U.S. patent application number 15/728012 was filed with the patent office on 2018-06-07 for stable glucagon peptide formulations.
The applicant listed for this patent is ZP OPCO, INC.. Invention is credited to Mahmoud Ameri, Yi Ao, Peter E. Daddona.
Application Number | 20180153798 15/728012 |
Document ID | / |
Family ID | 55347309 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180153798 |
Kind Code |
A1 |
Ameri; Mahmoud ; et
al. |
June 7, 2018 |
Stable Glucagon Peptide Formulations
Abstract
There is provided glucagon formulations suitable for preparing
coatings on microneedle patches for the transdermal delivery of
glucagon. The coated patches may be used for the treatment of low
blood sugar in diabetic patients. Also provided are glucagon loaded
patches, methods for their preparation, and methods of their
use.
Inventors: |
Ameri; Mahmoud; (Fremont,
CA) ; Daddona; Peter E.; (Menlo Park, CA) ;
Ao; Yi; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZP OPCO, INC. |
FREMONT |
CA |
US |
|
|
Family ID: |
55347309 |
Appl. No.: |
15/728012 |
Filed: |
October 9, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14930041 |
Nov 2, 2015 |
9782344 |
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15728012 |
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14466461 |
Aug 22, 2014 |
9173924 |
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14930041 |
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61868969 |
Aug 22, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/26 20130101;
A61M 37/0015 20130101; A61M 2037/0023 20130101; A61M 2037/0061
20130101; A61K 47/24 20130101; A61M 2037/0046 20130101; A61K 38/26
20130101; A61K 9/7023 20130101; A61K 47/183 20130101; A61M
2037/0053 20130101; A61K 9/0021 20130101; A61K 47/12 20130101; A61K
47/38 20130101; A61K 9/19 20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 9/19 20060101 A61K009/19; A61K 38/26 20060101
A61K038/26; A61K 47/12 20060101 A61K047/12; A61K 47/18 20060101
A61K047/18; A61K 47/24 20060101 A61K047/24; A61K 47/26 20060101
A61K047/26; A61K 47/38 20060101 A61K047/38; A61M 37/00 20060101
A61M037/00 |
Claims
1-29. (canceled)
30. A liquid pharmaceutical formulation, comprising glucagon or a
glucagon-like peptide and a stabilizing agent wherein the
stabilizing agent is present in an amount down to 8 fold less, by
weight, than the amount of glucagon or glucagon-like peptide.
31. The liquid pharmaceutical formulation of claim 30, further
comprising an amino acid, an organic acid, and a pharmaceutically
acceptable diluent.
32. The liquid pharmaceutical formulation of claim 30, wherein the
formulation has a pH which is not between about 4 and about 8.
33. The liquid pharmaceutical formulation of claim 30, wherein the
formulation has a pH between about 2.8 and about 3.2.
34. The liquid pharmaceutical formulation of claim 30, wherein the
stabilizing agent is a cationic surfactant or a neutral
surfactant.
35. The liquid pharmaceutical formulation of claim 34, wherein the
stabilizing agent is selected from the group consisting of
lyso-myristoyl phosphatidylcholine (LMPC), glucose, sucrose,
trehalose, dextrose, a derivative thereof substituted with a C8-C12
alkyl chain, and a combination of any of the foregoing.
36. The liquid pharmaceutical formulation of claim 31, wherein
glucagon or glucagon like peptide is present in amount of about
15-20% (w/w), wherein the stabilizing agent is present in an amount
of about 7.5-10% (w/w), wherein the amino acid is present in an
amount of about 3.75-5% (w/w) and the organic acid is present in an
amount of about 3.75-5% (w/w).
37. The liquid pharmaceutical formulation of claim 31, further
comprising a carboxymethyl cellulose.
38. The liquid pharmaceutical formulation of claim 31, wherein: a.
the amino acid is glutamine or glycine, b. the stabilizing agent is
selected from the group consisting of lyso-myristoyl
phosphatidylcholine (LMPC), glucose, sucrose, trehalose, dextrose,
a derivative thereof substituted with a C8-C12 alkyl chain, and a
combination of any of the foregoing, and c. the organic acid is
selected from the group consisting of methanoic acid, ethanoic
acid, tartaric acid, malonic acid, glycolic acid, malic acid,
gluconic acid, citric acid, caproic acid, benzoic acid, lactic
acid, propionic acid, sorbic acid, and a combination of any of the
foregoing.
39. The liquid pharmaceutical formulation of claim 38, further
comprising sodium carboxymethyl cellulose in a concentration from
about 0.1 mg/mL to about 1.0 mg/mL.
40. The liquid pharmaceutical formulation of claim 39, comprising
about 200 mg/mL glucagon, about 100 mg/mL LMPC, about 50 mg/mL
glutamine, and about 50 mg/mL tartaric acid.
41. The liquid pharmaceutical formulation of claim 39, comprising
about 200 mg/mL glucagon, about 100 mg/mL decanoyl sucrose (DS),
about 50 mg/mL glutamine, and about 50 mg/mL tartaric acid.
42. A pharmaceutical formulation, comprising: a. glucagon or a
glucagon-like peptide; b. a stabilizing agent selected from either
a cationic or neutral surfactant; c. an amino acid; and d. an
organic acid, wherein the amount of the cationic or neutral
surfactant is present in an amount which is greater than an amount
which is 8 fold less by weight than the amount of glucagon or
glucagon-like peptide.
43. The pharmaceutical formulation of claim 42 further comprising a
carboxymethyl cellulose.
44. The pharmaceutical formulation of claim 43, wherein the
carboxymethyl cellulose is sodium carboxymethyl cellulose.
45. The pharmaceutical formulation of claim 44, where the
formulation is a liquid formulation and the sodium carboxymethyl
cellulose is present in a concentration of from about 0.1 mg/ml to
about 1 mg/ml.
46. The pharmaceutical formulation of claim 44, wherein the sodium
carboxymethyl cellulose is present in a concentration of about 0.5
mg/ml.
47. The pharmaceutical formulation of claim 42, further comprising
trehalose.
48. The pharmaceutical composition of claim 47, wherein the
formulation is a liquid formulation, and the trehalose is present
in an amount of about 50 mg/ml.
49. The pharmaceutical formulation of claim 42, wherein the
surfactant is a phospholipid.
50. The pharmaceutical formulation of claim 49, wherein the
phospholipid is lyso-myristoyl phosphatidylcholine.
51. The pharmaceutical formulation of claim 42, wherein the
surfactant is decanoyl sucrose.
52. The pharmaceutical formulation of claim 42, wherein the amino
acid is selected from the group consisting of glutamine and
glycine.
53. The pharmaceutical formulation of claim 42, wherein the organic
acid is selected from the group consisting of methanoic acid,
ethanoic acid, tartaric acid, malonic acid, glycolic acid, malic
acid, gluconic acid, citric acid, caproic acid, benzoic acid,
lactic acid, propionic acid, and sorbic acid.
54. The pharmaceutical formulation of claim 42, wherein the organic
acid is tartaric acid.
55. The formulation of claim 42 wherein the formulation is a liquid
formulation comprising a pharmaceutically acceptable diluent, and
wherein the formulation has a pH which is not between 4 and 8.
56. The formulation of claim 55, comprising: a. 15-20% (w/w) of the
glucagon or glucagon like peptide; b. 7.5-10% (w/w) of the cationic
or neutral surfactant; c. 3.75-5% (w/w) of the amino acid; and d.
3.75-5% (w/w) of the organic acid.
57. The formulation of claim 56, wherein the pH of the liquid
formulation is between 2.8 and 3.2.
58. The formulation of claim 56, further comprising a property
selected from the group consisting of: a. the surfactant is a
phospholipid; b. the surfactant is lyso-myristoyl
phosphatidylcholine; c. the surfactant is decanoyl sucrose; d. the
amino acid is glutamine; e. the amino acid is glycine; f. the
organic acid is selected from the group consisting of methanoic
acid, ethanoic acid, tartaric acid, malonic acid, glycolic acid,
malic acid, gluconic acid, citric acid, caproic acid, benzoic acid,
lactic acid, propionic acid, and sorbic acid; g. the organic acid
is methanoic acid; and h. the organic acid is tartaric acid.
59. The formulation of claim 58, comprising: a. glucagon or
glucagon-like peptide at a concentration of about 200 mg/ml; b.
lyso-myristoyl phosphatidylcholine at a concentration of about 100
mg/ml; c. tartaric acid at a concentration of about 50 mg/ml; d.
glycine at a concentration of about 50 mg/ml; e. trehalose at a
concentration of about 50 mg/ml; and f. sodium carboxymethyl
cellulose at a concentration of about 0.5 mg/ml.
60. A medical device, comprising an array of microneedles having
coated thereon a solid composition according to claim 30.
61. The medical device of claim 60, wherein the solid composition
comprises: a. 40-50% (w/w) of the glucagon or a glucagon-like
peptide; b. 20-25% (w/w) of the cationic or neutral surfactant; c.
10-12.5% (w/w) of the amino acid; and d. 10-12.5% (w/w) of the
organic acid.
62. The medical device of claim 61, wherein the solid composition
further comprises a property selected from the group consisting of:
a. the surfactant is a phospholipid; b. the surfactant is
lyso-myristoyl phosphatidylcholine; c. the surfactant is decanoyl
sucrose; d. the amino acid is glutamine; e. the amino acid is
glycine; f. the organic acid is selected from the group consisting
of methanoic acid, ethanoic acid, tartaric acid, malonic acid,
glycolic acid, malic acid, gluconic acid, citric acid, caproic
acid, benzoic acid, lactic acid, propionic acid, and sorbic acid;
g. the organic acid is methanoic acid; and h. the organic acid is
tartaric acid.
63. The medical device of claim 62, wherein the solid composition
comprises: a. about 44% w/w glucagon or glucagon-like peptide; b.
about 22% w/w lyso-myristoyl phosphatidylcholine; c. about 11%
tartaric acid; d. about 11% glycine; e. about 11% trehalose; and f.
carboxymethyl cellulose.
64. An intracutaneous delivery system, comprising a patch having a
plurality of microprojections that are adapted to penetrate or
pierce the stratum corneum of a human subject, the microprojections
having a solid formulation coating thereon, wherein: a. the coating
comprises about 40-50% (w/w) of glucagon or a glucagon-like peptide
and about 20-25% (w/w) of a stabilizing agent, b. upon piercing the
stratum corneum layer of the skin, the coating is dissolved by body
fluids thereby releasing the glucagon or glucagon-like peptide into
the skin for absorption to the blood stream.
65. The system of claim 64, wherein the glucagon or glucagon-like
peptide is present in a therapeutic dose ranging from about 0.25 mg
to about 2.0 mg.
66. The system of claim 64, wherein the patch delivers from about
0.25 mg/patch to about 2.0 mg/patch of glucagon or glucagon-like
peptide.
67. The system of claim 64, wherein the patch delivers from about
0.5 mg/patch to about 1.0 mg/patch of glucagon or glucagon-like
peptide.
68. The system of claim 64, wherein the comprises a microprojection
array of about 2.5 cm.sup.2 to about 8 cm.sup.2.
69. The system of claim 64 used for treatment of low blood sugar in
a patient, wherein the patient may require the application of one
patch per occurrence.
70. The system of claim 64, wherein the patient may require the
application of several patches simultaneously or sequentially until
the blood sugar has reached a normal range of glucose serum
concentration.
71. The system of claim 64, wherein glucagon or glucagon-like
peptide is completely released from the patch within from about 1
minute to about 30 minutes of application.
72. A method for treating a patient having low blood sugar, wherein
the method comprises applying a patch having a plurality of
microprojections that are adapted to penetrate or pierce the
stratum corneum of the patient, the microprojections having a solid
formulation coating thereon, wherein: a. the coating comprises
about 40-50% (w/w) of glucagon or a glucagon-like peptide, about
20-25% (w/w) of a stabilizing agent, about 10-12.5% (w/w) of an
amino acid, and about 10-12.5% (w/w) of an organic acid, and b.
upon piercing the stratum corneum layer of the skin, the coating is
dissolved by body fluids thereby releasing the glucagon or
glucagon-like peptide into the skin for absorption to the blood
stream.
73. The method of claim 72, wherein blood serum Cmax of glucagon or
glucagon-like peptide is reached in less than about 30 minutes
following application of the patch to the skin of the patient.
74. The method of claim 73, wherein the blood serum Cmax of
glucagon or glucagon-like peptide reaches at least 5 ng/mL.
75. The method of claim 73, wherein the blood serum Cmax of
glucagon or glucagon-like peptide reaches about 20 ng/mL.
76. A method for treating a patient having low blood sugar, wherein
the method comprises applying a patch carrying a plurality of
microprojections that are adapted to penetrate or pierce the
stratum corneum of the patient, the microprojections having a solid
formulation coating thereon, wherein: a. the coating comprises
about 15-20% (w/w) of glucagon and about 7.5-10% (w/w) of a
stabilizing agent, about 3.75-5% (w/w) of an amino acid, and about
3.75-5% (w/w) of an organic acid, and b. upon piercing the stratum
corneum layer of the skin, the coating is dissolved by body fluids
thereby releasing the glucagon into the skin for absorption to the
blood stream.
77. The method of claim 76, wherein blood serum Cmax of glucagon is
reached in less that about 30 minutes following application of the
patch to the skin of the patient.
78. The method of claim 77, wherein the blood serum Cmax of
glucagon reaches at least 5 ng/mL.
79. The method of claim 77, wherein the blood serum Cmax of
glucagon reaches about 20 ng/mL.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/930,041, filed on Nov. 2, 2015, which is a continuation in
part of U.S. application Ser. No. 14/466,461, filed on Aug. 22,
2014, which claims the benefit of U.S. Provisional Application No.
61/868,969 filed on Aug. 22, 2013, the contents of which are
incorporated by reference herein in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to drug delivery, and more
particularly to formulations of glucagon for delivery through the
skin.
BACKGROUND ART
[0003] Glucagon is produced in humans by the pancreas. Glucagon
binds to specific receptors on liver cells and increases the
release of glucose in the blood stream. Thus, it is used in the
treatment of diabetes as a rescue medication when the blood sugar
level drops too low.
[0004] Glucagon is a short peptide having 29 amino acids and a
molecular weight of 3,483 kilodaltons (kDa). The sequence of amino
acid in glucagon is:
TABLE-US-00001 (SEQ ID NO: 1) His Ser Gln Gly Thr Phe Thr Ser Asp
Tyr Ser Lys 1 5 10 Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln
15 20 21 Trp Leu Met Asn Thr 25 29
[0005] Glucagon has a highly helical conformation in the
crystalline state, but forms a random coil in dilute solution with
about 15% alpha helix at the C-terminus. At higher concentrations
it generally precipitates and forms fibrils. Glucagon readily
dissolved in aqueous solution at pH below 3 or above 9, but
precipitates readily at pH between 4 and 8. Liquid formulations of
glucagon are highly unstable, and undergo hydrolysis and
deamidation at several positions (amino acid at position 3, 9, 15,
20, 21, and 24) and thus pharmaceutical preparations are generally
provided in dual containers: powders of glucagon in one side and a
liquid diluent in another. A solution of glucagon is then prepared
just prior to use. Procedures generally undertaken to mitigate the
instability of glucagon in liquid formulations include the use of
solid dispersions, aprotic solvents, surfactants, processes
conducted at low temperature, and packaging in dried form.
[0006] Dilute formulations have been prepared that are stable for
up to 6 days and are useful for delivery with a pump
(US2011/0097386). The concentration of glucagon in these
formulations is between 0.8 mg/mL to 5 mg/mL and the pH is between
4 and 7. Stabilizing agents are a combination of both low
concentrations of a surfactant such as 1 mg/ml lyso-myristoyl
phosphatidylcholine (LMPC) and high concentrations of saccharide
such as 45 mg/mL of glucose.
[0007] Additionally, Applicant describes herein a highly
concentrated liquid glucagon formulation that is stable for at
least 4 days with the utilization of Sodium Carboxymethyl Cellulose
(NaCMC) to prevent fibril and/or gelation of glucagon.
SUMMARY OF THE EMBODIMENTS
[0008] The invention comprises new formulations of glucagon
suitable for transdermal delivery. Stable liquid formulations are
described at high concentration of glucagon. These formulations do
not form gels or fibrils and can be readily deposited onto
substrates to form a uniform coating. Once deposited onto a
substrate and dried, the glucagon in the coatings has improved
stability over time. The glucagon can be readily reconstituted
(such as with bodily fluids) without forming a gel. These
formulations are thus suited for use as coatings on substrates such
as microneedle patches for the transdermal delivery of glucagon for
the treatment of low blood sugar.
[0009] In a first aspect of the invention, there is provided a
liquid pharmaceutical formulation comprising 15-20% (w/w) of
glucagon, 7.5-10% (w/w) of a stabilizing agent selected from either
a cationic or neutral surfactant, 3.75-5% (w/w) of an amino acid,
3.75-5% (w/w) of an organic acid, and a pharmaceutically acceptable
diluent; the formulation having a pH between 2 and 3. In some
embodiments, the surfactant is a phospholipid. In some embodiments,
the phospholipid is lyso-myristoyl phosphatidylcholine. In other
embodiments, the surfactant is decanoyl sucrose. In yet other
embodiments, the amino acid is selected from the group consisting
of glutamine and glycine. In some embodiments, the organic acid is
selected from the group consisting of methanoic acid, ethanoic
acid, tartaric acid, malonic acid, glycolic acid, malic acid,
gluconic acid, citric acid, caproic acid, benzoic acid, lactic
acid, propionic acid, and sorbic acid. In certain embodiments, the
pharmaceutical formulation has a viscosity in the range of 20-200
centipoise (cP).
[0010] In certain embodiments, the pharmaceutical formulation, the
surfactant is decanoyl sucrose, the amino acid is glutamine and the
organic acid is tartaric acid. In other embodiments, the surfactant
is decanoyl sucrose, the amino acid is glycine and the organic acid
is tartaric acid. In yet other embodiments, the surfactant is
lyso-myristoyl phosphatidylcholine, the amino acid is glutamine and
the organic acid is tartaric acid. Still in other embodiments, the
surfactant is lyso-myristoyl phosphatidylcholine, the amino acid is
glycine and the organic acid is tartaric acid.
[0011] In another aspect there is described herein a lyophilized
glucagon formulation that has the following composition, 44.5% w/w
glucagon, 22.2% w/w lyso-myristoyl phosphatidylcholine (LMPC),
11.1% w/w trehalose, 11.1% w/w tartaric acid and 11.1% w/w glycine.
The lyophilized glucagon formulation upon reconstitution with
de-ionized water, the liquid formulation comprises 0.5 mg/mL Sodium
Carboxymethylcellulose (NaCMC), to make a high concentration
glucagon formulation that is suitable for microneedle coating. The
liquid glucagon formulation has the following composition; 200
mg/mL glucagon, 100 mg/mL LMPC, 50 mg/mL trehalose, 50 mg/mL
tartaric acid, 50 mg/mL glycine and 0.5 mg/mL NaCMC. The pH of the
liquid formulation is between 2.8-3.2.
[0012] In another aspect described herein is a lyophilized in which
the amount of LMPC is between preferably 2-8 fold less than
glucagon. In another aspect the lyophilized glucagon formulation is
reconstitution with reconstituting media comprising de-ionized
water and an amount of NaCMC preferably less than 0.1 mg/mL and/or
less than or equal to 1 mg/mL.
[0013] In another aspect described herein is a liquid
pharmaceutical formulation comprising glucagon and lyso-myristoyl
phosphatidylcholine (LMPC), wherein the amount of LMPC is between 2
and 8 fold less than the amount of glucagon and further comprises
50 mg/mL trehalose, 50 mg/mL tartaric acid, 50 mg/mL glycine and
Sodium Carboxymethyl Cellulose (NaCMC), wherein the concentration
of NaCMC is greater than 0.1 mg/ml and less than 1 mg/ml, and
wherein the pH of said liquid pharmaceutical formulation is between
2.8 and 3.2. In one embodiment of this aspect, glucagon is at a
concentration of 200 mg/mL, and wherein LMPC is at a concentration
of 100 mg/mL. In another embodiment, the liquid pharmaceutical
formulation comprises NaCMC is at a concentration of 0.5 mg/mL.
[0014] In another aspect described herein is a solid pharmaceutical
formulation comprising glucagon and lyso-myristoyl
phosphatidylcholine (LMPC), wherein the amount of LMPC is between 2
and 8 fold less than the amount of glucagon and further comprises
11.1% w/w trehalose, 11.1% w/w tartaric acid and 11.1% w/w glycine.
In one embodiment of this aspect, the formulation comprises
comprising 44.5% w/w glucagon, 22.2% w/w LMPC, 11.1% w/w trehalose,
11.1% w/w tartaric acid and 11.1% w/w glycine.
[0015] Another aspect described herein is a medical device for the
delivery of a pharmaceutical agent through the skin, the device
comprising an array of microneedles having coated thereon a liquid
composition described herein. In one embodiment of this aspect, the
medical device carries a therapeutic dose of glucagon ranging from
of 0.5 mg to 1.0 mg.
[0016] In a second aspect of the invention, there is provided a
solid pharmaceutical formulation comprising 40-50% (w/w) of
glucagon or a glucagon-like peptide, 20-25% (w/w) of a stabilizing
agent selected from either a cationic or neutral surfactant,
10-12.5% (w/w) of an amino acid, 10-12.5% (w/w) of an organic acid;
the formulation having a pH between 2 and 3. In some embodiments,
the surfactant is a phospholipid. In some embodiments, the
phospholipid is lyso-myristoyl phosphatidylcholine. In other
embodiments, the surfactant is selected from the group consisting
of glucose, sucrose, trehalose, and dextrose substituted with a
C8-C12 alkyl chain. In other embodiments, the surfactant is
decanoyl sucrose. In some other embodiments, the amino acid is
selected from the group consisting of glutamine and glycine. In
some other embodiments, the organic acid is selected from the group
consisting of methanoic acid, ethanoic acid, tartaric acid, malonic
acid, glycolic acid, malic acid, gluconic acid, and citric
acid.
[0017] In yet other embodiments, the surfactant is decanoyl
sucrose, the amino acid is glutamine and the organic acid is
tartaric acid. In other embodiments, the surfactant is decanoyl
sucrose, the amino acid is glycine and the organic acid is tartaric
acid. In other embodiments, the surfactant is lyso-myristoyl
phosphatidylcholine, the amino acid is glutamine and the organic
acid is tartaric acid. In other embodiments, the surfactant is
lyso-myristoyl phosphatidylcholine, the amino acid is glycine and
the organic acid is tartaric acid.
[0018] In a third aspect of the invention, there is provided a
medical device for the delivery of a pharmaceutical agent through
the skin, the device comprising an array of microneedles having
coated thereon a liquid composition comprising 15-20% (w/w) of
glucagon, 7.5-10% (w/w) of a stabilizing agent selected from either
a cationic or neutral surfactant, 3.75-5% (w/w) of an amino acid,
3.75-5% (w/w) of an organic acid, and a pharmaceutically acceptable
diluent; the formulation having a pH between 2 and 3. In some
embodiments, the medical device carries a therapeutic dose of
glucagon of either 1 mg for an adult dose, or 0.5 mg for a
pediatric dose. In some embodiments, the surfactant is a
phospholipid. In some embodiments, the phospholipid is
lyso-myristoyl phosphatidylcholine. In other embodiments, the
surfactant is selected from the group consisting of glucose,
sucrose, trehalose, dextrose substituted with a C8-C12 alkyl chain.
In other embodiments, the surfactant is decanoyl sucrose. In some
other embodiments, the amino acid is selected from the group
consisting of glutamine and glycine. In some other embodiments, the
organic acid is selected from the group consisting of methanoic
acid, ethanoic acid, tartaric acid, malonic acid, glycolic acid,
malic acid, gluconic acid, and citric acid.
[0019] In a fourth aspect of the invention, there is provided a
medical device for the delivery of a pharmaceutical agent through
the skin, the device comprising an array of microneedles having
coated thereon a solid composition comprising 40-50% (w/w) of
glucagon or a glucagon-like peptide, 20-25% (w/w) of a stabilizing
agent selected from either a cationic or neutral surfactant,
10-12.5% (w/w) of an amino acid, 10-12.5% (w/w) of an organic acid;
the formulation having a pH between 2 and 3. In some embodiments,
the medical device carries a therapeutic dose of glucagon of either
1 mg for an adult dose, or 0.5 mg for a pediatric dose. In some
embodiments, the surfactant is a phospholipid. In some embodiments,
the phospholipid is lyso-myristoyl phosphatidylcholine. In other
embodiments, the surfactant is selected from the group consisting
of glucose, sucrose, trehalose, dextrose substituted with a C8-C12
alkyl chain. In other embodiments, the surfactant is decanoyl
sucrose. In some other embodiments, the amino acid is selected from
the group consisting of glutamine and glycine. In some other
embodiments, the organic acid is selected from the group consisting
of methanoic acid, ethanoic acid, tartaric acid, malonic acid,
glycolic acid, malic acid, gluconic acid, and citric acid.
[0020] In some embodiment, the solid formulation is such that once
applied to the skin of a patient the coating is dissolved by the
body fluids of the patient in less than 30 minutes. In other
embodiments, the coating is dissolved by the body fluids of the
patient in less than 20 minutes. In other embodiments, the coating
is dissolved by the body fluids of the patient in less than 10
minutes.
[0021] In a fifth aspect of the invention, there is provided a
process for coating a medical device comprising coating a liquid
pharmaceutical composition according to the invention described
herein onto a medical device and drying the pharmaceutical
composition.
[0022] In a sixth aspect of the invention, there is provided
methods of treating a patient having low blood sugar comprising
applying the medical device having a solid formulation applied
thereon according to the invention described herein to the skin of
the patient. In some embodiments, a blood serum C.sub.max of
glucagon is reached in less than 30 minutes. In other embodiments,
a blood serum C.sub.max of glucagon is reached in about 10 min. In
yet other embodiments, a blood serum C.sub.max of glucagon reaches
at least 5 ng/mL. In other embodiments, a blood serum C.sub.max of
glucagon reaches at least 10 ng/mL. In still other embodiments, a
blood serum C.sub.max of glucagon reaches about 20 ng/mL.
[0023] In other embodiments, a blood serum C.sub.max of glucagon of
at least 5 ng/mL is reached in less than 20 minutes following
application of the device to the skin of the patient. In yet other
embodiments, a blood serum C.sub.max of glucagon of at least 5
ng/mL is reached in about 10 minutes following application of the
device to the skin of the patient. In yet other embodiments, a
blood serum C.sub.max of glucagon of about 10 ng/mL is reached in
about 10 minutes following application of the device to the skin of
the patient. In still other embodiments, a blood serum
concentration of glucagon is less than 10 ng/mL at about 60 minutes
following application of the device to the skin of the patient.
[0024] In yet other embodiments, a blood serum C.sub.max of
glucagon of at least 5 ng/mL is reached in less than 20 minutes and
blood serum concentration of glucagon less than 10 ng/mL at about
40 minutes following application of the device to the skin of the
patient. In still other embodiments, a blood serum C.sub.max of
glucagon of at least 10 ng/mL is reached in less than 20 minutes
and blood serum concentration of glucagon less than 10 ng/mL at
about 30 minutes following application of the device to the skin of
the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The foregoing features of embodiments will be more readily
understood by reference to the following detailed description,
taken with reference to the accompanying drawings, in which:
[0026] FIG. 1 is a perspective view of a portion of one example of
a microneedle patch, according to the invention.
[0027] FIG. 2 is a perspective view of the microneedle patch shown
in FIG. 1 having a coating deposited on the microneedles, according
to the invention.
[0028] FIG. 3 is a side sectional view of a microneedle patch
having an adhesive backing, according to the invention.
[0029] FIG. 4 is a side sectional view of a retainer ring having a
microneedle patch disposed therein, according to the invention.
[0030] FIG. 5 is a perspective view of the retainer shown in FIG.
4.
[0031] FIG. 6 is an exploded perspective view of an applicator and
retainer, according to the invention.
[0032] FIG. 7 is an SEM photograph of microneedle patch coated with
glucagon according to embodiments of the invention.
[0033] FIGS. 8A and 8B are schematic representations of the process
of coating glucagon formulations onto microneedle patches according
to an embodiment of the invention.
[0034] FIG. 9 is a plot of time versus viscosity comparing time to
gelation of formulations of glucagon using various stabilizing
agents prepared according to Example 1.
[0035] FIG. 10 is a plot of time versus viscosity comparing time to
gelation of formulations of glucagon using various concentrations
of the stabilizing agents lyso-myristoyl phosphatidylcholine (LMPC)
prepared according to Example 2.
[0036] FIG. 11 is a plot of time versus viscosity comparing time to
gelation of formulations of glucagon with and without glutamine
prepared according to Example 3.
[0037] FIG. 12 is a plot of time versus viscosity comparing time to
gelation of formulations of glucagon with methanoic or tartaric
acid prepared according to Example 4.
[0038] FIG. 13 is a plot of time versus purity comparing stability
of formulations of glucagon with glutamine, LMPC and methanoic or
tartaric acid prepared according to example 5 at 25.degree. C. and
40.degree. C.
[0039] FIG. 14 is a plot of time versus purity comparing stability
of formulations of glucagon with glutamine, decanoyl sucrose and
methanoic or tartaric acid prepared according to Example 6 at
25.degree. C. and 40.degree. C.
[0040] FIG. 15 is a plot comparing the pharmacokinetics of
formulations of glucagon on the microneedle patch prepared
according to an embodiment of the invention versus subcutaneous
injection in the hairless guinea pig, detailed at Example 7.
[0041] FIG. 16 is a plot comparing purity over time for Formulation
1. The plot shows the stability of glucagon systems where the
titanium array was coated Formulation 1 with 0.5 mg of glucagon,
assembled with a polycarbonate retainer ring with a co-molded
desiccant and a 5 cm.sup.2 adhesive patch, and heat sealed in a
nitrogen-purged foil pouch.
[0042] FIG. 17 is a plot comparing purity over time for Formulation
2. The plot shows the stability of glucagon systems where the
titanium array was coated Formulation 1 with 0.5 mg of glucagon,
assembled with a polycarbonate retainer ring with a co-molded
desiccant and a 5 cm.sup.2 adhesive patch, and heat sealed in a
nitrogen-purged foil pouch.
[0043] FIG. 18 shows the Far UV (FUV) circular dichroism (CD)
spectra for glucagon extracted from Patch C and Patch D.
ZP-Glucagon patches were evaluated for glucagon fibrillation by CD
after glucagon dissolution. These CD spectra are consistent with a
glucagon peptide in a predominantly .alpha.-helical conformation as
a soluble trimer or soluble helical monomer, as opposed to the
.beta.-sheet rich structure in glucagon fibrils. As there is still
a significant amount of random coil structure, this is also
consistent with monomeric glucagon.
[0044] FIG. 19 is a plot of time versus viscosity comparing time to
gelation of formulations of lyophilized glucagon reconstituted with
NaCMC (0.5 mg/mL) (.diamond-solid.) and de-ionized water
(.box-solid.).
[0045] FIG. 20 is a plot of fluorescence against wavelength. An
increase in the intensity of fluorescence with time at wavelength
of 480 nm suggests fibril formation.
[0046] FIG. 21 is a plot of time versus viscosity comparing time to
gelation of formulations of lyophilized glucagon reconstituted with
NaCMC at concentrations of 0.5 mg/mL (.diamond-solid.), 0.2 mg/mL
(.box-solid.) and 0.1 mg/mL (.tangle-solidup.).
[0047] FIG. 22: Plot of time versus viscosity comparing time to
gelation of formulations of lyophilized glucagon reconstituted with
1 mg/mL NaCMC (.diamond-solid.) and 2 mg/mL HEC (.box-solid.).
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0048] Definitions. Unless defined otherwise, all technical and
scientific terms used in this description and the accompanying
claims have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0049] An alkyl saccharide according to the invention means a
compound comprising a carbohydrate moiety of the type
R--C.sub.xH.sub.2y+zO.sub.y, wherein x and y are integers ranging
from 3-12, z is a numeral ranging from -1 to 1, R may be an
hydrogen or a linear or branched C1-C22 alkyl or alkyl groups
saturated or partially unsaturated, including methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, octyl-, nonyl-, decyl-,
undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-,
hexadecyl-, heptadecyl-, octadecyl-, nondecyl-, eicosanyl-,
heneicosanyl-, docosanyl-, ethoyl-, propoyl-, butoyl-, pentoyl-,
hexoyl-, heptoyl-, capriloyl-, caproyl-, lauroyl-, myristoyl-,
palmitoyl-, stearoyl-, arachidoyl-, behenoyl-, myristoleoyl-,
palmitoleoyl-, oleoyl-, linoleoyl-, linolenoyl-, and
arachidoneoyl-; and the carbohydrate may be a moiety of glucose,
dextrose, maltose, galactose, lactose, sucrose, fructose, or
ribose. A preferred alkyl saccharide is decanoyl sucrose.
[0050] A cationic surfactant according to the invention means a
compound selected from a phosphatidylcholine and lyso
phosphatidylcholine, including lyso-myristoyl phosphocholine
(LMPC).
[0051] An organic acid means naturally occurring acids including
methanoic (formic), acetic, caproic, tartaric, citric, benzoic,
lactic, propionic, sorbic, malonic, malic, glycolic, and gluconic
acids.
[0052] Pharmaceutically acceptable diluent means water with or
without buffers, salts and the like.
[0053] The term "transdermal" means the delivery of an agent into
and/or through the skin for local or systemic therapy.
[0054] The term "transdermal flux" means the rate of transdermal
delivery.
[0055] The term "co-delivering" as used herein, means that a
supplemental agent(s) is administered transdermally either before
the agent is delivered, before and during transdermal flux of the
agent, during transdermal flux of the agent, during and after
transdermal flux of the agent, and/or after transdermal flux of the
agent. Additionally, two or more agents may be coated onto the
microprojections resulting in co-delivery of the agents.
[0056] The term "biologically active agent" or "active agent" as
used herein, refers to a composition of matter or mixture
containing a drug which is pharmacologically effective when
administered in a therapeutically effective amount.
[0057] The term "biologically effective amount" or "biologically
effective rate" shall be used when the biologically active agent is
a pharmaceutically active agent and refers to the amount or rate of
the pharmacologically active agent needed to effect the desired
therapeutic, often beneficial, result. The amount of agent employed
in the coatings will be that amount necessary to deliver a
therapeutically effective amount of the agent to achieve the
desired therapeutic result. In practice, this will vary widely
depending upon the particular pharmacologically active agent being
delivered, the site of delivery, the severity of the condition
being treated, the desired therapeutic effect and the dissolution
and release kinetics for delivery of the agent from the coating
into skin tissues. It is not practical to define a precise range
for the therapeutically effective amount of the pharmacologically
active agent incorporated into the microneedles and delivered
transdermally according to the methods described herein.
[0058] The term "stability" shall refer to the property of a
formulation to retain its purity level (% (w/w)), within 3% of its
starting purity level after a period of time, preferably 0-24
months, 0-12 months, or 0-6 months; at a temperature of
0-50.degree. C., preferably 4-42.degree. C., more preferably
25-40.degree. C.; and at a relative humidity (RH) of 0-100%,
preferably 25-85%, more preferably 60-75%. The term "microneedles"
refers to piercing elements which are adapted to pierce or cut
through the stratum corneum into the underlying epidermis layer, or
epidermis and dermis layers, of the skin of a living animal,
particularly a human. Typically the piercing elements have a blade
length of less than 500 .mu.m, and preferably less than 400 .mu.m.
The microprojections typically have a width of about 50 to 200
.mu.m and thickness of about 5 to 50 .mu.m. The microprojections
may be formed in different shapes, such as needles, hollow needles,
blades, pins, punches, and combinations thereof.
[0059] The term "microneedle array" or "microneedle patch" as used
herein, refers to a substrate carrying a plurality of microneedles
arranged in an array for piercing the stratum corneum. The
microneedle patch may be formed by etching or punching a plurality
of microneedles from a thin sheet and folding or bending the
microneedles out of the plane of the sheet to form a configuration
such as that shown in FIG. 1. The microneedle patch may also be
formed in other known manners, such as by forming one or more
strips having microneedle along an edge of each of the strip(s) as
disclosed in U.S. Pat. No. 6,050,988 of the ALZA Corporation, the
entire content of which is incorporated herein by reference. The
microneedle patch may also include hollow needles which hold a dry
pharmacologically active agent.
[0060] Liquid and solid dry formulations according to the invention
for application to microneedle patches are prepared according to
the general procedures of publication Pharm. Res. 27, 303-313
(2010). A liquid glucagon formulation containing a surfactant, an
amino acid and an organic acid are prepared according to the
following exemplary procedure. Three hundred mg of glucagon is
added to 1.5 mL of stock solution containing 50 mg/mL of tartaric
acid, 50 mg/mL of glutamine and 100 mg/mL of surfactant. The
resultant slurry is then mixed for 2-3 hours or until a clear
solution of the liquid formulation is obtained.
[0061] A liquid pharmaceutical formulation according to the
invention may contain 15-20% (w/w) of glucagon, 7.5-10% (w/w) of a
stabilizing agent selected from the group consisting of a cationic
or alkyl saccharide surfactant, 3.75-5% (w/w) of an amino acid;
3.75-5% (w/w) of an organic acid, and a pharmaceutically acceptable
diluent. The pH of the formulation is adjusted to between 2 and
3.
[0062] The dried pharmaceutical formulation on the coated, ready
for packaging, patches according to the invention may contain
40-50% (w/w) of glucagon, 20-25% (w/w) of a stabilizing agent
selected from either a cationic surfactant or an alkyl saccharide,
10-12.5% (w/w) of an amino acid, 10-12.5% (w/w) of an organic acid.
The pH of the formulation is 2 to 3.
[0063] The surfactant may be a cationic phospholipid such as
lyso-myristoyl phosphatidylcholine. The alkyl saccharide may be
sucrose with a C8-C12 alkyl chain such as decanoyl sucrose. The
amino acid may be glutamine or glycine. The organic acid may be
methanoic acid or tartaric acid.
[0064] Embodiments of the present invention provide a formulation
containing a biologically active agent, glucagon which when coated
and dried upon one or more microneedles of a microneedle patch as
shown in FIG. 1, forms a stable coating with enhanced
solubilization of the drug upon insertion into the skin for a fast
release into the blood stream of the patient and quick treatment
onset. Referring to FIG. 1, embodiments of the present invention
include a device 22 having a plurality of stratum corneum-piercing
microneedles 24 extending therefrom. The microneedles are adapted
to pierce through the stratum corneum into the underlying epidermis
layer, or epidermis and dermis layers, but do not penetrate so deep
as to reach the capillary beds and cause significant bleeding.
Referring to FIG. 2, the microneedles carry a coating 26 of the dry
formulation of the biologically active agent, glucagon. Upon
piercing the stratum corneum layer of the skin, the coating is
dissolved by body fluids (intracellular fluids and extracellular
fluids such as interstitial fluid) thereby releasing the
biologically active agent glucagon into the skin for absorption to
the blood stream.
[0065] The solid coating is obtained by drying a liquid formulation
on the microneedles, as described in U.S. Pat. No. 7,537,795 of the
ALZA Corporation, the entire content of which is incorporated
herein by reference herein. The liquid formulation is usually an
aqueous formulation. In a solid coating on a microneedle patch, the
drug is typically present in an amount of 1-2 mg per unit dose.
With the addition of formulating agents, the total mass of the
solid coating is less than 4 mg per unit dose. The microneedle
array 22 is usually present on an adhesive 30 with a backing 40 as
shown in FIG. 3, which is attached to a disposable polymeric
retainer ring 50 as shown in FIGS. 4 and 5. This assembly is
packaged individually in a pouch or a polymeric housing. The
microneedle patch 22 is applied to the skin of a patient with the
use of a deployment device 60, shown in FIG. 6, on which is mounted
the retainer ring 50 with the microneedle patch 22. The deployment
device is depressed, detaching the patch 22 from the retainer ring
and pushing the microneedles 24 into the skin of the patient.
Alternatively the patch can be mounted in a single use applicator
ready for patient application, as described in U.S. patent
application 61/864,857 filed Aug. 12, 2013.
[0066] Coated microneedle patches for delivery of glucagon may be
prepared as follows. Coatings on the microneedles can be formed by
a variety of known methods such as dip-coating or spraying.
Dip-coating consists of partially or totally immersing the
microneedles into a formulation prepared according to the
invention. Alternatively, the entire device can be immersed into
the formulation. In many instances, it may be preferable to only
coat the tips of the microneedles. Microneedles tip coating
apparatus and methods are disclosed in U.S. Pat. No. 6,855,372 of
the ALZA Corporation, the content of which is incorporated herein
by reference in its entirety.
[0067] A sketch of the process is shown in FIG. 8A. The coating
apparatus only applies coatings to the microneedles themselves and
not upon the substrate that the microneedles project from. A liquid
formulation 10 prepared according to the invention is placed into a
reservoir 12. A rotating drum 14 is partially submerged into the
liquid and is rotated. The liquid 10 forms a thin film on the
rotating drum 14. A blade 18 controls the thickness of the film to
match the length of the microneedles or adjust the dosage on the
patches. A sled 20 carrying the substrate 22 with the microneedles
24 is positioned on the drum 14 so that the microneedles 24 are
immersed or dipped into the film 16 (shown in the excerpt in FIG.
8B). As the drum 14 rotates, the substrate 22 moves from one side
to the other so as to coat the microneedles sequentially. The
process can be performed in a continuous manner feeding a series of
substrates to the apparatus. The process may be repeated to
increase the thickness of the coatings and thus vary the dosage of
the patches. This coating technique has the advantage of forming a
smooth coating that is not easily dislodged from the microneedles
during skin piercing. Other coating techniques such as microfluidic
spray or printing techniques can be used to precisely deposit a
coating on the microneedles 24.
[0068] Dosage of glucagon on the microneedle patches can be
controlled by varying a variety of features, such as the size of
the patch, the size of the microneedles, the thickness of the
coating on the microneedles, and the surface area of the coatings
on the microneedles. Patches may thus be prepared for the
transdermal delivery of glucagon at the following dose ranges and
doses: 0.25-2.0 mg/patch, preferably 0.5-1.0 mg/patch; patch size
can be 5 cm.sup.2 to 10 cm.sup.2 with a microneedle array of 2.5
cm.sup.2 to 8 cm.sup.2. Treatment of low blood sugar in a patient
may require the application of one patch per occurrence. It may
also require the application of several patches simultaneously or
sequentially until the sugar blood level has reached the normal
range of glucose serum concentration.
[0069] Techniques for the application of the patches to the skin
have been described in U.S. Pat. No. 6,855,131, the content of
which is incorporated herein by reference in its entirety. To apply
a microneedle patch according to the present invention, a sterile
foil package containing the glucagon-loaded patch in a single use,
ready to use applicator. The proximal end of the patch/applicator
system is pressed against the skin to activate the patch release
onto the skin. Within 1-30 minutes, the glucagon is completely
released from the patch. The patch may then be removed and
discarded.
[0070] Physical stabilization, especially minimizing the exposure
of the biologically active agent formulations over time to
oxidation and hydrolysis, is an important step in assuring efficacy
of the therapeutic agents, particularly when the mode of delivery
of the therapeutic agent is via a transdermal delivery device
having a plurality of microneedles coated with an agent containing
biocompatible coating.
[0071] Thus, the manufacture and/or packaging of the formulations
in a dry inert atmosphere or a partial vacuum, substantially free
of oxygen and water, substantially reduces or eliminates
undesirable deterioration of the biologically active agent.
[0072] The formulations of the present invention display superior
stability and are shown to retain substantial purity after storage
of up to six months when stored under various temperature and
relative humidity conditions. In addition, the formulations of the
present invention are shown to remain in predominantly
.alpha.-helical conformation as a soluble trimer or soluble or
soluble helical monomer, as opposed to the .beta.-sheet rich
structures found in glucagon fibrils. The superior stability and
limited fibril formation of the formulations presented herein offer
a significant improvement over currently available glucagon
therapeutics.
[0073] In one embodiment, the compositions of, and methods for
formulating and delivering, biologically active agents are
particularly suitable for transdermal delivery using a microneedle
delivery device, wherein the biologically active agents are
included in a biocompatible coating that is manufactured and/or
packaged in a dry inert atmosphere, preferably nitrogen or
argon.
[0074] In one embodiment, the compositions of, and methods for
formulating and delivering, biologically active agents are
particularly suitable for transdermal delivery using a microneedle
delivery device, wherein the biologically active agents are
included in a biocompatible coating that is coated on at least one
stratum corneum piercing microneedle, preferably a plurality of
stratum corneum piercing microneedles of a microneedle delivery
device, and manufactured and/or packaged in a dry inert atmosphere,
preferably nitrogen or argon.
[0075] In one embodiment, the compositions of, and methods for
formulating and delivering, biologically active agents are
particularly suitable for transdermal delivery using a microneedle
delivery device, wherein the biologically active agents are
included in a biocompatible coating that is manufactured and/or
packaged in a dry inert atmosphere, preferably nitrogen or argon,
and in the presence of a desiccant.
[0076] In one embodiment, the compositions of, and methods for
formulating and delivering, biologically active agents are
particularly suitable for transdermal delivery using a microneedle
delivery device, wherein the biologically active agents are
included in a biocompatible coating that is manufactured and/or
packaged in a foil lined chamber having a dry inert atmosphere,
preferably nitrogen and a desiccant.
[0077] In one embodiment, the compositions of, and methods for
formulating and delivering, biologically active agents are
particularly suitable for transdermal delivery using a microneedle
delivery device, wherein the biologically active agents are
included in a biocompatible coating that is manufactured and/or
packaged in a partial vacuum.
[0078] In one embodiment, the compositions of and methods for
formulating and delivering biologically active agents are
particularly suitable for transdermal delivery using a microneedle
delivery device, wherein the biologically active agents are
included in a biocompatible coating that is manufactured and/or
packaged in a dry inert atmosphere, preferably nitrogen or a
partial vacuum.
[0079] In one embodiment, the compositions of and methods for
formulating and delivering biologically active agents are
particularly suitable for transdermal delivery using a microneedle
delivery device, wherein the biologically active agents are
included in a biocompatible coating that is manufactured and/or
packaged in a foil lined chamber having a dry inert atmosphere,
preferably nitrogen and a desiccant.
EXAMPLES
Materials and General Procedures
[0080] Glucagon was acquired from BACHEM and was produced by
chemical synthesis at a purity of 98.8% (w/w). Formulations of
glucagon are prepared following the procedures of publication
Pharm. Res. 27, 303-313 (2010). A liquid formulation containing a
surfactant, an amino acid and an organic acid was prepared. Three
hundred mg of glucagon was added to 1.5 mL of stock solution
containing 50 mg/mL tartaric acid, 50 mg/mL glutamine and 100 mg/mL
surfactant. The resultant slurry was then mixed for 2-3 hours or
until a clear solution of the liquid formulation was obtained.
[0081] Physical stability testing of liquid glucagon formulations
was conducted utilizing a rheometer (model CVOR150, Bohlin
Instrument, Cranbury, N.J.) configured with a cone and plate
geometry (a cone angle of 1.degree. and radius 10 mm). Seventy
.mu.L of the glucagon liquid formulation was utilized for each
experiment. To determine the gel point of a particular glucagon
liquid formulation, the sample was sheared at 2667 s.sup.-1 and
viscosities were recorded every 30 seconds. Gelation point was
noted at the inflection of the viscosity versus time curve, i.e. at
the point where a rapid increase in viscosity was observed.
Example 1: Study of the Effect of Surfactants on the Physical
Stability of Glucagon Liquid Formulations
[0082] A summary of the various formulations is shown in Table 1
below. A comparison of the gelation profiles between the
formulations is shown in FIG. 9 and gelation point results are
shown in Table 1 below.
TABLE-US-00002 TABLE 1 Organic Formulation/ Glucagon Surfactant
Glutamine acid Gelation Surfactant (mg/mL) (mg/mL) (mg/mL) (mg/mL)
point (s) Decanoyl 200 100 N/A 50 1800 Sucrose LMPC 200 100 N/A 50
1800 Polysorbate 20 200 2 N/A 50 1300
[0083] For a liquid under a fixed shear rate, its viscosity should
be constant with time initially and then increase quickly,
indicating the point of gelation. The longer it takes to gel, the
lower the gelling tendency of a formulation. The viscosity profiles
for the three formulations incorporating decanoyl sucrose, LMPC or
polysorbate 20 as the surfactant are presented in FIG. 9. All three
formulations were sheared at shear rate of 2667 s.sup.-1 at
8.degree. C. The inflection point (where the viscosity begins to
increase) is 1800 seconds for both the decanoyl sucrose and LMPC
formulations and 1300 seconds for the polysorbate 20 formulation.
It suggests that the decanoyl sucrose and LMPC formulations are
less prone to gelation.
Example 2: Study of the Effect of the Concentration of the
Surfactant on the Physical Stability of Glucagon Liquid
Formulations
[0084] A summary of the various formulations is shown in Table 2
below. A comparison of the gelation profiles between the
formulations is shown in FIG. 10 and gelation point results are
shown in Table 2 below.
TABLE-US-00003 TABLE 2 Formulation/ Organic Glucagon: Glucagon
Surfactant Glutamine acid Gelation LMPC (mg/mL) (mg/mL) (mg/mL)
(mg/mL) point (s) 1:1 200 200 50 50 2200 1:0.5 200 100 50 50 2200
1:0.25 200 50 50 50 1200
[0085] Results:
[0086] The viscosity profiles for the three formulations
incorporating LMPC concentrations at 50 mg/mL, 100 mg/mL and 200
mg/mL are presented in FIG. 10. All three formulations were sheared
at shear rate of 2667 s.sup.-1 at 8.degree. C. The inflection point
(where the viscosity begins to increase) is 2200 seconds for
formulations containing LMPC concentrations of 100 mg/mL and 200
mg/mL (at constant glucagon concentration of 200 mg/mL) and 1200
seconds for the formulation containing LMPC at a concentration of
50 mg/mL. This suggests that a minimum LMPC concentration of 100
mg/mL is required (at a glucagon concentration of 200 mg/mL) is
required for a formulation that is less susceptible to
gelation.
Example 3: Study of the Effect of Glutamine on the Physical
Stability of Glucagon Liquid Formulations
[0087] A summary of the various formulations is shown in Table 3
below. A comparison of the gelation profiles between the
formulations is shown in FIG. 11 and gelation point results are
shown in Table 3 below.
TABLE-US-00004 TABLE 3 Organic Formulation/ Glucagon Surfactant
Glutamine acid Gelation Amino acid (mg/mL) (mg/mL) (mg/mL) (mg/mL)
point (s) Without 200 100 N/A 50 1400 With Glutamine 200 100 50 50
2200
[0088] Results:
[0089] The viscosity profiles for the two formulations evaluating
the effect of glutamine are presented in FIG. 11. The two
formulations were sheared at shear rate of 2667 s.sup.-1 at
8.degree. C. The inflection point (where the viscosity begins to
increase) is 2200 seconds for the formulation containing glutamine
and 1400 seconds for the formulation containing no glutamine. This
result indicates that glutamine is required for a formulation that
is less susceptible to gelation.
Example 4: Study of the Effect of the Organic Acid on the Physical
Stability of Glucagon Liquid Formulations
[0090] A comparison of the gelation profiles between the
formulations is shown in FIG. 12 and gelation point results are
shown in Table 4 below.
TABLE-US-00005 TABLE 4 Organic Glucagon LMPC Glutamine acid
Gelation Formulation (mg/mL) (mg/mL) (mg/mL) (mg/mL) point (s)
Methanoic acid 200 100 50 50 2200 Tartaric acid 200 100 50 50
2200
[0091] The viscosity profiles for the two formulations evaluating
the effect of tartaric acid and methanoic acid is presented in FIG.
12. The two formulations were sheared at shear rate of 2667
s.sup.-1 at 8.degree. C. The inflection point (where the viscosity
begins to increase) is 2200 seconds for both formulations. This
result indicates that methanoic acid and tartaric acid do not
adversely affect the physical stability of glucagon
formulations.
Example 5: Stability of Glucagon Coatings on Microneedle Patches,
Effect of the Organic Acid with LMPC
[0092] A summary of the various formulations is shown in Table 5
below.
[0093] Purity of the glucagon coating was measured at two
temperatures: 25.degree. C. and 40.degree. C. The comparison
between the formulations is shown in FIG. 13 and results are shown
in Tables 6 and 7 below. These results demonstrate the formulation
containing tartaric acid show greater stability after three months
than the formulation with methanoic acid at both 25.degree. C. and
40.degree. C. temperatures.
TABLE-US-00006 TABLE 5 Glucagon LMPC Glutamine Organic Formulation
(mg/mL) (mg/mL) (mg/mL) acid % A (Methanoic acid) 200 100 50 5 C
(tartaric acid) 200 100 50 5
TABLE-US-00007 TABLE 6 Formulation A Time % (w/w) purity (month)
25.degree. C. 40.degree. C. 0 99.1% .+-. 0.3 99.1 .+-. 0.3 1 98.4
.+-. 0.5 95.7 .+-. 1.5 3 97.5 .+-. 0.1 94.5 .+-. 0.6
TABLE-US-00008 TABLE 7 Formulation C Time % (w/w) purity (month)
25.degree. C. 40.degree. C. 0 99.6% .+-. 0.13 99.6 .+-. 0.13 1 99.6
.+-. 0.13 99.4 .+-. 0.02 3 99.6 .+-. 0.03 99.4 .+-. 0.06
Example 6: Stability of Glucagon Coatings on Microneedle Patches,
Effect of the Organic Acid with Decanoyl Sucrose
[0094] A summary of the various formulations is shown in Table 8
below.
[0095] Purity of the glucagon coating was measured at two
temperatures: 25.degree. C. and 40.degree. C. The comparison
between the formulations is shown in FIG. 14 and results are shown
in Tables 9 and 10 below. These results demonstrate the formulation
containing tartaric acid show greater stability after one month
than the formulation with methanoic acid at both temperatures.
TABLE-US-00009 TABLE 8 Decanoyl Organic Glucagon sucrose Glutamine
acid Formulation (mg/mL) (mg/mL) (mg/mL) % B (methanoic 200 100 50
5 acid) D (tartaric 200 100 50 5 acid)
TABLE-US-00010 TABLE 9 Formulation B Time % (w/w) purity (month)
25.degree. C. 40.degree. C. 0 99.5% .+-. 0.19 99.5 .+-. 0.19 1 98.2
.+-. 0.06 92.9 .+-. 0.31 3 92.9 .+-. 0.31 92.4 .+-. 0.17
TABLE-US-00011 TABLE 10 Formulation D Time % (w/w) purity (month)
25.degree. C. 40.degree. C. 0 99.7% .+-. 0.0 99.7 .+-. 0.0 1 99.6
.+-. 0.02 99.5 .+-. 0.05 3 99.4 .+-. 0.07 99.2 .+-. 0.06
Example 7: PK Study of Glucagon Coated Microneedle Patches Compared
to Subcutaneous Injection
[0096] In vivo glucagon delivery was performed in a hairless guinea
pig model under Institutional Animal Care and Use Committee (IACUC)
approved animal protocols. Formulations C and D were coated on
microneedle patches at 0.5 mg/3 cm.sup.2. Patches were applied to
the skin and removed after 1 hour. Subcutaneous (SC) glucagon
injection was prepared according to manufacturer's instructions
(Lilly-Glucagon Rescue Kit.RTM.). Both the patch and injection were
administered at a dose of 1 mg/kg.
[0097] As shown in FIG. 15, coatings of the formulation C and D
provide the fast and high release of glucagon as measured in the
serum levels. Table 11 shows that the bioavailability of glucagon
delivery with formulation C or D coated microneedle patch is 83%
and 86%, respectively of that observed with SC injection. This
indicates that the glucagon formulations can be efficiently
re-solubilized in the skin and glucagon delivered comparable to the
commercially available glucagon injection.
TABLE-US-00012 TABLE 11 Dose per kg Body Delivery Mean Mean Mean
Dose Weight Efficiency T.sub.max C.sub.max AUC.sub.t (ng Treatment
(.mu.g)/Animal (.mu.g/kg) (%) (minutes) (ng/ml) h/ml) SC injection
424 1000 N/A 9 95 91 ZP Glucagon Patch, 497 956 69 13 135 76 C 0.5
mg/3 cm.sup.2 ZP Glucagon Patch, 536 957 65 10 202 79 D 0.5 mg/3
cm.sup.2
[0098] The embodiments of the invention described above are
intended to be merely exemplary; numerous variations and
modifications will be apparent to those skilled in the art. All
such variations and modifications are intended to be within the
scope of the present invention as defined in any appended
claims.
Example 8: Stability of Glucagon Coatings on Microneedle Patch
[0099] Two glucagon liquid formulations were prepared. Formulation
1 comprises 200 mg/mL glucagon, 100 mg/mL LMPC
(1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine), 50 mg/mL
glutamine, and 50 mg/mL tartaric acid; Formulation 2 comprises 200
mg/mL glucagon, 100 mg/mL decanoyl sucrose (DS), 50 mg/mL
glutamine, and 50 mg/mL tartaric acid.
[0100] Each glucagon liquid formulation was coated on a microneedle
array using a dip coating method. After coating glucagon systems
were manufactured for stability studies, using patch components
involving a polycarbonate retainer ring with co-molded desiccant
and a 5 cm.sup.2 adhesive patch. The coated patch was heat sealed
in a nitrogen-purged foil pouch. The final systems were stored
under two conditions, 25.degree. C./60% relative humidity (RH) and
40.degree. C./75% RH. The coated patches were assessed for purity
at initial, 1-, 3-, and 6-month time points.
[0101] RP-HPLC was used to quantify purity of glucagon. Glucagon
related impurities were separated from native glucagon using an ACE
C18 column (3.0 mm ID.times.150 mm, 3 .mu.m) maintained at
45.degree. C. The eluted glucagon was detected by UV at 214 nm. The
mobile phase involved a gradient elution. Mobile phase A comprised
of 80% 0.15 M phosphate buffer at pH=2.7, 20% acetonitrile; mobile
phase B comprised of 60% water and 40% acetonitrile. Chromatography
was performed with an HPLC system (Waters 2695 Alliance, Milford
Mass.) provided with a binary pump, a thermostatted autosampler, a
thermostatted column compartment, and a 996 PAD. Data were
collected and analyzed using Empower.
[0102] The stability data is shown in FIGS. 16 and 17 for
Formulations 1 and 2 respectively. Table 12 summarizes the purity
data of the two formulations under the two storage conditions.
Under the accelerated conditions of 40.degree. C./75% RH and when
stored at 25.degree. C./60% RH, glucagon maintained excellent
stability within the study period.
TABLE-US-00013 TABLE 12 Summary of stability data of glucagon
coated systems Formulation Composition (Respective Amounts
Temperature Glucagon purity (% (w/w)) Formulation (mg/ml))
(.degree. C.) Initial 1 Month 3 Months 6 Months 1 Tartaric acid +
Glutamine + 25 99.6 .+-. 0.1 99.6 .+-. 0.1 99.6 .+-. 0.0 99.4 .+-.
0.14 LMPC 40 99.6 .+-. 0.1 99.4 .+-. 0.0 99.4 .+-. 0.1 99.0 .+-.
0.08 (50 mg/ml + 50 mg/ml + 100 mg/ml) 2 Tartaric acid + Glutamine
+ 25 99.7 .+-. 0.0 99.6 .+-. 0.0 99.4 .+-. 0.1 99.4 .+-. 0.02 DS 40
99.7 .+-. 0.0 9.5 .+-. 0.1 99.2 .+-. 0.1 98.8 .+-. 0.02 (50 mg/ml +
50 mg/ml + 100 mg/ml)
Example 9: Far-UV Circular Dichroism Spectra of Glucagon Coated
Patches
[0103] Patch C coated with formulation 1, and Patch D coated with
Formulation 2, were each placed into a separate extraction vessel
and 1.0 mL of dissolution solution was added to each vessel. Each
solution was then agitated for 2 minutes and a sample was taken for
circular dichroism (CD) spectroscopy. Each sample was also scanned
for OD280 (optical density at a wavelength of 280 nm) in a 1 mm
quartz cuvette.
[0104] NKTT120 Protein Concentration by OD280 Measurements
[0105] The protein concentration in solution was determined by
measurement of OD280. An extinction coefficient was calculated
based on the molecular weight and contributions by the aromatic
amino acids. All measurements were performed on a Varian Cary 100
UV/Vis spectrophotometer. The calculated molar extinction
coefficient of 8480 M.sup.-1 cm.sup.-1 was used to determine an
estimated specific extinction coefficient of E2 so, tcm=2.43 (based
on a molecular weight of 3482.8 kDa). Correction for light
scattering was made by subtracting the absorbance at 320 nm.
[0106] Circular Dichroism Spectroscopy
[0107] An Aviv Model 202 with a peltier controlled temperature
controlled cell was used to collect all CD spectra. All spectra
were collected at 25.degree. C. in quartz cuvettes. The quartz
cuvettes were tested using camphorsulfonic acid (CSA) to measure
accurate path lengths for all cells used. The 0.1 mm cell was
measured at 0.089 mm, and the 0.01 mm cell was measured at 0.0165
mm. All CD spectra are reported in units of mean residue
ellipticity (0) using a molecular weight of 3482.8 kDa and 29
residues.
[0108] The Far UV (FUV) circular dichroism (CD) spectra for
glucagon extracted from two patches (Patch C and Patch D), shown in
FIG. 18, are consistent with a glucagon peptide in a predominantly
.alpha.-helical conformation as a soluble trimer or soluble helical
monomer, as opposed to the .beta.-sheet rich structure in glucagon
fibrils. The spectra are also consistent with a peptide in a
soluble trimer or soluble helical monomer. As there is still a
significant amount of random coil structure, this is also
consistent with monomeric glucagon.
Example 10 the Incorporation of Sodium Salt of Carboxymethyl
Cellulose (NaCMC) to a Liquid Glucagon Formulation to Prevent
Fibril and/or Gelation
[0109] A lyophilized glucagon formulation that has the following
composition, 44.5% w/w glucagon, 22.2% w/w LMPC, 11.1% w/w
trehalose, 11.1% w/w tartaric acid and 11.1% w/w glycine was
reconstituted with de-ionized water containing 0.5 mg/mL NaCMC, to
make a high concentration glucagon formulation that is suitable for
microneedle coating. The liquid glucagon formulation had the
following composition; 200 mg/mL glucagon, 100 mg/mL LMPC, 50 mg/mL
trehalose, 50 mg/mL tartaric acid, 50 mg/mL glycine and 0.5 mg/mL
NaCMC. The pH of the liquid formulation is between 2.8-3.2.
[0110] To determine the gel point of a particular glucagon liquid
formulation, the sample was sheared at 2667 s-1 and viscosities
were recorded every 20 seconds. Gelation point was noted at the
inflection of the viscosity versus time curve, i.e. at the point
where a rapid increase in viscosity was observed. See FIG. 19 which
is a plot of time versus viscosity comparing time to gelation of
formulations of lyophilized glucagon reconstituted with NaCMC (0.5
mg/mL) and deionized water.
[0111] FIG. 19 shows that the lyophilized formulation that was
reconstituted with NaCMC did not gel within the testing period,
while the formulation that was reconstituted with de-ionized water
gelled in approximately 1300 seconds. Gelation point was noted at
the inflection of the viscosity versus time curve, i.e. at the
point where a rapid increase in viscosity was observed.
Example 11 Comparison of Coating Formulation Stability by
Thioflavin Fluorescence Assay of Lyo Formulations Reconstituted
w/Na-CMC and Water
[0112] A lyophilized glucagon formulation that has the following
composition, 44.5% w/w glucagon, 22.2% w/w LMPC, 11.1% w/w
trehalose, 11.1% w/w tartaric acid and 11.1% w/w glycine was
reconstituted with de-ionized water containing 0.5 mg/mL NaCMC or
de-ionized water. The liquid glucagon formulations had the
following compositions: [0113] 200 mg/mL glucagon, 100 mg/mL LMPC,
50 mg/mL trehalose, 50 mg/mL tartaric acid, 50 mg/mL glycine and
0.5 mg/mL NaCMC. [0114] 200 mg/mL glucagon, 100 mg/mL LMPC, 50
mg/mL trehalose, 50 mg/mL tartaric acid and 50 mg/mL glycine. The
two solutions were then stored at 2-8.degree. C. At each day an
aliquot of the two formulations were taken and thioflavin a dye
that specifically binds to amyloid fibrils was added. If fibrils
are detected there is an enhanced fluorescence or maxima around
wavelength 480 nm. The fluorescence was measured by SpectraMax M2e.
The results are illustrated in FIG. 20 as a plot of fluorescence
against wavelength. An increase in the intensity of fluorescence
with time at wavelength of 480 nm suggests fibril formation. The
thioflavin fluorescence assay did not detect fibrils for the
lyophilized glucagon formulation that was reconstituted with 0.5
mg/mL NaCMC within the testing period. For the lyophilized glucagon
formulation that was reconstituted with de-ionized water there was
an increase in fluorescence intensity at 480 nm after one day
storage, suggesting fibril formation. Later time points for the
glucagon formulation that was reconstituted with de-ionized water
were not collected as the formulation gelled on day 2 of the
study.
Example 12: Determining the Lowest Concentration of NaCMC that
Prevents Gelation
[0115] A lyophilized glucagon formulation that has the following
composition, 44.5% w/w glucagon, 22.2% w/w LMPC, 11.1% w/w
trehalose, 11.1% w/w tartaric acid and 11.1% w/w glycine was
reconstituted with 0.5 mg/mL NaCMC, 0.2 mg/mL NaCMC and 0.1 mg/mL
NaCMC. The liquid glucagon formulations had the following
compositions: [0116] 200 mg/mL glucagon, 100 mg/mL LMPC, 50 mg/mL
trehalose, 50 mg/mL tartaric acid, 50 mg/mL glycine, 0.5 mg/mL
NaCMC. [0117] 200 mg/mL glucagon, 100 mg/mL LMPC, 50 mg/mL
trehalose, 50 mg/mL tartaric acid, 50 mg/mL glycine, 0.2 mg/mL
NaCMC. [0118] 200 mg/mL glucagon, 100 mg/mL LMPC, 50 mg/mL
trehalose, 50 mg/mL tartaric acid, 50 mg/mL glycine, 0.1 mg/mL
NaCMC.
[0119] The pH of the liquid formulations was 2.9.
To determine the gel point of a particular glucagon liquid
formulation, the sample was sheared at 2667 s-1 and viscosities
were recorded every 20 seconds. Gelation point was noted at the
inflection of the viscosity versus time curve, i.e. at the point
where a rapid increase in viscosity was observed. FIG. 21 is a plot
of time versus viscosity comparing time to gelation of formulations
of lyophilized glucagon reconstituted with NaCMC at concentrations
of 0.5 mg/mL, 0.2 mg/mL and 0.1 mg/mL. The lyophilized formulations
that were reconstituted with NaCMC at 0.5 mg/mL and 0.2 mg/mL did
not gel within the testing period, while the formulation that was
reconstituted with 0.1 mg/mL NaCMC gelled in approximately 2200
seconds. The concentration of the NaCMC in the reconstituting media
is preferably >0.1 mg/mL and less than or equal to 1 mg/mL.
Example 13: Determining if Other Cellulose Based Polymers Prevent
Gelation of Glucagon
[0120] A lyophilized glucagon formulation that has the following
composition, 57.1% w/w glucagon, 14.3% w/w LMPC, 14.3% w/w sucrose,
14.3% w/w tartaric acid and 14.3% w/w glycine was reconstituted
with 1 mg/mL NaCMC or 2 mg/mL hydroxyethylcellulose (HEC). The
liquid glucagon formulations had the following compositions: [0121]
200 mg/mL glucagon, 50 mg/mL LMPC, 50 mg/mL sucrose, 50 mg/mL
tartaric acid, 50 mg/mL glycine, 1 mg/mL NaCMC. [0122] 200 mg/mL
glucagon, 50 mg/mL LMPC, 50 mg/mL sucrose, 50 mg/mL tartaric acid,
50 mg/mL glycine, 2 mg/mL HEC.
[0123] To determine the gel point of a particular glucagon liquid
formulation, the sample was sheared at 2667 s-1 and viscosities
were recorded every 20 seconds. Gelation point was noted at the
inflection of the viscosity versus time curve, i.e. at the point
where a rapid increase in viscosity was observed. FIG. 22 is a plot
of time versus viscosity comparing time to gelation of formulations
of lyophilized glucagon reconstituted with 1 mg/mL NaCMC and 2
mg/mL HEC. FIG. 22 shows that the lyophilized formulations that
were reconstituted with NaCMC at 1 mg/mL did not gel within the
testing period, while the formulation that was reconstituted with 2
mg/mL HEC gelled in approximately 1500 seconds.
Sequence CWU 1
1
1129PRTHomo sapiens 1His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser
Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp
Leu Met Asn Thr 20 25
* * * * *