U.S. patent application number 11/511951 was filed with the patent office on 2007-11-15 for process of preparing microspheres for sustained release having improved dispersibility and syringeability.
This patent application is currently assigned to Peptron Co., Ltd.. Invention is credited to Mi-Jin Baek, Mi-Young Baek, Yeon-Jin Chae, Ho-Il Choi, Jung-Soo Kim, Seong-Kyu Kim, Hee-Yong Lee, Eun-Ho Shin, Eun-Young Soel.
Application Number | 20070264341 11/511951 |
Document ID | / |
Family ID | 38278452 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264341 |
Kind Code |
A1 |
Lee; Hee-Yong ; et
al. |
November 15, 2007 |
Process of preparing microspheres for sustained release having
improved dispersibility and syringeability
Abstract
Disclosed is a process of preparing sustained release
microspheres, containing a biodegradable polymer as a carrier and a
drug, using spray drying. The process comprises preparing a
solution, suspension or emulsion containing a biodegradable
polymer, a drug and a solvent; spray drying the solution,
suspension or emulsion; and suspending spray-dried microspheres in
an aqueous solution containing polyvinyl alcohol to remove the
residual solvent and increase the hydrophilicity of the microsphere
surface. The process enables the preparation of microspheres having
high drug encapsulation efficiency, almost not having a toxicity
problem due to the residual solvent, and having good
syringeability. The microspheres prepared according to the present
invention release an effective concentration of a drug in a
sustained manner for a predetermined period when administered to
the body, and are thus useful in the treatment of diseases.
Inventors: |
Lee; Hee-Yong; (Daejeon,
KR) ; Kim; Jung-Soo; (Daejeon, KR) ; Shin;
Eun-Ho; (Daejeon, KR) ; Kim; Seong-Kyu;
(Daejeon, KR) ; Soel; Eun-Young; (Daejeon, KR)
; Baek; Mi-Jin; (Daejeon, KR) ; Baek;
Mi-Young; (Daejeon, KR) ; Chae; Yeon-Jin;
(Daejeon, KR) ; Choi; Ho-Il; (Daejeon,
KR) |
Correspondence
Address: |
Medlen & Carroll, LLP;Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
Peptron Co., Ltd.
Daejeon
KR
Daewoong Pharmaceutical Co., Ltd.
Kyunggi-do
KR
|
Family ID: |
38278452 |
Appl. No.: |
11/511951 |
Filed: |
August 29, 2006 |
Current U.S.
Class: |
424/486 |
Current CPC
Class: |
A61K 9/1647 20130101;
A61K 9/1635 20130101; A61K 31/765 20130101; A61K 9/1694 20130101;
A61P 43/00 20180101; A61K 38/09 20130101; A61K 38/31 20130101 |
Class at
Publication: |
424/486 |
International
Class: |
A61K 9/10 20060101
A61K009/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2006 |
KR |
10-2006-0042462 |
Claims
1. A process of preparing a sustained release microsphere,
comprising: spraying a solution, suspension or emulsion containing
a biodegradable polymer, a drug and a solvent into a dry chamber
and drying it using dry air to remove the solvent; and dispersing a
spray-dried microsphere in an aqueous solution containing polyvinyl
alcohol to remove a residual solvent and improve dispersibility of
the microsphere.
2. The process of preparing the sustained release microsphere
according to claim 1, wherein the biodegradable polymer is one or
more selected from the group consisting of polylactide,
polyglycolide, poly(lactide-co-glycolide), polyorthoester,
polyanhydride, polyhydroxybutyric acid, polycaprolactone,
polyalkylcarbonate, and derivatives thereof.
3. The process of preparing the sustained release microsphere
according to claim 1, wherein the drug is selected from among a
peptide and a protein.
4. The process of preparing the sustained release microsphere
according to claim 3, wherein the drug is, selected from among
luteinizing hormone releasing hormone (LHRH) derivatives,
somatostatin derivatives, and salts thereof.
5. The process of preparing the sustained release microsphere
according to claim 4, wherein the drug is selected from among
leuprolelin, goserelin, triptorelin, octreotide, and salts
thereof.
6. The process of preparing the sustained release microsphere
according to claim 1, wherein in the dispersing in the aqueous
solution containing polyvinyl alcohol, polyvinyl alcohol is coated
on the sustained release microsphere in an amount from 0.02% to
1.0% by weight.
7. The process of preparing the sustained release microsphere
according to claim 1, wherein in the dispersing in the aqueous
solution containing polyvinyl alcohol, the residual solvent in the
sustained release microsphere is additionally removed by more than
20%.
8. A drug-loaded sustained release microsphere, which is prepared
by atomizing a solution, suspension or emulsion containing a
biodegradable polymer, a drug and a solvent into droplets, drying
the atomized droplets to remove the solvent and obtain a dried
microsphere, and dispersing the dried microsphere in an aqueous
solution containing polyvinyl alcohol to coat polyvinyl alcohol on
the microsphere in an amount from 0.02% to 1.0% by weight.
9. The drug-loaded sustained release microsphere according to claim
8, wherein the drug is selected from among a peptide and a
protein.
10. The drug-loaded sustained release microsphere according to
claim 9, wherein the drug is selected from among luteinizing
hormone releasing hormone (LHRH) derivatives, somatostatin
derivatives, and salts thereof.
11. The drug-loaded sustained release microsphere according to
claim 10, wherein the drug is selected from among leuprolelin,
goserelin, triptorelin, octreotide, and salts thereof.
Description
TECHNICAL FIELD
[0001] The present invention relates to a process of preparing
sustained release microspheres comprising a biodegradable polymer
as a carrier and a drug. Such microspheres are an injectable
sustained-release formulation, which enables the sustained and
uniform release of a drug so as to maintain its biological activity
in the body upon subcutaneous or intramuscular injection.
BACKGROUND ART
[0002] A number of approaches have been used to encapsulate
bioactive agents into microspheres of polymers for sustained
release. Most of them are based on phase separation (U.S. Pat. No.
4,673,595, EP 52,510), cryopulverization after melt extrusion (U.S.
Pat. Nos. 5,134,122, 5,192,741, 5,225,205, 5,431,348, 5,439,688,
5,445,832 and 5,776,885), double emulsion evaporation (w/o/w,
water/oil/water) (U.S. Pat. Nos. 4,652,441, 4,711,782, 4,954,298,
5,061,492, 5,330,767, 5,476,663, 5,480,656, 5,611,971, 5,631,020
and 5,631,021), single emulsion evaporation (o/w, oil/water) (U.S.
Pat. Nos. 4,389,330 and 5,945,126; Shameem M, Lee Hee Yong, DeLuca
P. P., AAPS Pharmsci., 1 (3) article 7, 1999; Kostanski J. W.,
Pharm. Dev. Tech. 5, 585-596, 2000), and spray drying
(IE920956).
[0003] Phase separation encapsulation is a process for preparing
microspheres in which a biodegradable polymer is dissolved in an
excessive amount of an organic solvent, such as methylene chloride,
and a drug dissolved in a small amount of water is added to the
polymer solution with stirring. Silicon oil is then added to the
polymer-drug mixture at a constant rate to form embryonic
microspheres, and an excessive amount of a non-solvent, such as
trichlorofluoromethane, is added to the solution to extract the
organic solvent from the embryonic microspheres. The solidified
microspheres are recovered by filtration, and dried under pressure.
However, the phase separation method is problematic as follows.
Since the toxic solvent such as methylenechloride is not
sufficiently removed by drying under pressure, the residual solvent
reduces the stability of formulations, and may also be detrimental
to health when administered to the body. Also, the excessive use of
non-solvent, such as freon, hexane, heptane, cyclohexane, and
trichlorofluoromethane, for the solidification of embryonic
microspheres is not cost-effective upon mass production and causes
serious environmental contamination.
[0004] By contrast, cryopulverization after melt extrusion permits
minimal use of toxic solvents. This method is a process for
preparing microspheres in which a mixture of a biodegradable
polymer and a drug is melt-extruded through an extruder at a high
temperature and pulverized at a low temperature. The biodegradable
polymer-drug mixture may be obtained by homogeneously mixing a
polymer and a drug in a solvent, such as methylene chloride, with
an agitator, and removing the organic solvent using a rotary
evaporator or a vacuum dryer, or by cryo-milling at a low
temperature and sieving each powder and blending the two fine
powders. The latter case does not have the problem of the residual
toxic solvent because it does not employ a toxic solvent during
microsphere preparation. However, the procedure for preparing
microparticles does not exclude the possibility of an interaction
between the polymer and the drug and denaturation of the drug due
to high temperature and high pressure upon melt-extrusion, and
denaturation of the drug due to heat locally generated during
cryopulverization. This method is also difficult to use to make
microspheres having a uniform size, which are thus easy to
inject.
[0005] The two methods for preparing microspheres, in addition to
the problems of residual solvent, difficulty in mass production and
drug denaturation, have another disadvantage in that a
biodegradable polymer used for the sustainable release of a drug is
non-hydrophilic and thus poorly dispersible in an aqueous
suspension for injection.
[0006] Water-in-oil-in-water (w/o/w) double emulsion evaporation
has commonly been applied to encapsulate hydrophilic drugs, such as
peptides or proteins, into polymeric microspheres. In this W/O/W
method, a hydrophilic drug is dissolved in water, and this aqueous
phase is dispersed in an organic phase containing a biodegradable
polymer to yield a primary emulsion (water in oil). This primary
emulsion is again dispersed in a secondary aqueous phase containing
an emulsifier. Single emulsion evaporation (oil in water (o/w)) has
been commonly used in the encapsulation of lipophilic drugs. In
this O/W method, a drug and a biodegradable polymer are
co-dissolved in a mixture of suitable organic solvents (e.g.,
methanol and methylene chloride), and the resulting solution is
dispersed in an aqueous phase. In both emulsion evaporation
methods, as an organic solvent is removed by extraction or
evaporation during polymer dispersion in an aqueous phase, the
polymer decreases in solubility and is thus solidified to form
microspheres. In these methods, the technically important factor is
the encapsulation efficiency of bioactive drugs.
[0007] Most hydrophilic drugs leak in large amounts when dispersed
in an aqueous phase, resulting in low encapsulation efficiency. To
solve this problem, Okada et al. employed material such as gelatin
in the microsphere preparation based on double emulsion
evaporation. This material increased the viscosity of a primary
emulsion and decreased the diffusion rate of a drug (an LHRH
derivative) into a secondary emulsion, resulting in enhanced drug
encapsulation (Okada, H. and Toguchi, H., Crit. Rev. Ther. Drug
Carrier Syst., 12, 1-99, 1995). Similarly, the single emulsion
evaporation method also can enhance drug encapsulation by suitably
increasing the concentration of a biodegradable polymer (PLGA)
dissolved in an organic solvent phase. Typically, microspheres
prepared by double emulsion evaporation are more porous than those
prepared by single emulsion evaporation, and thus have increased
surface areas, leading to a relatively high initial release rate of
a drug.
[0008] However, the single and double emulsion evaporation methods
for preparing microspheres, like the phase separation method, have
the following disadvantages: difficulty in the removal of an
organic solvent used for dissolving a biodegradable polymer,
difficulty in mass production procedures due to changes in solvent
removal rate, allergic reactions to gelatin used for increasing the
viscosity of a primary emulsion, the possibility of a drug becoming
denatured and losing its activity due to strong shearing force
applied for making small microspheres during primary emulsion
preparation, limited drug encapsulation, and the like.
[0009] The spray drying method has also been used for preparing
finely atomized particles. In this method, typically, a solution of
a material to be dried, or a suspension or emulsion in which a
biodegradable polymer and a drug are homogenously dissolved, is
supplied to a nozzle, sprayed through the nozzle, and exposed to
heated air to evaporate the solvent used. In particular, in the
case of preparing sustained release microspheres, the drug release
rates of prepared microspheres greatly depend on the composition or
content of a biodegradable polymer, the type or content of an
additive, the composition of a solvent, and the like. In addition
to the above processing parameters, other parameters affecting the
morphology, size or properties of microspheres may be employed to
control the release rates of drugs, the parameters including the
type of a spray nozzle through which a spray solution is sprayed
(for example, a type that atomizes droplets using compressed air, a
type that atomizes droplets using centrifugal force when a spray
solution flows into a disc rotating at a high speed, a type that
atomizes droplets using ultrasonic waves generated when a vibrator
vibrates, etc.), supply rate of a spray solution, and temperature,
supplied amount and supply rate of dry air. In addition, the spray
drying method, unlike other methods for preparing sustained release
microspheres, is advantageous in that it provides a continuous
process, which facilitates microsphere production and thus
conversion from small-scale to large-scale production.
[0010] Although the spray drying method has the advantage of
permitting large-scale production of microspheres, it has
disadvantages as follows. The solvent used is not sufficiently
removed merely by spray drying. The residual solvent causes a
problem in the stability of the biodegradable polymer upon
long-term storage, leading to changes in drug release profiles of
microspheres. Another disadvantage of this method is that since
biodegradable polymers used for drug encapsulation are mostly
non-hydrophilic, microspheres prepared are not suspended well and
are thus difficult to accurately administer.
[0011] As described above, most conventional methods of preparing
sustained release microspheres employ a toxic solvent, and have
problems including residue of the toxic solvent used, the
microsphere size not being suitable for injection, poor
suspendability of microspheres, and difficult mass production.
[0012] The present inventors intended to provide a process for
preparing bioactive drug-loaded biodegradable polymer microspheres,
which are easy for mass production, by solving the aforementioned
problems.
DISCLOSURE OF THE INVENTION
[0013] It is therefore an object of the present invention to
provide a process for preparing sustained release microspheres
comprising a biodegradable polymer as a carrier and a bioactive
drug, the microspheres being easy for mass production, not
containing a residual toxic solvent, which is a problem of
conventional methods of preparing sustained release microspheres,
having high drug encapsulation efficiency, and having a uniform
size suitable for injection.
[0014] The present inventors conducted intensive studies, and found
that when microspheres, which are obtained by spray drying a
solution, suspension or emulsion containing a biodegradable
polymer, a drug and a solvent, are suspended in an aqueous solution
containing polyvinyl alcohol to remove the residual solvent, the
microspheres have improved suspendability and syringeability and
high drug encapsulation efficiency with no residual toxic solvent,
thereby leading to the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The above and other objects, features and other advantages
of the present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0016] FIGS. 1a and 1b show the dispersion/withdrawal rates and
syringeability of microspheres prepared in Preparation Example 1
according to the present invention in an aqueous solution, wherein,
after a test was carried out according to the method described in
Test Example 1, the withdrawal rates of microspheres in each tube
(FIG. 1a) and the deviation in each tube for the mean withdrawal
rate (FIG. 1b) were measured;
[0017] FIG. 2 shows the serum testosterone concentrations in male
SD rats (n=5) which were subcutaneously injected as described for
Test Example 5 with a single dosage of leuprolelin-loaded
microspheres prepared in Preparation Example 8; and
[0018] FIG. 3 shows the serum octreotide concentrations in male SD
rats (n=6) which were subcutaneously injected as described for Test
Example 6 with a single dosage of octreotide-loaded microspheres
prepared in Preparation Example 9.
BEST MODE FOR CARRYING OUT THE INVENTION
[0019] The present invention relates to a process of preparing
sustained release microspheres, in which a solution, suspension or
emulsion containing a biodegradable polymer, a drug and a solvent
is spray dried, and microspheres thus obtained are dispersed in an
aqueous solution containing polyvinyl alcohol, thereby more easily
removing residual solvent and improving suspendability of the
microspheres upon administration.
[0020] In detail, the present invention relates to a process of
preparing sustained release microspheres having high drug
encapsulation efficiency, almost no residual solvent, and improved
suspendability by dissolving and spray drying a biodegradable
polymer and a drug, suspending microspheres thus obtained in an
aqueous solution in which polyvinyl alcohol is dissolved, and
recovering, washing and freeze drying the microspheres.
[0021] In one aspect, the present invention provides a process of
preparing sustained release microspheres, comprising spraying a
solution, suspension or emulsion containing a biodegradable
polymer, a drug and a solvent into a dry chamber and drying it
using dry air to remove the solvent; and dispersing spray-dried
microspheres in an aqueous solution containing polyvinyl alcohol to
remove the residual solvent and improve dispersibility of the
microspheres.
[0022] The term "biodegradable polymer," as used herein, refers to
a polymer that slowly degrades when administered to the body, and
is thus not harmful to the body. Examples of such polymers include
polylactide (PLA), polyglycolide (PGA) and their copolymer,
poly(lactide-co-glycolide) (PLGA), polyorthoester, polyanhydride,
polyhydroxybutyric acid, polycaprolactone, polyalkylcarbonate, and
derivatives thereof.
[0023] The term "drug," as used herein, includes peptides having
biological activities, such as anticancer agents, antibiotics,
antipyretics, analgesics, anti-inflammatory agents, antitussive
expectorants, sedatives, antiulcer agents, antidepressants,
antiallergenic agents, antidiabetic agents, antihyperlipidemic
agents, antituberculous agents, hormonal agents, bone metabolic
agents, immunoinhibitors, angiogenesis inhibitors, contraceptives,
and vitamin-like agents. In particular, biologically active peptide
or protein drugs are preferred. Examples of oligopeptides having
biological activities include insulin, somatostatin and derivatives
thereof, growth hormone, prolactin, adrenocorticotropic hormone,
melanocyte-stimulating hormone, thyrotropin-releasing hormone and
salts and derivatives thereof, thyroid-stimulating hormone,
luteinizing hormone, follicle-stimulating hormone, vasopressin and
derivatives thereof, oxytocin, calcitonin, parathyroid hormone,
glucagon, gastrin, secretin, pancreozymin, cholecystokinin,
angiotensin, human placental lactogen, human chorionic
gonadotropin, and enkephalin and derivatives thereof. Examples of
polypeptides include endorphin, interferon (.alpha.-type,
.beta.-type, .gamma.-type), interleukin, tuftsin, thymopoietin,
thymosin, thymostimulin, thymic humoral factor (THF), serum thymic
factor and derivatives thereof, tumor necrosis factor, colony
stimulating factor (CSF), motilin, dynorphin, bombesin,
neurotensin, bradykinin, caerulein, urokinase, asparaginase,
kallikrein, substance P, nerve growth factor, blood coagulation
factor VIII and IX, lysozyme, polymyxin B, colistin, gramicidin,
bacitracin, protein synthesis-stimulating peptide, vasoactive
intestinal polypeptide, platelet-derived growth factor, growth
hormone-releasing factor, bone morphogenetic protein, epidermal
growth factor, and erythropoietin.
[0024] In the present invention, the aqueous solution containing
polyvinyl alcohol is used to more effectively remove the residual
solvent inside microspheres immediately after spray drying and to
disperse microspheres well in an injection solution upon
administration. This aqueous solution is removed through an
additional washing step, and finally remains in microspheres at an
amount less than 1%. Also, when microspheres are suspended in the
aqueous solution of polyvinyl alcohol to remove the residual
solvent, the removal speed of residual solvent may be controlled by
altering the temperature of the suspension. Using this feature,
upon small-scale production, the residual solvent may be removed
within a short time at room or ambient temperature. Upon
large-scale production, the suspension is maintained at low
temperature to remove the residual solvent at a slow speed, thereby
preventing deterioration of products due to extended handling time
of large quantities of products.
[0025] In an additional dispersal step, the content of polyvinyl
alcohol in the aqueous solution is preferably 0.01-20% (w/v), and
more preferably 0.05-10% (w/v). Polyvinyl alcohol has a molecular
weight of 3,000 to 300,000, preferably 5,000 to 100,000, and has a
hydration rate of 75% to 95%. The amount of polyvinyl alcohol
remaining on the surface of microspheres is preferably 0.02-1%
(w/v), and more preferably 0.05-0.5% (w/v).
[0026] The term "solvent," as used herein, refers to a material
that is able to dissolve a biodegradable polymer and/or a drug. An
appropriate solvent may be selected by those having ordinary skill
in the art according to the type of biodegradable polymer. Glacial
acetic acid is preferred.
[0027] The present invention includes a step of dispersing
microspheres in an aqueous solution of polyvinyl alcohol to improve
the dispersibility of microspheres. The dispersal step is carried
out for about one minute or longer to achieve maximal effects. The
preferred dispersal time is 5 min or longer.
[0028] In a patent application filed prior to the present invention
(Korean Pat. Application 10-2003-0023130), the present inventors
improved drug encapsulation efficiency and release profiles of
microspheres by homogenously dissolving a biodegradable polymer and
a drug using a non-toxic solvent and spray drying the resulting
solution, and suggested a spray drying method facilitating the mass
production of microspheres. This spray drying method has advantages
including easy mass production, low residual solvent, high drug
encapsulation efficiency, and ideal drug release profiles, but has
drawbacks including difficult dispersion and suspension of
sustained release microspheres obtained by spray drying in an
injection solution due to non-hydrophilicity of a polymer contained
in the microspheres, and requirement of an additional step for
removing the residual solvent to ensure the stability of
microspheres upon long-term storage. Thus, the present inventors
intended to solve these disadvantages in the present invention.
[0029] A conventional method of preparing microspheres by
spray-drying using two nozzles is disclosed in U.S. Pat. No.
5,622,657. To improve the dispersibility of polymeric microspheres,
which are apt to adhere to each other or aggregate when prepared by
spray drying, the cited invention provides a method of coating
drug-loaded polymeric microspheres with an aggregation-preventing
agent using two or more nozzles, in which a polymer solution
containing a biologically active substance and an aqueous solution
of an agent for preventing aggregation of microparticles are
sprayed separately from different nozzles at the same time. A
similar method is disclosed in Korean Pat. No. 0177309, the method
being characterized by spraying a dispersion in which a
water-soluble dispersing agent is dissolved in the direction
opposite to the spraying direction of a biodegradable polymer
solution containing an active ingredient and the flow direction of
dry air in order to coat a portion or all of sustained release
microparticles with the water-soluble dispersing agent. The cited
inventions intended to prevent microspheres from aggregating by
spraying an aqueous solution for preventing microsphere aggregation
immediately after microspheres are formed using spray drying, but
there are following disadvantages. Since microspheres are coated
with an aggregation-preventing agent immediately after being spray
dried, a toxic solvent used for dissolving a polymer for preparing
microspheres remains in microspheres in large quantities. In
addition, when microspheres are suspended in an injection solution
for administration, an excess amount of a dispersing agent is used.
Moreover, aggregation-preventing agents used in the cited
inventions, such as hydroxypropylcellulose, carboxymethylcellulose,
glycin, alanin, gelatin and collagen, do not improve the
suspendability in an injection solution or the syringeability,
resulting in non-uniform dosages.
[0030] On the other hand, in order to enhance the fluidity of
granules for tablet preparation or drug particles and improve the
solubility of drugs, a water-soluble dispersing agent, such as
hydroxypropylmethylcellulose, hydroxypropylcellulose, and polyvinyl
alcohol, may be spray dried together with a drug to be contained in
the resulting preparation in an amount of about 4 to 19 wt % (D.
Ermis, A. Yuksel, Preparation of spray-dried microspheres of
indomethacin and examination of the effects of coating on
dissolution rates, J. Microencapsulation, vol. 16, No. 3,
3,315-324(1999)). In the literature, the water-soluble dispersing
agents are used to enhance the solubility of poorly water-soluble
drugs, thereby dissolving the drugs within several hours, and
increase the fluidity of drug particles, thereby facilitating
processing such as tabletting. Polyvinyl alcohol has been
registered as a material liable to induce cancer when parenterally
administered to animals, and thus the residual amount thereof
should be controlled (Carcinogenic Studies on Water-Soluble and
Insoluble Macromolecules, Archives of Pathology, 67, 589-617,
1959). The present inventors found that when polyvinyl alcohol,
suspended in a polymer solution, is sprayed as described in the
above literature, the content of polyvinyl alcohol, which is
difficult to degrade in the body, increases, and the initial drug
release greatly increases. Such a high initial drug release may
cause side effects, and may lead to a reduced drug release
duration, thereby not guaranteeing suitable therapeutic
effects.
[0031] The present inventors prepared microspheres in which the
residual solvent is additionally removed and which have improved
syringeability, through a process comprising preparing microspheres
using a spray drying method developed by the present inventors
prior to the present invention, suspending the microspheres in an
aqueous solution containing polyvinyl alcohol, and washing and
recovering the microspheres.
[0032] The process of preparing biodegradable polymer microspheres
containing a drug having biological activity according to the
present invention comprises preparing microspheres by spray drying,
suspending the microspheres thus obtained in an aqueous solution
containing polyvinyl alcohol, and recovering, washing and drying
the microspheres, allows further removal of the residual solvent
and improves the suspendability of microspheres upon
administration, thereby leading to accurate administration of drugs
and effective treatment of diseases.
[0033] A better understanding of the present invention may be
obtained through the following examples which are set forth to
illustrate, but are not to be construed as the limit of the present
invention.
PREPARATION EXAMPLE 1
Preparation of Microspheres and a Post-Process Using Various
Dispersing Agents
[0034] PLGA microspheres were prepared using a spray dryer (SODEVA,
France) equipped with an ultrasonic nozzle (Sono-Tek, 120 kHz). A
biodegradable polymer, RG503H (Boehringer-Ingelheim, Germany), and
a drug, leuprolelin acetate (Polypeptide Laboratories, Denmark),
were used. 50 g of RG503H and 2.5 g of leuprolelin acetate were
homogeneously dissolved in 500 ml of glacial acetic acid (Yakuri
Pure Chemicals, Japan). The solution was transported at a flow rate
of 3 ml/min using a piston pump. The transported solution was
sprayed into the spray drier through an ultrasonic nozzle installed
in an upper part of a sprayer, and dried with dry air at
200.degree. C. Thereafter, microspheres recovered from a cyclone
were taken in a certain volume, weighed precisely, added at a
concentration of 50 mg/ml to an aqueous solution containing
distilled water and 1% (w/v) of a dispersing agent, and suspended
therein for one hour at room temperature using a magnetic agitator.
Dispersing agents used included polyvinyl alcohol (Sigma, P-8136),
polyvinylpyrrolidone (Sigma, PVP-360), human serum albumin (Sigma,
A-1654), polyethylene glycol (Yakuri Pure Chemicals, 28123), Tween
80 (Sigma, P-0343), poloxamer (Sigma, P-1300), sodium
carboxymethylcellulose (Sigma, C-5678), gelatin (Sigma, G-6650),
glycine (Sigma, G-7126), and mannitol (Sigma, M-8429). The
suspension was passed through a vacuum filter. Microspheres thus
collected were washed with distilled water two times and
freeze-dried.
TEST EXAMPLE 1
Evaluation of Withdrawal Rates and Syringeability of
Microspheres
[0035] The microspheres prepared in Preparation Example 1 were
dispersed in an aqueous solution, and assessed for withdrawal rates
into a syringe and syringeability. Each microsphere formulation was
placed into a beaker, and mixed with triple distilled water at a
concentration of 50 mg/ml. When microspheres were homogenously
dispersed using a magnetic agitator, 1 ml of the dispersion was
finely withdrawn into a 1-ml syringe fitted with a 21 gauge needle
(n=20). 1 ml of the microsphere suspension in the syringe was
transferred into a 1.5-ml Eppendorff tube and freeze-dried.
Microspheres recovered into the Eppendorff tube were assessed for
dry weight, and the results are given in Table 1, below.
TABLE-US-00001 TABLE 1 Withdrawal rates of microspheres according
to the type of dispersing agents Type of dispersing agents
Withdrawal rates (%) RSD (%) Microspheres immediately after 11.4
17.9 spray drying Distilled water 22.7 13.4 Polyvinyl alcohol 91.5
4.2 Polyvinylpyrrolidone 51.6 24.4 Human serum albumin 70.1 36.4
Polyethylene glycol 66.8 19.0 Tween 80 68.8 30.3 Poloxamer 24.2
56.8 Sodium carboxymethylcellulose 60.9 16.9 Gelatin 75.7 11.0
Glycine 44.7 39.0 Mannitol 46.5 25.5 Note: RSD (relative standard
deviation = [standard deviation of weight of recovered
microspheres/mean] .times. 100) indicates the deviation of
recovered mass.
[0036] As apparent in Table 1, microspheres immediately after spray
drying, which did not undergo a dispersal process, and microspheres
that underwent a dispersal process in distilled water not
containing a dispersing agent were found to decrease with respect
to withdrawal rate and homogeneity when drawn into a syringe and
injected from the syringe. Among several dispersing agents
mentioned in the literature studies, polyvinyl alcohol exhibited
the highest withdrawal rate, followed by gelatin, human serum
albumin and Tween 80. FIG. 1a shows the withdrawal rates and
homogeneity of microspheres, which did not undergo a dispersal
process or underwent a dispersal process using mannitol and
polyvinyl alcohol as dispersing agents, when a microsphere
suspension was drawn into a syringe and injected from the syringe
into a tube. FIG. 1b shows the deviation in each tube for the mean
withdrawal rate in a syringe when microspheres did not undergo a
dispersal process or underwent a dispersal process using polyvinyl
alcohol as a dispersing agent. As shown in FIGS. 1a and 1b,
microspheres had the highest withdrawal rate and the best
homogeneity when undergoing a dispersal process using polyvinyl
alcohol.
PREPARATION EXAMPLE 2
An Effect of Dispersal Time for Improving the Dispersibility of
Microspheres
[0037] PLGA microspheres were prepared using a spray dryer (SODEVA,
France) equipped with an ultrasonic nozzle (Sono-Tek, 120 kHz). A
biodegradable polymer, RG503H, and a drug, leuprolelin acetate,
were used. 40 g of RG503H and 4 g of leuprolelin acetate were
homogeneously dissolved in 400 ml of glacial acetic acid. The
solution was sprayed into a spray drier through an ultrasonic
nozzle at a flow rate of 3 ml/min, and dried with dry air at
200.degree. C. Thereafter, microspheres recovered from a cyclone
were taken in a predetermined amount, added at a concentration of
50 mg/ml to an aqueous solution containing 1% (w/v) polyvinyl
alcohol, and suspended therein for 1 min, 3 min, 5 min, 10 min, 1
hr, 3 hr and 6 hr at 25.degree. C. using a magnetic agitator. The
suspension was passed through a vacuum filter. Microspheres thus
collected were washed with distilled water two times, and
freeze-dried.
TEST EXAMPLE 2
Evaluation of Withdrawal Rates and Syringeability of
Microspheres
[0038] The microspheres prepared in Preparation Example 2 were
dispersed in an aqueous solution, and assessed for withdrawal rates
into a syringe and syringeability. This test was performed
according to the same method as in Test Example 1, and the results
are given in Table 2, below.
TABLE-US-00002 TABLE 2 Withdrawal rates of microspheres according
to dispersal time 1 min 3 min 5 min 10 min 1 hr 3 hr 6 hr
Withdrawal rates 71.4 80.0 86.1 89.6 89.4 90.2 90.3 (%) RSD (%)
13.6 11.6 7.8 5.5 5.9 4.9 4.8
[0039] As apparent in Table 2, a high withdrawal rate was observed
immediately after microspheres were suspended in a polyvinyl
alcohol solution. As the dispersal time was extended, microspheres
exhibited increased withdrawal rates and homogeneity when withdrawn
into a syringe and injected from the syringe. However, after a
certain suspension time, the withdrawal rates and homogeneity were
maintained at constant levels.
TEST EXAMPLE 3
Measurement of Residual Polyvinyl Alcohol Content
[0040] The microspheres prepared in Preparation Example 2 were
assessed for residual polyvinyl alcohol content. This assay was
performed by modifying a method described in Journal of Controlled
Release, 82 (2002), 105-114, as follows. 2 mg of a formulation,
which was accurately weighed, were placed into a glass vial, mixed
with 400 .mu.l of 0.5 N NaOH, and allowed to react in an oven at
60.degree. C. for 2-3 hrs to be completely dissolved (n=3). The
solution, in which microspheres were completely dissolved, was
neutralized with 180 .mu.l of 1 N HCR, supplemented with 600 .mu.l
of 0.65 M boric acid and 100 .mu.l of an I.sub.2/KI (0.05 M/0.15 M)
solution, and allowed to react for 20 min. Then, absorbance was
measured at 690 nm (UV). The results are given in Table 3,
below.
TABLE-US-00003 TABLE 3 Residual polyvinyl alcohol amounts according
to dispersal time 1 min 3 min 5 min 10 min 1 hr 3 hr 6 hr Residual
amount 0.02 0.04 0.05 0.07 0.12 0.22 0.38 (w/w)
[0041] As shown in Table 3, the residual amounts of polyvinyl
alcohol increased as the suspension time in the polyvinyl alcohol
solution increased.
[0042] When the results shown in Tables 2 and 3 were taken
together, the dispersibility and withdrawal rates of microspheres
improved immediately after microspheres were suspended and
dispersed in an aqueous solution of polyvinyl alcohol, but a
dispersal time of about 5 min or longer was needed for the optimal
effect. In this case, the amount of polyvinyl alcohol remaining on
the surface of microspheres was more than 0.05 wt %. This is a
critical factor in determining the withdrawal rate of microspheres
into a syringe.
COMPARATIVE PREPARATION EXAMPLE 1
Preparation of Microspheres by Co-Spraying with Polyvinyl
Alcohol
[0043] PLGA microspheres were prepared using a spray dryer (SODEVA,
France) equipped with an ultrasonic nozzle (Sono-Tek, 120 kHz). A
biodegradable polymer, RG503H, and a drug, leuprolelin acetate,
were used. To prepare a control microsphere formulation, 1.8 g of
RG503H and 0.2 g of leuprolelin acetate were homogeneously
dissolved in 90 ml of glacial acetic acid. The solution was
transported at a flow rate of 1.5 ml/min using a piston pump. The
transported solution was sprayed into the spray drier through an
ultrasonic nozzle installed at an upper part of a sprayer, and
dried with dry air at 200.degree. C. Thereafter, microspheres
recovered from a cyclone were taken in a predetermined amount,
added at a concentration of 50 mg/ml to an aqueous solution
containing 1% (w/v) polyvinyl alcohol as a dispersing agent, and
suspended therein for 1 hr at room temperature using a magnetic
agitator. The suspension was passed through a vacuum filter.
Microspheres thus collected were washed with distilled water two
times and freeze-dried.
[0044] A comparative microsphere formulation was prepared by spray
drying microspheres and polyvinyl alcohol at the same time in order
to compare the dispersal effect of co-spray dried polyvinyl alcohol
with that in the control formulation. 1.8 g of RG503H and 0.2 g of
leuprolelin acetate were homogeneously dissolved in 90 ml of
glacial acetic acid. The solution was transported at a flow rate of
1.5 ml/min using a piston pump. The transported solution was
sprayed into the spray drier through an ultrasonic nozzle installed
at an upper part of a sprayer, and dried with dry air at
200.degree. C, thereby yielding a comparative formulation.
[0045] The control and comparative microsphere formulations
prepared as described above were individually dispersed in an
aqueous solution, and assessed for withdrawal rates into a syringe
and syringeability. This assay was performed according to the same
method as in Test Example 1, and final dry weights of microspheres
in tubes were measured. The results are given in Table 4, below.
The residual polyvinyl alcohol amount of each microsphere
formulation was determined according to the same method as in Test
Example 3.
[0046] An in vitro release test was carried out, as follows. 10 mg
of each microsphere formulation were placed into a 1.5-ml tube,
mixed with 1 ml of phosphate buffered saline (PBS), and incubated
for 1 hr in an incubator at 37.degree. C. with agitation at 5 rpm
using a rotary shaker (SLRM-2M, Seoulin Bioscience). Each reaction
solution was centrifuged, and the supernatant was assessed for the
amounts of the peptide drug released from microspheres using a
fluorescence detector (Varian, Cary Eclipse; Ex: 280 nm, Em: 350
nm).
TABLE-US-00004 TABLE 4 Dispersibility, residual polyvinyl alcohol
amounts and initial drug release of control and comparative
microsphere formulations Residual polyvinyl Initial drug Withdrawal
RSD Formulation alcohol (%) release (%) rate (%) (%) Control 0.13
3.1 75.32 5.62 Comparative 0.52 15.5 68.18 13.13
[0047] As shown in Table 4, compared to the control microsphere
formulation undergoing a dispersal process using polyvinyl alcohol,
the comparative microsphere formulation not undergoing a suspending
process had a high residual polyvinyl alcohol concentration, but
exhibited decreased withdrawal rate and homogeneity when withdrawn,
into a syringe and injected therefrom, and a high initial drug
release rate.
TEST EXAMPLE 4
Evaluation of Residual Solvent Removal Rates According to Time of
Dispersal Using Polyvinyl Alcohol
[0048] Microspheres were prepared in and recovered from a cyclone
according to the same method as in Preparation Example 2, and
suspended in an aqueous solution of 1% polyvinyl alcohol at a
concentration of 50 mg/ml. While the aqueous solution was
maintained at 25.degree. C., the removal rates of residual acetic
acid were measured at given time points. The concentrations of
residual acetic acid in microspheres were determined as follows.
Microspheres were dissolved in methylene chloride (Junsei,
34355-0350), supplemented with an aqueous solution of 0.07%
phosphoric acid (Sigma, P-6560), and vigorously mixed therewith.
The mixture was centrifuged to separate a layer of the aqueous
phosphoric acid solution. This layer was recovered and assessed for
the amount of acetic acid using HPLC. HPLC was carried out using a
C18 column (5 .mu.m, 4.6.times.250 mm, 120 .ANG.). A mixture of
5-50% linear gradient of methanol (J. T. Baker, AH230-4) and 0.07%
phosphate buffer (pH 3.0) was used as a mobile phase having a flow
rate of 1.2 ml/min. Acetic acid was detected at 210 nm (UV), and
the results are given in Table 5, below. The residual acetic acid
content immediately after microspheres were prepared was 0.8 wt
%.
TABLE-US-00005 TABLE 5 Residual solvent removal rates according to
dispersal time Time (min) Acetic acid removal rates (%) 5 20.0 10
34.2 20 44.0 40 54.5 60 61.3 120 73.6 180 87.9
[0049] As shown in Table 5, the residual solvent removal rates
gradually increased with increasing time of suspension of
microspheres in an aqueous solution of polyvinyl alcohol to remove
the residual solvent. The final residual solvent was maintained at
less than 0.1 wt %.
PREPARATION EXAMPLE 3
Preparation of BSA-Loaded Microspheres by Spray Drying of W/O Type
Emulsion Containing BSA
[0050] 0.5 g of bovine serum albumin (BSA) (Sigma, A-7638) was
dissolved in distilled water, and homogeneously mixed with a
solution prepared by dissolving 9.5 g of RG502H in 95 ml of
methylene chloride, thereby yielding a W/O type emulsion. While the
emulsion was maintained in an emulsion state using an agitator, it
was supplied to a spray drier (Buchi-191) at a flow rate of 3
ml/min. Compressed air was supplied to a two-fluid nozzle at a flow
rate of 450 NL/h to dry sprayed atomized droplets using dry air at
80.degree. C. The recovered microspheres were suspended for 3 hrs
in an aqueous solution of 1% polyvinyl alcohol at a concentration
of 50 mg/ml with agitation using a magnetic agitator, washed with
distilled water, and freeze-dried. The microspheres thus prepared
had a mean particle size of 5.2 .mu.m, and the amount of polyvinyl
alcohol remaining on the surface of the microspheres was 0.93%
(w/w).
PREPARATION EXAMPLE 4
Preparation of BSA-Loaded Microspheres by Spray Drying of S/O Type
Emulsion Containing BSA
[0051] 1 g of BSA was finely pulverized in a mortar, and
homogeneously mixed with a solution prepared by dissolving 9 g of
RG502H in 90 ml of methylene chloride, thereby yielding an S/O type
emulsion. While the emulsion was maintained in an emulsion state
using, an agitator, it was supplied to a spray drier (Buchi-191) at
a flow rate of 3 ml/min. Compressed air was supplied to a two-fluid
nozzle at a flow rate of 450 NL/h to dry sprayed atomized droplets
using dry air at 80.degree. C. The recovered microspheres were
suspended for 3 hrs in an aqueous solution of 1% polyvinyl alcohol
at a concentration of 50 mg/ml with agitation using a magnetic
agitator, washed with distilled water, and freeze-dried. The
microspheres thus prepared had a mean particle size of 5.8 .mu.m,
and the amount of polyvinyl alcohol remaining on the surface of the
microspheres was 0.85% (w/w).
PREPARATION EXAMPLE 5
Preparation of Leuprolelin-Loaded Microspheres
[0052] 1 g of leuprolelin acetate and 9 g of RG502H were dissolved
in 90 ml of glacial acetic acid. The solution was supplied to a
spray drier (Buchi-191) at a flow rate of 2 ml/min. Compressed air
was supplied to a two-fluid nozzle at a flow rate of 500 NL/h to
dry sprayed atomized droplets using dry air at 120.degree. C. The
recovered microspheres were suspended for 3 hrs in an aqueous
solution of 1% polyvinyl alcohol at a concentration of 50 mg/ml
with agitation using a magnetic agitator, washed with distilled
water, and freeze-dried. The microspheres thus prepared had a mean
particle size of 5.1 .mu.m, and the amount of polyvinyl alcohol
remaining on the surface of the microspheres was 0.98% (w/w).
PREPARATION EXAMPLE 6
Preparation of Leuprolelin-Loaded Microspheres
[0053] 0.4 g of leuprolelin acetate and 9.6 g of R202H were
dissolved in 96 ml of glacial acetic acid. The solution was
supplied to a spray drier (SODEVA, France) at a flow rate of 3
ml/min, sprayed into a dry chamber using an ultrasonic nozzle
(Sono-Tek, 120 kHz), and dried using dry air at 200.degree. C. The
recovered microspheres were suspended for 3 hrs in an aqueous
solution of 1% polyvinyl alcohol at a concentration of 50 mg/ml
with agitation using a magnetic agitator, washed with distilled
water, and freeze-dried. The microspheres thus prepared had a mean
particle size of 23.4 .mu.m, and the amount of polyvinyl alcohol
remaining on the surface of the microspheres was 0.16% (w/w).
PREPARATION EXAMPLE 7
Preparation of Leuprolelin-Loaded Microspheres
[0054] 0.5 g of leuprolelin acetate and 9.5 g of R202H were
dissolved in 95 ml of glacial acetic acid. The solution was
supplied to a spray drier (SODEVA, France) at a flow rate of 3
ml/min, sprayed into a dry chamber using an ultrasonic nozzle
(Sono-Tek, 60 kHz), and dried using dry air at 200.degree. C. The
recovered microspheres were suspended for 3 hrs in an aqueous
solution of 1% polyvinyl alcohol at a concentration of 50 mg/ml
with agitation using a magnetic agitator, washed with distilled
water, and freeze-dried. The microspheres thus prepared had a mean
particle size of 32.6 .mu.m, and the amount of polyvinyl alcohol
remaining on the surface of the microspheres was 0.11% (w/w).
PREPARATION EXAMPLE 8
Preparation of Leuprolelin-Loaded Microspheres
[0055] 1.4 g of leuprolelin acetate, 0.86 g of RG504H and 7.74 g of
R202H were dissolved in 86 ml of glacial acetic acid. The solution
was supplied to a spray drier (SODEVA, France) at a flow rate of 3
ml/min, sprayed into a dry chamber using an ultrasonic nozzle
(Sono-Tek, 120 kHz), and dried using dry air at 200.degree. C. The
recovered microspheres were suspended for 90 min in an aqueous
solution of 1% polyvinyl alcohol at a concentration of 50 mg/ml
with agitation using a magnetic agitator, washed with distilled
water, and freeze-dried.
PREPARATION EXAMPLE 9
Preparation of Octreotide-Loaded Microspheres
[0056] 0.7 g of octreotide acetate and 9.3 g of RG502H were
dissolved in 186 ml of glacial acetic acid. The solution was
supplied to a spray drier (SODEVA, France) at a flow rate of 3
ml/min, sprayed into a dry chamber using an ultrasonic nozzle
(Sono-Tek, 120 kHz), and dried using dry air at 200.degree. C. The
recovered microspheres were suspended for 1 hr in an aqueous
solution of 1% polyvinyl alcohol at a concentration of 50 mg/ml
with agitation using a magnetic agitator, washed with distilled
water, and freeze-dried.
TEST EXAMPLE 5
[0057] The leuprolelin-loaded microspheres prepared in Preparation
Example 8 was suspended in a suspension (0.5% (w/w) sodium
carboxymethylcellulsoe, 5% (w/w) mannitol, 0.1% Tween 80), and
subcutaneously injected at a single dosage of 9 mg/kg (leuprolelin
acetate) into male SD rats (n=5, 195.+-.20 g). Blood samples were
collected from tail veins before drug administration, 30 min, 1 hr,
3 hr, 6 hr and 1, 2, 4 and 7 days after drug administration, and
every seven days for a period from day 8 to 90. Rats were
challenged with the microspheres at a dosage of 100 .mu.g/kg (based
on leuprolelin acetate) 28, 56 and 84 days after drug
administration in order to evaluate the sustained release of the
drug from the microspheres, and blood samples were collected before
drug administration and 3 and 24 hrs after drug administration.
Secondary drug administration was carried out 90 days after the
primary drug administration, and blood samples were collected as
described in the primary drug administration. The collected blood
samples were placed into 1.5-ml Eppendorff tubes, and centrifuged
for 10 min at 4.degree. C. and 12,000 rpm. The-obtained sera were
stored at -20.degree. C. Serum testosterone levels were measured
using a radio-immunoassay (RIA) Kit (DSL-10-4000, Diagnostic System
Laboratories, Inc., Webster, Tex., USA), and the results are given
in FIG. 2. The three challenges, which were performed every 28 days
after drug administration, and the secondary drug administration,
which was performed 90 days after drug administration, resulted in
inhibition of the rise of serum testosterone levels. These results
indicate that leuprolelin was continuously released over the test
period of 90 days.
TEST EXAMPLE 6
[0058] The octreotide-loaded microspheres prepared in Preparation
Example 9 were suspended in a suspension (0.5% (w/w) sodium
carboxymethylcellulsoe, 0.6% (w/w) mannitol), and subcutaneously
injected at a single dosage of 5 mg/kg (octreotide) to male SD rats
(n=6, 195.+-.20 g). Blood samples were collected from tail veins
before drug administration and 6 hrs and 1, 4, 7, 13, 21 and 31
days after drug administration. The collected blood samples were
placed into 1.5-ml Eppendorff tubes, and centrifuged for 10 min at
4.degree. C. and 12,000 rpm. The obtained sera were stored at
-20.degree. C. Serum octreotide concentrations were measured using
an enzyme immunoassay (EIA) kit (Bachem, S-1275, Peninsula
Laboratories Inc., USA). The results are given in FIG. 3. As shown
in FIG. 3, the octreotide drug was found to be continuously
released for a period of more than two weeks.
INDUSTRIAL APPLICABILITY
[0059] As described hereinbefore, the process of preparing
microspheres according to the present invention allows the
preparation of sustained release microspheres not having the
problems of conventional preparation methods of sustained release
microspheres, including toxicity due to residual solvent and poor
syringeability. The microspheres prepared according to the present
invention release an effective concentration of a drug in a
sustained manner for a predetermined period when administered to
the body, prevent rapid initial drug release, and reduce the
required administration frequency of a drug. Thus, the present
microspheres are useful in the treatment of diseases.
* * * * *