U.S. patent application number 09/810483 was filed with the patent office on 2002-01-24 for powder containing physiologically active peptide.
This patent application is currently assigned to JCR PHARMACEUTICALS CO., Ltd.. Invention is credited to Hanyu, Yoshinobu, Horie, Masato, Nishimuro, Satoshi, Okada, Mariko, Shindo, Chihiro, Yokoyama, Tetsuo.
Application Number | 20020009789 09/810483 |
Document ID | / |
Family ID | 27342741 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009789 |
Kind Code |
A1 |
Hanyu, Yoshinobu ; et
al. |
January 24, 2002 |
Powder containing physiologically active peptide
Abstract
A method is disclosed for stabilizing a physiologically active
peptide in a process of preparing a powder containing the
physiologically active peptide by drying an aqueous liquid
containing the physiologically active peptide, wherein the method
comprises adding to the aqueous liquid at least one compound
selected from the group consisting of a nonionic surfactant, a
water-soluble, nonionic, organic binder, hydrogenated lecithin, and
mannitol.
Inventors: |
Hanyu, Yoshinobu; (Kobe-shi,
JP) ; Okada, Mariko; (Ashiya-shi, JP) ;
Shindo, Chihiro; (Kobe-shi, JP) ; Nishimuro,
Satoshi; (Kobe-shi, JP) ; Yokoyama, Tetsuo;
(Kobe-shi, JP) ; Horie, Masato; (Kobe-shi,
JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1941 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
JCR PHARMACEUTICALS CO.,
Ltd.
Hyogo
JP
|
Family ID: |
27342741 |
Appl. No.: |
09/810483 |
Filed: |
March 19, 2001 |
Current U.S.
Class: |
435/189 ;
424/85.2; 424/94.4; 514/11.1; 514/11.3; 514/11.7; 514/11.9;
514/13.3; 514/14.6; 514/5.9; 514/7.7; 514/8.2; 514/8.4; 514/8.5;
514/9.1; 514/9.3; 514/9.5; 514/9.6 |
Current CPC
Class: |
A61K 9/1623 20130101;
A61K 38/27 20130101; A61K 9/0075 20130101; A61K 9/1617 20130101;
A61K 9/1635 20130101; A61K 9/1652 20130101 |
Class at
Publication: |
435/189 ; 514/2;
424/85.2; 424/94.4 |
International
Class: |
A61K 038/44; A61K
038/20; A61K 038/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2000 |
JP |
2000-78775 |
Oct 11, 2000 |
JP |
2000-310693 |
Jan 24, 2001 |
JP |
2001-15904 |
Claims
What is claimed is:
1. A method for stabilization of a physiologically active peptide
in a process of preparing a powder containing the physiologically
active peptide by drying an aqueous liquid containing the
physiologically active peptide, wherein the method comprises adding
to the aqueous liquid at least one compound selected from the group
consisting of a nonionic surfactant, a water-soluble, nonionic,
organic binder, hydrogenated lecithin, and mannitol.
2. A method for stabilization of a physiologically active peptide
in a process of preparing a powder containing the physiologically
active peptide by drying an aqueous liquid containing the
physiologically active peptide, wherein the method comprises adding
to the aqueous liquid mannitol and at least one compound selected
from the group consisting of a nonionic surfactant, a
water-soluble, nonionic, organic binder, and hydrogenated
lecithin.
3. A method for stabilization of a physiologically active peptide
in a process of preparing a powder containing the physiologically
active peptide by drying an aqueous liquid containing the
physiologically active peptide, wherein the method comprises adding
to the aqueous liquid at least one component selected from the
group consisting of a nonionic surfactant in an amount of 0.01-0.5%
by weight, a water-soluble, nonionic, organic binder in an amount
of 0.01-1% by weight, hydrogenated lecithin, and 1-50 parts by
weight of mannitol per one part by weight of the physiologically
active peptide.
4. A method for stabilization of a physiologically active peptide
in a process of preparing a powder containing the physiologically
active peptide by drying an aqueous liquid containing the
physiologically active peptide, wherein the method comprises adding
to the aqueous liquid 1-50 parts by weight of mannitol per one part
by weight of the physiologically active peptide and at least one
component selected from the group consisting of a nonionic
surfactant in an amount of 0.01-0.5% by weight, a water-soluble,
nonionic, organic binder in an amount of 0.01-1% by weight, and
hydrogenated lecithin.
5. The method of one of claims 1 to 4 wherein the water-soluble,
nonionic, organic binder is selected from the group consisting of
polyvinylpyrrolidone, a water-soluble, nonionic cellulose
derivative, and polyvinylalcohol.
6. The method of claim 5 wherein the water-soluble, nonionic
cellulose derivative is selected from the group consisting of
hydroxypropylcellulose, hydroxyethylcellulose, and
hydroxypropylmethylcellulose.
7. The method of one of claims 1 to 6 wherein the nonionic
surfactant is selected from the group consisting of polysorbate,
polyoxyethylenehydrogenated castor oil, and a poloxamer.
8. The method of one of claims 1 to 7 wherein drying of the aqueous
liquid is performed by spray drying, lyophilization or spray-freeze
drying, or by coating which may be fluid-bed coating, or performed
in fluid-bed granulation.
9. The method of one of claims 1 to 8 wherein the average size of
the particles making up the powder is 1-10 .mu.m.
10. The method of one of claims 1 to 9 wherein the physiologically
active peptide is selected from the group consisting of growth
hormones, insulins, calcitonins, erythropoietin, glucagon,
somatostatin, somatostatin derivatives, interferons, interleukins,
superoxide dismutase, urokinase, proteases, tumor necrosis factors,
colony-stimulating factors, kallikrein, lysozyme, fibronectin,
insulin-like growth factors, epidermal growth factor, fibroblast
growth factors, platelet-derived growth factor, nerve growth
factor, hepatocyte growth factor, vasculogenesis factors, and
anti-vasculogenesis factors.
11. The method of one of claims 1 to 9 wherein the physiologically
active peptide is human growth hormone or human insulin.
12. The method of one of claims 1 to 9 wherein the physiologically
active peptide is human growth hormone.
13. A method for preparation of a powder containing a
physiologically active peptide, wherein the method comprises
forming a powder by drying an aqueous liquid containing a
physiologically active peptide and at least one compound selected
from the group consisting of a nonionic surfactant, a
water-soluble, nonionic, organic binder, hydrogenated lecithin, and
mannitol.
14. A method for preparation of a powder containing a
physiologically active peptide, wherein the method comprises
forming a powder by drying an aqueous liquid containing the
physiologically active peptide, mannitol, and at least one compound
selected from the group consisting of a nonionic surfactant, a
water-soluble, nonionic, organic binder, and hydrogenated
lecithin.
15. A method for preparation of a powder containing a
physiologically active peptide, wherein the method comprises
forming a powder by drying an aqueous liquid containing the
physiologically active peptide and at least one component selected
from the group consisting of a nonionic surfactant in an amount of
0.01-0.5% by weight, a water-soluble, nonionic, organic binder in
an amount of 0.01-1% by weight, hydrogenated lecithin and 1-50
parts by weight of mannitol per one part by weight of the
physiologically active peptide.
16. A method for preparation of a powder containing a
physiologically active peptide, wherein the method comprises
forming a powder by drying an aqueous liquid containing the
physiologically active peptide, 1-50 parts by weight of mannitol
per one part by weight of the physiologically active peptide, and
at least one component selected from the group consisting of a
nonionic surfactant in an amount of 0.0 1-0.5% by weight, a
water-soluble, nonionic, organic binder in an amount of 0.01-1% by
weight, and hydrogenated lecithin.
17. The method for preparation of a powder containing a
physiologically active peptide of one of claims 13 to 16, wherein
the water-soluble, nonionic, organic binder is selected from the
group consisting of polyvinylpyrrolidone, a water-soluble, nonionic
cellulose derivative, and polyvinylalcohol.
18. The method for preparation of a powder containing a
physiologically active peptide of claim 17, wherein the
water-soluble, nonionic cellulose derivative is selected from the
group consisting of hydroxypropylcellulose, hydroxyethylcellulose,
and hydroxypropylmethylcellulose.
19. The method for preparation of a powder containing a
physiologically active peptide of one of claims 13 to 18, wherein
the nonionic surfactant is selected from the group consisting of
polysorbate, polyoxyethylenehydrogenated castor oil, and a
poloxamer.
20. The method for preparation of a powder containing a
physiologically active peptide of one of claims 13 to 19, wherein
drying of the aqueous liquid is performed by spray drying,
lyophilization or spray-freeze drying, or by coating which may be
fluid-bed coating, or performed in fluid-bed granulation.
21. The method for preparation of a powder containing a
physiologically active peptide of one of claims 13 to 20 wherein
the average size of the particles making up the powder is 1-10
.mu.m.
22. The method for preparation of a powder containing a
physiologically active peptide of one of claims 13 to 21, wherein
the physiologically active peptide is selected from the group
consisting of growth hormones, insulins, calcitonins,
erythropoietin, glucagon, somatostatin, somatostatin derivatives,
interferons, interleukins, superoxide dismutase, urokinase,
proteases, tumor necrosis factors, colony-stimulating factors,
kallikrein, lysozyme, fibronectin, insulin-like growth factors,
epidermal growth factor, fibroblast growth factors,
platelet-derived growth factor, nerve growth factor, hepatocyte
growth factor, vasculogenesis factors, and anti-vasculogenesis
factors.
23. The method for preparation of a powder containing a
physiologically active peptide of one of claims 13 to 21, wherein
the physiologically active peptide is human growth hormone or human
insulin.
24. The method for preparation of a powder containing a
physiologically active peptide of one of claims 13 to 21, wherein
the physiologically active peptide is human growth hormone.
25. A powder containing a physiologically active peptide, wherein
the powder is made up of particles comprising a physiologically
active peptide and mannitol at a weight proportion of 1:1 to
1:50.
26. The powder containing a physiologically active peptide of claim
25, wherein the particles further comprise per one part by weight
of the physiologically active peptide at least one component
selected from the group consisting of a nonionic surfactant in an
amount of 0.05-3 parts by weight, a water-soluble, nonionic,
organic binder in an amount of 0.05-6 parts by weight, and
hydrogenated lecithin.
27. The powder containing a physiologically active peptide of claim
25 or 26, wherein the average size of the particles is 1-10
.mu.m.
28. The powder containing a physiologically active peptide of one
of claims 25 to 27, for which drying of the aqueous solution was
performed by spray drying, spray-freeze drying, or
lyophilization.
29. The powder containing a physiologically active peptide of one
of claims 25 to 28, wherein the physiologically active peptide is
selected from the group consisting of growth hormones, insulins,
calcitonins, erythropoietin, glucagon, somatostatin, somatostatin
derivatives, interferons, interleukins, superoxide dismutase,
urokinase, proteases, tumor necrosis factors, colony-stimulating
factors, kallikrein, lysozyme, fibronectin, insulin-like growth
factors, epidermal growth factor, fibroblast growth factors,
platelet-derived growth factor, nerve growth factor, hepatocyte
growth factor, vasculogenesis factors, and anti-vasculogenesis
factors.
30. The powder containing a physiologically active peptide of one
of claims 25 to 28, wherein the physiologically active peptide is
human growth hormone or human insulin.
31. The powder containing a physiologically active peptide of one
of claims 25 to 28, wherein the physiologically active peptide is
human growth hormone.
32. An inhalant composition containing a physiologically active
peptide, wherein the inhalant composition comprises particles as
defined in one of claims 25 to 31.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a physiologically active
peptide-containing powder, and in particular to a physiologically
active peptide-containing powder in which contamination by
denatured peptides has been suppressed by stabilizing the
physiologically active peptide and thereby preventing its
denaturation from taking place in the process of forming a powder
by drying an aqueous liquid containing the physiologically active
peptide. The present invention further relates to a physiologically
active peptide-containing powder suitable for transpulmonary and
transnasal administration by inhalation.
BACKGROUND OF THE INVENTION
[0002] Administration of pharmaceutical products containing a
physiologically active peptide have been made, so far, by
injection. In this context, lyophilization has exclusively been
employed in the preparation of such pharmaceutical compositions.
Thus, for such pharmaceutical compositions, studies addressed to
the stabilization of their active components, physiologically
active peptides, have so far been focused on either the long-term
storage stability of the physiologically active peptides in a dry
state pharmaceutical compositions of the final products, or the
storage stability of the physiologically active peptides in liquids
which are prepared by dissolving the peptide-containing dry
compositions. For example, stabilization of calcitonin solutions is
disclosed in Japanese Unexamined Patent Publication Nos.
H07-179364, H07-188060 and H07-188061, and stabilization of
lyophilized growth hormone products is disclosed in Japanese
Unexamined Patent Publication Nos. H10-504531, H10-511965 and
H10-507183.
[0003] The reason why injection has been the sole way for
administering physiologically active peptides is that, when they
are orally administered, physiologically active peptides are
digested in the gastrointestinal tract. A practically applicable
new route for administration, if established, would provide a great
benefit to patients. Above all, in the case of active peptides
requiring lifelong administration such as growth hormone and
insulin, the conventional way of administration of injection has
been giving patients inconvenience and pain. For these
physiologically active peptides, therefore, establishment of a
route of administration other than injection has been longed for by
the patients.
[0004] On the other hand, those pharmaceutical compositions for
systemic administration of a drug are under investigation that are
intended either for transpulmonary absorption of a
pharmacologically active ingredient by inhalation (referred to as
an "inhalant composition" in the present specification) or for
absorption of such an ingredient through the nasal mucous membrane
by intranasal application, i.e. compositions for transnasal
administration, as compositions utilizing other, new administration
routes than those relied on by conventional pharmaceutical
compositions such as injections, oral preparations, suppositories
and the like. Inhalant compositions and compositions for transnasal
administration are not directly injected into the body, but they
are applied onto the surface of mucous membranes which are exposed
to the air such as membranes of the respiratory tract. Therefore,
their standards for microbiological quality control are not so
strict as those for injections. Thus, they may be produced not only
by a lyophilization apparatus but also by a fluid-bed granulation
apparatus, a spray drying apparatus, or a spray-freeze drying
apparatus. Concerning stabilization of active peptides in
production steps of pharmaceutical compositions using a fluid-bed
granulation apparatus, a spray drying apparatus, or a spray-freeze
drying apparatus, it is reported that stabilization is attained by
addition of an inhibitor of Maillard reaction (Japanese Unexamined
Patent Publication No. H10-505591). However, it is preferable, if
possible, that stabilization of a given active peptide in a
production process should be achieved by means of approved
pharmaceutical additives which are highly safe and have been used
for years. This is because such an additive would allow to expect
higher safety with regard to the final pharmaceutical product
obtained. It is also required that the absorption and transferal to
the blood of an physiologically active peptide is attained in
sufficient efficiency.
[0005] The present invention has as its objectives to provide a
method to improve stability of a physiologically active peptide in
a process of producing a powder by drying an aqueous liquid
containing the physiologically active peptide, as well as to
provide a physiologically active peptide-containing powder produced
by the method.
[0006] The present invention has as its further objectives to
provide a physiologically active peptide-containing powder
especially suited for absorption of the physiologically active
peptide by inhalation, and to provide an inhalant composition.
SUMMARY OF THE INVENTION
[0007] For production of a powder containing a physiologically
active peptide, the present inventors found that, in a process of
preparing a powder containing a physiologically active peptide by
drying an aqueous liquid containing the peptide, addition of
certain compounds to the aqueous liquid remarkably increases the
stability of the physiologically active peptides during the powder
preparation. In addition, the present inventors also found that
physiologically active peptides contained in the powder thus
prepared are efficiently absorbed into the blood when the powder is
transpulmonarily administered. The present invention was made on
the basis of these findings.
[0008] Thus, the present invention provides a method for
stabilization of a physiologically active peptide in a process of
preparing a powder containing the physiologically active peptide by
drying an aqueous liquid containing the physiologically active
peptide, wherein the method comprises adding to the aqueous liquid
at least one compound selected from the group consisting of a
nonionic surfactant, a water-soluble, nonionic, organic binder,
hydrogenated lecithin, and mannitol. In the method, a nonionic
surfactant, a water-soluble, nonionic, organic binder, hydrogenated
lecithin and mannitol serve as stabilizers in preparing a powder
containing a physiologically active peptide from an aqueous liquid
containing it. Thus, one or more of these compounds employed
suppress denaturation such as dimer formation in the process of
forming a powder from an aqueous liquid containing the peptide,
thereby enabling to prepare a physiologically active
peptide-containing powder which is substantially free of denatured
peptides.
[0009] The present invention further provides a method for
stabilization of a physiologically active peptide in a process of
preparing a powder containing the physiologically active peptide by
drying an aqueous liquid containing the physiologically active
peptide, wherein the method comprises adding to the aqueous liquid
mannitol and at least one compound selected from the group
consisting of a nonionic surfactant, a water-soluble, nonionic,
organic binder, and hydrogenated lecithin. This method enables, in
addition to the above-mentioned benefit, to prepare a powder
effecting especially efficient transpulmonary absorption of a
physiologically active peptide.
[0010] In the above methods for stabilization, with regard to a
nonionic surfactant or a water-soluble, nonionic, organic binder
added to the aqueous liquid, the concentration range where they
exhibit a potent stabilizing effect is 0.01-0.5 % by weight for a
nonionic surfactant and 0.01-1 % by weight for a water-soluble,
nonionic, organic binder. As for mannitol, it exhibits a potent
stabilizing effect when added in an amount of 1-50 parts by weight
per one part by weight of a physiologically active peptide.
[0011] In the above, it is more preferable that the nonionic
surfactant is selected from the group consisting of polysorbate,
polyoxyethylenehydrogenated castor oil, and a poloxamer
(polyoxyethylene polyoxypropylene block copolymer: Pluronic).
[0012] Also in the above, the water-soluble, nonionic, organic
binder is more preferably selected from the group consisting of
polyvinylpyrrolidone, a water-soluble, nonionic, cellulose
derivative and polyvinylalcohol.
[0013] Further, the water-soluble, nonionic, cellulose derivative
is more preferably selected from the group consisting of
hydroxypropylcellulose, hydroxyethylcellulose, and
hydroxypropylmethylcellulose.
[0014] The effects of these stabilizers are remarkable in the above
range, though they still have substantial effects somewhat outside
the ranges. A still more preferable concentration range for a
nonionic surfactant is 0.05-0.3 % by weight, where a particularly
potent stabilization effect is obtained. For a water-soluble,
nonionic, organic binder, a concentration range still more
preferable than the above is 0.02-0.5 % by weight, where a
particularly potent stabilization effect is obtained. As for
hydrogenated lecithin, its stabilizing effect is particularly
remarkable even at a concentration as low as 0.01% by weight. While
its effect peaks at concentrations of 0.5-1 % by weight, the effect
remains still remarkable outside this range, and even at 2% by
weight. Thus, the decline in its stabilizing effect is only limited
even when its concentration goes up beyond the peak concentration.
An upper limit concentration, therefore, is not clear over which
hydrogenated lecithin would substantially lose its stabilizing
effect. Its concentration, however, may be chosen as desired
considering ease of handling in production of the pharmaceutical
composition as there is no reason for using an unnecessarily large
amount of hydrogenated lecithin insofar as it exhibits a sufficient
effect as an additive. In general, the concentration of
hydrogenated lecithin is preferably in the range of about 0.005-4%
by weight, and more preferably in the range of 0.01-2% by weight.
In light that the total amount of the powder administered is to be
small insofar as it does not prevents easy handling, the weight
proportion of a physiologically active peptide to mannitol is more
preferably 1:1 to 1:40, further more preferably 1:1 to 1:30, still
more preferably 1:1 to 1:20, and most preferably 1:1 to 1:10. For
stabilization of an physiologically active peptide, any of the
above stabilizers may be used alone, or two or more of them may be
used in combination. When used in combination, they exhibit a still
more remarkable stabilizing effect than when one of them is used
alone, thus allowing to almost completely prevent the formation of
denatured peptide such as a dimer.
[0015] The present invention is characterized in that its uses, in
drying an aqueous liquid containing a physiologically active
peptide, a certain group of compounds that were found to stabilize
active peptides. The compounds can be used in a wide variety of
specific methods for drying. In the above, example of methods for
drying aqueous liquids include, but are not limited to, spray
drying, lyophilization and spray-freeze drying, and, furthermore, a
variety of methods which include a process of drying a solution by
spraying it, such as drying performed in fluid-bed granulation, in
a variety of coating method such as fluid-bed coating which allow
to coat the surface of core particles, as well as drying performed
in a granulation process in fluid-bed granulation involving coating
of, or attachment of materials to, the surface of core
particles.
[0016] Inhaled particles are more easily carried on the air flow
deep into the respiratory system when their average size is 1-10
.mu.m, and more preferably 2-5 .mu.m. When given such a size, the
particles of the physiologically active peptide-containing powder
obtained in a stable form by one of the above methods are easily
carried deep into the respiratory system by inhalation, allowing
efficient and relatively long-lasting transferal of the
physiologically active peptide into the circulating blood. Thus,
the present invention further provides one of the above method in
which the average size of the particles making up the powder is
1-10 .mu.m, and more preferably 2-5 .mu.m.
[0017] Examples of active peptides stabilized according to the
present invention include calcitonins, insulins, growth hormones,
erythropoietin, glucagon, somatostatin, somatostatin derivatives,
interferons (.alpha., .beta. or .gamma.), interleukins (I, II, III,
IV, V VI or VII), superoxide dismutase, urokinase, proteases, tumor
necrosis factors, colony-stimulating factors, kallikrein, lysozyme,
fibronectin, as well as a variety of factors regulating growth or
differentiation of cells such as insulin-like growth factors,
epidermal growth factor, fibroblast growth factors,
platelet-derived growth factor, nerve growth factor, hepatocyte
growth factor, vasculogenesis factors, and anti-vasculogenesis
factors. As active peptides share a common chemical structure that
they consist of two or more amino acids linked by peptide bonds,
the present invention is also applicable to a wide variety of other
active peptides than those enumerated above. Moreover, it does not
matter whether those peptides have been obtained by extraction from
natural sauces or produced by application of genetic recombination
technology, for such difference will not influence the basic
physicochemical characters of the peptides. Among the above
peptides, human growth hormone and human insulin are particularly
preferred peptides in the present invention, for they are such
peptides that patients have had to continue administering
themselves by subcutaneous injection for a long period of time
[0018] In addition to the above stabilizing methods, the present
invention provides a method for preparation of a powder containing
a physiologically active peptide. The method for preparation
comprises forming a powder by drying an aqueous liquid containing a
physiologically active peptide and a nonionic surfactant, a
water-soluble, nonionic, organic binder, hydrogenated lecithin,
and/or mannitol. By one or more of those stabilizers added to a
physiologically active peptide, denaturation such as dimer
formation is suppressed while the physiologically active peptide is
in the process of forming a powder from an aqueous liquid
containing the peptide. Thus, a physiologically active
peptide-containing powder is prepared which is substantially free
of denatured peptides.
[0019] The present invention further provides a method for
preparation of a powder containing a physiologically active
peptide, wherein the method comprises forming a powder by drying an
aqueous liquid containing the physiologically active peptide,
mannitol, and at least one compound selected from the group
consisting of a nonionic surfactant, a water-soluble, nonionic,
organic binder, and hydrogenated lecithin. This method for
preparation, in addition to the above-mentioned benefit, provides a
powder that effects especially efficient transpulmonary absorption
of a physiologically active peptide.
[0020] In the method for preparation above, the nonionic surfactant
is more preferably selected from the group consisting of
polysorbate, polyoxyethylenehydrogenated castor oil, and a
poloxamer (polyoxyethylene polyoxypropylene block copolymer:
Pluronic). The water-soluble, nonionic, organic binder is more
preferably selected from the group consisting of
polyvinylpyrrolidone, a water-soluble, nonionic, cellulose
derivative and polyvinylalcohol. The water-soluble, nonionic,
cellulose derivative is more preferably selected from the group
consisting of hydroxypropylcellulose, hydroxyethylcellulose, and
hydroxypropylmethyl-cellulose.
[0021] In the method for preparation above, preferable ranges of
the amount of the enumerated stabilizers when employed are the same
as those mentioned for them in the method for stabilization of
physiologically active peptides above. Therefore, a still more
preferable concentration range for a nonionic surfactant is
0.05-0.3% by weight, and, for a water-soluble, nonionic, organic
binder, a still more preferable concentration range is 0.02-0.5% by
weight. As for hydrogenated lecithin, an upper limit concentration
is not clear over which hydrogenated lecithin would substantially
lose its stabilizing effect. Its concentration, however, may be
chosen as desired considering ease of handling in production of the
pharmaceutical composition as there is no reason for using an
unnecessarily large amount of hydrogenated lecithin insofar as it
exhibits a sufficient effect as an additive. In general, the
concentration of hydrogenated lecithin is preferably in the range
of about 0.005-4% by weight, and more preferably in the range of
0.01-2% by weight. As to mannitol, the weight proportion of a
physiologically active peptide to mannitol is more preferably 1:1
to 1:40, further more preferably 1:1 to 1:30, still more preferably
1:1 to 1:20, and most preferably 1:1 to 1:10.
[0022] In the method for preparation above, example of methods for
drying aqueous liquids include, but are not limited to, spray
drying, lyophilization and spray-freeze drying, and fluid-bed
granulation, as well as a variety of coating method, such as
fluid-bed coating, which allow to coat the surface of core
particles, and fluid-bed granulation involving coating of, or
attachment of materials to, the surface of core particles.
[0023] In the method for preparation above, the average size of the
particles making up the powder is preferably 1-10 .mu.m, and more
preferably 2-5 .mu.m, when considering transpulmonary
administration of a physiologically active peptide.
[0024] The range of physiologically active peptides formed into a
powder by the method for preparation above is the same as already
mentioned with regard to the method for stabilization.
[0025] The present invention further provides a powder containing a
physiologically active peptide, wherein the powder is made up of
particles comprising a physiologically active peptide and mannitol
at a weight proportion of 1:1 to 1:50. In the powder, more
preferably, the particles making up the powder further comprise,
per one part by weight of the physiologically active peptide, at
least one component selected from the group consisting of a
nonionic surfactant in an amount of 0.05-3 parts by weight, a
water-soluble, nonionic, organic binder in an amount of 0.05-6
parts by weight, and hydrogenated lecithin. Such a powder effects
an efficient absorption of a physiologically active peptide through
a mucous membrane deep in the respiratory system.
[0026] Considering reduction of the total weight of the powder
inhaled per a predetermined amount of a physiologically active
peptide to be administered, the weight proportion of a
physiologically active peptide to mannitol in the particles above
is more preferably 1:1 to 1:40, further more preferably 1:1 to
1:30, still more preferably 1:1 to 1:20, and most preferably 1:1 to
1:10. The amount of a nonionic surfactant is more preferably
0.25-1.8 parts by weight per one part by weight of a
physiologically active peptide, in which range efficient absorption
of a physiologically active peptide is attained while suppressing
the amount of a nonionic surfactant employed. Likewise, the amount
of water-soluble, nonionic, organic binder is more preferably 0.1-3
parts by weight per one part by weight of a physiologically active
peptide.
[0027] In the above powder containing a physiologically active
peptide, the average size of the particles making up the powder is
preferably 1-10 .mu.m, and more preferably 2-5 .mu.m. By giving
such an average size to its particles, the powder becomes easily
carried deep into the respiratory system by inhalation, allowing
more efficient absorption of the physiologically active
peptide.
[0028] Method for preparation of the above powder containing a
physiologically active peptide is not limited. The powder may be
prepared, for example, by spray drying, spray-freeze drying or
lyophilization.
[0029] The range of physiologically active peptides in the above
powder containing a physiologically active peptide is the same as
already mentioned with regard to the method for stabilization.
[0030] The present invention further provides an inhalant
composition containing a physiologically active peptide, wherein
the inhalant composition comprises above-mentioned particles
containing a physiologically active peptide. The inhalant
composition may simply be such particles containing a
physiologically active peptide, or they may be either clusters
consisting of such particles loosely associated with one another or
composites consisting of such particles plus larger, inert carrier
particles (e.g. lactose) onto the surface of which the former
particles are loosely attached. Such loose clusters or composites
are constructed in an extent of looseness that, at the time of
inhaling the composition, they will be disintegrated upon release
from an inhalation device by the flow of air and the each fine
particle containing a physiologically active peptide will thereby
be liberated from the clusters or carriers into a separate particle
Preparation of such loose clusters or loose and coarse composite
particles can be prepared by any of a variety of methods well known
to those skilled in the art for bringing particles of the size of
one to several .mu.m making up a powder into a loose association
with one another or into a loose association onto larger, inert
carrier particles. Such loose clusters or loose and coarse
composite particles are intended to increase flowability of the
composition for improved ease of filling and accuracy of filling
amount in a process in which a unit dose of the inhalant
compositions is filled into each of predetermined containers like
capsules employed in a inhalation device. Therefore, once put in a
capsule, it is allowed that the whole or part of particles are
liberated to separate particles by external agitation and thus
forming a powder within the capsule.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 is a graph illustrating the effect of nonionic
surfactants.
[0032] FIG. 2 is a graph illustrating the effect of water-soluble,
nonionic, organic binders.
[0033] FIG. 3 is a graph illustrating the effect of hydrogenated
lecithin.
[0034] FIG. 4 shows blood concentration profiles of human growth
hormone in rat after transpulmonary administration of a human
growth hormone-containing powder and subcutaneous injection of the
same amount of the powder.
DETAILED DESCRIPTION OF THE INVENTION
[0035] In the present invention, the term an "aqueous liquid
containing a physiologically active peptide" includes not only a
simple aqueous solution of a physiologically active peptide but
also a solution of a physiologically active peptide further
containing one or more other components that do not adversely
affect the stability of the physiologically active peptide, e.g.,
buffering agents such as phosphates, pharmaceutically acceptable
salts such as sodium chloride, and diluents such as sorbitol.
[0036] As the method for stabilization of the present invention
stabilizes a physiologically active peptide dissolved in an aqueous
liquid in a process of evaporating water from the aqueous liquid,
its stabilization effect on a physiologically active peptide is not
affected even by drying the physiologically active peptide coated
on the surface of larger particles chemically inert to the active
peptide, such as lactose and the like. Such inert particles serve
as cores which carry on their surface a coat of the physiologically
active peptide mixed with one or more stabilizing agents
[0037] The powder of the present invention containing a
physiologically active peptide is based on the discovery that a
very efficient transpulmonary absorption is attained by employing
particles comprising a physiologically active peptide and mannitol.
Thus, any method of preparation may be chosen as desired for
preparing such powder containing a physiologically active peptide.
The method of the present invention for preparation of a powder
containing a physiologically active peptide is based also on the
discovery that mannitol has an effect of remarkably stabilizing a
physiologically active peptide in the process of forming a powder
by drying an aqueous liquid containing the peptide. Thus, drying of
an aqueous solution containing a physiologically active peptide and
mannitol may be performed by any conventional method as
desired.
[0038] In the present invention, an example of particularly
preferred physiologically active peptides is human growth hormone.
In the present invention, the term "human growth hormone" means not
only 22K hGH extractable from the pituitary of a human, which
consists 191 amino acids and has a molecular weight of 22,125, but
also 20K hGH, which lacks 15 amino acids corresponding amino acids
32-46. 20K hGH has a growth stimulating effect comparable to 22 K
hGH. In the present invention, the term "human growth hormone"
means not only these natural types of human growth hormones, but
also proteins which are produced by application of genetic
recombination technology and having a substantially comparable
effect to the natural human growth hormones. Examples of human
growth hormone produced by application of genetic recombination
technology include a N-terminal methionine-type hormone consisting
192 amino acids and variants which have part of their amino acids
deleted, substituted, added or inserted and having a comparable
activity to the natural types of human growth hormone.
EXAMPLES
[0039] When vigorously agitated in its aqueous solution, the
molecule of growth hormone (GH), among active peptides, readily
undergoes alteration in tertiary structure in contact with a
gas-liquid interface, resulting in the loss of its monomer and
leading to the formation of its dimer, polymer or insoluble
aggregates, as well as to the formation of deamidation products. As
the gas-liquid interface is expanded in a drying process such as
spray drying, it is necessary, particularly in the case of GH, to
manage to minimize its denaturation induced by this expansion of
the interface in the process of forming GH into a powder. Studies
were made as described below in search of a compound that
stabilizes GH. In the Examples and Control Examples below, human
growth hormone was chosen as a representative of active
peptides.
[0040] The human growth hormone employed in the Examples and
Control Examples below was a recombinant human growth hormone (in
which N-terminal methionine had been selectively deleted
enzymaticaly) which had the same amino acid sequence as the natural
human growth hormone consisting of 191 amino acids (22K hGH). In
addition, where the recombinant human growth hormone is identified
as "Growject Injection 4 IU", it indicates that a pharmaceutical
product (Growject Injection 4 IU: JCR pharmaceuticals, Co., Ltd.)
was employed there. The composition of Growject Injection 4 IU is
as follows. Where the recombinant human growth hormone is
specifically noted as a "bulk material", it indicates the pure
recombinant human growth hormone (produced by BTG), which is free
of any additives.
[0041] r-hGH Injection (Growject Injection 4 IU): (Formula)
1 (Formula) r-hGH 4 IU (1.7 mg) Disodium hydrogenphosphate 2.2 mg
Sodium dihydrogenphosphate 0.35 mg Sodium chloride 1.0 mg
D-mannitol 20.0 mg
[0042] Tests will be described below which were performed by spray
drying as a representative model of drying processes of an aqueous
liquid containing a physiologically active peptide. The apparatus
employed for spray drying was Spray Dryer SD-1000 (EYELA).
[0043] As an indicator of stabilization of the physiologically
active peptide r-hGH, recovery rate of the physiologically active
peptide monomer was employed, for it is considered to be the best
indicator of stabilization of the physiologically active peptide.
Calculation of recovery rate was done according to the following
equation, based on the concentration of the physiologically active
peptide in the aqueous liquid before drying (before spray drying)
and the content of recovered active peptide in the solution
prepared by reconstituting the obtained powder (spray dried
product) to the initial volume
[0044] Recovery rate of physiologically active peptide monomer
(%)=A.sub.P/A.sub.I.times.100 where:
[0045] A.sub.P=area of monomer peak on HPLC for spray dried
product, and
[0046] A.sub.I=area of monomer peak on HPLC before spray
drying.
CONTROL EXAMPLE 1
[0047] To each of fifteen vials of the r-hGH injection (Growject
Injection 4 IU) was added 1.0 ml of purified water to completely
dissolve the injection. The r-hGH solution thus obtained (15 vials:
15.0 ml) was spray-dried to obtain a dry powder. The conditions for
spray drying in the Spray Dryer SD-1000 were adjusted as
follows.
[0048] Spray Drying Conditions
[0049] Inlet temperature: 80.degree. C.
[0050] Atomizing pressure: 150 kPa
[0051] Dry air flow: 0.3 m.sup.3/min
[0052] Liquid feeder pump flow: 2.6 mL/min
[0053] The conditions for HPLC for determination of the monomer
content were as follows.
[0054] HPLC Conditions
[0055] Apparatus: LC10A (SHIMADZU CORPORATION)
[0056] Detector: UV (280 nm)
[0057] Analyzing column: TSK G3000SW.sub.XL
[0058] Column temperature: Room temperature
[0059] Mobile phase: 50 mM sodium dihydrogenphosphate, 50 mM
disodium hydrogenphosphate, 0.2 M sodium chloride.
[0060] Flow rate: 0.6 mL/min
[0061] Injection volume: 50 .mu.L
CONTROL EXAMPLE 2
[0062] Five sets of r-hGH injection (Growject injection 4IU) vials,
15 vials per set, were provided. To each of the vials was added 1.0
mL of purified water to completely dissolve the injection. The
r-hGH solution thus obtained (15.0 ml: 15 vials per set) was spray
dried to obtain a dry powder. The conditions for spray drying in
the Spray Dryer SD-1000 were different from those in Control
Example 1 and adjusted as follows. The HPLC conditions for
determination of the monomer content were the same as those in
Control Example 1.
[0063] Spray Drying Conditions
[0064] Inlet temperature: 90.degree. C.
[0065] Atomizing pressure: 100 kPa
[0066] Dry air flow: 0.2 m.sup.3/min
[0067] Fluid feeder pump flow: 2.6 mL/min
EXAMPLE 1
[0068] As solutions of a nonionic surfactant, aqueous solutions
containing Tween 20 at different concentrations (concentration:
0.01, 0.05, 0.1, 0.5, 1.0 and 2.0 w/w %) were prepared. Fifteen
vials of the r-hGH injection (Growject Injection 4IU) were provided
for each of the aqueous solutions containing Tween 20 at the
different concentrations. The aqueous solutions containing Tween 20
at different concentrations were added to corresponding 15 vials,
1.0 mL each, and the injection was completely dissolved. Thus
obtained r-hGH solutions containing Tween 20 at different
concentrations (15.0 mL: 15 vials for each Tween 20 concentration)
were spray-dried to obtain dry powders. The conditions for spray
drying and HPLC were the same as those in Control Example 1.
EXAMPLE 2
[0069] As solutions of a nonionic surfactant, aqueous solutions
containing HCO-60 (polyoxyethylenehydrogenated castor oil) at
different concentrations (concentration: 0.01, 0.05, 0.1, 0.5, 1.0
and 2.0 w/w %) were prepared. Fifteen vials of the r-hGH injection
(Growject Injection 4IU) were provided for each of the aqueous
solutions containing HCO-60 at different concentrations. The
aqueous solutions containing HCO-60 at different concentrations
were added to corresponding 15 vials, 1.0 mL each, and the
injection was completely dissolved. Thus obtained r-hGH solutions
containing HCO-60 at different concentrations (15.0 mL: 15 vials
for each HCO-60 concentration) were spray-dried to obtain dry
powders. The conditions for spray drying and HPLC were the same as
those in Control Example 1.
EXAMPLE 3
[0070] As solutions of a nonionic surfactant, aqueous solutions
containing Pluronic F68 (polyoxyethylene(160)polyoxypropylene(30)
glycol) at different concentrations (concentration: 0.01, 0.05,
0.1, 0.5, 1.0 and 2.0 w/w %) were prepared. Fifteen vials of the
r-hGH injection (Growject Injection 4IU) were provided for each of
the aqueous solutions containing Pluronic F68 at different
concentrations. The aqueous solutions containing Pluronic F68 at
different concentrations were added to corresponding 15 vials, 1.0
mL each, and the injection was completely dissolved. Thus obtained
r-hGH solutions containing Pluronic F68 at different concentrations
(15.0 mL: 15 vials for each Pluronic F68 concentration) were
spray-dried to obtain dry powders. The conditions for spray drying
and HPLC were the same as those in Control Example 1.
EXAMPLE 4
[0071] As solutions of a water soluble, nonionic, organic binder,
aqueous solutions containing Kollidone 17PF (polyvinylpyrrolidone:
BASF) at different concentrations (concentration: 0.01, 0.05, 0.1,
0.5, 1.0 and 2.0 w/w %) were prepared. Fifteen vials of the r-hGH
injection (Growject Injection 4IU) were provided for each of the
aqueous solutions containing Kollidone 17PF at different
concentrations. The aqueous solutions containing Kollidone 17PF at
different concentrations were added to corresponding 15 vials, 1.0
mL each, and the injection was completely dissolved. Thus obtained
r-hGH solutions containing Kollidone 17PF at different
concentrations (15.0 mL: 15 vials for each Kollidone 17PF
concentration) were spray dried to obtain dry powders. The
conditions for spray drying and HPLC were the same as those in
Control Example 1.
EXAMPLE 5
[0072] As a water soluble, nonionic, organic binder, aqueous
solutions containing Kollidone 12PF (polyvinylpyrrolidone: BASF) at
different concentrations (concentration: 0.01, 0.05, 0.1, 0.5, 1.0
and 2.0 w/w %) were prepared. Fifteen vials of the r-hGH injection
(Growject Injection 4IU) were provided for each of the aqueous
solutions containing Kollidone 12PF at different concentrations.
The aqueous solutions containing Kollidone 12PF at different
concentrations were added to corresponding 15 vials, 1.0 mL each,
and the injection was completely dissolved. Thus obtained r-hGH
solutions containing Kollidone 12PF at different concentrations
(15.0 mL: 15 vials for each Kollidone 12PF concentration) were
spray-dried to obtain dry powders. The conditions for spray drying
and HPLC were the same as those in Control Example 1.
EXAMPLE 6
[0073] As a water soluble, nonionic, organic binder, aqueous
solutions containing HPC-SSL (hydroxypropylcellulose: TOSOH) at
different concentrations (concentration: 0.01, 0.05, 0.1, 0.5 and
1.0 w/w %) were prepared. Fifteen vials of the r-hGH injection
(Growject Injection 4IU) were provided for each of the aqueous
solutions containing HPC-SSL at different concentrations. The
aqueous solutions containing HPC-SSL at different concentrations
were added to corresponding 15 vials, 1.0 mL each, and the
injection was completely dissolved. Thus obtained r-hGH solutions
containing HPC-SSL at different concentrations (15.0 mL: 15 vials
for each HPC-SSL concentration) were spray-dried to obtain dry
powders. The conditions for spray drying and HPLC were the same as
those in Control Example 1.
EXAMPLE 7
[0074] As solutions of a nonionic surfactant, aqueous solutions
containing Lecinol S-10E (hydrogenated lecithin: NIKKO CHEMICALS)
at different concentrations (concentration: 0.01, 0.05, 0.1, 0.5,
1.0 and 2.0 w/w %) were prepared. Fifteen vials of the r-hGH
injection (Growject Injection 4IU) were provided for each of the
aqueous solutions containing hydrogenated lecithin at different
concentrations. The aqueous solutions containing hydrogenated
lecithin at different concentrations were added to corresponding 15
vials, 1.0 mL each, and the injection was completely dissolved.
Thus obtained r-hGH solutions containing hydrogenated lecithin at
different concentrations (15.0 mL: 15 vials for each hydrogenated
lecithin concentration) were spray-dried to obtain dry powders. The
conditions for spray drying and HPLC were the same as those in
Control Example 1.
EXAMPLE 8
[0075] Aqueous solutions were prepared which contained HPC-SSL
(hydroxypropylcellulose) and a nonionic surfactant in combination
as indicated in the following table.
2TABLE 1 Aqueous Concentration of Nonionic surfactant and solution
No. HPC-SSL (w/w %) its concentration (w/w %) A 0.05 HCO-60 0.05 B
0.05 Pluronic F68 0.05 C 0.05 Pluronic F68 0.10 D 0.10 HCO-60 0.05
E 0.10 HCO-60 0.10
[0076] Fifteen vials of the r-hGH injection (Growject Injection
4IU) were provided for each of the aqueous solutions containing
HPC-SSL and a nonionic surfactant in different combinations. The
aqueous solutions were added to a corresponding set of 15 vials,
1.0 mL each, and the injection was completely dissolved. Thus
obtained r-hGH solutions (15.0 mL: per set of 15 vials for each
combination) were spray-dried to obtain dry powders. The conditions
for spray drying were the same as in Control Example 2, and HPLC
conditions were the same as those in Control Example 1.
[0077] Results of Analysis
[0078] FIG. 1 shows the results of HPLC analysis performed in
Control Example 1 and Examples 1-3.
[0079] As shown in the figure, the nonionic surfactants at
concentrations in certain ranges, respectively, remarkably
increased the recovery rate of the monomer of physiologically
active peptide r-hGH in the process of powder preparation from its
aqueous solutions. While the content of r-hGH monomer fell to the
order of 40% during powder preparation in Control Example 1, which
employed no nonionic surfactant, r-hGH monomer was maintained in
Examples 1-3, in contrast at much higher recovery rate in the
samples containing 0.01-0.5 w/w % nonionic surfactants in aqueous
solutions. The figure also shows that the stabilizing effect of the
respective nonionic surfactants peaked at their concentrations of
somewhere around 0.1 w/w %. Though lacking actually measured
values, it is also evident, for example, that the nonionic
surfactants at about 0.3 w/w % have higher stabilizing effects than
at 0.5 w/w %.
[0080] FIG. 2 shows the results of HPLC analysis performed in
Control Example 1 and Examples 4-6.
[0081] As shown in the figure, the water-soluble, nonionic, organic
binders markedly increased the recovery rate of the monomer of
physiologically active peptide r-hGH in the process of powder
preparation from its aqueous solutions. In Examples 4 (Kollidone
17PF) and 5 (Kollidone 12PF), improvement was noted at any of their
concentrations tested. Their stabilizing effect was particularly
potent up to a concentration of 1 w/w % and peaked at a
concentration of 0.1 w/w %. As for Example 6
(hydroxypropylcellulose), stabilizing effect was still more
remarkable than where the other binders were employed, showing a
r-hGH recovery rate of about 95% at a concentration of 0.1 w/w %,
where its effect peaked. In Example 6, hydroxypropylcellulose was
tested only up to the concentration of 1 w/w %. However, it is
largely evident that hydroxypropylcellulose would show a
stabilizing effect even at 2 w/w %. This is because its effect at 1
w/w % was much higher than the effects of the other organic binders
employed in Example 4 and 5 at the same concentration, and the
decline in its effect by increasing its concentration beyond the
peak is substantially not greater than the decline seen in the
graphs for Examples 4 or 5.
[0082] FIG. 3 shows the results of HPLC analysis performed in
Control Example 1 and Example 7.
[0083] As shown in the figure, hydrogenated lecithin, which was
employed in Example 7, exhibited a remarkably potent stabilizing
effect on r-hGH in any of the tested concentrations up to 2 w/w %.
In particular, even at 0.01 w/w %, i.e. the lowest concentration
tested, hydrogenated lecithin exhibited a stabilizing effect,
raising the r-hGH recovery rate to more than 70%. Beyond that
concentration and up to 0.2 w/w %, hydrogenated lecithin exhibited
still higher stabilizing effects. While it seems from the figure
that the effect of hydrogenated lecithin peaks at a concentration
somewhere around 0.5-1 w/w %, its effect declines only slightly by
increasing concentration beyond its peak. Therefore, there is no
doubt that hydrogenated lecithin has a remarkable stabilizing
effect in a concentration range much wider than tested above.
[0084] Following Table 2 shows the results of HPLC analysis for
Comparison Example 2 and Example 8.
3TABLE 2 Concentration Nonionic surfactant Aqueous of HPC-SSL and
its Monomer solution No. (w/w %) concentration (w/w %) recovery
rate (%) Control -- -- 64.04 .+-. 1.30 Example 2 Example 8 A 0.05
HCO-60 0.05 99.20 .+-. 1.16 Example 8 B 0.05 Pluronic F68 0.05
98.42 .+-. 0.61 Example 8 C 0.05 Pluronic F68 0.10 97.96 .+-. 1.34
Example 8 D 0.10 HCO-60 0.05 104.76 .+-. 0.68 Example 8 E 0.10
HCO-60 0.10 104.81 .+-. 0.17 n = 5, mean .+-. S.D.
[0085] As seen in Table 2, r-hGH was stabilized substantially
perfectly in the process of forming its aqueous solution into a
powder, by addition of both of hydroxypropylcellulose and a
nonionic surfactant to the aqueous solution. This indicates that a
combined use of both the water-soluble, nonionic organic
binder--hydroxypropylcellulose--and a nonionic surfactant provides
a higher stabilizing effect than by using them separately.
[0086] As seen from the results in Control Examples 1 and 2 and
Examples 1-8, stability of physiologically active peptide r-hGH in
a process of forming a powder from the aqueous solution of the
physiologically active peptide is remarkably improved by adding to
the solution; a nonionic surfactant such as polysorbate,
polyoxyethylenehydrogenated castor oil and poloxamer and the like;
a water-soluble, nonionic, organic binder such as
hydroxypropylcellulose and polyvinylpyrrolidone and the like; or
hydrogenated lecithin. Moreover, addition of two or more of these
components in combination further improves the stability of the
physiologically active peptide, leading to almost complete
stabilization.
EXAMPLE 9
[0087] Further studies were performed on the effect of mannitol,
either employed alone or in combination with other additives.
[0088] Materials
[0089] As GH, a recombinant human growth hormone (r-hGH) bulk
material was used. As stabilizers, D-mannitol, HPC-SSL and Pluronic
F68 were used.
[0090] Preparation of r-hGH Solution
[0091] According to the following formulas, r-hGH and additives
were weighed and dissolved in 15.0 mL of purified water to prepare
spray solutions. As a control, r-hGH alone was dissolved in 15.0 mL
of purified water to prepare a spray solution (Control Formula). In
the formulas, "% by weight" in parentheses indicates the ratio of
the weight of respective solid component to the weight of the solid
components as a whole.
4 (Formula M) r-hGH 29.25 mg (6.5% by weight) D-mannitol 420.75 mg
(93.5% by weight) Total 450.00 mg (Formula M-HP) r-hGH 29.25 mg
(6.5% by weight) D-mannitol 405.00 mg (90.0% by weight) HPC-SSL
15.75 mg (3.5% by weight) Total 450.00 mg (Formula M-P) r-hGH 29.25
mg (6.5% by weight) D-mannitol 405.00 mg (90.0% by weight) Pluronic
F68 15.75 mg (3.5% by weight) Total 450.00 mg
[0092] Spray Drying
[0093] As a spray dryer, EYELA SD- 1000 Spray Dryer were used. Dry
powders were prepared by spray-drying the above r-hGH solutions.
The conditions for spray drying was as follows.
[0094] Inlet temperature: 90.degree. C.
[0095] Dry air flow: 0.2 m.sup.3/min
[0096] Atomizing pressure: 100 kPa
[0097] Fluid feeder pump flow: 2.6 mL/min
[0098] HPLC/Monomer Content Determination
[0099] The conditions for HPLC for determination of r-hGH monomer
were as follows.
[0100] Apparatus: LC10A (SHIMADZU CORPORATION)
[0101] Sample amount: about 0.02 g/0.5 mL purified water
[0102] Detector: UV (280 nm)
[0103] Analyzing column: TSK G3000SW.sub.XL (TOSOH)
[0104] Column temperature: Room temperature
[0105] Mobile phase: 0.1 M sodium dihydrogenphosphate, 0.1 M
disodium hydrogenphosphate, 0.2 M sodium chloride.
[0106] Flowrate: 0.6 mL/min
[0107] Injection volume: 50 .mu.L
[0108] HPLC/Determination of the Content of Deamidation Product
[0109] The conditions for HPLC for determination of r-hGH
deamidation products were as follows.
[0110] Apparatus: LC10A (SHIMADZU CORPORATION)
[0111] Sample amount: about 0.02 g/0.5 mL purified water
[0112] Detector: UV (280 nm)
[0113] Analyzing column: Protein C4 column (VYDAC, Cat. No.
214ATP54)
[0114] Column temperature: 45.degree. C.
[0115] Mobile phase: 50 mM Tris-HCl (pH 7.5)/n-propanol (71:29)
buffer
[0116] Flow rate: 0.5 mL/min
[0117] Injection volume: 50 .mu.L
[0118] SDS-polyacrylamide Gel Electrophoresis
[0119] 1) Preparation of Samples
[0120] Solutions of about 0.04 mg/mL was prepared as samples. To
each 10 .mu.L of the solutions was added 10 .mu.L of water and 20
.mu.L of the sample buffer. As a standard sample, a solution of
about 1.6 mg r-hGH bulk material/mL was prepared, to 10 .mu.L of
which was added 10 .mu.L of water and 20 .mu.L of the sample
buffer.
[0121] 2) Preparation of Electrophoresis Buffer
[0122] (A) An electrophoresis buffer for 10.times.SDS-PAGE was
prepared by adding water to 30.3 g of Tris, 144 g of glycine and 10
g of SDS to make into volume of 1000 mL (for stock).
[0123] (B) An electrophoresis buffer for SDS-PAGE was prepared by
adding 900 mL of water to 100 mL of the electrophoresis buffer for
10.times.SDS-PAGE.
[0124] (C) A 0.25 M Tris-HCl buffer (pH 6.8) was prepared by adding
water to 30.25 g of Tris to make into volume of 800 mL, then
adjusting the pH of the solution to 6.8 with 6 N hydrochloric acid,
and making into volume of 1000 mL with water (preserved by
freezing).
[0125] (D) A sample buffer for SDS-PAGE was prepared by adding
water to 25 mL of 0.25 M Tris-HCl buffer (pH 6.8), 2 g of SDS, 5 g
of sucrose and 2 mg of bromphenol blue (BPB) to make 50 mL.
[0126] 3) SDS-PAGE
[0127] Using the samples and the buffer described above,
electrophoresis was carried out in a conventional manner at 20
mA/gel.
[0128] Results
[0129] The table below shows the results of the determination of
the contents of r-hGH monomer and deamidation products in the r-hGH
powders prepared above by freeze drying.
5TABLE 3 Content of Deamidation Formula r-hGH Recovery Rate (%)
Products (%) Control 68.5 7.3 M 80.5 4.2 M-HP 91.4 4.5 M-P 88.4 4.7
Bulk Material -- 3.1
[0130] As evident from the Table 3, r-hGH monomer recovery rate was
much higher in any of Formulas M, M-HP, M-P than in the control
formula: while the recovery rate of r-hGH in the control formula
was 68.5%, that was 80.5% in Formula M. In Formulas M-HP and M-P,
r-hGH recovery rate was still higher. The content of deamidation
products in any of the Formulas M, M-HP and M-P, which was lower
than that in the control formula, was substantially not different
from the proportion (3.1%) of deamidation products contained
originally in the bulk material employed. In the control formula,
in contrast, the content of deamidation products increased beyond
two times. The analysis by SDS-PAGE also showed electrophoretic
patters indicating that the purity of the peptide was higher in
Formula M than in the control formula, and that the purity in
Formula M-HP and M-P, in turn, was still higher than that in
Formula M.
EXAMPLE 10
Mannitol-containing r-hGH Powder for Transpulmonary Administration
for In Vivo Test
[0131] According to the following formula, r-hGH, HPC-SSL and
D-mannitol were weighed and dissolved in 90 mL of purified water to
obtain a spray solution. In the formula, "% by weight" in
parentheses indicates the ratio of the weight of respective solid
component to the weight of the solid components as a whole. In the
spray solution, the concentration of r-hGH, HPC-SSL and D-mannitol
is 0.27% by weight, 0.14% by weight and 2.92% by weight,
respectively. Spray drying and analyses were performed under the
same conditions as in Example 9.
6 (Formula) r-hGH 0.240 g (8.0% by weight) HPC-SSL 0.129 g (4.3% by
weight) D-mannitol 2.631 g (87.7% by weight) Total 3.000 g
[0132] The r-hGH dry powder prepared by spray drying was observed
by optical microscopy. Six hundred particles were randomly chosen
to measure their particle size. As a result, the particle size was
found to be 2.84.+-.0.83 .mu.m (mean .+-.SD, n=600). The areas of
the main peak (%) and the peak (%) for deamidation products were as
follows.
7 TABLE 4 Area of Monomer Area of Deamidation Products Peak (%)
Peak (%) Spray-dried 95.5 4.5 Product Standard 96.6 3.4
[0133] Pharmacokinetic Evaluation of GH Powder after Transpulmonary
Administration to Rats
[0134] The GH powder was administered to rats for pharmacokinetic
evaluation. The same amount of the GH powder as transpulmonarily
administered was dissolved in water and subcutaneously administered
to rats to compare its pharmacokinetics with that of
transpulmonarily administered GH.
[0135] Test Animals
[0136] Six male 9-week-old Wistar rats were used for transpulmonary
and subcutaneous injection, respectively.
[0137] GH Powder Tested
[0138] The r-hGH powder obtained in Example 10 above was used.
[0139] Administration of r-hGH
[0140] After fasting for a full day and night, the rats of the
transpulmonary administration group were anesthetized with
urethane. Two mg/kg rat body weight of the r-hGH powder was placed
in a transpulmonary administration device for rats (PennCentury).
The powder was discharged into the lungs of the rats through the
device's delivery tube inserted in the trachea by thrusting out 3
mL of air from a syringe connected to the device. The rats of the
subcutaneous administration group were also fasted for a full day
and night and then subcutaneously injected with the r-hGH powder
suspended in purified water in an amount equivalent to 2 mg/kg rat
body weight.
[0141] Blood Sampling and Processing
[0142] Blood sampling was performed just before the administration
of r-hGH and then 0, 15, 30, 60, 120, 240, 480 and 1440 minutes
thereafter. Blood was sampled from the cervical vein of restrained
rats. Blood sampling volume was 300 .mu.L at one time. Following
each blood sampling, the same amount (300 .mu.L) of physiological
saline was injected into the cervical vein. Blood samples were let
stand for one hour at room temperature and then overnight at
4.degree. C., and centrifuged (15,000 rpm, 10 minutes, 4.degree.
C.) to separate the sera.
[0143] Measurement of Blood r-hGH Concentration by GH-ELISA
[0144] An anti-hGH rabbit polyclonal antibody raised by a
conventional method was diluted and adjusted to the absorbance
OD280 of 0.02 with a 0.05 M Tris buffer. The solution was placed,
100 .mu.L each, in the wells of 96-well plates and incubated for
two hours at 37.degree. C. The plates were washed five times with a
0.01 M phosphate buffer (washing buffer). The wells of the plates
were filled with a block solution (Block Ace: Dainippon
Pharmaceutical Co., Ltd.) and let stand overnight at 4.degree. C.
The sera obtained above and r-hGH for a standard curve,
respectively, were diluted as needed with 10.times.Block Ace
aqueous solution and added to the wells of the washed plates, 100
.mu.L each, and preincubated for 2 hours at 37.degree. C.
[0145] Using the anti-hGH rabbit polyclonal antibody, a horseradish
peroxidase (HRP)-conjugated anti-hGH rabbit polyclonal antibody was
prepared in a conventional manner. The conjugated polyclonal
antibody was diluted 50,000 times with 10.times.Block Ace aqueous
solution and added to the wells of the washed plates, 100 .mu.L
each, and preincubated for 2 hours at 37.degree. C. After washing,
100 .mu.L each of TMB reagent (BIORAD) was added to the wells of
the plates and allowed to react for 10 minutes at room temperature.
The reaction was terminated by addition of 1 N sulfuric acid, and
absorbance was measured at 450 nm. A calibration curve was created
based on the absorbance for standard solutions, and the r-hGH
concentrations in the samples were derived from their absorbance
using the calibrative curve.
[0146] Results
[0147] Table 5 and FIG. 4 show r-hGH concentrations in the blood
after transpulmonary administration of the r-hGH powder or
subcutaneous injection of the r-hGH suspension.
8TABLE 5 Time after Blood r-hGH Concentration (ng/ml)
Administration Transpulmonary Subcutaneous (min) Administration
Injection 0 22.6 41.5 15 584.4 423.1 30 451.4 446.1 60 315.1 491.8
120 254.9 423.9 240 101.6 347.9 480 61.5 175.5 1440 34.7 51.3
[0148] As seen in Table 5 and FIG. 4, after transpulmonary
administration of the r-hGH powder prepared in the above example,
blood r-hGH concentration reached its peak of 584.4 ng/mL 15
minutes after the administration. The concentration then started to
decline but still remained at 34.7 ng/mL even 1440 minutes after
administration. The AUC (area under the curve representing blood
pharmacokinetics) up to 480 minutes after the administration was
128862 ng/mL.multidot.min for transpulmonary administration,
whereas that was 255826 ng/mL.multidot.min for subcutaneous
administration of the suspension containing the same amount of the
powder. As the r-hGH was transferred to the blood in unexpectedly
high efficiency after the subcutaneous injection of that
composition, the blood concentration of r-hGH following
transpulmonary administration was lower than that following its
subcutaneous injection, except for a period immediately after
pulmonary administration. However, the above results show that
r-hGH absorption after transpulmonary administration of the
composition was very high. In fact, the blood r-hGH concentration
after the transpulmonary administration of the very composition was
far higher than either of the blood r-hGH concentration after the
transpulmonary administration or subcutaneous administration of the
same amount of r-hGH suspension carried out in Control Example 3
below.
CONTROL EXAMPLE 3
[0149] According to the following formula, r-hGH and lactose were
weighed and dissolved in 120 mL of purified water to obtain a spray
solution. The concentration of r-hGH and lactose in the spray
solution is 0.20 w/w % and 2.30 w/w %, respectively.
[0150] Formula
[0151] r-hGH 0.240 g (8.0% by weight)
[0152] Lactose (monohydrate) 2.760 g (92.0% by weight)
[0153] Total 3.000 g
[0154] The above spray solution was spray-dried under the following
conditions.
[0155] Inlet temperature: 120.degree. C.
[0156] Dry air flow: 0.2 m.sup.3/min
[0157] Atomizing pressure: 100 kPa
[0158] Fluid feeder pump flow: 2.6 mL/min
[0159] Thus obtained stray-dried r-hGH powder was analyzed by the
same method as described above for the content of monomer and
deamidation products by HPLC, and subjected to SDS-PAGE. The area
of the monomer peak and that of the peak of deamidation products
are as shown in the following table, and the SDS-PAGE pattern
indicated high purity.
9 TABLE 6 Area for Deamidation Area for Monomer (%) Products (%)
Spray-dried 94.7 5.3 Product Standard 96.7 3.3
[0160] With this spray-dried product, r-hGH was transpulmonarily
administered or subcutaneously injected to male 9-week-old Wistar
rats, six animals per group, following the same dose and procedures
as indicated in "Pharmacokinetic Evaluation of GH Powder after
Transpulmonary Administration to Rats", and the pharmacokinetics
for r-hGH was determined. The results are shown in the table
below.
10TABLE 7 Time after Blood r-hGH Concentration (ng/ml)
Administration Transpulmonary Subcutaneous (min) Administration
Injection 0 5 6 15 147 46 30 129 52 60 85 62 120 70 75 240 58 71
480 48 21 1440 37 5
[0161] Natural human growth hormone, 22K hGH, is composed of 191
amino acids, with two S-S bonds within the molecule, whereas human
insulin is composed of 51 amino acids and has two S-S bonds within
the molecule. It is reasonably expected that transpulmonary
absorption demonstrated above with the powders containing human
growth hormone will occur also with human insulin, considering that
the far smaller molecule of human insulin compared with human
growth hormone will render the former more suitable for absorption
through mucous membranes and that it shares a structural similarity
with human growth hormone in light that they have two S-S bonds
within their molecule. Likewise, successful transpulmonary
absorption is expected to take place also with calcitonin (32 amino
acids) and somatostatin (28 amino acids), which are roughly of half
the size of human insulin, by forming them into the powder of the
present invention.
[0162] The present invention enables to remarkably stabilize a
physiologically active peptide in forming a powder by drying an
aqueous solution containing the physiologically active peptide,
thereby minimizing loss of the peptide in the process of powder
formation. As it is done employing additives approved as safety
ingredients in pharmaceutical products, the present invention also
enables to produce a powder stably retaining a physiologically
active peptide, without evoking unnecessary concerns on the safety
of such a product due to employed additives. The present invention
alto enables to provide physiologically active peptide-containing
powder in which content of dimers or other denatured peptide is
minimized, thereby making it easy to produce such types of
pharmaceutical compositions that are adapted to be applied to
mucous membranes in a powder form in order to introduce a drug into
the circulating blood, e.g. pharmaceutical compositions for
transnasal or transpulmonary administration. The present invention
further enables to provide inhalant compositions which allow
efficient transferal of growth hormone or insulin into the blood by
transpulmonary administration.
* * * * *