U.S. patent application number 11/748808 was filed with the patent office on 2007-11-15 for water-insoluble medicine.
This patent application is currently assigned to EBARA CORPORATION. Invention is credited to Tetsu Go, Kazuya Hirata, Akio Ishiguro, Hiroyuki KATO, Isao Umeda, Kazuo Watanabe.
Application Number | 20070264350 11/748808 |
Document ID | / |
Family ID | 38685436 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264350 |
Kind Code |
A1 |
KATO; Hiroyuki ; et
al. |
November 15, 2007 |
WATER-INSOLUBLE MEDICINE
Abstract
A water-insoluble medicine in the form of particulates, having
an average particle diameter of 50 to 200 nm; and a particulate
complex of the water-insoluble medicine and a polymer electrolyte,
having an average particle diameter of 50 to 250 nm.
Inventors: |
KATO; Hiroyuki;
(Yokohama-shi, JP) ; Umeda; Isao; (Yokohama-shi,
JP) ; Watanabe; Kazuo; (Tokyo, JP) ; Hirata;
Kazuya; (Kawasaki-shi, JP) ; Ishiguro; Akio;
(Tokyo, JP) ; Go; Tetsu; (Chiba-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
EBARA CORPORATION
Ohta-ku
JP
|
Family ID: |
38685436 |
Appl. No.: |
11/748808 |
Filed: |
May 15, 2007 |
Current U.S.
Class: |
424/489 ;
514/283; 977/906 |
Current CPC
Class: |
A61K 9/146 20130101;
A61K 9/5161 20130101; A61K 31/44 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/489 ;
514/283; 977/906 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/44 20060101 A61K031/44 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 2006 |
JP |
P2006-135677 |
Claims
1. A water-insoluble medicine in the form of particulates, having
an average particle diameter of 50 to 200 nm.
2. The water-insoluble medicine according to claim 1, which has at
least one multiple bond in the structure.
3. The water-insoluble medicine according to claim 1 or 2, which is
an anti-cancer drug.
4. The water-insoluble medicine according to claim 3, wherein said
anti-cancer drug is a camptothecin derivative.
5. The water-insoluble medicine according to claim 3, wherein said
anti-cancer drug is an ellipticine derivative.
6. The water-insoluble medicine according to claim 3, wherein said
anti-cancer drug is a podophyllotoxin derivative.
7. A particulate complex of the water-insoluble medicine of any one
of claims 1 to 6 with a polymer electrolyte, having an average
particle diameter of 50 to 250 mm.
8. The particulate complex according to claim 7, wherein said
polymer electrolyte is at least one member selected from the group
consisting of: biocompatible polymers including protamine, gelatin
A, collagen, albumin, casein, chitosan, poly-(L)-lysine,
carboxymethyl cellulose, alginate, heparin, hyaluronic acid,
chondroitin sulfate, gelatin B, carageenan, dextran sulfate, and
poly-(L)-glutamic acid; biopolymers including biodegradable
polymers, DNA, RNA, enzymes and antibodies; synthesized polymers
including polymethacrylic acid, polydiaryldimethylammonium; and
polymers in which such synthesized polymers are crosslinked with an
appropriate linker.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a fine-particulate,
water-insoluble medicine, and a complex of the same with a polymer
electrolyte. More particularly, the present invention is related to
a medicine in the form of ultrafine particles obtained by
irradiating a laser beam onto particles, and an ultrafine
particle-polymer electrolyte complex.
[0003] Priority is claimed on Japanese Patent Application No.
2006-135677, filed May 15, 2006, the content of which is
incorporated herein by reference.
[0004] 2. Description of Related Art
[0005] Medicines insoluble to solvents, such as anticancer drugs,
are insoluble to water and are hardly absorbed by cells, so that
the bioavailability thereof is low. Therefore, when these
water-insoluble medicines are used for injection, a solubilizer is
often added for the purpose of enhancing the water solubility of
the medicines to thereby improve the bioavailability thereof.
However, this solubilizer has toxicity problems.
[0006] For improving the intake of the water-insoluble medical
drugs by cells without using a solubilizer, the size of the drugs
can be reduced to ultrafine particles which can pass through the
cell membrane of the affected part. The size of ultrafine particles
which can pass through a cell membrane is considered to be 200 nm
or less.
[0007] As an organic substance is expected to exhibit interesting
improvement and changes in properties by size-reduction, various
methods for forming ultrafine particles of an organic compound have
been proposed. For example, a method has been disclosed in which an
organic compound dispersed in a solvent is irradiated with a laser
beam to thereby form ultrafine particles of the organic compound
(for example, see Japanese Unexamined Patent Application, First
Publication No. 2001-113159). In the method disclosed in Japanese
Unexamined Patent Application, First Publication No. 2001-113159,
an organic compound is irradiated with a beam having a wavelength
within the absorption band wavelength, so that thermal stress
cracking is caused by linear optical absorption at a relatively
weak chemical bond within the molecular structure, thereby forming
ultrafine particles. However, simultaneously with the formation of
ultrafine particles, it is highly possible that electronic
excitation occurs in some portions of the organic compound to cause
a photochemical reaction, such that the organic compound
decomposes. Especially when the organic compound is a medical drug
to be administered into a body, there is a danger that the
decomposition product may harmfully affect the body, and hence,
such a serious situation must be avoided.
[0008] For improving the method disclosed in Japanese Unexamined
Patent Application, First Publication No. 2001-113159, a method for
forming ultrafine particles has been proposed in which the organic
compound within the liquid to be treated is irradiated with a laser
beam having a wavelength longer than the absorption band (for
example, see Japanese Unexamined Patent Application, First
Publication No. 2004-267918). Further, a method for forming
ultrafine particles has been proposed in which a bulk crystal of an
organic compound dispersed in a poor solvent is irradiated with a
very short pulsed laser to induce ablation by non-linear
absorption, thereby pulverizing the bulk crystal (for example, see
Japanese Unexamined Patent Application, First Publication No.
2005-238342).
[0009] In these methods, crude particles of an organic compound
dispersed in a solvent within a transparent vessel are externally
irradiated with a laser beam having a wavelength longer than the
absorption band or a very short pulsed laser, thereby pulverizing
the organic compound within the solvent. These methods enable
formation of ultrafine particles of an organic compound under
relatively mild conditions, as compared to the method in which a
beam having a wavelength within the absorption band is linearly
absorbed. Therefore, in these methods, there is less danger of the
organic compound decomposing, and these methods were considered to
be suitable for formation of ultrafine particles of insoluble
organic compounds in small amounts, especially medical drugs.
[0010] However, although the principle of pulverization by laser
beam irradiation is assumed to be thermal stress cracking caused by
short-term heating by pulse energy, the laser energy absorption
properties of the drug and setting of the laser irradiation period
become important parameters for forming ultrafine particles of the
drug without causing deterioration. In a batchwise method, a laser
beam is irradiated onto the drug in a state where the drug is
dispersed in solvent within a vessel, or in a state where the drug
is being stirred in the vessel. Therefore, in a batchwise manner,
it was difficult to control various conditions, such as setting the
laser beam irradiation period and uniformly irradiating the laser
beam onto the dispersed particles. For example, certain particles
are irradiated with the laser beam many times, whereas other
particles are not irradiated at all. Therefore, formation of
ultrafine particles of a drug which have a uniform particle size
within a predetermined range and which are free from deterioration
so as to exhibit high bioavailability, has not been achieved at an
industrial scale.
SUMMARY OF THE INVENTION
[0011] An object of the present invention is to provide a
particulate, water-insoluble medicine which has a uniform particle
size within a predetermined range and which is unchanged in a drug
effect so as to exhibit high bioavailability, and a complex of such
particulate, water-insoluble medicine with a polymer
electrolyte.
[0012] In this situation, the inventors have performed extensive
and intensive studies in view of solving the above-mentioned
problems. As a result, they found that the above-mentioned problems
can be solved by using a technique for forming ultrafine particles
by the laser ablation method, while controlling the conditions for
the laser beam irradiation in detail. Based on this finding, the
present invention has been realized.
[0013] Accordingly, the present invention provides the following
items 1 to 8:
1. A water-insoluble medicine in the form of particulates, having
an average particle diameter of 50 to 200 nm.
2. The water-insoluble medicine according to item 1 above, which
has at least one multiple bond in the structure.
3. The water-insoluble medicine according to item 1 or 2 above,
which is an anti-cancer drug.
4. The water-insoluble medicine according to item 3 above, wherein
the anti-cancer drug is a camptothecin derivative.
5. The water-insoluble medicine according to item 3 above, wherein
the anti-cancer drug is an ellipticine derivative.
6. The water-insoluble medicine according to item 3 above, wherein
the anti-cancer drug is a podophyllotoxin derivative.
7. A particulate complex of the water-insoluble medicine of any one
of items 1 to 6 above with a polymer electrolyte, having an average
particle diameter of 50 to 250 nm
[0014] 8. The particulate complex according to item 7 above,
wherein the polymer electrolyte is at least one member selected
from the group consisting of: biocompatible polymers including
protamine, gelatin A, collagen, albumin, casein, chitosan,
poly-(L)-lysine, carboxymethyl cellulose, alginate, heparin,
hyaluronic acid, chondroitin sulfate, gelatin B, carageenan,
dextran sulfate, and poly-(L)-glutamic acid; biopolymers including
biodegradable polymers, DNA, RNA, enzymes and antibodies;
synthesized polymers including polymethacrylic acid,
polydiaryldimethylammonium; and polymers in which such synthesized
polymers are crosslinked with an appropriate linker.
[0015] The medicine of the present invention in the form of
ultrafine particles, and a complex of the same with a polymer
electrolyte can be manufactured as a colloidal dispersion which is
stable and free from contamination, so that they can be used for
various injectable formulations. Thus, the medicine of the present
invention in the form of ultrafine particles, and a complex of the
same with a polymer electrolyte can be directly injected into a
blood vessel. In an oral administration, only a small amount of the
drug can be delivered to the inside of the body because of the low
absorbability of the drug due to its water insolubility. Further,
in an oral administration, the drug is deteriorated by gastric
juices and enzymes, such that the drug effect is impaired. On the
other hand, the medicine of the present invention is injected into
a blood vessel, and the medicine is transferred at an extremely
high speed, so that the delivery of the medicine from the
administration part to the target part is extremely fast.
[0016] The medicine of the present invention in the form of
ultrafine particles, especially an anti-cancer drug, and a complex
of the same with a polymer electrolyte hardly pass through normal
vascular endothelial cells which have relatively narrow spaces
between the tissues, but are capable of passing through vascular
endothelial cells extending from tumor cells in which the spaces
between the tissues of the vascular endothelial cells are
relatively large, so as to be absorbed by the tumor cells. Thus,
the medicine of the present invention in the form of ultrafine
particles and a complex of the same with a polymer electrolyte
hardly pass through normal vascular endothelial cells during the
delivery thereof to the target part, so that normal cells are not
harmfully affected. Further, the dose of the drug can be suppressed
to a small amount, so that strong side-effect of the anti-cancer
drug can be suppressed.
[0017] In addition, the medicine of the present invention in the
form of ultrafine particles, especially an anti-cancer drug, and a
complex of the same with a polymer electrolyte has a high
probability of being absorbed by tumor cells, as compared to
conventional drugs. Therefore, the medicine and the complex are
hardly affected by individual difference in improvement of
bioavailability and absorption of the drug.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a general view of an apparatus for forming
ultrafine particles in a batchwise manner, which was used for
manufacturing the particulate, water-insoluble medicine and complex
according to the present invention.
[0019] FIG. 2 is a graph showing the pulse width and intensity of
the laser beam generated from the light source 10.
[0020] FIG. 3 is a general view of an apparatus for forming
ultrafine particles in a continuous manner, which was used for
manufacturing the particulate, water-insoluble medicine and complex
according to the present invention.
[0021] FIG. 4 is a schematic diagram showing an expanded,
perspective view of the microflow-channel introductory part 50 and
the microflow channel 60.
[0022] FIG. 5A is a cross-sectional view of the microflow-channel
introductory part 50 and the microflow channel 60.
[0023] FIG. 5B is a cross-sectional view showing the flow rate
distribution of the organic substance passing through the microflow
channel 60.
[0024] FIG. 5C is a cross-sectional view showing an embodiment in
which a transition part 64 is provided between the
microflow-channel introductory part 50 and the microflow channel
60.
[0025] FIG. 6 is a diagram showing one embodiment of a coating part
using a single microflow channel.
[0026] FIG. 7 is a diagram showing one embodiment of a coating part
using a multi-microflow channel.
[0027] FIG. 8 is a line diagram showing a general view of one
embodiment of the apparatus and method for coating particles used
in the present invention, following the flow of the
ultrafine-particle suspension and the polymer electrolyte
solution.
[0028] FIG. 9 is a diagram showing a comparison of absorption
spectra of ethanol solutions prior to and following
irradiation.
[0029] FIG. 10 is a liquid chromatogram of an ethanol solution of
ellipticine following irradiation with a laser beam.
[0030] FIG. 11 is a SEM image of ellipticine prior to
size-reduction.
[0031] FIG. 12 is a SEM image of ultrafine particles of
ellipticine.
[0032] FIG. 13 is a histogram of particle diameter distribution of
ultrafine particles of ellipticine.
[0033] FIG. 14A is a chromatogram of SN-38 prior to laser
irradiation (spreading solvent: ethanol) and results of HPLC
analysis.
[0034] FIG. 14B shows a chromatogram of supernatant of SN-38
suspension following laser irradiation (spreading solvent: ethanol)
and results of HPLC analysis.
[0035] FIG. 15 shows respective SEM images of SN-38 following laser
irradiation and centrifugal separation (2,000 rpm, 10 minutes), and
a histogram of particle diameter distribution (number of samples:
200, average particle diameter: 46 nm, CV value: 22%).
[0036] FIG. 16 is a graph showing changes in zeta potential by
addition of a polymer electrolyte to SN-38 nano particles.
[0037] FIG. 17 is a graph showing the anti-tumor effects of SN-38
nano particles, SN-38 nano particles-protamine sulfate, SN-38 nano
particles-chondroitin sulfate and irinotecan hydrochloride
(CPT-11), using human tumor tissue transplanted into nude mice.
[0038] FIG. 18A is a chromatogram of 10-hydroxy-camptothecin prior
to laser irradiation (spreading solvent: ethanol) and results of
HPLC analysis.
[0039] FIG. 18B shows a chromatogram of supernatant of
10-hydroxy-camptothecin suspension following laser irradiation
(spreading solvent: ethanol) and results of HPLC analysis.
[0040] FIG. 19 shows respective SEM images of
10-hydroxy-camptothecin following laser irradiation and centrifugal
separation (2,000 rpm, 10 minutes), and a histogram of particle
diameter distribution (number of samples: 150, average particle
diameter: 68 nm, CV value: 25%).
[0041] FIG. 20 is a graph showing the anti-tumor effects of
10-hydroxy-camptothecin nano particles and irinotecan hydrochloride
(CPT-11), using human tumor tissue transplanted into nude mice.
REFERENCE NUMERALS
[0042] 1 Vessel [0043] 2 Suspension [0044] 3 Stirrer [0045] 4 Laser
beam source [0046] 5 Laser beam [0047] 10 Light source [0048] 40
Pump (flow device) [0049] 50 Microflow-channel introductory part
[0050] 60 Microflow channel [0051] 64 Transition part [0052] 100
Apparatus for forming ultrafine particles [0053] 120 Polymer
membrane-shell coating part [0054] 122a Microflow channel for
ultrafine-particle suspension [0055] 122b Microflow channel for
polymer electrolyte solution [0056] 122c Merged microflow channel
[0057] 124 Tank for polymer electrolyte solution [0058] 140 Complex
collecting vessel
PREFERRED EMBODIMENTS OF THE PRESENT INVENTION
[0059] In the present invention, particles of a medicine are
size-reduced to ultrafine particles by irradiating with a laser
beam. In the present invention, the medicine to be size-reduced to
ultrafine particles may be a solid powder having an arbitrary size
and shape, such as a synthesized crude powder. However, in view of
easily forming ultrafine particles, enhancing the efficiency of the
formation of ultrafine particles and easily rendering the size of
the ultrafine particles of the medicine uniform, it is preferable
that the particles of the medicine be pulverized to fine particles
having an average particle diameter within a narrow range, for
example, from 1 to 100 .mu.m. The pulverization can be conducted by
any conventional method.
[0060] In the present invention, the medicine to be size-reduced to
ultrafine particles is a water-insoluble, particulate drug. In the
present invention, the term "water-insoluble" refers to the
solubility as prescribed in the Japanese pharmacopoeia, which is
defined as being "extremely hard to dissolve in water" or "hardly
dissolved in water".
[0061] Further, in the present invention, the medicine to be
size-reduced to ultrafine particles preferably has at least one
multiple bond in the structure. The reason for this is that, when
particles of the medicine are irradiated with a laser beam, the
multiple bond portions easily absorb the laser beam, and local
temperature elevation is rapidly caused at the portions where the
beam was absorbed. This temperature elevation occurs instantly
following the irradiation with the laser beam, so that temperature
difference is generated between the portions where the beam was
absorbed and the portions where the beam was not absorbed, and
hence, breaking of the particles occurs. In the present invention,
the term "multiple bond" refers to a conjugated or non-conjugated
double bond or triple bond.
[0062] Further, in the present invention, the medicine to be
size-reduced to ultrafine particles refers to a medical product as
prescribed by the Pharmaceutical Affairs Law, or a candidate
compound for a medicine which has been phased out at the human
clinical trial stage, or which is at a developmental phase in the
human clinical trial. Examples of water-insoluble medicines include
anti-cancer drugs, antifungal drugs, vitamins, painkillers and
anti-inflammatory agents.
[0063] It is particularly desirable that the particulate,
water-insoluble medicine of the present invention be an anti-cancer
drug. The reason for this is as follows. It is considered that the
spaces existing between tissues of vascular endothelial cells
extending from tumor cells are large as 50 nm or more, which is
larger than the spaces existing between tissues of normal vascular
endothelial cells. Therefore, a particulate, water-insoluble
anti-cancer drug having an average particle diameter of 50 to 200
nm can be suited for various injectable formulations, since such
anti-cancer drug can easily pass vascular endothelial cells
extending from tumor cells, but not normal vascular endothelial
cells.
[0064] In the present invention, the term "anti-cancer drug" refers
to a medical product which is prescribed by the Pharmaceutical
Affairs Law and which exhibits anti-cancer activities, or a
candidate compound for a medicine exhibiting anti-cancer
activities, which has been phased out at the human clinical trial
stage, or which is at a developmental phase in the human clinical
trial.
[0065] Examples of anti-cancer drugs usable in the present
invention include camptothecin and derivatives thereof, ellipticine
and derivatives thereof and podophyllotoxin and derivatives
thereof. The general structural formulas of these compounds are
shown below.
[0066] Camptothecin and derivatives thereof having a structure
represented by the structural formula shown below: ##STR1##
[0067] Ellipticine and derivatives thereof having a structure
represented by the structural formula shown below: ##STR2##
[0068] Podophyllotoxin and derivatives thereof having a structure
represented by the structural formula shown below: ##STR3##
[0069] Specific examples of camptothecin derivatives include
4(s)-ethyl-4-hydroxy-1H-pyrano[3',
4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione(camptothecin),
7-ethyl-10-hydroxycamptothecin (SN-38), 9-aminocamptothecin,
9-nitrocamptothecin
5(R)-ethyl-9,10-difluoro-1,4,5,13-tetrahydro-5-hydroxy-3H,15H-oxepino[3',-
4':6,7]indolizino[1,2-b]quinoline-3,15-dione (BN-80915)
[Anti-cancer Drugs (2001), 12(1), 9-[9], and
(9S)-9-ethyl-9-hydroxy-1-pentyl-1H,12H-pyrano[3'',
4'':6',7']indolizino[1',
2':6,5]pyrido[4,3,2-de]quinazoline-10,13(9H, 15H)-dione [Cancer
Chemotherapy and Biotherapy: Principle and Practice, second
edition, Lippincott-Ravenmeans, p. 463-484, (b)Biochim. Biophys.
Acta (1998), 1400(1-3), 107-[1,9], although the camptothecin
derivatives are not limited to these examples.
[0070] Specific examples of ellipticine derivatives include
ellipticine, 9-hydroxy-ellipticine and T-215 (TANABE SEIYAKU Co.
Ltd.), although the ellipticine derivatives are not limited to
these examples.
[0071] Specific examples of podophyllotoxin derivatives exhibiting
anti-cancer activities include podophyllotoxin, etoposide and
teniposide, although the podophyllotoxin derivatives are not
limited to these examples.
[0072] Water or alcoholic solution for dispersing the particulate
drug hardly dissolves the particulate drug to be size-reduced, does
not adversely affect human bodies, and does not absorb laser beams.
Examples of alcohols usable in the present invention include ethyl
alcohol, glycol and glycerol. The alcoholic solution is generally
an aqueous solution of 5% by weight or less of an alcohol.
[0073] The particulate, water-insoluble medicine of the present
invention has an average diameter of 50 to 200 nm. The average
diameter of the particles is a value obtained by measuring the
diameter of each particle using a microscope provided with a scale,
and dividing the sum of the particle diameters by the number of
particles.
[0074] The particulate, water-insoluble medicine is manufactured by
suspending a particulate anti-cancer drug in water or an alcoholic
solution, and irradiating the suspended drug with a laser beam to
form ultrafine particles thereof. More specifically, the
particulate, water-insoluble medicine is manufactured as follows.
Firstly, water or an alcoholic solution is charged into a vessel 1
shown in FIG. 1, and a particulate drug is mixed therewith to form
a suspension 2. The concentration of the suspension 2 varies
depending on the type and size of the particulate drug mixed, but
is generally from 1 to 10 mg/ml. Further, the vessel 1 may have any
shape as long as the face to be irradiated with the laser beam is
planar, but it is preferable that the vessel 1 is substantially
cuboid, and the size of the vessel 1 may be appropriately selected
depending on the amount of the drug to be treated. The material for
the vessel 1 need not be transparent as long as it is capable of
transmitting a laser beam, and any material capable of sustaining
the laser beam irradiation may be used. In general, the material
for the vessel 1 is quartz or glass.
[0075] The particulate medicine of the present invention which is
insoluble in water or the alcoholic solution and which has an
average diameter of 50 to 200 nm tends to agglomerate by the
surface energy thereof. Therefore, the ultrafine particles may be
subjected to an electrostatic interaction or hydrophobic
interaction with a polymer electrolyte having a charge opposite to
the ultrafine particles to form a complex, so as to manufacture the
particulate medicine in the form of a colloidal dispersion which is
stable and free from contamination.
[0076] In the present invention, the term "complex" refers to
ultrafine particles coated with one layer of a polymer electrolyte.
A complex preferably has an average diameter of 50 to 250 nm.
[0077] The manufacture of a complex is influenced by the ultrafine
particles as the core substance, and various conditions such as the
reaction period, concentration of the suspension and pH of the
suspension are determined in detail, depending on the core
substance. Therefore, the manufacture of a complex cannot be
defined in a single uniform way.
[0078] The thus manufactured complex of the particulate medicine
with a polymer electrolyte can be used for various injectable
formulations, and can be directly injected into a blood vessel. In
an oral administration, only a small amount of the drug can be
delivered to the inside of the body because of the low
absorbability of the drug due to its water insolubility. Further,
in an oral administration, the drug is deteriorated by gastric
juices and enzymes, such that the drug effect is impaired. On the
other hand, the medicine of the present invention is injected into
a blood vessel, and the medicine is transferred at an extremely
high speed, so that the delivery of the medicine from the
administration part to the target part is extremely fast.
[0079] The ultrafine particles formed by the laser irradiation tend
to agglomerate due to the surface energy thereof. That is, when the
concentration of the ultrafine particles within the suspension 2 is
too high, agglomeration is likely to occur. Therefore, the
concentration of the drug mixed within the suspension 2 cannot be
rendered too high. For this reason, it is preferable that a polymer
electrolyte having a charge opposite to the ultrafine particles be
added to the suspension 2 prior to irradiation of the laser beam.
In this manner, the ultrafine particles form a complex with the
polymer electrolyte. The complex does not have a surface energy as
high as the ultrafine particles. Therefore, the particles of the
complex do not agglomerate with each other, and are stably
suspended in water or a diluted alcohol. In other words, by adding
a polymer electrolyte to the suspension 2 in advance, the
concentration of the drug mixed with the suspension 2 can be
enhanced, and the amount of the drug to be treated can be
increased. As the polymer electrolyte to be added to the suspension
2 for this purpose, one or more types of polymer electrolytes may
be used. The concentration of the polymer electrolyte to be added
is generally from 1 to 10%.
[0080] The polymer electrolyte usable in the present invention is a
polymer having an ion-dissociable group which is typically a
polymer chain component or a substituent. In general, the number of
the ion-dissociable groups within the polymer electrolyte is a
number such that the polymer following the dissociation of the
ion-dissociable groups becomes water-soluble. In view of this, it
is considered that the polymer electrolyte includes ionomers which
have ion groups with a concentration insufficient to exhibit water
solubility, but has an electric charge sufficient for initiating
self-assembly. The polymer electrolyte is classified into polyacids
and polybase, depending on the type of the ion-dissociable group.
From a polyacid, a polyanion is generated by elimination of proton
upon dissociation, and the polyanion may be an inorganic polymer or
an organic polymer. Examples of polyacids include polyphosphoric
acid, polyvinylsulfuric acid, polyvinylsulfonic acid,
polyvinylphosphonic acid, polyacrylic acid, and salts thereof.
[0081] A polybase includes a group which is capable of taking up
protons for example by formation of salt by reacting with an acid.
Examples of polybases having an ion-dissociable group on the chain
position or side-chain position include polyethyleneimine,
polyvinylamine and polyvinylpyridine. The polybase forms a
polycation by taking up protons.
[0082] Examples of polymer electrolytes suitable for use in the
present invention include biocompatible polymers, biodegradable
polymers, biopolymers and synthesized polymers. A biocompatible
polymer is a polymer which is compatible with biotissues and organ
system without causing toxicity, damage, or rejection. A
biodegradable polymer is a generic term of polymers which are
decomposed in vivo or decomposed by action of microbes, and are
decomposed into water, carbon dioxide, methane and the like by
hydrolysis. A biopolymer is a generic term of polymeric compounds
which are synthesized in vivo.
[0083] Specific examples of biocompatible polymers include
protamine, gelatin A, collagen, albumin, casein, chitosan,
poly-(L)-lysine, carboxymethyl cellulose, alginate, heparin,
hyaluronic acid, chondroitin sulfate, gelatin B, carageenan,
dextran sulfate, and poly-(L)-glutamic acid. Specific examples of
biodegradable polymers include DNA, RNA, enzymes and antibodies.
Specific examples of synthesized polymers include polymethacrylic
acid, polydiaryldimethylammonium, and polymers in which such
synthesized polymers are crosslinked with an appropriate linker.
However, polymer electrolytes are not limited to these
examples.
[0084] The electric charge of the above-mentioned polymer
electrolyte can be changed to a positive charge or negative charge
by varying the pH. Therefore, the polymer electrolyte for use
changes, depending on various conditions.
[0085] The thus prepared suspension 2 is stirred with a stirrer 3,
which is preferably a magnetic stirrer, to thereby uniformly
disperse the drug and the polymer electrolyte.
[0086] The drug dispersed in water or a diluted alcohol is
irradiated with a laser beam 5 generated from a laser source 4,
which has a wavelength within the absorption band. The laser source
4 may be a laser source capable of continuously generating a laser
beam with a substantially constant intensity, or may be a laser
source capable of intermittently generating a laser beam such as a
pulsed laser beam.
[0087] The laser beam generated from the light source 4 may be
selected depending on the absorption wavelength of the drug to be
size-reduced. Examples of the laser beam include an ultraviolet
laser beam, a visible laser beam, a near-infrared laser beam or an
infrared laser beam. Examples of ultraviolet laser beams include
excimer lasers (193 nm, 248 nm, 308 nm, 351 nm), a nitrogen laser
(337 nm), and the third and fourth harmonics of a YAG laser (355
nm, 266 nm). Examples of visible laser beams include the second
harmonic of a YAG laser (532 .mu.m), an Ar ion laser (488 nm or 514
nm), and dye lasers. Examples of near-infrared lasers include
various semiconductor lasers, a titanium-sapphire laser, a YAG
laser and a glass laser. Further, by using any of the
above-exemplified lasers with an optical parametric oscillator, a
light ray having a desired wavelength within the range of
ultraviolet to infrared may be oscillated.
[0088] The laser beam generated from the light source 4 is
preferably a pulsed laser beam. FIG. 2 is a graph showing the pulse
width and intensity of the laser beam generated from the light
source 4. In the graph shown in FIG. 2, the horizontal axis
indicates time, and the vertical axis indicates the excitation
light intensity of the laser beam generated from the light source
4. As shown in FIG. 2, the laser beam generated from the light
source 4 is a pulsed laser beam. That is, the light source 4
generates a laser beam intermittently, so as to alternately repeat
an on-state in which a laser beam is generated and an off-state in
which a laser beam is not generated. It is particularly desirable
to use a laser beam in which the intensity changes in a pulsewise
manner. Hereafter, one pulse of a laser beam is referred to as a
"pulsed beam". When a pulsed laser beam is used, one pulsed beam
effects one irradiation.
[0089] The excitation light intensity P of the laser beam generated
from the light source 4 is preferably from 1 to 1,000 mJ/cm.sup.2,
more preferably 30 to 300 mJ/cm.sup.2. Further, the pulse period T
between a pulsed beam and a subsequent pulsed beam (adjacent pulsed
beams) is preferably from 0.1 to 1,000 Hz. Here, a "pulse period"
means the period from the start of a pulsed beam to the start of a
subsequent (adjacent) pulsed beam, or the period from the end of a
pulsed beam to the end of a subsequent (adjacent) pulsed beam
Furthermore, the pulse width s of respective pulsed beams is
preferably from 10.sup.15 to 10.sup.-6 seconds. Here, a "pulse
width" means the period from the start of a pulsed beam to the end
of the pulsed beam.
[0090] When a pulsed laser beam is used, one irradiation of the
drug is effected by one pulsed beam. In the present specification,
the period during which a laser beam can be irradiated onto a
target drug is referred to as "irradiation period tL". As shown in
FIG. 2, when the irradiation period tL is long, it includes both of
the on- and off-states of the pulsed laser beam. In the off-state,
no laser beam is generated, whereas in the on-state, a laser beam
is generated and irradiated onto a target drug. Thus, even when a
certain period includes an off-state, that period is regarded as
the irradiation period tL if it also includes an on-state in which
a laser beam can be irradiated onto a target drug.
[0091] More specifically, when a target drug is allowed to flow
into the irradiation region of the laser beam, remain in the
irradiation region for a long period, and then come out of the
irradiation region, the drug gets irradiated with a pulsed beam a
plurality of times. That is, when the drug is allowed to flow in
this manner, the above-mentioned irradiation period tL is regarded
as the period during which the target drug is present in the
irradiation region of the laser beam. As described above, ultrafine
particles of the drug can be formed by irradiating the drug with a
laser beam. For forming ultrafine particles having a desired size,
the number of irradiations of the drug with a pulsed beam can be
determined. The number of irradiations with a pulsed beam can be
changed by adjusting the above-mentioned pulse period T, the flow
rate of the drug, etc. Thus, in the irradiation region of the laser
beam, the drug is irradiated with a pulsed beam at least once. The
irradiation region of the laser beam is the region where the laser
beam is irradiated during the on-state.
[0092] As described above, with respect to the irradiation period,
it is preferable that a pulsed beam of a short period in the order
of nano seconds be irradiated a plurality of times. Further, by
changing the above-mentioned pulse width s, the particle diameter
of the ultrafine particles of the drug can be controlled.
[0093] As in the present embodiment, when ultrafine particles are
formed in a batchwise manner using a stirring vessel, the drug is
irradiated with the laser beam a plurality of times while stirring.
For this reason, the drug may be irradiated with the laser beam too
many times, such that the formed ultrafine particles have an
average diameter of less than 50 nm, or that the drug may be
deteriorated. Therefore, the total irradiation period of the laser
beam is an important factor.
[0094] The total irradiation period of the laser beam varies
depending on the stirring rate, the size of the drug, the laser
beam source, the pulse width, the beam intensity, and the like, but
is generally from a few seconds to a few minutes.
[0095] When a polymer electrolyte is not added in advance, it is
necessary that a polymer electrolyte be added immediately after the
stop of the laser beam irradiation to form complexes, thereby
stably suspending the ultrafine particles in water or the diluted
alcohol.
[0096] The thus obtained colloidal solution containing the
complexes may be either diluted with an appropriate solvent or
concentrated, so that it becomes usable as an injection having a
desired concentration.
[0097] Further, when the complexes are desired to be obtained in
the form of a solid, the colloidal solution is passed through a
filter to separate the solid contents, followed by washing, and
optionally drying. Alternatively, the water or diluted alcohol
within the colloidal solution may be vaporized to collect the solid
contents, followed by washing, and optionally drying. In the latter
case, as the solvent, an alcohol, liquid nitrogen or liquid helium
is preferable.
[0098] Hereinabove, explanation has been given of a method for
manufacturing the medicine of the present invention in a batchwise
manner. Next, explanation is given of a method for manufacturing
the medicine of the present invention in a continuous manner.
[0099] FIG. 3 shows an apparatus 100 for forming ultrafine
particles of a drug, which is usable in manufacturing the medicine
of the present invention in a continuous manner.
[0100] As shown in FIG. 3, the supply part 20 is a vessel for
storing a drug suspension which is water or a diluted alcohol
having the drug mixed therein.
[0101] The supply part 20 has a predetermined volume. Further, the
supply part 20 is preferably sealable so that the concentration of
the supplied drug suspension does not change.
[0102] At a lower portion of the supply part 20, a conduit 30 is
connected, and the supply part 20 communicates with the conduit 30.
The drug suspension charged into the supply part 20 can be
discharged to the conduit 30.
[0103] As shown in FIG. 3, the conduit 30 is provided with a pump
40. The pump 40 supplies the drug suspension to the microflow
channel 60 described below. Herein, the term "microflow channel"
means a flow channel which is formed by precise processing and
which has a width of micron order. The pump 40 is capable of
controlling the flow of the drug suspension to a desired flow rate.
Especially when the drug suspension is continuously passed through
the microflow channel 60, it is desirable that the pump 40 be
capable of controlling the flow of the drug suspension to a
constant flow rate. On the other hand, when the drug suspension is
intermittently passed through the microflow channel 60, it is
desirable that the pump 40 be capable of stopping or allowing the
flow of the drug suspension at a desired timing. When the flow of
the drug suspension is stopped, a laser beam generated from the
light source 10 can be reliably irradiated onto the drug.
[0104] As shown in FIG. 3, the conduit 30 has connected thereto a
microflow-channel introductory part 50, and communicates with the
microflow-channel introductory part 50. By driving the pump 40, the
drug suspension charged into the supply part 20 can be supplied to
the microflow-channel introductory part 50 via the conduit 30.
[0105] The microflow-channel introductory part 50 has a
substantially cuboid shape The microflow-channel introductory part
50 temporarily stores the drug suspension supplied from the supply
part 20, so as to render uniform the flow rate of the drug
suspension flowing through the microflow channel 60 described
below. The volume of the microflow-channel introductory part 50 can
be appropriately selected, depending on the type of the drug
suspension to be treated, and the flow rate generated by the pump
40.
[0106] In the example described above, the shape of the
microflow-channel introductory part 50 is substantially cuboid.
However, the shape of the microflow-channel introductory part 50 is
not particularly limited, as long as the microflow-channel
introductory part 50 is capable of rendering the flow rate of the
drug suspension passing through the microflow channel 60 (described
below) substantially uniform. For example, the microflow-channel
introductory part 50 may be formed of a curved surface, such as a
substantially cylindrical shape. The shape of the microflow-channel
introductory part 50 can be appropriately selected depending on the
flow rate of the drug suspension passing through the microflow
channel 60, and the type and size of the drug within the
suspension.
[0107] As shown in FIG. 4, the microflow-channel introductory part
50 has connected thereto the microflow channel 60, and communicates
with the microflow channel 60. As shown in FIG. 4, the
microflow-channel introductory part 50 has a discharge face 52. At
substantially the center of the discharge face 52, an opening 54 is
formed. The microflow channel 60 is connected to this opening 54.
By this configuration, the drug suspension can be supplied from the
microflow-channel introductory part 50 to the microflow channel 60
via the opening 54.
[0108] By irradiating the laser beam generated from the light
source 10 onto the drug suspension passing through the microflow
channel 60, ultrafine particles of the drug can be formed.
[0109] The microflow channel 60 has a long cuboid shape, and the
cross-section taken along the plane perpendicular to the lengthwise
direction of the microflow channel 60 is substantially square. It
is preferable that the length ML of a side of this square (see FIG.
4) is 1.1 to 200 times the diameter of the drug, more
advantageously 3 to 60 times. By setting the length ML of a side of
the square within this range, the flow of the drug within the
microflow channel 60 can be smoothed, so that clogging of the
microflow channel 60 by the drug can be avoided, and the laser beam
can be accurately irradiated onto the drug.
[0110] The shape of the microflow channel 60 is not particularly
limited to cuboids, as long as the portion to be irradiated with
the laser beam generated from the light source 10 (the portion
located in the irradiation region LR described below) is even.
[0111] Further, in the example described above, the cross-section
taken along the plane perpendicular to the lengthwise direction of
the microflow channel 60 is substantially square. However, the
cross-section may be a rectangle or the like, as long as the laser
beam can be accurately irradiated onto the drug.
[0112] The microflow channel 60 is made of a transparent material
such as a quartz glass, which is capable of transmitting the laser
beam generated from the light source 10.
[0113] As shown in FIG. 4, the laser beam LA generated from the
light source 10 is irradiated onto a portion of the upper face 62
of the microflow channel 60. The irradiation region LR of the laser
beam LA (the region indicated with oblique lines in FIG. 4) is
substantially circular. By making the diameter dL of the
irradiation region LR longer than the length ML of the shortwise
direction of the upper face 62, the laser beam can be
satisfactorily irradiated into the microflow channel 60.
[0114] By driving the above-mentioned pump 40, the drug suspension
is allowed to flow into the microflow channel 60 from the
microflow-channel introductory part 50, and the drug passes through
the microflow channel 60. Drawing attention to a certain drug, the
drug passes through the microflow channel 60 following the flow of
the suspension to arrive at the irradiation region LR of the laser
beam LA. The drug is present in the irradiation region LR for a
while, and then, the drug comes out of the irradiation region
LR.
[0115] When a pulsed laser beam is used and the drug is present in
the irradiation region LR for a long time, the drug gets irradiated
with a pulsed beam a plurality of times. As described above, when
the drug is allowed to pass through the microflow channel 60, the
irradiation period tL can be regarded as the period during which
the drug is present in the irradiation region LR.
[0116] As described above, by irradiating the drug with the laser
beam, ultrafine particles of the drug can be formed. The number of
irradiations of the drug with a pulsed beam can be determined, so
as to form ultrafine particles having a desired size. The number of
irradiations with a pulsed beam can be changed by adjusting the
above-mentioned pulse period T or the flow rate of the drug. Thus,
in the irradiation region LR of the laser beam, the drug is
irradiated with a pulsed beam at least once.
[0117] It is preferable that the cross-sectional area SA (see FIG.
4) of the microflow-channel introductory part 50 be two or more
times of the cross-sectional area SB (see FIG. 4) taken along the
plane perpendicular to the lengthwise direction of the microflow
channel 60. In general, the flow rate VL of the drug passing
through the microflow channel 60 tends to exhibit a distribution
(hereafter, referred to as a "flow rate distribution") such that
the flow becomes slowest near the walls of the microflow channel
and fastest near the center line of the microflow channel (see FIG.
5B). When such a flow rate distribution is generated, the drug
flowing near the walls of the microflow channel 60 exhibits a low
flow rate, so that the period during which the drug is present in
the irradiation region LR becomes long. On the other hand, in such
a case, the drug flowing near the center line of the microflow
channel 60 exhibits a high flow rate, so that the period during
which the drug is present in the irradiation region LR becomes
short. That is, when a pulsed laser beam is irradiated, the number
of pulsed beam irradiations of the drug flowing near the walls of
the microflow channel 60 becomes large, whereas the number of
pulsed beam irradiations of the drug flowing near the center line
of the microflow channel 60 becomes small. Thus, the amount of the
size-reduction treatment of the drug varies depending on the
position at which the drug flows. Therefore, it is possible that
the size of the ultrafine particles of the drug becomes
heterogeneous. By making the cross-sectional area SA of the
microflow-channel introductory part 50 two times or more of the
cross-sectional area SB of the microflow channel 60, the flow rate
distribution of the drug can be rendered substantially uniform, so
that heterogeneity in the size of the ultrafine particles of the
drug can be prevented.
[0118] As described above, by driving the above-mentioned pump 40,
the drug suspension is allowed to flow into the microflow channel
60 from the microflow-channel introductory part 50. The flow rate
VL (see FIG. 5A) of the drug passing through the microflow channel
60 preferably satisfies the relation VL<K.times.dL/tL. Here, the
flow rate VL is the flow rate of the drug which is in a state where
the above-mentioned flow rate distribution is not observed, or in a
state where the above-mentioned flow rate distribution can be
disregarded. By setting the flow rate VL of the drug within the
above-mentioned range, the drug can be irradiated with the laser
beam without any excess or deficiency of irradiation, so that the
drug can be reduced to a desired size. For example, when the drug
is irradiated with a pulsed laser beam, the irradiation can be
performed an appropriate number of times.
[0119] Here, dL represents the diameter of the laser beam upon
irradiation (see FIG. 4), tL represents the above-mentioned
irradiation period (see FIG. 2), and K represents a constant which
can be determined within the range of 1 to 0.1, depending on the
type of drug. K is not particularly limited to the above-mentioned
range, and is preferably set such that the number of pulsed beam
irradiations of the drug passing the irradiation region LR becomes
sufficient for forming ultrafine particles of the drug. The thus
formed ultrafine particles of the drug have a size of 50 to 200
nm.
[0120] FIG. 5A is a cross-sectional view of the microflow-channel
introductory part 50 and the microflow channel 60. In the microflow
channel 60, with respect to the portion to be irradiated with the
laser beam LA, it is preferable that the distance from the
discharge face 52 of the microflow-channel introductory part 50 to
the center LC of the irradiation region LR of the laser beam LA be
no more than 10.times.Dm (see FIG. 5A). Here, Dm is the hydraulic
diameter of the microflow channel 60, and Dm is equal to
4.times.(cross-sectional area SB of the microflow channel
60)/(Perimeter of the cross-section of the microflow channel 60).
For example, when the cross-section of the microflow channel 60 is
a square having a length of ML on each side, Dm is calculated as
follows: Dm=4.times.ML.sup.2/4ML=ML The above-mentioned flow rate
distribution of the drug passing through the microflow channel 60
tends to increase as the drug flows away from the discharge face 52
of the microflow-channel introductory part 50. Therefore, by
setting the portion to be irradiated with the laser LA within the
above-mentioned range, the drug can be irradiated with the laser
beam LA before the flow rate distribution of the drug passing
through the microflow channel 60 becomes large. As a result, the
ultrafine particles of the drug can be reliably formed, and the
size of the ultrafine particles can be rendered substantially
uniform.
[0121] In the example described above, the microflow channel 60 is
directly connected to the opening 54 formed in the
microflow-channel introductory part 50. However, as shown in FIG.
5C, a transition part 64 may be formed between the
microflow-channel introductory part 50 and the microflow channel
60. The transition part 64 is formed in a manner such that the
cross-section thereof becomes smaller as it becomes further from
the opening 54 formed in the microflow-channel introductory part
50. The transition part 64 formed in this manner functions as an
approach section, so as to render the flow rate of the drug flowing
into the microflow channel 60 closer to being uniform. The shape of
the transition part 64 can be appropriately selected depending on
the flow rate and viscosity of the drug suspension.
[0122] As shown in FIG. 3, the microflow channel 60 has a
collecting part 70 connected thereto, and communicates with the
collecting part 70. The collecting part 70 is a vessel for storing
the drug suspension which has been irradiated with the laser beam
within the microflow channel 60.
[0123] It is preferable that the collecting part 70 be provided
with an agglomeration prevention device 72. The agglomeration
prevention device 72 includes a piezoelectric transducer which
applies ultrasonic waves to the drug suspension stored in the
collecting part 70. By the propagation of the ultrasonic waves to
the drug suspension, the ultrafine particles of the drug can be
prevented from agglomeration. The intensity and wavelength of the
ultrasonic waves generated from the agglomeration prevention device
72 can be appropriately selected depending on the size of the
ultrafine particles of the drug and the type of the drug. In the
example described above, the agglomeration prevention device 72
generates ultrasonic waves to effect the prevention of
agglomeration. However, any other agglomeration prevention device
which is capable of preventing agglomeration of the ultrafine
particles of the drug may be used.
[0124] In the vessel of the collecting part 70, a magnetic-drive
impeller 74 may be provided. The magnetic-drive impeller 74 can be
rotated by applying a magnetic field from the outside of the
collecting part 70. By rotating the magnetic-drive impeller 74, the
drug suspension stored within the collecting part 70 can be
stirred, thereby preventing agglomeration of the ultrafine
particles of the drug. The size and number of revolutions of the
magnetic-drive impeller 74 can be appropriately selected depending
on the size of the ultrafine particles of the drug and the type of
the drug.
[0125] Subsequently, with respect to the ultrafine particles of the
drug obtained in the manner as described above, the electric charge
is measured. The electric charge is measured by a zeta
potentiometer. The zeta potential may be positive or negative,
depending in the core substance. The ultrafine particles are
subjected to an electrostatic interaction or hydrophobic
interaction with one or more polymer electrolytes having a charge
opposite to the ultrafine particles to form a complex, thereby
preventing agglomeration of the ultrafine particles.
[0126] Explanation is given below of a method and apparatus for
producing such complex.
[0127] One embodiment of a polymer membrane shell-coating part in
which the drug suspension and a polymer electrolyte solution are
used to form a polymer electrolyte membrane shell on the outer
surface of the ultrafine particles is explained, with reference to
FIGS. 6 and 7. The polymer membrane shell-coating part is one of
the main components of the apparatus for coating ultrafine
particles used in the present invention.
[0128] In the present embodiment, coating is performed by merging
the flow of the drug suspension with the flow of the polymer
electrolyte solution. Further, in the present embodiment, both of
the drug suspension and the polymer electrolyte solution are passed
through a microflow channel. FIG. 6 shows an embodiment of a
polymer membrane shell-coating part using a single microflow
channel in which the drug suspension and the polymer electrolyte
solution are respectively passed through microflow channels which
merge together. FIG. 7 shows an embodiment of a polymer membrane
shell-coating part using a multi-microflow channel which is
provided with a plurality of single microflow channels shown in
FIG. 6.
[0129] Firstly, an explanation is given below of the embodiment of
a polymer membrane shell-coating part using a single microflow
channel as shown in FIG. 6. A discharge microflow channel 76 is
provided at a lower portion of the backside of the collecting part
70 shown in FIG. 3 (i.e., lower portion of the face opposite to the
face where inlet from the microflow channel 60 is provided). Here,
this microflow channel is effective in preventing the agglomeration
of particles size-reduced by the apparatus for forming ultrafine
particles. It is especially preferable to set the width of the
microflow channel slightly larger than the maximum diameter of the
ultrafine particles flowing within the suspension. However, in view
of the fluctuation of particle diameter and precision in producing
the microflow channel, the width of the microflow channel is
preferably set in the range of 1.1 to 500 times, more preferably 50
to 500 times of the maximum diameter of the particles flowing.
[0130] Further, the microflow channel through which the polymer
electrolyte solution is passed can be set at the same size as the
above-mentioned microflow channel through which the
ultrafine-particle suspension is passed. The polymer electrolyte
solution passed through the microflow channel contains a polymer
electrolyte having a charge opposite to that of the outermost layer
of the ultrafine particles which are bonded to or coated with the
polymer electrolyte. Namely, when the outermost layer of the
ultrafine particles contained in the suspension has a negative
charge, a cationic polymer electrolyte solution having a positive
charge is passed through the microflow channel. Likewise, when the
outermost layer of the ultrafine particles contained in the
suspension has a positive charge, an anionic polymer electrolyte
solution having a negative charge is passed through the microflow
channel.
[0131] The angle at which the microflow channels merge can be
selected from acute angles to obtuse angles. The angle at which the
microflow channels merge is preferably from 0 to 180 degrees, more
preferably from 0 to 5 degrees especially for a multi microflow
channel.
[0132] As explained above, in each of the polymer membrane
shell-coating parts, an ultrafine-particle suspension and a polymer
electrolyte having a charge opposite to that of the outermost layer
of the ultrafine particles are used. Therefore, the ultrafine
particles and the polymer electrolyte are attracted to each other
by electrostatic force by simply merging the flow of the suspension
and the flow of the polymer electrolyte solution, so that strong
membrane shells can be easily formed.
[0133] Although an example using a single microflow channel is
illustrated in FIG. 6, a multi microflow channel may be used to
perform the coating of the polymer membrane shell in the same
manner as mentioned above, thereby enabling a production of complex
with high productivity.
[0134] Next, a general explanation is given of the apparatus and
method for sequentially producing complex from the
ultrafine-particle suspension contained in the collecting part 70,
with reference to the line diagram shown in FIG. 8.
[0135] The apparatus used in the present invention for coating
ultrafine particles is mainly composed of a polymer membrane-shell
coating part 120. Further, the polymer membrane-shell coating part
120 is mainly composed of: an ultrafine-particle suspension vessel
70 (the above-mentioned collecting part 70) for containing a
suspension of ultrafine particles prior to coating; a microflow
channel 122a for ultrafine-particle suspension; a microflow channel
122b for polymer electrolyte solution; a merged microflow channel
122c which is formed by merging of the microflow channel 122a with
the microflow channel 122b; a tank 124 for polymer electrolyte
solution, where a polymer electrolyte solution is stored; a complex
collecting vessel 140 for collecting the polymer membrane
shell-coated ultrafine particles following coating treatment (i.e.,
complex formed); pumps; conduits; and valves.
[0136] For the sake of simplifying the figure, the microflow
channel used in the polymer membrane shell-coating part 120 is
shown in the form of single microflow channel. However, in
practice, a multi-microflow channel having the required number of
microflow channels corresponding to the production rate of
ultrafine particles can be used.
[0137] Next, an explanation is given following the flow of the
ultrafine-particle suspension. The ultrafine-particle suspension is
stored in the particle suspension vessel 70. Taking example of a
case where water is used as a solvent, ultrafine particles are
suspended in water, and the outer surfaces of the ultrafine
particles are ionized in water to exhibit a positive or negative
charge. For sake of simplicity, explanation is given of a case
where the outer surfaces of the ultrafine particles have a negative
charge. In this case, a cationic polymer electrolyte solution is
used as the polymer electrolyte.
[0138] In this state, using a pump 114, the suspension of the
ultrafine particles prior to coating is transferred from the
ultrafine-particle suspension vessel 70 to the microflow channel
122a for ultrafine-particle suspension provided within the polymer
membrane shell-coating part 120. Likewise, using a pump 126, the
cationic polymer electrolyte solution stored in a tank 124 for
polymer electrolyte solution is transferred to the microflow
channel 122b for polymer electrolyte solution.
[0139] Then, the microflow channel 122a for ultrafine-particle
suspension and the microflow channel 122b for polymer electrolyte
solution merge together to form a merged microflow channel 122c. In
the merged microflow channel 122c, the ultrafine-particle
suspension and the cationic polymer electrolyte solution are mixed
together, whereby the outer surfaces of the ultrafine particles
contact the cationic polymer electrolyte to form cationic membrane
shells, thereby obtaining complexes having cationic membrane
shells.
[0140] Finally, a cationic mixture of the ultrafine-particle
suspension and the cationic polymer electrolyte solution containing
the complexes formed is transferred to the complex collecting
vessel 140.
[0141] In the case where the outer surfaces of the ultrafine
particles have a positive charge, complexes can be formed in
substantially the same manner as described above, except that an
anionic polymer electrolyte solution is used as the polymer
electrolyte.
[0142] Hereinbelow, the present invention will be described in more
detail with reference to the Examples.
EXAMPLES
Example 1
[0143] (1) Conditions for Laser Irradiation
[0144] Using Nd.sup.3+: YAG laser (Continuum, Surelite), a laser
beam was generated by an Optical Parametric Oscillator (OPO) system
(Continuum, SureliteOPO). The intensity of the laser beam was
adjusted using an attenuation plate and an attenuator. The area of
beam irradiation was estimated by irradiating a laser beam to a
photosensitive paper provided at the front face of the quartz
cell.
[0145] Laser: Repetition frequency: 10 Hz [0146] Pulse width: 7
ns
[0147] Excitation wavelength: 355 nm
[0148] Irradiation area: 0.28 cm.sup.2
[0149] Size-reduction of ellipticine: [0150] Intensity of laser
beam: 100 mJ/cm.sup.2 [0151] Total irradiation time: 10 seconds
[0152] (2) Ellipticine Sample
[0153] As a test sample, ellipticine (Fluka, >99%) was used,
which was roughly pulverized to about 1 .mu.m. As a solvent, a
deionized water was used.
[0154] In the formation of ultrafine particles, 75 ml of a
suspension of the test sample which had been irradiated with
ultrasonic waves (SHARP, UT-205, high frequency: maximum of 200 W)
was used. 3 ml of this test particle suspension was measured and
charged into a quartz cell (1.times.1.times.5 cm.sup.3) having an
optical path length of 1 cm, and the quartz cell was irradiated
with a laser beam while stirring with a magnetic stirrer.
[0155] The ultrafine particles formed were immediately coated with
a polymer electrolyte added in advance for the purpose of
stabilizing the ultrafine particles and preventing the ultrafine
particles from agglomerating. Therefore, the laser beam was
irradiated onto (a) a sample having a polymer electrolyte added
thereto and (b) a sample having no polymer electrolyte added
thereto, and a comparison was made between sample (a) and sample
(b).
[0156] (a) Ellipticine+Polymer Electrolyte+Aqueous Dispersion
[0157] Polymer electrolyte: protamine (concentration:
1.times.10.sup.-2 g/ml)
[0158] Ellipticine 4.1.times.10.sup.-3 M (1.0 mgml.sup.-1) as an
anti-cancer drug was dispersed in water while irradiating with a
laser beam. The resulting suspension was allowed to stand for 1
hour, and then the supernatant formed was evaluated.
[0159] Separately from the above, ellipticine 4.1.times.10.sup.-3 M
(1.0 mgml.sup.-1) was dispersed in water without irradiating a
laser beam. The supernatant of the resulting suspension was used as
a control.
[0160] The concentration of the supernatant was estimated from the
absorbance.
[0161] (b) Ellipticine+Aqueous Dispersion
[0162] Ellipticine 1.5.times.10.sup.-4 M (3.6.times.10.sup.-2
gl.sup.-1) was dispersed in water while irradiating a laser beam.
The supernatant following the irradiation was evaluated.
[0163] Separately from the above, ellipticine 1.5.times.10.sup.-4 M
(3.6.times.10.sup.-2 gl.sup.-1) was dispersed in water without
irradiating a laser beam. The supernatant of the resulting
suspension was used as a control.
[0164] The concentration of the supernatant was estimated from the
absorbance.
[0165] As a result, it was found that the concentration of the
supernatant of the suspension following irradiation of the laser
beam in the presence of the polymer electrolyte was more than 100
times the supernatant of the suspension in the absence of the
polymer electrolyte.
[0166] Concentration of the supernatant of the suspension following
irradiation of laser beam in the presence of the polymer
electrolyte: >1.8.times.10.sup.-5 g/ml.
[0167] (3) Evaluation of Ultrafine Particles
[0168] (3-1) Evaluation of the purity of the ultrafine particles
formed was performed in the following manner.
[0169] From the suspension following irradiation, the supernatant
was taken out, and the solvent was vaporized by using a vacuum
pump. Then, ethanol was added to the residue, and the resultant was
analyzed by ultraviolet and visible ray spectroscopy (SHIMADZU,
UV-3100. HITACHI, F-4500) and liquid chromatography (SHIMADZU,
SPD-10).
[0170] FIG. 9 shows a comparison of absorption spectra--ethanol
solution prior to and following irradiation. In FIG. 9, absorption
spectrum (solid line) of unirradiated ellipticine ethanol solution
and absorption spectrum (dotted line) of the ethanol solution
following irradiation (100 mJ/cm.sup.2, 10 seconds) are shown. From
FIG. 9, almost no difference is observed between the absorption
spectrum (solid line) of unirradiated ellipticine ethanol solution
and the absorption spectrum (dotted line) of the ethanol solution
following irradiation. From this result, it is understood that the
ultrafine particles of ellipticine formed by laser beam irradiation
are hardly decomposed.
[0171] FIG. 10 is a chromatogram of the ethanol solution of
ellipticine following laser beam irradiation. From FIG. 10, it is
also understood that the ultrafine particles of ellipticine formed
by laser beam irradiation are hardly decomposed.
[0172] (3-2) FIG. 11 is a SEM image of ellipticine prior to
size-reduction treatment, and FIG. 12 is a SEM image of ultrafine
particles of ellipticine (ellipticine following size-reduction
treatment). The average diameter of the particles prior to the
size-reducing treatment is about 1 .mu.m, which is the limit size
achieved by size-reduction using machines.
[0173] Observation was performed by FEI, Strata DB235-51. See
attached microphotographs.
[0174] (3-3) FIG. 13 is a histogram of the particle diameter
distribution of the ultrafine particles of ellipticine.
[0175] From FIG. 13, almost all of the complexes of the present
invention have a particle size distribution within the range of 70
to 130 nm, which meant that the particle size was uniform. The
average diameter was 100 nm. The average diameter was determined by
measuring the diameter of each particle using a microscope provided
with a scale, and dividing the sum of the particle diameters by the
number of particles.
[0176] Observation was performed by FEI, Strata DB235-51.
Measurement was performed by MALVERN zeta sizer Nano-ZS.
[0177] In the present example, the thickness of the coating was 3
to 4 nm. Therefore, the thickness of the coating could be
disregarded from the entire particle size.
[0178] (3-4) Cytotoxicity Test
[0179] Ellipticine which had been size-reduced by laser beam
irradiation (concentration: 2 .mu.g/ml) was diluted with the
below-mentioned culturing solution, and test samples having
concentrations of 1 .mu.g/ml, 0.5 .mu.g/ml, 0.25 .mu.g/ml and 0.125
.mu.g/ml were prepared. MCF-7 (MEM-culture medium) and L-1210
(RPMI-1640 culture medium) tumor cells were used as target cells.
The cytotoxicity was evaluated by counting the number of viable
cells following 24 hours of culturing, using Cell Counting Kit-8.
More specifically, WST-8 (U.S. Pat. No. 2,757,348) as an indicate
for dehydrogenase activities was used, and the color at 450 nm was
evaluated.
Viability(%)=(A.sub.samples-A.sub.blank)/(A.sub.no-samples-A.sub.blank)10-
0%
[0180] wherein A is the absorbance at a wavelength of 450 nm
exhibiting UV properties, A.sub.samples is the absorbance as
measured when a sample was present, A.sub.no-samples is the
absorbance as measured when a sample was not present but the
polymer electrolyte was present, and A.sub.blank is the absorbance
as measured when only the culture medium was present.
[0181] The 50% inhibiting activities on the cells were as
follows.
[0182] MCF-7 cells: 0.21 .mu.g/ml
[0183] L-1210 cells: 0.09 .mu.g/ml
[0184] The control could not be evaluated because ellipticine is
insoluble in water. The values indicated in prior art documents
cannot be directly compared with the present invention because an
organic solvent such as DMSO is used.
[0185] From the results shown above, it has been proved that the
ultrafine particles of an anti-cancer drug according to the present
invention and a complex of the same with a polymer electrolyte
exhibits an inhibiting activity to tumor cells, and that the
ultrafine particles of an anti-cancer drug and the complex have a
drug effect.
Example 2
[0186] Preparation of SN-38 Nano Particle
[0187] 0.01N HCl was diluted by 100 folds to obtain an aqueous
solution of hydrochloric acid exhibiting a pH value of 4.0. To 20
ml of this solution was added 60 mg of SN-38 and the resultant was
subjected to an ultrasonic treatment for 2 or more hours, to
thereby obtain a suspension. Then, 2.0 ml of the suspension was
measured out while stirring the suspension with a magnetic stirrer,
and charged into a quartz cell having an optical path length of 1
cm. Then, 1 ml of the aqueous solution of hydrochloric acid
exhibiting a pH value of 4.0 was further charged into the quartz
cell, thereby obtaining a suspension having an SN-38 concentration
of 2 mg/ml. Subsequently, the suspension was irradiated with a
laser beam (420 nm excitation, 80 mJ/cm.sup.2, 100 minutes) while
thoroughly stirring with a magnetic stirrer. After the irradiation,
the suspension was allowed to stand for 1 day at room temperature,
and the supernatant of the resulting suspension was taken out and
analyzed by absorbance spectroscopy, HPLC, and measurement of
particle size distribution and SEM. As a result, it was found that
nanosizing had proceeded without chemical decomposition of SN-38
caused by the laser beam irradiation under the above-mentioned
conditions (see FIGS. 14A, 14B and FIG. 15). The yield of the nano
particles formed was 50% or more, and the concentration was 1
mg/ml.
[0188] Preparation of SN-38 Nano Particles-Protamine Sulfate and
SN-38 Nano Particles-Chondroitin Sulfate
[0189] For the purpose of stabilizing the SN-38 nano particles
(preventing self-agglomeration of SN-38 nano particles), protamine
sulfate and chondroitin sulfate were respectively added to two
separate samples of the above-mentioned supernatant having a
concentration of 1 mg/ml, in an amount sufficient for rendering the
zeta potential of the surface of the nanoparticles a predetermined
value (more specifically, 10 mg/ml protamine sulfate (pH4) and 10
mg/ml chondroitin sulfate (pH4) were respectively added to two
separate 1 mg/ml suspensions of SN-38 ultrafine particles in an
amount of 30 wt %, based on the weight of the suspension of SN-38
ultrafine particles), so as to adjust the zeta potential to +19.9
mV and -47.2 mV, respectively (see FIG. 16).
[0190] Cytotoxicity Test of SN-38 Nano Particles
[0191] The cytotoxicity of the SN-38 nano particles was evaluated
by counting the number of viable cells following 24 hours of
culturing in the same nm er as in the cytotoxicity test of
ellipticine as described in item (3-4) of Example 1. The 50%
inhibiting activity for MCF-7 cells was 100 nM. The DMSO solution
and water suspension of SN-38 which were not irradiated with a
laser beam and which were used as controls each exhibited a 50%
inhibiting activity of 500 nM and 2,000 nM.
[0192] From the results above, it was shown that the nano-sized
sample exhibited high ability of intracellular transport, as
compared to the control.
[0193] Comparison of Anti-Tumor Effect of SN-38 Nano Particles,
SN-38 Nano Particles-Protamine Sulfate, SN-38 Nano
Particles-Chondroitin Sulfate and Irinotecan Hydrochloride (CPT-11)
Using Nude Mice-Transplanted Human Tumor
[0194] Test Laboratory
[0195] Name: EXPERIMENTAL CANCER CHEMOTHERAPY RESEARCH LAB., Co.,
LTD.
[0196] Address: Hakushima3-13-1 Minou-shi Osaka-fu Japan
[0197] Materials and Method
[0198] 1. Test Substances
[0199] SN-38 nano particles
[0200] SN-38 nano particles-protamine sulfate
[0201] SN-38 nano particles-chondroitin sulfate
[0202] Preservation conditions: The test substances were placed in
an air-tightly sealed vessel and shielded, and preserved at room
temperature (23.degree. C.)
[0203] Control drug: irinotecan hydrochloride (CPT-11)
[0204] 2. Human Cancer Strain Used
[0205] Gastric cancer H-23, 323th passage moderately
differentiated-type adenocarcinoma
[0206] 3. Test Animal
[0207] BALB/cAJcl-nu nude mouse (male, Clea Japan Inc.)
[0208] 4. Transplantation Method
[0209] A nude mouse having tumor cells transplanted was killed by
cervical dislocation, and the subcutaneously passage-cultured tumor
cells were extracted. From the extracted tumor cells, the capsule
and necrotic portion were removed, and the resultant washed with
RPMI medium. Thereafter, substantially uniform cubes having sides
of 2 to 3 mm were cut out as tumor specimens, and the tumor
specimens were transplanted subcutaneously onto the backs of
6-week-old mice using a trocar (transplantation day: Day 0).
[0210] 5. Experiment Method
[0211] Using vernier calipers, the maximum diameter (L), the
transverse diameter (W) crossing the maximum diameter (L), and the
thickness (D) were measured to the 0.5 mm scale. When the estimated
volume of the tumor as determined by the formula:
V=1/2.times.L.times.W.times.D became about 70 mm.sup.3 (7 days
after transplantation), a control group and treatment group were
set as 5 mice per each group, and the average values of the
estimated tumor volumes and the standard deviations of the
respective groups were set to be substantially equal. Then,
administration to each of the groups was started.
[0212] The experiment was completed after 4 weeks from the starting
day of administration. The tumor cell diameter was measured twice a
week, and the weight was measured at the time of administration, so
as to monitor the state of tumor cell proliferation and effect of
drug administration, as well as any other physical changes. The
mice were kept in a small vinyl isolator throughout the experiment,
except for when they were moved to a clean bench through a sleeve
to perform tumor cell transplantation, administration or weight
measurement.
[0213] 6. Dose and Administration Schedule
[0214] (1) Control (No drug treatment)
[0215] (2) CPT-11 60 mg/kg (i.v.) q4d.times.4 4 times in total
[0216] (3) SN-38 nano particles 10 mg/kg (i.v.) q4d.times.4 4 times
in total
[0217] (4) SN-38 nano particles-protamine 3 mg/kg (i.v.)
q4d.times.4 4 times in total
[0218] (5) SN-38 nano particles-chondroitin sulfate 10 mg/kg (i.v.)
q4d.times.4 4 times in total
[0219] 7. Preparation of Sample Solution
[0220] Each of SN-38 nano particles (1 mg/ml), stock solution of
SN-38 nano particles-chondroitin sulfate (1 mg/ml) (for 10 mg/kg
treatment group) and SN-38 nano particles-protamine sulfate (1
mg/ml) were respectively diluted with distilled water for injection
to obtain 0.3 mg/ml sample solutions (for 3 mg/kg treatment group).
Further, just before administration, 27% NaCl solution was added
with a volume ratio of 1:30. CPT-11 was diluted with physiological
saline to a concentration of 3 mg/ml.
[0221] 8. Administration Method of Sample Solution
[0222] To each of the SN-38 nano particles, SN-38 nano
particles-protamine and SN-38 nano particles-chondroitin sulfate
treatment groups, the sample solution was administered within 25
minutes from the addition of NaCl. The sample solution was
intravenously administered once a day in an amount of 0.11 ml per
10 g of the mouse weight, and the administration was performed once
every 4 days and 4 times in total (q4d.times.4).
[0223] With respect to the CPT-11 treatment group, 3 mg/ml sample
solution was intravenously administered twice a day (60 mg/kg
treatment group), in an amount of 0.1 ml per 10 g of the mouse
weight, and the administration was performed once every 4 days and
4 times in total (q4d.times.4). Further, with respect to the
control group, no drug administration was performed.
[0224] 9. Evaluation of Drug Effect
[0225] On the final day of the experiment, tumor cells were
extracted from the control group (C) and the treatment group (T).
From the average weight of the tumor cells, the tumor-proliferation
inhibiting efficiency (IR) was determined by the formula shown
below. IR of below 58% was evaluated as "non-effective",
IR.gtoreq.58% was evaluated as "effective", and IR.gtoreq.80% was
evaluated as "significantly effective". IR=(1-T/C).times.100(%)
[0226] The statistical significance between the weights of tumor
cells of each group was determined by Student's T test
(two-tailed).
[0227] Further, the average estimated tumor volumes of the control
group (C) and the treatment group (T) were measured sequentially
during the experiment, and the tumor volume IR was determined in
the similar manner as mentioned above, to thereby determine the
maximum proliferation inhibiting efficiency (max. IR) during the
experiment. Furthermore, when the average estimate tumor volume at
the end of the experiment was larger than that at the time of
administration, it was evaluated as having size-reducing
effect.
[0228] The influence of the drug on the host was evaluated by
considering the change in weight and expression of symptoms.
[0229] Results
[0230] A study was made by comparing the tumor proliferation
inhibiting effect of each of SN-38 nano particles, SN-38 nano
particles-protamine sulfate and SN-38 nano particles-chondroitin
sulfate with that of CPT-11, using 323th passage of nude-mouse
transplanted human gastric cancer H-23 (moderately
differentiated-type adenocarcinoma).
[0231] 1. Tumor Proliferation Inhibiting Effect
[0232] 1) CPT-11 60 mg/kg Treatment Group
[0233] As the number of times performing the administration
increased, the tumor volume IR increased. 2 days after the 4th
administration (d21), the maximum tumor proliferation inhibiting
efficiency (max. IR) during the experiment became 69.1%, and hence,
a drug effect was confirmed. However, thereafter, the tumor volume
IR gradually decreased, and the tumor volume IR on the final day of
the experiment (d35) was 22.4%, and hence, the drug effect was
evaluated as non-effective. With respect to the tumor weight, no
statistical significance against the control group was observed by
the t-test.
[0234] 2) SN-38 Nano Particles 10 mg/kg Treatment Group
[0235] 4 days after the first administration (d11), the tumor
volume IR became 57.7% which was the maximum value during the
treatment. 3 days after the second administration (d14), tendency
of tumor-cell size-reduction was exclusively observed among all
treatment groups. As a result, the tumor volume IR was found to be
59.9%, and drug effect was exclusively confirmed among all
treatment groups only after the second administration. 3 days after
the third administration (d18), the tumor volume IR became higher
as 73.4%. 2 days after the fourth administration (d21), the max. IR
of 74.7% was observed, and the tumor proliferation inhibiting
effect was significant. 9 days after the fourth administration
(d28), the tumor volume IR was 61.1%, and drug effect was
exclusively confirmed among all treatment groups. However, the drug
effect gradually decreased, and the tumor weight IR on the final
day of the experiment (d35) was 45.9%, and hence, the drug effect
was evaluated as non-effective. Nevertheless, by the t-test
regarding the tumor weight, statistical significance of p<1%
against the control group was observed, which was p<5% higher
than the CPT-11 60 mg/kg treatment group.
[0236] 3) SN-38 Nano Particles-Protamine Sulfate 3 mg kg Treatment
Group
[0237] 3 days after the third administration (d18), the tumor
volume IR was 57.5%, and hence, a tumor proliferation inhibiting
effect slightly higher than the CPT-11 60 mg/kg treatment group was
observed.
[0238] 2 days after the fourth administration (d21), the max. IR of
62.1% was observed, and hence, a drug effect was confirmed.
Thereafter, the tumor volume IR gradually decreased. On the final
day of the experiment (d35), the tumor weight IR was 38.9%, and
hence, the drug effect was evaluated as non-effective.
Nevertheless, by the t-test regarding the tumor weight, statistical
significance of p<1% against the control group was observed.
Further, the IR was advantageous over the CPT-11 60 mg/kg treatment
group.
[0239] 4) SN-3 8 Nano Particles-Chondroitin Sulfate 10 mg/kg
Treatment Group
[0240] 3 days after the third administration (d18), the tumor
volume IR was 65.3%, and hence, a drug effect was confirmed.
Specifically, the second high tumor proliferation inhibiting effect
following the tumor proliferation inhibiting effect of the SN-38 10
mg/kg treatment group was confirmed. 2 days after the fourth
administration (d21), the max. IR of 65.4% was observed. However,
thereafter, the IR rapidly decreased. On the final day of the
experiment (d35), the tumor weight IR was 28.5%, and hence, the
drug effect was evaluated as non-effective. Nevertheless, by the
t-test regarding the tumor weight, statistical significance of
p<5% against the control group was observed.
[0241] Conclusion
[0242] The SN-38 nano particles 10 mg/kg treatment group exhibited
an apparently high tumor proliferation inhibiting effect as
compared to the CPT-11 treatment group. Further, the CPT-11
treatment group exhibited marked lowering of the tumor
proliferation inhibiting effect by stopping administration. On the
other hand, although the SN-38 nano particles 10 mg/kg treatment
group exhibited lowering of the tumor proliferation inhibiting
effect by stopping administration, the degree of lowering was much
smaller than the CPT-11 treatment group. Furthermore, in the SN-38
nano particles 10 mg/kg treatment group, no weight reduction was
observed, and no serious side-effect was observed.
[0243] The SN-38 nano particles-protamine sulfate 3 mg/kg treatment
group and the SN-38 nano particles-chondroitin sulfate 10 mg/kg
treatment group both exhibited a high tumor proliferation
inhibiting effect, as compared to the CPT-11 treatment group.
Especially up to 3 days after the third administration (d18) and
between d32 to d35 (final day of the experiment), the tumor
proliferation inhibiting effect was advantageous over the CPT-11
treatment group (see FIG. 17).
Example 3
[0244] Preparation of 10-Hydroxy-Camptothecin Nano Particles
[0245] A suspension of 10-hydroxy-camptothecin with a concentration
of 0.5 mg/ml was prepared in the same manner as in the preparation
of SN-38 nano particles.
[0246] Subsequently, the suspension was irradiated with a laser
beam (430 nm excitation, 40 mJ/cm.sup.2, 60 minutes) while
thoroughly stirring with a magnetic stirrer. After the irradiation,
the suspension was subjected to centrifugal separation, and the
supernatant of the resulting suspension was taken out and analyzed
by absorbance spectroscopy, HPLC, and measurement of particle size
distribution and SEM (see FIGS. 18A, 18B and 19). As a result, it
was found that the yield of the nano particles formed was 50% or
more, and the concentration was 0.25 mg/ml.
[0247] Cytotoxicity Test of 10-Hydroxy-Camptothecin Nano
Particles
[0248] The cytotoxicity of the 10-hydroxy-camptothecin nano
particles was evaluated by counting the number of viable cells
following 72 hours of culturing in the same manner as in the
cytotoxicity test of ellipticine as described in item (3-4) of
Example 1. The 50% inhibiting activity for MCF-7 cells was 100 nM.
The DMSO solution and water suspension of 10-hydroxy-camptothecin
which were unirradiated with a laser beam and which were used as
controls each exhibited a 50% inhibiting activity of 100 nM and 500
nM.
[0249] From the results above, it was shown that the nano-sized
sample exhibited high ability of intracellular transport, as
compared to the control.
[0250] Comparison of Anti-Tumor Effect of 10-Hydroxy-Camptothecin
Nano Particles and Irinotecan Hydrochloride (CPT-1) Using Nude
Mice-Transplanted Human Tumor
[0251] Test Laboratory
[0252] Name: EXPERIMENTAL CANCER CHEMOTHERAPY RESEARCH LAB., Co.,
LTD.
[0253] Address: Hakushima3-13-1 Minou-shi Osaka-fu Japan
[0254] Materials and Method
[0255] 1. Test Substances
[0256] 10-hydroxy-camptothecin nano particles
[0257] Control drug: irinotecan hydrochloride (CPT-11)
[0258] 2. Human Cancer Strain Used
[0259] Gastric cancer H-23, moderately differentiated-type
adenocarcinoma
[0260] 3. Test Animal
[0261] BALB/cAJcl-nu nude mouse (male, Clea Japan Inc.)
[0262] 4. Transplantation Method
[0263] A nude mouse having tumor cells transplanted was killed by
cervical dislocation, and the subcutaneously passage-cultured tumor
cells were extracted. From the extracted tumor cells, the capsule
and necrotic portion were removed, and the resultant washed with
RPMI medium. Thereafter, substantially uniform cubes having sides
of 2 to 3 mm were cut out as tumor specimens, and the tumor
specimens were transplanted subcutaneously onto the backs of
5-week-old mice using a trocar (transplantation day: Day 0).
[0264] 5. Experiment Method
[0265] Using vernier calipers, the maximum diameter (L), the
transverse diameter (W) crossing the maximum diameter (L), and the
thickness (D) were measured to the 0.5 mm scale. When the estimated
volume of the tumor as determined by the formula:
V=1/2.times.L.times.W.times.D became about 100 mm.sup.3 (7 days
after transplantation), a control group and treatment group were
set as 5 mice per each group, and the average values of the
estimated tumor volumes and the standard deviations of the
respective groups were set to be substantially equal. Then,
administration to each of the groups was started.
[0266] The experiment was ended after 4 weeks from the starting day
of administration. The tumor cell diameter was measured twice a
week, and the weight was measured at the time of administration, so
as to monitor the state of tumor cell proliferation and effect of
drug administration, as well as any other physical changes. The
mice were kept in a small vinyl isolator throughout the experiment,
except for when they were moved to a clean bench through a sleeve
to perform tumor cell transplantation, administration or weight
measurement.
[0267] 6. Dose and Administration Schedule
[0268] (1) Control: physiological saline 0.1 ml/10 g of mouse
weight (i.v.) d7, d11, d14 3 times in total
[0269] (2) CPT-11 60 mg/kg (i.v.) d7, d11, d14 3 times in total
[0270] (3) 10-hydroxy-camptothecin nano particles 5 mg/kg (i.v.)
d7, d8, d11, d12, d14, d15 6 times in total
[0271] (4) 10-hydroxy-camptothecin nano particles 2.5 mg/kg (i.v.)
d7, d8, d11, d12, d14, d15 6 times in total
[0272] 7. Preparation of Sample Solution
[0273] 1.8% NaCl solution was added to 10-hydroxy-camptothecin nano
particle solution (0.25 mg/ml) to obtain a 0.125 mg/ml sample
solution. CPT-11 was diluted with physiological saline to a
concentration of 3 mg/ml.
[0274] 8. Administration Method of Sample Solution
[0275] To each of the 10-hydroxy-camptothecin nano particle
treatment groups, the 0.125 mg/ml sample solution was administered
within 25 minutes from the preparation thereof. Specifically, the
sample solution was intravenously administered to the 5 mg/kg
treatment group twice a day in an amount of 0.2 ml per 10 g of the
mouse weight, and once a day in an amount of 0.2 ml per 10 g of the
mouse weight to the 2.5 mg/kg treatment group. The administration
was performed 6 times in total on d7, d8, d11, d12, d14, d15.
[0276] With respect to the CPT-11 treatment group, 3 mg/ml sample
solution was intravenously administered twice a day (60 mg/kg
treatment group), in an amount of 0.1 ml per 10 g of the mouse
weight. The administration was performed 3 times in total on d7,
d11 and d14. Further, with respect to the control group,
physiological saline was administered 3 times in the same manner as
in the CPT-11 treatment group.
[0277] 9. Evaluation of Drug Effect
[0278] On the final day of the experiment, tumor cells were
extracted from the control group (C) and the treatment group (T).
From the average weight of the tumor cells, the tumor-proliferation
inhibiting efficiency (IR) was determined by the formula shown
below. IR of below 58% was evaluated as "non-effective",
IR.gtoreq.58% was evaluated as "effective", and IR.gtoreq.80% was
evaluated as "significantly effective". IR=(1-T/C).times.100(%)
[0279] The statistical significance between the weights of tumor
cells of each groups was determined by Student's T test
(two-tailed).
[0280] Further, the average estimated tumor volumes of the control
group (C) and the treatment group (T) were measured sequentially
measured during the experiment, and the tumor volume IR was
determined in the similar manner as mentioned above, to thereby
determine the maximum proliferation inhibiting efficiency (max. IR)
during the experiment. Furthermore, when the average estimate tumor
volume at the end of the experiment was larger than that at the
time of administration, it was evaluated as having size-reduction
effect.
[0281] The influence of the drug on the host was evaluated by
considering the change in weight and the expression of
symptoms.
[0282] Results
[0283] A study was made by comparing the tumor proliferation
inhibiting effect of 10-hydroxy-camptothecin nano particles with
that of CPT-11, using 317th passage of nude-mouse transplanted
human gastric cancer H-23 (moderately differentiated-type
adenocarcinoma).
[0284] 1. Tumor Proliferation Inhibiting Effect
[0285] 1) CPT-11 60 mg/kg Treatment Group
[0286] Significant tumor proliferation inhibiting effect was
observed from the start of administration. At the time of the third
administration (d14), the tumor volume IR was 60.7%, and hence,
drug effect was confirmed. Further, 4 days after the third
administration (d18), the max. IR became 69.0%. By the t-test
regarding the tumor weight, statistical significance of p<1%
against the control group was observed. However, thereafter, the
proliferation rate of tumor cells increased, and the IR after 3
days (d21) became 50.7%, and hence, the drug effect was evaluated
as non-effective. The proliferation rate increased even more, and
the tumor volume IR after 18 days from the start of administration
(d25) became lower than the CPT-11 30 mg/kg treatment group in
which proliferation had reached a peak. As a result, the surfaces
of the tumor cells were ulcerated. The tumor volume IR on the
finish day of the experiment (d35) was 14.1%. The tumor weight IR
was 14.1%, and hence, the drug effect was evaluated as
non-effective. Further, no statistical significance against the
control group was observed by the t-test regarding the tumor
weight.
[0287] 2) 10-Hydroxy-Camptothecin Nano Particles 5 mg/kg Treatment
Group
[0288] From the start of administration, size-reduction of tumor
cells was observed, and the tumor volume IR on the third
administration day (d11) was 63.2%, and hence, drug effect was
exclusively observed among all treatment groups. Thereafter,
proliferation gradually started again, but a significant inhibiting
effect was observed as compared to the control group, and the tumor
volume IR was enhanced. 3 days after the sixth administration
(d18), the tumor volume IR was 85.4% (max. IR), and hence, a
significant drug effect was observed. By the t-test regarding the
tumor volume, statistical significance of p<1% against the
control group was observed. However, thereafter, the proliferation
rate increased and the tumor volume IR gradually decreased.
Nevertheless, the tumor volume IR after 10 days from the end of the
sixth administration (d25) was 62.3%, and hence, drug effect was
exclusively observed among all treatment groups. Thereafter, the
proliferation of the control group reached a peak, and hence, the
tumor proliferation rate of the 10-hydroxy-camptothecin nano
particles treatment groups markedly increased, and the tumor volume
IR rapidly decreased. The tumor volume IR on the final day of the
experiment (d35) was 23.1%. The tumor weight IR was 19.4%, and
hence, the drug effect was evaluated as non-effective. Further, no
statistical significance against the control group was observed by
the t-test regarding the tumor weight.
[0289] 3) 10-Hydroxy-Camptothecin Nano Particles 2.5 mg/kg
Treatment Group
[0290] From the start of administration, the proliferation rate of
tumor cells was moderate, and a significant proliferation
inhibiting effect was observed. 2 days after the fourth
administration (d14), the tumor volume IR was 68.0%, and hence,
drug effect was confirmed. 3 days after the sixth administration
(d18), the max. IR of 76.4% was observed. By the t-test regarding
the tumor volume, statistical significance of p<1% against the
control group was observed. The tumor volume IR after further 3
days (d21) was 68.0%, and hence, drug effect was confirmed. During
the last stage of the experiment, the number of animals having the
surface of the tumor cells ulcerated increased. Therefore, the
proliferation of tumor cells reached a peak, and the tumor volume
IR became higher than the 5 mg/kg treatment group. The tumor volume
IR on the final day of the experiment (d35) was 28.7%. The tumor
weight IR was 25.5%, and hence, the drug effect was evaluated as
non-effective, but the tumor weight IR was highest of all treatment
groups. However, no statistical significance against the control
group was observed by the t-test regarding the tumor weight.
[0291] 2. Side-Effects
[0292] With respect to the maximum weight loss (max. wt. loss) of
the treatment groups, CPT-11 60 mg/kg treatment group was 0.4%
which was very small, and no weight loss was observed in the
10-hydroxy-camptothecin nano particles 2.5 mg/kg treatment group
from the first day of the experiment.
[0293] In the 10-hydroxy-camptothecin nano particles 5 mg/kg
treatment group, a moderate max. wt. loss of 9.1% was observed 3
days after the sixth administration (d15), and soft stool was
confirmed with respect to one mouse (No. 5) among the 5 mice of the
group. However, after further 3 days (d21), the weight loss
recovered, and the weight continued to recover up to the final day
of the experiment. No other significant side-effects were observed.
In the control group, although the average weight loss was small at
the start of the experiment, tendency of weight loss was observed
at the last stage of the experiment, despite the fact that the
weight of proliferated tumor cells was included. Especially, 1
mouse (No. 2) among the 5 mice of the group suffered marked weight
loss, and was the only mouse among the 30 mice (i.e, all of the
treated mice) to have a weight lower than that at the start of the
experiment, as measured on the final day of the experiment. The
body state of the control group and the CPT-11 60 mg/kg treatment
group was not good.
[0294] 3. Comparison of 10-Hydroxy-Camptothecin Nano Particles and
CPT-11
[0295] From a comparison of the CPT-11 60 mg/kg treatment group and
the 10-hydroxy-camptothecin nano particles 5 mg/kg treatment group,
it was confirmed that both groups exhibit the max. IR after 2 or 3
days from the administration (d18). The max. IR of the CPT-11
treatment group was 69.0% (effective), and the
10-hydroxy-camptothecin nano particles treatment group was 85.4%
(significant effect). By the t-test regarding the estimate tumor
volume, 10-hydroxy-camptothecin nano particles treatment group was
advantageous by p<5% of statistical significance. Although no
statistical significance was observed on the final day of the
experiment (d35), the IR of the CPT-11 treatment group was 8.9%,
whereas the 10-hydroxy-camptothecin nano particles treatment group
was 19.4%, which meant that the 10-hydroxy-camptothecin nano
particles treatment group exceeded the CPT-11 treatment group.
[0296] Even the 10-hydroxy-camptothecin nano particles 2.5 mg/kg
treatment group exhibited max. IR of 76.4% on d18, which was nearly
"significant effect", and the tumor weight IR on the final day of
the experiment (d35) was 25.5%, which was higher than that of the
CPT-11 60 mg/kg treatment group.
[0297] Conclusion
[0298] From the studies of the anti-tumor effect using nude
mice-transplanted human gastric cancer H-23, it was found that the
tumor proliferation inhibiting effect of the
10-hydroxy-camptothecin nano particles treatment groups was
advantageous over that of the CPT-11 treatment group (see FIG.
20).
INDUSTRIAL APPLICABILITY
[0299] The water- or alcoholic solution-insoluble anti-cancer drug
in the form of ultrafine particles according to the present
invention, and the complex of the same with a polymer electrolyte,
are usable as an injection in which bioavailability is improved,
and side-effects are suppressed.
[0300] Therefore, the present invention is expected to be applied
to therapy for targeting specific tissues, using a polymer
electrolyte which recognizes adhesion factors or any other specific
cell tissue surface.
[0301] Further, by the present invention, it is expected that an
anti-cancer agent can be outwardly effused over a long period, so
as to suppress adverse side-effects caused when a drug is
introduced into a body at once in a large amount.
[0302] Furthermore, the present invention can be expected to
provide a safe and effective cancer therapy in which the drug is
prepared to have a particle size within the range of 50 to 200 nm.
Therefore, the drug can be selectively taken in by tumor cells with
an enhancement permeability and retention (ERP) effect.
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