liquor
itself be suitable for administration to the patient, or that the
mother liquor can be made suitable for administration by dilution
with a suitable diluent. The term diluent will be understood to
mean a solution comprised of an aqueous solvent in which is
dissolved various pharmaceutically acceptable excipients, including
without limitation, a buffer, an isotonicity agent, zinc, a
preservative, protamine, and the like.
[0406] In addition to the protein, derivatized protein, divalent
cation, complexing compound, and hexamer-stabilizing compound,
pharmaceutical compositions adapted for parenteral administration
in accordance with the present invention may employ additional
excipients and carriers such as water miscible organic solvents
such as glycerol, sesame oil, aqueous propylene glycol and the
like. When present, such agents are usually used in an amount less
than about 2.0% by weight based upon the final formulation. For
further information on the variety of techniques using conventional
excipients or carriers for parenteral products, please see
Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing
Company, Easton, Pa., USA (1985), which is incorporated herein by
reference.
[0407] In the broad practice of the present invention, it is also
contemplated that a formulation may contain a mixture of the
microcrystals and a soluble fraction of a protein selected from
insulin, derivatized insulin, insulin analogs, and derivatized
insulin analogs. Examples of such pharmaceutical compositions
include sterile, isotonic, aqueous saline solutions of insulin, an
insulin analog, a derivatized insulin, or a derivatized insulin
analog, buffered with a pharmaceutically acceptable buffer and
pyrogen-free. Preferred for the soluble phase are insulin or a
rapid-acting insulin analog, such as, LysB28, ProB29-human insulin,
or AspB28-human insulin. Such mixtures are designed to provide a
combination of meal-time control of glucose levels, which is
provided by the soluble insulin, and basal control of glucose
levels, which is provided by the insoluble insulin. The ratio of
total protein (protein plus derivatized protein) in the insoluble
phase and total protein in the soluble phase is in the range of
about 9:1 to about 1:9. A preferred range of this ratio is from
about 9:1 to about 1:1, and more preferably, about 7:3. Other
ratios are 1:1, and 3:7.
[0408] The following preparations and examples illustrate and
explain the invention. The scope of the invention is not limited to
these preparations and examples. Reference to "parts" for solids
means parts by weight. Reference to "parts" for liquids means parts
by volume. Percentages, when used to express concentration, mean
mass per volume (.times.100). All temperatures are degrees
Centigrade (.degree. C.). "TRIS" refers to
2-amino-2-hydroxymethyl-1,3,-propanediol. The 1000 part-per-million
(ppm) zinc solution was prepared by diluting 1.00 mL of a 10,000
ppm zinc atomic absorption standard solution [Ricca Chemical
Company, zinc in dilute nitric acid] with water to a final volume
of 10.00 mL.
[0409] In many of the preparations described below, the yield of
precipitates and crystals was estimated. The yield estimate relied
on determination of the amount of total protein in the precipitate
or crystal, and on an estimate of the amount of the same initially
in solution. To determine the amount of total protein, samples of
re-dissolved precipitate or crystal, and of the supernatant above
the precipitate or crystals, were analyzed by reversed-phase
gradient HPLC, as described below.
[0410] Briefly, the analytical system relied on a C8 reversed-phase
column, at 23.degree. C. The flow rate was 1.0 mL/min and UV
detection at 214 nm was used. Solvent A was 0.1% (vol:vol)
trifluroacetic acid in 10:90 (vol:vol) acetonitrile:water. Solvent
B was 0.1% (vol:vol) trifluroacetic acid in 90:10 (vol:vol)
acetonitrile:water. The development program was (minutes, % B):
(0.1,0); (45.1,75); (50.1,100); (55,100); (57,0); (72,0). All
changes were linear. Other analytical systems could be devised by
the skilled person to achieve the same objective.
[0411] To prepare for the HPLC analysis, aliquots of the well-mixed
suspensions were dissolved by diluting with either 0.01 N HCl or
0.03 N HCl. Results of HPLC analysis of these solutions permitted
calculation of total protein. Aliquots of the suspensions were
centrifuged for approximately 5 minutes in an Eppendorf 5415C
microcentrifuge at 14,000 rpm. The decanted supernatant was diluted
with either 0.01 N or 0.1 N HCl and analyzed by HPLC. The
precipitate was washed by re-suspending in Dulbecco's phosphate
buffered saline (without calcium or magnesium) and re-pelleted by
centrifugation. The buffer was decanted and the solid was
re-dissolved in 0.01 N HCl. The re-dissolved precipitate was
analyzed by HPLC.
[0412] HPLC was used to confirm the presence of the expected
proteins in the acidified suspension, re-dissolved precipitate, and
supernatant and also to determine protein concentrations. The
retention times of peaks in the chromatograms of the re-dissolved
precipitates were compared with the retention times observed for
protamine and the active compounds used to make the formulations.
The agreement between retention times was always good, showing that
protamine, protein, and derivatized proteins were actually
incorporated into the microcrystals. Concentrations of protein and
derivatized protein were determined by comparing the appropriate
peak areas to the areas of a standard. A 0.22 mg/mL solution of
derivatized insulin was used as the standard. A standard containing
protamine was run, but only for the purpose of determining the
retention time. Protamine concentration was not quantitated.
[0413] In many of the preparations described below, a standard
spectrophotometric assay was used to determine how rapidly the
crystals dissolved in Dulbecco's phosphate buffered saline (pH 7.4)
at room temperature. Significant deviations from the procedure
described immediately below are noted where appropriate in the
descriptions of the preparations. A spectrophotometer suitable for
measuring in the ultraviolet range, and equipped with a 1 cm
cuvette and a magnetic cuvette stirrer was used for all the
dissolution assays. The cuvette, containing a small stir bar and
3.00 mL of phosphate buffered saline (PBS), was put into the cell
compartment of the spectrophotometer. The instrument was set to 320
nm and zeroed against the same buffer. Then 4.0 microliters of a
well suspended formulation, usually having a total concentration
approximately equivalent to a U50 formulation, or about 1.6 to 1.8
mg/mL, was added to the cuvette. After waiting 1.0 minute for
mixing, the optical density at 320 nm was recorded. Since the
proteins involved in this work do not absorb light at 320 nm, the
decrease in optical density was due to reduction in light
scattering as the crystals dissolved. The time for the optical
density to drop to half of its initial value is typically reported
(t1/2). As a control, 2.0 microliters of U100 Humulin.RTM. N (i.e.,
human insulin NPH, which is also known as human NPH insulin) was
added to 3.00 mL of PBS buffer, and the optical density at 320 nm
monitored as above. The dissolution half-time (t1/2) for the
Humulin.RTM. N formulation was about 6 minutes.
PREPARATION 1
9:1 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Insulin
[0414] A dry powder of B29-N.epsilon.-octanoyl-LysB29 human insulin
(0.7 parts by mass) and a dry powder of humen insulin (6.3 parts by
mass) are dissolved in 1000 parts by volume of an aqueous solvent
composed of 50 mM TRIS, 0.1 M trisodium citrate, and 10 mg/ml
phenol at pH 7.6. To this solution is added 75 parts of a 15.3 mM
solution of zinc chloride. The pH is adjusted to 7.6 with 1 N HCl
and/or 1 N NaOH. This solution is filtered through a 0.22 micron,
low-protein binding filter. A second solution is prepared by
dissolving 7 parts by mass of protamine sulfate in 10,000 parts by
volume of water then filtering through-a 0.22 micron, low-protein
binding filter. Equal volumes of the solution containing insulin
and acylated insulin and of the protamine sulfate solution are
combined. Initially, an amorphous precipitate forms. This
suspension is allowed to stand for about 24 hours at room
temperature (typically about 22.degree. C.). The amorphous
precipitate converts to a co-crystalline microcrystalline
solid.
PREPARATION 2
3:1 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Insulin
[0415] The procedure of Preparation 1 is followed, except that 1.75
parts by mass of a dry powder of B29-N.epsilon.-octanoyl-LysB29
human insulin and 5.25 parts by mass of a dry powder of human
insulin are used. After equal volumes of the solution containing
insulin and acylated insulin and of the protamine sulfate solution
are combined, an amorphous precipitate forms. This suspension is
allowed to stand for about 24 hours at room temperature (typically
about 22.degree. C.). The amorphous precipitate converts to a
co-crystalline microcrystalline solid.
PREPARATION 3
Formulation of 3:1 Co-Crystals of Human Insulin and
B29-N.epsilon.-octanoyl-Human Insulin
[0416] The co-crystalline microcrystals prepared by the method of
Preparation 1 are separated from the supernatant and are recovered
by conventional solid/liquid separation methods, such as,
filtration, centrifugation, or decantation. The recovered
co-crystalline microcrystals are then suspended in a solution
consisting of 25 mM TRIS, 5 mg/ml phenol, and 16 mg/ml glycerol, pH
7.8, so that the final concentration of insulin activity is
about.100 U/mL.
PREPARATION 4
1:1 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Insulin
[0417] The procedure of Preparation 1 is followed, except that 3.5
parts by mass of a dry powder of B29-N.epsilon.-octanoyl-LysB29
human insulin and 3.5 parts by mass of a dry powder of human
insulin are used. After equal volumes of the solution containing
insulin and acylated insulin and of the protamine sulfate solution
are combined, an amorphous precipitate forms. This suspension is
allowed to stand for about 24 hours at room temperature (typically
about 22.degree. C.) The amorphous precipitate converts to a
co-crystalline microcrystalline solid.
PREPARATION 5
1:3 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Insulin
[0418] The procedure of Preparation 1 is followed, except that 5.25
parts by mass of a dry powder of B29-N.epsilon.-octanoyl-LysB29
human insulin and 1.75 parts by mass of a dry powder of human
insulin are used. After equal volumes of the solution containing
insulin and acylated insulin and of the protamine sulfate solution
are combined, an amorphous precipitate forms. This suspension is
allowed to stand for about 24 hours at room temperature (typically
about 22.degree. C.). The amorphous precipitate converts to a
co-crystalline microcrystalline solid.
PREPARATION 6
3:1 Co-Crystals of Human Insulin and B29-N.epsilon.-hexanoyl-Human
Insulin
[0419] The procedure of Preparation 1 is followed, except that 1.75
parts by mass of a dry powder of B29-N.epsilon.-hexanoyl-LysB29
human insulin and 5.25 parts by mass of a dry powder of human
insulin are used. After equal volumes of the solution containing
insulin and acylated insulin and of the protamine sulfate solution
are combined, an amorphous precipitate forms. This suspension is
allowed to stand for about 24 hours at room temperature (typically
about 22.degree. C.). The amorphous precipitate will convert to a
co-crystalline microcrystalline solid.
PREPARATION 7
3:1 Co-Crystals of Human Insulin and B29-N.epsilon.-butyrl-Human
Insulin
[0420] The procedure of Preparation 1 is followed, except that 1.75
parts by mass of a dry powder of B29-N.epsilon.-butyryl-LysB29
human insulin and 5.25 parts by mass of a dry powder of human
insulin are used. After equal volumes of the solution containing
insulin and acylated insulin and of the protamine sulfate solution
are combined, an amorphous precipitate forms. This suspension is
allowed to stand for about 24 hours at room temperature (typically
about 22.degree. C.). The amorphous precipitate will convert to a
co-crystalline microcrystalline solid.
PREPARATION 8
Co-Crystalline Microcrystals of
Protamine-Zinc-B29-N.epsilon.-octanoyl-Hum- an Insulin-Human
Insulin
[0421] B29-N.epsilon.-octanoyl-LysB29 human insulin (20.1 mg) was
dissolved in 1 mL of a solvent composed of 0.1 N HCl. Human insulin
(19.3 mg) was dissolved in 1 mL of a solvent composed of 0.1 N HCl.
Five solutions comprising different ratios of
B29-N.epsilon.-octanoyl-LysB29 human insulin to human insulin were
prepared by combining volumes of each solution in the ratios shown
below.
4TABLE 3 Volumes of solutions of human insulin and of
B29-N.epsilon.-octanoyl-human insulin used to prepare precipitates
and microcrystals. Ratio of Human Insulin to Acylated Human Insulin
Volume (.mu.1) 1:0 3:1 1:1 1:3 0:1 Human insulin solution 400 300
200 100 0 B29-N.epsilon.-octanoyl-human 0 100 200 300 400 insulin
solution
[0422] To each of these five solutions, 1.6 mL of a solvent
composed of 50 mM TRIS buffer, 0.1 M trisodium citrate, and 10
mg/mL phenol at pH 7.6 was added. To each of the five solutions,
0.15 ml of a 15.3 mM solution of zinc chloride was added. Each of
the resulting five solutions were adjusted to a pH of 7.6 with 1 N
NaOH. Each of the resulting five solutions were filtered through a
0.22 micron, low-protein binding filter. An additional solution was
prepared by dissolving 3.50 mg of protamine sulfate in 10 mL of
water then filtered through a 0.22 micron, low-protein binding
filter. A volume of 1.9 mL of each of the five solutions and 1.9 mL
of the protamine sulfate solution were combined respectively, in
each of the five solutions resulting in the immediate appearance of
an amorphous precipitate. These five solutions were allowed to
stand for 24 hours at room temperature (approximately 22.degree.
C.). This procedure resulted in the formation of a
white-to-off-white microcrystalline solid in each of the five
solutions.
PREPARATION 9
9:1 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Insulin
[0423] A dry powder of B29-N.epsilon.-octanoyl-LysB29 human insulin
(0.7 parts by mass) is dissolved in 100 parts by volume of an
aqueous solvent composed of 50 mM TRIS, 0.1 M trisodium citrate,
and 10 mg/ml phenol at pH 7.6. To this solution is added 7.5 parts
of a 15.3 mM solution of zinc chloride. A second solution is
prepared wherein a dry powder of human insulin (6.3 parts by mass)
is dissolved in 900 parts by volume of an aqueous solvent composed
of 50 mM TRIS, 0.1 M trisodium citrate, and 10 mg/ml phenol at pH
7.6. To this solution is added 67.5 parts of a 15.3 mM solution of
zinc chloride. The acylated insulin solution and the insulin
solution are combined together and stirred to insure mixing of the
two solutions. This solution is filtered through a 0.22 micron,
low-protein binding filter. A protamine solution is prepared by
dissolving 7 parts by mass of protamine sulfate in 10,000 parts by
volume of water then filtering through a 0.22 micron, low-protein
binding filter. Equal volumes of the acylated insulin solution and
of the protamine sulfate solution are combined. An amorphous
precipitate forms. This suspension is allowed to stand for about 24
hours at room temperature (typically about 22.degree. C.). The
amorphous precipitate will convert to a co-crystalline
microcrystalline solid.
PREPARATION 10
3:1 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Isulin
[0424] A dry powder of B29-N.epsilon.-octanoyl-LysB29 human insulin
(1.75 parts by mass) is dissolved in 250 parts by volume of an
aqueous solvent composed of 50 mM TRIS, 0.1 M trisodium citrate,
and 10 mg/ml phenol at pH 7.6. To this solution is added 18.75
parts of a 15.3 mM solution of zinc chloride. A second solution is
prepared wherein a dry powder of human insulin (5.25 parts by mass)
is dissolved in 750 parts by volume of an aqueous solvent composed
of 50 mM TRIS, 0.1 M trisodium citrate, and 10 mg/ml phenol at pH
7.6. To this solution is added 56.25 parts of a 15.3 mM solution of
zinc chloride. The acylated insulin solution and the insulin
solution are combined together and stirred to insure mixing of the
two solutions. This solution is filtered through a 0.22 micron,
low-protein binding filter. A protamine solution is prepared by
dissolving 7 parts by mass of protamine sulfate in 10,000 parts by
volume of water then filtering through a 0.22 micron, low-protein
binding filter. Equal volumes of the acylated insulin solution and
of the protamine sulfate solution are combined. An amorphous
precipitate forms. This suspension is allowed to stand for about 24
hours at room temperature (typically about 22.degree. C.). The
amorphous precipitate converts to a co-crystalline microcrystalline
solid.
PREPARATION 11
1:1 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Insulin
[0425] A dry powder of B29-N.epsilon.-octanoyl-LysB29 human insulin
(3.5 parts by mass) is dissolved in 500 parts by volume of an
aqueous solvent composed of 50 mM TRIS, 0.1 M trisodium citrate,
and 10 mg/ml phenol at pH 7.6. To this solution is added 1.75 parts
of a 15.3 mM solution of zinc chloride. A second solution is
prepared wherein a dry powder of human insulin (3.5 parts by mass)
is dissolved in 500 parts by volume of an aqueous solvent composed
of 50 mM TRIS, 0.1 M trisodium citrate, and 10 mg/ml phenol at pH
7.6. To this solution is added 37.5 parts of a 15.3 mM solution of
zinc chloride. The acylated insulin solution and the insulin
solution are combined together and stirred to insure mixing of the
two solutions. This solution is filtered through a 0.22 micron,
low-protein binding filter. A protamine solution is prepared by
dissolving 7 parts by mass of protamine sulfate in 10,000 parts by
volume of water then filtering through a 0.22 micron, low-protein
binding filter. Equal volumes of the acylated insulin solution and
of the protamine sulfate solution are combined. An amorphous
precipitate forms. This suspension is allowed to stand for about 24
hours at room temperature (typically about 22.degree. C.). The
amorphous precipitate converts to a co-crystalline microcrystalline
solid.
PREPARATION 12
1:3 Co-Crystals of Human Insulin and B29-N.epsilon.-octanoyl-Human
Insulin
[0426] A dry powder of B29-N.epsilon.-octanoyl-LysB29 human insulin
(5.25 parts by mass) is dissolved in 750 parts by volume of an
aqueous solvent composed of 50 mM TRIS, 0.1 M trisodium citrate,
and 10 mg/ml phenol at pH 7.6. To this solution is added 56.25
parts of a 15.3 mM solution of zinc chloride. A second solution is
prepared wherein a dry powder of human insulin (1.75 parts by mass)
is dissolved in 250 parts by volume of an aqueous solvent composed
of 50 mM TRIS, 0.1 M trisodium citrate, and 10 mg/ml phenol at pH
7.6. To this solution is added 18.75 parts of a 15.3 mM solution of
zinc chloride. The acylated insulin solution and the insulin
solution are combined together and stirred to insure mixing of the
two solutions. This solution is filtered through a 0.22 micron,
low-protein binding filter. A protamine solution is prepared by
dissolving 7 parts by mass of protamine sulfate in 10,000 parts by
volume of water then filtering through a 0.22 micron, low-protein
binding filter. Equal volumes of the acylated insulin solution and
of the protamine sulfate solution are combined. An amorphous
precipitate forms. This suspension is allowed to stand for about 24
hours at room temperature (typically about 22.degree. C.). The
amorphous precipitate converts to a co-crystalline microcrystalline
solid.
PREPARATION 13
Co-Crystals of Human Insulin and B29-N.epsilon.-hexanoyl-Human
Insulin
[0427] An acidic solution of B29-N.epsilon.-hexanoyl-human insulin
was prepared by dissolving 12.3 mg of B29-N.epsilon.-hexanoyl-human
insulin in 0.3 mL of 0.1 N HCl. An acidic solution of human insulin
was prepared by dissolving 4.6 mg of human insulin (zinc crystals)
in 0.1 mL of 0.1 N HCl. The two solutions were combined giving a
total volume of 0.4 mL. This resulting solution was stirred for
approximately 5 minutes. To this resulting solution was added, with
stirring, 0.150 mL of a 1000 ppm zinc(II) solution. A
crystallization diluent was prepared comprising 32 mg/mL glycerol,
50 mM tris buffer, 10 mg/mL phenol, 100 mM trisodium citrate, at a
pH of 7.6. To the insulin solution was added 1.6 mL of the
crystallization diluent. The pH of the solution was adjusted to
7.59 using 1 N NaOH and 1 N HCl. The solution was filtered through
a 0.22 micron, low protein binding filter. A protamine solution was
prepared by dissolving 7.47 mg of protamine sulfate in 10 mL of
water. Two milliliters (2 mL) of the protamine solution was added
to 2 mL of the insulin solution. The resulting solution was allowed
to stand undisturbed for 18 hours at a controlled temperature of
25.degree. C.
[0428] Microscopic inspection (at 18 hours) revealed that
crystallization had occurred and that the preparation yielded
uniform, single, rod-like crystals possessing approximate average
lengths of 3 microns.
[0429] Four milliliters (4 mL) of the crystal formulation produced
above after 18 hours were allowed to stand undisturbed overnight,
and the crystals sedimented completely. The supernatant was then
removed, and replaced with 4 mL of a diluent comprising 16 mg/mL
glycerol, 20 mM tris buffer, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol,
40 mM trisodium citrate, pH 7.6. The crystals were then
resuspended, and allowed to sediment again. This procedure was
carried out three times except that on the third occasion the
supernatant was replaced with only 3 mL of diluent.
[0430] The dissolution rate of the crystals was measured by placing
0.005 mL of the uniformly suspended formulation into 3 mL of
Dulbecco's phosphate buffered saline (without calcium or magnesium)
in a 1 cm path length square quartz cuvette at a temperature of
.sub.22.degree. C. This solution was stirred at a constant rate
using a magnetic cuvette stirrer. Absorbance measurements at 320 nm
were taken at 1 minute intervals. The absorbance at 320 nm
corresponds to the light scattered by the insoluble particles
present in the aqueous suspension. Consequently, as the
microcrystals dissolve, the absorbance approaches zero. The time
required for the 0.005 mL of this formulation to dissolve was
greater than 150 minutes. The time required for dissolution of a
0.005 mL sample of U100 commercial Humulin N to dissolve when
subjected to the same conditions was about 10 minutes.
[0431] The amount of total protein in the formulation was analyzed
by HPLC to quantitate the total potency. The total potency refers
to the total concentration of human insulin and
B29-N.epsilon.-hexanoyl-human insulin. An aliquot (0.050 mL) of the
fully resuspended formulation was dissolved in 0.950 mL of 0.01 N
HCL, and subjected to HPLC analysis, as described below. The total
potency determined from this analysis was 4.54 mg/mL.
[0432] For HPLC analyses, the following conditions were used: a
C8-reversed phase column; constant 23.degree. C.; 1.0 mL/min,
detection at 214 nm; solvent A=10% acetonitrile (vol/vol) in 0.1%
aqueous trifluoroacetic acid; solvent B=90% acetonitrile (vol/vol)
in 0.1% aqueous trifluoroacetic acid; linear gradients (0.1 min, 0%
B; 45.1 min, 75% B; 50.1 min, 100% B; 55 min 100% B; 57 min, 0% B;
72 min, 0% B) . Standards were prepared by dissolving bulk insulin
and bulk acyl insulin in 0.01 N HCl. The concentration of each
standard was determined by UV spectroscopy. A solution of 1.0 mg/ml
of human insulin in a 1 cm cuvette was assumed to have an
absorbance of 1.05 optical density units at the wavelength maximum
(approximately 276 nm). This corresponds to a molar extinction
coefficient of 6098. Acylated insulins were assumed to have the
same molar extinction coefficient as human insulin. The solutions
calibrated by UV were then diluted to get standards at 0.220,
0.147, 0.073, and 0.022 mg/mL. The standards were run on HPLC and a
standard curve of area vs. concentration was obtained.
[0433] The supernatant was analyzed to determine the total
concentration of soluble human insulin and
B29-N.epsilon.-hexanoyl-human insulin present in the formulation.
To 0.040 mL of the supernatant, were added 0.160 mL of 0.01 N HCl.
The acidified supernatant was analyzed by HPLC, as described above.
The concentration of soluble human insulin and
B29-N.epsilon.-hexanoyl-human insulin in the supernatant was
determined to be 0.07 mg/mL.
[0434] The ratios of B29-N.epsilon.-hexanoyl-human insulin and
human insulin in the crystal were determined by sedimenting an
aliquot (0.100 mL) of the formulation using a bench-top centrifuge,
decanting the supernatant, resuspending the crystals in 0.400 mL of
Dulbecco's phosphate-buffered saline, recentrifuging, removing the
supernatant, and finally dissolving the crystals in 1.50 mL of 0.01
N HCl. The HPLC analysis described above was performed. The result
of this analysis was 84.2% B29-N.epsilon.-hexanoyl-human insulin
and 15.8% human insulin.
PREPARATION 14
Co-Crystal Suspension Formulation Comprising Human Insulin and
B29-N.epsilon.-decanoyl-Human Insulin
[0435] An acidic solution of B29-N.epsilon.-decanoyl-human insulin
was prepared by dissolving 10.4 mg of B29-N.epsilon.-decanoyl-human
insulin in 0.25 mL of 0.1 N HCl. An acidic solution of human
insulin was prepared by dissolving 30.3 mg of human insulin (zinc
crystals) in 0.75 mL of 0.1 N HCl. The two solutions were combined,
giving a total volume of 1 mL. This resulting solution was stirred
for approximately 5 minutes. To this solution was added, with
stirring, 0.305 mL of a 1000 ppm zinc(II) solution. To the
resulting solution, was added 4 mL of a crystallization diluent (40
mg/mL glycerol, 50 mM tris buffer, 4 mg/mL m-cresol, 1.625 mg/mL
phenol, 100 mM trisodium citrate, pH 7.4). The pH of the resulting
solution was adjusted to 7.58. This solution was filtered through a
0.22 micron, low protein binding filter. Five milliliters (5 mL) of
protamine solution (37.6 mg of protamine sulfate in 50 mL of water)
was added to 5 mL of the filtered solution. The resulting solution
was allowed to stand undisturbed for 63 hours at a controlled
temperature of 25.degree. C.
[0436] Microscopic inspection (at 63 hours) revealed that
crystallization had occurred, and that the preparation had yielded
uniform, single, rod-like crystals possessing approximate average
lengths of 8 microns.
[0437] The dissolution rate of the crystals was measured by placing
0.006 mL of the uniformly suspended crystal formulation into 3 mL
of Dulbecco's phosphate buffered saline (without calcium or
magnesium) in a 1 cm path length square quartz cuvette at a
temperature of 22.degree. C. The time required for the 0.006 mL of
this crystal formulation to dissolve was greater than 300 minutes.
The time required for a 0.005 mL sample of U100 commercial Humulin
N to dissolve under the same conditions was about 10 minutes.
[0438] To prepare for HPLC analysis, the crystals were sedimented
by allowing the formulation to stand undisturbed. Eight milliliters
(8 mL) of the supernatant were then removed, and were replaced with
8 mL of a diluent [16 mg/mL glycerol, 20 mM tris buffer, 1.6 mg/mL
m-cresol, 0.65 mg/mL phenol, 40 mM trisodium citrate, pH 7.6]. The
co-crystals were then resuspended. This procedure was carried out
in the same way three times, except that on the third occasion, the
8 mL of supernatant was replaced with 7 mL of diluent.
[0439] Potency of the crystal formulation, and in the supernatant,
was analyzed by HPLC, essentially as described in Preparation 13.
The total potency determined from this analysis was 3.87 mg/mL. The
concentration of soluble human insulin and
B29-N.epsilon.-decanoyl-human insulin in the supernatant was
determined to be 0.06 mg/mL. The proportions of human insulin and
B29-N.epsilon.-decanoyl-human insulin in the crystal phase were
determined by the procedure of Preparation 13 to be 74.3% human
insulin, and 25.7% B29-N.epsilon.-decanoyl-human insulin.
[0440] A particle sizing measurement was performed on a sample of
the formulation utilizing a particle sizing instrument (Multisizer
Model IIE, Coulter Corp., Miami, Fla. 33116-9015). To perform this
measurement, 0.25 mL of the crystal formulation was added to 100 mL
of a diluent consisting 14 mM dibasic sodium phosphate, 16 mM
glycerol, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, pH 7.4. The
instrument aperture tube orifice size was 50 microns. Particle size
data was collected for 50 seconds. This measurement showed that the
mean particle diameter of the crystals was approximately 6 microns
with an approximately normal distribution encompassing a range of
particle sizes from approximately 2 microns to approximately 9
microns. This result is similar to the particle size distribution
of commercial NPH determined using an analogous method [DeFelippis,
M. R., et al. J. Pharmaceut. Sci. 87:170-176 (1998)].
PREPARATION 15
Co-Crystal Suspension Formulation Comprising Human Insulin and
B29-N.epsilon.-octanoyl-Human Insulin
[0441] An acidic solution of B29-N.epsilon.-octanoyl-human insulin
was prepared by dissolving 30.3 mg of B29-N.epsilon.-octanoyl-human
insulin in 0.75 mL of 0.1 N HCl. An acidic solution of human
insulin was prepared by dissolving 59.7 mg of human insulin (zinc
crystals) in 1.5 mL of 0.1 N HCl. An aliquot (0.25 mL) of the human
insulin solution was combined with the 0.75 mL
B29-N.epsilon.-octanoyl-human insulin solution, giving a total
volume of 1 mL, which was stirred for approximately 5 minutes. To
this was added, with stirring, 0.365 mL of a 1000 ppm zinc(II)
solution. To the insulin plus zinc solution was added 4 mL of
crystallization diluent (40 mg/mL glycerol, 35 mM sodium phosphate
dibasic buffer, 4 mg/mL m-cresol, 1.625 mg/mL phenol, 15 mM
trisodium citrate, pH 7.4). The pH of the resulting solution was
adjusted to 7.60. The solution was filtered through a 0.22 micron,
low protein binding filter. Five milliliters (5 mL) of protamine
solution (37.9 mg of protamine sulfate in 50 mL of water) was added
to 5 mL of the filtered insulin plus zinc solution. The resulting
solution was allowed to stand undisturbed for 48 hours at a
controlled temperature of 25.degree. C.
[0442] Microscopic inspection (at 48 hours) revealed that
crystallization had occurred and that the preparation had yielded
uniform, single, rod-like crystals possessing approximate average
lengths of 5 microns.
[0443] To prepare for HPLC analysis and dissolution testing, the
crystals were sedimented by allowing the formulation to stand
undisturbed. Eight milliliters (8 mL) of the supernatant were then
removed and replaced with 8 mL of a diluent [16 mg/mL glycerol, 14
mM sodium phosphate dibasic buffer, 1.6 mg/mL m-cresol, 0.65 mg/mL
phenol, 6 mM trisodium citrate, pH 7.6]. The crystals were then
resuspended. This procedure was carried out in the same way three
times, except that on the third occasion the 8 mL of supernatant
was replaced with 7 mL of diluent.
[0444] The dissolution rate was determined essentially as described
in Preparation 13, above. The approximate time required for 0.005
mL of the present formulation to dissolve was more than 300
minutes. The time required for a 0.005 mL sample of U100 commercial
Humulin N to dissolve under the same conditions was about 10
minutes.
[0445] Total potency, and potency in the supernatant, were
determined by HPLC, essentially as described in Preparation 13. The
total potency was 3.44 mg/mL. The concentration of soluble human
insulin and B29-N.epsilon.-octanoyl-human insulin in the crystal
formulation was determined to be 0.01 mg/mL. The proportions of
human insulin and B29-N.epsilon.-octanoyl-human insulin in the
crystal phase were determined, essentially by the procedure of
Preparation 13, to be 25.5% human insulin, and 74.5%
B29-N.epsilon.-octanoyl-human insulin.
[0446] The mean particle diameter of the crystals, determined as
described in Preparation 14, was approximately 6 microns, with an
approximately normal distribution, encompassing a range of particle
sizes from approximately 2 microns to approximately 12 microns.
This result is similar to the particle size distribution of
commercial NPH as reported in DeFelippis, M. R., et al. supra.
PREPARATION 16
Three Co-Crystal Formulations Compared with an Insulin
Formulation
[0447] An acidic solution of B29-N.epsilon.-octanoyl-human insulin
was prepared by dissolving 24.18 mg of
B29-N.epsilon.-octanoyl-human insulin in 0.6 mL of 0.1 N HCl. An
acidic solution of human insulin was prepared by dissolving 41.1 mg
of human insulin (as zinc crystals) in 1 mL of 0.1 N HCl. Four 0.4
mL solutions were prepared by combining different volumes of the
B29-N.epsilon.-octanoyl-human insulin and human insulin solutions
as indicated below in Table 4.
5TABLE 4 Preparation of formulations of microcrystals Formulation D
C B A Nominal Mass Percent of B29-N.epsilon.- 75 50 25 0
octanoyl-human insulin Volume of B29-N.epsilon.-octanoyl-human 300
200 100 0 insulin solution added (.mu.L) Volume of human insulin
solution 100 200 300 400 added (.mu.L)
[0448] To each of the four 0.4 mL solutions, 0.15 mL of a 1000 ppm
zinc(II) solution was added. To each of the four 0.55 mL solutions,
1.6 mL of a crystallization diluent (50 mM tris buffer, 10 mg/mL
phenol, 100 mM trisodium citrate, with a pH of 7.6) were added.
Each of the four solutions was adjusted to pH 7.6 with small
quantities of 1 N NaOH and 0.1 N HCl. Each solution was filtered
through a 0.22 micron, low protein binding filter. Two milliliters
(2 mL) of each of the four protein solutions were combined with 2
mL of protamine solution (7.34 mg of protamine sulfate in 10 mL of
water). In each case, a precipitate formed immediately. These four
4 mL suspensions were allowed to stand undisturbed at room
temperature (approximately 22.degree. C.) for 16 hours.
[0449] Microscopic inspection (at 16 hours) revealed that each of
the four preparations had yielded uniform, single, rod-like
crystals with approximate average lengths of about 10 microns.
[0450] Each 4 mL formulation was transferred to a test tube and
centrifuged in a bench-top centrifuge at 3000 rpm for 20 minutes to
fully sediment the crystals. For each formulation, 3 mL of the
supernatant were removed and replaced with 3 mL of a diluent (25 mM
tris buffer, 5 mg/mL phenol, 16 mg/mL glycerol, pH 7.4). The
crystals were then resuspended. This procedure was carried out
three times except that on the third occasion the 3 mL of
supernatant was replaced with 2.5 mL of diluent for each
formulation.
[0451] Each of the four formulations was analyzed by HPLC to
quantitate the total potencies of the formulations and the
compositions of the respective crystals, essentially as described
above. The total potency refers to the total concentration of human
insulin and B29-N.epsilon.-octanoyl-human insulin. The total
potency and percentage of B29-N.epsilon.-octanoyl-human insulin
were determined by analyzing an aliquot of the uniformly suspended
formulation. The supernatant was analyzed to determine the total
concentration of soluble human insulin and soluble
B29-N.epsilon.-octanoyl-human insulin present in each formulation.
The results of these analyses are presented below. Dissolution
times were determined as described above in Preparation 13.
6TABLE 5 Characteristics of formulations of microcrystals.
Formulation D C B A NPH B29-N.epsilon.-octanoyl-human 77.7 51.4
23.7 0 -- insulin in crystal (%) Human insulin in 22.3 48.6 76.3
100 -- crystal (%) Total potency (mg/mL) 3.21 3.48 3.38 3.43 --
Supernatant potency <0.01 <0.01 <0.01 <0.01 -- (mg/mL)
Dissolution Time (min) 300 120 50 20 10
PREPARATION 17
Preparation of Insoluble Compositions
[0452] The following is an outline of another method that was used
to prepare precipitates and micro-crystals of the present
invention. The outline is to be read together with the data in
Table 6, below.
[0453] A measured mass of a derivatized protein, prepared as
described herein, was dissolved in 0.6 mL of 0.1 N HCl. A measured
mass of a protein was dissolved in 0.2 mL of 0.1 N HCl (zinc
crystals of human insulin or LysB28, Pro29-human insulin analog).
The two solutions were thoroughly mixed together by stirring for
five to ten minutes. A volume (0.32 mL) of an aqueous solution
containing 1000 ppm Zn(II) and a volume (3.2 mL) of a diluent
solution (about 50 mM Tris reagent, about 10 mg/mL phenol, about 16
mg/mL glycerol, and about 29.5 mg/mL trisodium citrate) were added
to the mixture of the two proteins. The pH of the resulting
solution was adjusted to about 7.6 (7.55-7.64) using 1 N HCl or 1 N
NaOH. The pH-adjusted solution was filtered through a 0.22 micron,
low-protein binding filter. To four milliliters of the filtrate was
added four milliliters of a solution of protamine in water (about
37.3 mg protamine sulfate per 100 mL, range 37.18-37.48).
Precipitate formed immediately upon adding the protamine solution.
The preparation was allowed to stand undisturbed at 25.degree. C.
Dissolution tests were carried out as previously described. Under
the same conditions, insulin NPH dissolved in about 6 minutes.
7TABLE 6 Preparation of insoluble compositions. Protein Lys (B28),
Pro (B29)-human insulin analog Protein 4.28 4.02 3.84 3.96 4.15
mass (mg) Derivatized B29-acylated-human insulin protein
Derivatizing butanoyl pentanoyl hexanoyl nonanoyl decanoyl group
Derivatized 11.90 12.1 12.08 12.13 12.20 protein mass (mg) Crystal
rod-like rod-like rod-like rod-like rod-like shape Yield (%) >80
>90 >90 >90 >90 Derivatized 77.5 75.1 77 76.5 76.6
protein in insoluble phase (%) Dissolution 23-24 31 54 67 37-38
time (min) Protein human insulin Protein 12.09 12.18 12.12 12.21
12.27 mass (mg) Derivatized B28-acylated- A1, B28-diacylated-
protein Lys (B28), Pro (B29)-human Lys (B28), Pro (B29)- insulin
analog human insulin Derivatizing butanoyl hexanoyl octanoyl
dibutanoyl dihexanoyl Derivatized 4.39 4.21 4.28 4.11 4.23 protein
mass (mg) Crystal rod-like rod-like rod-like rod-like rod-like
shape Yield (%) >80 >90 >90 >90 >90 Derivatized 27.6
25 27 26.8 24.1 protein in insoluble phase (%) Dissolution 16-17
10-11 27-28 10-11 20-21 time (min) Protein 4.26 4.26 4.13 4.23
12.09 mass(mg) Derivatized B29-acylated-human insulin protein
Derivatizing butanoyl pentanoyl hexanoyl nonanoyl tetra- group
decanoyl Derivatized 12.39 12.39 12.03 12.06 4.16 protein mass (mg)
Crystal shape rod-like rod-like rod-like rod-like rod-like Yield
(%) >90 72 >90 >90 >90 Derivatized 76.3 76.5 75 72.5
25.07 protein in insoluble phase (%) Dissolution 45-46 62-63 77-78
77-78 61 time (min) Protein mass 12.19 (mg) Derivatized
B29-acylated-human insulin protein Derivatizing hexa-decanoyl group
Derivatized 3.99 protein mass (mg) Crystal shape rod-like Yield (%)
>90 Derivatized 24.7 protein in insoluble phase (%) Dissolution
71-72 time (min)
[0454] The following is an outline of another method that was used
to prepare precipitates and micro-crystals of the present
invention. The outline is to be read together with the data in
Table 7, below.
[0455] A measured mass of a derivatized protein, prepared as
described herein, was dissolved in 3.2 mL of diluent solution
(about 50 mM Tris reagent, about 10 mg/mL phenol, about 16 mg/mL
glycerol, and about 29.5 mg/mL trisodium citrate). A measured mass
of a protein was dissolved in 0.6 mL of 0.1 N HCl (zinc crystals of
human insulin or LysB28, Pro29-human insulin analog). The two
solutions were thoroughly mixed together by stirring for five to
ten minutes. The pH of the resulting solution was adjusted to about
7.6 (7.55-7.64) using 1 N HCl or 1 N NaOH. The pH-adjusted solution
was filtered through a 0.22 micron, low-protein binding filter. To
a volume of the filtrate was added an equal volume of a solution of
protamine in water (about 37.3 mg protamine sulfate per 100 mL,
range 37.18-37.48). Precipitate formed immediately upon adding the
protamine solution. The preparation was allowed to stand
undisturbed at 25.degree. C. Dissolution tests were carried out as
previously described. Under the same conditions, insulin NPH
dissolved in about 6 minutes.
8TABLE 7 Preparation of insoluble compositions. Protein human
insulin Protein mass 12.19 11.87 12.15 12.28 12.22 (mg) Derivatized
A1, B29-diacyl-human insulin A1, B28- B29-acyl- Protein diacyl-
AspB28 LysB28, human ProB29 insulin human analog insulin
Derivatizing dioctanoyl di- didecanoyl dioctanoyl octanoyl group
nonanoyl Derivatized 4.29 4.07 4.06 4.29 3.98 protein mass (mg)
Crystal rod-like rod-like rod-like rod-like rod-like shape Yield
(%) >90 >90 >90 >90 >90 Derivatized 25.2 24.6 26.6
27.4 24.4 protein in insoluble phase (%) Dissolution 73-74 25 31-32
47-48 43-44 time (min)
[0456] The following is an outline of another method that was used
to prepare precipitates and micro-crystals of the present
invention. The outline is to be read together with the data in
Table 8, below.
[0457] A measured mass of a derivatized protein, prepared as
described herein, was dissolved in a measured volume of 0.1 N HCl.
A measured mass of a protein was dissolved in a measured volume of
0.1 N HCl (zinc crystals of human insulin or LysB28, Pro29-human
insulin analog). Measured volumes of each of the two solutions were
thoroughly mixed together by stirring for five to ten minutes.
Measured volumes of an aqueous solution containing 1000 ppm Zn(II)
and of a diluent solution (about 50 mM Tris reagent, about 10 mg/mL
phenol, about 32 mg/mL glycerol, and about 30 mg/mL trisodium
citrate dihydrate, pH 8.47) were added to the mixture of the two
proteins. The pH of the resulting solution was adjusted to about
7.6 (7.58-7.63) using 1 N HCl or 1 N NaOH. The pH-adjusted solution
was filtered through a 0.22 micron, low-protein binding filter. To
two milliliters of the filtrate was added two milliliters of a
solution of protamine in water (about 37.5 mg protamine sulfate per
100 mL). Precipitate formed immediately upon adding the protamine
solution. The preparation was allowed to stand undisturbed at
25.degree. C. Dissolution tests were carried out as previously
described. Under the same conditions, insulin NPH dissolved in
about 6 minutes.
9TABLE 8 Preparation of insoluble compositions. Protein human
insulin Protein mass 11.3 11.3 33.6 33.6 16.5 (mg) Volume of 0.1
0.57 0.57 1.68 1.68 0.83 N HCl Derivatized B29-actylated-human
insulin protein Derivatizing 2-methy- 2-ethyl- 4-methyl- 3-methyl-
do- group hexanoyl hexanoyl octanoyl decanoyl decanoyl Derivatized
6.07 6.3 6.12 2.12 6.3 protein mass (mg) Volume of 0.1 0.3 0.3 0.3
0.1 0.3 N HCl mL of protein 0.10 + 0.30 0.10 + 0.30 0.10 + 0.30
0.30 + 0.10 0.10 + 0.30 solution combined with mL of derivatized
protein solution mL 1000 ppm 0.152 0.152 0.112 0.096 0.152 zinc mL
diluent 1.6 1.6 1.6 1.6 1.6 added Crystal shape rod-like rod-like
rod-like rod-like small- irregular Yield (%) >90 >90 >80
Derivatized 74.1 75.5 81.9 protein in insoluble phase (%)
Dissolution 116 236 40 time (min) Protein human insulin Protein
mass 22.3 22.3 22.3 22.3 (mg) Volume of 0.1 1.12 1.12 1.12 1.12 N
HCl Derivatized B29-acylated- B29-acylated- B29-acylated-
B29-acylated- protein rabbit pork sheep beef insulin insulin
insulin insulin Derivatizing octanoyl octanoyl hexanoyl hexanoyl
group Derivatized 6.21 6.07 6.07 6.21 protein mass (mg) Volume of
0.1 0.3 0.3 0.3 0.3 N HCl mL of protein 0.1 + 0.3 0.1 + 0.3 0.1 +
0.3 0.1 + 0.3 solution combined with mL of derivatized protein
solution mL 1000 ppm 0.152 0.152 0.152 0.152 zinc mL diluent 1.6
1.6 1.6 1.6 added Crystal shape rod-like rod-like small-irregular
rod-like Yield (%) >90 >90 >90 >90 Derivatized 74.6
74.7 75.4 75.9 protein in insoluble phase (%) Dissolution 111
>300 227 >300 time (min) Protein human insulin Protein mass
33.6 2.4 22.6 (mg) Volume of 0.1 1.68 0.1 1.12 N HCl Derivatized
B29-acylated- B29-acylated B29-acylated- protein Gly (A21), Arg
(b31), human insulin desThr (B30)- Arg (B32)- human insulin human
insulin analog analog Derivatizing decanoyl 1,4- octanoyl group
dichlorophenyl- thio-acetyl Derivatized 2.23 5.1 6.06 protein mass
(mg) Volume of 0.1 0.1 0.3 0.3 N HCl mL of protein 0.3 + 0.1 0.1 +
0.3 0.1 + 0.3 solution combined with mL of derivatized protein
solution mL 1000 ppm 0.096 0.152 0.152 zinc mL diluent 1.6* 1.6 1.6
added Crystal shape rod-like small, irregular rod-like crystals
Yield (%) >90 >90 >90 Derivatized 20.4 71.8 74.2 protein
in insoluble phase (%) Dissolution 63 66 >300 time (min) *2.096
mL of 37.5 mg/100 mL protamine sulfate solution were added before
the diluent was added. The pH was adjusted after adding the
diluent.
PREPARATION 18
Preparation of an Amorphous Suspension
[0458] A measured mass (13.84 mg of protein) of solid
B28-tetradecanoyl-Lys(B28), Pro(B29) human insulin analog was
dissolved in 0.375 mL of 0.1 N HCl. A measured mass of zinc human
insulin (7.40 mg protein) was dissolved in 207 microliters of 0.1 N
HCl. An aliquot (125 .mu.L) of the insulin solution (containing
4.47 mg of human insulin) was added to the solution of
B28-tetradecanoyl-Lys(B28), Pro(B29)-human insulin analog. A volume
(180 .mu.L) of 1000 ppm zinc and 2.0 mL of diluent (1.6 mg/mL
phenol, 4 mg/mL m-cresol, 40 mg/mL glycerol, 5 mg/mL anhydrous
sodium dibasic phosphate, 7.5 mg/mL trisodium phosphate dihydrate,
pH 7.6) were added. The pH was increased from 5.6 to 8.0 with 100
microliters of 1N NaOH and back to 7.59 with 20 microliters of 1N
HCl and 1N NaOH. The concentration of B28-tetradecanoyl-Lys(B28),
Pro(B29) human insulin analog was 4.94 mg/mL and the human insulin
concentration was 1.60 mg/mL. The solution was passed through a
0.22 micron, low-protein binding filter and refrigerated overnight.
The next morning, the solution had no precipitate present. To 2.50
mL of the solution was added 2.88 mL of a protamine solution (0.75
mg/mL of solid protamine sulfate dissolved in water). An amorphous
precipitate formed when the protamine was added.
[0459] After adding the protamine, the concentration of
B28-tetradecanoyl-Lys(B28), Pro(B29) human insulin analog and human
insulin in the soluble phase was again determined. Samples for HPLC
analysis were prepared promptly after the protamine was added. From
peak retention times, HPLC analysis showed that the insoluble
material in the suspension contained protamine,
B28-tetradecanoyl-Lys(B28), Pro(B29) human insulin analog, and
human insulin. The concentration of B28.-tetradecanoyl-Lys(B28),
Pro(B29) human insulin analog in the soluble phase was 2.30 mg/mL
and the concentration of human insulin was 0.74 mg/mL.
[0460] The concentrations of B28-tetradecanoyl-Lys(B28), Pro(B29)
human insulin analog and human insulin in acidified samples of the
suspension, supernatant, and precipitate were determined and are
tabulated below. They are in reasonable agreement with expected
values. Protamine concentrations were not quantitated.
10TABLE 9 Preparation of insoluble compositions. Concentration
(mg/mL) B28-tetradecanoyl- Mass Lys (B28), Pro (B29) human human
Percent Sample insulin analog insulin (%) Suspension 2.52 0.79 76.2
Supernatant 0.006 0.057 8.9 Precipitate 2.29 0.60 79.2
PREPARATION 19
Preparation of Amorphous Suspensions
[0461] The following is an outline of another method used for
preparing precipitates of the present invention. The method was
used to prepare formulations of amorphous precipitates of insulin
with each of three derivatized proteins:
B29-N.epsilon.-octanoyl-human insulin;
B29-N.epsilon.-nonanoyl-human insulin; and
B28-N.epsilon.-octanoyl-LysB28- , ProB29-human insulin analog.
[0462] A measured mass of solid derivatized protein was dissolved
in 3 mL of 0.1 N HCl to produce a solution containing approximately
16 mg/mL derivatized protein. A measured mass of zinc human insulin
crystals (73 mg, of which 67.17 mg was protein) was dissolved in
4.198 mL of 0.1 N HCl to produce a solution containing
approximately 16 mg/mL insulin). Three milliliters of the solution
of derivatized protein and one milliliter of the insulin solution
were combined and thoroughly mixed. Measured volumes of a 1000 ppm
zinc solution (1.137 mL) and of a diluent (16 mL, containing, per
mL: 1.625 mg phenol, 4 mg m-cresol, 40 mg glycerol, 5 mg anhydrous
sodium dibasic phosphate, 7.5 mg trisodium citrate dihydrate, pH
7.6) were added. The pH was adjusted to about 7.6 (7.58-7.61) using
5 N NaOH and 5 N HCl solutions. The volume added during pH
adjustment was from 0.11 to 0.12 mL. The solution was passed
through a 0.22 micron, low-protein binding filter and refrigerated
overnight. The next morning, the solution had no precipitate
present. The solution was comprised of protein and derivatized
protein (approximately a 1:3 mass ratio), and the total protein
concentration was equivalent to about 85 units per milliliter. Just
prior to testing in rats, equal volumes of the solution and of a
solution of protamine sulfate (0.352 mg/mL) were combined and mixed
thoroughly. An amorphous precipitate formed immediately. A sample
of the suspension formulation containing the amorphous precipitate
was promptly injected into test animals. After mixing with
protamine, the concentration of total protein was about 42.4
units/ml.
PREPARATION 20
Gly(A21), Arg(B31), Arg(B32)-Human Insulin Analog
[0463] Gly(A21)Arg(B31)Arg(B32)-human insulin was obtained from an
E. coli fermentation in which a Gly(A21)-human proinsulin precursor
molecule was overexpressed into inclusion bodies. A portion (94.7
g) of inclusion bodies was solubilized in 500 mL of 6 M guanidine
hydrochloride containing 0.1 M TRIS, 0.27 M sodium sulfite, and 0.1
M sodium tetrathionate, pH 10.5 at room temperature. The pH was
quickly lowered to 8.8 with 12 N HCl. After vigorously stirring in
an open container for 45 minutes the pH was lowered to 2.1 with
phosphoric acid and the sample centrifuged overnight at 4.degree.
C. The supernatant was decanted and stored at 4.degree. C. for
additional processing. The pellet was re-extracted with 200 mL of
additional pH 10.5 solution (see above) and then centrifuged for 3
hours at 4.degree. C. This and the previously obtained supernatant
were each diluted 4.times.with 100 mM sodium phosphate, pH 4,
precipitating the product and other acidic components. After
allowing the precipitate to settle, most of the supernatant was
decanted and discarded. The resulting suspension was centrifuged,
followed by decanting and discarding of additional supernatant,
leaving wet pellets of the crude Gly(A21)-human proinsulin
S-sulfonate precursor. The pellets were solubilized in 1.5 liters
of 7 M deionized urea, adjusting the pH to 8 with 5 N NaOH and
stirring over several hours at 4.degree. C. Salt (NaCl) was then
added to achieve 1 M concentration and the sample was loaded onto a
XAD-7 column (14 cm.times.20 cm, Toso-Haas, Montgomeryville, Pa.),
previously flushed with 50% acetonitrile/50% 50 mM ammonium
bicarbonate, 10% acetonitrile/90% 50 mM ammonium bicarbonate, and
finally with 7 M deionized urea/1M NaCl/20 mM TRIS, pH 8. Once
loaded, the column was pumped with 4.5 liters of a 7 M deionized
urea/1 M NaCl/20 mM TRIS, pH 8 solution, followed by 2.8 liters of
50 mM ammonium bicarbonate/1 M NaCl, and 6.5 liters of 50 mM
ammonium bicarbonate. The column was eluted with a linear gradient
of acetonitrile in 50 mM ammonium bicarbonate, while monitoring the
eluant by UV at 280 nm. The peak of interest, partially purified
Gly(A21)-human proinsulin S-sulfonate precursor, was collected,
lyophilized, and subjected to a folding/disulfide bond procedure as
follows. A quantity (5.4 g) of the precursor was dissolved in 3
liters of 20 mM glycine, pH 10.5, 4.degree. C. Then, 15 mL of 240
mM cysteine HCl were added with stirring, while maintaining the pH
at 10.5 and the temperature at 4.degree. C. The reaction solution
was stirred gently at 4.degree. C. for 27 hours and then quenched
by lowering the pH to 3.1 with phosphoric acid. Acetonitrile (155
mL) was added, and the solution was then loaded onto a 5.times.25
cm C4 reversed-phase column previously pumped with 60%
acetonitrile/40% water/0.1% TFA and equilibrated in 10%
acetonitrile/90% water/0.1% TFA. Once loaded the column was pumped
with 1 liter of 17.5% acetonitrile/82.5% water/0.1% TFA, then
eluted with a linear gradient of acetonitrile in 0.1% TFA while
monitoring at 280 nm. Selected fractions were pooled and
lyophilized with a recovery of 714 mg. For conversion of the
proinsulin precursor to the desired insulin analog, 697 mg of the
Gly(A21) human proinsulin precursor were dissolved in 70 mL 50 mM
ammonium bicarbonate, then chilled to 4.degree. C., pH 8.3. A
volume (0.14 mL) of a 1 mg/mL solution of pork trypsin (Sigma
Chemical Company, St. Louis, Mo.) in 0.01 N HCl was added to the
sample solution which was stirred gently at 4.degree. C. for about
24 hours. An additional 0.14 mL of the trypsin solution was added
to the reaction solution which was then stirred for an additional
21 hours, 45 minutes. The reaction was quenched by lowering the pH
to 3.2 with 0.7 mL glacial acetic acid and 0.3 mL phosphoric acid.
The quenched Gly(A21)Arg(B31)Arg(B32)-human insulin sample solution
from the tryptic cleavage reaction was diluted 4.times.with 30%
acetonitrile/70% 50 mM acetic acid, pH 3.1, and loaded onto a
1.times.30 cm S HyperD F (Biosepra, Marlborough, Mass.) cation
exchange column previously pumped with 30% acetonitrile/70% 50 mM
acetic acid/500 mM NaCl, pH 3.3, and equilibrated in 30%
acetonitrile/70% 50 mM acetic acid. Once loaded the column was
pumped with about 50 mL of 30% acetonitrile/70% 50 mM acetic acid,
then eluted with a linear gradient of NaCl in 30% acetonitrile/50
mM acetic acid while monitoring the eluant at 276 nm. Selected
fractions containing the Gly(A21)Arg(B31)Arg(B32)-human insulin
were pooled, diluted 3.times.with purified water and loaded onto a
2.2.times.25 cm C4 reversed-phase column (Vydac, Hesperia, Calif.)
previously pumped with 60% acetonitrile/40% water/0.1% TFA, then
10% acetonitrile/90% water/0.1% TFA. Once loaded, the column was
pumped with about 200 mL of 10% acetonitrile/90% water/0.1% TFA,
then eluted with a linear gradient of acetonitrile in 0.1% TFA.
Selected fractions were pooled and lyophilized giving a recovery of
101 mg. Analytical HPLC revealed a purity of greater that 95% main
peak. Electrospray mass spectroscopy (ESMS) analysis of the
purified protein yielded a molecular weight of 6062.9 (6063.0,
theory).
PREPARATION 21
Des(B30)-Human Insulin
[0464] Des(B30)-human insulin was prepared from human proinsulin by
controlled tryptic hydrolysis. A mass (2 g) of human proinsulin
biosynthesized in recombinant E. coli and purified by conventional
methods [Frank, B. H., et al., in PEPTIDES:
Synthesis-Structure-Function. Proceedings of the Seventh American
Peptide Symposium, Rich, D. H. and Gross, E. (Eds.), Pierce
Chemical Company, Rockford, pp. 729-738, 1981; also, Frank, B. H.,
U.S. Pat. No. 4,430,266, issued Feb. 7, 1984, each of which is
incorporated by reference] were dissolved in 400 mL of 0.1 M, pH
7.5 HEPES buffer. After addition of 8 mL of 1 M CaCl.sub.2 (in
water) and pH adjustment to 7.5 with 5 N NaOH, 2 mL of a 10 mg/mL
solution of pork trypsin (Sigma) in 0.01 N HCl were transferred to
the sample solution while gently stirring. The reaction solution
was allowed to stir at ambient temperature for 2 hours and 42
minutes, at which time it was transferred to a 37.degree. C.
environment while stirring occasionally. After 1 hour and 45
minutes at 37.degree. C. the enzymatic reaction was quenched by
lowering the pH to 3.0 with phosphoric acid and the temperature to
4.degree. C. for storage. Subsequently, the solution was brought to
room temperature and diluted with 50 mL acetonitrile, then to a
final volume of 500 mL with purified water, then loaded onto a
2.5.times.58 cm CG-161 (Toso-Haas) column previously pumped with 1
c.v. (column volume) of 40% acetonitrile/60% 0.1 M ammonium
sulfate, pH 2.5, and 2 c.v. of 10% acetonitrile/90% 0.1 M ammonium
sulfate, pH 2.5. Once loaded, the column was pumped with 1 c.v. of
10% acetonitrile/90% 0.1 M ammonium sulfate, pH 2.5. The column was
eluted with a linear gradient of acetonitrile in 0.1 M ammonium
sulfate, pH 2.5, while monitoring the eluant at 276 nm. The peak of
interest, partially purified des(B30)-human insulin, was collected
by pooling selected fractions. This pooled sample of partially
purified des(B30)-human insulin was diluted to 1.28 liters with
purified water, pH 3.5, and applied to a 1.times.29 cm S HyperD F
(Biosepra) cation exchange column previously pumped with 1 c.v. of
30% acetonitrile/70% 0.1% TFA/0.5 M NaCl, pH 1.9, and 2 c.v. of 30%
acetonitrile/70% 0.1% TFA, pH 2.3. Once loaded the column was
pumped with 1 c.v. 30% acetonitrile/70% 0.1% TFA, pH 2.3, then
eluted with a linear gradient of NaCl in 30% acetonitrile/70% 0.1%
TFA, pH 1.9 to 2.3, while monitoring the eluant at 276 nm. Selected
fractions containing the purified des(B30)-human insulin were
pooled, diluted 2.5.times.with purified water and loaded onto a
35-c.c. C8 SepPak (Waters, Milford, Mass.) previously cleaned and
primed with 2 c.v. of acetonitrile, 2 c.v. of 60% acetonitrile/40%
0.1% TFA, and 2 c.v. of 10% acetonitrile/90% 0.1% TFA. Once loaded
the SepPak was flushed with 3 c.v. of 10% acetonitrile/90% 0.1% TFA
and then eluted with 2 c.v. of 60% acetonitrile/40% 0.1% TFA. The
lyophilized eluant yielded 500 mg. An analytical HPLC assay
suggested greater than 95% main peak. Electrospray mass
spectroscopy (ESMS) analysis of the purified protein yielded a
molecular weight of 5706.5 (5707, theory).
PREPARATION 22
Rabbit Insulin
[0465] Rabbit insulin was prepared as described in Chance, R. E.,
et al. [Proinsulin, Insulin, C-Peptide, Baba, S., et al. (Eds.),
Excezpta Medica, Amsterdam-Oxford, pp. 99-105 (1979)].
PREPARATION 23
Asp(B28)-Human Insulin Analog
[0466] Asp(B28)-human insulin was prepared and purified essentially
according to the teaching of examples 31 and 32 of Chance, R. E.,
et al. (U.S. Pat. No. 5,700,662, issued Dec. 23, 1997) which is
expressly incorporated herein by reference. Des(B23-30)-human
insulin [Bromer, W. W. and Chance, R. E., Biochim. Biophys. Acta,
133:219-223 (1967), which is incorporated herein by reference] and
a synthetic octapeptide Gly-Phe-Phe-Tyr-Thr-Asp-Lys(Tfa)-Thr were
condensed using trypsin-assisted semisynthesis, purified by gel
filtration and reversed-phased HPLC, treated with 15% ammonium
hydroxide (v/v) for four hours at ambient temperature to remove the
trifluoroacetate (Tfa) blocking group from Lys(B29), purified by
reversed-phase HPLC, and lyophilized.
PREPARATION 24
Syntheses of Derivatized Proteins
[0467] The following is an outline of the syntheses of certain
derivatized proteins used to prepare the precipitates and
microcrystals of the present invention. The outline is to be read
together with the data in Table 10, below.
[0468] A measured mass of purified insulin or of an insulin analog
was dissolved in a measured volume of dimethylsulfoxide (DMSO) with
stirring. Then, a measured volume of tetramethylguanidine
hydrochloride (TMG) was added and the solution mixed thoroughly. In
a separate container, a measured mass of an N-acyl-succinimide
(NAS) was dissolved in a measured volume of DMSO. A measured volume
of the second solution was added to the first solution. The
reaction was carried out at room temperature, and the progress of
the reaction was monitored by analyzing samples of the reaction
mixture using HPLC. The reaction was quenched by adding a measured
volume of ethanolamine, and then acidifying to pH 2-3.
[0469] The reaction mixture was then subjected to purification
using reversed-phase chromatography alone, or using a combination
of cation exchange chromatography followed by reversed-phase
chromatography. The reversed-phase purification was carried out
using an FPLC.RTM. system (Pharmacia) with UV detection at 214 nm
or at 280 nm, a fraction collector, 2.2.times.25 cm or 5.times.30
cm C18 column, 2.5 or 5 mL/min flow rate, at room temperature. The
liquid phases were mixtures of Solution A [0.1% trifluroacetic acid
(TFA) in 10:90 acetonitrile:water (vol:vol)] and Solution B [0.1%
trifluroacetic acid (TFA) in 70:30 acetonitrile:water (vol:vol)]
appropriate to elute and separate the species of interest.
Typically, the column was equilibrated and loaded while in 100%
Solution A. Then, a linear gradient to some proportion of Solution
B was used to separate the reaction products adequately. Fractions
containing product were pooled. The development of purification
methods is within the skill of the art.
[0470] Table 10 below provides experimental data, according to the
outline above, for the synthesis of the derivatized proteins that
were used to prepare various embodiments of the present invention.
The starting proteins were prepared as described above, or
according to conventional methods. Conventional purification was
used to provide highly purified starting proteins for the syntheses
described below. The synthesis of insulin, insulin analogs, and
proinsulin is within the skill of the art, and may be accomplished
using recombinant expression, semisythesis, or solid phase
synthesis followed by chain combination. The purification of
synthesized proteins to a purity adequate to prepare the
derivatives used in the present invention is carried out by
conventional purification techniques.
[0471] Molecular weight of the purified derivatives was confirmed
by mass spectrometry via electrospray mass analysis (ESMS).
Assignment of the acylation site was based either on a
chromatographic analysis ("HPLC"), or on an N-terminal analysis
("N-terminal"), or both.
11TABLE 10 Summary of synthesis of various derivatized proteins.
Starting protein human insulin human insulin human insulin protein
mass (mg) 141.3 1,080 120 DMSO (mL) 42 30 36 TMG (.mu.L) 30.5 233
25.9 NAS acyl chain n-hexanoyl n-octanoyl n-dodecanoyl Mass of NAS
(mg) 7.76 85.7 9.22 Volume of DMSO 1.0 1.0 0.701 (mL) Volume of NAS
0.494 0.785 0.701 solution added (mL) Reaction time (min) 40 105 40
Ethanolamine volume 20 100 120 (.mu.L) Total yield (%) 40 33 36
Mol. Wt. (theory) 5906.0 5933.9 5990.0 Mol. Wt. (ESMS) 5906.8
5933.9 5990.0 HPLC Purity (%) 96 94 98 Acylation site N.epsilon.
N.epsilon. N.epsilon. (HPLC) Acylation site N.epsilon. N.epsilon.
N.epsilon. (N-terminal) protein mass (mg) 194 2040 2050 DMSO (mL)
60 62 58 TMG (.mu.L) 41.9 441 443 NAS acyl chain n-tetradecanoyl
n-butyryl n-hexanoyl Mass of NAS (mg) 23.4 269.3 209 Volume of DMSO
1.0 1.0 2.0 (mL) Volume of NAS 0.756 0.29 1.44 solution added (mL)
Reaction time (min) 20 30 30 Ethanolamine volume 5 100 100 (.mu.L)
Total yield (%) 45 27* 22 Mol. Wt. (theory) 6018.1 5877.8 5905.9
Mol. Wt. (ESMS) 6018.2 5877.8 5906.0 HPLC Purity (%) 98 94 93
Acylation site N.epsilon. N.epsilon. N.epsilon. (HPLC) Acylation
site N.epsilon. -- -- (N-terminal) *purification involved first
reversed-phase HPLC, then cation exchange HPLC, then reversed-phase
HPLC
[0472] The following is an outline of the synthesis of additional
derivatized proteins. The outline is to be read together with the
data in Table 11, below, to provide full synthetic schemes.
[0473] A measured mass of purified insulin or of an insulin analog
was dissolved by adding to it a measured volume of 50 mM boric
acid, pH 2.57. A measured volume of acetonitrile, equal to the
volume of boric acid solution, was then added slowly with stirring.
The "solvent" volume is the sum of the volumes of the boric acid
and acetonitrile. The pH of the solution was adjusted to between
10.2 and 10.5 using NaOH. In a separate container, a measured mass
of an N-acyl-succinimide ("NAS") was dissolved in a measured volume
of DMSO. A measured volume of the second solution was added to the
first solution. The reaction was carried out at room temperature,
the pH was maintained above 10.2 as necessary, and the progress of
the reaction was monitored by analyzing samples of the reaction
mixture using HPLC. The reaction was quenched by acidifying to pH
2-3. The reaction mixture was then subjected to purification using
a reversed-phase chromatography system as described above.
[0474] Table 11 provides experimental data, according to the
outline above, for the synthesis of the derivatized proteins that
were used to prepare various embodiments of the present invention.
Molecular weight of the purified derivatives was confirmed by mass
spectrometry via electrospray mass analysis (ESMS). Assignment of
the acylation site was based either on a chromatographic analysis
("HPLC"), or on an N-terminal analysis ("N-terminal"), or both.
12TABLE 11 Summary of synthesis of various derivatized proteins.
Starting protein human insulin human insulin human insulin protein
mass (mg) 2,170 2,420 2,250 solvent (mL) 200 240 200 NAS acyl chain
n-butyryl n-pentanoyl n-octanoyl Mass of N-acyl-succinimide 108.7
1155 173 (mg) Volume of DMSO (mL) 1.0 5 1.0 Volume of NAS 0.955
0.719 0.81 solution added (mL) Reaction time (min) 40 40 40 Total
yield (%) 25 12 37 Mol. Wt. (theory) 5877.8 5891.8 5933.9 Mol. Wt.
(ESMS) 5877.7 5891.9 5933.8 HPLC Purity (%) 96 95 96 Acylation site
N.epsilon. N.epsilon. N.epsilon. (HPLC) protein mass (mg) 1,960
2,750 1,040 solvent (mL) 200 200 200 NAS acyl chain n-nonanoyl
n-dodecanoyl n-tetradecanoyl Mass ot N-acyl-succinimide 145.8 19.9
102.3 (mg) Volume of DMSO (mL) 1.0 1.0 1.0 Volume of NAS 0.887
0.771 0.885 solution added (mL) Reaction time (min) 40 30 35 Total
yield (%) 35 14 39 Mol. Wt. (theory) 5947.9 5990.0 6018.1 Mol. Wt.
(ESMS) 5948.1 5989.9 6018.1 HPLC purity (%) 94 93 94 Acylation site
N.epsilon. N.epsilon. N.epsilon. (HPLC) sheep Starting protein
insulin beef insulin pork insulin protein mass 312 275 200 solvent
(mL) 100 100 100 NAS acyl chain n-hexanoyl n-hexanoyl n-octanoyl
Mass of N-acyl-succinimide 27.2 19.9 16.4 (mg) Volume of DMSO (mL)
1.0 1.0 1.0 Volume of NAS 0.644 0.771 0.764 solution added (mL)
Reaction time (min) 45 30 82 Total yield (%) 31 50 41 Mol. Wt.
(theory) 5801.7 5831.8 5903.9 Mol. Wt. (ms) 5801.8 5831.7 5903.9
HPLC Purity (%) 96 96 96 Acylation site N.epsilon. N.epsilon.
N.epsilon. (HPLC) rabbit des (B30)-human AspB28-human Starting
protein insulin insulin insulin Protein mass (mg) 211.4 205.3 132.3
Solvent (mL) 100 20 20 NAS acyl chain n-octanoyl n-octanoyl
n-octanoyl Mass of N-acyl-succinimide 16.8 21.5 11.5 (mg) Volume of
DMSO (mL) 1.0 0.5 1.0 Volume of NAS 0.786 0.303 0.715 solution
added (mL) Reaction time (min) 57 40 85 Total yield (%) 39 47 32
Mol. Wt. (theory) 5919.9 5833.6 5951.9 Mol. Wt. (ms) 5920.0 5832.7
5952.2 HPLC Purity (%) 95 96 94 Acylation site N.epsilon.
N.epsilon. N.epsilon. (HPLC) GlyA21, ArgB31, ArgB32-human des
(B27)- insulin human insulin Starting protein analog human insulin
analog Protein mass (mg) 86.2 134.8 44.8 Solvent (mL) 10 20 7 NAS
acyl chain n-octanoyl 2-methyl-hexanoyl n-octanoyl Mass of
N-acyl-succinimide 22.4 749 3.6 (mg) Volume of DMSO (mL) 0.5 4.93*
1.0 Volume of NAS 0.115 0.052 0.993 solution added (mL) Reaction
time (min) 40 45 40 Total yield (%) 45 45 53 Mol. Wt. (theory)
6189.2 5919.9 5832.8 Mol. Wt. (ms) 6189.2 5919.9 5832.9 HPLC Purity
(%) 97 96 93 Acylation site N.epsilon. N.epsilon. N.epsilon. (HPLC)
Starting protein human insulin human insulin human insulin Protein
mass (mg) 160 147.7 2.080 Solvent (mL) 20 20 200 NAS acyl chain
4-methyl-octanoyl 3-methyl-decanoyl n-octanoyl Mass of
N-acyl-succinimide 715 22.5 146.7 (mg) Volume of DMSO (mL) 4.97*
1.0* 1.0* Volume of NAS 0.0734 0.609 0.884 solution added (mL)
Reaction time (min) 60 45 40 Total yield (%) 54 38 5.3** Mol. Wt.
(theory) 5947.9 5976.0 6060.1 Mol. Wt. (ms)*** 5947.8 5976.2 6060.5
HPLC Purity (%) 96 96 92 Acylation site N.epsilon. N.epsilon.
A1-N.alpha., N.epsilon. (HPLC) des (B30) - human insulin Starting
protein analog human insulin human insulin Protein mass (mg) 205.3
1.960 2.110 Solvent (mL) 20 200 200 NAS acyl chain n-octanoyl
n-nonanoyl n-decanoyl Mass of N-acyl-succinimide 21.5 145.8 150.5
(mg) Volume of DMSO (mL) 0.5 1.0 1.0 Volume of NAS 0.089 0.0887
0.975 solution added (mL) Reaction time (min) 40 40 60 Total yield
(%) 11.0 11.5 11.1 Mol. Wt. (theory) 5959.5 6088.2 6116.2 Mol. Wt.
(ms) 5959.3 6088.3 6116.4 HPLC Purity (%) 96 92 92 Acylation site
A1-N.alpha., N.epsilon. A1-N.alpha., N.epsilon. A1-N.alpha.,
N.epsilon. (HPLC) *Dissolved in acetonitrile instead of DMSO.
**Yield of the A1-N.alpha., B29-N.epsilon.-diacyl-human insulin
derivative ***Determined by Matrix-Assisted Laser Desorption
Ionization (MALDI) mass spectroscopy instead of electrospray mass
spectroscopy
[0475] The following is a general outline of a synthetic scheme to
produce additional derivatized proteins. In a specific instance,
the outline is to be read together with the data in Table 12,
below, to a provide full synthetic scheme for a particular
derivatized protein. A measured mass of purified insulin or insulin
analog was dissolved by adding to it a measured volume DMSO. The pH
of the solution was adjusted with 10 equivalents of
tetramethylguanidine. In a separate container, a measured mass of
an N-acyl-succinimide ("NAS") was dissolved in a measured volume of
DMSO. A measured volume of the second solution was added to the
first solution to provide a 1.9 fold molar excess of
N-acyl-succinimide. The reaction was carried out at room
temperature and the progress of the reaction was monitored by
analyzing samples of the reaction mixture using HPLC. The reaction
was quenched with 20 microliters of ethanolamine, chilled in
ice/water bath and diluted 2.1 times with 0.1N HCl. The reaction
mixture was then subjected to desalting on reversed phase
chromatography column using the following protocol: 1) the column
was wetted with 100% acetonitrile, then was washed using three to
four column volumes of 0.1% TFA/70% acetonitrile (Buffer B); and
finally was washed using four to five column volumes of 0.1%
TFA/10% acetonitrile (Buffer A); 2) diluted reaction mixtures were
loaded, and the column was again washed with five to six column
volumes of Buffer A; and 3) the derivatized protein was eluted by
passing five to six column volumes of Buffer B through the column.
The fluid collected during elution was frozen, then lyophilized.
The lyophilized crude product (86.1 mg) was then subjected to
re-purification using a reversed-phase chromatography system as
described above.
[0476] Table 12 provides experimental data, according to the
outline above, for the synthesis of the derivatized proteins that
were used to prepare various embodiments of the present invention.
Molecular weight of the purified derivatives was confirmed by mass
spectrometry via electrospray mass analysis (ESMS). Assignment of
the acylation site was based either on a chromatographic analysis
("HPLC").
13TABLE 12 Summary of synthesis of various derivatized proteins.
Starting protein Human Insulin protein mass (mg) 179 DMSO to
dissolve 20 human insulin (mL) NAS acyl chain 1,4-
dichlorophenylthio- acetyl Mass of N-acyl-succinimide 30 (mg)
Volume of DMSO 1.0 (mL) Volume of NAS 0.412 solution added (mL)
Reaction time 50 (min) Total yield (%) 32.2* Mol. Wt. (theory)
6026.78 Mol. Wt. (ESMS) 6026.9 HPLC Purity (%) 96 Acylation site
N.epsilon. (HPLC) *Yield calculation based on the desalted and
lyophilized crude product weight.
EXPERIMENT 1
Time Action of Co-crystals in Dogs
[0477] The time-action of three co-crystal compositions of the
present invention was determined in normal dogs that received a
constant infusion of somatostatin to create a transient diabetic
state. The first co-crystal formulation, comprising human insulin
and B29-N.epsilon.-octanoyl-human insulin, was prepared essentially
as described for Formulation D in Preparation 16 above, and was
administered subcutaneously at a dose of 3 nmol/kg ("8753"). The
second co-crystal formulation, comprising human insulin and
B29-N.epsilon.-octanoyl-human insulin, was prepared as described
for Formulation D in Preparation 16 above, and was administered
subcutaneously at a dose of 2.5 nmol/kg (%8752.5n). Finally, a
third co-crystal formulation, comprising human insulin and
B29-N.epsilon.-decanoyl-human insulin, was prepared as described in
Preparation 14 above, and was administered subcutaneously at a dose
of 2.5 nmol/kg ("0252.5"). The data were compared to that observed
in the same model after administration of Humulin N (2.0 nmol/kg
"NPH"), Beef/Pork Ultratlente insulin (3 nmol/kg, "BP-UL"), and
saline.
[0478] Experiments were conducted in overnight-fasted, chronically
cannulated, conscious male and female beagles weighing 10-17 kg
(Marshall Farms, North Rose, N.Y.). At least ten days prior to the
study, animals were anesthetized with isoflurane (Anaquest,
Madison, Wis.), and silicone catheters attached to vascular access
ports (V-A-P.TM., Access Technologies, Norfolk Medical, Skokie,
Ill.) were inserted into the femoral artery and femoral vein. The
catheters were filled with a glycerol/heparin solution (3:1, v/v;
final heparin concentration of 250 KIU/ml; glycerol from Sigma
Chemical Co., St. Louis, Mo., and heparin from Elkins-Sinn, Inc.,
Cherry Hill, N.J.) to prevent catheter occlusion, and the wounds
were closed. Kefzol (Eli Lilly & Co., Indianapolis, Ind.) was
administered pre-operatively (20 mg/kg, IV and 20 mg/kg, I.M.), and
Keflex was administered post-operatively (250 mg, p.o. once daily
for seven days) to prevent infections. Torbugesic (1.5 mg/kg, I.M.)
was administered post-operatively to control pain.
[0479] Blood was drawn just prior to the study day to determine the
health of the animal. Only animals with hematocrits above 38% and
leukocyte counts below 16,000/mm.sup.3 were used (hematology
analyzer: Cell-Dyn 900, Sequoia-Turner, Mountain view, Calif.).
[0480] The morning of the experiment, the ports were accessed
(Access Technologies, Norfolk Medical, Skokie, Ill.); the contents
of the catheters were aspirated; the catheters were flushed with
saline (Baxter Healthcare Corp., Deerfield, Ill.); the dog was
placed in a cage; and extension lines (protected by a stainless
steel tether and attached to a swivel system [Instech Laboratories,
Plymouth Meeting, Pa.]) were attached to the port access lines.
[0481] Dogs were allowed at least 10 minutes to acclimate to the
cage environment before an arterial blood sample was drawn for
determination of fasting insulin, glucose, and glucagon
concentrations (time=-30 minutes). At this time, a continuous, IV
infusion of cyclic somatostatin (0.65 .mu.g/kg/min; BACHEM
California, Torrance, Calif.) was initiated and continued for the
next 30.5 hours. Thirty minutes after the'start of infusion (time=0
minutes), an arterial blood sample was drawn, and a subcutaneous
bolus of test substance, or vehicle, was injected in the dorsal
aspect of the neck. Arterial blood samples were taken every 3 hours
thereafter for the determination of plasma glucose and insulin
concentrations and every 6 hours for determination of plasma
glucagon concentrations. The entire study lasted 30 hours.
[0482] Arterial blood samples were collected in vacuum blood
collection tubes containing disodium EDTA (Terumo Medical Corp.,
Elkton, Md.) and immediately placed on ice. A portion of the blood
sample (1.5 ml) was transferred to a polypropylene tube containing
40 .mu.l of aprotinin (10,000 KIU/ml; Trasylol, Miles, Inc.,
Diagnostics Division, Kankakee, Ill.) in preparation for the
determination of the plasma glucagon concentration. The samples
were centrifuged, and the resulting plasma was transferred to
polypropylene test tubes and stored on ice for the duration of the
study.
[0483] Plasma glucose concentrations were determined the day of the
study using a glucose oxidase with a commercial glucose analyzer.
Samples for other assays were stored at -80.degree. C. until time
for analysis. Insulin concentrations were determined using a double
antibody radioimmunoassay. Glucagon concentrations were determined
using a radioimmunoassay kit (LINCO Research, Inc., St. Charles,
Mo.).
[0484] At the conclusion of the experiment, the catheters were
flushed with fresh saline, treated with Kefzol (20 mg/kg), and
filled with the glycerol/heparin mixture; antibiotic (Keflex; 250
mg) was administered p.o. To minimize the number of animals being
used and to allow pairing of the data base when possible, animals
were studied multiple times. Experiments in animals being restudied
were carried out a minimum of one week apart.
[0485] The 1:3 co-crystal of human insulin and
B29-N.epsilon.-octanoyl-hum- an insulin had a time-action 9 hours
longer than NPH human insulin (24 hours vs. 15 hours) for the
higher dose (8753), and a time-action 6 hours longer than NPH human
insulin (21 hours vs. 15 hours) for the lower dose (8752.5). The
time-action was determined by statistically comparing the mean
glucose levels with those of the control group (saline). The
glucose profiles for the 1:3 co-crystal formulations of human
insulin and B29-N.epsilon.-octanoyl-human insulin were more like
that expected of a basal insulin than was the profile for NPH-human
insulin. The 1:3 co-crystals also had greater activity and a more
desirable glucose profile than did Beef/Pork Ultralente insulin.
The reduction in blood glucose, compared with the control (saline),
that the co-crystal formulations caused persisted longer than that
caused by this Beef/Pork Ultralente insulin.
[0486] The 3:1 co-crystal formulation of human insulin and
B29-N.epsilon.-decanoyl-human insulin (10252.5) had a time-action 9
hours longer than NPH human insulin (24 hours vs. 15 hours). The
difference was significant statistically (p<0.05). In the same
animals, the 3:1 co-crystal formulation of human insulin and
B29-N.epsilon.-decanoyl-human insulin (2.5 nmol/kg, SC) had a
time-action 6 hours longer than either Humulin U or Beef U
preparations (24 hours vs. 18 hours). The differences were also
significant statistically (p<0.05). The glucose profile for the
co-crystal formulations was more like that expected of a basal
insulin than was the profile for NPH-human insulin. Furthermore,
the variability in time-action among the dogs was the least when
the 3:1 co-crystal was administered.
[0487] In conclusion, these data demonstrate that the co-crystals
of the present invention are effective for controlling glucose
levels for protracted periods of time in dogs. They also support a
conclusion that the co-crystals of the present invention will be
effective for overnight glucose control of patients with type 2
diabetes or as the basal arm of basal/bolus insulin therapy for
patients with type 1 or type 2 diabetes. They also suggest that
these preparations may produce less variable responses than
commercially available insulin preparations.
EXPERIMENT 2
Time Action of Co-Crystals in Pigs
[0488] Studies were performed on normal, conscious female pigs
weighing 17-25 kg. An arterial (carotid or femoral) catheter was
surgically pre-implanted for sampling along with jugular venous
lines for the administration of somatostatin. Prior to experiments,
the cannulated pigs were fasted 22-24 hours. Subcutaneous insulin
injections were given in the soft skin behind the ear at a dosage
of 3.0 nmol/kg (0.5 unit/kg). Somatostatin was administered
concurrently at 0.3 .mu.g/kg/min (dissolved in 0.9% NaCl containing
1% human serum albumin, Miles Canada, Etobicoke, ON) to suppress
endogenous insulin secretion. Near normoglycemia was maintained by
infusing 20% dextrose at a variable rate, with frequent monitoring
of glucose concentrations. The plasma glucose levels were
determined on fresh plasma samples the day of the study using a
glucose oxidase method with a commercial glucose analyzer.
[0489] In a euglycemic clamp study, a formulation of the present
microcrystals comprised of 1:3
insulin:B29-N.epsilon.-octanoyl-human insulin was administered
subcutaneously at a dose of 0.5 U/kg (equivalent to about 3
nmol/kg) at the start of the study (time 0) to five pigs. The rate
of glucose infusion required to maintain euglycemia (set
point=about 90 mg/dL) was determined continuously. A control group
received Humulin N (U100) by subcutaneous administration at the
same dose (n=6). A concomitant infusion of somatostatin (0.3
.mu.g/kg/min) was maintained for the entire duration of the
experiment. For the microcrystals of the present invention, the
glucose infusion rate increased steadily over the first two hours,
reaching a maximum of about 7 mg/kg/min. From then, until about
17.5 hours, the glucose infusion rate decreased fairly steadily to
about 0.5 mg/kg/min. For most of the time between 17.5 hours and
the end of the study at 24 hours, the glucose infusion rate
remained between about 0.5 and about 2 mg/kg/min.
[0490] By contrast, the mean glucose infusion rate in the control
group (Humulin NPH) increased steadily, reaching a maximum of about
14 mg/kg/min at about 3 hours after administration. Thereafter, the
infusion rate decreased to about 7 mg/kg/min by about 4.5 hours,
and to about 5 mg/kg/min by 13 hours after administration. No
further data were taken for the control group. These results are
consistent with a conclusion that the microcrystalline formulation
comprised of insulin and B29-N.epsilon.-octanoyl-human insulin in a
1:3 molar ratio has a flatter glucodynamic profile than does
insulin NPH.
EXPERIMENT 3
Time Action of Co-Crystals in Rats
[0491] A formulation of the present microcrystals comprised of 1:3
insulin:B29-N.epsilon.-octanoyl-human insulin was tested in
BBDP/Wor rats, a genetically-characterized animal model, maintained
by, and available from, the University of Massachusetts Medical
Center (Worchester, Mass.) in connection with Biomedical Research
Models, Inc. (Rutland, Mass.). The DPBB/Wor rat line is
diabetes-prone, and exhibits insulin-dependent (autoimmune)
diabetes mellitus. All preparations were administered
subcutaneously at a dose of 0.9 U/100 g body weight.
[0492] Male BBDP/Wor rats, aged 4-5 months and maintained on a
long-acting insulin (PZI), were randomly assigned to five
experimental groups, A, B, C, D and E. Group A (n=22) was treated
for three days with a U40 human insulin ultralente (Humulin UL);
group B (n=18) was treated for 3 days with a U40 preparation of
Iletin Ultralente (65% beef insulin, 35% pork insulin); group C
(n=10) was treated for three days with a formulation of
microcrystals comprised of 1:3
insulin:B29-N.epsilon.-octanoyl-human insulin, prepared as
described above; group D (n=21) was treated with a formulation of
microcrystals comprised of 100% B29-N.epsilon.-octanoyl-hu- man
insulin; and group E received U40 beef-pork PZI insulin (PZI). Each
rat was given daily injections of its group's formulation for the
two days before blood glucose was determined, and on the day that
the blood glucose was determined.
[0493] Blood was obtained half an hour before administering the
test formulations. Samples of the formulations were injected at
11:30 A.M. Blood was obtained by nicking the tail (not
anaesthetized). The samples were stored briefly on ice, then were
centrifuged, and glucose determined using a Beckman II glucose
analyzer. Blood samples were obtained just prior to administering
the test formulations, and at 2, 4, 6, 8, 12, 16, 20, and 24 hours
after administration. Considering adequate control to be indicated
by blood glucose levels less than 200 mg/dL, the preparations
provided about 9.5 hours (Humulin UL), about 12 hours (Iletin U),
about 15.5 hours (the present invention), about 20.5 hours (100%
B29-N.epsilon.-octanoyl-human insulin), and about 21.5 hours (PZI)
of control. Therefore, the microcrystal formulation of the present
invention controlled blood glucose longer than both Humulin UL and
Iletin U and for a shorter period of time than did either the 100%
B29-N.epsilon.-octanoyl- -human insulin microcrystal preparation or
the PZI preparation.
EXPERIMENT 4
Time Actions of Amorphous Precipitates in Rats
[0494] Formulations of amorphous precipitates comprised of 1:3
insulin:B29-N.epsilon.-octanoyl-human insulin, 1:3
insulin:B28-N.epsilon.-octanoyl-Lys(B28), Pro(B29)-human insulin,
and 1:3 insulin:B29-N.epsilon.-nonanoyl-human insulin, prepared as
described in Preparation 19, were tested in BBDP/Wor rats. All
preparations were administered subcutaneously at a dose of 0.9
U/100 g body weight.
[0495] Male BBDP/Wor rats, aged 4-5 months and maintained on a
long-acting insulin (PZI), were randomly assigned to five
experimental groups, A, B, C, D, and E. Group A (n=7) was treated
with a preparation of U40 NPH (Humulin N). Group B (n=8) was
treated with a U42.4 preparation of 1:3
insulin:B29-N.epsilon.-octanoyl-human insulin. Group C (n=8) was
treated with a U42.6 preparation of 1:3
insulin:B28-N.epsilon.-octanoyl-Lys(B28), Pro(B29)-human insulin.
Group D(n=8) was treated with a U42.7 preparation of 1:3
insulin:B29-N.epsilon.-nonanoyl-human insulin. Group E was treated
with U40 beef-pork PZI insulin (PZI).
[0496] Blood was obtained half an hour before administering the
test formulations. Animals were injected subcutaneously (0.9 U/100
g body weight) at 11:30 A.M. Blood was obtained by nicking the tail
(not anaesthetized). The samples were stored briefly on ice, then
centrifuged, and glucose was determined using a commercial glucose
analyzer. Blood samples were obtained just prior to administering
the test formulations, and at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, and 24 hours after administration. Considering adequate control
to be indicated by blood glucose levels less than 200 mg/dL, the
preparations provided about 7.5 hours (1:3
insulin:B28-N.epsilon.-octanoyl-Lys(B28), Pro(B29)-human insulin),
about 9 hours (NPH), about 15.5 hours (1:3
insulin:B29-N.epsilon.-octanoyl-human insulin), about 16 hours (1:3
insulin:B29-N.epsilon.-nonanoyl-human insulin) and about 22.5 hours
(PZI) of control.
[0497] The principles, preferred embodiments and modes of operation
of the present invention have been described in the foregoing
specification. The invention which is intended to be protected
herein, however, is not to be construed as limited to the
particular forms disclosed, since they are to be regarded as
illustrative rather than restrictive. Variations and changes may be
made by those skilled in the art without departing from the spirit
of the invention.
* * * * *