U.S. patent application number 13/504783 was filed with the patent office on 2013-05-23 for composition, method, and kit for alpha-1 proteinase inhibitor.
This patent application is currently assigned to GRIFOLS THERAPEUTICS INC.. The applicant listed for this patent is Deborah Barnette, Bret Coldren, Jianxin Guo, Anthony Klos, Mark Manning. Invention is credited to Deborah Barnette, Bret Coldren, Jianxin Guo, Anthony Klos, Mark Manning.
Application Number | 20130131192 13/504783 |
Document ID | / |
Family ID | 43970713 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130131192 |
Kind Code |
A1 |
Guo; Jianxin ; et
al. |
May 23, 2013 |
COMPOSITION, METHOD, AND KIT FOR ALPHA-1 PROTEINASE INHIBITOR
Abstract
The present invention provides compositions comprising API and
at least one amino acid, in particular a liquid API formulation
comprising amino acids, and methods and kits related thereto. These
amino acids when incorporated into the API composition afford
stability to the API formulation.
Inventors: |
Guo; Jianxin; (Clayton,
NC) ; Klos; Anthony; (Raleigh, NC) ; Coldren;
Bret; (Research Triangle Park, NC) ; Barnette;
Deborah; (Cary, NC) ; Manning; Mark;
(Johnstown, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Guo; Jianxin
Klos; Anthony
Coldren; Bret
Barnette; Deborah
Manning; Mark |
Clayton
Raleigh
Research Triangle Park
Cary
Johnstown |
NC
NC
NC
NC
CO |
US
US
US
US
US |
|
|
Assignee: |
GRIFOLS THERAPEUTICS INC.
|
Family ID: |
43970713 |
Appl. No.: |
13/504783 |
Filed: |
November 2, 2010 |
PCT Filed: |
November 2, 2010 |
PCT NO: |
PCT/US10/55135 |
371 Date: |
July 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61257711 |
Nov 3, 2009 |
|
|
|
Current U.S.
Class: |
514/778 ;
514/777; 514/784 |
Current CPC
Class: |
A61K 31/195 20130101;
A61P 1/16 20180101; A61P 1/00 20180101; A61K 47/26 20130101; A61K
45/06 20130101; A61P 11/00 20180101; A61K 38/17 20130101; A61K
47/02 20130101; A61K 31/185 20130101; A61K 9/19 20130101; A61P
43/00 20180101; A61K 47/22 20130101; A61K 38/16 20130101; A61K
9/0019 20130101; A61K 47/20 20130101; A61K 31/185 20130101; A61K
2300/00 20130101; A61K 31/195 20130101; A61K 2300/00 20130101; A61K
38/16 20130101; A61K 2300/00 20130101; A61K 38/17 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/778 ;
514/784; 514/777 |
International
Class: |
A61K 47/18 20060101
A61K047/18; A61K 47/20 20060101 A61K047/20; A61K 47/22 20060101
A61K047/22 |
Claims
1. A composition comprising: (a) an alpha 1-proteinase inhibitor
(API); and (b) at least one amino acid.
2. The composition of claim 1, wherein the at least one amino acid
is present in the composition in a total amino acid amount
sufficient such that the API retains at least 50% of its activity
when the composition is exposed to a temperature for a period of
time.
3. The composition of claim 2, wherein the temperature is about
5.degree. C. to about 40.degree. C.
4. The composition of claim 2, wherein the time is at least about 6
months.
5. The composition of claim 1, wherein the at least one amino acid
is a neutral or a hydrophilic amino acid.
6. The composition of claim 1, wherein the at least one amino acid
is selected from the group consisting of alanine, threonine,
serine, hydroxyproline, glycine, proline, leucine, and
histidine.
7. The composition of claim 1, wherein the amino acid is
alanine.
8. The composition of claim 1, wherein the at least one amino acid
is present in the composition in a total amino acid amount of about
0.01 M to about 3 M.
9. The composition of claim 1 further comprising one or more
excipients.
10. The composition of claim 1, wherein the one or more excipients
is selected from the group consisting of: sucrose, mannitol,
glycerol, sorbitol, dextran, trehalose, hydroxyethyl starch (HES),
and 1,2-propanediol.
11. The composition of claim 1, wherein the composition further
comprises a pharmaceutically acceptable carrier.
12. The composition of claim 1, wherein the composition is suitable
for intravenous administration to a subject.
13. The composition of claim 1, wherein the at least one amino acid
is present in the composition in a total amino acid amount
sufficient to maintain total protein aggregation at less than about
5% following exposure of the composition to a temperature for a
period of time.
14. The composition of claim 13, wherein the temperature is about
25.degree. C., wherein the period of time is about 6 months.
15. The composition of claim 13, wherein the temperature is about
5.degree. C., wherein the period of time is about 6 months.
16. The composition of claim 15, wherein the at least one amino
acid is present in the composition in a total amino acid amount
sufficient to maintain total protein aggregation at less than about
2% following exposure of the composition to the temperature for the
period of time.
17. The composition of claim 13, wherein the pH of the composition
is about 6.3 to about 7.4.
18. The composition of claim 13, wherein the pH of the composition
is about 6.7 to about 7.1.
19. The composition of claim 1, wherein the composition is in
lyophilized form.
20. A kit comprising the composition of claim 1 as a pharmaceutical
composition suitable for intravenous administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Application No. 61/257,711 filed Nov. 3, 2009, which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions comprising
alpha 1-proteinase inhibitor (API), in particular stable API
compositions. The present invention also relates to methods and
kits for providing API to a subject, in particular for providing to
the subject a therapeutically or prophylactically effective amount
of API.
BACKGROUND OF THE INVENTION
[0003] API deficiency is a relatively common genetic disorder that
predisposes affected individuals to liver disease and/or pulmonary
emphysema. The most common type of API deficiency termed protease
inhibitor type Z (PiZ), is transmitted as an autosomal recessive
trait and affects approximately 1 in 1700 live births in most
Northern European and North American populations. The PiZ mutation
is a single nucleotide substitution that results in a single amino
acid substitution (glutamate 342 to lysine).
[0004] Human API has 394 amino acids, one cysteine residue, and
three carbohydrate side chains, giving an overall molecular weight
of 52 kDa. A single reactive center loop (RCL) is located at a
Met-Ser sequence at position 358-359. The tertiary structure of API
contains 8 well-defined .alpha.-helices (A-H) and 3 large
.beta.-sheets (A-C). It is believed that serpins function via
irreversible "suicide-substrate" mechanism. Upon cleavage by a
protease, the serpin undergoes a conformational change, whereby the
RCL is cleaved and inserted into the center of the A .beta.-sheet,
and the protease is translocated to the distal end of the molecule.
This structural transition results in stabilization of the serpin
and extensive distortion of the protease's tertiary structures,
which inactivates its catalytic machinery. It is believed that the
biological activity of API can be effected by, e.g., chemical
modifications, including inter- or intramolecular polymerization,
oxidation, complex formation, and/or cleavage by non-specific
proteinases.
[0005] It is believed that the major physiological function of API
is the inhibition of neutrophil elastase, cathepsin G, and
proteinase 3. The API produced in individuals with PiZ API
deficiency is functionally active, although there may be a decrease
in its specific elastase inhibitory capacity. The predominant site
of API synthesis is the liver, however, it is also synthesized in
extrahepatic cell types including macrophages, intestinal
epithelial cells and intestinal Paneth cells.
[0006] The pathogenesis of lung injury in API deficiency is
attributable to the marked reduction in available API activity. API
has been found to constitute greater than 90% of the neutrophil
elastase inhibitor activity in pulmonary alveolar lavage fluid.
Thus, it appears that the destructive lung disease seen in many
individuals with API deficiency is due to a perturbation in the net
balance between elastase and API within the lungs. The uninhibited
activity of neutrophil elastase, cathepsin G, and proteinase 3, in
turn, results in slow destruction of the connective tissue
integrity of the lungs. This destruction of connective tissue leads
to over distension and a reduction in the retractive force of the
lungs which results in decreased expiratory airflow. Smoking
exacerbates the problem by causing oxidative inactivation of what
API is present.
[0007] At present, treatment options for individuals with
pathologies associated with API deficiency are limited. Liver
disease associated with API deficiency has been treated by
orthotopic liver transplantation. Somatic gene therapy to replace
the defective API gene has been discussed, but has yet to be
successfully used.
[0008] There remains a need, therefore, for API compositions that
are stable and that provide desirable API plasma bioavailability
levels following administration to a subject.
SUMMARY OF THE INVENTION
[0009] There is now provided, in one aspect, a composition
comprising: (a) an alpha 1-proteinase inhibitor (API); and (b) at
least one amino acid.
[0010] In another aspect, the present invention provides methods
for preparing the composition.
[0011] In other aspects, kits comprising the composition are
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph showing the aggregation profiles of liquid
API formulations shown in Table 1 of Example 1.
[0013] FIG. 2 is a graph showing the aggregation profiles of API
formulations containing alanine, sucrose, or sucrose/alanine.
[0014] FIG. 3 is a graph showing the aggregation profiles of API
formulations containing alanine, mannitol, or mannitol/alanine.
[0015] FIG. 4 is a graph showing the aggregation profiles of API
formulations containing alanine, sucrose, or trehalose.
[0016] FIG. 5 is a graph showing the effect of amino acid
concentration on aggregation of API formulations after incubating
at 40.degree. C. for one week.
[0017] FIG. 6 is a graph showing the effect of amino acid
concentration on aggregation of API formulations after incubating
at 40.degree. C. for two weeks.
[0018] FIG. 7 is a graph showing the aggregation profiles of API
formulations containing alanine with or without different
concentrations of an oxygen scavenger.
[0019] FIG. 8 is a graph showing potency profiles of API
formulations described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0020] FIG. 9 is a graph showing nephelometry (NTU) profiles of API
formulations described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0021] FIG. 10 is a graph showing specific activity profiles of API
formulations described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0022] FIG. 11 is a graph showing deamidation profiles of API
formulations described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0023] FIG. 12 is a graph showing aggregation increase profiles of
API formulations described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0024] FIG. 13 is a graph showing aggregation profiles of API
formulations as described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0025] FIG. 14 is a graph showing oligomer level profiles of API
formulations described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0026] FIG. 15 is a graph showing monomer level profiles of API
formulations described in Example 6 at 5.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0027] FIG. 16 is a graph showing potency profiles of API
formulations described in Example 6 at 25.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0028] FIG. 17 is a graph showing NTU profiles of API formulations
described in Example 6 at 25.degree. C.: "Glycine" denotes
Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine" denotes
Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0029] FIG. 18 is a graph showing specific activity profiles of API
formulations described in Example 6 at 25.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0030] FIG. 19 is a graph showing deamidation profiles of API
formulations described in Example 6 at 25.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0031] FIG. 20 is a graph showing aggregation increase profiles of
API formulations described in Example 6 at 25.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0032] FIG. 21 is a graph showing aggregation profiles of API
formulations described in Example 6 at 25.degree. C.: "Glycine"
denotes Formulation 1 (API (50 mg/ml)/Glycine (250 mM)); "Alanine"
denotes Formulation 2 (API (50 mg/ml)/L-Alanine (250 mM));
"Alanine/Sucrose" denotes Formulation 3 (API (50 mg/ml)/L-Alanine
(125 mM)/Sucrose (125 mM)); and "NaCl" denotes Formulation 4 (API
(50 mg/ml)/NaCl (250 mM)).
[0033] FIG. 22 is a graph showing the oligomer profile of API
formulations at 25.degree. C. "Glycine" denotes Formulation 1 (API
(50 mg/ml)/Glycine (250 mM)); "Alanine" denotes Formulation 2 (API
(50 mg/ml)/L-Alanine (250 mM)); "Alanine/Sucrose" denotes
Formulation 3 (API (50 mg/ml)/L-Alanine (125 mM)/Sucrose (125 mM));
and "NaCl" denotes Formulation 4 (API (50 mg/ml)/NaCl (250
mM)).
[0034] FIG. 23 is a graph showing the monomer profile of API
formulations at 25.degree. C. "Glycine" denotes Formulation 1 (API
(50 mg/ml)/Glycine (250 mM)); "Alanine" denotes Formulation 2 (API
(50 mg/ml)/L-Alanine (250 mM)); "Alanine/Sucrose" denotes
Formulation 3 (API (50 mg/ml)/L-Alanine (125 mM)/Sucrose (125 mM));
and "NaCl" denotes Formulation 4 (API (50 mg/ml)/NaCl (250
mM)).
[0035] FIG. 24 is a 2D-contour plot for pH and protein
concentration for 1 week, 2 weeks, and 3 weeks aggregate in an API
solution at 40.degree. C.
[0036] FIG. 25 is a pH and aggregation interaction plot of an API
solution at 40.degree. C. for 1 week, 2 week, and 3 weeks.
[0037] FIG. 26 shows the pH effect on aggregation in an API
solution at 40.degree. C. for 1 week, 2 week, and 3 weeks.
[0038] FIG. 27 shows the protein concentration effect on
aggregation profile in an API solution at 40.degree. C. for 1 week,
2 week, and 3 weeks.
DETAILED DESCRIPTION
[0039] The present invention is based upon the discovery that a
relatively stable API containing composition can be prepared by
also including in the composition one or more amino acids. In
particular embodiments, the present invention encompasses a stable
API containing pharmaceutical composition suitable for providing
API for administration to a subject for therapeutic or prophylactic
effects. Non-limiting examples of disorders and diseases associated
with API (e.g., lower than normal plasma API levels) and for which
the present embodiments can be especially useful include, without
limitation, lung disease such as chronic obstructive pulmonary
disease (COPD) (e.g., emphysema), liver disease, vascular disease
(e.g., intracranial aneurysms, arterial fibromuscular dysplasia,
severe bleeding disorders, and hypertension), panniculitis, eye
disease (e.g., anterior uveitis), systemic necrotizing vasculitis,
and Wegener's granulomatosis.
I. Composition
[0040] In one aspect, the present invention provides a composition
comprising:
[0041] (i) API; and
[0042] (ii) at least one amino acid.
[0043] In some embodiments, the composition consists of or consists
essentially of the API and at least one amino acid.
[0044] The composition of the present invention can be provided in
a relatively pure form. For example, in some embodiments, the
composition can be characterized as being of a pharmaceutical
grade.
[0045] The composition can be in the form of a solid, a liquid, or
a semi-solid. For example, the composition can be a liquid
formulation including, but not limited to, an aqueous solution,
aqueous suspension, oil suspension, etc. Alternatively, the
composition can be a lyophilized preparation, dry powder, solid
particles, etc. Other examples, without limitation, include
colloidals, emulsions, gels, and ointments.
[0046] In one embodiment, the composition is a liquid formulation,
preferably an aqueous solution. In another embodiment, the
composition is suitable for pharmaceutical use, for example a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent.
[0047] In one embodiment, the composition is a pharmaceutical
composition comprising the API, the amino acid, and a
pharmaceutically acceptable carrier. As used herein
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, and the like that are
physiologically compatible. The type of carrier can be selected
based upon the intended route of administration. In some
embodiments, the carrier is suitable for administering by way of,
but not limited to, intravenous, inhalation, parenteral,
subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or transdermal
means. Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the preparation of
sterile injectable solutions or dispersion.
[0048] In some embodiments, the composition is sterile and stable
under one or more conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure. For example, sterile
injectable solutions can be prepared by filtered sterilization.
Dispersions can be prepared by incorporating the API into a sterile
vehicle which contains a dispersion medium. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which provides a composition comprising a powder of
the API and the amino acid plus any additional desired ingredient,
preferably from a previously sterile-filtered solution thereof.
[0049] Exemplary diluents (e.g., for use for dilution,
reconstitution, etc. of the composition) can include, but are not
limited to, sterile water, bacteriostatic water for injection
(BWFI), a pH buffered solution (e.g. phosphate-buffered saline),
sterile saline solution, and Ringer's solution or dextrose
solution. The diluent optionally contains a preservative. The
amount of preservative employed is determined by assessing
different preservative concentrations for compatibility with API
and preservative efficacy testing.
[0050] Inhalation therapy involves the administration of a drug in
an aerosol form to the respiratory tract. Aerosol delivery is based
on the concept that delivery to the deep lung regions (alveoli),
which account of 95% of lung epithelia, can significantly enhance
the transport of the protein through the epithelial membrane if the
molecule is bioavailable. The liquid aerosols can be generated by
nebulizing solutions of the API. Alternatively, solid particles can
be either in the form of a powder suspended in a propellant which
is administered from a metered dose inhaler or simply as a powder
that is administered from a dry powder inhaler. For example, solid
particle aerosols can be prepared by lyophilizing (e.g.,
freeze-drying) the API from solution and then milling or grinding
the lyophilized drug to the desired particle size distribution for
pulmonary administration. Another technique involves spray drying,
which is a dehydration process that utilizes heat from a hot gas
stream (usually air) to evaporate dispersed droplets created by
atomization of a continuous liquid feed. Using these methods, the
composition comprising API can be dried into fine particles,
preferably with suitable types and amounts of excipients such as
that can act as water-replacing agents.
[0051] Pressurized metered dose inhalers are now well known in the
art. Any pressurized metered dose inhaler that is suitable for
application of drugs to the lungs or nose of a patient can be used.
The aerosol formulation and metering valve can be selected to
provide a therapeutically or prophylactically effective amount of
the API.
A. API
[0052] The term "API," as used herein, is intended to be broad
unless specifically stated otherwise. The term refers to all
naturally occurring polymorphs of API. The term also includes
functional fragments of API, chimeric proteins comprising API or
functional fragments thereof, homologs obtained by analogous
substitution of one or more amino acids of API, and species
homologs. The term also refers to all API polypeptides that are a
product of recombinant DNA technology including an API that is a
product of transgenic technology. For example, the gene coding for
API can be inserted into a mammalian gene encoding a milk whey
protein in such a way that the DNA sequence is expressed in the
mammary gland as described in, e.g., U.S. Pat. No. 5,322,775, which
is herein incorporated by reference for its teaching of a method of
producing a proteinaceous compound. The term also refers to all API
proteins synthesized chemically by methods known in the art such
as, e.g., solid-phase peptide synthesis. The term also refers to
API prepared from plasma. The term also refers to API that can be
obtained commercially. The API can correspond to a human or a
non-human API.
[0053] In one embodiment, the API is plasma-derived API. In another
embodiment, the API is prepared from Cohn Fraction IV-1 paste. In
other embodiments, the API is prepared from an albumin-depleted
plasma fraction, a Cohn V precipitate, or a pre-purified API
preparation fraction. U.S. Pat. No. 6,974,792 is herein
incorporated by reference for its teaching of a method of preparing
API.
[0054] In other embodiments, the API is recombinant API. Amino acid
and nucleotide sequences for API and/or production of recombinant
API are described by, e.g., U.S. Pat. Nos. 4,711,848; 4,732,973;
4,931,373; 5,079,336; 5,134,119; 5,218,091; 6,072,029; and Wright
et al., Biotechnology, 9:830 (1991); and Archibald et al., PNAS,
87:5178 (1990), each of which is herein incorporated by reference
for its teaching of API sequences, recombinant API, and/or
recombinant expression of API.
[0055] In one embodiment, the composition is characterized as
comprising an API having a greater than 90% purity. In other
embodiments, the API has a purity greater than 95%, preferably at
least about 99%. In some embodiments, at least about 50%,
illustratively, about 50% to about 100%, about 60% to about 90%,
about 70% to about 80% of all API in the composition is active
API.
[0056] In one embodiment, the composition comprises a
therapeutically effective amount of API. A "therapeutically
effective amount" refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic
result, such as, for example, reduction or inhibition of emphysema
associated with congenital deficiency of API. A therapeutically
effective amount of API can vary according to factors such as the
disease state, age, sex, and weight of the individual subject, and
the ability of the API to elicit a desired response in the subject.
A therapeutically effective amount also can be one in which any
toxic or detrimental effects of API are outweighed by the
therapeutically beneficial effects.
[0057] In other embodiments, the composition comprises a
prophylactically effective amount of API. A "prophylactically
effective amount" refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired prophylactic
result, such as, for example, preventing or inhibiting emphysema
associated with congenital deficiency of API. A prophylactically
effective amount can be determined as described above for the
therapeutically effective amount.
[0058] One factor that may be considered when determining a
therapeutically or prophylactically effective amount of API is the
concentration of functionally active API expressed in a biological
compartment of the subject receiving the treatment, such as, for
example, in the lower respiratory tract or the epithelial lining
fluid (ELF) of the subject. Another factor that also may be
considered when determining a therapeutically or prophylactically
effective amount of API is the pharmacology (e.g.,
pharmacokinetics) of the API.
[0059] The pharmacokinetics of API refers to the concentration or
levels of API in the subject's blood. Pharmacokinetic parameters or
measures of API levels in the blood include the area under the
curve (AUC), C.sub.min (i.e., trough), and C.sub.max. The AUC is
the total exposure of API in the subject's blood over a fixed
dosing period (e.g., 8, 12, and 24 hours). The C.sub.min (i.e.,
trough level) is the lowest blood level of API during a fixed
dosing period. The C.sub.max is the highest or peak level achieved
by API in the subject's blood over a fixed dosing period.
[0060] In one embodiment, the therapeutically or prophylactically
effective amount that is administered is sufficient to achieve or
maintain an API blood (e.g., plasma) trough level above a target
threshold level. In one embodiment, the target threshold level is
at least about 10 mg/dL, illustratively, about 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, and 200 mg/dL.
[0061] In another embodiment, the therapeutically or
prophylactically effective amount is sufficient to achieve or
maintain an API blood trough level of at least about 50 mg/dL. In
other embodiments, the therapeutically or prophylactically
effective amount of API is sufficient to maintain a blood API
trough level of at least about 80 mg/dL.
[0062] In one embodiment, the concentration of API in the
composition is at least about 0.1 mg/ml, illustratively, at least
about: 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, and 1000
mg/ml. In one embodiment, the specific activity of API in the
composition is at least about 0.05 mg active API per mg total
protein, illustratively, at least about 0.05, 0.1, 0.2 mg, 0.3 mg,
0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, or more active API per mg total
protein.
[0063] In some embodiments, the concentration of the API contained
in the composition is, on a weight percentage basis (w/v), at least
about 1%, illustratively, about 1 to about 60%, about 5 to about
55%, about 10 to about 45%, about 20 to about 30% (w/v).
B. Amino Acid
[0064] It is to be understood that the amino acids specifically
recited herein are for illustrative purposes only and are not
intended to be limiting the amino acids that can be used in
accordance with the present invention. Other amino acids not
specifically recited herein can be readily categorized based on
their observed physical and chemical properties. Further, some of
the amino acids recited herein have been exemplified in terms of
the genetically encoded amino acids, however, the amino acid need
not be restricted to the genetically encoded amino acids but also
can include genetically non-encoded amino acids. For example, the
amino acid also can include naturally occurring non-encoded amino
acids and synthetic amino acids (e.g., .beta.-alanine,
hydroxyproline). Also contemplated are L-enantiomeric amino acids
(L-amino acids) and D-amino acids as well as free amino acid forms
and/or physiologically acceptable salt forms and/or mixtures
thereof.
[0065] Table 1 lists some non-limiting examples of amino acids.
TABLE-US-00001 TABLE 1 Amino Acids. Amino Acid Three Letter Code
Single Letter Code glycine Gly G alanine Ala A valine Val V leucine
Leu L isoleucine Ile I methionine Met M phenylalanine Phe F
tryptophan Trp W proline Pro P serine Ser S threonine Thr T
cysteine Cys C tyrosine Tyr Y asparagine Asn N glutamine Gln Q
lysine Lys K arginine Arg R histidine His H aspartic acid Asp D
glutamic acid Glu E
[0066] In one embodiment, the composition comprises at least one
amino acid each selected from the group consisting of: Ala (A), Thr
(T), hydroxyproline, Gly (G), Ser (S), Gln (Q), Phe (F), Trp (W),
Met (M), acetyl cysteine, Leu (L), Ile (I), Val (V), Lys (K), Pro
(P), Tyr (Y), His (H), Glu (E), Asn (N), Asp (D), and Arg (R).
[0067] In another embodiment, the at least one amino acid each is
selected from the group consisting of Ala (A), Thr (T),
hydroxyproline, Gly (G), and Ser (S).
[0068] In other embodiments, the at least one amino acid is Ala (A)
or Gly (G).
[0069] In some embodiments, the amino acid is a neutral amino acid
or a hydrophilic amino acid. In another embodiment, a hydrophilic
amino acid is an amino acid that is characterized as exhibiting a
hydrophobicity of less than zero according to a consensus
hydrophobicity scale.
[0070] In one embodiment, the at least one amino acid is present in
the composition in a total amino acid amount that is sufficient to
provide a stable API composition, for example as determined
relative to an API composition not having the at least one amino
acid.
[0071] In another embodiment, the total amino acid amount is an
amount sufficient such that the API retains at least about 5% of an
API activity for a period of time at a temperature, illustratively,
at least about 5% to about 100%, about 10% to about 95%, about 20%
to about 90%, about 30% to about 80%, about 40% to about 70%, and
about 50% to about 60% of an API activity for a period of time at a
temperature. In some embodiments, the period of time and/or the
temperature is characterized as a storage time and/or temperature
(e.g., shelf-life). In one embodiment, the period of time is at
least about: 1 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3
months, 4 months, 5 months, 6 months, 8 months, 1 year, 2 years,
and 3 years. In another embodiment, the temperature is equal to or
greater than about: -120.degree. C., -70.degree. C., -20.degree.
C., 0.degree. C., 5.degree. C., 25.degree. C., 37.degree. C.,
40.degree. C., and 50.degree. C.
[0072] In one embodiment, the API activity can be determined by
inhibition of porcine pancreatic elastase as described by, e.g.,
Coan et al., Vox Sang., 48:333 (1985), which is herein incorporated
by reference in its entirety. In some embodiments, the API activity
can be assessed as potency or as toxicity by an in vitro, in vivo,
and/or ex vivo determination (e.g. therapeutic index, LD.sub.50,
ED.sub.50, etc.) in an animal. For example, following serial
dilutions from a bulk or stock solution, or reconstitution of a
lyophilized, or vacuum dried API containing composition, the API
present in the reconstituted or aqueous composition has (in the
presence of the at least one amino acid and/or the total at least
one amino acid amount) greater than at least about 5% to about 100%
of the potency or toxicity that the biologically active API had
prior to being incorporated into the composition.
[0073] In other embodiments, stability of the API can be determined
by, for example, determining degradation of the API including, but
not limited to, formation of oxidation, proteolytic, and/or
deamidation products, changes in molecular weight distribution,
changes in potency, changes in functionality, increases in
insoluble matter such as aggregates or modification of the product
due to the presence of excipients, such as non-enzymatic glycation.
In addition, stability can be determined as a change in
concentration due to, for example, volatilization, adsorption,
chemical modification, and the like. Accordingly, in some
embodiments, storage stable API compositions are those compositions
that show less than 5, 10, 15, 20, 25, 30, 40, 50, 60, 70% API
degradation. Storage stable compositions also can show any
combination of these characteristics.
[0074] In another embodiment, the total amino acid amount of the
composition is an amount sufficient to increase a melting point of
API relative to a composition without the at least one amino acid.
One of ordinary skill in the art can determine the melting point,
for example using a differential scanning calorimeter (DSC). In one
embodiment, the total amino acid amount of the composition is
sufficient to raise the melting point by at least about:
0.01.degree. C., 0.1.degree. C., 1.degree. C., 1.2.degree. C.,
1.4.degree. C., 1.6.degree. C., 1.8.degree. C., and 2.degree.
C.
[0075] In other embodiments, the total amino acid amount of the
composition, after storage of the composition at a period of time
at a temperature, is an amount sufficient such that the percentage
of monomeric API in the composition is at least about 50%,
illustratively, about 50% to about 100%, about 60% to about 99%,
about 98%, about 70% to about 98%, about 80% to about 95%, and
about 85% to about 90%. In one embodiment, the period of time is at
least about: 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 2
months, 3 months, 4 months, 5 months, 6 months, 8 months, 1 year, 2
years, and 3 years. In another embodiment, the temperature is equal
to or greater than about: -120.degree. C., -70.degree. C.,
-20.degree. C., 0.degree. C., 5.degree. C., 25.degree. C.,
37.degree. C., 40.degree. C., and 50.degree. C.
[0076] In another embodiment, the total amino acid amount of the
composition is an amount sufficient such that, after storage of the
composition for a period of time at a temperature, the percentage
of protein aggregates in the composition is less than or equal to
about: 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,
0.5%, and 0%. In one embodiment, the period of time is at least
about: 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 2 months,
3 months, 4 months, 5 months, 6 months, 8 months, 1 year, 2 years,
and 3 years. In another embodiment, the temperature is equal to or
greater than about: -120.degree. C., -70.degree. C., -20.degree.
C., 0.degree. C., 5.degree. C., 25.degree. C., 37.degree. C.,
40.degree. C., and 50.degree. C.
[0077] Protein aggregation levels in a composition can be
determined by one of ordinary skill in the art, for example by
using size-exclusion high-performance liquid chromatography
(SE-HPLC).
[0078] In one embodiment, the total amino acid amount that is
present in the composition is at least about 0.01 M,
illustratively, about 0.01 to about 3 M, about 0.03 to about 1 M,
about 0.05 to about 0.5 M, and about 0.1 to about 0.3 M.
[0079] In another embodiment, the total amino acid amount that is
present in the composition is at least about 0.01 M, at least about
0.03, or at least about 0.3 M.
[0080] In other embodiments, the composition comprises alanine at a
total alanine concentration of at least about 0.01 M alanine,
illustratively, about 0.01 to about 1 M and about 0.01 to about
0.75 M alanine. In some embodiments, the at least one amino acid is
alanine, wherein the composition does not contain an amino acid
other than the alanine.
[0081] In one embodiment, the composition comprises threonine at a
total threonine concentration of at least about 0.01 M threonine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M threonine. In some embodiments, the at least one amino acid is
threonine, wherein the composition does not contain an amino acid
other than the threonine.
[0082] In another embodiment, the composition comprises
hydroxyproline at a total hydroxyproline concentration of at least
about 0.01 M hydroxyproline, illustratively, about 0.01 to about 1
M and about 0.1 to about 0.3 M hydroxyproline. In some embodiments,
the at least one amino acid is hydroxyproline, wherein the
composition does not contain an amino acid other than the
hydroxyproline.
[0083] In one embodiment, the composition comprises glycine at a
total glycine concentration of at least about 0.01 M glycine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M glycine. In some embodiments, the at least one amino acid is
glycine, wherein the composition does not contain an amino acid
other than the glycine.
[0084] In other embodiments, the composition comprises serine at a
total serine concentration of at least about 0.01 M serine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M serine. In some embodiments, the at least one amino acid is
serine, wherein the composition does not contain an amino acid
other than the serine.
[0085] In one embodiment, the composition comprises glutamine at a
total glutamine concentration of at least about 0.01 M glutamine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M glutamine. In some embodiments, the at least one amino acid is
glutamine, wherein the composition does not contain an amino acid
other than the glutamine.
[0086] In other embodiments, the composition comprises
phenylalanine at a total phenylalanine concentration of at least
about 0.01 M phenylalanine, illustratively, about 0.01 to about 1 M
and about 0.1 to about 0.3 M phenylalanine. In some embodiments,
the at least one amino acid is phenylalanine, wherein the
composition does not contain an amino acid other than the
phenylalanine.
[0087] In one embodiment, the composition comprises tryptophan at a
total tryptophan concentration of at least about 0.01 M tryptophan,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M tryptophan. In some embodiments, the at least one amino acid is
tryptophan, wherein the composition does not contain an amino acid
other than the tryptophan.
[0088] In another embodiment, the composition comprises methionine
at a total methionine concentration of at least about 0.01 M
methionine, illustratively, about 0.01 to about 1 M and about 0.1
to about 0.3 M methionine. In some embodiments, the at least one
amino acid is methionine, wherein the composition does not contain
an amino acid other than the methionine.
[0089] In other embodiments, the composition comprises acetyl
cysteine at a total acetyl cysteine concentration of at least about
0.01 M acetyl cysteine, illustratively, about 0.01 to about 1 M and
about 0.1 to about 0.3 M acetyl cysteine. In some embodiments, the
at least one amino acid is acetyl cysteine, wherein the composition
does not contain an amino acid other than the acetyl cysteine.
[0090] In one embodiment, the composition comprises leucine at a
total leucine concentration of at least about 0.01 M leucine,
illustratively, about 0.01 to about 1 M and about 0.03 to about
0.15 M leucine. In some embodiments, the at least one amino acid is
leucine, wherein the composition does not contain an amino acid
other than the leucine.
[0091] In another embodiment, the composition comprises isoleucine
at a total isoleucine concentration of at least about 0.01 M
isoleucine, illustratively, about 0.01 to about 1 M and about 0.1
to about 0.3 M isoleucine. In some embodiments, the at least one
amino acid is isoleucine, wherein the composition does not contain
an amino acid other than the isoleucine.
[0092] In other embodiments, the composition comprises valine at a
total valine concentration of at least about 0.01 M valine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M valine. In some embodiments, the at least one amino acid is
valine, wherein the composition does not contain an amino acid
other than the valine.
[0093] In one embodiment, the composition comprises lysine at a
total lysine concentration of at least about 0.01 M lysine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M lysine. In some embodiments, the at least one amino acid is
lysine, wherein the composition does not contain an amino acid
other than the lysine.
[0094] In another embodiment, the composition comprises proline at
a total proline concentration of at least about 0.01 M proline,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M proline. In some embodiments, the at least one amino acid is
proline, wherein the composition does not contain an amino acid
other than the proline.
[0095] In other embodiments, the composition comprises serine at a
total tyrosine concentration of at least about 0.01 M tyrosine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M tyrosine. In some embodiments, the at least one amino acid is
tyrosine, wherein the composition does not contain an amino acid
other than the tyrosine.
[0096] In one embodiment, the composition comprises histidine at a
total histidine concentration of at least about 0.01 M histidine,
illustratively, about 0.01 to about 1 M and about 0.1 to about 0.3
M histidine. In some embodiments, the at least one amino acid is
histidine, wherein the composition does not contain an amino acid
other than the histidine.
[0097] In another embodiment, the composition comprises glutamic
acid at a total glutamic acid concentration of at least about 0.01
M glutamic acid, illustratively, about 0.01 to about 1 M and about
0.1 to about 0.3 M glutamic acid. In some embodiments, the at least
one amino acid is glutamic acid, wherein the composition does not
contain an amino acid other than the glutamic acid.
[0098] In other embodiments, the composition comprises asparagine
at a total asparagine concentration of at least about 0.01 M
asparagine, illustratively, about 0.01 to about 1 M and about 0.1
to about 0.3 M asparagine. In some embodiments, the at least one
amino acid is asparagine, wherein the composition does not contain
an amino acid other than the asparagine.
[0099] In one embodiment, the composition comprises aspartic acid
at a total aspartic acid concentration of at least about 0.01 M
aspartic acid, illustratively, about 0.01 to about 1 M and about
0.1 to about 0.3 M aspartic acid. In some embodiments, the at least
one amino acid is aspartic acid, wherein the composition does not
contain an amino acid other than the aspartic acid.
[0100] In another embodiment, the composition comprises arginine at
a total arginine concentration of at least about 0.01 M arginine,
illustratively, about 0.01 to about 1 M and about 0.17 to about 0.3
M arginine. In some embodiments, the at least one amino acid is
arginine, wherein the composition does not contain an amino acid
other than the arginine.
C. Excipients
[0101] In other embodiments, the present invention provides a
composition comprising:
[0102] (i) an API;
[0103] (ii) at least one amino acid, wherein the composition
further comprises one or more excipients. Non-limiting examples of
excipients include polyols, sugars, surfactants, antioxidants,
salts (e.g., inorganic salts), metal chelators, polymers (e.g.,
polyethylene glycol (PEG), cyclodextrin, dextran), urea, guanidium
chloride, polypeptides (e.g., antibodies), and combinations
thereof. Accordingly, one of skill in the art will understand that
the term "excipients" as used herein is intended to be broad and
can include such agents as commonly used by one of ordinary skill
in the art to prepare protein containing compositions.
[0104] In some embodiments, the excipient is a polyol or a sugar.
For example, the polyol or sugar can be, but is not limited to,
sucrose, mannitol, trehalose, sorbitol, sorbose, melezitose,
glycerol, fructose, mannose, maltose, lactose, arabinose, xylose,
ribose, rhamnose, galactose, glucose, xylitol, erythritol,
threitol, raffinose, and/or the like.
[0105] In one embodiment, the one or more excipients each is
selected from the group consisting of: sucrose, mannitol, glycerol,
sorbitol, dextran, trehalose, hydroxyethyl starch (HES), saccharin
sodium, polyethylene glycol (PEG) (e.g., PEG 3350, PEG 2000, PEG
8000), and 1,2-propanediol. In another embodiment, the excipient is
sucrose or mannitol.
[0106] In other embodiments, the total amount of sucrose, if
present in the composition, is less than 20% (w/v), illustratively,
about 0% to about 15% (w/v), about 2.5% to about 7.5%, and about 4%
to about 5% (w/v). In one embodiment, the composition does not
contain sucrose.
[0107] The total amount of each excipient and/or the total
excipient amount of the composition can vary, for example depending
on the type and/or amount of excipients used and/or the type and/or
amount of other components present in the composition comprising
the API.
[0108] In one embodiment, the composition comprises a sugar at a
total sugar concentration of at least about 0.01 M sugar,
illustratively, about 0.01 to about 1 M and about 0.125 to about
0.75 M sugar. In some embodiments, the sugar is sucrose. In other
embodiments, the sugar is sucrose, wherein the composition does not
contain a sugar other than the sucrose.
[0109] In another embodiment, the composition comprises a polyol at
a total polyol concentration of at least about 0.01 M polyol,
illustratively, about 0.01 to about 1 M and about 0.25 to about
0.75 M polyol. In some embodiments, the polyol is mannitol. In
other embodiments, the polyol is mannitol, wherein the composition
does not contain a polyol other than the mannitol.
[0110] In other embodiments, the composition comprises a polyol at
a total polyol concentration of at least about 0.25% (w/v) polyol,
illustratively, about 0.25 to about 5% (w/v) polyol. In some
embodiments, the polyol is glycerol. In other embodiments, the
polyol is glycerol, wherein the composition does not contain a
polyol other than the glycerol.
[0111] In one embodiment, the composition comprises a polyol at a
total polyol concentration of at least about 0.01 M polyol,
illustratively, about 0.01 to about 1 M and about 0.27 to about
0.55 M polyol. In some embodiments, the polyol is sorbitol. In
other embodiments, the polyol is sorbitol wherein the composition
does not contain a polyol other than the sorbitol.
[0112] In another embodiment, the composition comprises a polymer
at a total polymer concentration of at least about 0.25% (w/v)
polymer, illustratively, about 0.25 to about 5% (w/v) polymer. In
some embodiments, the polyol is dextran. In other embodiments, the
polymer is dextran, wherein the composition does not contain a
polymer other than the dextran.
[0113] In other embodiments, the composition comprises a sugar at a
total sugar concentration of at least about 0.01 M sugar,
illustratively, about 0.01 to about 1 M and about 0.15 to about
0.75 M sugar. In some embodiments, the sugar is trehalose. In other
embodiments, the sugar is trehalose, wherein the composition does
not contain a sugar other than the trehalose.
[0114] In one embodiment, the composition comprises HES at a total
HES concentration of at least about 0.25% (w/v) HES,
illustratively, about 0.25 to about 5% (w/v) HES.
[0115] In another embodiment, the composition comprises saccharin
sodium at a total saccharin sodium concentration of at least about
0.01 M saccharin sodium, illustratively, about 0.01 to about 1 M
and about 0.15 to about 0.75 M saccharin sodium.
[0116] In other embodiments, the composition comprises a PEG at a
total PEG concentration of at least about 0.25% (w/v) PEG,
illustratively, about 0.25 to about 10% (w/v) PEG, and about 1 to
about 5% (w/v) PEG. In some embodiments, the PEG is PEG 3350. In
other embodiments, the PEG is about 5% (w/v) PEG 3350, wherein the
composition does not contain a PEG other than the PEG 3350.
[0117] In one embodiment, the composition comprises 1,2-propanediol
at a total 1,2-propanediol concentration of at least about 0.01 M
1,2-propanediol, illustratively, about 0.01 to about 1 M and about
0.125 to about 0.3 M 1,2-propanediol.
[0118] Surfactants can include, without limitation, e.g., block
polymers of polyethylene and polypropylene glycol (e.g., Pluronic
F68); or polyethylene glycol sorbitan monolaurates (e.g., Tween
80), polyoxyethylenesorbitan monooleates (e.g., Tween 20), and/or
the like. In one embodiment, the surfactant is Pluronic F68 or
Tween 80 (i.e., Polysorbate 80).
[0119] In one embodiment, the composition further comprises a
surfactant at a total surfactant concentration of at least about
0.0025% (v/v) surfactant, illustratively, about 0.0025 to about 1%
(v/v) surfactant, about 0.025 to about 0.1% (v/v) surfactant, and
about 0.05 to about 0.075% (v/v) surfactant. In some embodiments,
the surfactant is Pluronic F68 or Tween 80.
[0120] In another embodiment, the present invention provides a
composition comprising:
[0121] (i) an API;
[0122] (ii) at least one amino acid, wherein the composition
further comprises a salt, preferably an inorganic salt. Without
limitation, examples of inorganic salts include sodium chloride or
sodium phosphate.
[0123] In one embodiment, the inorganic salt is present in the
composition in a total inorganic salt amount of at least 0.01 M,
illustratively, about 0.01 to about 0.5 M, about 0.05 to about 0.4
M, and about 0.1 to about 0.15 M inorganic salt. In some
embodiments, the inorganic salt is sodium chloride, sodium
phosphate, or a combination thereof.
[0124] As used herein, the term "antioxidant" refers to an agent
that reduces or prevents oxidation. "Antioxidants" include, but are
not limited to, methionine, glutathione, acetylcysteine, ascorbic
acid, butyl hydroxyl anisol (BHA), butyl hydroxyl toluene (BHT),
and maleic acid.
[0125] In one embodiment, the present invention provides a
composition comprising:
[0126] (i) an API;
[0127] (ii) at least one amino acid, wherein the composition
further comprises an antioxidant. In some embodiments, the
antioxidant is methionine. In other embodiments, the antioxidant is
glutathione. In another embodiment, the antioxidant is present in
the composition in a total antioxidant amount of at least about
0.01% (w/v), illustratively, about 0.01 to about 5% (w/v), and
about 0.05 to about 0.7% (w/v).
[0128] Non-limiting examples of a metal chelator include
ethylenediaminetetraacetic acid (EDTA), (0.1-0.5%), [ethylenebis
(oxyethylenenitrilo)] tetraacetic acid (EGTA),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
hydroxyethlene triamine diacetic acid (HEDTA), or salts thereof. In
one embodiment, the metal chelator is a divalent metal chelator. In
another embodiment, the metal chelator is EDTA, EGTA, BAPTA, HEDTA,
or salts thereof. In some embodiments, the composition comprises a
combination of metal chelators. In other embodiments, the metal
chelator is present in the composition at a total chelator amount
of at least about 0.01% (w/v), illustratively, about 0.01 to about
1% (w/v), about 0.05 to about 0.5% (w/v), and about 0.1 to about
0.25%.
[0129] In another embodiment, the present invention provides a
composition comprising:
[0130] (i) an API;
[0131] (ii) at least one amino acid, wherein the composition
further comprises a metal chelator.
[0132] Accordingly, in other embodiments, the present invention
provides a composition comprising:
[0133] (a) API; and
[0134] (b) an amino acid,
wherein the composition further comprises, or optionally further
comprises, one or more excipients.
[0135] A variety of different API containing compositions are
provided in accordance with the present invention. The active
ingredient within such formulations is API, which can be human or
non-human recombinant API but, as indicated above, also may include
API extracted from plasma sources. Although the API can be present
by itself as the sole active ingredient, the API also may be
present with one or more additional active ingredient.
II. Method for Preparing the Composition
[0136] In other aspects, the present invention provides a method
for preparing a composition comprising API and at least one amino
acid.
[0137] In one embodiment, the method comprises dialyzing a first
composition comprising API to remove a salt, if present in the
composition.
[0138] For example, the first composition can be an API containing
composition prepared in or contacted with a buffer, wherein the
first composition comprises the salt. The composition can be, but
is not limited, to a composition obtained from a bulk manufacturing
process for preparing API from plasma (e.g., human plasma) or
recombinant API preparations.
[0139] In one embodiment, the first composition comprises API in a
phosphate buffer (e.g., 20 mM sodium phosphate, pH 7), wherein
sodium chloride is present in the buffer, e.g., at about 100 mM
NaCl. In some embodiments, dialyzing is preformed using
ultrafiltration/diafiltration (UF/DF).
[0140] In some embodiments, the method further comprises providing
at least one amino acid to the composition following the step of
dialyzing. Excipients and/or any other reagents, etc. may be added
along with the amino acid, following amino acid addition, or
subsequent to amino acid addition.
[0141] In other embodiments, the method comprises dialyzing a first
composition comprising API against a buffer (e.g., a phosphate
buffer 20 mM, pH 7) comprising the at least one amino acid and/or
excipients and/or any other reagent.
[0142] In one embodiment, the method further comprises maintaining
the pH of the API containing composition at about 6.0 to about 8.0,
preferably about 6.6 to about 7.4. For example, in another
embodiment, a buffer salt can be added first to achieve the
effective pH range, followed by addition of amino acids and/or
excipients and/or any other reagents/ingredients. In other
embodiments, amino acids and/or excipients, and or other reagents
can be combined for adding to the API containing composition.
III. KIT
[0143] In other aspects, the present invention further provides a
kit, e.g., a kit for treating a disease or condition associated API
expression or activity. In one embodiment, the kit comprises a
composition in accordance with the present invention, in particular
a pharmaceutical composition comprising an API and an amino acid;
and, optionally, instruction for use in treating or preventing the
disease or condition associated API, for example a disease or
condition associated with or caused by lower than normal plasma API
levels.
[0144] In other embodiments, separate containers are provided by
the kit, wherein at least one container comprises the API
containing composition, and, in one or more further containers, a
different composition, in particular a composition comprising an
active ingredient other than API. Optionally, the kit can contain
still further pharmaceutically acceptable carriers and diluents in
combination with the active ingredients, and/or, separate therefrom
in a separate container.
[0145] The present invention will be illustrated in more detail by
way of Examples, but it is to be noted that the invention is not
limited to the Examples.
EXAMPLES
Example 1
Stability of Liquid Formulations Comprising API
[0146] An accelerated stability study was performed in order to
determine the stability of liquid formulations comprising API.
[0147] API prepared from a fraction IV-1 manufacturing and macro
bench lot was subjected to dialysis against phosphate buffers (20
mM, pH 7) to remove salt and then concentrated to 100 mg/ml.
Following dialysis, various API formulations containing other
ingredients were obtained by adding a 2-fold solution containing
the other ingredient(s). The final API formulation was then tested
for API stability by adding about 2 ml of the final formulation
into a 10 ml tubing vial, which was then incubated in a
temperature-controlled chamber at 40.degree. C. for .about.3-4
weeks. Samples were collected every week to determine aggregate
levels using SEC-HPLC.
[0148] The result are shown in FIG. 1 and summarized in Table
2.
TABLE-US-00002 TABLE 2 Percent aggregate levels of API liquid
formulations. Formulation % Aggregation at 40.degree. C. (50 mg/ml
API) Week 0 Week 1 Week 2 Week 3 API only 0.1 1.6 3.9 5.7 API +
0.1M NaCl 0.2 1.8 4.0 6.9 API + 0.3M Hydroxyproline 0.3 1.1 2.5 4.3
API + 0.3M Serine 0.3 1.0 2.2 4.2 API + 0.3M Glycine 0.2 1 2.2 4.0
API + 0.3M Alanine 0.2 1 2.1 3.8 API + 0.3M Mannitol 0.2 1 2.2 3.6
API + 0.3M Threonine 0.3 1 2 3.6 API + 0.3M Sucrose 0.2 0.7 1.5
2.6
[0149] As shown in FIG. 1 and Table 2, the formulation containing
API alone and the formulation containing 0.1 M NaCl exhibited the
most amount of aggregation. The API formulation containing sucrose
was the most stable with the least amount of aggregates forming
over time.
[0150] The accelerated stability study also was conducted at
40.degree. C. with liquid API formulations containing API (50
mg/mL); API (50 mg/mL) plus alanine (0.25M) or sucrose (0.25M); and
API (50 mg/mL) plus alanine (0.125M)/sucrose (0.125M).
[0151] As shown in FIG. 2, alanine alone conferred a degree of
stability to an API containing composition as determined by percent
aggregate levels over time. Moreover, combining a limited amount of
sucrose with alanine also provided a degree of stability, which was
greater than alanine alone.
Example 2
Stability of Liquid Formulations Comprising API
[0152] The accelerated stability study described in Example 1 also
was conducted at 40.degree. C. with liquid API formulations
containing API (50 mg/mL); API (50 mg/mL) plus alanine (0.25M) or
mannitol (0.25M); and API (50 mg/mL) plus alanine (0.125M)/mannitol
(0.125M).
[0153] As shown in FIG. 3, alanine alone conferred a degree of
stability to an API containing composition as determined by percent
aggregate levels over time. Moreover, combining a limited amount of
mannitol with alanine also provided a degree of stability, which
was greater than alanine alone.
Example 3
Stability of Liquid Formulations Comprising API
[0154] The accelerated stability study described in Example 1 also
was conducted at 40.degree. C. to determine the stabilization
effect of sucrose, trehalose, and alanine at various
concentrations. In order to assess the stabilization effects of
sucrose, trehalose and alanine, ten formulations containing these
ingredients at 0.25 M, 0.5 M, and 0.75 M, respectively, were
prepared and aseptically filled into 5 ml tubing vials. The
aggregation of these samples at 40.degree. C. was tested for 4
weeks.
[0155] The results are shown in FIG. 4. The starting aggregate
level was 0. During the 4 week incubation, no aggregation of API
was detected in 0.75 M sucrose or 0.75 M trehalose formulations. No
aggregation was detected at the first week at 40.degree. C. API
without the presence of the various ingredients aggregated at the
first week and the aggregate level was highest at each time point.
Aggregate level decreased with the increase in the concentration of
the added ingredients. Trehalose provided the best stabilization
effect against aggregation, followed by sucrose and alanine.
Example 4
Stability of Liquid Formulations Comprising API
[0156] The accelerated stability study described in Example 1 also
was conducted at 40.degree. C. to determine the effect of the
concentration of alanine or glycine on API formulations. Alanine or
glycine at 0 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, or 0.3 M were added
to API liquid formulations and the aggregate levels compared at
different time intervals at 40.degree. C. All formulations started
with about 1.3-1.4% aggregate levels.
[0157] FIG. 5 shows the aggregate levels in API liquid solutions
formulated with different concentrations of alanine or glycine
after incubating at 40.degree. C. for 1 week. The aggregate level
is reduced with the increase in the alanine or glycine
concentration. FIG. 6 shows the aggregate levels at 2 weeks. The
results agree with the 1 week data. The aggregate level is reduced
with the increase in alanine or glycine concentration. Aggregation
in 3 and 4 weeks also indicate that the aggregation is less with
more alanine or glycine added (data not shown).
Example 5
Stability of Liquid Formulations Comprising API
[0158] The accelerated stability study described in Example 1 also
was conducted at 40.degree. C. to determine the effect of oxygen
scavengers on aggregation. Thus, liquid formulations containing API
(50 mg/ml) and alanine (0.25 M) were prepared that further
contained 0.3%, 0.5%, and 0.7% methionine.
[0159] FIG. 7 shows the aggregation profile at 40.degree. C. The
effect of methionine against aggregation is minimal. No significant
reduction in aggregate level is observed.
Example 6
Stability of Liquid Formulations Comprising API
[0160] A bulk solution comprising alpha-1 proteinase inhibitor
(API) (prepared from human plasma; 65 mg/ml potency) was obtained
in 20 mM sodium phosphate buffer containing 100 mM sodium chloride.
Ultrafiltration/diafiltration (UF/DF) was applied to dialyze the
bulk solution (30 L) into phosphate buffer (20 mM, pH 7), using 240
L of phosphate buffer to reach the end point. Amino acids and/or
other ingredients were added afterwards and the final protein
concentration was adjusted to 50 mg/ml. The final formulation
solution was filtered through a 0.22 .mu.m filter and filled under
a laminar flow hood. Filling volume was 20.0 ml. The vials were
oversealed and labeled.
[0161] Formulation 1 contained 250 mM glycine; Formulation 2
contained 250 mM L-alanine; Formulation 3 contained 125 mM
L-alanine and 4.2% sucrose; and Formulation 4 contained 100 mM
sodium chloride. For each formulation, API was 50 mg/ml and the
buffer was 20 mM sodium phosphate buffer (mixture of monobasic and
dibasic heptahydrate). For a lyophilized product, additional assays
including solubility time/visual appearance after reconstitution
and cake moisture were performed.
[0162] Samples were placed on stability at 5.degree. C. and
25.degree. C. The following tests were conducted for initial (time
0) sample per formulation: appearance, fibrils, potency, pH,
SEC-HPLC, measurement of light absorbance at A.sub.280,
Nephelometry (NTU) measurement, protein composition by capillary
zone electrophoresis (CZE), deamidation and dynamic light
scattering (DLS). Determination of sodium was performed by Atomic
Absorption Spectrophotometry; determination of sucrose was
performed by ion chromatography; nephelometry: turbidity
determinations were performed using the HACH Ratio Turbidimeter;
and quantitative determination of API was preformed using a 96-well
plate based potency assay.
[0163] Table 3 shows the results from initial analytical
testing.
TABLE-US-00003 TABLE 3 Results of initial analytical tests.
Formulation Lyophi- Test 1 2 3 4 lized Appearance (Protein None
None None None .dagger. particles only) Appearance L{circumflex
over ( )}coord 96.45 96.37 96.38 96.03 96.2 By CIE a{circumflex
over ( )}coord -0.61 -0.6 -0.56 -0.76 -0.72 b{circumflex over (
)}coord 2.55 2.52 2.44 2.81 2.78 NTU 2.89 2.83 2.62 4.77 4.71 pH
6.97 7.00 7.03 6.92 7.01 Alpha potency 51.39 52.57 50.55 52.89
51.72 (mg/mL) A.sub.280 AU 25.3 25.3 24.9 25.6 24.5 Specific
activity 2.03 2.08 2.03 2.07 2.11 Alpha HPLC Region 1 1.78 1.75
1.65 2.04 1.44 (% area) Region 2 6.45 6.43 6.43 6.42 6.51 Region 3
3.27 3.17 3.13 3.14 3.44 Region 4 0 0 0 0 0 Region 5 88.44 88.57
88.73 88.33 88.52 Fragment 0.07 0.09 0.07 0.08 0.11 area CZE Gamma
0 0 0 0 0 (% area) Beta 0 0 0 0 0 Alpha 2 2.9 3.3 3.0 3.2 3.4 Alpha
1 97.1 96.7 97.1 96.8 96.7 Albumin 0 0 0 0 0 Sodium (mEq/L) 51.07
51.33 51.08 153.83 152.31 Chloride (mEq/L) n/a n/a n/a 96.0 88.0
Phosphate (mEq/L) 1472.4 1472.3 1445.6 1511.7 1765.3 Deamidation (%
area) 5.9 6.4 6.4 6.7 n/a DLS 1 Radius (nm) 3.7 3.6 n/a 3.9 n/a %
Mass 99.9 99.9 n/a 99.9 n/a 2 Radius (nm) 23 25 n/a 25.1 n/a % Mass
0.1 0.1 0 0.1 n/a 3 Radius (nm) n/a n/a n/a n/a n/a % Mass n/a n/a
n/a n/a n/a 4 Radius (nm) 188 n/a 365.3 n/a n/a % Mass 0 n/a 0 n/a
n/a .dagger. Off white cake, no protein particles, clear, 120 (pale
yellow), solubilization time: 1 min.
[0164] 5.degree. C. stability data: At 5.degree. C., few fibrils
were observed in API formulated with alanine/sucrose (formulation
3) at 3 and 6 months. No fibril was noted in any other formulations
for any time point. No significant change was observed in potency
(FIG. 8), NTU (FIG. 9), pH, specific activity (FIG. 10), DLS and
deamidation measurement (FIG. 11). The final container precision
for potency (BCS) is 5%. The intermediate precision for NTU and
deamidation are 3.0% and 18.5% respectively. The difference
observed is within assay variability.
[0165] Size Exclusion chromatography (SEC-HPLC) separates the
sample's protein based on the molecule's hydrodynamic volume. In
SEC-HPLC, molecules with a large hydrodynamic volume have minimal
(if any) interaction with the column's packing and elute off at the
column's void volume (V.sub.0). Molecules with a smaller
hydrodynamic volume interact more with the column's packing and
elute off the column after the void volume.
[0166] According to the stability data, API formulated with NaCl
(Formulation 4) displayed slightly higher aggregate level compared
to other formulations as determined by SEC-HPLC. At 6 months, the
aggregate level increased by 0.66%, 0.58%, 0.43% and 0.42% in NaCl
(Formulation 4), glycine (Formulation 1), alanine (Formulation 2)
and alanine/sucrose (Formulation 3) samples (FIGS. 12 and 13),
respectively; the oligomer peaks appeared to remain unchanged (FIG.
14); and the monomer levels decreased by 0.87%, 0.61%, 0.57% and
0.65%, respectively (FIG. 15).
[0167] 25.degree. C. stability data: At 25.degree. C., no fibril
was observed in all formulations at 6 months. Little change was
observed by potency (FIG. 16), pH and DLS. NTU increased with NaCl
reaching the highest value 7.88 (FIG. 17).
[0168] The specific activity decreased by 0.2-0.3 in all
formulations over 6 months (FIG. 18). This decrease can be
attributed to increase in the A.sub.280 absorbance, which increased
from 25 AU to approximately 28 AU in all formulations over 6 months
(FIG. 19).
[0169] Deamidation might occur in the bulk production process,
which may explain the approximately 6% deamidation level of the
starting material. Deamidation increased from initial average of 6%
to 20% in 6 months (FIG. 19). Even though Formulation 4 exhibits
slightly lower deamidation level (17%) relative to other
formulations (approximately 20%), the difference is within the
assay variability of 18.5%.
[0170] Aggregate level and oligomer level increased (FIGS. 21-23).
Monomer levels decreased (FIG. 23) over time. The decrease in
monomer level is comparable to the sum of aggregate and oligomer
increase. In 6 months, the aggregate level increased by 3.52%,
3.12%, 2.6% and 2.25% in Formulation 4, Formulation 1, Formulation
2, and Formulation 3, respectively (FIG. 20).
[0171] After incubation at 5.degree. C. for 6 months, no obvious
change was observed by potency, NTU, pH, specific activity, DLS and
deamidation measurements. There were slight increases (<1%) in
aggregate level and corresponding decreases in monomer level. These
results show stability of API formulations at 5.degree. C. for 6
months.
[0172] Following incubation at 25.degree. C. for 6 months,
significant increase in aggregate level and NTU was observed in
Formulation 4 API plus NaCl). Glycine, alanine, and alanine/sucrose
reduced aggregation and NTU with alanine/sucrose being the most
effective stabilizer. Deamidation increased dramatically. These
results indicate that liquid formulations comprising API are
subject to aggregation and deamidation at 25.degree. C., and that
amino acids provide protection against aggregation. Extrapolation
from aggregation data at 5.degree. C. (FIG. 13) shows the predicted
time to reach 5% aggregate level is .about.25 months for
Formulation 4, 44 months for Formulation 2, and 51 months for
Formulation 3. The amino acids are therefore expected to confer
significant protein stabilization, and increase shelf life
substantially.
Example 7
Melting Points of Liquid Formulations Comprising API
[0173] DSC was performed to measure the transition midpoints
(T.sub.m) of various API formulations in order to determine their
melting points.
[0174] API formulations containing API (50 mg/ml) were prepared
containing various ingredients. Briefly, API solutions were
dialyzed against phosphate buffers (20 mM, pH 7) containing each of
the ingredients listed in Table 4. The results are shown in Table
4.
TABLE-US-00004 TABLE 4 Melting points of API formulations.
Ingredient Concentration (mM) Melting Points (.degree. C.) -- 0
61.54 Alanine 300 64.10 Threonine 300 64.36 Hydroxyproline 300
64.07 Glycine 300 64.43 Serine 300 64.37 Sucrose 300 64.29
Glutamine 30 62.13 Phenylalanine 30 62.02 Tryptophan 30 61.26
Methionine 30 61.32 Acetyl cysteine 30 63.91 Leucine 30 62.24
Isoleucine 30 62.50 Valine 300 62.70 Lysine 30 63.61 Arginine 300
62.88
[0175] The melting point of the API protein in pH 7 phosphate
buffer is 61.54.degree. C. An increase in melting point can be
served as an indicator for the increase in the conformational
stability of the API protein.
Example 8
Effect of pH and Protein Concentration on Liquid Formulations
Comprising API
[0176] To determine the effect of pH and protein concentration on
aggregation, API solutions prepared at different pH and protein
concentrations were incubated at 40.degree. C., and the aggregation
data were subjected to E-CHIP analysis. The protein concentration
was calculated by measuring light absorbance at 280 nm where
A280/0.53 corresponds to a concentration value of 50 mg/ml. The
aggregation data at 40.degree. C. were determined for the trials
listed in Table 5.
TABLE-US-00005 TABLE 5 Studies regarding the effect of pH and
protein concentration on the aggregation of API formulations. Trial
API concentration (mg/ml) pH 3 50 6.3 9A 50 6.7 9B 50 6.7 6 25 7.1
4 50 7.1 8 25 6.3 5 75 7.1 7 75 6.3 9C 50 6.7 9D 50 6.7 1 25 6.7 2
75 6.7 9E 50 6.7
[0177] The E-CHIP model suggested the optimum pH for minimum
aggregation at 50 mg/ml API is 6.9 (FIG. 24). Aggregation was
observed at pH 6.3 and the aggregate amount was much less at pH 6.7
and 7.1 (FIG. 25). Aggregation decreased with the increase in pH
and reached a minimum value at pH 6.9 (FIG. 26). FIG. 27 shows that
the aggregation increased almost linearly with protein
concentration.
* * * * *