DESCRIPTION
MICROPARTICLE AND PHARMACEUTICAL COMPOSITION THEREOF
TECHNICAL FIELD
[0001]
The present invention relates to a microparticle comprising an agglomerate of particles containing
hydrophilic active substances, and a pharmaceutical composition thereof. Particularly, the invention
relates to a microparticle and a pharmaceutical composition thereof as so-called drug delivery system.
More particularly, for example, the invention relates to a microparticle effectively containing a protein,
a peptide drugs, a nucleic acid drugs, and the like of hydrophilic property and large molecular weight,
and a pharmaceutical composition thereof.
BACKGROUND ART
[0002]
Particulate preparations having drugs enclosed in fine particles called nanoparticle, microparticle,
nanosphere, microsphere, or microcapsule are developed, and are attempted to be used as
sustained-release agents for drugs.
[0003]
Particulate preparations using polymer compounds as the base include fine particles composed of
biodegradable polylactic acid or poly (lactic acid-glycolic acid). In these particulate preparations, it
is hard to encapsulate a protein or a peptide drug of hydrophilic property and large molecular weight
while maintaining the bioactivity. In addition, when administering in the human body, it is known
that the drug is massively released in a short time, and this phenomenon is called an initial burst.

[0004]
As fine particles composed of a polymer of covalent bonding of saccharide and poly (hydroxy acid),
patent literature 1 discloses a microcapsule for carrying a pharmacologically active substance
composed of a reaction product of polyol and polylactic acid. In this technique, polysaccharides are
not used, and nothing is mentioned about inclusion of peptide or protein. The microcapsule
manufactured by a spray drying method released the encapsulated drug by 62% in 24 hours. This
release speed is too fast, and the microcapsule can be hardly applied as a sustained-release agent for a
drug.
[0005]
Patent literature 2 and non patent literature 1 disclose a nanoparticle or a nanoparticle composed of a
material having a biodegradable polymer grafted in polysaccharides, but these literatures mention
nothing about a microparticle composed of nanoparticles. Patent literature 2 discloses, for example,
a double emulsion method already cited in other literatures, as a manufacturing method of a
microparticle for encapsulating a hydrophilic active substance, but there is no specific description, and
inclusion of a drug into a particle, or release of a drug from a particle is not realized. Non-patent
document 1 discloses a microparticle encapsulating an albumin manufactured by the double emulsion
method, but the encapsulation efficiency to the included amount of the albumin is 53% or less, and the
low encapsulation efficiency of the hydrophilic active substance has a problem in the manufacturing
cost.
[0006]
Patent literature 3 discloses a fine particle containing an amphiphilic polymer composed of
polysaccharides and an aliphatic polyester, more specifically a fine particle composed of an inner
nucleus of polysaccharides, a hydrophobic outer layer of aliphatic polyester, and a surface modifier
bonded to the hydrophobic outer layer. This fine particle does not have an agglomeration structure of
fine particles, and specific examples are not shown about particles of particle diameter of micrometer

units. The encapsulation efficiency of the hydrophilic substance is 50% or less, and this low
encapsulation efficiency is a similar problem as in the case above.
[0007]
Patent literature 4 discloses a nanoparticle of average particle diameter of less than 300 nm, composed
of a naturally derived polymer of dextran, but specific examples are not shown. This is not an
agglomeration structure of fine particles, and the average particle diameter is hundreds of nanometers,
and the drug is likely to diffuse from the site of administration, and it is not preferred as a
sustained-release agent.
[0008]
As the polymer for forming particles, patent literature 5 and patent literature 6 disclose and suggest
use of an amphiphilic block polymer having a hydrophilic portion such as polyethylene glycol, and a
hydrophobic portion such as poly(lactic acid-glycolic acid). Micelle particles using such amphiphilic
block polymer are usually hydrophobic in the inside, and hydrophilic in the outer layer, and they are
suited to containment of hydrophobic low molecular weight drugs, but not suited to containment of
hydrophilic active substances such as protein or peptide.
[0009]
Patent literature 7 and non patent literature 2 disclose attempts to contain a protein in a particle using
an amphiphilic block polymer, but the amount of the drug to be contained is small, or the initial burst
is large, and so far the manufacturing technology of particles having properties suited as
sustained-release injection of a hydrophilic drug is not established yet.
Patent literature 1: Japanese Patent Application Publication No. 8-19226
Patent literature 2: Japanese Translation of PCT International Application Publication No.
2004-521152
Patent literature 3: WO2006/095668

Patent literature 4: Japanese Translation of PCT International Application Publication No. 10-511957
Patent literature 5: Japanese Translation of PCT International Application Publication No.
2004-513154
Patent literature 6: Japanese Translation of PCT International Application Publication No.
2004-514734
Patent literature 7: Japanese Translation of PCT International Application Publication No.
2000-501084
Non-patent literature 1: Yuichi Oya and 3 others, "Encapsulation and/or Release Behavior of Bovine
Serum Albumin within and from Polylactide-Grafted Dextran Microspheres" (Macromolecular
Bioscience, 2004, vol. 4, pp. 458-463).
Non-patent literature 2: Anshu Yang and 5 others, "Tumor necrosis factor alpha blocking peptide
loaded PEG-PLGA nanopeptides: Preparation and in vitro evaluation" (International Journal of
Pharmaceutics, 2007, vol. 331, pp. 123-132).
DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0010]
As mentioned above, microparticles using a polymer have been developed, and it is hence a primary
object of the invention to present a microparticle capable of encapsulating a hydrophilic active
substance efficiently, and more particularly a microparticle capable of releasing the encapsulated drug
at an appropriate speed, without causing significant initial burst.
MEANS FOR SOLVING THE PROBLEMS

[0011]
The present inventors intensively accumulated studies to solve the problems, and have finally
completed the invention.
[0012]
That is, the invention relates to a microparticle comprising an agglomerate of hydrophilic active
substance containing particles, which particle comprises an amphiphilic polymer composed of a
hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharides or
polyethylene glycol, and a hydrophilic active substance, or more particularly a microparticle
comprising agglomerate of hydrophilic active substance containing particles, which particle has a
hydrophilic segment of an amphiphilic polymer in the inside and has an outer layer of the hydrophobic
segment of the amphiphilic polymer, and a manufacturing method thereof, and a pharmaceutical
composition thereof.
EFFECTS OF THE INVENTION
[0013]
The microparticle of the invention is capable of encapsulating a hydrophilic active substance
efficiently, and releasing the hydrophilic active substance at an appropriate speed in the human body,
and is hence usable as a novel DDS preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014]
[Fig. 1] shows drug release from microparticles encapsulating human growth hormone.
[Fig. 2] shows drug release from dextran-PLGA microparticles encapsulating human insulin.
[Fig. 3] shows an SEM image of dextran-PLGA microparticles.

[Fig. 4] shows an SEM image of polyethylene glycol-poly(epsilon-caprolactone) microparticle.
[Fig. 5] shows time-course changes of blood drug concentration in mouse administered human growth
hormone-encapsulating particles subcutaneously.
[Fig. 6] shows time-course changes of blood drug concentration in mouse administered human growth
hormone-encapsulating microparticles subcutaneously.
[Fig. 7] shows changes of body weight in mouse administered human growth hormone-encapsulating
microparticles subcutaneously.
[Fig. 8] shows time-course changes of blood IGF-1 concentration in mouse administered human
growth hormone-encapsulating microparticles subcutaneously.
[Fig. 9] shows drug release in a buffer solution of Exendin-4-encapsulating microparticles.
[Fig. 10] shows time-course changes of blood drug concentration in mouse administered
Exendin-4-encapsulating microparticles subcutaneously.
[Fig. 11] shows drug release from human growth hormone-encapsulating associated particles
microparticles.
[Fig. 12] shows the relation between particle diameter and amount of dimethyl carbonate added at the
time of preparation of S/O/W type emulsion.
[Fig. 13] shows results of enclosure rate entrappement efficiency of FD40 encapsulating
microparticles.
[Fig. 14] shows an SEM image of microparticle powder prepared from PEG-PLGA polymer (5k-10k).
[Fig. 15] shows an SEM image of microparticle powder prepared from PEG-PLGA polymer (5k-61k).
[Fig. 16] shows release behavior of FD40 from FD40-encapsulating microparticles.
[Fig. 17] shows release behavior of drug from human insulin-encapsulating microparticles.

[Fig. 18] shows time-course changes of blood drug concentration in mouse administered human
growth hormone-encapsulating microparticles subcutaneously.
[Fig. 19] shows time-course changes of blood drug concentration in mouse administered human
growth hormone-encapsulating microparticles subcutaneously.
[Fig. 20] shows time-course changes of blood IGF-1 concentration in mouse administered human
growth hormone-encapsulating microparticles subcutaneously.
[Fig. 21] shows time-course changes of blood pharmacokinetics in mouse administered
Exendin-4-encapsulating microparticles subcutaneously.
[Fig. 22] shows the relation between particle diameter and amount of dimethyl carbonate added at the
time of preparation of S/O/W type emulsion.
BEST MODE FOR CARRYING OUT THE INVENTION
[0015]
In the invention, it is characterized by forming a microparticle by agglomeration of hydrophilic active
substance containing particles which particle comprises an amphiphilic polymer and a hydrophilic
active substance. Herein, the agglomeration is bonding of two or more particles by way of
inter-particle force or other substance, and forming of a set. The inter-particle force is not specified
particularly, but usable examples include hydrophobic interaction, hydrogen bond, and van der Waals
force. The agglomeration is not limited to a state of mutual contact of particles, but substances
having an affinity for particles may be present among particles, or particles may distribute in a matrix.
As the substances having affinity for particles or the matrix, a polymer is preferred. In the invention,
by agglomeration of the hydrophilic active substance containing particles, as compared with a single
particle, the effect that the encapsulation efficiency of the hydrophilic active substance is higher is
attained. The particle diameter of the hydrophilic active substance containing particles to be
associated is variable.

[0016]
Microparticles are particles having the particle diameter ranging from sub-microns to sub-millimeters.
In the invention, the average particle diameter of microparticles is not particularly limited, but in the
case of administration of the microparticles by injection to the human body, the greater the average
particle diameter, the larger is the syringe needle, and the patient's burden is increased, and therefore
from the viewpoint of lowering of the patient's burden, it is preferred to be in a range of 1 urn to 50
urn. The average particle diameter of microparticles may be determined by image analysis by using
a scanning electron microscope.
[0017]
The number of agglomerations of hydrophilic active substance containing particles for composing a
microparticle is preferred to be in a range from 10 to the seventh power of 10, more preferably in a
range from the fifth power of 10 to the seventh power of 10. The number of agglomerations is
calculated from the average particle diameter of hydrophilic active substance containing particles and
the average particle diameter of microparticles.
[0018]
In the invention, the amphiphilic polymer is composed of a hydrophobic segment of poly(hydroxy
acid) and a hydrophilic segment of polysaccharides or polyethylene glycol. Herein, the amphiphilic
property is a state having both hydrophilic and hydrophobic properties, and as for the hydrophilic
property, when solubility in water is higher in a certain segment than in other segments, such segment
is said to be hydrophilic. A hydrophilic segment is preferred to be soluble in water, but if hardly
soluble, it is hydrophilic if solubility in water is higher than other segments. A certain segment is
said to be hydrophobic if solubility in water is lower than other parts. A hydrophobic segment is
preferred to be insoluble in water, but if soluble, it can be hydrophobic if solubility in water is lower
than other segments.
[0019]

Specific examples of poly(hydroxy acid) of the amphiphilic polymer include polyglycolic acid,
polylactic acid, poly(2-hydroxy butyric acid), poly(2-hydroxy valeric acid), poly(2-hydroxy caproic
acid), poly(2-hydroxy capric acid), poly(malic acid), and derivatives and copolymers of these high
molecular compounds. However, since microparticles of the invention are desired to have no
significant effects at the time of administration in human body, the poly(hydroxy acid) of amphiphilic
polymer is also preferred to be a biocompatible high polymer. The biocompatible high polymer is a
substance not having significant effects on the human body when administered, and more specifically
the LD50 is preferred to be 2,000 mg/kg or more by oral administration of the high polymer in rat.
[0020]
As poly(hydroxy acid) of the biocompatible high polymer, a copolymer of polylactic acid, and
polyglycolic acid, or poly(lactic acid-glycolic acid) is preferred. When the poly(hydroxy acid) is a
poly(lactic acid-glycolic acid), the composition ratio of the poly(lactic acid-glycolic acid) (lactic
acid/glycolic acid) (mol/mol%) is not particularly limited as far as the objects of the invention are
achieved, but the ratio is preferably 10/0 to 30/70, or more preferably 60/40 to 40/60.
[0021]
When the hydrophilic segment of the amphiphilic polymer is polysaccharides, examples of the
polysaccharides may include cellulose, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid,
pullulan, or dextran, and dextran is most preferable.
[0022]
The amphiphilic polymer is preferably composed by graft polymerization of graft chain(s) of
poly(hydroxy acid) in a main chain of polysaccharide. Herein, the average molecular weight of the
main chain of polysaccharide is preferably 1,000 to 100,000, or more preferably 2,000 to 50,000, and
the average molecular weight of the poly(hydroxy acid) is preferably 500 to 100,000, or more
preferably 1,000 to 10,000. The value of average molecular weight of poly(hydroxy acid) to the

average molecular weight of polysaccharides is preferably 0.01 times to 100 times, more preferably
0.02 times to 10 times, or most preferably 0.02 times to 1 times.
[0023]
The number of graft chains of poly(hydroxy acid) bonded with the main chain of polysaccharides is
preferably 2 to 50. The number of graft chains may be determined from the average molecular
weight of graft type amphiphilic polymer, main chain of polysaccharides, and graft chain of
poly(hydroxy acid).
[0024]
When the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, the amphiphilic
polymer is preferred to be a block polymer of polyethylene glycol and poly(hydroxy acid). In the
invention, the term "block" refers to a portion segment of a polymer molecule, consisting of at least
five or more monomer units, and being different in chemical structure or configuration between a
portion segment and other adjacent portion segment, and a polymer formed of two or more blocks
coupled straightly is called a block polymer. Each block forming a block polymer may comprise two
or more monomer units, that is, a random, alternating, or gradient polymer may be formed. When
the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, the amphiphilic polymer is
preferred to be a block polymer coupling one each of polyethylene glycol and polyhydroxy acid.
[0025]
When the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, specific examples
of the polyethylene glycol to be used include straight-chain or branched polyethylene glycol or its
derivatives, and a preferred example of polyethylene glycol derivative is polyethylene glycol
monoalkyl ether. The alkyl group of the polyethylene glycol monoalkyl ether is a straight-chain or
branched alkyl group having 1 to 10 carbon atom(s), and a branched alkyl group having 1 to 4 carbon
atom(s) is more preferable, and methyl, ethyl, propyl, and iso-propyl groups are particularly desired.
[0026]

The average molecular weight of the polyethylene glycol is not particularly limited, but is preferably
2,000 to 15,000, more preferably 2,000 to 12,000, even more preferably 4,000 to 12,000, and
particularly preferably 5,000 to 12,000.
[0027]
When the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, the average
molecular weight of the poly(hydroxy acid) is not particularly limited, but is preferably 5,000 to
200,000, more preferably 15,000 to 150,000, or even more preferably 20,000 to 100,000. The value
of average molecular weight of poly(hydroxy acid) to the average molecular weight of polyethylene
glycol is preferably 1.0 times or more, more preferably 2 times or more, most preferably 4 times or
more, and particularly preferably 4 times or more to 25 times or less.
[0028]
In this description, the average molecular weight refers to the number-average molecular weight
unless otherwise specified, and the number-average molecular weight is an average molecular weight
calculated by a method not considering weighting of magnitude of a molecule, and the average
molecular weight of amphiphilic polymer, polysaccharides, and polyethylene glycol can be obtained
as the molecular weight converted into polystyrene or pullulan measured by gel permeation
chromatography (GPC). The average molecular weight of poly(hydroxy acid) can be determined
from the ratio of peak integral value of terminal residue and peak integral value of others than terminal
residue as measured by nuclear magnetic resonance ('H-NMR) measurement.
[0029]
The amphiphilic polymer composed of polysaccharides and poly(hydroxy acid) used in the invention
may be synthesized in any one of the known methods, and as far as a reversed-phase emulsion can be
formed, the synthesizing method is not specified, and it can be manufactured, for example, in any one
of the following methods (1), (2), and (3).

(1) In the presence of a tin catalyst, a hydroxy acid activating monomer is added to polysaccharides
to carry out a polymerization reaction, and poly (hydroxy acid) is further added, and a graft type
amphiphilic polymer is manufactured [Macromolecules, 31, 1032-1039 (1998)].
(2) The hydroxyl group of partially non-protected polysaccharides of which majority of hydroxy
group is protected by a substituent is activated by a base, a hydroxy acid activating monomer is added
to form graft chain(s) composed of poly(hydroxy acid), and finally the protective group is removed,
and a graft type amphiphilic polymer is manufactured [Polymer, 44, 3927-3933, (2003)].
(3) In polysaccharides, a copolymer of poly(hydroxy acid) is added to execute condensation
reaction by using a dehydrating agent and/or a functional activating agent, and a graft type
amphiphilic polymer is manufactured [Macromolecules, 33, 3680-3685 (2000)].
[0030]
The amphiphilic polymer composed of polyethylene glycol and poly(hydroxy acid) used in the
invention may be synthesized in any one of the known methods, and as far as a reversed-phase
emulsion can be formed, the synthesizing method is not specified, and for example, in the presence of
a tin catalyst, a hydroxy acid activating monomer is added to polyethylene glycol to carry out a
polymerization reaction to form poly(hydroxy acid), and an amphiphilic block polymer is
manufactured [Journal of Controlled Release, 71, 203-211 (2001)].
[0031]
The structure of the hydrophilic active substance containing particle comprising the amphiphilic
polymer and hydrophilic bioactive substance is not particularly limited, but as far as the hydrophilic
active substance containing particle has a hydrophilic segment of an amphiphilic polymer in the inside,
and has an outer layer of a hydrophobic segment of an amphiphilic polymer, it is preferable because
the contained hydrophilic active substance can be maintained more stably.
[0032]

When the hydrophilic active substance containing particle is a particle having a hydrophilic segment
of an amphiphilic polymer in the inside, and having an outer layer of a hydrophobic segment of an
amphiphilic polymer, it is one of the preferred embodiments if a surface modifier is bonded to the
outer layer of poly(hydroxy acid). Herein, bonding may be either non-covalent bonding or covalent
bonding. Non-covalent bonding is preferably hydrophobic interaction, but may include electrostatic
interaction, hydrogen bond, or van der Waals force, or a combination thereof. In non-covalent
bonding, the hydrophobic outer layer of fine particles containing the amphiphilic polymer, and the
hydrophobic portion of a surface modifier described below may be preferably bonded to each other by
hydrophobic interaction. In this case, the dispersant of fine particles is particularly preferred to be
water, buffer solution, physiological saline, surface modifier aqueous solution, or fine particle
dispersant of hydrophilic solvent.
[0033]
The surface modifier is preferably a compound capable of stabilizing the water-oil interface of S/O/W
type emulsion, or the oil-oil emulsion interface of S/O1/02 type emulsion, and more preferably a
compound having properties for enhancing the colloid stability of microparticles. The surface
modifier may be one type or a mixture of plural types. Herein, the property of enhancing the colloid
stability means to prevent or delay aggregation of microparticles in the solvent.
[0034]
In the present invention, the surface modifier is preferred to be an amphiphilic compound or a
hydrophilic polymer.
[0035]
The hydrophilic polymer of the surface modifier of the invention is preferably any one selected from
the group consisting of polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene
imine, polyacrylic acid, polymethacrylic acid, poly-1,3-dioxolane, 2-methacryloyl oxyethyl

phosphoryl choline polymer, poly-1,3,6-trioxane, polyamino acid, peptide, protein, saccharides, and
analogs thereof.
[0036]
Analogs of the hydrophilic polymer may include a surfactant having a hydrophilic polymer partially
modified by a hydrophobic group such as a long-chain alkyl, but are not particularly limited to this.
[0037]
As a polyethylene glycol analog of a surface modifier of the invention, it is preferred to use Pluronic
(registered trademark) commercially available by BASF or its equivalents.
[0038]
As a polyamino acid of a surface modifier of the invention, polyaspartic acid, polyglutamic acid, or
their analogs may be preferably used. An analog introducing a long-chain alkyl group in part of
polyaspartic acid or polyglutamic acid is particularly preferable.
[0039]
As a peptide of a surface modifier of the invention, a basic peptide may be used.
[0040]
As a protein of a surface modifier of the invention, gelatin, casein, or albumin is preferred for
enhancing the dispersion performance of particles. As a protein, an antibody is one of preferred
examples.
[0041]
As saccharides of a surface modifier of the invention, monosaccharides, oligosaccharides, and
polysaccharides are preferable. As polysaccharides, cellulose, chitin, chitosan, gellan gum, alginic
acid, hyaluronic acid, pullulan, and dextran are preferable. Particularly, cholesteryl pullulan is
preferable in view of better dispersibility of particles. Analogs of any one selected from the group

consisting of cellulose, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, pullulan, and
dextran are preferable.
[0042]
As the surface modifier, these examples of peptide, protein, and saccharides are particularly preferred
to be analogs partly modifying the hydrophobic group of a long-chain alkyl, or analogs modifying the
hydrophilic polymer or the amphiphilic compound.
[0043]
In the surface modifier of the invention, the amphiphilic compound includes a lipid as one of the
preferred examples.
[0044]
In the surface modifier of the invention, the amphiphilic compound includes a surfactant as one of the
preferred examples. Preferred examples of the surfactant include: nonionic active agents such as
polyoxyethylene-polypropylene glycol copolymer, sucrose fatty acid ester, polyethylene glycol fatty
acid ester, polyoxyethylene sorbitan mono-fatty acid ester, polyoxyethylene sorbitan di-fatty acid ester,
polyoxyethylene glycerin mono-fatty acid ester, polyoxyethylene glycerin di-fatty acid ester,
polyglycerin fatty acid, polyoxyethylene castor oil, polyoxyethylene hardened castor oil; alkyl sulfates
such as lauryl sodium sulfate, lauryl ammonium sulfate, stearyl sodium sulfate; or lecithin.
[0045]
In the invention, the hydrophilic active substance is exemplified by low molecular compound, protein,
peptide, DNA, RNA, or modifying nucleic acid. Even a hydrophobic drugs may be contained in the
microparticle of the invention if made hydrophilic by using a solubilizing agent. The solubilizing
agent herein preferably includes cyclodextrin and its analogs.
[0046]

The protein or the peptide used in the invention as the hydrophilic active substance is not particularly
limited, but a bioactive protein or a bioactive peptide is preferred. The bioactive protein or the
bioactive peptide include peptide hormone, cytokine, enzyme protein, or antibody. And specific
examples include: GLP-1 receptor antagonist peptide such as Exendin-4, parathyroid hormone (PTH),
calcitonin, insulin, insulin-like growth factor, angiotensin, glucagon, GLP-1; bombesin, motilin,
gastrin, growth hormone, prolactin (luteotropic hormone), gonadotropin (gonadotropic hormone),
thyrotropic hormone, adrenocorticotropic hormone (ACTH), ACTH derivative (ebiratide), melanocyte
stimulating hormone, follicle stimulating hormone (FSH), sermorelin, vasopressin, oxytocin, protirelin,
leuteinizing hormone (LH), corticotropin, secretin, somatropin, thyrotropin (thyroid stimulating
hormone), stomatostatin, gonadotropin releasing hormone (GnRH), G-CSF, erythropoetin (EPO),
thrombopoetin (TPO), megakaryocyte potentiator, HGF, EGF, VEGF, interferon a, interferon P,
interferon y, interleukins, FGF (fibroblast growth factor), BMP (bone marrow proteins), thymic humor
factor (THF), serum thymic factor (FTS), superoxide dimustase (SOD), urokinase, lisozyme, tissue
plasminogen activator, asparakinase, kallikrein, Ghrelin, adiponectin, leptin, atrial sodium diuretic
peptide, atrial sodium diuretic factor, cerebral sodium diuretic peptide (BNP), conantokin G,
dynorphin, endorphin, Kyotorphin, enkephalin, neurotensin, angiostin, bradykinin, substance P, kalidin,
hemoglobin, protein C, VIIa factor, glycocerebrosidase, streptokinase, staphylokinase, thymosin,
pancreozimine, cholecistokinin, human placenta lactogen, tumor necrosis factor (TNF), polymixin B,
cholistine, gramicidin, bacitracin, thymopoetin, bombecin, cerulein, thymostimulin, secretin, resistin,
hepcidin, neuropetide Y, neuropeptide S, cholecistokinine-pancreozimine (CCK-PZ), brain-derived
nutrient factor (BDNF), vaccine, and the like. These bioactive proteins or bioactive peptides may be
natural proteins or peptides, or derivatives modified in part of their sequence, or compounds modified
by polyethylene glycol or sugar chain.
[0047]

When the hydrophilic active substance is DNA, RNA, or modifying nucleic acid, it may be any one of
cationic surfactant, cationic lipid, cationic polymer, or other compounds complexed with the analogs
thereof.
[0048]
In the invention, saccharides used as the hydrophilic active substance include hyaluronic acid, heparin,
dextran sulfate, dextran or FITC labeled dextran (for example, FD40, etc.).
[0049]
The invention also relates to a method for manufacturing the microparticle formed by agglomeration
of the hydrophilic active substance containing particles, the method comprising:
(a) a step of forming a reversed-phase emulsion by mixing an aqueous solvent containing the
hydrophilic active substance and a water-immiscible organic solvent dissolving the amphiphilic
polymer,
(b) a step of obtaining a solid content containing the hydrophilic active substance by removing the
solvent from the reversed-phase emulsion, and
(c) a step of introducing the solid content or a dispersion liquid containing the solid content into a
liquid phase containing the surface modifier.
[0050]
In the method for manufacturing the microparticle formed by agglomeration of the hydrophilic active
substance containing particles of the invention, the reversed-phase emulsion is formed by adding an
aqueous solvent containing the hydrophilic active substance to a water-immiscible organic solvent
dissolving an amphiphilic polymer and mixing them. If necessary, it is possible to use, for example,
an agitating device such as magnetic stirrer, a turbine agitating device, a homogenizer, or a membrane
emulsifying device provided with a porous film. The water-immiscible organic solvent in the
invention is an organic solvent of which solubility in water is 30 g (water-immiscible organic solvent)/

100 ml (water) or less, and other organic solvents of which solubility in water is higher than the
specified value are characterized as water-miscible organic solvents.
[0051]
As the aqueous solution at step (a), water or water solution containing a water-soluble substance is
used. The water-soluble substance may be any one of inorganic salts, saccharides, organic salts,
amino acid, and the like.
[0052]
The property of water immiscible organic solvent at step (a) is not particularly limited, but it is
preferably a solvent capable of dissolving poly(hydroxyl acid) as the hydrophobic segment of the
amphiphilic polymer, and hardly dissolving or not dissolving the hydrophilic segment. The
water-immiscible organic solvent is preferred to be dissipated and removed by freeze-drying or the
like, and is preferred to be 0.1 g (water-immiscible organic solvent)/ 100 ml (water) or less. Specific
examples of the water-immiscible organic solvent include ethyl acetate, isopropyl acetate, butyl
acetate, dimethyl carbonate, diethyl carbonate, methylene chloride, and chloroform. The ratio of the
water-immiscible organic solvent to the aqueous solvent is preferably 1,000: 1 to 1:1, more preferably
100:3 to 3:1. The concentration of the amphiphilic polymer in the water-immiscible organic solvent
varies with the type of the water immiscible organic solvent or the amphiphilic polymer, but is
preferably 0.01 to 90% (w/w), more preferably 0.1 to 50% (w/w), or even more preferably 1 to 20%
(w/w).
[0053]
At step (a), in the process of forming a reversed-phase emulsion by the aqueous solvent containing the
hydrophilic active substance and the water-immiscible organic solvent dissolving the amphiphilic
polymer, depending on the pharmacological purpose, a reversed-phase emulsion may be formed by
using a water-immiscible organic solvent dissolving two or more types of amphiphilic polymer.
[0054]

At step (a), in the process of forming a reversed-phase emulsion by the aqueous solvent containing the
hydrophilic active substance and the water-immiscible organic solvent dissolving the amphiphilic
polymer, in order to assist formation of the reversed-phase emulsion and to form a uniform and fine
reversed-phase emulsion, an assisting agent may be added. Such assisting agent may be preferably a
compound selected from the group consisting of alkyl alcohol having 3 to 6 carbon atoms, alkyl amine
having 3 to 6 carbon atoms, and alkyl carboxylic acid having 3 to 6 carbon atoms. The structure of
alkyl chain of these assisting agents is not specified particularly, and either straight-chain structure or
branched structure may be applicable, or saturated alkyl or non-saturated alkyl may be usable. In the
invention, in particular, tert-butanol, iso-butanol, and pentanol are preferred as the assisting agent.
[0055]
The average particle diameter of the reversed-phase emulsion at step (a) is variable with the particle
diameter of the desired microparticle of the invention, and is not particularly limited, but to
manufacture a microparticle for a pharmaceutical preparation which is one of the applications of the
microparticle of the invention, the upper limit of the average particle diameter is preferably 50 µm,
more preferably 5 µm, even more preferably 500 nm, particularly preferably 150 nm, and most
preferably 100 nm. The lower limit of the average particle diameter of the reversed-phase emulsion
is preferably 10 nm, or more preferably 50 nm.
[0056]
Next, in the manufacturing method of a microparticle, it is important to include step (b) of obtaining a
solid content containing the hydrophilic active substance by removing the solvent from the
reversed-phase emulsion obtained at step (a).
[0057]
At step (b), the method of removing the solvent from the reversed-phase emulsion is not particularly
limited, but may include, for example, heating, in-vacuo drying, dialysis, freeze-drying, centrifugal
operation, filtration, re-sedimentation, and a combination thereof.

[0058]
Among these methods of removing the solvent from the reversed-phase emulsion, freeze-drying is
particularly preferred because it is small in structural changes due to uniting of particles in the
reversed-phase emulsion, and is capable of avoiding degeneration due to high temperature of the
hydrophilic active substance. The condition and the apparatus of freeze-drying include a freezing
process and a drying process at reduced pressure, and the process is particularly preferred to consist of
preliminary freezing step as an ordinary method of freeze-drying, a primary drying step at reduced
pressure and low temperature, and a secondary drying step at reduced pressure. For example, by
cooling and freezing below the melting point of aqueous solution and water immiscible organic
solvent, for composing a reversed-phase emulsion, and then drying at reduced pressure, a freeze-dried
hydrophilic active substance containing solid content is obtained. The temperature of preliminary
freezing may be determined properly by experiment considering from the solvent composition, and is
generally preferred to be -20°C or less. The degree of reduced pressure in the drying process may be
determined properly by experiment considering from the solvent composition, and is generally
preferred to be 3,000 Pa or less, or more preferably 500 Pa or less, for shortening of the drying time.
For freeze-drying, it is preferred to employ a freeze-drying apparatus for laboratory having a cold trap
and connectable to a vacuum pump, or a rack type vacuum freeze-drying apparatus used in
manufacture of pharmaceutical preparations, and after preliminary freezing by using liquid nitrogen or
refrigerant, drying at reduced pressure is executed at cooled temperature or room temperature by using
a vacuum pump or other pressure reducing device.
[0059]
The solid content containing the hydrophilic active substance obtained at step (b) is obtained as an
aggregate of hydrophilic active substance containing particles comprising the amphiphilic polymer,
which aggregate conforms to the structure of the reversed-phase emulsion. Herein, the aggregate is
an irregular mass gathering fine particles by inter-particle force, and is clearly distinguished in shape
from the microparticle of the invention. The average particle diameter of the hydrophilic active

substance containing fine particles for forming this aggregate is variable with the particle diameter of
the desired microparticle of the invention, and is not particularly limited, but to manufacture a
microparticle for a pharmaceutical preparation which is one of the applications of the microparticle of
the invention, the upper limit of the average particle diameter is preferably 50 urn, more preferably 5
µm, most preferably 500 nm, especially 150 nm, particularly 100 nm. The lower limit of the average
particle diameter of the hydrophilic active substance containing fine particles is preferably 10 nm, or
more preferably 50 nm.
[0060]
In the method for manufacturing the microparticle of the invention, it is important to include step (c)
of introducing the solid content containing the hydrophilic active substance or a dispersion liquid
containing the solid content in a liquid phase containing the surface modifier.
[0061]
At step (c), the method of introducing the solid content or the dispersion liquid containing the solid
content in a liquid phase containing the surface modifier includes, for example, a method of adding the
solid content in a liquid phase containing the surface modifier, and a method of dispersing the solid
content once in a dispersion medium, and adding the obtained dispersion liquid (solid-in-oil (S/O)
suspension) in a liquid phase containing the surface modifier.
[0062] When dispersing the solid content containing the hydrophilic active substance once in a
dispersion medium, the dispersion medium is not particularly limited, but is preferably a solvent
capable of dissolving poly(hydroxy acid), but not dissolving substantially the hydrophilic segment
composing the amphiphilic polymer, for the purpose of sustaining the hydrophilic active substance
containing particle structure composed of the amphiphilic polymer having the structure of the
reversed-phase emulsion for composing the hydrophilic active substance containing solid content.
The solvent capable of dissolving poly(hydroxy acid), but not dissolving substantially the hydrophilic
segment is a solvent of which solubility of hydrophilic segment in the solvent is 50 mg/mL or less,
preferably 10 mg/mL or less.

[0063]
The dispersion medium may be either water-immiscible organic solvent or water miscible organic
solvent as far as having the features mentioned above, and the water-immiscible organic solvent is
more preferable. Specific examples of the water-immiscible organic solvent capable of dissolving
poly(hydroxy acid) of amphiphilic polymer, but not dissolving substantially in the hydrophilic
segment include ethyl acetate, isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate,
methylene chloride, chloroform, dioxane, toluene, and xylene.
[0064]
The dispersion medium for dispersing the solid content containing the hydrophilic active substance
may contain various additives soluble in the dispersion medium, for the purpose of controlling the
releasing speed of the hydrophilic active substance due to decomposition or disintegration of the
hydrophilic active substance containing particles, for example, an acidic compound, a basic compound,
an amphiphilic polymer, or a biodegradable polymer.
[0065] The liquid phase at step (c) is preferably capable of dissolving the surface modifier, and is
higher in boiling point than the hydrophilic active substance containing solid content dispersion
medium, and may include any one of aqueous solvent, water-immiscible organic solvent, and water
miscible organic solvent. Herein, the aqueous solvent is water, or water solution containing a water
soluble component, and the water soluble component includes, or example, inorganic salts,
saccharides, organic salts, and amino acids; the water-immiscible organic solvent includes, for
example, silicone oil, sesame oil, soybean oil, corn oil, cotton seed oil, coconut oil, linseed oil, mineral
oil, castor oil, hardened castor oil, liquid paraffin, n-hexane, n-heptane, glycerol, and oleic oil; and the
water miscible organic solvent includes, for example, glycerin, acetone, ethanol , acetic acid,
dipropylene glycol, triethanol amine, and triethylene glycol. In the invention, the liquid phase at step
(c) is preferably an aqueous solvent or a water miscible organic solvent. When the liquid phase is an
aqueous solvent, and the dispersion medium is a water-immiscible organic solvent, the suspension
obtained at step (c) is a so-called solid-in-oil-water (S/O/W) type emulsion, and when the liquid phase

is water-immiscible organic solvent or water miscible organic solvent, and is not miscible in the
dispersion medium, it is a solid-in-oil-in-oil (S/O1/02) type emulsion.
[0066]
The ratio by volume of the liquid phase to the dispersion medium for dispersing the hydrophilic active
substance containing solid content is generally 1,000:1 to 1:1,000, or preferably 100:1 to 1:100.
[0067]
The concentration of the surface modifier in the liquid phase of the invention is variable with the type
of the surface modifier, and is preferably 0.01 to 90% (w/v), more preferably 0.1 to 50% (w/v), or
even more preferably 5 to 10% (w/v).
[0068]
The surface modifier may be bonded to a poly(hydroxy acid) outer layer of the amphiphihc polymer
of the microparticle of the invention, and the bonding amount in this case is preferably 0.0001% to 1%
of the weight of the microparticle.
[0069]
In the liquid phase at step (c), in addition to the surface modifier, various additives may be added
depending on the pharmacological purpose, such as buffer agent, antioxidant, salt, polymer, or sugar.
[0070]
At step (c), it is also preferred to add inorganic salts in the liquid phase. Inorganic salts are preferred
to be alkaline metal salt or alkaline earth metal salt, and sodium chloride is particularly preferable.
The concentration of inorganic salts in the liquid phase is preferably 0 to 1 M, more preferably 10 mM
to 1 M, or even more preferably 10 mM to 100 mM.
[0071]
At step (c), in order to manufacture a microparticle of a smaller particle size, the formed
solid-in-oil-in-water (S/O/W) type emulsion or solid-in-oil-in-oil (S/Ol/02) type emulsion may be

processed by an emulsifying operation. The emulsifying method is not particularly limited as far as a
stable emulsion can be manufactured. For example, the method includes an agitating method, or a
method by using a high-pressure homogenizer, or a high-speed homo-mixer.
[0072]
At step (c), when the dispersion liquid obtained by dispersing the solid content containing the
hydrophilic active substance once in the dispersion medium is added in the liquid phase containing the
surface modifier, by removing the dispersion medium, a desired suspension of the microparticle
formed by agglomeration of the hydrophilic active substance containing particles is obtained. The
method of removing the dispersion medium is not particularly limited, but may include methods of
drying in liquid, dialysis, freeze-drying, centrifugal operation, filtration, and re-sedimentation, and
drying in liquid or freeze-drying may be particularly preferred. At step (c), when an aqueous solvent
is used as the liquid phase, an aqueous dispersant of the microparticle is obtained in this process.
[0073]
By removing the liquid phase from the microparticle dispersant obtained in this process, the
microparticle of the invention can be obtained. The method of removing the liquid phase is not
particularly limited, but may preferably include methods of distilling-away by evaporation, dialysis,
freeze-drying, centrifugal operation, and filtration.
[0074]
Fields of application of the microparticle obtained in the invention are wide, and versatile, and it is
particularly used as a pharmaceutical preparation. When the microparticle of the invention is used as
the pharmaceutical preparation, aside from microparticles, various pharmacological useful additives
may be contained, and usable additives include buffer agent, antioxidant, salt, polymer, or sugar.
[0075]
When the microparticle of the invention is used as a pharmaceutical preparation, the method of
administration includes, for example, oral administration and parental administration, and the parental

administration is preferred. The parental administration includes hypodermic administration,
intramuscular administration, enteric administration, pulmonary administration, local administration
(nose, skin, eye), and body cavity administration, and the hypodermic and intramuscular injections are
preferred in particular. The dose and the number of times of administration of the pharmaceutical
preparation of the invention in the body of the patient may be properly selected depending on the
hydrophilic active substance, route of administration, age and body weight of the patient, or severity
of the symptom, but usually a dose of 0.1 µg to 100 mg, preferably 1 µg to 10 mg is administered per
day per adult person.
EXAMPLES
[0076]
Examples are shown below, but the invention is not limited to the examples described herein.
[0077]
Example 1 Synthesis of dextran-polylactic acid (PLA)
1.1 Synthesis of TMS-dextran (compound 1)
Dextran (NACALAI TESQUE, INC. NAKARAI standard special grade conforming product,
number-average molecular weight: 13000, 5.0 g) was added to formamide (100 ml), and heated to
80°C. In this solution, 1,1,1,3,3,3-hexamethyldisilazane (100 ml) was added by dropping for 20
minutes. After dropping, the solution was stirred for 2 hours at 80°C. After completion of the
reaction, the reaction solution was returned to room temperature, and the solution was separated into
two layers by a dispensing funnel. The upper layer was concentrated at reduced pressure, and
methanol (300 ml) was added, and the obtained solid content was filtered and dried, and TMS-dextran
(11.4 g) was obtained as white solid content.
[0078]

1.2 Synthesis of TMS-dextran-PLA (compound 2)
Compound 1 (0.5 g) and tert-butoxy potassium (35 mg) were dried for 1 hour at reduced pressure, and
tetrahydrofurane (20 ml) was added, and the mixture was stirred for 1 hour at room temperature. In
this solution, tetrahydrofurane (20 ml) solution of (L)-lactide (4.49 g) was dropped, and the mixture
was stirred for 5 minutes. After completion of reaction, the solvent was concentrated at reduced
pressure, and purified by reprecipitation by a chloroform-methanol system, and TMS-dextran-PLA
(1.9 g) was obtained as white solid content.
[0079]
1.3 Synthesis of dextran-PLA (compound 3)
In chloroform (24 ml) solution of compound 2 (1.9 g), methanol (10.8 ml) and 12N hydrochloric acid
(1.2 ml) were added, and stirred for 30 minutes at room temperature. The solvent was distilled away
at reduced pressure, and the residue was dissolved in chloroform (10 ml), and dropped into diethyl
ether cooled to 0°C, and the product was deposited. The deposition matter was filtered away, and
concentrated at reduced pressure, and dextran-PLA (1.6 g) was obtained. The weight-average
molecular weight of this polymer was 48720, and the number-average molecular weight was 43530.
(Measurement by GPC: column Toso TSK-gel α-5000 x 2, DMF system solvent, detector RI, standard
product, pullulan). The average molecular weight of the graft chain of this polymer determined by
1H-NMR measurement was 2300. The number of graft chains was 10 to 12.
[0080]
Example 2 Synthesis of dextran-poly (lactic acid-glycolic acid) (PLGA)
2.1 Synthesis of TMS-dextran-PLGA (compound 4, compound 5, compound 6)
Compound 1 (0.5 g) and tert-butoxy potassium (35 mg) were dried for 1 hour at reduced pressure, and
tetrahydrofurane (10 ml) was added, and the mixture was stirred for 1 hour at room temperature. In
this solution, tetrahydrofurane (15 ml) solution of (DL)-lactide (1.12 g) and glycolide (0.9 g) was
dropped, and the mixture was stirred for 5 minutes. After completion of reaction, the solvent was

concentrated at reduced pressure, and purified by reprecipitation by a chloroform-methanol system,
and TMS-dextran-PLGA (1.96 g) was obtained as white solid content (compound 4). In the same
manner, by the charging amount of (DL)-lactide (0.784 g) and glycolide (0.63 g), compound 5 was
synthesized, and by the charging amount of (DL)-lactide (1.12 g) and glycolide (0.9 g), compound 6
was synthesized.
[0081]
2.2 Synthesis of dextran-PLGA (compound 7, compound 8, compound 9)
In chloroform (14 ml) solution of compound 4 (1.96 g), methanol (6.3 ml) and 12N hydrochloric acid
(0.7 ml) were added, and stirred for 30 minutes at room temperature. The solvent was distilled away
at reduced pressure, and the residue was dissolved in chloroform (10 ml), and dropped into diethyl
ether cooled to 0°C, and the product was deposited. The deposition matter was filtered away, and
concentrated at reduced pressure, and dextran-PLGA (1.25 g) was obtained (compound 7). From
compounds 5 and 6, dextran-PLGA products were obtained as the same manner except that
trifluoroacetic acid was used (compound 8, compound 9). The weight-average molecular weight and
the number-average molecular weight of the polymer of compounds 7 to 9 were determined by GPC
measurement (column Toso TSK-gel α-5000 x 2, DMF system solvent, detector RI, standard product,
pullulan). The average molecular weight of the graft chain and the number of graft chains were
determined by 1H-NMR measurement.
[0082]
As for compound 7, the weight-average molecular weight was 43,820, the number-average molecular
weight was 33,422, the graft chain molecular weight was 1,900, and the number of graft chains was 7
to 10.
[0083]

As for compound 8, the weight-average molecular weight was 94,088, the number-average molecular
weight was 81,250, the graft chain molecular weight was 3,250, and the number of graft chains was
21.
[0084]
As for compound 9, the weight-average molecular weight was 137,695, the number-average molecular
weight was 109,630, the graft chain molecular weight was 6,442, and the number of graft chains was
15.
[0085]
Example 3. Preparation method of microparticles encapsulating human growth hormone (hGH)
5 mg of dextran-polylactic acid (PLA) of example 1 (average molecular weight of dextran is 13,000,
average molecular weight of PLA is 2,300, number of graft chains of PLA is 10 to 12, compound 3) or
dextran-poly (lactic acid-glycolic acid) (PLGA) of example 2 (average molecular weight of dextran:
13,000, average molecular weight of PLGA is 19,000, number of graft chains of PLGA 7 to 10,
compound 7) was dissolved in 100 µl of dimethyl carbonate to prepare a polymer solution of 50
mg/ml. In this polymer solution, 20 µl of tert-butanol was added, and 20 µl of 2 mg/ml hGH
aqueous solution was dropped, and stirred by vortex to prepare a reversed-phase emulsion. This
reversed-phase emulsion was frozen preliminarily, and was freeze-dried by using a freeze-drying
apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of-45°C, and degree of
vacuum of 20 Pa, for 24 hours. The obtained solid content was dispersed in 200 ul of dimethyl
carbonate to prepare an S/O suspension. This S/O suspension was dropped in 2 ml of aqueous
solution containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred and
emulsified in a vortex mixer to prepare S/O/W type emulsion. From this S/O/W type emulsion, the
water-immiscible organic solvent was removed by drying in liquid, and a microparticle dispersion
liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45 °C, and degree of vacuum of 20 Pa, for 24 hours, and

hGH-encapsulating microparticle powder was obtained. The obtained microparticles were observed
by a scanning electron microscope (SEM: HITACHI, S-4800), and the average particle diameter was
calculated, and the average particle diameter of the microparticles was 4.0 µm.
[0086]
Example 4. Measurement of drug encapsulation efficiency of microparticles encapsulating human
growth hormone (hGH)
20 mg of microparticles encapsulating human growth hormone prepared in the method of example 3
by using dextran-PLA (compound 3) or dextran-PLGA (compound 7) polymer was weighed by using
a 1.5 ml Eppendorf tube, and was dissolved in 1 ml of buffer solution A (PBS containing 0.1% bovine
serum albumin, 0.1% Pluronic F-68 (a registered trademark of BASF), and 0.02% sodium azide), and
was centrifuged for 10 minutes at 18,000 x g, and was separated into particles (precipitation) and a
supernatant. The supernatant was collected in other tube, and the particles were suspended again in 1
ml of buffer solution, and the centrifugal operation and the separation into particles and a supernatant
were conducted again in the same conditions. This cleaning operation was repeated once more (total
three times of centrifugal operation), and the human growth hormone concentration of each
supernatant collected by the centrifugal operations was measured by using an ELISA kit
(manufactured by R&D Systems). From the charged amount of hGH at the time of preparation of
particles (particle weight 20 mg), the hGH total amount of three supernatants by centrifugal operations
was subtracted, and the encapsulation efficiency was calculated according to the formula below.
[0087]

[0088]
In dextran-PLA microparticles or dextran-PLGA microparticles, the encapsulation efficiency of hGH
was 92.6% in dextran-PLA microparticles, and 85.7% in dextran-PLGA microparticles, and it was
proved that the protein drug can be encapsulated at a high efficiency in both particles.

[0089]
Example 5. Analysis of in-vitro drug release speed from microparticles encapsulating human growth
hormone (hGH)
The microparticles centrifuged three times in example 4 were suspended and dispersed in 1.2 ml of
buffer solution A. From this solution, a part (40 µl) was transferred into other tube, and was
centrifuged for 10 minutes at 18,000 x g to precipitate the particles, and 30 ul of supernatant was
collected in a different tube (0-hour sample). The remaining particle suspension was put in a 1.5 ml
Eppendorf tube, and was rolled and mixed slowly in an incubator at 37°C, by using a rotator at a speed
of 6 rpm. From this solution, a small portion (40 µl) was dispensed at specific time intervals, and the
supernatant was separated similarly by centrifugal operation. In the supernatant sample collected at
each time, the hGH concentration was measured by using the ELISA kit, and the release amount (%)
was calculated in the formula below.
[0090]

[0091]
Fig. 1 shows time-course changes of drug release from microparticles manufactured by using
dextran-PLA or dextran-PLGA polymer. In both particles, initial burst was hardly observed, and the
drug was released linearly in proportion to the lapse of time, and a favorable profile was observed.
The time required for 50% release of the drug was about 1 month in the dextran-PLA microparticle,
and about 1 week in the dextran-PLGA microparticle, and it was suggested that the release speed can
be controlled by selecting the type of poly (hydroxy acid).
[0092]
Example 6. Preparation method of microparticles encapsulating human insulin

5 mg of dextran-PLA (average molecular weight of dextran is 13,000, average molecular weight of
PLA is 2,300, number of graft chains of PLA is 10 to 12, compound 3) or dextran-PLGA (average
molecular weight of dextran is 13,000, average molecular weight of PLGA is 19,000, number of graft
chains of PLGA 7 to 10, compound 7) was dissolved in 100 µl of dimethyl carbonate to prepare a
polymer solution of 50 mg/ml. In this polymer solution, 20 µl of tert-butanol was added, and 20 µl
of 2 mg/ml human insulin aqueous solution was dropped, and stirred by vortex to prepare a
reversed-phase emulsion. This reversed-phase emulsion was frozen preliminarily by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45°C, and degree of vacuum of 20 Pa, for 24 hours. The obtained solid
content was dispersed in 200 µl of dimethyl carbonate to prepare an S/O suspension. This S/O
suspension was dropped in 2 ml of aqueous solution containing 10% Pluronic F-68 (a registered
trademark of BASF), and was stirred and emulsified in a vortex mixer to prepare an S/O/W type
emulsion. From this S/O/W type emulsion, the water-immiscible organic solvent was removed by
drying in liquid, and a microparticle dispersion liquid was obtained. The microparticle dispersion
liquid was preliminarily frozen by liquid nitrogen, and was freeze-dried by using a freeze-drying
apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of -45 °C, and degree of
vacuum of 20 Pa, for 24 hours, and human insulin-encapsulating microparticle powder was obtained.
The obtained microparticles were observed by a scanning electron microscope (SEM: HITACHI,
S-4800), and the average particle diameter was calculated, and the average particle diameter was 6.4
µm in the microparticles obtained from compound 3, and 5.3 µm in the microparticles obtained from
compound 7.
[0093]
Example 7. Measurement of drug encapsulation efficiency of microparticles encapsulating human
insulin
20 mg of microparticles encapsulating human insulin prepared in the method of example 6 by using
dextran-PLGA (compound 7) polymer was weighed by using a 1.5 ml Eppendorf tube, and was

dissolved in 1 ml of buffer solution A (PBS containing 0.1% bovine serum albumin, 0.1% Pluronic
F-68 (a registered trademark of BASF), and 0.02% sodium azide), and was centrifuged for 10 minutes
at 18,800 x g, and was separated into particles (precipitation) and a supernatant. The supernatant was
collected in other tube, and the particles were suspended again in 1 ml of buffer solution, and the
centrifugal operation and the separation into particles and a supernatant were conducted again in the
same conditions. This cleaning operation was repeated once more (total three times of centrifugal
operation), and the human insulin concentration of each supernatant collected by the centrifugal
operations was measured by sandwich ELISA method. From the charged amount of human insulin at
the time of preparation of particles (per particle weight 20 mg), the human insulin total amount of
three supernatants by centrifugal operations was subtracted, and the encapsulation efficiency was
calculated according to the formula below.
[0094]

[0095]
In dextran-PLA microparticles or dextran-PLGA microparticles, the encapsulation efficiency of human
insulin was 75.7%, and it was proved that the drug can be encapsulated at a high efficiency.
[0096]
Example 8. Analysis of in-vitro drug release speed from microparticles encapsulating human insulin
The microparticles centrifuged three times in example 7 were suspended and dispersed in 1.2 ml of
buffer solution A. From this solution, a part (40 µl) was transferred into other tube, and was
centrifuged for 10 minutes at 18,800 x g to precipitate the particles, and 30 ul of supernatant was
collected in a different tube (0-hour sample). The remaining particle suspension was put in a 1.5 ml
Eppendorf tube, and was rolled and mixed slowly in an incubator at 37°C, by using a rotator at a speed
of 6 rpm. From this solution, a small portion (40 µl) was dispensed at specific time intervals, and the

supernatant was separated similarly by centrifugal operation. In the supernatant sample collected at
each time, the human insulin concentration was measured by the sandwich ELISA method, and the
release amount (%) was calculated in the formula below.
[0097]

[0098]
Fig. 2 shows time-course changes of human insulin release. Initial burst was hardly observed, and
the drug was released linearly in proportion to the lapse of time, and a favorable profile was observed.
The time required for 50% release of the drug was about 6 days.
[0099]
Example 9. Time-course changes of microparticle morphology
5 mg of microparticles encapsulating hGH prepared in example 3 was weighed in an Eppendorf tube,
and dispersed in 1 ml of Milli-Q, and was centrifugally separated for 30 minutes at 13,000 rpm, and
deprived of the supernatant, and dispersed again in 1 ml of Milli-Q, and centrifugally separated, and
the microparticles were cleaned. In the microparticle suspension solution incubated for a specified
time, 1 ml of Milli-Q was added, and the solution was centrifugally separated for 30 minutes at 13,000
rpm, deprived of the supernatant, and dispersed again in 1 ml of Milli-Q, and centrifugally separated,
and the microparticles were cleaned. The microparticles obtained after cleaning were dispersed in
100 ul of Milli-Q, and 3 ul of the microparticle dispersion liquid was dropped on a silicon substrate,
and let stand at room temperature for 10 minutes, and dried for 3 hours in a desiccator. Then, using
an ion sputtering device (HITACHI, E-1030), platinum was deposited on the sample surface
(deposition time 15 seconds), and the microparticle shape and the surface state were observed by a
scanning electron microscope (SEM: HITACHI, S-4800), at an acceleration voltage of 1 kV and a high
probe current.

[0100]
As shown in Fig. 3, right after manufacture, the surface was smooth and spherical, and the particles
were obviously deformed after incubation for 13 days at 37°C, many pores were formed, and it was
proved that the particles were decomposed gradually along with the progress of release of the drug.
[0101]
Comparative example 1
5 mg of polyethylene glycol-poly (epsilon-caprolactone) (average molecular weight of polyethylene
glycol is 5,000, average molecular weight of poly (epsilon-caprolactone) is 37,000) was dissolved in
100 ul of dimethyl carbonate to prepare a polymer solution of 50 mg/ml. In this polymer solution, 20
ul of tert-butanol was added, and 20 ul of 2 mg/ml hGH aqueous solution was dropped, and stirred by
vortex to prepare a reversed-phase emulsion. This reversed-phase emulsion was frozen preliminarily
by liquid nitrogen, and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE
DRYER FD-1000), at trap cooling temperature of-45°C, and degree of vacuum of 20 Pa, for 24 hours.
The obtained solid content was dispersed in 200 ul of dimethyl carbonate to prepare an S/O
suspension. This S/O suspension was dropped in 2 ml of aqueous solution containing 10% Pluronic
F-68 (a registered trademark of BASF), and was stirred and emulsified in a vortex mixer to prepare an
S/O/W type emulsion. From this S/O/W type emulsion, the water-immiscible organic solvent was
removed by drying in liquid, and a microparticle dispersion liquid was obtained. The microparticle
dispersion liquid was preliminarily frozen by liquid nitrogen, and was freeze-dried by using a
freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of-45°C,
and degree of vacuum of 20 Pa, for 24 hours, and hGH-encapsulating microparticle powder was
obtained. The obtained microparticles were observed by a scanning electron microscope (SEM:
HITACHI, S-4800), and the average particle diameter was calculated, and the average particle
diameter of the microparticles was 8.0 µm.
[0102]

5 mg of the prepared microparticle powder encapsulating hGH was weighed by using an Eppendorf
tube, and was dispersed in 1 ml of Milli-Q, and was centrifuged for 30 minutes at 13,000, deprived of
the supernatant, and dispersed again in 1 ml of Milli-Q, and centrifugally separated similarly, and the
microparticles were cleaned. The microparticles obtained after cleaning were dispersed in 100 ul of
Milli-Q, and 5 ul of the microparticle dispersion liquid was dropped on a silicon substrate, and let
stand at room temperature for 10 minutes, and dried for 3 hours in a desiccator. Then, using an ion
sputtering device (HITACHI, E-1030), platinum was deposited on the sample surface (deposition time
15 seconds), and the microparticle shape and the surface state were observed by a scanning electron
microscope (SEM: HITACHI, S-4800), at an acceleration voltage of 1 kV and a high probe current.
[0103]
As shown in Fig. 4, different from the dextran-PLGA microparticle in example 9, after incubation for
21 days at 37°C, the particles were hardly changed morphologically, and there was a problem in
releasing performance of hydrophilic active substance.
[0104]
Example 10. Hypodermic administration of microparticles encapsulating human growth hormone
(hGH) in mouse
25 mg of dextran-polylactic acid (PLA) (average molecular weight of dextran is 13,000, average
molecular weight of PLA is 2,300, number of graft chains of PLA is 10 to 12, compound 3) or
dextran-poly (lactic acid-glycolic acid) (PLGA) (average molecular weight of dextran is 13,000,
average molecular weight of PLGA 19,000, number of graft chains of PLGA is 7 to 10, compound 7)
was dissolved in 500 ul of dimethyl carbonate to prepare a polymer solution of 50 mg/ml. In this
polymer solution, 100 ul of tert-butanol was added, and 250 ul of 10 mg/ml hGH aqueous solution
was dropped, and stirred by vortex to prepare a reversed-phase emulsion. This reversed-phase
emulsion was frozen preliminarily, and was freeze-dried by using a freeze-drying apparatus (EYELA,
FREEZE DRYER FD-1000), at trap cooling temperature of-45°C, and degree of vacuum of 20 Pa, for
24 hours. The obtained solid content was dispersed in 1 ml of dimethyl carbonate to prepare an S/O

suspension. This S/O suspension was dropped in 10 ml of aqueous solution containing 10% Pluronic
F-68 (a registered trademark of BASF), and was stirred and emulsified in a vortex mixer to prepare an
S/O/W type emulsion. From this S/O/W type emulsion, the water-immiscible organic solvent was
removed by drying in liquid, and a microparticle dispersion liquid was obtained. The microparticle
dispersion liquid was preliminarily frozen by liquid nitrogen, and was freeze-dried by using a
freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of-45°C,
and degree of vacuum of 20 Pa, for 24 hours, and hGH-encapsulating microparticle powder was
obtained. The obtained microparticles were observed by a scanning electron microscope (SEM:
HITACHI, S-4800), and the average particle diameter was calculated, and the average particle
diameter was 4.9 µm in the microparticles obtained from compound 3, and 4.2 urn in the
microparticles obtained from compound 7.
[0105]
300 mg of the prepared microparticles was suspended and dispersed in 3 ml of phosphate
physiological buffer solution (PBS), and centrifuged for 5 minutes at 80 x g to precipitate
microparticles, and the supernatant was transferred into other tube. The supernatant was centrifuged
again for 5 minutes at 80 x g to precipitate the remaining particles, and the supernatant was removed.
By re-dispersing in 1 ml of PBS after first time of centrifugal precipitation and second time of
centrifugal precipitation, the same centrifugal cleaning operation was repeated three times, and the
growth hormone not encapsulated in the microparticles were removed. Finally, the precipitation was
dispersed again in 200 µl of PBS, and an administration solution was obtained. The growth hormone
amount encapsulated in dextran-PLA microparticle and dextran-PLGA microcapsule was measured by
an ELISA kit and the concentration in the cleaning solution was determined, and subtracted from the
charged amount, and the amount encapsulated in 300 mg of particles administered per mouse was
determined, and the dextran-PLA microparticle was 590 ug, and the dextran-PLGA microparticles was
536 µg.
[0106]

This solution was injected hypodermically at two positions in the back of 10-week male Balb/C
mouse, and the blood was sampled at specific time intervals from the caudal vein. In the sampled
blood, heparin of final concentration of 3.3IU/ml was added, and plasma was collected by centrifugal
separation for 5 minutes at 5,000 rpm, and the concentration of growth hormone in plasma was
measured by using an ELISA kit.
[0107]
By way of comparison, a non-granulated human growth hormone protein solution (700 ug/0.2 ml) was
hypodermically administered in mouse, and the blood was sampled similarly.
[0108]
In order to suppress antibody production by administration of human growth hormone, which is a
dissimilar protein for mouse, three days before administration of the particle, an immunosuppressant
Tacrolimus hydrate (Astellas) was hypodermically administered by 26 µg/mouse, and thereafter 13
µg/mouse was hypodermically administered at the time of the drug administration, and 3 days and
7days later.
[0109]
Fig. 5 shows time-course changes of concentration of human growth hormone in plasma. In the
mouse administered non-granulated drug, the blood level in 1 hour after administration was very high,
more than 5,000 ng/ml, and then dropped suddenly, to a level before administration in a day. On the
other hand, in the mouse administered the microparticle drug prepared by using dextran-PLA polymer,
a transient elevation of blood level right after administration was suppressed to 200 ng/ml or less, and
for seven consecutive days, the blood level was sustained at high levels. In dextran-PLA
microparticles, transient elevation of concentration after administration was not observed at all, and a
nearly specific blood concentration was maintained for seven days, and an excellent sustained-release
performance was observed.
[0110]

Example 11. Hypodermic administration of microparticles encapsulating human growth hormone
(hGH) in mouse (pharmacological activity evaluation)
2 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (average molecular weight of dextran is
13,000, average molecular weight of PLGA is 1,900, number of graft chains of PLGA is 7 to 10,
compound 7) was dissolved in 500 ul of dimethyl carbonate to prepare a polymer solution of 50
mg/ml. In this polymer solution, 100 ul of tert-butanol was added, and 250 µl of 10 mg/ml hGH
aqueous solution was dropped, and stirred by vortex to prepare a reversed-phase emulsion. This
reversed-phase emulsion was frozen preliminarily by liquid nitrogen, and was freeze-dried by using a
freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of-45°C,
and degree of vacuum of 20 Pa, for 24 hours. The obtained solid content was dispersed in 1 ml of
dimethyl carbonate to prepare an S/O suspension. This S/O suspension was dropped in 10 ml of
aqueous solution containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred and
emulsified in a vortex mixer to prepare an S/O/W type emulsion. From this S/O/W type emulsion,
the water-immiscible organic solvent was removed by drying in liquid, and a microparticle dispersion
liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45°C, and degree of vacuum of 20 Pa, for 24 hours, and
hGH-encapsulating microparticle powder was obtained. The obtained microparticles were observed
by a scanning electron microscope (SEM: HITACHI, S-4800), and the average particle diameter was
calculated, and the average particle diameter of the obtained microparticles was 4.1 µm.
[0111]
300 mg of the prepared microparticles was suspended and dispersed in 3 ml of phosphate
physiological buffer solution (PBS), and particles were precipitated by centrifugal separation for 5
minutes at 80 x g, and a supernatant was transferred in other tube. The supernatant was centrifugally
separated again for 5 minutes at 80 x g, and the remaining particles were precipitated, and the
supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation

were combined, and dispersed again in 1 ml of PBS, and similarly a third centrifugal operation was
conducted, and the growth hormone not encapsulated in the particles was removed. Finally, the
precipitation was dispersed again in 200 µl of PBS to prepare an administration solution.
[0112]
This solution was hypodermically injected in the back of 8-week-old pituitary gland extracted mouse
(from Japan SLC), and the blood was sampled at specific intervals from the caudal vein. In the
sampled blood, heparin of final concentration of 3.3 IU/ml was added, and centrifuged for 5 minutes at
5,000 rpm, and the plasma was collected, and the growth hormone concentration in plasma and the
mouse IGF-1 concentration were measured by ELISA method.
[0113]
By way of comparison, a non-granulated human growth hormone protein solution (700 µg/0.2 ml) was
hypodermically administered in mouse, and the blood was sampled similarly.
[0114]
In order to suppress antibody production by administration of human growth hormone, which is a
foreign protein for mouse, three days before administration of the particle, an immunosuppressant
Tacrolimus hydrate (Astellas) was hypodermically administered by 26 ug/mouse, and thereafter 13
fig/mouse was hypodermically administered at the time of the drug administration, and 3 days and
7days later.
[0115]
Fig. 6 shows time-course changes of concentration of human growth hormone in plasma. In the
mouse administered non-granulated drug, the blood level in 1 hour after administration was very high,
and then dropped suddenly, to a level before administration in two days. On the other hand, in the
dextran-PLGA microparticle, a transient concentration elevation right after administration was
suppressed low, and for ten consecutive days after administration, the concentration in plasma was
sustained at high levels. At this time, the body weight changes of mouse are shown in Fig. 7. In the

mouse administered the growth hormone alone, the body weight increased was suppressed at about
5%, but in the mouse administered the dextran-PLGA microparticles, the body weight increased about
20%.
[0116]
Fig. 8 shows the IGF-1 concentration in plasma. The IGF-1 concentration in plasma is correlated
with the human growth hormone concentration in blood, and in the mouse administered the
dextran-PLGA microparticles, high levels were maintained for ten days after administration.
[0117]
Example 12. Analysis of drug release speed in buffer solution from microparticles encapsulating
Exendin-4 (GLP-1 receptor agonist)
25 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (average molecular weight of dextran is
13,000, average molecular weight of PLGA is 3,250 (compound 8) or 6,442 (compound 9), number of
graft chains of PLGA is 21 (compound 8) or 15 (compound 9) was dissolved in 500 ul of dimethyl
carbonate to prepare a polymer solution of 50 mg/ml. In this polymer solution, 100 ul of tert-butanol
was added, and 250 µ1 of 10 mg/ml Exendin-4 (synthesized by commission with Sigma Genosys) was
dropped, and stirred by vortex to prepare a reversed-phase emulsion. This reversed-phase emulsion
was frozen preliminarily by liquid nitrogen, and was freeze-dried by using a freeze-drying apparatus
(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of-45°C, and degree of vacuum of
20 Pa, for 24 hours. The obtained solid content was dispersed in 1 ml of dimethyl carbonate to
prepare an S/O suspension. This S/O suspension was dropped in 10 ml of aqueous solution
containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred and emulsified in a
vortex mixer to prepare an S/O/W type emulsion. From this S/O/W type emulsion, the
water-immiscible organic solvent was removed by drying in liquid, and a microparticle dispersion
liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45°C, and degree of vacuum of 20 Pa, for 24 hours, and

Exendin-4-encapsulating microparticle powder was obtained. The obtained microparticles were
observed by a scanning electron microscope (SEM: HITACHI, S-4800), and the average particle
diameter was calculated, and the average particle diameter was 4.3 urn in compound 8, and 4.5 µm in
compound 9.
[0118]
These microparticles were cleaned three times according to the method in example 4, and were
suspended and dispersed in 1.2 ml of buffer solution A. From this solution, a part (40 ul) was
transferred into other tube, and was centrifuged for 10 minutes at 18,000 x g to precipitate the particles,
and 30 ul of supernatant was collected in a different tube (0-hour sample). The remaining particle
suspension was put in a 1.5 ml Eppendorf tube, and was rolled and mixed slowly in an incubator at
37°C, by using a rotator at a speed of 6 rpm. From this solution, a small portion (40 ul) was
dispensed at specific time intervals, and the supernatant was separated similarly by centrifugal
operation. In the supernatant sample collected at each time, the Exendin-4 concentration was
measured by the ELISA method, and the release amount (%) was calculated in the formula below.
[0119]

[0120]
Fig. 9 shows time-course changes of drug release from microparticles manufactured by using each
dextran-PLGA polymer. In both microparticles, initial burst was hardly observed, and the drug was
released linearly in proportion to the lapse of time, and a favorable profile was observed.
[0121]
Example 13. Hypodermic administration of microparticles encapsulating Exendin-4 (GLP-1 receptor
agonist) in mouse

300 mg of microparticles in example 12 was suspended and dispersed in 3 ml of phosphate
physiological buffer solution (PBS), and the microparticles were precipitated by centrifugal operation
for 5 minutes at 80 x g, and a supernatant was transferred in other tube. The supernatant was
centrifugally separated again for 5 minutes at 80 x g, and the remaining particles were precipitated,
and the supernatant was removed. The first centrifugal precipitation and the second centrifugal
precipitation were combined, and dispersed again in 1 ml of PBS, and similarly a third centrifugal
operation was conducted, and the Exendin-4 not encapsulated in the particles was removed. Finally,
the precipitation was dispersed again in 200 µl of PBS to prepare an administration solution.
[0122]
This solution was hypodermically injected in the back of 8-week-old SCID mouse
(CB17/lcr-Prkdcscid/CrlCrlk) (from Crea Japan Inc.), and the blood was sampled at specific intervals
from the caudal vein. In the sampled blood, heparin of final concentration of 3.3 IU/ml was added,
and centrifuged for 5 minutes at 5,000 rpm, and the plasma was collected, and the Exendin-4
concentration in plasma was measured by ELISA method. By way of comparison, a non-granulated
Exendin-4 solution (700 µg/0.2 ml) was hypodermically administered in mouse, and the blood was
sampled similarly.
[0123]
Fig. 10 shows time-course changes of concentration of Exendin-4 in plasma. In the mouse
administered non-granulated drug, the blood level in 1 hour after administration was very high, and
then dropped suddenly, to a level before administration. On the other hand, in the dextran-PLGA
microparticle, a transient concentration elevation after administration was suppressed low, and for five
consecutive weeks after administration, the concentration in plasma was sustained at high levels.
[0124]
Example 14. Synthesis of dextran-poly (lactic acid-glycolic acid) (PLGA)
14.1 Synthesis of TMS-dextran-PLGA (compound 10, compound 11, compound 12, compound 13)

Compound 1 (0.5 g) and tert-butoxy potassium (35 mg) were dried for 1 hour at reduced pressure, and
tetrahydrofurane (10 ml) was added, and the mixture was stirred for 1 hour at room temperature. In
this solution, tetrahydrofurane (15 ml) solution of (DL)-lactide (0.558 g) and glycolide (0.45 g) was
dropped, and the mixture was stirred for 5 minutes. After completion of reaction, the solvent was
concentrated at reduced pressure, and purified by reprecipitation by a chloroform-methanol system,
and TMS-dextran-PLGA (1.96 g) was obtained as white solid content (compound 10).
[0125]
In a similar method, by the charging amount of (DL)-lactide (0.67 g) and glycolide (0.54 g),
compound 11 was synthesized.
[0126]
In a similar method, by the charging amount of (DL)-lactide (0.781 g) and glycolide (0.629 g),
compound 12 was synthesized.
[0127]
In a similar method, by the charging amount of (DL)-lactide (1.123 g) and glycolide (0.9 g),
compound 13 was synthesized.
[0128]
14.2 Synthesis of dextran-PLGA (compound 14, compound 15, compound 16, compound 17)
In chloroform solution (10 mL) of compound 10, trifluoroacetic acid (1 mL) was added, and stirred for
30 minutes at room temperature. The solvent was distilled away at reduced pressure, and the residue
was dissolved in chloroform (10 ml), and dropped into diethyl ether cooled to 0°C, and the product
was deposited. The deposition matter was filtered away, and concentrated at reduced pressure, and
dextran-PLGA (0.44 g) was obtained (compound 14).
[0129]

From compounds 11, 12, and 13, dextran-PLGA products were obtained by a similar method
(compound 5, compound 16, compound 17). The weight-average molecular weight and the
number-average molecular weight of the polymer of compounds 14 to 17 were determined by GPC
measurement (column Toso TSK-gel a-5000 x 2, DMF system solvent, detector RI, standard product,
pullulan). The average molecular weight of the graft chain and the number of graft chains were
determined by 'H-NMR measurement.
[0130]
As for compound 14, the weight-average molecular weight was 99,462, the number-average molecular
weight was 85,101, the graft chain number-average molecular weight was 2,167, and the number of
graft chains was 33.
[0131]
As for compound 15, the weight-average molecular weight was 107,779, the number-average
molecular weight was 92,134, the graft chain number-average molecular weight was 3,127, and the
number of graft chains was 25.
[0132]
As for compound 16, the weight-average molecular weight was 121,281, the number-average
molecular weight was 101,873, the graft chain number-average molecular weight was 3,000, and the
number of graft chains was 30.
[0133]
As for compound 17, the weight-average molecular weight was 144,838, the number-average
molecular weight was 122,151, the graft chain number-average molecular weight was 4,864, and the
number of graft chains was 22.
[0134]
Example 15. Preparation method of microparticles encapsulating human growth hormone (hGH)

5 mg of each dextran-poly (lactic acid-glycolic acid) (dextran-PLGA polymer, compounds 14 to 17) of
example 14 was dissolved in 100 ul of dimethyl carbonate to prepare a polymer solution of 50 mg/ml.
In this polymer solution, 20 ul of tert-butanol was added, and 50 ul of 1 mg/ml hGH aqueous solution
was dropped, and stirred by vortex to prepare a reversed-phase emulsion. This reversed-phase
emulsion was frozen preliminarily by liquid nitrogen, and was freeze-dried by using a freeze-drying
apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of -45 °C, and degree of
vacuum of 20 Pa, for 24 hours. The obtained solid content was dispersed in 200 ul of dimethyl
carbonate to prepare an S/O suspension. This S/O suspension was dropped in 2 ml of aqueous
solution containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred and
emulsified in a vortex mixer to prepare an S/O/W type emulsion. From this S/O/W type emulsion,
the water-immiscible organic solvent was removed by drying in liquid, and a microparticle dispersion
liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45 °C, and degree of vacuum of 20 Pa, for 24 hours, and
hGH-encapsulating microparticle powder was obtained. The obtained microparticles were observed
by a scanning electron microscope (SEM: HITACHI, S-4800), and the average particle diameter was
calculated, and the average particle diameter of the microparticles was within a range of 1.0 to 10 µm.
[0135]
Example 16. Measurement of drug encapsulation efficiency of microparticles encapsulating human
growth hormone (hGH)
20 mg of microparticles encapsulating human growth hormone prepared in the method of example 15
by using each dextran-PLGA polymer (compounds 14 to 17) was weighed by using a 1.5 ml
Eppendorf tube, and was dissolved in 1 ml of buffer solution A (PBS containing 0.1% bovine serum
albumin, 0.1% Pluronic F-68 (a registered trademark of BASF), and 0.02% sodium azide), and was
centrifuged for 10 minutes at 18,000 x g, and was separated into particles (precipitation) and a
supernatant. The supernatant was collected in other tube, and the particles were suspended again in 1

ml of buffer solution, and the centrifugal operation and the separation into particles and a supernatant
were conducted again in the same conditions. This cleaning operation was repeated once more (total
three times of centrifugal operation), and the human growth hormone concentration of each
supernatant collected by the centrifugal operations was measured by using an ELISA kit
(manufactured by R&D Systems). From the charged amount of hGH at the time of preparation of
particles (particle weight 20 mg), the hGH total amount of three supernatants by centrifugal operations
was subtracted, and the encapsulation efficiency was calculated according to the formula below.
[0136]

[0137]
In dextran-PLGA microparticles, the encapsulation efficiency of hGH was 87.5% in microparticles of
compound 14, 94.2% in microparticles of compound 15, 95.7% in microparticles of compound 16, and
97.5% in microparticles of compound 17, and it was proved that the protein drug can be encapsulated
at a high efficiency in all microparticles.
[0138]
Comparative example 2
Manufacture of particles encapsulating growth hormone and measurement of drug encapsulation
efficiency
10 mg dextran-poly (lactic acid-glycolic acid)(PLGA) (compound 14 or compound 17) was dissolved
in 2 mL of ethyl acetate to prepare a polymer solution. In this polymer solution, 100 uL of 0.5
mg/mL hGH aqueous solution was dropped, and stirred. After stirring operation, the solution was
added to 20 mL of dioxane. The solvent was evaporated, and the solution was concentrated to about
2 mL, and the particle dispersion liquid was added to water containing 500 mg Pluronic F-68 (a

registered trademark of BASF). The sample was freeze-dried, and 1 mL of water is added to 50 mg
of the sample, and the particles were dispersed again, and non-associated hydrophilic active substance
containing particles were obtained. The average particle diameter of the particles was measured by a
dynamic light scatter method by using an apparatus ELS-Z (manufactured by Otsuka Denshi), and the
drug encapsulation efficiency was determined same as in example 16.
[0139]
As a result, in the particles of compound 14, the average particle diameter was 190.5 nm, and the
encapsulation efficiency was 73%, and in the particles of compound 17, the average particle diameter
was 197.5 nm, and the encapsulation efficiency was 70%, and the encapsulation efficiency was lower
than in the microparticles of example 16.
[0140]
Example 17. Analysis of in-vitro drug release speed from microparticles encapsulating human
growth hormone (hGH)
Particles cleaned three times in example 16 were suspended and dispersed in 1.2 ml of buffer solution
A. From this solution, a part (40 µl) was transferred into other tube, and was centrifuged for 10
minutes at 18,000 x g to precipitate the particles, and 30 µl of supernatant was collected in a different
tube (0-hour sample). The remaining particle suspension was put in a 1.5 ml Eppendorf tube, and
was rolled and mixed slowly in an incubator at 37°C, by using a rotator at a speed of 6 rpm. From
this solution, a small portion (40 µl) was dispensed at specific time intervals, and the supernatant was
separated similarly by centrifugal operation. In the supernatant sample collected at each time, the
hGH concentration was measured by the ELISA kit, and the release amount (%) was calculated in the
formula below.
[0141]



[0142]
Fig. 11 shows time-course changes of drug release from microparticles manufactured in example 15.
In these microparticles, initial burst was hardly observed, and the drug was released linearly in
proportion to the lapse of time, and a favorable profile was observed. The time required for 50%
release of the drug was about 6 days in microparticles of compound 14, about 9 days in microparticles
of compound 15, about 16 days in microparticles of compound 16, and about 1 month in
microparticles of compound 17, and it was suggested that the release speed could be controlled by
changing the charged amount of lactide and glycolide at the time of synthesis of TMS-dextran-PLGA.
[0143]
Example 18. Preparation method of microparticles encapsulating fluoresceine labeled dextran
(FD40) different in particle diameter
5 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (compound 7) of example 2 was dissolved in
100 µl of dimethyl carbonate to prepare a polymer solution of 50 mg/ml. In this polymer solution, 20
µl of tert-butanol was added, and 20 µl of 1 mg/ml FD40 aqueous solution was dropped, and stirred by
vortex to prepare a reversed-phase emulsion. This reversed-phase emulsion was frozen preliminarily
by liquid nitrogen, and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE
DRYER FD-1000), at trap cooling temperature of -45°C, and degree of vacuum of 20 Pa, for 24 hours.
The obtained solid content was dispersed in 50 ul, 100 ul, 200 ul, 350 ul, 500 ul, 1 ml, 2 ml, and 6 ml
of dimethyl carbonate to prepare an S/O suspension. This S/O suspension was dropped in 2 ml of
aqueous solution containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred and
emulsified in a vortex mixer to prepare an S/O/W type emulsion. From this S/O/W type emulsion,
the water immiscible organic solvent was removed by drying in liquid, and a microparticle dispersion
liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45°C, and degree of vacuum of 20 Pa, for 24 hours, and FD40

-encapsulating microparticle powder was obtained. The obtained microparticles were observed by a
scanning electron microscope (SEM: HITACHI, S-4800), and the average particle diameter was
calculated.
[0144]
Fig. 12 shows the correlation between the average particle diameter and the amount of dimethyl
carbonate added at the time of preparation of S/O/W type emulsion. In a range from 50 µl to 500 µl,
along with increase of dimethyl carbonate amount, decline of the average particle diameter was
observed. From 500 ul to 6 ml, almost no difference was observed in the average particle diameter.
[0145]
Example 19. Synthesis of PEG-PLGA polymer (PEG2k series)
Polyethylene glycol monomethyl ether (manufactured by NOF Corp., SUNBRIGHT MEH-20H,
number-average molecular weight: 1,862, Mw/Mn = 1.03), (DL)-lactide, and glycolide were mixed in
the specified composition shown in Table 1, and heated at 140°C. After stirring for 20 minutes, tin
octylate (II) was added (by 0.05 wt.% to polyethylene glycol monomethyl ether), and stirred for 3
hours at 180°C. The reaction solution was returned to room temperature, and was dissolved in
chloroform (to a concentration of about 100 mg/ml), and precipitated again and refined in diethyl ether
cooled at 0°C, and the obtained solid content was filtered, decompressed, and dried, and PEG-PLGA
polymer was obtained as white or pale brown solid content. The number-average molecular weight
of this polymer was determined by 'H-NMR (Table 1).
[0146]
[Table 1]
Table 1. Raw material charged amount and reaction results of synthesis of PEG-PLGA polymer
(PEG2k series)

[0147]
Example 20. Synthesis of PEG-PLGA polymer (PEG5k series)
Polyethylene glycol monomethyl ether (manufactured by NOF Corp., SUNBRIGHT MEH-20H,
number-average molecular weight: 5,128, Mw/Mn = 1.02), (DL)-lactide, and glycolide were mixed in
the specified composition shown in Table 2, and heated at 140°C. After stirring for 20 minutes, tin
octylate (II) was added (by 0.05 wt.% to polyethylene glycol monomethyl ether), and stirred for 3
hours at 180°C. The reaction solution was returned to room temperature, and was dissolved in
chloroform (to a concentration of about 100 mg/ml), and precipitated again and refined in diethyl ether
cooled at 0°C, and the obtained solid content was filtered, decompressed, and dried, and PEG-PLGA
polymer was obtained as white or pale brown solid content. The number-average molecular weight
of this polymer was determined by 1H-NMR (Table 2).
[0148]
[Table 2]
Table 2. Raw material charged amount and reaction results of synthesis of PEG-PLGA polymer
(PEG5k series)


[0149]
Example 21. Synthesis of PEG-PLGA polymer (PEG10k series)
Polyethylene glycol monomethyl ether (manufactured by NOF Corp., SUNBRIGHT MEH-10H,
number-average molecular weight: 9,975, Mw/Mn = 1.02), (DL)-lactide, and glycolide were mixed in
the specified composition shown in Table 3, and heated at 140°C. After stirring for 20 minutes, tin
octylate (II) was added (by 0.05 wt.% to polyethylene glycol monomethyl ether), and stirred for 3
hours at 180°C. The reaction solution was returned to room temperature, and was dissolved in
chloroform (to a concentration of about 100 mg/ml), and precipitated again and refined in diethyl ether
cooled at 0°C, and the obtained solid content was filtered, decompressed, and dried, and PEG-PLGA
polymer was obtained as white or pale brown solid content. The number-average molecular weight
of this polymer was determined by 1H-NMR (Table 3).

[0150]
[Table 3]
Table 3. Raw material charged amount and reaction results of synthesis of PEG-PLGA polymer
(PEG 10k series)

[0151]
Example 22. Preparation method of FD40-encapsulating microparticles
5 mg of PEG-PLGA polymer prepared in examples 19 to 21 was dissolved in 100 ul of dimethyl
carbonate to prepare a polymer solution of 50 mg/ml. In this polymer solution, 20 ul of tert-butanol
was added, a specified amount of 10 mg/ml FD40 aqueous solution as shown in Table 4 was added,
and stirred to prepare a reversed-phase emulsion. This reversed-phase emulsion was frozen
preliminarily by liquid nitrogen, and was freeze-dried by using a freeze-drying apparatus (EYELA,
FREEZE DRYER FD-1000), at trap cooling temperature of-45°C, and degree of vacuum of 20 Pa, for
24 hours. The obtained solid content was dispersed in 200 ul of dimethyl carbonate to prepare an
S/O suspension. This S/O suspension was dropped in 2 ml of aqueous solution containing 10%
Pluronic F-68 (a registered trademark of BASF), and was stirred and emulsified in a vortex mixer to
prepare an S/O/W type emulsion. From this S/O/W type emulsion, the water-immiscible organic
solvent was removed by drying in liquid, and a microparticle dispersion liquid was obtained. The
microparticle dispersion liquid was preliminarily frozen by liquid nitrogen, and was freeze-dried by

using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of
-45°C, and degree of vacuum of 20 Pa, for 24 hours, and FD40-encapsulating microparticle powder
was obtained, and a part thereof was observed by a scanning electron microscope (SEM: HITACHI,
S-4800), and the average particle diameter was calculated (Table 4). SEM images of the powder
prepared from the PEG-PLGA polymer of 5k to 10k are shown in Fig. 14, and SEM images of the
powder prepared from the PEG-PLGA polymer of 5k to 61k are shown in Fig. 15.
[0152]
[Table 4]


[0153]
Example 23. Measurement of encapsulation efficiency of FD40-encapsulating microparticles
Microparticle (5 mg) encapsulating FD40 prepared in the method of example 22 by using the
PEG-PLGA polymer wasweighed by using a 1.5 ml Eppendorf tube, and was dispersed in Milli-Q (1
ml), and centrifuged for 30 minutes, and separated into a supernatant containing non-encapsulated
FD40 and FD40-encapsulating particles, and collected. The collected FD40-encapsulating particles
were dissolved in N,N-dimethyl formamide (250 ul), and the particles were disintegrated. The
supernatant containing non-encapsulated FD40 and N,N-dimethyl formamide solution (50 ul)
containing encapsulated FD40 were added to Milli-Q (3 ml) individually, and stirred well, and FD40
was quantitatively determined by using a fluorescent spectrophotometer (HORIBA, Fluoro MAX-3,
excitation wavelength 495 nm, fluorescent wavelength 520 nm), and the encapsulation efficiency in
the whole collection volume was calculated.
[0154]
Fig. 13 shows the encapsulation efficiency of FD40 in microparticles prepared from PEG-PLGA
polymer. In all series of 2k, 5k, 10k of molecular weight of PEG, when the molecular weight of PLG
was high, the encapsulation efficiency tended to be high. In particular, in the PEG5k series, at
5k-65k, the encapsulation efficiency was very high, being about 90%. The encapsulation efficiency
was about 55% in 10k-95k (PLGA/PEG = 9.5) nearly at a same molecular weight ratio as 5k-47k
*OKGA/PEG = 9.4) of high encapsulation efficiency (about 80%), the encapsulation efficiency was
about 55%.
[0155]
Comparative example 3. Manufacture of particles encapsulating FD40
10 mg of PEG-PLGA polymer (5k-61k) was dissolved in 2 mL of ethyl acetate to prepare a polymer
solution. In this polymer solution, 100 µL of 2 mg/mL growth hormone solution was dropped, and

stirred. After stirring operation, the solution was added to 20 mL of dioxane. The solvent was
evaporated, and concentrated to about 2 mL, and the particle dispersion liquid was added to water
containing 500 mg Pluronic F-68 (a registered trademark of BASF). The sample was freeze-dried,
and 1 mL of water is added to 50 mg of the sample, and the particles were dispersed again, and
non-associated hydrophilic active substance containing particles were obtained. The average particle
diameter of the particles was measured by a dynamic light scatter method by using an apparatus
ELS-Z (manufactured by Otsuka Denshi), and the drug encapsulation efficiency was determined same
as in example 23.
[0156]
As a result, the encapsulation efficiency of FD40 was 48%, the average particle diameter was 203.8
nm, and the encapsulation efficiency was lower than in the microparticles of example 23.
[0157]
Example 24. Analysis of in-vitro FD40 release speed from microparticles encapsulating FD40
In order to evaluate the relation between the sustained-release behavior and the length of PLGA chain
for composing the PEG-PLGA polymer particle, release behavior was evaluated in particles of 5k-23k,
5k-32.5k, 5k-47k, and 5k-61k, out of the microparticles encapsulating the FD40 prepared in example
22.
[0158]
Microparticles were, right after preparation, stored in freeze-dried state at -30°C, and returned to
normal temperature before use. Exactly 20 mg of particle powder was weighed, and put in a 1.5 ml
tube (Eppendorf tube), and 1 ml of assay buffer was added (0.02% sodium azide, 0.1% Pluronic F-68
(a registered trademark of BASF), and 0.1% bovine serum albumin added PBS solution), and stirred
firmly by a touch mixer and suspended. Then, using Hitachi high-speed centrifugal machine
(CF16RX), the solution was centrifuged for 10 minutes at 18,900 x g, and 950 |il of supernatant
fraction containing non-encapsulated FD40 was removed, and 950 µl of assay buffer was added again,

and the particles were suspended and centrifuged, and the particle cleaning operation was repeated in a
total of three times.
[0159]
In the particles cleaned three times, 950 ul of assay buffer was added once more, and the particles
were suspended, and 100 µl each was dispensed in a 1.5 ml tube. In each tube, 900 µl of assay buffer
was added to obtain a total solution of 1ml, which was incubated in an incubator at 37°C while being
rotated at 10 rpm by means of a rotator. Each incubated tube was centrifuged sequentially for 10
minutes at 18900 x g, and 950 ul of supernatant was dispended, and stored at 4°C until the time of
measurement of fluorescent intensity.
[0160]
The fluorescent intensity of the sampled solution was measured by using 3 ml disposal cuvette
(KARTELL) and HORIBA Fluoro MAX-3, at excitation wavelength of 494 nm and fluorescent
wavelength of 512 nm, and the sustained-release ratio was determined from the ratio of the amount of
FD40 used in preparation of particles.
[0161]
Fig. 16 shows the release amount of FD40 from the various microparticles determined by the release
evaluation. The axis of abscissas denotes the incubation time, and the axis of ordinates represents the
release ratio to the charged amount. In 5k-23k particles short in the PLGA chain, about 40% of the
charged amount was released within 1 day in the initial period of incubation, and in one month, almost
all amount was released except of the portion of initial burst. By contrast, as the length of the PLGA
chain becomes longer, the initial release amount decreased, and in microparticles of 5k-61k, the
release amount in a first day of initial period of 10% or less.
[0162]
Example 25. Measurement of drug encapsulation efficiency of microparticles encapsulating human
insulin

Using the PEG-PLGA polymer (5k-61k) prepared in example 20, microparticles encapsulating human
insulin were prepared in the same method as in example 22. Obtained microparticles (20 mg) were
weighed by using a 1.5 ml Eppendorf tube, and dissolved in 1 ml of buffer solution A (PBS containing
0.1% bovine serum albumin, 0.1% Pluronic F-68 (a registered trademark of BASF), and 0.02%
sodium azide), and were centrifuged for 10 minutes at 18,800 x g, and separated into particles
(precipitation) and a supernatant. The supernatant was collected in other tube, and the particles were
suspended again in 1 ml of buffer solution A, and the centrifugal operation and the separation into
particles and a supernatant were conducted again in the same conditions. This cleaning operation
was repeated once more (total three times of centrifugal operation), and the human insulin
concentration of each supernatant collected by the centrifugal operations was measured by sandwich
ELISA method.
[0163]
The sandwich ELISA method was conducted in the following procedure. Anti-human insulin
monoclonal antibody (manufactured by Fitzgerald, clone No. E6E5) was immobilized on an ELISA
plate (Maxisorp of Nunc Corp.) at concentration of 5 µg/ml, and 50 uL of ELISA buffer solution (0.1
M Tris chlorate buffer solution containing 0.25% BSA and 0.05% Tween 20, pH 8.0) and 50 uL of
measurement sample or standard sample diluted in ELISA diluting solution (PBS containing 0.25%
BSA and 0.05% Tween 20) were added, and the solution was reacted at room temperature by shaking
for 1 hour. The plate was cleaned three times in a cleaning solution (PBS containing 0.05% Tween
20), and the unreacted reagent was removed, and 0.5 µg/ml of biotin-labeled antihuman monoclonal
antibody (manufactured by Fitzgerald, clone No. D4B8), and strepto-avidin-HRP conjugate
(manufactured by Zymed) were added, and the solutions were reacted at room temperature by shaking
for 1 hour and 15 minutes. After each reaction, the plate was cleaned three times in a cleaning
solution (PBS containing 0.05% Tween 20), and the unreacted reagent was removed. Finally, the
substrate of HRP was added, and the HRP enzyme activity of the combined conjugate was determined

by colorimetry, and on the basis of the working curve prepared from color development of standard
insulin, the insulin concentration in the sample was determined.
[0164]
From the charged amount of human insulin at the time of preparation of particles (per particle weight
20 mg), the human insulin total amount of three supernatants by centrifugal operations was subtracted,
and the encapsulation efficiency was calculated according to the formula below.
[0165]

[0166]
The average particle diameter of the obtained microparticles was 4.7 urn. The encapsulation
efficiency of human insulin in microparticles was 86.75, and it was proved that the protein drug could
be contained at a high efficiency.
[0167]
Example 26. Analysis of in-vitro drug release speed from microparticles encapsulating human
insulin
The microparticles centrifuged three times in example 25 were suspended and dispersed in 1.0 ml of
buffer solution A. From this solution, 0.1 ml each was dispensed in ten Eppendorf tubes (1.5 ml
capacity), and 0.9 ml of buffer solution A was added in each tube, and diluted 10 times. Right after
dilution, one tube was centrifuged for 10 minutes at 18,800 x g to precipitate the particles, and a
supernatant was collected in a different tube (0-hour sample). The remaining nine tubes were rolled
and mixed slowly in an incubator at 37°C, by using a rotator at a speed of 6 rpm. At specific time
intervals, each tube was similarly centrifuged, and the supernatant was separated. In the supernatant
sample collected at each time, the insulin concentration was measured by the sandwich ELISA method,
and the insulin release amount (%) was calculated in the formula below.

[0168]

[0169]
Fig. 17 shows time-course changes of insulin release. Along with the lapse of time, the drug was
released gradually, and the release speed increased after 30 days, and the majority of the drug was
released in about 60 days.
[0170]
Example 27. Hypodermic administration of microparticles encapsulating human growth hormone
(hGH) in mouse
25 mg of PEG-PLGA polymer was dissolved in 500 ul of dimethyl carbonate to prepare a polymer
solution of 50 mg/ml. In this polymer solution, 100 ul of tert-butanol was added, and 250 ul of 10
mg/ml hGH aqueous solution was dropped, and stirred by vortex to prepare a reversed-phase emulsion.
This reversed-phase emulsion was frozen preliminarily by liquid nitrogen, and was freeze-dried by
using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of
-45°C, and degree of vacuum of 20 Pa, for 24 hours. The obtained solid content was dispersed in 1
ml of dimethyl carbonate to prepare an S/O suspension. This S/O suspension was dropped in 10 ml
of aqueous solution containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred
and emulsified in a vortex mixer to prepare an S/O/W type emulsion. From this S/O/W type
emulsion, the water-immiscible organic solvent was removed by drying in liquid, and a microparticle
dispersion liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by
liquid nitrogen, and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER
FD-1000), at trap cooling temperature of -45 °C, and degree of vacuum of 20 Pa, for 24 hours, and
hGH-encapsulating microparticle powder was obtained. The average particle diameter of the
obtained microparticles was 6.0 µm.

[0171]
300 mg of the prepared microparticles was suspended and dispersed in 3 ml of phosphate
physiological buffer solution (PBS), and centrifuged for 5 minutes at 80 x g to precipitate
microparticles, and a supernatant was transferred into other tube. The supernatant was centrifuged
again for 5 minutes at 80 x g to precipitate the remaining particles, and the supernatant was removed.
The first centrifugal precipitation and the second centrifugal precipitation were combined, and
dispersed again in 1 ml of PBS, and the same centrifugal cleaning operation was repeated three times
in total, and the growth hormone not encapsulated in the microparticles were removed. Finally, the
precipitation was dispersed again in 200 µl of PBS, and an administration solution was obtained.
The growth hormone amount encapsulated in PEG-PLGA particles was measured by an ELISA kit,
and subtracted from the charged amount, and the amount encapsulated in 300 mg of particles
administered per mouse was determined, and 700 µg of PEG-PLGA microparticles was obtained.
[0172]
This solution was injected hypodermically at two positions in the back of 10-week male Balb/C
mouse, and the blood was sampled at specific time intervals from the caudal vein. In the sampled
blood, heparin of final concentration of 3.3 IU/ml was added, and plasma was collected by centrifugal
separation for 5 minutes at 5,000 rpm, and the concentration of growth hormone in plasma was
measured by using an ELISA kit.
[0173]
By way of comparison, a non-granulated human growth hormone protein solution (700 µg/0.2 ml) was
hypodermically administered in mouse, and the blood was sampled similarly.
[0174]
In order to suppress antibody production by administration of human growth hormone, which is a
foreign protein for mouse, three days before administration of the particle, an immunosuppressant
Tacrolimus hydrate (Astellas) was hypodermically administered by 26 µg/mouse, and thereafter 13

µg/mouse was hypodermically administered at the time of the drug administration, and 3 days and
7days later.
[0175]
Fig. 18 shows time-course changes of concentration of human growth hormone in plasma. In the
mouse administered non-granulated drug, the blood level in 1 hour after administration was very high,
more than 5,000 ng/ml, and then dropped suddenly, to a level before administration in a day. On the
other hand, in the mouse administered the microparticle drug prepared by using PEG-PLGA polymer,
a transient elevation of blood level right after administration was suppressed to 100 ng/ml or less, and
for seven consecutive days, the blood level was sustained at high levels.
[0176]
Example 28. Manufacture of microparticles adding salt to liquid phase in step (c)
In 100 ul of 50 mg/ml PEG-PLGA polymer (5k-61k)/dimethyl carbonate solution, 20 µl of
tert-butanol was added, and 20 µl of 10 mg/ml FD40 aqueous solution was added, and the mixture was
stirred to prepare a reversed micelle (W/O emulsion) solution. The obtained solution was frozen
preliminarily by liquid nitrogen, and was freeze-dried overnight by using a freeze-drying apparatus,
and a solid content containing FD40 was obtained. In the obtained solid content containing FD40,
200 µl of dimethyl carbonate was added, and stirred for 10 second by vortex to prepare an S/O
suspension, and it was dropped in 2 ml of aqueous solution containing 10% Pluronic F-68 (a registered
trademark of BASF) together with sodium chloride at specified concentration (0 M, 10 mM, 50 mM, 1
M), and was stirred and emulsified by vortex for 30 seconds to prepare an S/O/W type emulsion
solution. From the obtained S/O/W type emulsion solution, the water-immiscible organic solvent
was removed by using an evaporator (evacuated to 30 hPa, and evacuated and distilled away for 5
minutes) to prepare a water disperse matter of microparticles containing FD40. The disperse aqueous
solution of microparticles containing FD40 was frozen preliminarily by liquid nitrogen, and was
freeze-dried overnight by using a freeze-drying apparatus, and FD40 containing microparticle powder
was obtained. The obtained microparticles were observed by a scanning electron microscope (SEM:

HITACHI, S-4800), and the average particle diameter was calculated, and in all sodium chloride
concentration conditions, the average particle diameter of microparticles was 6.5 µm.
[0177]
20 mg of the obtained FD40 containing microparticle powder was weighed, and dispersed in 1 ml of
PBS buffer solution (containing 0.1% Pluronic F-68 (a registered trademark of BASF), 0.1% BSA,
and 0.01% sodium azide), and centrifuged (14,000 rpm, 10 minutes). After collection of the
supernatant, the microparticles were suspended again in 1 ml of PBS buffer solution, and centrifuged,
and the microparticles were cleaned further two more times. The cleaned microparticles were
suspended again in 1 ml of PBS buffer solution, dispended by 900 µl each in 1.5 ml Eppendorf tubes,
and 900 ul of PBS buffer solution was added, and the solution was incubated at 37°C, and samples
were collected after 24 hours. The collected samples were centrifuged for 10 minutes at 14,000 rpm,
and FD40 contained in the supernatant was measured by using a fluorescent spectrophotometer
(HORIBA, Fluoro MAX-3, excitation wavelength 495 nm, fluorescent wavelength 520 nm), and the
release amount was calculated. The amount of FD40 in the supernatant collected at the time of
cleaning was measured similarly, and the encapsulation efficiency was calculated from the charged
amount.
[0178]
The encapsulation efficiency was 73%, 97%, 84%, and 82% at sodium chloride concentrations of 0 M,
10 mM, 50 mM, and 1 M. The release amount in 1 day was 14%, 7%, 15%, and 11% at sodium
chloride concentrations of 0 M, 10 mM, 50 mM, and 1 M, and at the sodium chloride concentration of
10 mM, the encapsulation efficiency was highest, and the release amount in 1 day (initial burst) was
least.
[0179]
Example 29. Hypodermic administration of microparticles encapsulating human growth hormone
(hGH) in mouse (pharmacological activity evaluation)

25 mg each of PEG-PLGA polymer (5k-55k) and PEG-PLGA polymer (5k-105k) of example 20 was
dissolved in 500 µl of dimethyl carbonate to prepare a polymer solution of 50 mg/ml. In this
polymer solution, 100 µl of tert-butanol was added, and 250 µl of 10 mg/ml hGH aqueous solution
was dropped, and stirred by vortex to prepare a reversed-phase emulsion. This reversed-phase
emulsion was frozen preliminarily by liquid nitrogen, and was freeze-dried by using a freeze-drying
apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of-45°C, and degree of
vacuum of 20 Pa, for 24 hours. The obtained solid content was dispersed in 1 ml of dimethyl
carbonate to prepare an S/O suspension. This S/O suspension was dropped in 10 ml of aqueous
solution containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred and
emulsified in a vortex mixer to prepare an S/O/W type emulsion. From this S/O/W type emulsion,
the water-immiscible organic solvent was removed by drying in liquid, and a microparticle dispersion
liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45°C, and degree of vacuum of 20 Pa, for 24 hours, and
hGH-encapsulating microparticle powder was obtained. The obtained microparticles were observed
by a scanning electron microscope (SEM: HITACHI, S-4800), and the average particle diameter was
calculated, and the average particle diameter of the obtained microparticles was 4.2 µm in the
microparticles from PEG-PLGA polymer (5k-55k) (5k-55k microparticles), and 7.5 µm in the
microparticles from PEG-PLGA polymer (5k-105k) (5k-105k microparticles).
[0180]
300 mg each of the microparticles prepared above was suspended and dispersed in 3 ml of phosphate
physiological buffer solution (PBS), and particles were precipitated by centrifugal separation for 5
minutes at 80 x g, and a supernatant was transferred in other tube. The supernatant was centrifugally
separated again for 5 minutes at 80 x g, and the remaining particles were precipitated, and the
supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation
were combined, and dispersed again in 1 ml of PBS, and similarly a third centrifugal operation was

conducted, and the growth hormone not encapsulated in the particles was removed. Finally, the
precipitation was dispersed again in 200 µl of PBS to prepare an administration solution.
[0181]
This solution was hypodermically injected in the back of 8-week-old pituitary gland extracted ICR
mouse (from Japan SLC), and the blood was sampled at specific intervals from the caudal vein. In
the sampled blood, heparin of final concentration of 3.3 IU/ml was added, and centrifuged for 5
minutes at 5,000 rpm, and the plasma was collected, and the growth hormone concentration in plasma
and the mouse IGF-1 concentration were measured by ELISA method.
[0182]
By way of comparison, a non-granulated human growth hormone protein solution (700 µg/0.2 ml) was
hypodermically administered in mouse, and the blood was sampled similarly.
[0183]
In order to suppress antibody production by administration of human growth hormone, which is a
foreign protein for mouse, three days before administration of the particle, an immunosuppressant
Tacrolimus hydrate (Astellas) was hypodermically administered by 26 ug/mouse, and thereafter 13
ug/mouse was hypodermically administered at the time of the drug administration, and twice a week
thereafter.
[0184]
Fig. 19 shows time-course changes of concentration of human growth hormone in plasma. In the
mouse administered non-granulated drug, the blood level in 1 hour after administration was very high,
and then dropped suddenly, to a level before administration in one day. On the other hand, in the
mouse administered the microparticle drug manufactured by using PEG-PLGA polymer, a transient
concentration elevation right after administration was suppressed low, about 1/100 of the level in the
mouse administered non-granulated drug, and for more than nine consecutive days after administration,
the blood level was sustained at high levels.

[0185]
Fig. 20 shows the IGF-1 concentration in plasma during this time period. The IGF-1 concentration in
plasma was elevated after administration in both 5k-55k microparticles and 5k-105k microparticles,
and high levels were maintained for 7 days in 5k-55k microparticles, and more than 14 days in
5k-105k microparticles.
[0186]
Example 30. Hypodermic administration of microparticles encapsulating Exendin-4 (GLP-1 receptor
agonist) in mouse
25 mg of PEG-PLGA polymer (5k-61k) in example 20 was dissolved in 500 ul of dimethyl carbonate
to prepare a polymer solution of 50 mg/ml. In this polymer solution, 100 ul of tert-butanol was
added, and 250 ul of 10 mg/ml Exendin-4 (synthesized by commission with Sigma Genosys) was
dropped, and stirred by vortex to prepare a reversed-phase emulsion. This reversed-phase emulsion
was frozen preliminarily by liquid nitrogen, and was freeze-dried by using a freeze-drying apparatus
(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of-45°C, and degree of vacuum of
20 Pa, for 24 hours. The obtained solid content was dispersed in 1 ml of dimethyl carbonate to
prepare an S/O suspension. This S/O suspension was dropped in 10 ml of aqueous solution
containing 10% Pluronic F-68 (a registered trademark of BASF), and was stirred and emulsified in a
vortex mixer to prepare an S/O/W type emulsion. From this S/O/W type emulsion, the
water-immiscible organic solvent was removed by drying in liquid, and a microparticle dispersion
liquid was obtained. The microparticle dispersion liquid was preliminarily frozen by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of -45°C, and degree of vacuum of 20 Pa, for 24 hours, and
Exendin-4-encapsulating microparticle powder was obtained. The obtained microparticles were
observed by a scanning electron microscope (SEM: HITACHI, S-4800), and the average particle
diameter was calculated, and the average particle diameter of microparticles was 6.0 urn.
[0187]

300 mg of the prepared microparticles was suspended and dispersed in 3 ml of phosphate
physiological buffer solution (PBS), and particles were precipitated by centrifugal separation for 5
minutes at 80 x g, and a supernatant was transferred in other tube. The supernatant was centrifugally
separated again for 5 minutes at 80 x g, and the remaining particles were precipitated, and the
supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation
were combined, and dispersed again in 1 ml of PBS, and similarly a third centrifugal operation was
conducted, and the Exendin-4 not encapsulated in the particles was removed. Finally, the
precipitation was dispersed again in 200 µl of PBS to prepare an administration solution.
[0188]
This solution was injected hypodermically at two positions in the back of 10-week male Balb/C
mouse (from Japan SLC), and the blood was sampled at specific time intervals from the caudal vein.
In the sampled blood, heparin of final concentration of 3.3 IU/ml was added, and plasma was collected
by centrifugal separation for 5 minutes at 5,000 rpm, and the concentration of growth hormone in
plasma was measured by the ELISA method.
[0189]
By way of comparison, a non-granulated Exendin-4 solution (700 ug/0.2 ml) was hypodermically
administered in mouse, and the blood was sampled similarly.
[0190]
In order to suppress antibody production by administration of Exendin-4, which is a dissimilar protein
for mouse, three days before administration of the particle, an immunosuppressant Tacrolimus hydrate
(Astellas) was hypodermically administered by 26 µg/mouse, and thereafter 13 µg/mouse was
hypodermically administered at the time of the drug administration, and twice a week thereafter.
[0191]
Fig. 21 shows time-course changes of Exendin-4 concentration in plasma. In the mouse administered
non-granulated drug, the blood level in 1 hour after administration was very high, and then dropped

suddenly, to a level before administration in a day. On the other hand, in the mouse administered the
microparticle drug prepared by using PEG-PLGA polymer, a transient elevation of blood level right
after administration was suppressed to about less than 1/100, and the blood level was sustained at high
levels for a month.
[0192]
Example 31. Preparation of microparticles encapsulating fluoresceine labeled dextran (FD40)
different in particle diameter
5 mg of PEG-PLGA polymer (5k-55k) in example 20 was dissolved in 100 ul of dimethyl carbonate to
prepare a polymer solution of 50 mg/ml. In this polymer solution, 20 ul of tert-butanol was added,
and 20 ul of 1 mg/ml FD40 aqueous solution was dropped, and stirred by vortex to prepare a
reversed-phase emulsion. This reversed-phase emulsion was frozen preliminarily by liquid nitrogen,
and was freeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at
trap cooling temperature of-45°C, and degree of vacuum of 20 Pa, for 24 hours. The obtained solid
content was dispersed in 50 µl, 200 ul, and 500 ul of dimethyl carbonate to prepare an S/O suspension.
This S/O suspension was dropped in 2 ml of aqueous solution containing 10% Pluronic F-68 (a
registered trademark of BASF), and was stirred and emulsified in a vortex mixer to prepare an S/O/W
type emulsion. From this S/O/W type emulsion, the water-immiscible organic solvent was removed
by drying in liquid, and a microparticle dispersion liquid was obtained. The microparticle dispersion
liquid was preliminarily frozen by liquid nitrogen, and was freeze-dried by using a freeze-drying
apparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of -45 °C, and degree of
vacuum of 20 Pa, for 24 hours, and FD40-encapsulating microparticle powder was obtained. The
obtained microparticles were observed by a scanning electron microscope (SEM: HITACHI, S-4800),
and the average particle diameter was calculated.
[0193]
Fig. 22 shows the correlation between the average particle diameter and the amount of dimethyl
carbonate added at the time of preparation of S/O/W type emulsion. In a range from 50 ul to 500 ul,

along with increase of dimethyl carbonate amount, decline of the average particle diameter was
observed.
INDUSTRIAL APPLICABILITY
[0194]
A microparticle of the invention releases a hydrophilic active substance at an appropriate speed in the
human body, and is useful as a DDS pharmaceutical preparation.

We Claim:
[1] A microparticle, comprising an agglomerate of hydrophilic active substance containing particles,
which particle comprises an amphiphilic polymer composed of a hydrophobic segment of
poly(hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol, and a
hydrophilic active substance.
[2] The microparticle according to claim 1, wherein the hydrophilic active substance containing
particle has a hydrophilic segment of amphiphilic polymer in the inside, and has an outer layer of a
hydrophobic segment of amphiphilic polymer.
[3] The microparticle according to claim 1 or 2, wherein the amphiphilic polymer is a graft type
amphiphilic polymer composed of a polysaccharide main chain and poly(hydroxy acid) graft chain(s).
[4] The microparticle according to claim 3, wherein the polysaccharide main chain is dextran.
[5] The microparticle according to claim 1 or 2, wherein the amphiphilic polymer is a block
polymer composed of polyethylene glycol and poly(hydroxy acid).
[6] The microparticle according to claim 5, wherein the average molecular weight of polyethylene
glycol is 2,000 to 15,000.
[7] The microparticle according to claim 5 or 6, wherein the ratio of the average molecular weight
of poly(hydroxy acid) to the average molecular weight of polyethylene glycol is 4 or more.
[8] The microparticle according to any one of claims 1 to 7, wherein the poly(hydroxy acid) is
poly(lactic acid-glycolic acid).
[9] The microparticle according to any one of claims 1 to 8, wherein the average particle diameter
is 1 to 50 µm
[10] The microparticle according to any one of claims 1 to 8, wherein the hydrophilic active
substance is a peptide or a protein.

[11] A method for manufacturing a microparticle comprising:
(a) a step of forming a reversed-phase emulsion by mixing an aqueous solvent containing the
hydrophilic active substance and a water-immiscible organic solvent dissolving an amphiphilic
polymer,
(b) a step of obtaining a solid content containing a hydrophilic active substance by removing the
solvent from the reversed-phase emulsion, and
(c) a step of introducing the solid content or a dispersion liquid containing the solid content into a
liquid phase containing a surface modifier.
[12] The method according to claim 11, wherein the solvent is removed from the reversed-phase
emulsion by a freeze-drying method.
[13] The method according to claim 11 or 12, wherein the dispersion medium of the dispersion liquid
containing the solid content is a solvent capable of dissolving poly(hydroxy acid) and being 10 mg/mL
or less in the solubility of a hydrophilic segment for composing an amphiphilic polymer.
[14] The method according to any one of claims 11 to 13, wherein the liquid phase is either an
aqueous solvent or a water miscible organic solvent.
[15] A pharmaceutical preparation comprising the microparticle of any one of claims 1 to 10.

A microparticle, comprising an agglomerate of a hydrophilic active substance containing particle,
which particle comprises an amphiphilic polymer composed of a hydrophobic segment of poly
(hydroxy acid) and a hydrophilic segment of polysaccharides or polyethylene glycol, and a
hydrophilic active substance, is characterized by an efficient inclusion of the hydrophilic active
substance, and a release of the hydrophilic active substance at an appropriate speed in the human body,
and is hence very useful as a DDS pharmaceutical preparation.