U.S. patent application number 09/767144 was filed with the patent office on 2001-05-31 for complex of human growth hormone and zinc and use.
Invention is credited to Iwasa, Susumu, Misaki, Masafumi, Yamagata, Yutaka.
Application Number | 20010002263 09/767144 |
Document ID | / |
Family ID | 17359239 |
Filed Date | 2001-05-31 |
United States Patent
Application |
20010002263 |
Kind Code |
A1 |
Yamagata, Yutaka ; et
al. |
May 31, 2001 |
Complex of human growth hormone and zinc and use
Abstract
The present invention provides a complex of human growth hormone
and zinc containing human growth hormone and zinc at a molar ratio
of about 1:1.6 to about 1:2.4, and a sustained-release preparation
which comprises the complex of human growth hormone and zinc and a
biodegradable polymer and which has a high entrapment ratio of
human growth hormone and exhibits a stable sustained-release
suppressing the initial burst.
Inventors: |
Yamagata, Yutaka; (Kobe,
JP) ; Misaki, Masafumi; (Takarazuka, JP) ;
Iwasa, Susumu; (Kyotanabe, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W. SUITE 800
WASHINGTON
DC
20006
US
|
Family ID: |
17359239 |
Appl. No.: |
09/767144 |
Filed: |
January 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09767144 |
Jan 23, 2001 |
|
|
|
09151783 |
Sep 11, 1998 |
|
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|
Current U.S.
Class: |
424/486 ;
514/11.4; 514/16.4; 514/16.9; 514/5.1; 530/399 |
Current CPC
Class: |
C07K 14/61 20130101;
Y10S 530/839 20130101; A61P 5/06 20180101; A61K 38/27 20130101;
A61K 9/1647 20130101 |
Class at
Publication: |
424/486 ;
530/399; 514/6 |
International
Class: |
A61K 038/16; A61K
009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 26, 1997 |
JP |
261251/1997 |
Claims
What is claimed is:
1. A complex of human growth hormone and zinc containing human
growth hormone and zinc at a molar ratio of about 1:1.6 to about
1:2.4.
2. A complex of claim 1, which is water-soluble.
3. A complex of claim 1, wherein the mean particle diameter of the
complex is less than about 10 .mu.m.
4. A sustained-release preparation, which comprises the complex of
claim 1 and a biodegradable polymer.
5. A preparation of claim 4, wherein the biodegradable polymer is
an aliphatic polyester.
6. A preparation of claim 5, wherein the aliphatic polyester is a
polymer of lactic acid and glycolic acid.
7. A preparation of claim 6, wherein the content ratio of a
copolymer of lactic acid and glycolic acid is 100/0 to 40/60 (mole
%).
8. A preparation of claim 5, wherein the weight-average molecular
weight of the aliphatic polyester is about 3,000 to about
20,000.
9. A preparation of claim 5, wherein the aliphatic polyester is a
salt of polyvalent metal.
10. A preparation of claim 9, wherein the polyvalent metal is
zinc.
11. A preparation of claim 4, wherein the preparation is a
microcapsule.
12. A preparation of claim 12, wherein the microcapsule is for
injection.
13. A preparation of claim 4, wherein the initial burst ratio of GH
is less than about 50%.
14. A method for producing a complex of human growth hormone and
zinc, which comprises mixing human growth hormone and zinc salt at
a molar ratio of about 1:1.6 to about 1:2.4.
15. A method for producing micronized human growth hormone, which
comprises forming a complex of human growth hormone and zinc
containing human growth hormone and zinc at a molar ratio of about
1:1.6 to about 1:2.4 and atomizing them.
16. Use of a complex of human growth hormone and zinc containing
human growth hormone and zinc at a molar ratio of about 1:1.6 to
about 1:2.4 for producing a sustained-release preparation
containing human growth hormone.
17. A method for producing a sustained-release preparation
containing human growth hormone, which comprises dispersing a
complex of human growth hormone and zinc containing human growth
hormone and zinc at a molar ratio of about 1:1.6 to about 1:2.4 in
an oil phase containing a biodegradable polymer to make a
solid-in-oil emulsion, adding the solid-in-oil emulsion to water
phase to make a solid-in-oil-in-water emulsion, and then in-water
drying the solid-in-oil-in-water emulsion.
18. A pharmaceutical composition which comprises an effective
amount of the complex of claim 1 in admixture with a
pharmaceutically acceptable carrier or diluent.
19. A pharmaceutical composition for treating or preventing
pituitary drawfism, which comprises an effective amount of the
complex of claim 1 in admixture with a pharmaceutically acceptable
carrier or diluent.
Description
[0001] The present invention relates to a complex of human growth
hormone and zinc containing human growth hormone and zinc at a
molar ratio of about 1:1.6 to about 1:2.4, a sustained-release
preparation which comprises the complex of human growth hormone and
zinc and a biodegradable polymer, and so on.
BACKGROUND ART
[0002] In recent years, human growth hormone (hereinafter
abbreviated as GH) has been produced on a large scale by utilizing
genetic engineering technology, and is used widely, for example,
being clinically applied to Turner's syndrome, infantile chronic
renal diseases, achondroplasia and adult GH hyposecretion as well
as pituitary dwarfism. Further, applications for osteoporosis in an
aging society and static heart diseases are expected.
[0003] Since GH is usually administered by intramuscular or
subcutaneous injection repeatedly and over a long term, due to
consideration of stability in body, a significant physical burden
on patients is a problem. For instance, in the case of pituitary
dwarfism, a daily subcutaneous administration to infants or young
patients over a long period of time ranging from a few months to at
least 10 years is practiced. On one hand, development of a
sustained-release preparation containing GH, which is medicinally
effective with an administration ranging from once every few weeks
to few months, has been reported (S.T.P. Pharma. Sci., 4(6), pages
437-441, 1994; Nature Med., 2(7), pages 795-799, 1996; J. Pharm.
Exp. Ther., 281, pages 1431-1439, 1997,; WO 94/12158; WO 95/29664;
WO 97/01331).
BRIEF EXPLANATION OF DRAWINGS
[0004] FIG. 1 shows the changes in particle size distribution
according to the changes in the composition ratio of GH and zinc in
a complex of GH and zinc.
[0005] FIG. 2 shows the changes in solubility of a complex
according to the changes of the composition ratio of GH and zinc in
a complex of GH and zinc.
DETAILED DESCRIPTION OF INVENTION
[0006] Current sustained-release preparation containing GH has been
produced by a method which comprises making GH in water (in water
phase) and dispersing the water phase in organic solvent (in oil
phase) containing a biodegradable polymer to make a water-in-oil
emulsion. But, in this method, GH is remarkably denaturalized in
the production process or in the shelves and a sufficient
entrapment ratio and release is not obtained. On one hand, a method
which comprises dispersing GH powder into organic solvent (in oil
phase) containing a biodegradable polymer to make a solid-in-oil
dispersion is not appropriate for producing a sustained-release
preparation on a large scale, since it is necessary to maintain
stability by spraying the solid-in-oil dispersion into liquid
nitrogen. Furthermore, since GH is not fine particle and is usually
used after atomizing, the activity of GH is remarkably lowered by
atomization GH and it is difficult to make a solid-in-oil
dispersion containing GH at a high content.
[0007] Thus, it is very difficult to maintain stability of GH and
micronize GH in the process for producing preparations. Further, it
is very difficult that a sustained-release preparation containing
GH at high content is produced on a large-scale, without lowering
activity of GH while maintaining quality and stability.
[0008] Therefore, a clinically useful preparation comprising GH
which overcomes the above problem and has constant release over a
long period of time, and a method for producing the
sustained-release preparation on a large scale at high yield, have
been desired.
[0009] The present inventors made extensive and intensive studies
and as a result, made a complex of GH and zinc containing GH and
zinc at a molar ratio of about 1:1.6 to about 1:2.4 for the first
time. Further, they found that the complex is substantially
water-soluble and the micronization of the complex is easier than
GH itself, without lowering activity of GH, and when the obtained
complex of GH and zinc having small particle diameter is used to
produce sustained-release preparation, sustained-release
preparation with assured stability and without denaturalizing GH in
a process, which has an enhanced entrapment of GH and an
improvement of the release, can be produced on a large scale.
[0010] Namely, the present invention provides
[0011] (1) a complex of human growth hormone and zinc containing
human growth hormone and zinc at a molar ratio of about 1:1.6 to
about 1:2.4,
[0012] (2) a complex of the above (1), which is water-soluble,
[0013] (3) a complex of the above (1), wherein the mean particle
diameter of the complex is less than about 10 .mu.m,
[0014] (4) a sustained-release preparation, which comprises the
complex of the above (1) and a biodegradable polymer,
[0015] (5) a preparation of the above (4), wherein the
biodegradable polymer is an aliphatic polyester,
[0016] (6) a preparation of the above (5), wherein the aliphatic
polyester is a polymer of lactic acid and glycolic acid,
[0017] (7) a preparation of the above (6), wherein the content
ratio of a polymer of lactic acid and glycolic acid is 100/0 to
40/60 (mole %),
[0018] (8) a preparation of the above (5), wherein the
weight-average molecular weight of the aliphatic polyester is about
3,000 to about 20,000,
[0019] (9) a preparation of the above (5), wherein the aliphatic
polyester is a salt of polyvalent metal,
[0020] (10) a preparation of the above (9), wherein the polyvalent
metal is zinc,
[0021] (11) a preparation of the above (4), wherein the preparation
is a microcapsule,
[0022] (12) a preparation of the above (11), wherein the
microcapsule is for injection,
[0023] (13) a preparation of the above (4), wherein the initial
burst ratio of GH is less than about 50%,
[0024] (14) a method for producing a complex of human growth
hormone and zinc, which comprises mixing human growth hormone and
zinc salt at a molar ratio of about 1:1.6 to about 1:2.4,
[0025] (15) a method for producing micronized human growth hormone,
which comprises forming a complex of human growth hormone and zinc
containing human growth hormone and zinc at a molar ratio of about
1:1.6 to about 1:2.4 and atomizing them,
[0026] (16) use of a complex of human growth hormone and zinc
containing human growth hormone and zinc at a molar ratio of about
1:1.6 to about 1:2.4 for producing a sustained-release preparation
containing human growth hormone,
[0027] (17) a method for producing a sustained-release preparation
containing human growth hormone, which comprises dispersing a
complex of human growth hormone and zinc containing human growth
hormone and zinc at a molar ratio of about 1:1.6 to about 1:2.4 in
an oil phase containing a biodegradable polymer to make a
solid-in-oil emulsion, adding the solid-in-oil emulsion to water
phase to make a solid-in-oil-in-water emulsion, and then in-water
drying the solid-in-oil-in-water emulsion,
[0028] (18) a pharmaceutical composition which comprises the
complex of the above (1), and
[0029] (19) a pharmaceutical composition for treating or preventing
pituitary drawfism, which comprises the complex of the above
(1).
[0030] GH used in the present invention may be any type, for
example, natural type (extracted products, etc.) or genetic
recombinant type GH (Nature Vol.281, page 544 (1979), Vol.293, page
408(1981), Proc. Natl. Acad. Sci. USA, Vol. 80, page 397 (1983),
Biotechnol., Vol. 5, page 161 (1981), etc.), and genetic
recombinant type GH is preferred in safety and quality. Further, in
the present invention, muteins, derivatives, analogous and active
fragments of GH may be used as GH (J. Biol. Chem., Vol. 253, page
2679 (1978), B.B.R.C., Vol. 92, page 511 (1980), Endocrinol., Vol.
109, page 1301(1981),Protein Eng. Vol. 3,page 49(1989), etc.).
[0031] Complex of GH and zinc in the present invention may be
produced by any kinds of method, for example, methods generally
used to production for complex, as far as molar ratio of GH and
zinc ranges from about 1:1.6 to about 1:2.4. The said complex of GH
and zinc is usually produced by bringing GH to contact with a
water-soluble zinc salt. This reaction for contact is preferably
employed in a solvent, for example, aqueous-solvent. Reaction time
ranges from 1 minute to 1 hour. Reaction temperature ranges from
4.degree. C. to 37.degree. C. Water-soluble zinc salts used in this
method, include salts of zinc and inorganic acids, salts of zinc
and organic acids and so on. Inorganic acids include hydrochloric
acid, sulfuric acid, nitric acid, thiocyanic acid and so on and
organic acids include aliphatic carboxylic acids, aromatic acids
and so on.
[0032] Examples of aliphatic carboxylic acids used as organic
acids, are aliphatic monocarboxylic acids, aliphatic dicarboxylic
acids, and aliphatic tricarboxylic acids. These aliphatic
carboxylic acids may be saturated or unsaturated. The aliphatic
carboxylic acid is preferably an aliphatic carboxylic acid having 2
to 9 carbon atoms.
[0033] Examples of aliphatic monocarboxylic acids are saturated
aliphatic monocarboxylic acids having 2 to 9 carbon atoms (e.g.,
acetic acid, propionic acid, butyric acid, valeric acid, caproic
acid, enanthic acid, caprylic acid, pelargonic acid, caprynic acid
etc.) and unsaturated aliphatic monocarboxylic acids having 2 to 9
carbon atoms (e.g., acrylic acid, propiolic acid, methacrylic acid,
crotonic acid, isocrotonic acid etc.).
[0034] Examples of aliphatic dicarboxylic acids are saturated
aliphatic dicarboxylic acids having 2 to 9 carbon atoms (e.g.,
malonic acid, succinic acid, glutaric acid, adipic acid, pimelic
acid etc.) and unsaturated aliphatic dicarboxylic acids having 2 to
9 carbon atoms (e.g., maleic acid, fumaric acid, citraconic acid,
mesaconic acid etc.).
[0035] Examples of aliphatic tricarboxylic acids are saturated
aliphatic tricarboxylic acids having 2 to 9 carbon atoms (e.g.,
tricarballylic acid, 1,2,3-butanetricarboxylic acid etc.).
[0036] The above-mentioned aliphatic carboxylic acids may have 1 or
2 hydroxyl groups. Such aliphatic carboxylic acids include glycolic
acid, lactic acid, glyceric acid, tartronic acid, malic acid,
tartaric acid, citric acid and so on.
[0037] The aliphatic carboxylic acid is preferably an aliphatic
monocarboxylic acid, more preferably an aliphatic monocarboxylic
acid having 2 to 9 carbon atoms, and still more preferably a
saturated aliphatic monocarboxylic acid having 2 or 3 carbon atoms.
Examples of particularly preferable aliphatic carboxylic acids
include acetic acid and so on.
[0038] Examples of aromatic carboxylic acids used as the
above-organic acid are benzoic acid and salicylic acid, with
preference given to benzoic acid.
[0039] For example, a complex of GH and zinc in the present
invention is produced by mixing GH and a water-soluble zinc salt at
a mixing ratio (molar ratio) of about 1:1.6 to about 1:2.4,
preferably about 1:1.8 to about 1:2.2 in a aqueous solvent (e.g.,
aqueous solutions containing ethanol, acetonitril or acetone at a
concentration (for example, about 1 to about 10% (W/W)) which do
not exert an adverse influence on the solubility of GH and a
water-soluble zinc salt, preferably water). The said complex may be
a compound (complex salt, double salt, salt, and organic metal
compound etc.) formed by intermolecular binding between GH and zinc
or an mixture of compounds being differed in binding patterns. The
composition ratio (molar ratio) of GH and zinc in the complex of GH
and in the present invention is within scope of about 1:1.6 to
about 1:2.4, preferably about 1:1.8 to about 1:2.2, more preferably
about 1:2. In complex of GH and zinc of the present invention,
though it is preferred that all of GH and zinc contained at molar
ratio of about 1:1.6 to about 1:2.4 form complex, GH and/or zinc
which do not form complex may be contained.
[0040] The pH of the aqueous solution resulting from the above
mixing must be such that the bioactivity of GH is not affected, and
such that each solubility of GH and zinc salt is not lowered in
excess. Although the mixing procedure is normally conducted in
distilled water, it may be conducted in water adjusted to be weakly
acidic, neutral, or weakly alkaline pH (pH 6 to 9) as necessary.
Concentration of GH and water-soluble zinc salt in the water may
range within each solubility.
[0041] The thus-obtained complex of GH and zinc in water is
substantially water-soluble since no precipitate is visible found
in the water. Substantially water-soluble complex of GH and zinc
means that solubility of complex in 1 ml of water (pH 6 to 8) at
normal temperature is more than about 2 mg.
[0042] This complex of GH and zinc in water is used for producing a
pharmaceutical composition, preferably a sustained-release
preparation after being vacuum dried or lyophylized and
micronized.
[0043] The obtained powder of complex of GH and zinc is
fine-grained and is easier to handle than bulky powder of GH free
from zinc, and is very useful for producing a sustained-release
preparation on a large scale. For example, complex of GH and zinc
can be obtained as powder whose mean particle diameter is less than
about 10 .mu.m, preferably about 4 to about 7 .mu.m.
[0044] In the case where the complex is dispersed into an organic
solvent containing hereinafter-mentioned biodegradable polymer, the
particle of small diameter is very useful for an enhanced
entrapment ratio of GH and an improved release. For example, an
entrapment ratio of GH in the sustained-release preparation is
preferably more than about 90% and concerning a sustained-release
of GH, a initial burst ratio of GH is preferable less than about
50%.
[0045] The content of the complex of GH and zinc in the
sustained-release preparation of the present invention is normally
about 0.1% (W/W) to about 40% (W/W), preferably about 1% (W/W) to
about 20% (W/W).
[0046] The biodegradable polymer is exemplified by high-molecular
polymers being slightly soluble or insoluble in water, such as
aliphatic polyesters (e.g., homopolymers, copolymers or mixtures
thereof synthesized from one or more .alpha.-hydroxycarboxylic
acids such as glycolic acid, lactic acid, hydroxybutyric acid
etc.), hydroxydicarboxylic acids such as malic acid etc.,
hydroxytricarboxylic acids such as citric acid etc. and others,
poly-.alpha.-cyanoacrylic acid esters, polyamino acids such as
poly-.gamma.-benzyl-L-glutamic acid and so on. These may be used in
mixture at appropriate ratios. The type of polymerization may be
random, block or graft.
[0047] The biodegradable polymer is preferably an aliphatic
polyester (e.g., a homopolymer, copolymer or mixture thereof
synthesized from one or more .alpha.-hydroxycarboxylic acids such
as glycolic acid, lactic acid, hydroxybutyric acid etc.,
hydroxydicarboxylic acids such as malic acid etc.,
hydroxytricarboxylic acids such as citric acid etc. and
others).
[0048] Among the above-mentioned aliphatic polyesters, homopolymers
or copolymers synthesized from one or more
.alpha.-hydroxycarboxylic acids (e.g. , glycolic acid, lactic acid,
hydroxybutyric acid etc.) are preferred from the viewpoint of
reliable biodegradability and biocompatibility. More preferably,
the aliphatic polyester is a copolymer synthesized from one or more
.alpha.-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid,
hydroxybutyric acid etc.). Also, these copolymers may be used in
mixture.
[0049] Although the above-described .alpha.-hydroxycarboxylic acid
may be of the D-, L- or D,L-configuration, it is preferable that
the ratio of the D-/L-configuration (mole%) falls within the range
from about 75/25 to about 25/75. The ratio of the
D-/L-configuration (mole %) is more preferably about 60/40 to about
30/70.
[0050] Examples of copolymers of the above-described
.alpha.-hydroxy-carboxylic acid include copolymers of glycolic acid
with another .alpha.-hydroxy acid, which is preferably lactic acid,
2-hydroxybutyric acid etc.
[0051] The .alpha.-hydroxycarboxylic acid copolymer is preferably a
lactic acid-glycolic acid copolymer, a 2-hydroxybutyric
acid-glycolic acid copolymer etc., more preferably, the
.alpha.-hydroxycarboxylic acid copolymer is a lactic acid-glycolic
acid copolymer etc.
[0052] With respect to the lactic acid-glycolic acid copolymer
(hereinafter generally called lactic acid-glycolic acid polymer or
PLGA), it is preferable that the content ratio (lactic
acid/glycolic acid ratio, hereinafter called L/G) (mole/mole %) be
about 100/0 to about 40/60. The content ratio is more preferably
about 90/10 to about 45/55, and more preferably about 80/20 to
about 45/55. The weight-average molecular weight of the lactic
acid-glycolic acid copolymer is about 3,000 to about 20,000,
preferably about 3,000 to about 16,000 more preferably about 3,000
to about 14,000.
[0053] Also, the degree of dispersion of the lactic acid-glycolic
acid copolymer (weight-average molecular weight/number-average
molecular weight) is preferably about 1.2 to about 4.0, more
preferably about 1.5 to about 3.5.
[0054] The lactic acid-glycolic acid copolymer can be synthesized
by a known process, such as the method described in Japanese Patent
Unexamined Publication No. 28521/1986. It is preferable that the
copolymer be synthesized by catalyst-free dehydration
polymerization condensation.
[0055] With respect to the 2-hydroxybutyric acid-glycolic acid
copolymer, it is preferable that glycolic acid account for about 10
to about 75 mole % and 2-hydroxybutyric acid for the remaining
portion. More preferably, glycolic acid accounts for about 20 to
about 75 mole %, and still more preferably about 30 to about 70
mole %. The weight-average molecular weight of the 2-hydroxybutyric
acid-glycolic acid copolymer is preferably about 2,000 to about
20,000. The degree of dispersion of the 2-hydroxybutyric
acid-glycolic acid copolymer (weight-average molecular
weight/number-average molecular weight) is preferably about 1.2 to
about 4.0, more preferably about 1.5 to about 3.5. A
2-hydroxybutyric acid-glycolic acid copolymer can be synthesized by
a known process, such as that described in Japanese Patent
Unexamined Publication Nos. 28521/1986 and 112465/1993. It is
preferable that the copolymer be synthesized by catalyst-free
dehydration polymerization condensation.
[0056] Preferable example homopolymers of the above-described
.alpha.-hydroxycarboxylic acid include homopolymer of lactic acid.
The weight-average molecular weight of the homopolymer of lactic
acid is about 3,000 to about 20,000, preferably about 3,000 to
about 14,000.
[0057] A homopolymer of lactic acid can be synthesized by a known
process, such as that described in Japanese Patent Unexamined
Publication No. 28521/1986. It is preferable that the homopolymer
be synthesized by catalyst-free dehydration polymerization
condensation.
[0058] The above-described 2-hydroxybutyric acid-glycolic acid
copolymer may be used in a mixture with polylactic acid. Although
the polylactic acid may be of the D- or L-configuration or a
mixture thereof, it is preferable that the ratio of the
D-/L-configuration (mole %) fall within the range from about 75/25
to about 20/80. The ratio of the D-/L-configuration (mole %) is
more preferably about 60/40 to about 25/75, and still more
preferably about 55/45 to about 25/75. The weight-average molecular
weight of polylactic acid is preferably about 1,500 to about
20,000, more preferably about 1,500 to about 10,000. Also, the
degree of dispersion of the polylactic acid is preferably about 1.2
to about 4.0, more preferably about 1.5 to about 3.5.
[0059] For producing polylactic acid, two methods are known:
ring-opening polymerization of lactide, a dimer of lactic acid, and
dehydration polymerization condensation of lactic acid. For
obtaining a polylactic acid of relatively low molecular weight for
the present invention, direct dehydration polymerization
condensation of lactic acid is preferred. This method is, for
example, described in Japanese Patent Unexamined Publication No.
28521/1986.
[0060] When a 2-hydroxybutyric acid-glycolic acid copolymer and
polylactic acid are used in mixture, their mixing ratio is about
10/90 to about 90/10 (% by weight). The mixing ratio is preferably
about 20/80 to about 80/20, and more preferably about 30/70 to
about 70/30.
[0061] In the present specification, weight-average molecular
weight is defined as the molecular weight obtained by gel
permeation chromatography (GPC) with 9 polystyrenes as reference
substances with respective weight-average molecular weights of
120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and 162.
Number-average molecular weight based on GPC measurement is also
calculated. The degree of dispersion is calculated from the
weight-average molecular weight and the number-average molecular
weight. Measurements is taken using a GPC column KF804L.times.2
(produced by Showa Denko) and an RI monitor L-3300 (produced by
Hitachi, Ltd.) with chloroform as the mobile phase.
[0062] The above-described copolymer synthesized by catalyst-free
dehydration polymerization condensation, usually has a terminal
carboxyl group.
[0063] In the present invention, the biodegradable polymer
preferably has a terminal carboxyl group.
[0064] A biodegradable polymer having a terminal carboxyl group is
a polymer in which the number-average molecular weight by GPC
determination and that by terminal group determination almost
agree.
[0065] By terminal group quantitation, number-average molecular
weight is calculated as follows:
[0066] About 1 to 3 g of the biodegradable polymer is dissolved in
a mixed solvent of acetone (25 ml) and methanol (5 ml), and the
solution is quickly titrated with a 0.05 N alcoholic solution of
potassium hydroxide while being stirred at room temperature with
phenolphthalein as an indicator to determine the terminal carboxyl
group content; the number-average molecular weight based on
terminal group quantitation is calculated using the following
equation: Number-average molecular weight based on terminal group
quantitation=20,000 A/B
[0067] A: Weight mass (g) of the biodegradable polymer
[0068] B: Amount (ml) of the 0.05 N alcoholic solution of potassium
hydroxide added until the titration end point is reached
[0069] For example, in the case of a polymer having a terminal
carboxyl group synthesized from one or more .alpha.-hydroxy acids
by catalyst-free dehydration polymerization condensation, the
number-average molecular weight based on GPC measurement and the
number-average molecular weight based on terminal group
quantitation almost agree. On the other hand, in the case of a
polymer having essentially no terminal carboxyl group synthesized
from a cyclic dimer by ring-opening polymerization using a
catalyst, the number-average molecular weight based on terminal
group quantitation is significantly higher than the number-average
molecular weight based on GPC determination. This difference makes
it possible to clearly differentiate a polymer having a terminal
carboxyl group from a polymer having no terminal carboxyl
group.
[0070] While the number-average molecular weight based on terminal
group quantitation is an absolute value, the number-average
molecular weight based on GPC determination is a relative value
that varies depending on various analyti-cal conditions (e.g., kind
of mobile phase, kind of column, reference substance, slice width
chosen, baseline chosen etc.); it is therefore difficult to have an
absolute numerical representation of the latter. However, the fact
that the number-average molecular weight based on GPC determination
almost agrees the number-average molecular weight based on terminal
group quantitation means that the number-average molecular weight
based on terminal group quantitation falls within the range from
about 0.5 to about 2 times, preferably from about 0.8 to about 1.5
times as high as the number-average molecular weight based on GPC
determination. Also, the fact that the number-average molecular
weight based on terminal group quantitation is significantly higher
than the number-average molecular weight based on GPC determination
means that the number-average molecular weight based on terminal
group quantitation is about 2 times or more as high as the
number-average molecular weight based on GPC determination.
[0071] Also, as the biodegradable polymer of the present invention,
a metal salt (also referred to as complex) of the above-described
biodegradable polymer is preferably used. For instance, a
polyvalent metal salt of each kind of biodegradable polymer
disclosed in WO 97/01331, preferably divalent metal salt,
especially zinc salt of lactic acid-glycolic acid copolymer is
preferably used. Biodegradable polymers can be produced by a
process described in WO 97/01331 and the modifications.
[0072] In the case that the polyvalent metal salt of biodegradable
polymer is zinc, the polymer may be produced by reacting
biodegradable polymer with zinc oxide in an organic solvent.
[0073] In the said process, biodegradable polymer and zinc oxide
are first allowed to exist together in an organic solvent to
prepare a solution of complex of a biodegradable polymer and zinc
oxide in the organic solvent. Although the concentration of
biodegradable polymer in the solution varies depending on the
molecular weight and the kinds of the organic solvent, and it is,
for instance, about 0.1 to about 80% (W/W), preferably about 1 to
about 70% (W/W) and more preferably about 2 to about 60% (W/W).
Although the amount of zinc oxide added varies depending on the
kinds of the organic solvent, it is, for instance, about 0.001 to
about 2% (W/W), preferably about 0.01 to about 1.5% (W/W) and more
preferably about 0.1 to about 1% (W/W), based on the amount of the
biodegradable polymer.
[0074] Regarding the order of addition of the biodegradable polymer
and zinc oxide to the organic solvent, zinc oxide both in a powder
state or in a dispersed state in the organic solvent can be added
to a solution of the biodegradable polymer in the organic solvent,
conversely, a solution of the biodegradable polymer in the organic
solvent can be added to a dispersion of zinc oxide in the organic
solvent. Further, the organic solvent can be added after the
biodegradable polymer and zinc oxide both in a powder state are
admixed.
[0075] The conditions to produce a solution of complex of a
biodegradable polymer and zinc oxide, such as complex of PLGA and
zinc oxide, from a biodegradable polymer and zinc oxide can be
changed according to the kinds of the biodegradable polymer used,
the particle diameter of zinc oxide, the kinds of the organic
solvent, and these composition ratio. When PLGA is, for example,
employed as a polymer, a complex of PLGA and zinc oxide can be
obtained by the above reaction usually at about 0 to about
30.degree. C., preferably about 2 to about 25.degree. C., for about
1 to about 168 hours, preferably about 12 to about 96 hours, more
preferably about 24 to about 72 hours. The production of complex of
PLGA and zinc oxide in the present invention can be confirmed
visibly since zinc oxide which is in a dispersed state at the time
of addition dissolves in the organic solvent to give a clear
solution, and the reaction time is not limited to the above ranges
and can be determined using turbidity as an index.
[0076] Although this reaction proceeds simply by the co-presence of
PLGA and zinc oxide in the organic solvent, the reaction carried
out under stirring or shaking is advantageous to reduction of the
reaction time. Further, the reaction carried out under
ultrasonication is equally preferred. As the reaction temperature
becomes higher, the reaction time becomes shorter.
[0077] The thus obtained complex of biodegradable polymer and zinc
oxide is applied to the next process, preferably as a solution in
an organic solvent, or if necessary as a solid after removal of the
organic solvent.
[0078] The sustained-release preparation of the present invention
is produced by removing the organic solvent from dispersion,
preferably a solid-in-oil dispersion in which a complex of GH and
zinc containing at molar ratio of about 1:1.6 to about 1:2.4,
preferably as powder, is dispersed into a solution of a
biodegradable polymer (hereafter also means "biodegradable polymer"
including metal salt of biodegradable polymer) in an organic
solvent (oil phase). Methods of producing a sustained-release
preparation include the in-water drying method, phase separation
method, spray drying method, and modifications thereof.
[0079] Methods of producing a sustained-release preparation, e.g.,
microcapsules, are described below.
[0080] (a) In-water drying method (S/O/W method)
[0081] In this method, a solution of a biodegradable polymer in an
organic solvent is first prepared. The organic solvent used to
produce the sustained-release preparation of the present invention
preferably has a boiling point not higher than 120.degree. C. Such
organic solvents include halogenated hydrocarbons (e.g.,
dichloromethane, chloro-form, carbon tetrachloride etc.), alcohols
(e.g., ethanol, methanol), acetonitrile and so on. These may be
used in mixture at appropriate ratios. The organic solvent is
preferably dichloromethane and acetonitrile, and still more
preferably dichloromethane. The concentration of the biodegradable
polymer in the organic solvent solution is normally about 0.01 to
about 80% (W/W), preferably about 0.1 to about 70% (W/W), and more
preferably about 1 to about 60% (W/W), depending on the molecular
weight of the biodegradable polymer, kinds of organic solvent and
so on.
[0082] To the organic solvent solution (oil phase) of the
biodegradable polymer thus obtained, a complex of GH and zinc is
added or dispersed. In this operation, the amount of complex of GH
and zinc added is set so that the complex of GH and zinc weight
ratio to biodegradable polymer is up to about 0.4, preferably about
0.2. As a method of dispersing, powder of complex of GH and zinc
may be added and dispersed uniformly, and lyophilized bulk of
complex of GH and zinc is added directly and dispersed uniformly by
atomizing and mixing in an oil phase.
[0083] The organic solvent suspension (S/O type dispersion) thus
prepared is added to an aqueous phase (water phase) to form an
S/O/W type emulsion using a turbine type mechanical stirrer,
ultrasonic equipment or the like, followed by evaporation of the
solvent in the oil phase, to yield microcapsules. The volume of the
aqueous phase is normally chosen over the range of about 1 to about
10,000 times, preferably about 5 to about 2,000 times, and more
preferably about 10 to about 1,000 times, the volume of the oil
phase.
[0084] An emulsifier may be added to the external aqueous phase.
The emulsifier may be any one, as long as it is capable of forming
a stable S/O/W type emulsion. Examples of such emulsifiers include
anionic surfactants, nonionic surfactants, polyoxyethylene castor
oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol,
carboxymethyl cellulose, lecithin, gelatin, hyaluronic acid and so
on. These may be used in combination as appropriate. The emulsifier
concentration in the external aqueous phase is preferably about
0.001 to about 20% (W/V), more preferably about 0.01 to about 10%
(W/V), and still more preferably about 0.05 to about 5% (W/V).
[0085] The thus obtained microcapsules are recovered by
centrifugation or filtration, washed with distilled water to remove
the emulsifier etc. adhering to the surface of microcapsules,
redispersed in distilled water, and lyophilized. Then, if
necessary, water and the organic solvent in the microcapsules are
further removed by heating. The heating may be conducted under
reduced pressure. Regarding the heating conditions, heating and
drying are conducted at a temperature not lower than a glass
transition temperature of the biodegradable polymer and not so high
as to cause aggregation of each microcapsule particle. The heating
and drying are conducted preferably at a temperature ranging from
the glass transition temperature of the biodegradable polymer to a
temperature which is about 30.degree. C. higher than the glass
transition point obtained using a differential scanning calorimeter
when the temperature is increased at a rate of about 10 to about
20.degree. C. per minute.
[0086] (b) Phase separation method (Coacervation method)
[0087] In this method, a coacervating agent is gradually added to
the above described S/O type dispersion under stirring to
precipitate and solidify microcapsules. The amount of the
coacervating agent used is about 0.01 to about 1,000 times by
volume, preferably about 0.05 to about 500 times by volume,
especially preferably about 0.1 to about 200 times by volume. Any
coacervating agent can be used, as long as it is a polymeric,
mineral oil or vegetable oil compound miscible with the organic
solvent for dissolution of a biodegradable polymer and it does not
dissolve the biodegradable polymer used. Specifically, examples of
such coacervating agents include silicone oil, sesame oil, soybean
oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral
oil, n-hexane and n-heptane. Two or more of these may be used in
combination. The thus obtained microcapsule are recovered by
filtration, and washed repeatedly with heptane etc. to remove the
coacervating agent. Further, washing is conducted in the same
manner as in the above (a), followed by lyophilization.
[0088] (c) Spray-drying method
[0089] In this method, the above described S/O type dispersion is
sprayed via a nozzle into the drying chamber of a spray drier to
volatilize the organic solvent in fine droplets in a very short
time to yield microcapsules. Examples of the nozzle include, for
instance, a two-fluid nozzle type, a pressure nozzle type and a
rotary disc type. It is also advantageous, if necessary , to spray
an aqueous solution of the above-described antiaggregation agent
via another nozzle in order to prevent aggregation of each
microcapsule particle. The thus obtained microcapsule is washed in
the same manner as in the above (a), if necessary followed by
heating (if necessary under reduced pressure) to remove water and
the organic solvent.
[0090] The sustained-release preparation of the present invention
is preferably used in the form of fine particles. This is so that
the sustained-release preparation does not cause undue pain to the
patient when administered via an injection needle for ordinary
subcutaneous or intramuscular injection. The mean particle diameter
of the sustained-release preparation, for example, is about 0.1 to
about 300 .mu.m, preferably about 1 to about 150 .mu.m, and more
preferably about 2 to about 100 .mu.m.
[0091] In the present specification, the microcapsule may be a fine
particle (called microspheres) comprising active ingredient
(complex of GH and zinc) and a base of microcapsule (biodegradable
polymer). Typically, they include microcapsules containing one core
of active ingredient in one particle, or microcapsules containing
many cores of active ingredient in one particle.
[0092] The sustained-release preparation of the present invention,
for example, can be administered as microcapsules, in the form of
various dosage forms of non-oral preparations (e.g., intramuscular,
subcutaneous or visceral injections or indwellable preparations,
nasal, rectal or uterine transmucosal preparations etc.) or oral
preparations (e.g., capsules such as hard capsules, soft capsules
etc., solid preparations such as granules and powders etc., liquid
preparations such as suspensions etc.).
[0093] In the present invention, the sustained-release preparation
is preferably used for injection. When the sustained-release
preparation is a microcapsule, for instance, it can be prepared as
an aqueous suspension by suspending microcapsules in water, along
with a dispersing agent (e.g., surfactants such as polysorbate
(Tween 80, Bio Rad) and HCO-60 (Nikko Chemicals), polysaccharides
such as carboxymethyl cellulose, sodium alginate and sodium
hyaluronate etc.), a preservative (e.g., methyl paraben, propyl
paraben etc.), an isotonizing agent (e.g., sodium chloride,
mannitol, sorbitol, glucose etc.), etc., to yield a
sustained-release preparation for injection of practical use.
Alternatively, the sustained-release preparation of the present
invention is prepared as an oily suspension by dispersing
microcapsules, along with a vegetable oil such as sesame oil or
corn oil with or without a phospholipid such as lecithin, or a
medium-chain fatty acid triglyceride (e.g., MIGLYOL 812, Huls A. G.
(Marl, Germany)), to yield a sustained-release preparation for
injection of practical use.
[0094] When the sustained-release preparation is a microcapsule,
for instance, its mean particle size is chosen over the range from
about 0.1 to about 300 .mu.m as long as the requirements concerning
degree of dispersion and needle passage are met, when it is to be
used as an injectable suspension. Preferably, the particle size
falls within the range from about 1 to about 150 .mu.m, more
preferably about 2 to about 100 .mu.m.
[0095] The above-described microcapsule can be prepared as a
sterile preparation, without limitation, for example, by the method
in which the entire production process is sterile, the method in
which gamma rays is used as sterilant, and the method in which an
antiseptic is added.
[0096] The sustained-release preparation of the present invention
is of low toxicity and can be safely used in mammals (e.g., humans,
bovines, swines, dogs, cats, mice, rats, rabbits etc.).
[0097] The sustained-release preparation of the present invention
is useful for treating or preventing adult GH hyposecretion,
Turner's syndrome, pituitary dwarfism, chronic renal diseases,
achondroplasia, adult hypopituitarism, Down syndrome, Silver
syndrome, hypochondroplasia, juvenile chronic arthritis and static
heart diseases etc.
[0098] Depending on duration of the release, target disease,
subject animal species and other factors, the dose of the
sustained-release preparation may be set at any level, as long as
the effective concentration of GH in the body is maintained. For
instance, when the sustained-release preparation is designed for
two weeks release and administered to patients for pituitary
dwarfism, the dose of an effective ingredient can be chosen from
the range of preferably about 0.01 to about 5 mg/kg body weight,
more preferably about 0.03 to about 1 mg/kg body weight, per an
adult, administered once every two weeks.
[0099] The complex of GH and zinc of the present invention is with
low toxicity and while the complex of GH and zinc can be
administrated as it is, it is usually administered in the form of
composition formulated by a conventional method using
pharmaceutical acceptable carriers or diluents for pharmaceutical
compositions adequately selected from excipients (e.g. calcium
carbonate, kaolin, sodium hydrogencarbonate, lactose, corn starch,
crystalline cellulose, talc, fine granulated sugar and porous
substance), binders (e.g. dextrin, gum, .alpha.-starch, gelatin,
hydroxypropyl cellulose, hydroxypropylmethyl cellulose and furran),
thickener (e.g. natural rubbers and cellulose derivatives),
disintegrants (e.g. carboxymethyl cellulose calcium, closcarmellose
sodium, clospovidone, low-substituted hydroxypropyl cellulose and
partial .alpha.-starch), solvents (e.g. water for injection,
physiological saline, ringels solution, alcohol, propylene glycol,
sesame oil and corn oil), dispersing agent (e.g. Tween 80, HCO 60,
carboxymethyl cellulose and sodium alginate), suspending agents
(e.g. sodium lauryl sulfate and benzalkonium chloride), dissolution
acids (e.g. polyethylene glycol, propylene glycol, D-mannitol,
benzyl benzoate, ethanol, torisaminomethane, triethanolamine,
sodium carbonate and sodium citrate), anesthetizing agent (e.g.
benzylalcohol), buffers (e.g. phosphate, acetate, carbonate and
citrate), lubricants (e.g. magnesium stearate, calcium stearate,
talc, starch and sodium benzoate), colorants (e.g. tar pigment,
caramel, iron sesquioxide, titanium oxide and riboflavins),
falvoring agents (e.g. sweeteners and perfume), stabilizers (e.g.
sodium sulfite and ascorbic acid) and preservatives (e.g. parabens
and sorbic acid) in adequate amounts respectively. The
pharmaceutical composition in the present invention which may
contain the above-mentioned carriers or diluents for pharmaceutical
compositions contains effective amounts of the complex of GH and
zinc for preventing and treating the above-diseases in a similar
way to the above-sustained-release preparation. The content of the
complex of GH and zinc of the present invention in the
pharmaceutical composition ranges usually from about 0.1 to about
100 weight % relative to the whole weight of the pharmaceutical
composition.
[0100] Although the sustained-release preparation may be stored at
normal temperature or in a cold place, it is preferable to store it
in a cold place. Normal temperature and a cold place as mentioned
herein are as defined by the Pharmacopoeia of Japan, specifically,
15 to 25.degree. C. for normal temperatures and under 15.degree. C.
for cold places.
EXAMPLES
[0101] The present invention will be explained in more detail by
the following Examples, Reference Examples, Comparative Examples
and Experimental Examples. The scope of the present invention is
not intended to be restricted to them. In the present specification
and Examples, amino acid abbreviations are based on those
recommended by the IUPAC-IUB Commission on Biochemical
Nomenclature, or those used commonly in the related field, and as
shown below. In the case that amino acids have optical isomers,
they represent L-type unless otherwise specified.
[0102] SDS: Sodium dodecylsulfate
[0103] Gly: Glycine
[0104] Ala: Alanine
[0105] Val: Valine
[0106] Leu: Leucine
[0107] Ile: Isoleucine
[0108] Ser: Serine
[0109] Thr: Threonine
[0110] Cys: Cystein
[0111] Met: Methionine
[0112] Glu: Glutamic acid
[0113] Gln: Glutamine
[0114] Asp: Aspartic acid
[0115] Asn: Asparagine
[0116] Lys: Lysine
[0117] Arg: Arginine
[0118] His: Histidine
[0119] Phe: Phenylalanine
[0120] Tyr: Tyrosine
[0121] Trp: Tryptophan
[0122] Pro: Proline
[0123] Asx: Asp+Asn
[0124] Glx: Glu+Gln
Reference Example 1
[0125] Construction of expression vector for GH using T7
promoter
[0126] The structure gene of GH was isolated as about 0.75 kb of
EcoRI-EcoRV fragment from plasmid pHGH107 (ATCC 31538 or ATCC
40011) described in Japanese Patent Publication No. 12996/1994. On
the other hand, T7 promoter and ampicillin resistant gene were
isolated as about 4.6 kb of NdeI-BamHI fragment from pET-3C
[Rosenberg et al., Gene, 56, 125 (1987)]. Both of the two fragments
were treated with T4 DNA polymerase (DNA blunting kit; Takara
Shuzo, Inc.) and ligated with T4 DNA ligase, followed by
introduction into Escherichia coli JM109 and selection of
ampicillin resistant transformant. From the obtained 12 colonies,
plasmids were prepared and digested with PstI. As a result, it was
found that GH gene was inserted in a correct direction in the
plasmids from the 6 colonies. The plasmid obtained from one
transformant among the 6 colonies was named as pTGA201.
Reference Example 2
[0127] Expression of Met-GH in Escherichia coli
[0128] Escherichia coli JM109 was transformed with .lambda. phage
(Studie, Supura) having RNA polymerase gene of T7 phage.
Thereafter, into the obtained Escherichia coli JM109 (DE3), GH
expression vector pTGA201 obtained in Reference Example 1 was
introduced to obtain Escherichia coli JM109 (DE3)/pTGA201.
[0129] Escherichia coli JM109 (DE3)/pTGA201 was inoculated into a
flask of 2 liter capacity containing 1 liter of LB medium [1%
peptone, 0.5% yeast extract, 0.5% sodium chloride] and 50 .mu.g/ml
ampicillin and then subjected to rotary shaking cultivation at
30.degree. C. for 16 hours. The resultant culture liquid was then
transferred to a 50 liter jar fermentor containing 20 liter of LB
medium [0.02% antiforming agent (New Pole LB-625; San-yo Kasei
Kogyo), 50 .mu.g/ml ampicillin], after which it was subjected to
cultivation under aeration and agitation at 37.degree. C. for 6
hours. The resultant culture liquid was then transferred to a 500
liter jar fermentor containing 360 liter of a liquid production
medium [1.68% sodium hydrogen phosphate, 0.3% potassium dihydrogen
phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.024%
magnesium sulfate, 0.02% antiforming agent (New Pole LB-625),
0.0005% thiamine hydrochloride, 1.5% glucose, 1.5% casamino acid],
after which it was subjected to cultivation under aeration and
agitation at 37.degree. C. When the Klett value was about 500, 5.95
mg/L/minute of isopropyl-.beta.-D-thiogalactopyronoside (IPTG) was
added to the medium and the cultivation was further continued for 4
hours. The culture liquid was centrifuged to obtain about 4.5 kg of
wet cells which were frozen at -80.degree. C.
[0130] The above-described transformant Escherichia coli JM109
(DE3)/pTGA201 has been deposited FERM BP-5632 at the NIBH (National
Institute of Bioscience and Human-Technology) and IFO 16001 at the
IFO (Institute Fermentation Osaka).
Reference Example 3
[0131] Activation of Met-GH
[0132] Two kg of wet cells obtained in Reference Example 2 was
dissolved in 6 1 of 50 mM Tris-HCl and guanidine hydrochloride (pH
8.0), followed by centrifugation (10000 rpm, 120 minutes). To 6
liters of the resultant supernatant, was added 18 liter of a
solution (pH 8.0) containing 50 mM Tris-HCl, 0.28 mM GSSG and 0.7 M
Arg to adjust pH 8.0, followed by standing at 4.degree. C. for 5
days to continue activation of Met-GH.
Reference Example 4
[0133] Purification of Met-GH
[0134] The solution obtained in Reference Example 3 was subjected
to salting-out and concentration by Pellicon cassette system (PTGC
membrane; Millipore Corporation) and with adding a solution (pH
8.0) of 20 mM Tris-HCl and 2.5 M urea until electric conduction
became not more than 10 mS. The obtained concentrate was
centrifuged (10000 rpm, 60 minutes) to obtain 5 liter of
supernatant. The supernatant was loaded on DEAE-Toyopearl 650 M
column (20 cm.PHI..times.84 cm, Tosoh) equilibrated with a solution
(pH 8.0) of 20 mM Tris-HCl and 2.5 M urea, followed by adsorption
and washing. The column was eluted with using a linear
concentration gradient consisting of 0-25% solution B (B=20 mM
Tris-HCl, 2.5 M urea, 1 M NaCl, pH 8.0) at 300 ml/minute of flow
rate for 100 minutes. The eluted solution containing Met-GH of 10
liter was again subjected to salting-out and concentration by
Pellicon cassette system (PTGC membrane; Millipore). The
concentrated solution was passed through DEAE-5PW column (21
cm.PHI..times.30 cm, Tosoh) using HPLC method (Gilson HPLC system;
Gilson). The column was eluted using a pH gradient consisting of
70-85% solution B (A=50 mM Tris-HCl and 2.5 M urea (pH 8.0); B=50
mM MES [2-(N-morpholino)ethane sulfonate] and 2.5 M urea (pH 4.0))
at 320 ml/minute of flow rate for 70 minutes. To the obtained
Met-GH fraction 6 liter, was added 2 M Tris-HCl (pH 7.8) to adjust
to pH 7.2, followed by salting-out and concentration by Pellicon
cassette system (PTGC membrane; Millipore) to obtain 9,979 mg of
Met-GH.
Reference Example 5
[0135] Removal of N-terminal Met
[0136] To 1650 ml solution of Met-GH obtained in Reference Example
4, was added 413 ml of a solution containing 35 mM copper sulfate,
2.5 M glyoxylic acid and 6 M pyridine and the mixture was stirred
and allowed to stand at 25.degree. C. for 1 hour. The reaction
solution was passed at a flow rate of 3 liter/h through Sephadex
G-25 column (11.3 cm.PHI..times.125 cm, Pharmacia) equilibrated
with a solution (pH 8.0) of 20 mM Tris-HCl and 2.5 M urea and the
column washed with the same solution to collect the fraction of
diketone derivative of Met-GH. The eluted fraction was directly
added to a 4 liter solution of 4 M acetic acid, 4 M sodium acetate,
80 mM o-phenylenediamine and 3 Murea with stirring. After the
elution, the reaction solution (8 liters) was allowed to stand at
4.degree. C. for 3 days. The solution was subjected to salting-out
by Pellicon cassette system (PTGC membrane; Millipore). The
concentrated solution (4 liters) was passed at a flow rate of 3
liter/h through Sephadex G-25 column (11.3 cm.PHI..times.140 cm,
Pharmacia) equilibrated with a solution (pH 8.0) of 20 mM Tris-HCl
and 2.5 M urea to collect the fraction of GH (4.7 liter). The
obtained fraction was passed through a DEAE-5 PW column (21
cm.PHI..times.30 cm, Tosoh) using HPLC method (Gilson HPLC system;
Gilson). The column was eluted using a pH gradient consisting of
70-85 % solution B (A=50 mM Tris-HCl and 2.5 M urea (pH 8.0); B=50
mM MES [2-(N-morpholino)ethane sulfonate] and 2.5 M urea (pH 4.0))
at a flow rate of 320 ml/minute for 70 minutes to collect 10 liter
fraction of GH. To the obtained GH fraction was added 500 ml
solution of 2 M Tris-HCl (pH 7.8) to adjust to pH 7.2, followed by
concentration with Minitan II (PTGC membrane; Millipore). The
concentrated solution 500 ml was passed at a flow rate of 2 liter/h
through Sephacryl S-100 column (11.3 cm.PHI..times. 50 cm,
Pharmacia) equilibrated with distilled water to collect the GH
fraction 1651 ml, and followed by filtration with Millipack 60
(Millipore) to obtain GH solution 1487 ml (3309 mg of GH).
Reference Example 6
[0137] Determination of Feature of GH
[0138] (a) Analysis with SDS-polyacrylamide gel electrophoresis
[0139] To the GH solution obtained in Reference Example 5 was added
the same volume of Sample buffer [Laemmli, Nature, 227, 680 (1970)]
containing 100 mM DTT, and the mixture was heated at 95.degree. C.
for 2 minutes, followed by electrophoresis with Multi Gel 10/20
(Daiichi Pure Chemicals). After electrophoresis, the gel was
stained with Coomassie brilliant blue and only one single band at
about 22 kd of the purified protein was obtained.
[0140] (b) Analysis of amino acid composition
[0141] Analysis of amino acid was used for the determination of
amino acid composition was done with an amino acid analyzer
(L-8500A, Hitachi). The amino acid composition of GH obtained
agreed with that predicted from cDNA sequence of GH (Table 1).
1TABLE 1 Analysis of amino acid composition Number of residues
Values predicted from Amino acid per 1 mole cDNA sequence of GH Asx
20.2 20 Thr.sup.1) 10.0 10 Ser.sup.1) 16.7 18 Glx 27.0 27 Pro 8.1 8
Gly 8.2 8 Ala 7.6 7 Cys.sup.2) N.D. 4 Val 7.0 7 Met 3.0 3 Ile 7.7 8
Leu 27.9 26 Tyr 8.1 8 Phe 12.7 13 His 3.2 3 Lys 8.9 9 Arg 10.9 11
TrP 0.8 1 Acid hydrolysis (6N HCl, 4% thioglycolic acid,
110.degree. C., Mean value of those obtained after 24 and 48 hours
of hydrolysis) .sup.1)Value extrapolated on the assumption that
hydrolysis time was 0 hours. .sup.2)Undetected Analysis was carried
out using about 20 .mu.g of GH.
[0142] (c) Analysis of N-terminal amino acid sequence
[0143] The N-terminal amino acid sequence of GH was determined
using a gas-phase protein sequencer (Applied Biosystems, 477A
model). The N-terminal amino acid sequence of GH obtained agreed
with that predicted from cDNA sequence of GH (Table 2).
2TABLE 2 Analysis of N-terminal amino acid sequence
PTH.sup.1)-amino acid amino acid predicted from Residue No.
detected (pmol) cDNA sequence of GH 1 Phe (949) Phe 2 Pro (404) Pro
3 Thr (422) Thr 4 Ile (744) Tle 5 Pro (283) Pro 6 Leu (514) Leu 7
Ser (136) Ser 8 Arg (36) Arg 9 Leu (377) Leu 10 Phe (408) Phe 11
Asp (77) Asp 12 Asn (230) Asn 13 Ala (435) Ala 14 Met (334) Met 15
Leu (398) Leu 16 Arg (67) Arg 17 Ala (488) Ala 18 His (30) His 19
Arg (42) Arg 20 Leu (406) Leu .sup.1)phenylthiohydantoin Analysis
was carried out using 1 nmol of GH.
[0144] (d) Analysis of C-terminal amino acid
[0145] Analysis of C-terminal amino acid was done by determination
of C-terminal amino acid with amino acid analyzer (L-8500A,
Hitachi). The C-terminal amino acid of GH obtained agreed with that
predicted from cDNA sequence of GH (Table 3).
3TABLE 3 Analysis of C-terminal amino acid C-terminal amino acid
Yield (%) Phe 52 Vapor-phase hydrazinolysis (100.degree. C., 3.5
hours) Analysis was carried out using 18 nmol of GH.
[0146] (e)Determination of GH activity
[0147] GH purified and obtained in Reference Example 5 had a cell
growth enhancing activity to Nb 2 cells according to the method
described in Journal of Clinical Endocrinology and Metabolism, 51,
1058 (1980) almost similar to a standard product (Chemicon
International, USA).
Example 1
[0148] (1)Production of complex of GH and zinc
[0149] To 120 ml of aqueous solution of genetic recombinant type GH
(2 mg/ml) obtained according to Reference Example 5, 0.5 ml of two
kinds of aqueous solution of zinc acetate whose concentration was
set so that the molar ratio of zinc to 1 mole of GH was 1.8 and
2.0, were added independently and were lyophilized to yield complex
of GH and zinc (about 230 mg).
[0150] (2)Production of microcapsule containing GH
[0151] 1.89 g of lactic-glycolic acid copolymer (lactic
acid/glycolic acid=50/50, average molecular weight converted into
polystyrene=12,000, viscosity=0.145 dl/g) was dissolved in 4 ml of
dichloromethane, and after 10 mg of zinc oxide was added, it was
stirred (60 rpm) at 25.degree. C. to be dissolved completely. To
this solution of polymer in an organic solution, 100 mg of complex
of GH and zinc obtained in the above (1) was added and was atomized
by Polytron (Kinematica). The obtained S/O dispersion was added to
800 ml of 0.1% aqueous solution of polyvinyl alcohol, and stirred
and emulsified by homomixer. After drying in water for 2 hours, it
was washed with distilled water and lyophilized to yield
microcapsules containing GH (1.07 g (1:1.8), 0.91 g (1:2.0)).
Comparative Example 1
[0152] (1) Production of GH powder
[0153] To 120 ml of aqueous solution of genetic recombinant type GH
(2 mg/ml) obtained according to Reference Example 5, 0.5 ml of
various kinds of aqueous solution of zinc acetate whose
concentration was set so that the molar ratio of zinc to 1 mole of
GH were 0, 3.0, 4.0, 5.0 and 6.0, were added independently and was
lyophilized to yield GH powder (about 230 mg).
[0154] (2) Production of microcapsule containing GH
[0155] By the same method of Example 1-(2), microcapsule containing
GH (0.95 g (1:0), 1.23 g (1:3), 1.13 g (1:4), 1.2 g (1:5), 1.27 g
(1:6)) was obtained by using the GH powder described in the
above-described (1).
Experimental Example 1
[0156] By using microcapsules produced in Example 1- (2) and
Comparative Example 1-(2), the following experiments were
conducted.
[0157] (1)Content of GH
[0158] 300 .mu.l of acetonitrile was added to 4 mg of microcapsules
produced in Example 1-(2) or Comparative Example 1- (2), the base
lactic acid-glycolic acid copolymer was dissolved in it, and 700
.mu.l of aqueous solution which contained 0.02% bovine serum
albumin-0.05% trifluoro acetic acid was added by stirring to elute
GH. In the supernatant obtained by the centrifugation, the content
of GH in the microcapsule was determinated by high performance
liquid chromatography. The result is shown in Table 4.
[0159] As illustrated by Table 4, more than 90% entrapment ratio of
GH was obtained in the case of using the complex of the present
invention which contains GH and zinc at a molar ratio of 1.8 or 2.0
of zinc to 1 mole of GH.
[0160] It is clear that the sustained-release preparation
containing a complex of GH and zinc in the present invention has an
excellent entrapment ratio of GH.
[0161] (2) Release in vivo
[0162] Microcapsules produced in Example 1-(2) and Comparative
Example 1- (2) were subcutaneously administered to imunosuppressed
SD rats (male, 6 weeks old) in the amount of 6 mg GH/rat. Blood was
collected periodically and the serum concentration was assayed with
radioimuunoassay kit (Ab beads HGH:Eiken Kagaku). Immunosuppressed
SD rats were obtained by administering Prograph.RTM. (Fujisawa
Pharm.) in the amount of 0.4 mg/rat on 3 days before administration
of microcapsule and in the amount of 0.2 mg/rat on the
administration day and 4, 7, 11 and 14 days after the
administration. The amount of GH released on the first day and 1-18
days after the administration of microcapsule were calculated based
on pharmaco kinetic parameters (AUC and clearance) obtained by the
changes of serum GH concentration. The initial burst amount GH was
obtained by ratio of the amount of GH released during the first day
after the administration to the amount of GH administered. The
result is shown in the Table 4.
[0163] As shown in Table 4, in microcapsules containing the complex
of GH and zinc at a molar ratio of 1:1.8 or 1:2.0, the initial
burst amount of GH (amount released until 1 day (24 hours) after
administration) was low (less than about 30%) and the amount of GH
release after 1 day (amount released from 1 day after
administration (24 hours to 18 days after administration)) was high
(more than about 50%). In the microcapsule produced in Comparative
Example 1-(2), an effective sustained release was not obtained
because the initial burst amount was high and the amount of release
after it was low.
[0164] It is clear that the sustained-release preparation
containing a complex of GH and zinc in the present invention has an
excellent sustained-release.
4TABLE 4 initial GH/zinc amount of GH burst micro- molar entrapment
release ratio capsule ratio ratio (%) 1 day 1-18 days (%) Example 1
1:1.8 92 1.60 2.31 26.7 1:2.0 92 1.23 2.68 20.5 Comparative 1:0 88
3.26 1.96 54.3 Example 1c 1:3.0 73 4.32 1.95 72.0 1:4.0 75 4.83
1.97 80.5 1:5.0 70 5.99 1.44 83.2 1:6.0 73 4.46 1.91 74.3
Experimental Example 2
[0165] To 2 ml of aqueous solution of genetic recombinant type GH
(2 mg/ml) obtained according to Reference Example 5, 50 .mu.l of
various kinds of aqueous solution of zinc acetate whose
concentration was set so that the molar ratio of zinc to 1 mole of
GH were 0, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 3.0, 4.0, 5.0 and 6.0,
were added independently and lyophilized. The obtained lyophilized
powder was dispersed in 2 ml of dichloromethane, atomized and
micronized by Vortex mixer. The distribution of particle size was
determined by using laser diffraction apparatus of determining
distribution of particle size (SALD2000A; Shimazu). The results are
shown in FIG. 1. In FIG. 1, particle size(mean particle
diameter:.mu.m) is shown by .circle-solid..
[0166] As shown by FIG. 1, the complex of GH and zinc at a molar
ratio of 1:1.6 to 1:2.4 in the present invention showed the mean
particle diameter being less than 5 .mu.m.
[0167] It is clear that the complex of GH and zinc in the present
invention is fine particle.
Experimental Example 3
[0168] To 1 ml of aqueous solution of genetic recombinant type GH
(2 mg/ml) obtained according to Reference Example 5, 25 .mu.l of
various kinds of aqueous solution of zinc acetate whose
concentration was set so that the molar ratio of zinc to 1 mole of
GH were 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 were added
independently, and was centrifugated for 5 minutes at 12,000
rpm/minute. The obtained supernatant was filtered through 0.45
.mu.m filter and the concentration of GH was quantitatively
analyzed by high performance liquid chromatography to calculate the
ratio formed of water-insoluble complex (ratio of GH in
water-insoluble complex to total amount of GH added (%)). The
result is shown in FIG. 2. In FIG. 2, the ratio of water-insoluble
complex formed is shown by .largecircle..
[0169] As shown by FIG. 2, in the case that the zinc molar ratio to
1 mole of GH is less than 2.0, the co-existing water-insoluble
component was less than 30% and the complex was substantially
water-soluble.
[0170] It is clear that the complex of GH and zinc in the present
invention is substantially water-soluble.
* * * * *