U.S. patent application number 11/013354 was filed with the patent office on 2005-05-12 for sustained-release composition of drugs encapsulated in microparticles of hyaluronic acid.
This patent application is currently assigned to LG LIFE SCIENCES, LTD, INC.. Invention is credited to Kim, Myung-Jin, Kim, Sun-Jin, Kwon, Oh-Ryong.
Application Number | 20050100605 11/013354 |
Document ID | / |
Family ID | 34557561 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100605 |
Kind Code |
A1 |
Kim, Myung-Jin ; et
al. |
May 12, 2005 |
Sustained-release composition of drugs encapsulated in
microparticles of hyaluronic acid
Abstract
A sustained-release drug composition consisting essentially of
microparticles of hyaluronic acid having a high molecular weight or
an inorganic salt thereof and a protein or peptide drug encased in
said microparticles, wherein the average size of said
microparticles ranges from 0.1 to 40 .mu.m.
Inventors: |
Kim, Myung-Jin; (Daejeon,
KR) ; Kim, Sun-Jin; (Seoul, KR) ; Kwon,
Oh-Ryong; (Daejeon, KR) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
LG LIFE SCIENCES, LTD, INC.
|
Family ID: |
34557561 |
Appl. No.: |
11/013354 |
Filed: |
December 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11013354 |
Dec 17, 2004 |
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10205954 |
Jul 26, 2002 |
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10205954 |
Jul 26, 2002 |
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09596593 |
Jun 19, 2000 |
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09596593 |
Jun 19, 2000 |
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09194653 |
Nov 30, 1998 |
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Current U.S.
Class: |
424/468 ;
424/130.1; 424/85.1; 424/85.2; 424/85.4; 424/94.64; 514/11.2;
514/11.4; 514/14.6; 514/5.9; 514/54; 514/8.5; 514/8.8; 514/9.6 |
Current CPC
Class: |
Y10S 514/951 20130101;
A61K 9/1652 20130101 |
Class at
Publication: |
424/468 ;
424/085.1; 514/012; 424/130.1; 424/085.2; 514/054; 424/094.64;
514/003; 424/085.4 |
International
Class: |
A61K 038/48; A61K
039/395; A61K 009/22; A61K 009/14; A61K 038/19; A61K 038/22; A61K
038/21 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 1, 1997 |
KR |
1997-12046 |
Claims
What is claimed is:
1. A sustained-release drug composition consisting essentially of
microparticles of hyaluronic acid having a high molecular weight or
an inorganic salt thereof and a protein or peptide drug encased in
said microparticles, wherein the average size of said
microparticles ranges from 0.1 to 40 .mu.m.
2. The composition of claim 1, wherein the molecular weight of
hyaluronic acid ranges from 1,000,000 to 3,200,000.
3. The composition of claim 1 further comprising a stabilizer.
4. The composition of claim 1, wherein the average size of
microparticle ranges from 1 to 10 .mu.m.
5. The composition of claim 1, wherein the drug is selected from
the group consisting of human growth hormone, bovine somatotropin,
porcine somatotropin, growth hormone releasing hormone, growth
hormone releasing peptide, granulocyte-colony stimulating factor,
granulocyte macrophage-colony stimulating factor, macrophage-colony
stimulating factor, erythropoietin, bone morphogenetic protein,
interferon, insulin, atriopeptin-III, monoclonal antibody, TNF,
macrophage activating factor, interleukin, tumor denaturing factor,
insulin-like growth factor, epidermal growth factor, tissue
plasminogen activator, urokinase and a mixture thereof.
6. The composition of claim 1, wherein the inorganic salt is
selected from the group consisting of sodium, potassium, lithium,
calcium, ammonium, magnesium, zinc, copper and cobalt salts of
hyaluronic acid.
7. The composition of claim 3, wherein the stabilizer is selected
from the group consisting of a polysaccharide, protein, amino acid,
lipid, fatty acid, polyethyleneglycol, inorganic salt, surfactant
and a mixture thereof.
8. An injection formulation comprising the sustained-release
composition of claim 1 dispersed in an injection medium.
9. The injection formulation of claim 8 further comprising a
dispersant or preservative.
10. The injection formulation of claim 8, wherein the injection
medium is selected from the group consisting of a buffered aqueous
solution, ethanol, propyleneglycol, polyethyleneglycol, vegetable
oil, mineral oil, squalene, cod liver oil, mono-, di- and
tri-glyceride and a mixture thereof.
11. The injection formulation of claim 10, wherein the vegetable
oil is selected from the group consisting of corn oil, olive oil,
soy bean oil, safflower oil, cottonseed oil, peanut oil, sesame
oil, coconut oil, castor oil and a mixture thereof.
12. An aerosol formulation comprising the sustained-release
composition of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part (CIP) application
of U.S. Ser. No. 09/596,593 filed on Jun. 19, 2000, now abandoned,
which is a CIP application of U.S. Ser. No. 09/194,653 filed on
Nov. 30, 1998, now abandoned.
FIELD OF THE INVENTION
[0002] The present invention relates to a sustained-release
composition of a protein or peptide drug encapsulated in solid
microparticles of high molecular weight hyaluronic acid or its
salt; and to an injection formulation containing same.
BACKGROUND OF THE INVENTION
[0003] Protein or peptide drugs are usually administered by
injection because of their sluggish absorption via oral
administration. Once injected, their in vivo activities last only a
short period of time and, for this reason, repeated injections must
be administered when a long-term treatment is required. For
example, treatment of children suffering from pituitary growth
hormone deficiency is carried out by daily injections of
recombinant human growth hormone over a period of more than 6
months. Accordingly, a sustained-release formulation which does not
require cumbersome daily administrations is highly desirable in
such applications.
[0004] A typical sustained-release formulation of a protein or
peptide drug, e.g., human growth hormone, is prepared by
encapsulating the drug in microparticles of a biodegradable polymer
matrix material, which slowly releases the drug as the matrix
material undergoes in vivo degradation. In this line, extensive
studies have been conducted to develop biodegradable polymers
suitable for use in sustained-release drug formulations, and
biodegradable polyesters such as polylactide, polyglycolide,
poly(lactide-co-glycolide), poly-ortho-ester and polyanhydride have
been found to be effective in such use [M. Chasin and R. Langer, et
al., Biodegradable Polymers as Drug Delivery System, Mercel Dekker
(1990) and J. Heller, Adv. Drug Del. Rev., 10, 163 (1993)].
[0005] Other studies have also been carried out to develop a
sustained-release drug formulation using natural polymer materials
such as gelatin, collagen, chitosan, carboxymethyl cellulose,
alginate and hyaluronic acid. A natural polymer generally forms a
gel when placed in an aqueous environment and this type of highly
viscous gel matrix, through which the drug diffuses very slowly,
has been used in formulating sustained-release drug
compositions.
[0006] For example, U.S. Pat. No. 5,416,071 discloses a
sustained-release injection formulation of erythropoietin employing
a gel containing 0.01% to 3% hyaluronic acid; Japanese Patent
Publication No. 1-287041(1989) describes a sustained-released
injection formulation of insulin employing a gel formed with 1%
hyaluronic acid; and Japanese Patent Publication No. 2-00213(1990)
reports a sustained-release formulation of calcitonin, elcanonine
or human growth hormone employing a gel containing 5% hyaluronic
acid. Similarly, Meyer et al., have developed a sustained-release
formulation of granulocyte colony stimulating factor employing a
gel containing 0.5 to 4% hyaluronic acid [James Meyer, et al., J.
Controlled Release, 35, 67 (1995)].
[0007] However, administration of such formulations by injection
requires the use of a large bore syringe-needle because a gel
containing a few % hyaluronic acid has a high-viscosity in the
order of 10.sup.7 centipoise. Moreover, as the injected gel gets
diluted by body fluid, its drug retaining ability is rapidly
diminished, and as a result, the sustaining of the drug release
lasts no more than 1 day. For instance, Japanese Patent Publication
No. 1-287041(1989) discloses that when a sustained-release
injection formulation insulin containing 1% hyaluronic acid was
administered to rabbits, the therapeutic effect of suppressing the
blood glucose level did not last more than 24 hours. Also, the drug
concentration in blood was reported to decrease to less than
{fraction (1/10)} of the initial concentration in less than 24
hours when test animals were injected with a formulation of 2%
hyaluronic acid containing granulocyte colony stimulating factor
[James Meyer, et al., J. Controlled Release, 35, 67(1995)] or a
formulation of 1.5% hyaluronic acid containing interferon-.alpha.
and plasma protein (U.S. Pat. No. 5,416,017). Accordingly, a
sustained release drug formulation based on hyaluronic acid gels
has the serious drawback that the drug release cannot be maintained
for more than 24 hours.
[0008] Natural hyaluronic acid or an inorganic salt thereof
dissolves only in water. Hyaluronic acid-benzyl ester HYAFF.TM., on
the other hand, dissolves not in water but in an organic solvent,
e.g., dimethylsulfoxide. Drug compositions, comprising solid
microparticles of such hydrophobic hyaluronic acid derivatives and
drugs encased therein have been prepared by the conventional
emulsion-solvent extraction method [N. S. Nightlinger, et al.,
Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 22nd, Paper
No. 3205 (1995); L. Ilum, et al., J. Controlled Rel., 29, 133
(1994)]. Such preparation is typically carried out as follows: A
protein drug is dispersed in a dimethylsulfoxide solution of
hyaluronic acid-benzyl ester and the dispersion thus obtained is
added to a mineral oil to form an emulsion. An organic solvent,
e.g., ethylacetate, is added to the emulsion to extract
dimethylsulfoxide; and microparticles consisting of the drug and
hyaluronic acid-benzyl ester are recovered therefrom.
[0009] However, this method has the problem that the protein drug
may be denatured through its contact with the organic solvent or
with hydrophobic hyaluronic acid-benzyl ester. In fact, a
microparticular composition of granulocyte macrophage-colony
stimulating factor (GM-CSF) prepared by using a fully esterified
hyaluronic acid derivative was reported to release only about 25%
of GM-CSF during the first few days and none after 17 days [N. S.
Nightlinger, et al., proceed. Intern. Symp. Control. Rel. Bioact.
Mater., 22nd, Paper No. 3205(1995)]. In this case, a major portion
of the protein drug was lost, most likely due to denaturation
thereof through its interaction with hyaluronic acid-benzyl ester
and/or the organic solvent.
SUMMARY OF THE INVENTION
[0010] Accordingly, it is an object of the present invention to
provide an improved sustained-release composition of a protein or
peptide drug.
[0011] In accordance with one aspect of the present invention,
there is provided a sustained-release drug composition consisting
essentially of microparticles of hyaluronic acid having a high
molecular weight or an inorganic salt thereof and a protein or
peptide drug encased in said microparticles, wherein the average
size of said microparticles ranges from 0.1 to 40 .mu.m.
BRIEF DESCRIPTION OF DRAWINGS
[0012] The above and other objects and features of the present
invention will become apparent from the following description of
the invention taken in conjunction with the following accompanying
drawings, wherein:
[0013] FIG. 1 displays the time-dependent changes in the released
amount of human growth hormone (hGH) in vitro;
[0014] FIGS. 2A and 2B demonstrate the stability of the
sustained-release composition of the present invention containing
hGH by reversed phase high performance liquid chromatography (A:
hGH released from the formulation of the present invention; and B:
aqueous hGH Control);
[0015] FIGS. 3A and 3B illustrate the stability of the
sustained-release composition of the present invention containing
hGH by size exclusion chromatography (A: hGH released from the
formulation of the present invention and B: aqueous hGH
Control);
[0016] FIG. 4 compares the time-dependent changes in the weight
gain pattern of dwarf rats treated with the inventive
sustained-release formulation of human growth hormone with those of
conventional formulations;
[0017] FIG. 5 contrasts the time-dependent changes in the weight
gain pattern of dwarf rats treated with the inventive
sustained-release formulation of human growth hormone with those of
conventional formulations;
[0018] FIG. 6 depicts the time-dependent changes in the
concentration of human growth hormone (hGH) in blood; and
[0019] FIG. 7 describes the time-dependent changes in the weight
gain pattern of dwarf rats treated with the inventive
sustained-release formulation of human growth hormone in comparison
with those of conventional formulations.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The sustained-release composition of the present invention
comprises solid microparticles of hyaluronic acid having a high
molecular weight or a salt thereof and a protein or peptide drug
encapsuled in said particles. This inventive composition is
superior to conventional formulations based on hyaluronic acid gels
in terms of release characteristics and easiness in handling: that
is, an injection formulation prepared using the inventive
microparticular composition is easier to inject because of its low
viscosity, and the composition releases the drug in vivo at a
constant rate over a longer period of time.
[0021] Further, the inventive composition is advantageous in that
the denaturation of the drug does not occur until 100% thereof is
released from the composition.
[0022] The microparticular composition of the present invention
having an average particle size ranging from 0.1 to 40 .mu.m,
preferably from 0.1 to 10 .mu.m may be prepared by spray-drying or
freeze-drying an aqueous solution containing a protein or peptide
drug and hyaluronic acid having a high molecular weight or its
salt. If desired, a stabilizer may be added to the solution.
[0023] Exemplary drugs that may be used in preparing the solid
microparticular composition of this invention include human growth
hormone, bovine somatotropin, porcine somatotropin, growth hormone
releasing hormone, growth hormone releasing peptide,
granulocyte-colony stimulating factor, granulocyte
macrophage-colony stimulating factor, macrophage-colony stimulating
factor, erythropoietin, bone morphogenetic protein, interferon,
insulin, atriopeptin-III, monoclonal antibody, TNF, macrophage
activating factor, interleukin, tumor denaturing factor,
insulin-like growth factor, epidermal growth factor, tissue
plasminogen activator and urokinase.
[0024] Hyaluronic acid having a high molecular weight which may be
suitably used in the present invention is hyaluronic acid having a
molecular weight of more than 1,000,000, preferably 1,000,000 to
3,200,000, and more preferably 1,000,000 to 2,000,000.
[0025] Representative inorganic salts of hyaluronic acid that may
be used in preparing the solid microparticular composition of this
invention include sodium, potassium, lithium, calcium, ammonium,
magnesium, zinc, copper and cobalt salts.
[0026] Some of the stabilizers usable in the present invent include
polysaccharide, protein, amino acid, lipid, fatty acid,
polyethylene glycol, inorganic salt and surfactant.
[0027] The microparticular sustained-release composition of the
present invention may contain a protein or peptide drug in an
amount ranging from 1 to 90 wt % based on the weight of the
composition, and optionally, a stabilizer in an amount ranging form
1 to 90 wt % based on the weight of the composition.
[0028] The sustained-release injection formulation of the present
invention is prepared by dispersing in an injection medium the
microparticular sustained-release composition of the present
invention in an amount ranging from 0.01 to 10 wt % based on the
weight of the injection formulation. If desired, a dispersant or
preservative may be added thereto. Typical injection media that may
be used in the injection formulation of the present invention
include a buffered aqueous solution, ethanol, propyleneglycol,
polyethyleneglycol, vegetable oil, mineral oil, squalene, cod liver
oil, monoglyceride, diglyceride and triglyceride or a mixture
thereof.
[0029] Exemplary vegetable oils are corn oil, olive oil, soy bean
oil, sunflower oil, cotton seed oil, peanut oil, sesame oil and a
mixture thereof.
[0030] Further, an aerosol formulation containing the
microparticular sustained release composition of the present
invention may be prepared. The aerosol formulation of the present
invention thus prepared may be applied to the nose or bronchi
mucous membrane wherein the microparticular composition releases
the drug in a controlled manner.
[0031] The following Examples and Test Examples are intended to
further illustrate the present invention without limiting its
scope.
EXAMPLE 1
Preparation of Microparticle
[0032] To a 5 mM phosphate buffered saline (PBS) containing 2 mg/ml
of human growth hormone (hGH), Tween 80 was added to a
concentration of 0.01 wt %. Sodium hyaluronate having a molecular
weight of 1,000,000 was added thereto-to a concentration of 2
mg/ml. The resulting solution was supplied to a spray-dryer (Buchi
190) at a rate of 3 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 3.0
.mu.m.
EXAMPLE 2
Preparation of Microparticle
[0033] To a 5 mM PBS containing 1 mg/ml of hGH, Tween 80 was added
to a concentration of 0.01 wt %. Sodium hyaluronate having a
molecular weight of 2,000,000 was added thereto to a concentration
of 1 mg/ml. The resulting solution was supplied to a spray-dryer
(Buchi 190) at a rate of 2 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 2.0
.mu.m.
EXAMPLE 3
Preparation of Microparticle
[0034] To a 5 mM PBS containing 0.1 mg/ml of hGH, Tween 80 was
added to a concentration of 0.01 wt %. Sodium hyaluronate having a
molecular weight of 2,000,000 was added thereto to a concentration
of 0.9 mg/ml. The resulting solution was supplied to a spray-dryer
(Buchi 190) at a rate of 3 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 2.0
.mu.m.
TEST EXAMPLE 1
In vitro Release-Test
[0035] The microparticles prepared in Examples 1, 2 and 3 were
respectively suspended in a buffer (150 mM sodium chloride, 10 mM
phosphate and 0.05% sodium azide, pH 7.4) so that a concentration
of hGH becomes 1.0 mg/ml. The dispersion thus obtained was put in
oven and the release of hGH was tested in a 37.degree. C. stirrer.
At the predetermined sampling time, the resulting dispersion was
centrifuged at 800 g for 10 min. to obtain a supernatant and a
fraction of the supernatant corresponding to {fraction (1/10)} of
the entire dispersion was removed therefrom. An equal amount of the
buffer was added to the dispersion and the release-test was
continued at 37.degree. C.
[0036] The concentration of hGH in the supernatant fraction was
measured by Lowry method and high performance liquid chromatography
(HPLC) in order to determine the amount of released hGH relative to
the time. The results are shown in FIG. 1.
[0037] FIG. 1 displays the time-dependent changes in the released
amount of hGH in vitro. As shown in FIG. 1, the rate of hGH release
is slower as the molecular weight of hyaluronic acid is higher and
the content of hGH is lower. In deed, the microparticle obtained in
Example 3 shows slowest release rate. These results show that the
period of sustained-release of the drug can be controlled by
regulating the molecular weight of hyaluronic acid, the content of
hGH and the like. Moreover, the microparticles prepared in the
present invention exhibit constant rate of in vitro until 70% of
hGH is released, without initial burst release.
TEST EXAMPLE 2
Stability of hGH in Microparticle
[0038] In order to confirm whether the hGH in the inventive
microparticles is identical with the aqueous hGH used for the
preparation of the microparticles, hGH released from the
microparticles in the in vitro release test was assayed by
employing reversed-phase high performance liquid chromatography
(RP-HPLC) and size exclusion chromatography (SEC).
[0039] The denaturation of hGH due to the oxidation and deamidation
can be confirmed by RP-HPLC and the results are shown in FIGS. 2A
and 2B.
[0040] FIGS. 2A and 2B demonstrate the stability of the
sustained-release composition of the present invention containing
hGH by RP-HPLC, wherein FIG. 2A is the RP-HPLC profile of hGH
released from the formulation of the present invention and FIG. 2B,
aqueous hGH Control.
[0041] The denaturation of hGH due to the aggregation can be
confirmed by SEC and the results are shown in FIGS. 3A and 3B.
[0042] FIGS. 3A and 3B demonstrate the stability of the
sustained-release composition of the present invention containing
hGH by SEC, wherein FIG. 3A is the SEC profile of hGH released from
the formulation of the present invention and FIG. 3B, aqueous hGH
Control.
[0043] As shown in FIGS. 2A, 2B, 3A and 3B, hGH released from the
inventive compositions is identical with the aqueous hGH Control
and the content of hGH monomer is more than 95%. These results show
that the denaturation of hGH does not occurred during the
preparation of the inventive microparticle compositions and the
release thereof at 37.degree. C.
TEST EXAMPLE 3
In vivo Release-Test
[0044] Dwarf rats having the heredity of low growth hormone
secretion were employed in a test to examine the sustained-release
property of the microparticle of the present invention.
[0045] The sustained-release microparticle prepared in Example 1
was dispersed in a mixture of propyleneglycol and ethanol
(7:3(v/v)) so that the concentration of hGH became 5 mg/ml. The
resulting dispersion was diluted with a buffered aqueous solution
(150 mM NaCl and 10 mM phosphate, pH 7.4) so that a concentration
of hGH became 0.5 mg/ml.
[0046] Eighteen heads of seven week-old dwarf rats having an
average body weight of 103 g were divided into three groups, each
consisting of six rats. The rats of the first group were
administered by a subcutaneous injection with 0.1 ml of the
microparticle dispersion prepared above (corresponding to 50 .mu.g
of hGH) daily for a period of two weeks (Experimental group). The
rats of the second group were administered with Eutropin.RTM., a
commercially available hGH formulation for aqueous injection, under
the same condition (Comparative group). The rats of the third group
were not administered with hGH (non-treated Control group). The
rats were weighted every day to examine the change in their body
weight.
[0047] FIG. 4 compares the time-dependent changes in the weight
gain pattern, the rats of the Experimental group, the Comparative
group and the Control group.
[0048] As shown in FIG. 4, the rats of the Experimental group
exhibited a continuous weight gain over a period of 2 weeks, which
is larger than that of the Comparative group and, the Control
group. These results show that the inventive microparticle
formulation is more effective than the conventional formulations
owing to its sustained-release property.
TEST EXAMPLE 4
In vivo Release-Test
[0049] The sustained-release microparticle prepared in Example 2
was dispersed in a cottonseed oil so that the concentration of hGH
became 1.5 mg/ml.
[0050] Twenty-four heads of seven week-old dwarf rats having an
average body weight of 105 g were divided into four groups, each
consisting of six rats. The rats of the first group were
administered by a subcutaneous injection with 0.1 ml of the
microparticle dispersion prepared above (corresponding to 150 .mu.g
of hGH) every three days for a period of two weeks (Experimental
group). The rats of the second group were administered with
Eutropin.RTM., under the same condition (Comparative group 1). The
rats of the third group were administered with Eutropino
corresponding to 50 .mu.g of hGH daily for a period of two weeks
(Comparative group 2). The rats of the fourth group were not
administered with hGH (non-treated Control group). The rats were
weighted every day to examine the change in their body weight.
[0051] FIG. 5 contrasts the time-dependent changes in the weight
gain patterns of the Experimental group, the Comparative groups and
the Control group.
[0052] As shown in FIG. 5, the rats of the Experimental group
exhibited a larger weight gain than the rats of the Comparative
groups and the Control group. The rats of Comparative group 1 shows
significant weight gain at day 1, however, they exhibits lower
weight gain than the rats of the Control group at days 2 and 3
after the administration. The rats of the Experimental group and
Comparative group 2 show continuous weight gain. These results show
that the inventive microparticle formulation has an effective
sustained-release property remaining at least for 3 days.
TEST EXAMPLE 5
In vivo Release-Test
[0053] The sustained-release microparticle prepared in Example 2
was dispersed in a cottonseed oil so that the concentration of hGH
become 1.5 mg/ml. Eight rabbits having an average body weight of
2.5 kg were divided into two groups, each consisting of four
rabbits. The rabbits of one group were administered by an injection
with the microparticle dispersion prepared above in an amount
corresponding to 3,700 .mu.g of hGH (Experimental group). The
rabbits of the other group were not administered with hGH (Control
group).
[0054] After the administration, blood samples were taken from the
rabbits daily over a period of 6 days.
[0055] The amount of hGH in the blood samples was quantified by RIA
(radio-immuno assay) method.
[0056] FIG. 6 depicts the time-dependent changes in the
concentration of human growth hormone in blood.
[0057] As shown in FIG. 6, the amount of hOH in blood was
maintained in a range of from 0 to 11 ng/ml for 4 days after the
administration and then was gradually reduced after day 5. This
result shows that the inventive microparticle composition have a
constant release rate during 4 days and the release rate was
gradually reduced thereafter. This result agrees with the result of
Test Example 1 wherein the in vivo release rate of hGH is linear
until 70% of hGH is release. In contrast, the concentration of hGH
in blood of the Control group was under the concentration
detectable by RIA method (1 ng/ml) and accordingly can be
ignored.
EXAMPLE 4
Preparation of Microparticle and In vitro Release-Test
[0058] (Step 1) Preparation of Microparticle
[0059] To a 5 mM PBS containing 2 mg/ml of bovine somatotropin
(bST), Tween 80 was added to a concentration of 0.01 wt %. Sodium
hyaluronate having a molecular weight of 1,000,000 was added
thereto to a concentration of 2mg/ml. The resulting solution was
supplied to a spray-dryer (Buchi 190) at a rate of 3 ml/min. to
prepare microparticles. The temperature of the influx air to the
spray dryer was 85.degree. C. The mean diameter of the
microparticles thus obtained was 3.0 .mu.m.
[0060] (Step 2) In vitro Release-Test
[0061] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released bST was tested in
accordance with the method of Test Example 2.
[0062] The released bST was quantified and qualified by way of
conducting SEC. As a result, bST was released more than 85% for 72
hours and the denaturation of bGH did not occur.
EXAMPLE 5
Preparation of Microparticle and In vitro Release-Test
[0063] (Step 1) Preparation of Microparticle
[0064] To a 5 mM PBS containing 2 mg/ml of porcine somatotropin
(pST), Tween 80 was added to a concentration of 0.01 wt %. Sodium
hyaluronate having a molecular weight of 1,000,000 was added
thereto to a concentration of 2 mg/ml. The resulting solution was
supplied to a spray-dryer (Buchi 190) at a rate of 3 ml/min. to
prepare microparticles. The temperature of the influx air to the
spray dryer was 85.degree. C. The mean diameter of the
microparticles thus obtained was 3.0 .mu.m.
[0065] (Step 2) In vitro Release-Test
[0066] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released pST was tested in
accordance with the method of Test Example 2.
[0067] The released pST was quantified and qualified by way of
conducting SEC. As a result, pST was released more than 90% for 72
hours and the denaturation of pST did not occur.
EXAMPLE 6
Preparation of Microparticle and In vitro Release-Test
[0068] (Step 1) Preparation of Microparticle
[0069] To a 5 mM PBS containing 0.4 mg/ml of granulocyte
macrophage-colony stimulating factor (GM-CSF), Tween 80 was added
to a concentration of 0.01 wt %. Sodium hyaluronate having a
molecular weight of 1,000,000 was added thereto to a concentration
of 1.6 mg/ml. The resulting solution was supplied to a spray-dryer
(Buchi 190) at a rate of 3 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 3.0
.mu.m.
[0070] (Step 2) In vitro Release-Test
[0071] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released GM-CSF was tested
in accordance with the method of Test Example 2.
[0072] The released GM-CSF was quantified and qualified by way of
conducting SEC. As a result, GM-CSF was released more than 92% for
72 hours and the denaturation of GM-CSF did not occur.
EXAMPLE 7
Preparation of Microparticle and In vitro Release-Test
[0073] (Step 1) Preparation of Microparticle
[0074] To a 5 mM PBS containing 1000 IU/ml of erythropoietin (EPO)
and 0.5 mg/ml of serum albumin, Tween 80 was added to a
concentration of 0.01 wt %. Sodium hyaluronate having a molecular
weight of 1,000,000 was added thereto to a concentration of 2.5
mg/ml. The resulting solution was supplied to a spray-dryer (Buchi
190) at a rate of 3 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 3.5
.mu.m.
[0075] (Step 2) In vitro Release-Test
[0076] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released EPO was tested in
accordance with the method of Test Example 2.
[0077] The released EPO was quantified and qualified by way of
conducting SEC. As a result, EPO was released more than 70% for 72
hours and the denaturation-of EPO did not occur.
EXAMPLE 8
Preparation of Microparticle and In vitro Release-Test
[0078] (Step 1) Preparation of Microparticle
[0079] To a 5 mM PBS containing 2.times.10.sup.5 IU/ml of
interferon-.alpha., 0.2 mg/ml of D-mannitol and 0.2 mg/ml of serum
albumin, Tween 80 was added to a concentration of 0.01 wt %. Sodium
hyaluronate having a molecular weight of 1,000,000 was added
thereto to a concentration of 2.5 mg/ml. The resulting solution was
supplied to a spray-dryer (Buchi 190) at a rate of 3 ml/min. to
prepare microparticles. The temperature of the influx air to the
spray dryer was 105.degree. C. The mean diameter of the
microparticles thus obtained was 3.5 .mu.m.
[0080] (Step 2) In vitro Release-Test
[0081] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released interferon-a was
tested in accordance with the method of Test Example 2.
[0082] The released interferon-a was quantified and qualified by
way of conducting RP-HPLC. As a result, interferon-a was released
more than 90% for 72 hours and the denaturation of
interferon-.alpha. did not occur.
EXAMPLE 9
Preparation of Microparticle and In vitro Release-Test
[0083] (Step 1) Preparation of Microparticle
[0084] To a 5 mM PBS containing 2.times.10.sup.5 IU/ml of
interferon-.gamma., 0.2 mg/ml of glycine and 0.2 mg/ml of serum
albumin, Tween 80 was added to a concentration of 0.01 wt %. Sodium
hyaluronate having a molecular weight of 1,000,000 was added
thereto to a concentration of 2.5 mg/ml. The resulting solution was
supplied to a spray-dryer (Buchi 190) at a rate of 3 ml/min. to
prepare microparticles. The temperature of the influx air to the
spray dryer was 105.degree. C. The mean diameter of the
microparticles thus obtained was 3.5 .mu.m.
[0085] (Step 2) In vitro Release-Test
[0086] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released interferon-.gamma.
was tested in accordance with the method of Test Example 2.
[0087] The released interferon-.gamma. was quantified and qualified
by way of conducting RP-HPLC. As a result, interferon-.gamma. was
released more than 85% for 72 hours and the denaturation of
interferon-.gamma. did not occur.
EXAMPLE 10
Preparation of Microparticle and In vitro Release-Test
[0088] (Step 1) Preparation of Microparticles
[0089] To a 10 mM PBS containing 20 IU/ml of insulin, Tween 80 was
added to a concentration of 0.01 wt %. Sodium hyaluronate having a
molecular weight of 1,000,000 was added thereto to a concentration
of 2 mg/ml. The resulting solution was supplied to a spray-dryer
(Buchi 190) at a rate of 3 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 3.0
.mu.m.
[0090] (Step 2) In vitro Release-Test
[0091] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released insulin was tested
in accordance with the method of Test Example 2.
[0092] The released insulin was quantified and qualified by way of
conducting RP-HPLC. As a result, insulin was released more than 95%
for 72 hours and the denaturation of insulin did not occur.
EXAMPLE 11
Preparation of Microparticle and In vitro Release-Test
[0093] (Step 1) Preparation of Microparticle
[0094] To a 5 mM PBS containing 2 mg/ml of insulin-like growth
factor, Tween 80 was added to a concentration of 0.01 wt %. Sodium
hyaluronate having a molecular weight of 1,000,000 was added
thereto to a concentration of 2 mg/ml. The resulting solution was
supplied to a spray-dryer (Buchi 190) at a rate of 3 ml/min. to
prepare microparticles. The temperature of the influx air to the
spray dryer was 85.degree. C. The mean diameter of the
microparticles thus obtained was 3.0 .mu.m.
[0095] (Step 2) In vitro Release-Test
[0096] An in vitro release-test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1 and the stability of the released insulin-like
growth factor was tested in accordance with the method of Test
Example 2.
[0097] The released insulin-like growth factor was quantified and
qualified by way of conducting RP-HPLC. As a result, insulin-like
growth factor was released more than 90% for 72 hours and the
denaturation of insulin-like growth factor did not occur.
COMPARATIVE EXAMPLE 1
Preparation of Gel Formulation and In vitro Release-Test
[0098] (Step 1) Preparation of Gel Formulation
[0099] To a 5 mM PBS containing 2.3 mg/ml of hGH, sodium
hyaluronate having a molecular weight of 2,000,000 was added to a
concentration of 20 mg/ml to obtain 2% hyaluronate gel formulation
containing hGH.
[0100] (Step 2) In vitro Release-Test
[0101] The gel formulation prepared in Step 1 was tested by the
procedure of Test Example 1. As a result, 100% of hGH was released
within 1 hour. This result shows that the gel formulation releases
a drug within a shorter period than the inventive microparticles
because it is easily diluted by water.
COMPARATIVE EXAMPLE 2
Preparation of Gel Formulation and In vivo Release-Test
[0102] (Step 1) Preparation of Gel Formulation
[0103] To a 5 mM PBS containing 1.5 mg/ml of hGH, sodium
hyaluronate having a molecular weight of 2,000,000 was added to a
concentration of 20 mg/ml to obtain a non-fluid gel formulation
containing hGH.
[0104] 1 ml of the gel formulation thus obtained was dispersed in 2
ml of cottonseed oil and the mixture was homogenized to form an
emulsion.
[0105] (Step 2) In vitro Release-Test
[0106] Twenty-four heads of seven week-old dwarf rats having an
average body weight of 95 g were divided into four groups, each
consisting of 6 rats. The rats of one group were administered by a
subcutaneous injection with the 0.3 ml of the emulsion prepared in
Step 1 (corresponding to 150 .mu.g of hGH) (Group 1).
[0107] In order to compare the efficiency of the emulsion
formulation with other formulations, the rats of another two groups
were administered with a dispersion containing the
sustained-release microparticles prepared in Example 2 dispersed in
a cottonseed oil so that the concentration of hGH became 150 .mu.g
(Group 2); Eutropin.RTM. corresponding to 150 .mu.g of hGH (Group
3), respectively. The rats of the last group were not administered
with hGH formulation (Control group). After the administration, the
changes in the weight gain of the rats were observed for 6
days.
[0108] FIG. 7 describes the time-dependent changes in the weight
gain patterns of dwarf rats treated with the inventive
sustained-release formulation of human growth hormone in comparison
with those of conventional formulations. As a result, the
time-dependent changes in the weight gain pattern of dwarf rats
treated with the hyaluronate gel formulation were similar to the
Eutropino group. That is, the body weight of dwarf rats treated
with the hyaluronate gel formulation was reduced 2 or 3 days after
the administration and was similar to that of the rats of the
Control group thereafter. However, rats of the group treated with
the inventive formulation exhibited continuous weight gain higher
than the other groups by 150% during 6 days.
COMPARATIVE EXAMPLE 3
Preparation of Microparticle Formulation using Sodium-Carboxymethyl
Cellulose and In vivo and In vitro Release-Test
[0109] (Step1) Preparation of Microparticle Formulation
[0110] To a 5 mM PBS containing 0.2 mg/ml of hGH, Tween 80 was
added to a concentration of 0.01 wt %. Sodium-carboxymethyl
cellulose (Na--CMC, medium viscosity grade) was added thereto to a
concentration of 1.8 mg/ml. The resulting solution was supplied to
a spray-dryer (Buchi 190) at a rate of 3 ml/min. to prepare
microparticles. The temperature of the influx air to the spray
dryer was 85.degree. C. The mean diameter of the microparticles
thus obtained was 3.0 .mu.m.
[0111] (Step 2) In vitro Release-Test
[0112] The microparticle formulation prepared in Step 1 was tested
by the procedure of Test Example 1 and the results are listed in
Table 1.
1 TABLE 1 Time(hour) 0 1 3 5 7 24 48 72 144 Released Amount 0 32 40
48 52 57 63 65 65 of hGH(%)
[0113] As shown in Table 1, the time-dependent changes in the in
vitro release pattern of the microparticle formulation prepared in
Step 1 differ from that of the inventive microparticle. That is, it
exhibited the ill-balanced release pattern that more than 30% of
hGH was released during initial 1 hour, another 30% was released
until 48 hours, and then release of hGH hardly occurred thereafter.
These results show that the release pattern of a drug becomes
ill-balanced by the interaction between the protein drug and the
matrix, and possibility of the denaturation of the drug is very
high, when a natural carbohydrate polymer having a hydrophobicity
stronger than hyaluronic acid is used as a matrix material.
[0114] (Step 3) In vivo Release-Test
[0115] The microparticle formulation prepared in Step 1 was
dispersed in a cottonseed oil. The resulting dispersion was
administered to 7 week-old dwarf rats in an amount of 300 .mu.g of
hGH per head and a non-administered group were used as a Control
group. The weight gains of rats were measured over a period of 7
days and the results are listed in Table 2 as an accumulated weight
gain.
2 TABLE 2 Time(day) day 1 day 2 day 3 day 4 day 5 day 6 day 7
Control group 0.6 0.8 3.3 5.5 7.6 6.7 7.4 Na-CMC 5.2 3.3 6.4 8.3
10.5 9.4 9.0 formulation group
[0116] As shown in Table 2, the rats treated with the microparticle
prepared in Step 1 exhibited the weight gain pattern similar to
that of the hyaluronate-gel formulation in Comparative Example 2.
That is, they exhibited the weight gain only at day 1 and the body
weight thereof was reduced at day 2. Further, they exhibited the
weight gain rate lower than the Control group thereafter, and
finally show a weight gain similar to the Control group at day 7.
These results show that the Na--CMC formulation has an inferior
release property and titer than the inventive hyaluronate
microparticle, although Na--CMC is a natural carbohydrate polymer
as hyaluronic acid is.
COMPARATIVE EXAMPLE 4
Preparation of Microparticle Formulation using Hyaluronic
Acid-Benzyl Ester and In vivo and In vitro Release-Test
[0117] (Step 1) Preparation of Microparticle Formulation
[0118] Natural hyaluronic acid and benzyl alcohol were chemically
reacted to produce hyaluronic acid-benzyl ester and then
microparticles containing hGH was prepared as described below.
[0119] To a 5 mM PBS containing 2 mg/ml of hGH, Tween 80 was added
to a concentration of 0.01 wt %. The resulting solution was
supplied to a spray-dryer (Buchi 190) at a rate of 3 ml/min. to
prepare microparticles. The temperature of the influx air to the
spray dryer was 85.degree. C. The mean diameter of the
microparticles thus obtained was 2.5 .mu.m.
[0120] The particles thus obtained was dispersed in
dimethylsulfoxide (DMSO) containing 6% of hyaluronic acid-benzyl
ester and the resulting dispersion was added to mineral oil
containing a surfactant, Aracel ATM (ICI, U.S.A.), and the mixture
was homogenized to form a microemulsion. The resulting
microemulsion consists of a continuous phase of mineral oil and a
dispersive phase of hyaluronic acid-benzyl ester/DMSO solution
containing hGH dispersed therein.
[0121] Ethyl acetate was added to the microemulsion thus obtained
with stirring and then DMSO was extracted with ethyl acetate and
hyaluronic acid-benzyl ester becomes hardened to produce hyaluronic
acid-benzyl ester particles containing hGH particle. The mean
diameter of the final particles thus obtained was 5.5 .mu.m and the
content of hGH was 45%.
[0122] (Step 2) In vivo Release-Test
[0123] The microparticles prepared in step 1 were tested by the
procedure of Test Example 1 and the results are listed in Table
3.
3 TABLE 3 Time(hour) 0 1 3 5 7 24 48 72 144 Released Amount 0 15 21
23 25 27 28 30 30 of hGH(%)
[0124] As shown in Table 3, in the microparticle prepared by
endowing hydrophobicity to the natural hyaluronic acid by using
hyaluronic acid-benzyl ester, release of hGH was hardly occurred
after initial 5 hours. The reason why hGH was not released is that
the interaction between the protein drug (hGH) and hyaluronic
acid-benzyl ester matrix is too strong.
[0125] (Step 3) In vivo Release-Test
[0126] The microparticles prepared in step 1 were dispersed in a
cottonseed oil. The resulting dispersion was administered to 7
week-old dwarf rats in an amount of 300 .mu.g of hGH per head and a
non-administered group were used as a Control group. The weight
gains of rats were measured over a period of 7 days and the results
are listed in Table 4 as an accumulated weight gain.
4 TABLE 4 Time(day) Day 1 day 2 day 3 day 4 day 5 day 6 day 7 A 1.2
2.3 3.6 5.7 6.6 7.3 8.2 B 3.6 2.7 5.4 6.3 7.1 8.4 8.0 A: Control
group B: hyaluronic acid-benzyl ester microparticle formulation
group
[0127] As shown in Table 4, the hyaluronic acid-benzyl ester
microparticle formulation has hardly an efficiency after day 1.
COMPARATIVE EXAMPLE 5
Preparation of Microparticle and In vitro Release-Test
[0128] (Step 1) Preparation of Gel Formulation
[0129] To a 5 mM PBS containing 1 mg/ml of hGH, Tween 80 was added
to a concentration of 0.01 wt %. Sodium hyaluronate having a
molecular weight of 100,000 was added to a concentration of 1
mg/ml. The resulting solution was supplied to a spray-dryer (Buchi
190) at a rate of 3 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 3.0
.mu.m.
[0130] (Step 2) In vitro Release-Test
[0131] An in vitro release test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1. The result showed that 100% of hGH was released
within 1 hour. Thus, the microparticle prepared by using sodium
hyaluronate having a low molecular weight releases the drug within
a much shorter period than the inventive microparticles because it
is readily solubilized in water.
COMPARATIVE EXAMPLE 6
Preparation of Microparticle and In vitro Release-Test
[0132] (Step 1) Preparation of Gel Formulation
[0133] To a 5 mM PBS containing 1 mg/ml of hGH, Tween 80 was added
to a concentration of 0.01 wt %. Sodium hyaluronate having a
molecular weight of 500,000 was added to a concentration of 1
mg/ml. The resulting solution was supplied to a spray-dryer (Buchi
190) at a rate of 3 ml/min. to prepare microparticles. The
temperature of the influx air to the spray dryer was 85.degree. C.
The mean diameter of the microparticles thus obtained was 3.0
.mu.m.
[0134] (Step 2) In vitro Release-Test
[0135] An in vitro release test was conducted by employing the
microparticles prepared in Step 1 in accordance with the method of
Test Example 1. The result showed that 100% of hGH was released
within 1 hour. Thus, the microparticle prepared by using sodium
hyaluronate having a low molecular weight releases the drug within
a much shorter period than the inventive microparticles because it
is readily solubilized in water.
[0136] While the invention has been described with respect to the
above specific embodiments, it should be recognized that various
modifications and changes may be made and also fall within the
scope of the invention as defined by the claims that follow.
* * * * *