U.S. patent application number 14/683674 was filed with the patent office on 2015-09-24 for method for coating fine particles with lipid membrane.
The applicant listed for this patent is KYOWA HAKKO KIRIN, CO., LTD.. Invention is credited to Shiro AKINAGA, Takeshi IWATA, Yasuki KATO, Hiroko KUSANO, Takaaki UOCHI, Masahiro YAMAUCHI.
Application Number | 20150265545 14/683674 |
Document ID | / |
Family ID | 18786007 |
Filed Date | 2015-09-24 |
United States Patent
Application |
20150265545 |
Kind Code |
A1 |
KATO; Yasuki ; et
al. |
September 24, 2015 |
METHOD FOR COATING FINE PARTICLES WITH LIPID MEMBRANE
Abstract
A method for coating fine particles with lipid membrane,
characterized in that, the rate of a polar organic solvent in an
aqueous solution containing the polar organic solvent where the
fine particles are dispersed and lipid is dissolved, is
decreased.
Inventors: |
KATO; Yasuki; (Sunto-gun,
JP) ; YAMAUCHI; Masahiro; (Sunto-gun, JP) ;
KUSANO; Hiroko; (Sunto-gun, JP) ; IWATA; Takeshi;
(Sunto-gun, JP) ; UOCHI; Takaaki; (Sunto-gun,
JP) ; AKINAGA; Shiro; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KYOWA HAKKO KIRIN, CO., LTD. |
Tokyo |
|
JP |
|
|
Family ID: |
18786007 |
Appl. No.: |
14/683674 |
Filed: |
April 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12656487 |
Feb 1, 2010 |
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14683674 |
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10398222 |
Jul 3, 2003 |
7678415 |
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PCT/JP01/08759 |
Oct 4, 2001 |
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12656487 |
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Current U.S.
Class: |
427/2.14 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 9/1271 20130101; A61K 9/127 20130101; A61K 9/5015 20130101;
A61K 31/7088 20130101; A61K 9/5123 20130101; A61K 9/146 20130101;
A61K 9/5192 20130101; A61K 9/141 20130101; A61K 47/6911 20170801;
B01J 13/12 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 47/48 20060101 A61K047/48; A61K 31/7088 20060101
A61K031/7088; A61K 9/127 20060101 A61K009/127 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 4, 2000 |
JP |
305065/2000 |
Claims
1-19. (canceled)
20. A method for coating a complex of a nucleic acid and a liposome
with closed vesicles of a lipid bilayer, the liposome comprising
cationic lipid, the complex obtained by mixing the nucleic acid and
the liposome in water, which consists of the steps of (i) and (ii):
(i) suspending the complex in an aqueous solution comprising:
water, polar organic solvent(s) selected from an alcohol, a glycol
and a polyalkylene glycol, and lipid(s) selected from phospholipid,
glyceroglycolipid, sphingoglycolipid, cholesterol and synthetic
lipid, wherein the complex has a diameter of 10 nm to 1,000 nm;
wherein the lipid(s) is dissolved in the aqueous solution; and
wherein the aqueous solution does not comprise chloroform, and (ii)
adding water to the aqueous solution in which the complex is
suspended, so as to decrease the vol % concentration of the polar
organic solvent(s) in the aqueous solution, whereby the complex is
coated with closed vesicles of the lipid bilayer formed of the
lipid(s).
21. A method for coating a complex of a nucleic acid and a liposome
with closed vesicles of a lipid bilayer, the liposome comprising
cationic lipid the complex obtained by mixing the nucleic acid and
the liposome in water, which consists of the steps of (i), (ii),
(iii) and (iv): (i) suspending the complex in liquid A comprising:
water, and polar organic solvent(s) selected from an alcohol, a
glycol and a polyalkylene glycol, wherein the complex has a
diameter of 10 nm to 1,000 nm, and wherein the aqueous solution
does not comprise chloroform, (ii) dissolving lipid(s) selected
from phospholipid, glyceroglycolipid, sphingoglycolipid,
cholesterol and synthetic lipid, in liquid B consisting essentially
of: polar organic solvent(s) selected from an alcohol, a glycol and
a polyalkylene glycol, and optionally water, (iii) mixing the
liquid A and the liquid B, to obtain liquid C in which the complex
is suspended, and (iv) adding water to the liquid C so as to
decrease the vol % concentration of the polar organic solvent(s),
to obtain liquid D, and whereby the complex is coated with closed
vesicles of the lipid bilayer formed of the lipid(s).
22. The method according to claim 21, wherein the concentrations of
the polar organic solvent(s) in the liquid A and the liquid B are
60 to 90 vol %.
23. The method according to claim 22, wherein the concentration of
the polar organic solvent(s) in the liquid D is 50 vol % or
less.
24. The method according to claim 20, wherein the complex contains
a water-soluble polymer derivative.
25. The method according to claim 21, wherein the complex contains
a water-soluble polymer derivative.
26. The method according to claim 20, wherein the liposome
comprises a phospholipid and a dextran sulfate sodium salt.
27. The method according to claim 21, wherein the liposome
comprises a phospholipid and a dextran sulfate sodium salt.
28. The method according to claim 20, wherein the alcohol is
ethanol.
29. The method according to claim 21, wherein the alcohol is
ethanol.
30. The method according to claim 20, wherein the glycol is a
propylene glycol.
31. The method according to claim 21, wherein the glycol is a
propylene glycol.
32. The method according to claim 20, wherein the polyalkylene
glycol is polyethylene glycol.
33. The method according to claim 21, wherein the polyalkylene
glycol is polyethylene glycol.
34. The method according to claim 24, wherein the water-soluble
polymer derivative is one or more member(s) selected from
polyethylene glycolated lipid, a polyethylene glycol alkyl ether, a
polyethylene glycol castor oil derivative, a polyethylene glycol
sorbitan fatty acid ester, a polyethylene glycol stearate, a
copolymer of ethylene glycol with propylene glycol and a glycerol
ester.
35. The method according to claim 20, wherein the lipid(s) is one
or more member(s) selected from phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,
phosphatidylglycerol, phosphatidylinositol,
lysophosphatidylcholine, sphingomyelin, polyethylene glycolated
phospholipid, egg yolk lecithin, soybean lecithin and hydrogenated
phospholipid.
36. The method according to claim 21, wherein the lipid(s) is one
or more member(s) selected from phosphatidylcholine,
phosphatidylethoanolamine, phosphatidylserine, phosphatidic acid,
phosphatidylglcerol, phosphatidylinositol, lysophosphatidylcholine,
sphingomyelin, polyethylene glycolated phospholipid, egg yolk
lecithin, soybean lecithin and hydrogenated phospholipid.
37. The method according to claim 20, wherein the lipid(s) is a
mixture of egg yolk phosphatidylcholine (EggPC) and
1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethyleneglyco-
l 2000) (PEG-DSPE).
38. The method according to claim 21, wherein the lipid(s) is a
mixture of egg yolk phosphatidylcholine (EggPC) and
1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethyleneglyco-
l 2000) (PEG-DSPE).
39. The method according to claim 20, wherein the polar organic
solvent(s) is ethanol.
40. The method according to claim 21, wherein the polar organic
solvent(s) is ethanol.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for coating fine
particles with lipid membrane.
BACKGROUND ART
[0002] It has widely known that a drug is enclosed in fine
particles to enhance the effect of the drug, and that the fine
particles include, for example, liposome, fat emulsion, etc. Their
clinical applications are carried out mainly by injections,
particularly by intravascular administration. The fine particles
administered to blood vessel have been known to interact with blood
components and, as a result of the interaction, the fine particles
themselves or the drug is destructed (disintegrated), or the fine
particles are opsonized whereupon they are removed from the blood
(they are removed as an extraneous substance by a
reticuloendothelial system). In order to prevent that removal,
modification of liposome with polyethylene glycol, for example, has
been studied [Stealth Liposomes, ed. by D. D. Lasic and F. Martin,
CRC Press Inc., Florida, 93-102 (1995)].
[0003] Further, in order to deliver a nucleic acid such as
oligonucleotide, DNA and RNA to target cells, a complex of a
nucleic acid with liposome comprising lipid containing cationic
lipid (hereinafter, referred to as cationic lipid liposome), a
basic polymer such as poly-L-lysine and polyamideamine have been
frequently used. However, it has been known that, when a complex of
cationic lipid liposome with a nucleic acid is intravenously
administered, it is quickly distributed from blood to liver, lung,
etc. [Biochim. Biophys. Acta, 1281, 139-149 (1996); J. Controlled
Release, 41, 121-130 (1996)]. On the other hand, S. Li, et al. has
reported that, when mouse serum is contacted with a cationic lipid
liposome/DNA complex, an increase in the size of the complex,
aggregation, disintegration of liposome, and release and
disintegration of DNA has taken place [Gene Therapy, 5, 930-937
(1998); Gene Therapy, 6, 585-594 (1999)]. In order to solve those
problems, modification of cationic lipid liposome with polyethylene
glycol was studied, and O. Meyer, et al. prepared a complex of
oligodeoxynucleotide (ODN) with cationic lipid liposome containing
polyethylene glycol phosphatidylethanolamine (PEG-PE) [J. Biol.
Chem., 273, 15621-15627 (1998)]. However, when it was contacted
with a 50% aqueous solution of human plasma for 4 hours, 35% of ODN
were dissociated. In order to reduce the dissociation, D. Stuart
and T. Allen previously dissolved the cationic lipid liposome in
chloroform, mixed the resulting solvent with an aqueous ODN
solution and methanol, and transferred a cationic lipid
liposome/ODN complex to chloroform layer, and subjecting to
centrifugal separation. They further took out the chloroform layer,
added thereto PEG lipid, neutral lipid and water to form W/O
emulsion. They have tried to enclose ODN inside the liposome
completely by forming the W/O emulsion in a manner similar to a
reverse phase evaporation method of F. Szoka, et al. [Biochim.
Biophys. Acta, 1463, 219-229 (2000)]. In recent years, however, the
use of chloroform is not considered to be desirable in view of
safety. Further, D. McPhail, et al. prepared a vesicle-in-vesicle
where chitosan vesicle is placed in liposome by adding a vesicle
suspension (chitosan vesicle) of palmitoylchitosan and cholesterol
to a thin layer of egg yolk phosphatidylcholine and cholesterol
[Int. J. Pharmaceutics, 200, 73-86 (2000)]. However, there is no
description about the enclosing efficiency and, when guessed from
the preparation method, the enclosing efficacy was presumed to be
as low as about a few percent, which is presumed to cause a problem
in its practical use. From such viewpoints as well, convenient and
highly efficient enclosure of fine particles by closed vesicle is
very useful when application to medical treatment is aimed.
[0004] Further, there are some cases where many peptides and
proteins which are useful in medical care are quickly decomposed in
living body by enzyme or the like or are removed from living body
as a result of generation of an antibody by frequent
administrations, whereby their effect is no longer exerted.
Therefore, with an object of enhancing the stability of those
peptides and proteins in living body, it has been attempted to
enclose them into liposome. As the means of enclosing them into
liposome, there have been known, for example, a liposome
preparation method by Bangham, et al. [J. Mol. Biol., 13, 238
(1965)], an ethanol injection method [J. Cell Biol., 66, 621
(1975)], a French press method [FEBS Lett., 99, 210 (1979)], a
freeze-thaw method [Arch. Biochem. Biophys., 212, 186 (1981)], a
reverse phase evaporation method [Proc. Natl. Acad. Sci. USA, 75,
186 (1981)], a pH gradient method (Japanese Patent No. 2,572,554;
Japanese Patent No. 2,659,136; etc.) and the like. For
low-molecular compounds, a pH gradient method is appropriate and
improved method thereof has been devised as well. However, with
regard to peptides and proteins, invariably efficient enclosing has
not been achieved yet and, in the case of fluorescence-labeled
insulin, it was enclosed to an extent of about 5 to 40% but no
insulin was enclosed at all [Int. J. Pharmaceutics, 179, 85-95
(1999)]. In order to enhance the therapeutic effect by peptides and
proteins, it is in demand to develop a method whereby peptides and
proteins are efficiently enclosed within closed vesicles.
BRIEF SUMMARY OF THE INVENTION
[0005] An object of the present invention is to provide a safe,
convenient and efficient method for coating fine particles with
lipid membrane in order to stabilize a drug etc. contained in the
said fine particles. When the fine particles are enclosed in closed
vesicles comprising lipid bilayer or multilayer membrane using the
said coating method, affection by components in living body
particularly in blood or in gastrointestinal tract and by
reticuloendothelial system and also affection during storage period
by each of the fine particles or by a dispersing medium wherein the
fine particles are dispersed can be suppressed.
[0006] The present inventors have found that a complex comprising a
water-soluble drug and cationic lipid formed due to electrostatic
interaction is not soluble in an aqueous solution of ethanol and
that, although phospholipid is soluble in an aqueous solution of
ethanol having a high concentration of ethanol, it forms a liposome
due to formation of lipid membrane in an aqueous solution of
ethanol having a low concentration of ethanol. As a result of
further intensive investigations, it has been found that a complex
of a drug with lipid can be coated with a lipid membrane comprising
polyethylene glycolated lipid and phospholipids, when a
water-soluble polymer derivative such as polyethylene glycolated
lipid is previously added to a complex of a water-soluble drug with
lipid, the mixture is dispersed in an aqueous solution of ethanol
having a high concentration of ethanol, polyethylene glycolated
lipid and phospholipid are dissolved in the resulting liquid and
then the content of ethanol is gradually reduced.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows a complex of a nucleic acid and a liposome.
FIG. 1 also shows the complex with closed vesicles of a lipid
bilayer obtained in Examples 7 to 18. Examples 7-13 and 17 utilize
PEG-DSPE for the lipid bilayer, while Examples 14, 15, 16 and 18
use polyoxyethylene hydrogenated castor oil 60, CREMOPHOR and Tween
80 in place of PEG-DSPE for the lipid bilayer.
[0008] FIG. 2 shows the amount of F-PS distributed to tumor.
Abscissa shows time (hour(s)) after administration of the
preparation, while ordinate shows the amount of F-PS in tumor.
[0009] FIG. 3 shows the concentration of FD in plasma. Abscissa
shows time (hour(s)) after administration of the preparation, while
ordinate shows the concentration of FD in plasma.
[0010] FIG. 4 shows the concentration of F-PS in plasma. Abscissa
shows time (hour(s)) after administration of the preparation, while
ordinate shows the concentration of F-PS in plasma.
[0011] FIG. 5 shows the concentration of F-PS in plasma. Abscissa
shows time (hour(s)) after administration of the preparation, while
ordinate shows the concentration of F-PS in plasma.
[0012] Symbols used in FIG. 2 to FIG. 5 have the following
meanings.
[0013] FIG. 2
[0014] - -: group to which the preparation 7 was administered
[0015] -.largecircle.-: group to which the preparation f was
administered
[0016] FIG. 3
[0017] - -: group to which the preparation 1 was administered
[0018] -.diamond.-: group to which the preparation a was
administered
[0019] -.DELTA.-: group to which the preparation b was
administered
[0020] FIG. 4
[0021] -: group to which the preparation 7 was administered
[0022] -.diamond.-: group to which the preparation 8 was
administered
[0023] -.diamond-solid.-: group to which the preparation 9 was
administered
[0024] -.DELTA.-: group to which the preparation 10 was
administered
[0025] -.tangle-solidup.-: group to which the preparation 11 was
administered
[0026] -.quadrature.-: group to which the preparation 12 was
administered
[0027] -.box-solid.-: group to which the preparation 13 was
administered
[0028] -+-: group to which the preparation f was administered
[0029] FIG. 5
[0030] - -: group to which the preparation 7 was administered
[0031] -+-: group to which the preparation f was administered
[0032] -.diamond-solid.-: group to which the preparation g was
administered
DETAILED DESCRIPTION OF THE INVENTION
[0033] Thus, the present invention relates to the following (1) to
(19).
[0034] (1) A method for coating fine particles with lipid membrane,
which comprises coating fine particles with lipid membrane by
decreasing the rate of a polar organic solvent in an aqueous
solution containing the polar organic solvent where the fine
particles are dispersed and lipid is dissolved.
[0035] (2) A method for coating fine particles with lipid membrane,
which comprises coating fine particles with lipid membrane by
dispersing fine particles in an aqueous solution containing a polar
organic solvent (liquid A), dissolving lipid in a polar organic
solvent or an aqueous solution containing a polar organic solvent
which is the same as or different from the above aqueous solution
containing a polar organic solvent (liquid B), mixing the liquid A
and the liquid B into liquid C, and decreasing the rate of a polar
organic solvent in the liquid C to obtain liquid D.
[0036] (3) The method for coating fine particles with lipid
membrane according to the above (2), wherein the liquid B is a
solution which is prepared by dissolving a water-soluble polymer
derivative (I) together with the lipid.
[0037] (4) The method for coating fine particles with lipid
membrane according to the above (2) or (3), wherein the
concentrations of the polar organic solvent in the liquid A and the
liquid B are 30% or more.
[0038] (5) The method for coating fine particles with lipid
membrane according to the above (2) or (3), wherein the
concentrations of the polar organic solvent in the liquid A and the
liquid B are 60 to 90%.
[0039] (6) The method for coating fine particles with lipid
membrane according to the above (5), wherein the concentration of
the polar organic solvent in the liquid D is 50% or less.
[0040] (7) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (6), wherein the
fine particles are those containing a water-soluble polymer
derivative which is the same as or different from the water-soluble
polymer derivative (I) recited in the above (3).
[0041] (8) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (7), wherein the
fine particles are those containing one or more member(s) selected
from a drug, lipid assembly, liposome, fine particles in the
emulsion, natural polymer, synthetic polymer, metal colloid,
cationic lipid, anionic lipid and a fine particle preparation.
[0042] (9) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (7), wherein the
fine particles are those containing a drug.
[0043] (10) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (7), wherein the
fine particles comprise a complex of a drug with one or more
member(s) selected from lipid assembly, liposome, fine particles in
the emulsion, natural polymer, synthetic polymer, metal colloid,
cationic lipid, anionic lipid and a fine particle preparation.
[0044] (11) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (7), wherein the
fine particles comprise a complex of a drug with cationic
lipid.
[0045] (12) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (7), wherein the
fine particles comprise a complex of a drug with anionic lipid.
[0046] (13) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (7), wherein the
fine particles comprise a complex of a drug, liposome containing
phospholipid and a dextran sulfate sodium salt.
[0047] (14) The method for coating fine particles with lipid
membrane according to any one of the above (8) to (13), wherein the
drug is a drug selected from a peptide, a protein, a nucleic acid,
a low-molecular compound, a saccharide and a polymer compound.
[0048] (15) The method for coating fine particles with lipid
membrane according to any one of the above (1) to (14), wherein the
polar organic solvent is one or more member(s) selected from an
alcohol, a glycol and a polyalkylene glycol.
[0049] (16) The method for coating fine particles with lipid
membrane according to the above (15), wherein the alcohol is
ethanol.
[0050] (17) The method for coating fine particles with lipid
membrane according to the above (15) or (16), wherein the glycol is
a propylene glycol.
[0051] (18) The method for coating fine particles with lipid
membrane according to any one of the above (15) to (17), wherein
the polyalkylene glycol is polyethylene glycol.
[0052] (19) The method for coating fine particles with lipid
membrane according to any one of the above (3) to (18), wherein the
water-soluble polymer derivative is one or more member(s) selected
from polyethylene glycolated lipid, a polyethylene glycol alkyl
ether, a polyethylene glycol castor oil derivative, a polyethylene
glycol sorbitan fatty acid ester, a polyethylene glycol stearate, a
copolymer of ethylene glycol with propylene glycol and a glycerol
ester.
[0053] Examples of the polar organic solvent in the aqueous
solution containing the polar organic solvent used in the present
invention are an alcohol such as methanol, ethanol, n-propanol,
isopropanol, n-butanol, isobutanol and tert-butanol; a glycol such
as glycerol, ethylene glycol and propylene glycol; and polyalkylene
glycol such as polyethylene glycol.
[0054] With regard to the thing which constitutes the fine
particles used in the present invention, there is no particular
limitation and its examples are a drug, lipid assembly, liposome,
fine particles in emulsion, natural polymer, synthetic polymer,
metal colloid, cationic lipid, anionic lipid, a fine particle
preparation and a water-soluble polymer derivative. They may be
used independently, as a complex where two or more of them are
combined, or as a complex where one or more of them and another
compound are combined.
[0055] To be specific, an example of the above-mentioned complex is
a complex of drug with one or more member(s) selected from lipid
assembly, liposome, fine particles in emulsion, natural polymer,
synthetic polymer, metal colloid, cationic lipid, anionic lipid and
a fine particle preparation and, to be more specific, it is a
complex of a nucleic acid with cationic lipid formed due to
electrostatic interaction; a complex of a nucleic acid with
positive-charged polymer such as poly-L-lysine; a complex of a
basic protein having a high isoelectric point with anionic lipid
such as phosphatidic acid or negative-charged polymer such as
styrene-maleic acid; a complex of an acidic protein with
positive-charged polymer such as cationic lipid and poly-L-lysine;
etc.
[0056] With regard to the drug, its examples are substances having
a pharmacological activity such as a protein including enzyme, a
peptide, a nucleic acid including gene, a low-molecular compound, a
saccharide and a polymer compound. Examples of the protein
including enzyme and the peptide are bradykinin, angiotensin,
oxytocin, vasopressin, adrenocorticotropin (ACTH), calcitonin,
insulin, glucagon, cholecystokinin, .beta.-endorphin, melanocyte
inhibiting factor, melanocyte stimulating hormone, gastrin
antagonist, neurotensin, somatostatin, brucine, cyclosporine,
enkephalin, transferrin, RGD (Arg-Gly-Asp) peptide, thyroid
hormone, growth hormone, gonadotropic hormone, luteinizing hormone
(LHRH), asparaginase, arginase, uricase, carboxypeptidase,
glutaminase, superoxide dismutase (SOD), tissue plasminogen
activator (t-PA), streptokinase, interleukin, interferon, muramyl
dipeptide, thymopoietin, granulocyte colony stimulating factor
(G-CSF), granulocyte microphage colony stimulating factor (GM-CSF),
erythropoietin (EPO), thrombopoietin (TPO), trypsin inhibitor,
lysozyme, epidermal growth factor (EGF), insulin-like growth factor
(IGF), nerve growth factor (NGF), platelet-derived growth factor
(PDGF), transforming growth factor (TGF), endothelial cell growth
factor (ECGF), fibroblast growth factor (FGF), glial growth factor
(GGF), thymosin and a specific antibody (such as anti-EGF receptor
antibody); examples of the nucleic acid including gene are nucleic
acids such as antisense oligonucleotide, sense oligonucleotide, DNA
and RNA; examples of the low-molecular compound are
.epsilon.-aminocaproic acid, arginine hydrochloride, potassium
L-aspartate, tranexamic acid, bleomycin sulfate, vincristine
sulfate, cefazolin sodium, cephalothin sodium, citicoline,
cytarabine, gentamicin sulfate, vancomycin hydrochloride, kanamycin
sulfate and amikacin sulfate; examples of the saccharide are sodium
chondroitin sulfate, heparin sodium and dextran fluorescein; and
examples of the polymer compound are sodium polyethylene sulfonate,
DIVEMA (copolymer of divinyl ether with maleic anhydride) and
SMANCS (bonded product of a styrene-maleic anhydride copolymer with
neocarzinostatin).
[0057] Examples of the lipid assembly are spherical micelle,
spherical reversed micelle, sausage-shaped micelle, sausage-shaped
reversed micelle, plate-shaped micelle, plate-shaped reversed
micelle, hexagonal I, hexagonal II and associated product
comprising two or more lipid molecules.
[0058] Examples of the lipid constituting the liposome are
phospholipid, glyceroglycolipid, sphingoglycolipid, cholesterol and
cationic lipid, and phospholipid is preferably used. Such lipid may
be modified by nonionic detergent such as Polysorbate 80, Pluronic
F68 and sorbitan monolaurate (e.g., Span 20); cationic detergent
such as benzalkonium chloride; anionic detergent such as sodium
lauryl sulfate; polysaccharide such as dextran or a derivative
thereof; a polyoxyethylene derivative such as polyoxyethylene
lauryl alcohol and polyethylene glycol (PEG); etc.
[0059] Examples of the phospholipid are natural or synthetic
phospholipids such as phosphatidylcholine (soybean
phosphatidylcholine, egg yolk phosphatidylcholine, distearoyl
phosphatidylcholine, dipalmitoyl phosphatidylcholine, etc.),
phosphatidylethanolamine (distearoyl phosphatidylethanolamine,
dipalmitoyl phosphatidylethanolamine, etc.), phosphatidylserine,
phosphatidic acid, phosphatidylglycerol, phosphatidylinositol,
lysophosphatidylcholine, sphingomyelin, polyethylene glycolated
phospholipid, egg yolk lecithin, soybean lecithin and hydrogenated
phospholipid.
[0060] Examples of the glyceroglycolipid are sulfoxyribosyl
glyceride, diglycosyl diglyceride, digalactosyl diglyceride,
galactosyl diglyceride and glycosyl diglyceride.
[0061] Examples of the sphingoglycolipid are galactosyl
cerebroside, lactosyl cerebroside and ganglioside.
[0062] Examples of the cationic lipid are 1,2-dioleoyl-3-trimethyl
ammonium propane (DOTAP),
N-(2,3-dioleyloxypropan-1-yl)-N,N,N-trimethylammonium chloride
(DOTMA), 2,3-dioleyloxy-N-[2
(sperminecarboxyamido)ethyl]-N,N-dimethyl-1-propanaminium
trifluoroacetate (DOSPA),
1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide
(DMRIE), 1,2-dioleoyloxypropyl-3-diethylhydroxyethylammonium
bromide (DORIE) and
3.beta.-[N--(N',N'-dimethylaminoethyl)carbamoyl]-cholesterol
(DC-Chol).
[0063] In liposome, those lipids each is used either independently
or jointly. When they are used jointly, the lipids used are, for
example, lipids comprising at least two components selected from
hydrogenated soybean phosphatidylcholine, polyethylene glycolated
phospholipid and cholesterol; lipids comprising at least two
components selected from distearoyl phosphatidylcholine,
polyethylene glycolated phospholipid and cholesterol; lipids
comprising egg yolk phosphatidylcholine and DOTAP; lipids
comprising egg yolk phosphatidylcholine, DOTAP and polyethylene
glycolated phospholipid; lipids comprising egg yolk
phosphatidylcholine, DOTAP, cholesterol and polyethylene glycolated
phospholipid; etc.
[0064] Further, in the preparation of liposome, sterol or the like
such as cholesterol as a membrane stabilizer, tocopherol or the
like as an antioxidant and stearylamine, dicetyl phosphate,
ganglioside and cationic lipid such as DOTMA [Proc. Natl. Acad.
Sci. USA, 84, 7413-7417 (1987)], dioctadecylamidoglycyl spermine
(DOGS) [Proc. Natl. Acad. Sci. USA, 86, 6982-6986 (1989)], DMRIE or
DORIE [Methods, 5, 67-75 (1993)] and DC-Chol [Biochem. Biophys.
Res. Commun., 179, 280-285 (1991)] as a charging substance may also
be added, if necessary, together with lipid.
[0065] There is no particular limitation for the fine particles in
the emulsion, and their specific examples are fine particles
contained in all kinds of O/W emulsion and W/O/W emulsion such as a
fat emulsion, an emulsion comprising nonionic detergent and soybean
oil, a lipid emulsion and a lipid nanosphere.
[0066] There is no particular limitation for the natural polymer,
and its specific examples are albumin, dextran, chitosan,
deoxyribonucleic acid and the like.
[0067] There is no particular limitation for the synthetic polymer
substance and its specific examples are poly-L-lysine,
polyethyleneimine, polyaspartic acid, a copolymer of styrene with
maleic acid, a copolymer of isopropylacrylamide with
acrylpyrrolidone, PEG-modified dendrimer, polylactic acid,
polylactic acid polyglycolic acid, polyethylene glycolated
polylactic acid, dextran sulfate and a salt thereof.
[0068] Here, the salt includes metal salt, ammonium salt, organic
amine addition salt, amino acid addition salt, etc. Examples of the
metal salt are alkaline metal salt such as lithium salt, sodium
salt and potassium salt; alkaline earth metal salt such as
magnesium salt and calcium salt; aluminum salt; zinc salt; etc.
Examples of the ammonium salt are ammonium salt,
tetramethylammonium salt, etc.; examples of the organic amine
addition salt are addition salts of morpholine, piperidine, etc.;
and examples of the amino acid addition salt are addition salts of
glycine, phenylalanine, aspartic acid, glutamic acid, lysine,
etc.
[0069] Examples of the metal colloid are metal colloids containing
gold, silver, platinum, copper, rhodium, silica, calcium, aluminum,
iron, indium, cadmium, barium, lead, etc.
[0070] With regard to the cationic lipid, the same ones as those
mentioned above may be exemplified.
[0071] Examples of the anionic lipid are phosphatidylserine,
phosphatidylglycerol and phosphatidylinositol.
[0072] Examples of the fine particle preparation are microsphere,
microcapsule, nanocrystal and lipid nanoparticle polymer
micelle.
[0073] With regard to the fine particles, those where a drug is
enclosed in liposome may be preferably exemplified.
[0074] With regard to the size of the fine particles, an average
particle size thereof is preferably from several nm to several tens
.mu.m and, more preferably, from 10 nm to 1,000 nm.
[0075] There is no particular limitation for the water-soluble
polymer derivative used in the present invention, and its examples
are polyethylene glycolated lipid such as PEG-PE,
1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethyleneglyco-
l 2000) (PEG-DSPE); polyethylene glycol alkyl ether; a
polyoxyethylene castor oil derivative such as polyoxyethylene
hydrogenated castor oil 60 and Cremophor EL; a polyethylene glycol
sorbitan fatty acid ester such as polyoxyethylene sorbitan
monooleate (Tween 80); a polyethylene glycol stearic acid ester; a
copolymer of ethylene glycol with propylene glycol; a glycerol
ester; a polyethyleneimine derivative; a polyvinyl alcohol
derivative; a polyacrylic acid derivative; a polyacrylamide
derivative; a dextran derivative; a polyglycerol derivative; a
chitosan derivative; a polyvinylpyrrolidone derivative; a
polyaspartic amide derivative; a poly-L-lysine derivative; a mannan
derivative; and a pullulan derivative.
[0076] Examples of the lipid used for the lipid membrane in the
present invention are phospholipid, glyceroglycolipid,
sphingoglycolipid, cholesterol and synthetic lipid, and
phospholipid is particularly preferably used. With regard to the
phospholipid, glyceroglycolipid and sphingoglycolipid, the same
ones as mentioned above may be exemplified and, with regard to the
synthetic lipid, there may be exemplified fluorine-added
phosphatidylcholine, fluorine-added detergent and dialkylammonium
bromide.
[0077] With regard to the method for the preparation of an aqueous
solution containing a polar organic solvent in which the fine
particles are dispersed and the lipid is dissolved to be used in
the present invention, there may be exemplified a method where fine
particles are dispersed in an aqueous solution containing a polar
organic solvent (liquid A), lipid is dissolved in a polar organic
solvent or an aqueous solution containing a polar organic solvent
which is the same as or different from the said aqueous solution
containing a polar organic solvent (liquid B) and then the liquid A
and the liquid B are mixed.
[0078] With regard to the method for coating the fine particles
with lipid membrane according to the present invention, the
following method may be specifically exemplified.
[0079] (step 1) The fine particles are dispersed (suspended) in an
aqueous solution containing a polar organic solvent, preferably in
an aqueous solution containing an alcohol such as ethanol;
[0080] (step 2) The lipid which is dissolved in an aqueous solution
containing a polar organic solvent which is the same as or
different from the aqueous solution containing the said polar
organic solvent or, preferably in the same aqueous solution
containing the polar organic solvent or in the polar organic
solvent is added to the suspension prepared in the step 1, followed
by mixing. At that time, a water-soluble polymer derivative may be
further added thereto and there is no particular limitation for the
amount of the water-soluble polymer derivative to be added here;
and
[0081] (step 3) Small amounts of water are added to the mixed
solution prepared in the step 2, dialysis is carried out or the
polar organic solvent is evaporated so as to decrease the rate of
the polar organic solvent in the mixed solution whereby the
dissolved lipid (lipid and the water-soluble polymer derivative, in
case the water-soluble polymer derivative is added in the step 2)
is accumulated on the surface of the fine particles, a lipid
membrane is formed on the surface of the fine particles and closed
vesicles in which the fine particles are enclosed are prepared.
[0082] There is no particular limitation for the rate of the polar
organic solvent in the aqueous solution containing the polar
organic solvent used in the method of the present invention so far
as the conditions that the fine particles are present without being
dissolved, and that the components constituting the lipid membrane
coating the fine particles are dissolved is satisfied and, although
the rate varies depending upon the solvent and the fine particles
used and the type of the lipid used, it is preferably 30% or more
and, more preferably, it is 60 to 90%. Further, with regard to the
rate of the polar organic solvent in the mixed solution after
decreased during the above-mentioned step 3, there is no particular
limitation so far as the rate is within such an extent that the
dissolved lipid (lipid and the water-soluble polymer derivative, in
case the water-soluble polymer derivative is added in the step 2)
is/are accumulated on the surface of the fine particles whereby a
lipid membrane can be formed on the surface of the fine particles
and, preferably, concentration of the polar organic solvent in the
aqueous solution is 50% or less.
[0083] Although the rate of the fine particles used in the present
invention to the aqueous solution containing the polar organic
solvent or to the preparation obtained by the method of the present
invention is not particularly limited so far as the said fine
particles can be coated with the lipid membrane, it is preferably 1
.mu.g/mL to 1 g/mL or, more preferably, 0.1 to 500 mg/mL.
[0084] Although the rate of the lipid used in the present invention
to the aqueous solution containing the polar organic solvent or to
the preparation obtained by the method of the present invention is
not particularly limited so far as the fine particles can be coated
therewith, it is preferably 1 .mu.g/mL to 1 g/mL or, more
preferably, 0.1 to 400 mg/mL.
[0085] Independently of the type of the fine particles used, it is
basically possible to coat the fine particles with the lipid
membrane by the same method as mentioned above.
[0086] With regard to the polar organic solvent and the lipid used
in the present invention, commercially available ones may be
used.
[0087] The fine particles used in the present invention are
available from the market or may be manufactured by known
methods.
[0088] For example, for the manufacture of liposome constituting
the fine particles, a known method for the preparation of liposome
may be applied. With regard to a known method for the preparation
of liposome, there may be exemplified a liposome preparation method
by Bangham, et al. [J. Mol. Biol., 13, 238 (1965)], an ethanol
injection method [J. Cell. Biol., 66, 621 (1975)], a French press
method [FEBS Lett., 99, 210 (1979)], a freeze-thaw method [Arch.
Biochem. Biophys., 212, 186 (1981)], a reverse phase evaporation
method [Proc. Natl. Acad. Sci. USA, 75, 4194 (1978)] and a pH
gradient method (Japanese Patent No. 2,572,554; Japanese Patent No.
2,659,136; etc.).
[0089] Improvement in quality of liposome surface by nonionic
detergent, cationic detergent, anionic detergent, polysaccharide or
a derivative thereof, a polyoxyethylene derivative, etc. may also
be carried out optionally, and those liposomes where quality of the
surface is improved may also be used as fine particles of the
present invention [Stealth Liposomes, ed. by D. D. Lasic and F.
Martin, CRC Press Inc., Florida, 93-102 (1995)].
[0090] With regard to a solution for suspending the liposome in the
manufacture of liposome constituting the fine particles, there may
be used acid, alkali, various kinds of buffers, physiological
saline, amino acid infusion solution, etc. in addition to water. It
is also possible to add an antioxidant such as citric acid,
ascorbic acid, cysteine and ethylenediamine tetraacetic acid (EDTA)
to a liposome suspension. It is further possible to add glycerol,
glucose, sodium chloride, etc. as an isotonizing solution.
[0091] It is also possible that a drug and lipid are dissolved in
an organic solvent such as ethanol, that the solvent is evaporated
therefrom, a physiological saline or the like is added to the
residue and that the mixture is shaken and stirred to form
liposome.
[0092] A water-soluble polymer derivative may be added when the
drug is enclosed in liposome, when a complex of the liposome with
the drug is formed or after that.
[0093] There is no particular limitation for an average particle
size of the liposome and it may be freely selected upon demand.
Examples of a method for adjusting the average particle size are an
extrusion method and a method where a big multilamellar liposome
(MLV) is mechanically pulverized using Manton-gaulin,
microfluidizers, etc. [R. H. Muller, S. Benita, B. Bohm (ed.),
"Emulsion and Nanosuspensions for the Formulation of Poorly Soluble
Drugs", High-Pressure Homogenization Techniques for the Production
of Liposome Dispersions: Potential and Limitations, M. Brandl,
267-294 (1998) (Scientific Publishers, Stuttgart, Germany)].
[0094] A method for the formation of a complex combined by two or
more members selected from a drug constituting the fine particles,
lipid assembly, liposome, fine particles in emulsion, natural
polymer, synthetic polymer, metal colloid, cationic lipid, anionic
lipid, a fine particle preparation and a water-soluble polymer
derivative may be a method where the drug is just mixed with lipid,
polymer, etc. in water and, at that time, particle size selecting
step, aseptic step, etc. may be further added if necessary. It is
also possible that formation of the complex is carried out in
various solvents such as acetone and ether.
[0095] With regard to the fine particles used in the present
invention and/or the lipid membrane which coats the fine particles
obtained by the present invention, it is possible that the surface
thereof is modified by a protein such as an antibody, a saccharide,
glycolipid, an amino acid, a nucleic acid and various low-molecular
compounds and polymer compounds, or that such a substance is just
added to the fine particles and/or lipid membrane followed by
using.
[0096] For example, in order to apply to targeting, it is possible
that the coated membrane is further subjected to a surface
modification of the lipid membrane using a protein (including an
antibody), a peptide, a fatty acid, etc. [Stealth Liposomes, ed. by
D. D. Lasic and F. Martin, CRC Press Inc., Florida, 93-102,
(1995)].
[0097] The fine particles coated with the lipid membrane obtained
by the method of the present invention may be used as they are, or
depending upon the purpose of use, storage condition, etc., they
may be freeze-dried after addition of excipient such as mannitol,
lactose, trehalose, maltose or glycine thereto. Freeze preservation
after addition of a freeze-preserving agent such as glycerol is
also possible. It is further possible that granulation, drying,
etc. are carried out together with an appropriate excipient to
manufacture an oral preparation such as capsules, tablets or
granules.
[0098] The fine particles coated with the lipid membrane obtained
by the method of the present invention may be suspended using acid,
alkali, various buffers, physiological saline, amino acid
transfusion solution, etc. in addition to water. It is also
possible that an antioxidant such as citric acid, ascorbic acid,
cysteine or EDTA is added to a liposome suspension. It is further
possible to add glycerol, glucose, sodium chloride or the like as
an isotonizing agent.
[0099] The preparation obtained by the method of the present
invention is generally used as injections but it is also possible
to use after preparing into oral preparations, nasal drops,
ophthalmic solutions, percutaneous preparations, suppositories,
inhalation preparations or the like.
[0100] The preparation obtained by the present invention may be
used with such an object as stabilization of the drug in living
body components such as blood components or in blood or
gastrointestinal tracts, reduction of side effect, increase in
accumulation property to target organ such as tumor, improvement in
absorption per os or via mucous membrane, etc.
[0101] Examples and Comparative Examples of the present invention
will be shown hereunder.
BEST MODE FOR CARRYING OUT THE INVENTION
Example 1
[0102] Distilled water (3 mL) was added to 10 mg of dextran
fluorescein anionic (FD) (manufactured by Molecular Probes), 60 mg
of DOTAP (manufactured by Avanti) and 24 mg of PEG-DSPE
(manufactured by Avanti), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times
and then a polycarbonate membrane filter of 0.1 .mu.m for 20 times
and 4 mL of ethanol was added thereto. After that, to this
suspension was added a solution prepared by dissolving 240 mg of
egg yolk phosphatidylcholine (EggPC) and 50 mg of PEG-DSPE in 1 mL
of ethanol. Distilled water (92 mL) was gradually added to the
suspension so that the concentration of ethanol was adjusted to 5%
or lower. The resulting liposome suspension was subjected to an
ultracentrifugal separation (110,000.times.g at 25.degree. C. for 1
hour) and the supernatant liquid was removed. A phosphate-buffered
saline (PBS) was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 1) was
obtained.
[0103] When an average particle size of the liposome was measured
by means of a dynamic light scattering (DLS) [A model ELS-800,
Otsuka Electronics, Ltd.; hereinafter the same one was used], it
was found to be 134 nm.
Example 2
[0104] Distilled water (3 mL) was added to 10 mg of FD, 60 mg of
DOTAP and 24 mg of PEG-DSPE, followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times
and then a polycarbonate membrane filter of 0.1 .mu.m for 20 times
and 4 mL of ethanol was added thereto. After that, to this
suspension was added a solution prepared by dissolving 120 mg of
EggPC and 25 mg of PEG-DSPE in 1 mL of ethanol. Distilled water (92
mL) was gradually added to this suspension so that the
concentration of ethanol was adjusted to 5% or lower. The resulting
liposome suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 2) was
obtained.
[0105] When an average particle size of the liposome was measured
by means of DLS, it was found to be 179 nm.
Example 3
[0106] Distilled water (3 mL) was added to 10 mg of FD, 60 mg of
DOTAP and 24 mg of PEG-DSPE, followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times
and then a polycarbonate membrane filter of 0.1 .mu.m for 20 times
and 4 mL of ethanol was added thereto. After that, to this
suspension was added a solution prepared by dissolving 60 mg of
EggPC and 12.5 mg of PEG-DSPE in 1 mL of ethanol. Distilled water
(92 mL) was gradually added to this suspension so that the
concentration of ethanol was adjusted to 5% or lower. The resulting
liposome suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 3) was
obtained.
[0107] When an average particle size of the liposome was measured
by means of DLS, it was found to be 184 nm.
Example 4
[0108] Distilled water (3 mL) was added to 10 mg of FD and 60 mg of
DOTAP, followed by shaking and stirring in a vortex mixer. The
suspension was passed, at room temperature, through a polycarbonate
membrane filter of 0.4 .mu.m for 20 times and then a polycarbonate
membrane filter of 0.1 .mu.m for 20 times and 4 mL of ethanol was
added thereto. After that, to this suspension was added a solution
prepared by dissolving 240 mg of EggPC and 74 mg of PEG-DSPE in 1
mL of ethanol. Distilled water (92 mL) was gradually added to this
suspension so that the concentration of ethanol was adjusted to 5%
or lower. The resulting liposome suspension was subjected to an
ultracentrifugal separation (110,000.times.g at 25.degree. C. for 1
hour) and the supernatant liquid was removed. PBS was added thereto
followed by subjecting to re-suspending so as to adjust the total
lipid concentration to 30 mg/mL, whereupon a liposome suspension
(Preparation 4) was obtained.
[0109] When an average particle size of the liposome was measured
by means of DLS, it was found to be 131 nm.
Example 5
[0110] Distilled water (3 mL) was added to 10 mg of FD and 60 mg of
DOTAP, followed by shaking and stirring in a vortex mixer. The
suspension was passed, at room temperature, through a polycarbonate
membrane filter of 0.4 .mu.m for 20 times and then a polycarbonate
membrane filter of 0.1 .mu.m for 20 times and 4 mL of ethanol was
added thereto. After that, to this suspension was added a solution
prepared by dissolving 240 mg of EggPC in 1 mL of ethanol.
Distilled water (92 mL) was gradually added to this suspension so
that the concentration of ethanol was adjusted to 5% or lower. The
resulting liposome suspension was subjected to an ultracentrifugal
separation (110,000.times.g at 25.degree. C. for 1 hour) and the
supernatant liquid was removed. PBS was added thereto followed by
subjecting to re-suspending so as to adjust the total lipid
concentration to 30 mg/mL, whereupon a liposome suspension
(Preparation 5) was obtained.
[0111] When an average particle size of the liposome was measured
by means of DLS, it was found to be 458 nm.
Example 6
[0112] Distilled water (1.49 mL) and 0.01 mL of an aqueous solution
of sodium hydroxide (1 mol/L) were added to 2.5 mg of fluorescein
isothiocyanate (FITC)-labeled insulin (F-Ins), 30 mg of DOTAP and
12 mg of PEG-DSPE, followed by shaking and stirring in a vortex
mixer. The suspension was passed, at room temperature, through a
polycarbonate membrane filter of 0.4 .mu.m for 20 times and then a
polycarbonate membrane filter of 0.1 .mu.m for 20 times and 2 mL of
ethanol was added thereto. After that, to this suspension was added
a solution prepared by dissolving 120 mg of EggPC and 25 mg of
PEG-DSPE in 0.5 mL of ethanol. Distilled water (46 mL) was
gradually added to this suspension so that the concentration of
ethanol was adjusted to 5% or lower. The resulting liposome
suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 6) was
obtained.
[0113] When an average particle size of the liposome was measured
by means of DLS, it was found to be 132 nm.
Example 7
[0114] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and
additionally a polycarbonate membrane filter of 0.05 .mu.m for 20
times. To this suspension (0.5 mL) was added 0.25 mL of a solution
prepared by dissolving fluorescein isothiocyanate-bound
phosphorothioate (F-PS) (manufactured by Sci Media) in distilled
water to adjust to 15 mg/mL, followed by adding 1 mL of ethanol
thereto. To this suspension was further added 0.25 mL of a solution
of EggPC/PEG-DSPE/ethanol (120 mg/25 mg/mL). Distilled water (23
mL) was gradually added to this suspension so that the
concentration of ethanol was adjusted to 5% or lower. The resulting
liposome suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension was obtained. PEG-DSPE
(ratio of PEG-DSPE to EggPC is 50:120 (weight:weight)) was
dissolved in a small amount of ethanol (4% by volume of the
liposome suspension) followed by mixing with the liposome
suspension and heating at 70.degree. C. for 2 minutes. PBS was
added thereto so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 7) was
obtained.
[0115] When an average particle size of the liposome was measured
by means of DLS, it was found to be 111 nm.
Example 8
[0116] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and
additionally a polycarbonate membrane filter of 0.05 .mu.m for 20
times. To this suspension (0.4 mL) was added 0.2 mL of a solution
prepared by dissolving F-PS in distilled water to adjust to 10
mg/mL followed by adding 0.8 mL of ethanol thereto. To this
suspension was further added 0.2 mL of a solution of
EggPC/PEG-DSPE/ethanol (240 mg/50 mg/mL). Distilled water (18.4 mL)
was gradually added to this suspension so that the concentration of
ethanol was adjusted to 5% or lower. The resulting liposome
suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 8) was
obtained.
[0117] When an average particle size of the liposome was measured
by means of DLS, it was found to be 112 nm.
Example 9
[0118] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and
additionally a polycarbonate membrane filter of 0.05 .mu.m for 20
times. To this suspension (0.4 mL) was added 0.2 mL of a solution
prepared by dissolving F-PS in distilled water to adjust to 15
mg/mL followed by adding 0.8 mL of ethanol thereto. To this
suspension was further added 0.2 mL of a solution of
EggPC/PEG-DSPE/ethanol (240 mg/50 mg/mL). Distilled water (18.4 mL)
was gradually added to this suspension so that concentration of
ethanol was adjusted to 5% or lower. The resulting liposome
suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 9) was
obtained.
[0119] When an average particle size of the liposome was measured
by means of DLS, it was found to be 137 nm.
Example 10
[0120] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and
additionally a polycarbonate membrane filter of 0.05 .mu.m for 20
times. To this suspension (0.4 mL) was added 0.2 mL of a solution
prepared by dissolving F-PS in distilled water to adjust to 17.5
mg/mL followed by adding 0.8 mL of ethanol thereto. To this
suspension was further added 0.2 mL of a solution of
EggPC/PEG-DSPE/ethanol (240 mg/50 mg/mL). Distilled water (18.4 mL)
was gradually added to this suspension so that the concentration of
ethanol was adjusted to 5% or lower. The resulting liposome
suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 10) was
obtained.
[0121] When an average particle size of the liposome was measured
by means of DLS, it was found to be 132 nm.
Example 11
[0122] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and
additionally a polycarbonate membrane filter of 0.05 .mu.m for 20
times. To this suspension (0.4 mL) was added 0.2 mL of a solution
prepared by dissolving F-PS in distilled water to adjust to 20
mg/mL followed by adding 0.8 mL of ethanol thereto. To this
suspension was further added 0.2 mL of a solution of
EggPC/PEG-DSPE/ethanol (240 mg/50 mg/mL). Distilled water (18.4 mL)
was gradually added to this suspension so that the concentration of
ethanol was adjusted to 5% or lower. The resulting liposome
suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 11) was
obtained.
[0123] When an average particle size of the liposome was measured
by means of DLS, it was found to be 164 nm.
Example 12
[0124] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and
additionally a polycarbonate membrane filter of 0.05 .mu.m for 20
times. To this suspension (0.25 mL) was added 0.125 mL of a
solution prepared by dissolving F-PS in distilled water to adjust
to 15 mg/mL followed by adding 0.5 mL of ethanol thereto. To this
suspension was further added 0.125 mL of a solution of
EggPC/PEG-DSPE/ethanol (240 mg/100 mg/mL). Distilled water (11.5
mL) was gradually added to this suspension so that the
concentration of ethanol was adjusted to 5% or lower. The resulting
liposome suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 12) was
obtained.
[0125] When an average particle size of the liposome was measured
by means of DLS, it was found to be 122 nm.
Example 13
[0126] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and
additionally a polycarbonate membrane filter of 0.05 .mu.m for 20
times. To this suspension (0.25 mL) was added 0.125 mL of a
solution prepared by dissolving F-PS in distilled water to adjust
to 15 mg/mL followed by adding 0.5 mL of ethanol thereto. To this
suspension was further added 0.125 mL of a solution of
EggPC/PEG-DSPE/ethanol (360 mg/150 mg/mL). Distilled water (11.5
mL) was gradually added to this suspension so that the
concentration of ethanol was adjusted to 5% or lower. The resulting
liposome suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS was added thereto followed by subjecting to
re-suspending so as to adjust the total lipid concentration to 30
mg/mL, whereupon a liposome suspension (Preparation 13) was
obtained.
[0127] When an average particle size of the liposome was measured
by means of DLS, it was found to be 165 nm.
Example 14
[0128] DOTAP (30 mg/mL) and PEG-DSPE (12 mg/mL) suspensions were
shaken and stirred in a vortex mixer. The suspension was passed, at
room temperature, through a polycarbonate membrane filter of 0.4
.mu.m for 20 times, a polycarbonate membrane filter of 0.1 .mu.m
for 20 times and additionally a polycarbonate membrane filter of
0.05 .mu.m for 20 times to give a suspension of liposome where
particle size was around 80 nm. Under stirring by a stirrer, 125
.mu.L of a 15 mg/mL aqueous solution of F-PS was added to 250 .mu.L
of the suspension followed by adding 0.5 mL of ethanol thereto. To
this suspension was further added 125 .mu.L of a solution where 120
mg of EggPC and 25 mg of polyoxyethylene hydrogenated castor oil 60
(manufactured by NOF CORPORATION) were dissolved in 1 mL of
ethanol. Distilled water (11.5 mL) was gradually added to this
suspension so that the concentration of ethanol was adjusted to 5%
or lower. The resulting liposome suspension was subjected to an
ultracentrifugal separation (110,000.times.g at 25.degree. C. for 1
hour) and the supernatant liquid was removed. PBS (40 .mu.L) was
added thereto followed by subjecting to re-suspending and further
diluting with PBS, whereupon a liposome suspension of 3 mg/mL of
F-PS was obtained. PEG-DSPE (ratio of PEG-DSPE to EggPC is 50:120
(weight:weight)) was dissolved in a small amount (about 1/25 by
volume of the liposome suspension) of ethanol. Each of the liposome
suspension and the ethanolic solution of PEG-DSPE was heated at
70.degree. C. for 2 minutes. Then the liposome suspension was added
to the ethanolic solution of PEG-DSPE and, after mixing, the
mixture was heated at 70.degree. C. for 2 minutes and cooled with
water. To the resulting liposome suspension was added PBS to dilute
so that the total lipid concentration was adjusted to 30 mg/mL,
whereupon a liposome suspension (Preparation 14) was obtained.
[0129] When an average particle size of the liposome was measured
by means of DLS, it was found to be 116 nm.
Example 15
[0130] DOTAP (30 mg/mL) and PEG-DSPE (12 mg/mL) suspensions were
shaken and stirred in a vortex mixer. The suspension was passed, at
room temperature, through a polycarbonate membrane filter of 0.4
.mu.m for 20 times, a polycarbonate membrane filter of 0.1 .mu.m
for 20 times and additionally a polycarbonate membrane filter of
0.05 .mu.m for 20 times to give a suspension of liposome where
particle size was around 80 nm. Under stirring by a stirrer, 250
.mu.L of a 15 mg/mL aqueous solution of F-PS was added to 500 .mu.L
of the suspension followed by adding 1 mL of ethanol thereto. To
this suspension was further added 250 .mu.L of a solution where 120
mg of EggPC and 25 mg of Cremophor EL (manufactured by Sigma) were
dissolved in 1 mL of ethanol. Distilled water (23 mL) was gradually
added to this suspension so that the concentration of ethanol was
adjusted to 5% or lower. The resulting liposome suspension was
subjected to an ultracentrifugal separation (110,000.times.g at
25.degree. C. for 1 hour) and the supernatant liquid was removed.
PBS (40 .mu.L) was added thereto followed by subjecting to
re-suspending and further diluting with PBS, whereupon a liposome
suspension of 3 mg/mL of F-PS was obtained. PEG-DSPE (ratio of
PEG-DSPE to EggPC is 50:120 (weight:weight)) was dissolved in a
small amount (about 1/25 by volume of the liposome suspension) of
ethanol. Each of the liposome suspension and the ethanolic solution
of PEG-DSPE was heated at 70.degree. C. for 2 minutes. Then the
liposome suspension was added to the ethanolic solution of PEG-DSPE
and, after mixing, the mixture was heated at 70.degree. C. for 2
minutes and cooled with water. To the resulting liposome suspension
was added PBS to dilute so that the total lipid concentration was
adjusted to 30 mg/mL, whereupon a liposome suspension (Preparation
15) was obtained.
[0131] When an average particle size of the liposome was measured
by means of DLS, it was found to be 140 nm.
Example 16
[0132] DOTAP (30 mg/mL) and PEG-DSPE (12 mg/mL) suspensions were
shaken and stirred in a vortex mixer. The suspension was passed, at
room temperature, through a polycarbonate membrane filter of 0.4
.mu.m for 20 times, a polycarbonate membrane filter of 0.1 .mu.m
for 20 times and additionally a polycarbonate membrane filter of
0.05 .mu.m for 20 times to give a suspension of liposome where
particle size was around 80 nm. Under stirring by a stirrer, 120
.mu.L of a 15 mg/mL aqueous solution of F-PS was added to 250 .mu.L
of the suspension followed by adding 0.5 mL of ethanol thereto. To
this suspension was further added 125 .mu.L of a solution where 25
mg of Tween 80 was dissolved in 1 mL of an ethanolic solution of
120 mg/mL EggPC. Distilled water (11.5 mL) was gradually added to
this suspension so that the concentration of ethanol was adjusted
to 5% or lower. The resulting liposome suspension was subjected to
an ultracentrifugal separation (110,000.times.g at 25.degree. C.
for 1 hour) and the supernatant liquid was removed. PBS (40 .mu.L)
was added thereto followed by subjecting to re-suspending and
further diluting with PBS, whereupon a liposome suspension of 3
mg/mL of F-PS was obtained. PEG-DSPE (ratio of PEG-DSPE to EggPC is
50:120 (weight:weight)) was dissolved in a small amount (about 1/25
by volume of the liposome suspension) of ethanol. Each of the
liposome suspension and the ethanolic solution of PEG-DSPE was
heated at 70.degree. C. for 2 minutes. Then the liposome suspension
was added to the ethanolic solution of PEG-DSPE and, after mixing,
the mixture was heated at 70.degree. C. for 2 minutes and cooled
with water. To the resulting liposome suspension was added PBS to
dilute so that the total lipid concentration was adjusted to 30
mg/mL, whereupon a liposome suspension (Preparation 16) was
obtained.
[0133] When an average particle size of the liposome was measured
by means of DLS, it was found to be 151 nm.
Example 17
[0134] DOTAP (30 mg/mL) and PEG-DSPE (12 mg/mL) suspensions were
shaken and stirred in a vortex mixer. The suspension was passed, at
room temperature, through a polycarbonate membrane filter of 0.4
.mu.m for 20 times, a polycarbonate membrane filter of 0.1 .mu.m
for 20 times and additionally a polycarbonate membrane filter of
0.05 .mu.m for 20 times to give a suspension of liposome where
particle size was around 80 nm. Under stirring by a stirrer, 125
.mu.L of a 15 mg/mL aqueous solution of F-PS was added to 250 .mu.L
of the suspension followed by adding 0.5 mL of ethanol thereto. To
this suspension was further added 125 .mu.L of a solution where 120
mg of EggPC, 120 mg of Span 20 (manufactured by Kanto Kagaku) and
50 mg of PEG-DSPE were dissolved in 2 mL of ethanol. Distilled
water (11.5 mL) was gradually added to this suspension so that the
concentration of ethanol was adjusted to 5% or lower. The resulting
liposome suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS (40 .mu.L) was added thereto followed by
subjecting to re-suspending and further diluting with PBS,
whereupon a liposome suspension of 3 mg/mL of F-PS was obtained.
PEG-DSPE (ratio of PEG-DSPE to EggPC is 50:120 (weight:weight)) was
dissolved in a small amount (about 1/25 by volume of the liposome
suspension) of ethanol. Each of the liposome suspension and the
ethanolic solution of PEG-DSPE was heated at 70.degree. C. for 2
minutes. Then the liposome suspension was added to the ethanolic
solution of PEG-DSPE and, after mixing, the mixture was heated at
70.degree. C. for 2 minutes and cooled with water. To the resulting
liposome suspension was added PBS to dilute so that the total lipid
concentration was adjusted to 30 mg/mL, whereupon a liposome
suspension (Preparation 17) was obtained.
[0135] When an average particle size of the liposome was measured
by means of DLS, it was found to be 131 nm.
Example 18
[0136] DOTAP (30 mg/mL) and PEG-DSPE (12 mg/mL) suspensions were
shaken and stirred in a vortex mixer. The suspension was passed, at
room temperature, through a polycarbonate membrane filter of 0.4
.mu.m for 20 times, a polycarbonate membrane filter of 0.1 .mu.m
for 20 times and additionally a polycarbonate membrane filter of
0.05 .mu.m for 20 times to give a suspension of liposome where
particle size was around 80 nm. Under stirring by a stirrer, 250
.mu.L of a 15 mg/mL aqueous solution of F-PS was added to 500 .mu.L
of the suspension followed by adding 1 mL of ethanol thereto. To
this suspension was further added 250 .mu.L of a solution where 120
mg of EggPC and 12.5 mg of Cremophor EL (manufactured by Sigma)
were dissolved in 1 mL of ethanol. Distilled water (23 mL) was
gradually added to this suspension so that the concentration of
ethanol was adjusted to 5% or lower. The resulting liposome
suspension was subjected to an ultracentrifugal separation
(110,000.times.g at 25.degree. C. for 1 hour) and the supernatant
liquid was removed. PBS (40 .mu.L) was added thereto followed by
subjecting to re-suspending and further diluting with PBS,
whereupon a liposome suspension of 3 mg/mL of F-PS was obtained.
PEG-DSPE (ratio of PEG-DSPE to EggPC is 50:120 (weight:weight)) was
dissolved in a small amount (about 1/25 by volume of the liposome
suspension) of ethanol. Each of the liposome suspension and the
ethanolic solution of PEG-DSPE was heated at 70.degree. C. for 2
minutes. Then the liposome suspension was added to the ethanolic
solution of PEG-DSPE and, after mixing, the mixture was heated at
70.degree. C. for 2 minutes and cooled with water. To the resulting
liposome suspension was added PBS to dilute so as to adjust the
total lipid concentration to 30 mg/mL, whereupon a liposome
suspension (Preparation 18) was obtained.
[0137] When an average particle size of the liposome was measured
by means of DLS, it was found to be 133 nm.
Example 19
[0138] To 589 mg of egg yolk lecithin (manufactured by QP
CORPORATION) was added 11.8 mL of a 50 mmol/L aqueous solution of
potassium dihydrogen phosphate, followed by shaking and stirring in
a vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.1 .mu.m for 30 times.
The suspension (40 .mu.L) was mixed with a mixed solution
comprising 1 mL of an aqueous solution of 1 mg/mL of G-CSF
(manufactured by Kyowa Hakko; Nartograstim; genetically recombined
human G-CSF mutant) and 1 mL of an aqueous solution of 2 mg/mL of
sodium dextransulfate (manufactured by Merck), and pH value of the
mixed solution was adjusted to 4 using aqueous solutions of
hydrochloric acid of 1 mol/L and 0.1 mol/L (manufactured by Kanto
Kagaku). To 500 .mu.L of this mixed solution were added 753 .mu.L
of ethanol and then 80 .mu.L of an ethanolic solution of 50 mg/mL
of egg yolk lecithin. Distilled water (7 mL) was gradually added to
this suspension so that the concentration of ethanol was adjusted
to 10% or lower. The resulting liposome suspension was subjected to
an ultracentrifugal separation (146,000.times.g at 25.degree. C.
for 1 hour) and the supernatant liquid was removed. Water was added
thereto followed by subjecting to re-suspending so as to make the
total amount 500 .mu.L, whereupon a liposome suspension
(Preparation 19) was obtained.
[0139] When an average particle size of the liposome was measured
by means of DLS, it was found to be 316 nm.
Example 20
[0140] To 588 mg of egg yolk lecithin and 205 mg of PEG-DSPE was
added an appropriate amount of ethanol at room temperature to
dissolve them and then ethanol was evaporated, followed by drying
in vacuo. 11.8 mL of a 50 mmol/L aqueous solution of potassium
dihydrogen phosphate was added thereto followed by shaking and
stirring in a vortex mixer. The suspension was passed, at room
temperature, through a polycarbonate membrane filter of 0.1 .mu.m
for 30 times. The suspension (40 .mu.L) was mixed with a mixed
solution comprising 1 mL of an aqueous solution of 1 mg/mL of G-CSF
and 1 mL of an aqueous solution of 2 mg/mL of sodium
dextransulfate, and pH value of the mixed solution was adjusted to
4 using aqueous solutions of hydrochloric acid of 1 mol/L and 0.1
mol/L. To 500 .mu.L of this mixed solution was added 753 .mu.L of
ethanol, and then 80 .mu.L of ethanol containing 50 mg/mL of egg
yolk lecithin and 16.7 mg/mL of PEG-DSPE was added thereto.
Distilled water (7 mL) was gradually added to this suspension so
that the concentration of ethanol was adjusted to 10% or lower. The
resulting liposome suspension was subjected to an ultracentrifugal
separation (146,000.times.g at 25.degree. C. for 1 hour) and the
supernatant liquid was removed. Water was added thereto followed by
subjecting to re-suspending so as to make the total amount 500
.mu.L, whereupon a liposome suspension (Preparation 20) was
obtained.
[0141] When an average particle size of the liposome was measured
by means of DLS, it was found to be 316 nm.
Example 21
[0142] To 598 mg of egg yolk lecithin and 68.8 mg of sucrose fatty
acid ester (manufactured by Mitsubishi Chemical) was added an
appropriate amount of ethanol at room temperature to dissolve them
and then ethanol was evaporated, followed by drying in vacuo. 12.0
mL of a 50 mmol/L aqueous solution of potassium dihydrogen
phosphate was added thereto followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.1 .mu.m for 30 times.
The suspension (40 .mu.L) was mixed with a mixed solution
comprising 1 mL of an aqueous solution of 1 mg/mL of G-CSF and 1 mL
of an aqueous solution of 2 mg/mL of sodium dextransulfate, and pH
value of the mixed solution was adjusted to 4 with aqueous
solutions of hydrochloric acid of 1 mol/L and 0.1 mol/L. To 500
.mu.L of this mixed solution was added 753 .mu.L of ethanol, and
then 80 .mu.L of ethanol where 50 mg/mL of egg yolk lecithin and
4.5 mg/mL of sucrose fatty acid ester were dissolved therein was
added thereto. Distilled water (7 mL) was gradually added to this
suspension so that the concentration of ethanol was adjusted to 10%
or lower. The resulting liposome suspension was subjected to an
ultracentrifugal separation (146,000.times.g at 25.degree. C. for 1
hour) and the supernatant liquid was removed. Water was added
thereto followed by subjecting to re-suspending so as to make the
total amount 500 .mu.L, whereupon a liposome suspension
(Preparation 21) was obtained.
[0143] When an average particle size of the liposome was measured
by means of DLS, it was found to be 242 nm.
Example 22
[0144] To 150 mg of egg yolk lecithin and 60 mg of PEG-DSPE was
added 5 mL of distilled water, followed by shaking and stirring in
a vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.08 .mu.m for 20 times.
The suspension (571 .mu.L) was mixed with a mixed solution
comprising 119 .mu.L of an aqueous solution of potassium dihydrogen
phosphate containing 1.4 mg/mL of G-CSF and 167 .mu.L of an aqueous
solution of 2 mg/mL of sodium dextransulfate, and pH value of the
mixed solution was adjusted to 4 with aqueous solutions of
hydrochloric acid of 1 mol/L and 0.1 mol/L. To 0.5 mL of this mixed
solution was added 666 .mu.L of ethanol, and then 167 .mu.L of a
solution where 120 mg of egg yolk phosphatidylcholine (EggPC;
manufactured by NOF CORPORATION) and 25 mg of PEG-DSPE were
dissolved in 1 mL of ethanol was added thereto. Distilled water (47
mL) was gradually added to this suspension so that the
concentration of ethanol was adjusted to 5% or lower. The resulting
liposome suspension was subjected to an ultracentrifugal separation
(146,000.times.g at 4.degree. C. for 1 hour) and the supernatant
liquid was removed. Distilled water was added thereto followed by
subjecting to re-suspending, whereupon a liposome suspension
(Preparation 22) was obtained.
[0145] When an average particle size of the liposome was measured
by means of DLS, it was found to be 126 nm.
Example 23
[0146] To 150 mg of egg yolk lecithin and 60 mg of PEG-DSPE was
added 5 mL of distilled water followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.08 .mu.m for 20 times
and then a polycarbonate membrane filter of 0.03 .mu.m for 7 times.
The suspension (286 .mu.L) was mixed with 286 .mu.L of a mixed
solution comprising 119 .mu.L of an aqueous solution of potassium
dihydrogen phosphate containing 1.4 mg/mL of G-CSF and 167 .mu.L of
an aqueous solution of 2 mg/mL of sodium dextransulfate, and pH
value of the mixed solution was adjusted to 4 with aqueous
solutions of hydrochloric acid of 1 mol/L and 0.1 mol/L. To 0.25 mL
of this mixed solution was added 333 .mu.L of ethanol, and then 84
.mu.L of a solution where 120 mg of egg yolk phosphatidylcholine
and 25 mg of PEG-DSPE were dissolved in 1 mL of ethanol was added
thereto. Distilled water (23.5 mL) was gradually added to this
suspension so that the concentration of ethanol was adjusted to 5%
or lower. The resulting liposome suspension was subjected to an
ultracentrifugal separation (146,000.times.g at 4.degree. C. for 1
hour) and the supernatant liquid was removed. Re-suspending was
carried out by adding an aqueous solution of potassium dihydrogen
phosphate, whereupon a liposome suspension (Preparation 23) was
obtained.
[0147] When an average particle size of the liposome was measured
by means of DLS, it was found to be 146 nm.
Example 24
[0148] To 150 mg of egg yolk lecithin and 60 mg of PEG-DSPE was
added 5 mL of distilled water followed by shaking and stirring in a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.08 .mu.m for 20 times
and then a polycarbonate membrane filter of 0.05 .mu.m for 20
times. The suspension (428 .mu.L) was mixed with a mixed solution
comprising 89 .mu.L of an aqueous solution of potassium dihydrogen
phosphate containing 1.4 mg/mL of G-CSF and 125 .mu.L of an aqueous
solution of 2 mg/mL of sodium dextransulfate, and pH value of the
mixed solution was adjusted to 4 with aqueous solutions of
hydrochloric acid of 1 mol/L and 0.1 mol/L. To 0.5 mL of this mixed
solution was added 666 .mu.L of ethanol, and then 167 .mu.L of a
solution where 120 mg of egg yolk phosphatidylcholine and 25 mg of
PEG-DSPE were dissolved in 1 mL of ethanol was added thereto.
Distilled water (47 mL) was gradually added to this suspension so
that the concentration of ethanol was adjusted to 5% or lower. The
resulting liposome suspension was subjected to an ultracentrifugal
separation (146,000.times.g at 4.degree. C. for 1 hour) and the
supernatant liquid was removed. Re-suspending was carried out by
adding an aqueous solution of potassium dihydrogen phosphate,
whereupon a liposome suspension (Preparation 24) was obtained.
[0149] When an average particle size of the liposome was measured
by means of DLS, it was found to be 134 nm.
Example 25
[0150] To 150 mg of egg yolk lecithin and 60 mg of PEG-DSPE was
added 5 mL of distilled water, followed by shaking and stirring in
a vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.08 .mu.m for 20 times
and then a polycarbonate membrane filter of 0.05 .mu.m for 20
times. The suspension (500 .mu.L) was mixed with a mixed solution
comprising 20 .mu.L of a PBS solution containing 1,000,000 units/mL
of interferon .alpha.-2b (manufactured by Research Diagnostics),
150 .mu.L of an aqueous solution of 2 mg/mL of sodium
dextransulfate and 80 .mu.L of distilled water, and pH value of the
mixed solution was adjusted to 4 with aqueous solutions of
hydrochloric acid of 1 mol/L and 0.1 mol/L. To 0.5 mL of this mixed
solution was added 666 .mu.L of ethanol, and then 167 .mu.L of a
solution where 120 mg of egg yolk phosphatidylcholine and 25 mg of
PEG-DSPE were dissolved in 1 mL of ethanol was added thereto.
Distilled water (47 mL) was gradually added to this suspension so
that the concentration of ethanol was adjusted to 5% or lower. The
resulting liposome suspension was subjected to an ultracentrifugal
separation (146,000.times.g at 4.degree. C. for 1 hour) and the
supernatant liquid was removed. Re-suspending was carried out by
adding an aqueous solution of potassium dihydrogen phosphate,
whereupon a liposome suspension (Preparation 25) was obtained.
[0151] When an average particle size of the liposome was measured
by means of DLS, it was found to be 164 nm.
Comparative Example 1
[0152] EggPC (215 mg), 61 mg of DOTAP and 54 mg of cholesterol
(Chol) were dissolved in chloroform and the solvent was evaporated
therefrom in vacuo. To the resulting thin layer was added 5 mL of
an aqueous solution of FD (2 mg/mL). The suspension was passed, at
room temperature, through a polycarbonate membrane filter of 0.4
.mu.m for 20 times and then a polycarbonate membrane filter of 0.1
.mu.m for 20 times. PBS was added thereto so as to adjust the total
lipid concentration to 30 mg/mL, whereupon a liposome suspension
(Preparation a) was obtained.
[0153] When an average particle size of the liposome was measured
by means of DLS, it was found to be 134 nm.
Comparative Example 2
[0154] EggPC (215 mg), 61 mg of DOTAP and 54 mg of Chol were
dissolved in chloroform and the solvent was evaporated therefrom in
vacuo. To the resulting thin layer was added mL of an aqueous
solution of FD (2 mg/mL). The suspension was passed, at room
temperature, through a polycarbonate membrane filter of 0.4 .mu.m
for 20 times and then a polycarbonate membrane filter of 0.1 .mu.m
for 20 times. An ethanolic solution of PEG-DSPE with a
concentration of 1471 mg/mL (0.05 mL) was added thereto followed by
heating at 70.degree. C. for 5 minutes, whereupon the surface of
the liposome was coated with PEG. PBS was added thereto so as to
adjust the total lipid concentration to 30 mg/mL, whereupon a
liposome suspension (Preparation b) was obtained.
[0155] When an average particle size of the liposome was measured
by means of DLS, it was found to be 143 nm.
Comparative Example 3
[0156] Hydrogenated soybean phosphatidylcholine (HSPC) (300 mg),
100 mg of Chol and 100 mg of PEG-DSPE were dissolved in chloroform
and the solvent was evaporated therefrom in vacuo. To the resulting
thin layer was added 8 mL of an aqueous solution of FD (18.8
mg/mL). The suspension was passed, at 70 C..degree., through a
polycarbonate membrane filter of 0.4 .mu.m for 4 times and then a
polycarbonate membrane filter of 0.1 .mu.m for 10 times. The
resulting liposome suspension was subjected to a ultracentrifugal
separation (110,000.times.g at 25.degree. C. for 1 hour) and the
supernatant liquid was removed. Re-suspending was carried out by
adding thereto PBS so as to adjust the total lipid concentration to
30 mg/mL, whereupon a liposome suspension (Preparation c) was
obtained.
[0157] When an average particle size of the liposome was measured
by means of DLS, it was found to be 149 nm.
Comparative Example 4
[0158] EggPC (215 mg), 61 mg of DOTAP and 54 mg of Chol were
dissolved in chloroform and the solvent was evaporated therefrom in
vacuo. To the resulting thin layer were added mL of an aqueous
solution of FD (2 mg/mL). The suspension was passed, at room
temperature, through a polycarbonate membrane filter of 0.4 .mu.m
for 20 times and then a polycarbonate membrane filter of 0.1 .mu.m
for 20 times. The resulting liposome suspension was subjected to a
ultracentrifugal separation (110,000.times.g at 25.degree. C. for 1
hour) and the supernatant liquid was removed. Re-suspending was
carried out by adding thereto PBS so as to adjust the total lipid
concentration to 30 mg/mL, whereupon a liposome suspension
(Preparation d) was obtained.
[0159] When an average particle size of the liposome was measured
by means of DLS, it was found to be 141 nm.
Comparative Example 5
[0160] EggPC (215 mg), 61 mg of DOTAP and 54 mg of Chol were
dissolved in chloroform and the solvent was evaporated therefrom in
vacuo. To the resulting thin layer was added mL of an aqueous
solution of FD (2 mg/mL). The suspension was passed, at room
temperature, through a polycarbonate membrane filter of 0.4 .mu.m
for 20 times and then a polycarbonate membrane filter of 0.1 .mu.m
for 20 times. An ethanolic solution of PEG-DSPE with a
concentration of 1471 mg/mL (0.05 mL) was added thereto followed by
heating at 70.degree. C. for 5 minutes, whereupon the surface of
the liposome was coated with PEG. The resulting liposome suspension
was subjected to a ultracentrifugal separation (110,000.times.g at
25.degree. C. for 1 hour) and the supernatant liquid was removed.
Re-suspending was carried out by adding thereto PBS so as to adjust
the total lipid concentration to 30 mg/mL, whereupon a liposome
suspension (Preparation e) was obtained.
[0161] When an average particle size of the liposome was measured
by means of DLS, it was found to be 139 nm.
Comparative Example 6
[0162] F-PS was dissolved in PBS whereupon an F-PS solution
(Preparation f) was obtained.
Comparative Example 7
[0163] Mixing was carried out to prepare DOTAP/PEG-DSPE/distilled
water (30 mg/12 mg/mL), followed by shaking and stirring by a
vortex mixer. The suspension was passed, at room temperature,
through a polycarbonate membrane filter of 0.4 .mu.m for 20 times,
a polycarbonate membrane filter of 0.1 .mu.m for 20 times and a
polycarbonate membrane filter of 0.05 .mu.m for 20 times. To 0.5 mL
of the suspension was added 0.25 mL of an aqueous solution where
F-PS was dissolved in distilled water in an amount of 15 mg/mL,
whereupon a liposome suspension (Preparation g) in which the total
lipid concentration was 28 mg/mL was obtained.
[0164] When an average particle size of the liposome was measured
by means of DLS, it was found to be 108 nm.
[0165] Now the advantages of the present invention will be
illustrated by the following Test Examples.
Test Example 1
[0166] The preparations 1 to 5 prepared in Examples 1 to 5 and the
preparations a to e prepared in Comparative Examples 1 to 5 were
subjected to an ultracentrifugal separation (110,000.times.g at
25.degree. C. for 1 hour). In order to determine the quantity of
the FD in each preparation and the FD in the supernatant liquid
after the ultracentrifugal separation, fluorescent intensity at
excitation wavelength of 485 nm and fluorescence wavelength of 530
nm was measured using a fluorescent microplate reader. Further,
phosphatidylcholine (PC) in liposome of each preparation was
quantified by means of an enzymatic method using a Phospholipid C
Test Wako (manufactured by Wako Pure Chemical). Total lipid
concentration was calculated based on the PC concentration in view
of a laid-in rate. Enclosing efficiency of FD into liposome per
lipid and enclosing rate by ultracentrifugal separation were
calculated by the following expressions (1) and (2).
Enclosing efficiency per Lipid (mg FD/mg total
lipid)=(A.sup.1-C.sup.1)/B.sup.1 (1)
in which
[0167] A.sup.1: amount of FD in each preparation (mg/mL)
[0168] B.sup.1: amount of total lipid in each preparation
(mg/mL)
[0169] C.sup.1: amount of FD in the supernatant liquid (mg/mL)
Enclosing Rate by Ultracentrifugal Separation
(%)=[(A.sup.1-C.sup.1)/B.sup.1]/(D.sup.1/E.sup.1).times.100 (2)
in which
[0170] A.sup.1: amount of FD in each preparation (mg/mL)
[0171] B.sup.1: amount of total lipid in each preparation
(mg/mL)
[0172] C.sup.1: amount of FD in the supernatant liquid (mg/mL)
[0173] D.sup.1: laid-in amount of FD (mg/mL) in Examples 1 to 5 or
in Comparative Examples 1 to 5
[0174] E.sup.1: laid-in amount of total lipid (mg/mL) in Examples 1
to 5 or in Comparative Examples 1 to 5
[0175] The result is shown in Table 1 and Table 2.
TABLE-US-00001 TABLE 1 Enclosing Efficiency per Lipid Enclosing
Efficiency (mg FD/mg total lipid) Preparation 1 0.015 Preparation 2
0.031 Preparation 3 0.063 Preparation 4 0.019 Preparation 5 0.027
Preparation a 0.021 Preparation b 0.009 Preparation c 0.036
Preparation d 0.011 Preparation e 0.007
TABLE-US-00002 TABLE 2 Enclosing Rate by Ultracentrifugal
Separation Enclosing Rate (%) Preparation 1 54 Preparation 2 72
Preparation 3 97 Preparation 4 72 Preparation 5 81 Preparation a 70
Preparation b 36 Preparation c 12 Preparation d 38 Preparation e
26
Table 1 shows that the liposomes of the preparations 1 to 5 have
high enclosing efficiency as same as those of preparations a and c,
however the (amount of FD)/(total lipid amount) in a laying-in
stage is not taken into consideration. The laid-in amounts in the
manufacture of the preparations 1 to 5 and the preparations a to e
are different and, since large amount of FD for the lipid is used
for the manufacture particularly in the case of the preparation c,
it can be said that the liposomes of the preparations 1 to 5 and
the preparation a are the liposome where the amount of FD for the
total lipid amount is large when the laid-in amount is taken into
consideration.
[0176] The enclosing rate as shown in Table 2 is the percentage
obtained by dividing the result of Table 1 by (amount of FD)/(total
lipid amount) in a laying-in stage, and it reflects the (amount of
FD)/(total lipid amount) in a laying-in stage. Table 2 shows that
enclosing rate of FD is high in the liposomes of the preparations 1
to 5 and a, while the enclosing rate is low in the liposomes of the
preparations b to e. This means that, in the liposomes of the
preparations b to e, there are many free FD which is not enclosed
and, during the preparing process and analyzing process for
enclosing rate, the free FD is removed. From the result, it is
understood that, in the laid-in FD, an amount of the FD which is
included in the liposome or is mildly bound to (electrostatically
adsorbed with) the liposome surface is small and the effectively
utilized FD is small in the preparations b to e.
Test Example 2
[0177] PBS (1.98 mL) was added to and mixed with each 0.02 mL of
the preparations 1 to 5 manufactured in Examples 1 to 5 and the
preparations a to e manufactured in Comparative Examples 1 to 5,
whereupon a sample solution was produced. Immediately after mixing,
0.5 mL of the sample solution was subjected to gel filtration
(Sepharose CL-4B; .phi. 10 mm.times.20 cm; mobile phase: PBS;
amount of the sample added: 2 mL; amount of fraction collected:
about 2 mL). Liposome fraction and a fraction of the component
which is not enclosed in the liposome were separated and
fluorescence intensity of the eluate was measured by the same
manner as in Test Example 1. Enclosing rate by the gel filtration
was calculated by the formula (3).
Enclosing rate (%) by the gel
filtration=[F.sup.1/(F.sup.1+G.sup.1)].times.100 (3)
in which
[0178] F.sup.1: amount of FD in the liposome fraction (mg)
[0179] G.sup.1: amount of FD in the fraction of the component which
is not enclosed in the liposome (mg)
[0180] The result is shown in Table 3.
TABLE-US-00003 TABLE 3 Enclosing Rate by Gel Filtration Enclosing
Rate (%) Preparation 1 61 Preparation 2 71 Preparation 3 75
Preparation 4 65 Preparation 5 55 Preparation a 13 Preparation b 16
Preparation c 82 Preparation d 26 Preparation e 70
[0181] In the gel filtration, not only free FD but also FD which is
gently bound to (electrostatically adsorbed with) the surface of
liposome are removed. Further, when the stability of the
preparation is poor, a part of FD existing in the inner aqueous
phase leaks out. Table 3 shows that the enclosing rates of liposome
in the preparations 1 to 5, c and e are high. This means FD is
abundantly present in the inner aqueous phase of the liposome.
However, in Table 3, (amount of FD)/(amount of total lipid) in a
laying-in stage is not taken into consideration and, in the
preparation c, manufacturing is done using a lot of FD for the
lipid, whereby the enclosing rate is naturally high. On the other
hand, in the preparations a, b and d, the values are low and FD is
not substantially included in the liposome, whereby it can be said
that they are the preparations having poor stability.
[0182] Table 2 shows a manufacturing property, while Table 3 shows
the stability of liposome. From Table 2 and Table 3, it is apparent
that the preparation a has a good manufacturing property but is not
well stable and, on the contrary, the preparations c and e are
stable but manufacturing property is poor. Furthermore, it is
apparent that the preparations b and d have poor manufacturing
property and stability, while the preparations 1 to 5 have good
manufacturing property and stability. That shows that FD is
substantially enclosed inside the liposome or, in other words, the
FD-cationic lipid complex is coated with lipid in the preparations
1 to 5.
Test Example 3
[0183] The preparation 6 prepared in Example 6 was subjected to an
ultracentrifugal separation (110,000.times.g at 25.degree. C. for 1
hour). In order to determine the quantity of the F-Ins in the
preparation 6 and the F-Ins in the supernatant liquid after the
ultracentrifugal separation, fluorescent intensity at excitation
wavelength of 485 nm and fluorescence wavelength of 530 nm was
measured using a fluorescent microplate reader. Further, PC in
liposome of the preparation was quantified by means of an enzymatic
method using a Phospholipid C-Test Wako (manufactured by Wako Pure
Chemical). Total lipid concentration was calculated based on the PC
concentration in view of a laid-in rate. Enclosing efficiency of
F-Ins into liposome per lipid and enclosing rate by
ultracentrifugal separation were calculated by the following
expressions (4) and (5).
Enclosing Efficiency per Lipid (mg F-Ins/mg total
lipid)=(A.sup.2-C.sup.2)/B.sup.2 (4)
in which
[0184] A.sup.2: amount of F-Ins in the preparation 6 (mg/mL)
[0185] B.sup.2: amount of total lipid in the preparation 6
(mg/mL)
[0186] C.sup.2: amount of F-Ins in the supernatant liquid
(mg/mL)
Enclosing Rate by Ultracentrifugal Separation
(%)=[(A.sup.2-C.sup.2)/B.sup.2]/(D.sup.2/E.sup.2).times.100 (5)
in which
[0187] A.sup.2: amount of F-Ins in the preparation 6 (mg/mL)
[0188] B.sup.2: amount of total lipid in the preparation 6
(mg/mL)
[0189] C.sup.2: amount of F-Ins in the supernatant liquid
(mg/mL)
[0190] D.sup.2: laid-in amount of F-Ins (mg/mL) in Example 6
[0191] E.sup.2: laid-in amount of total lipid (mg/mL) in Example
6
[0192] The result is shown in Table 4 and Table 5.
TABLE-US-00004 TABLE 4 Enclosing Efficiency per Lipid Enclosing
Efficiency (mg F-Ins/mg total lipid) Preparation 6 0.010
TABLE-US-00005 TABLE 5 Enclosing Rate by Ultracentrifugal
Separation Enclosing Rate (%) Preparation 6 73
[0193] Table 4 shows that, in the preparation 6, the enclosing
efficiency of F-Ins into the liposome per lipid is high in spite of
the fact that the laid-in amount of F-Ins per lipid is small.
[0194] With regard to the enclosing rate by ultracentrifugal
separation, the measurement is conducted in such a respect that the
F-Ins which is gently bound to the liposome surface is enclosed.
Table 5 shows that in the liposome of the preparation 6, the
enclosing rate is high in the liposome of the preparation 6 as same
as in the cases of liposomes of the preparations 1 to 5.
Test Example 4
[0195] PBS (1.98 mL) was added to and mixed with 0.02 mL of the
preparation 6 manufactured in Example 6, whereupon a sample
solution was produced. Immediately after mixing, 0.5 mL of the
sample solution was subjected to gel filtration (Sepharose CL-4B;
.phi. 10 mm.times.20 cm; mobile phase: PBS; amount of the sample
added: 2 mL; amount of fraction collected: about 2 mL). Liposome
fraction and a fraction of the component which is not enclosed in
the liposome were separated and fluorescence intensity of the
eluate was measured by the same manner as in Test Example 3.
Enclosing rate by the gel filtration was calculated by the formula
(6).
Enclosing rate (%) by the gel
filtration=[F.sup.2/(F.sup.2+G.sup.2)].times.100 (6)
in which
[0196] F.sup.2: amount of F-Ins in the liposome fraction (mg)
[0197] G.sup.2: amount of F-Ins in the fraction of the component
which is not enclosed in the liposome (mg)
[0198] The result is shown in Table 6.
TABLE-US-00006 TABLE 6 Enclosing Rate by Gel Filtration Enclosing
Rate (%) Preparation 6 73
[0199] Enclosing rate by gel filtration is measured after removal
of F-Ins which is gently bound to the liposome surface. Table 6
shows that, in the case of liposome of the preparation 6, enclosing
rate is high and F-Ins is abundantly present in the inner aqueous
phase of the liposome as same as in the cases of the preparations 1
to 5. Thus, it can be said that the preparation 6 is a preparation
having good stability where F-Ins is substantially included in the
liposome, showing that a complex of F-Ins with lipid is coated with
lipid.
Test Example 5
[0200] The preparations 7 to 13 prepared in Examples 7 to 13 were
subjected to an ultracentrifugal separation (110,000.times.g at
25.degree. C. for 1 hour). In order to determine the quantity of
the F-PS in each preparation and the F-PS in the supernatant liquid
after the ultracentrifugal separation, fluorescent intensity at
excitation wavelength of 485 nm and fluorescence wavelength of 530
nm was measured using a fluorescent microplate reader. Further, PC
in liposome of each preparation was quantified by means of an
enzymatic method using a Phospholipid C-Test Wako (manufactured by
Wako Pure Chemical). Total lipid concentration was calculated based
on the PC concentration in view of a laid-in rate. Enclosing
efficiency of F-PS into liposome per lipid and enclosing rate by
ultracentrifugal separation were calculated by the following
expressions (7) and (8).
Enclosing Efficiency per Lipid (mg F-PS/mg total
lipid)=(A.sup.3-C.sup.3)/B.sup.3 (7)
in which
[0201] A.sup.3: amount of F-PS in each preparation (mg/mL)
[0202] B.sup.3: amount of total lipid in each preparation
(mg/mL)
[0203] C.sup.3: amount of F-PS in the supernatant liquid
(mg/mL)
Enclosing Rate by Ultracentrifugal Separation
(%)=[(A.sup.3-C.sup.3)/B.sup.3]/(D.sup.3/E.sup.3).times.100 (8)
in which
[0204] A.sup.3: amount of F-PS in each preparation (mg/mL)
[0205] B.sup.3: amount of total lipid in each preparation
(mg/mL)
[0206] C.sup.3: amount of F-PS in the supernatant liquid
(mg/mL)
[0207] D.sup.3: laid-in amount (mg/mL) of F-PS in Examples 7 to
13
[0208] E.sup.3: laid-in amount (mg/mL) of total lipid in Examples 7
to 13
[0209] The result is shown in Table 7 and Table 8.
TABLE-US-00007 TABLE 7 Enclosing Efficiency per Lipid Enclosing
Efficiency (mg F-PS/mg total lipid) Preparation 7 0.058 Preparation
8 0.034 Preparation 9 0.031 Preparation 10 0.050 Preparation 11
0.059 Preparation 12 0.030 Preparation 13 0.024
TABLE-US-00008 TABLE 8 Enclosing Rate by Ultracentrifugal
Separation Enclosing Rate (%) Preparation 7 109 Preparation 8 126
Preparation 9 76 Preparation 10 107 Preparation 11 111 Preparation
12 86 Preparation 13 97
[0210] Table 7 shows that, in the preparations 7 to 13, the
enclosing efficiency of F-PS per lipid is high.
[0211] In the enclosing rate by ultracentrifugal separation, the
measurement is conducted in such a respect that the F-PS which is
gently bound to the liposome surface is also enclosed. Table 8
shows that the liposomes of the preparations 7 to 13 are liposomes
where the enclosing rate of F-PS is high.
Test Example 6
[0212] PBS (1.98 mL) was added to and mixed with 0.02 mL of the
preparations 7 to 9, 11 and 12 manufactured in Examples to 9, 11
and 12, whereupon a sample solution was produced. Immediately after
mixing, 0.5 mL of the sample solution was subjected to gel
filtration (Sepharose CL-4B; .phi. 10 mm.times.20 cm; mobile phase:
PBS; amount of the sample added: 2 mL; amount of fraction
collected: about 2 mL). Liposome fraction and a fraction of the
component which is not enclosed in the liposome were separated and
fluorescence intensity of the eluate was measured by the same
manner as in Test Example 5. Enclosing rate by the gel filtration
was calculated by the formula (9).
Enclosing rate (%) by the gel
filtration=[F.sup.3/(F.sup.3+G.sup.3)].times.100 (9)
in which
[0213] F.sup.3: amount of F-PS in the liposome fraction (mg)
[0214] G.sup.3: amount of F-PS in the fraction of the component
which is not enclosed in the liposome (mg)
[0215] The result is shown in Table 9.
TABLE-US-00009 TABLE 9 Enclosing Rate by Gel Filtration Enclosing
Rate (%) Preparation 7 84 Preparation 8 96 Preparation 9 94
Preparation 11 90 Preparation 12 89
Enclosing rate by gel filtration was measured after removal of F-PS
which is gently bound to the liposome surface. Table 9 shows that,
in the case of liposome of the preparations 7 to 9, 11 and 12,
enclosing rate is high and F-PS is abundantly present in the inner
aqueous phase of the liposome. Thus, it can be said that the
preparations 7 to 9, 11 and 12 are the preparations having good
stability where F-PS is substantially included in the liposome,
showing that a complex of F-PS with lipid is coated with lipid.
Test Example 7
[0216] Comparison was made to investigate distribution of F-PS to
tumor. Tumor piece of 2 mm square from human renal cancer cell
Caki-1 was transplanted under the skin of right body of six-week
old nude mouse (CLEA Japan) of a BALB/cA Jcl-nu strain. After 20
days from the transplantation, mice in which tumor volume reached
102 to 349 mm.sup.3 were grouped in such a manner that each group
comprised 3 mice, and the preparation 7 manufactured in Example 7
and the preparation f manufactured in Comparative Example 6 each
was administered from tail vein of the mouse using a syringe (26 G;
1 mL; manufactured by Terumo) at the dose of 25 mg F-PS/kg. Tumor
was excised from time to time, the tumor was homogenized and the
fluorescence intensity in the homogenate was measured by the same
manner as in Test Example 5. Distribution of F-PS to tumor was
evaluated by calculating the F-PS amount in tumor (.mu.g/g) at each
time point.
[0217] The result is shown in FIG. 2.
[0218] FIG. 2 shows that, in the mice to which the preparation 7
was administered, the amount of F-PS distributed to tumor increases
as compared with the mice to which the preparation f was
administered.
Test Example 8
[0219] The preparation 1 manufactured in Example 1 was subjected to
a morphological observation using electron microscope. The
preparation was added to a 1% aqueous solution of ammonium
molybdate (adjusted to pH 7.3 using ammonia) so as to adjust the
total lipid concentration to 0.5 mg/mL. This was dropped onto a
mesh (400 mesh; Nisshin EM) to which collodion membrane was
attached and, after about 1 minute, excessive water was absorbed
with filter paper followed by drying. An observation was made using
a transmission electron microscope (type H-7000; Hitachi) with an
accelerating voltage of 75 kV.
[0220] The result was that, in the preparation 1, there were a lot
of particles having therein particle whose form is different from
that of the membrane which surrounded the outside. Thus, this
result shows that the inner particles are coated with a lipid
bilayer.
Test Example 9
[0221] The preparation 1 manufactured in Example 1 and the
preparations a and b manufactured in Comparative Examples 1 and 2
were administered (dose: corresponding to 10 mg of total lipid per
kg) to CD(SD)IGS male rats under urethane anesthetization (body
weight: 200 to 300 g; one group comprised 2 or 3 rats) from the
femoral vein. Blood was collected from time to time using a
heparin-treated syringe from the jugular vein and centrifuged
(10,000.times.g at 4.degree. C. for 5 minutes). Thereafter, the
fluorescent intensity of FD in the plasma was measured by the same
manner as in Test Example 1 and FD concentration in the plasma was
calculated.
[0222] The result is shown in FIG. 3.
[0223] FIG. 3 shows that, in the rats to which the preparation 1
was administered, level in the concentration of FD in the plasma
was high as compared with the rats to which the preparation a or b
was administered. Thus, it is shown that, due to the coating of
FD-lipid complex with lipid, retention in blood is improved.
Test Example 10
[0224] The preparations 7 to 13 manufactured in Examples 7 to and
the preparation f manufactured in Comparative Example 6 were
administered (does: corresponding to 10 mg of the total lipid per
kg) to CD(SD)IGS male rats (body weight: 200 to 300 g; one group
comprised 2 rats) under urethane anesthetization from the femoral
vein. Blood was collected from time to time using a heparin-treated
syringe from the jugular vein and centrifuged (10,000.times.g at
4.degree. C. for 5 minutes). Thereafter, the fluorescent intensity
of F-PS in the plasma was measured by the same manner as in Test
Example 5 and F-PS concentration in the plasma was calculated.
[0225] The result is shown in FIG. 4.
[0226] FIG. 4 shows that, in the rats to which the preparations 7
to 13 were administered, the level in the concentration of F-PS in
the plasma was high as compared with the rats to which the
preparation f was administered.
Test Example 11
[0227] The preparation 7 manufactured in Example 7 and the
preparations f to g manufactured in Comparative Examples 6 to 7
were administered (dose: corresponding to 10 mg of total lipid per
kg) to BALB/cA Jcl male mice (body weight: 20 to 30 g; one group
comprised 2 or 3 mice) from the tail vein. Under ether
anesthetization, blood was collected from femoral vein using a
heparin-treated syringe and centrifuged (10,000.times.g at
4.degree. C. for 5 minutes). Thereafter, fluorescence intensity of
F-PS in the plasma was measured by the same manner as in Test
Example 5 and F-PS concentration in the plasma was calculated.
[0228] The result is shown in FIG. 5.
[0229] FIG. 5 shows that, in the mice to which the preparations f
to g were administered, F-PS quickly disappeared from the plasma
after its administration, while in the mice to which the
preparation 7 was administered, the level in the concentration of
F-PS in the plasma was high.
Test Example 12
[0230] The preparations 13 to 18 manufactured in Examples 13 to 18
were subjected to ultracentrifugal separation (110,000.times.g at
25.degree. C. for 1 hour). In order to determine the quantity of
the F-PS in each preparation and the F-PS in the supernatant liquid
after the ultracentrifugal separation, fluorescent intensity at
excitation wavelength of 485 nm and fluorescence wavelength of 530
nm was measured using a fluorescent microplate reader. Enclosing
rate of F-PS to liposome by ultracentrifugal separation was
calculated by the following expression (10).
Enclosing Rate by Ultracentrifugal Separation
(%)=[(A.sup.4-B.sup.4)]/B.sup.4.times.100 (10)
[0231] A.sup.4: amount of F-PS in each preparation (mg/mL)
[0232] B.sup.4: amount of F-PS in the supernatant liquid
(mg/mL)
[0233] The result is shown in Table 10.
TABLE-US-00010 TABLE 10 Enclosing Rate by Ultracentrifugal
Separation Enclosing Rate (%) Preparation 13 98 Preparation 14 95
Preparation 15 97 Preparation 16 98 Preparation 17 95 Preparation
18 99
Test Example 13
[0234] Fetal Bovine serum (FBS) (7.92 mL) was added to and mixed
with each 0.08 mL of the preparations 7 and 14 to 18 manufactured
in Examples 7 and 14 to 18, whereupon a sample solution was
produced. Immediately after mixing (0 hour) and after being allowed
to stand at 37.degree. C. for 6 to 168 hours, samplings were
conducted and 0.5 mL of each of the sample solutions was subjected
to gel filtration (Sepharose CL-4B; .phi. 10 mm.times.20 cm; mobile
phase: PBS; amount of the sample added: 2 mL; amount of fraction
collected: about 2 mL). Liposome fraction and a fraction of the
component which was not enclosed in the liposome were separated. In
order to quantify the F-PS in each fraction, fluorescence intensity
at excitation wavelength of 485 nm and fluorescence wavelength of
530 nm was measured using a fluorescent microplate reader. Amount
of F-PS leaked out from the liposome was calculated by the
expression (11).
Amount of F-PS leaked out (%)=[B.sup.5/(A.sup.5+B.sup.5)].times.100
(11)
[0235] A.sup.5: amount of F-PS in the liposome fraction (mg)
[0236] B.sup.5: amount of F-PS in the component which is not
enclosed in the liposome (mg)
[0237] The result is shown in Table 11.
TABLE-US-00011 TABLE 11 Leakage of F-PS from Liposome in FBS (%
Leakage) Time (Hour(s)) 0 6 24 48 72 96 168 Preparation 7 17 28 41
51 52 -- 64 Preparation 14 30 47 55 63 62 -- 65 Preparation 15 35
48 54 57 -- -- 72 Preparation 16 29 44 56 55 -- -- 62 Preparation
17 15 21 26 -- -- 43 47 Preparation 18 27 43 51 55 -- -- 57
[0238] Table 11 shows the F-PS in the preparations 7 and 14 to 18
leaks from the liposome with a lapse of time. Thus, when the fine
particles are coated with lipid membrane using the method of the
present invention, fine particles of a drug or the like enclosed in
the lipid membrane are leaked out after accumulating at the target
site in the body, whereupon achievement of sustained pharmaceutical
effect is now possible.
Test Example 14
[0239] Gel filtration of the preparations 19 to 21 manufactured in
Examples 19 to 21 was carried out. A liposome suspension (0.5 mL)
was used as a sample solution and added to a gel filtration column
(Sepharose CL-4B; .phi. 10 mm.times.20 cm; mobile phase: PBS;
amount of the sample added: 0.5 mL; amount of the fraction
collected: about 1.5 mL). Liposome fraction and free G-CSF fraction
were separated, and each of them was concentrated by means of a
centrifugal evaporation. Distilled water was added to the
concentrated liposome fraction to make the total volume 2 mL. To
200 .mu.L of the liposome suspension were added 50 .mu.L of a 50
mmol/L phosphate buffer of pH 7 containing 10% of sodium
laurylsulfate and 150 .mu.L of distilled water, followed by
stirring. Further, 400 .mu.L of 2-propanol was added thereto
followed by stirring so that liposome was completely destroyed, and
then 800 .mu.L of the following mobile phase I was added thereto
and mixed. A centrifugal separation (10,000.times.g for 5 minutes)
was carried out and the supernatant liquid was subjected to an HPLC
analysis. To the concentrated free G-CSF fraction were added a 50%
aqueous solution of acetonitrile containing 2% of Pluronic F 127
(manufactured by Sigma) and 0.5% of trifluoroacetic acid (TFA) so
as to make the total volume 5 mL, which was then subjected to an
HPLC analysis.
[0240] Column: YMCpack ODS-AM, .phi. 6.0 mm.times.15 cm
[0241] Mobile phase: I 50% acetonitrile containing 0.5% TFA [0242]
II 80% acetonitrile containing 0.5% TFA
[0243] Rate of the liquid II after initiation of the analysis was
made 0% during 0 to 5 minute(s), linearly increased within 0% to
100% during 5 to 35 minutes and made 100% during 35 to 45
minutes.
[0244] Detection: 280 nm
[0245] Temperature for analysis: 30.degree. C.
[0246] Flow rate: 1 mL/minute
[0247] Injected amount: 200 .mu.l,
[0248] Enclosing rate of G-CSF enclosed in each liposome was
calculated by the expression (12).
Enclosing Rate (%)=A.sup.6/(A.sup.6+B.sup.6).times.100 (12)
[0249] A.sup.6: amount of G-CSF in liposome fraction (.mu.g)
[0250] B.sup.6: amount of G-CSF in free G-CSF fraction (.mu.g)
[0251] The result is shown in Table 12.
TABLE-US-00012 TABLE 12 Enclosing Rate Enclosing Rate (%)
Preparation 19 100 Preparation 20 100 Preparation 21 100
[0252] Table 12 shows that the preparations 19 to 21 are those
where no free G-CSF separated by gel filtration is contained
therein.
INDUSTRIAL APPLICABILITY
[0253] In accordance with the present invention, safe, convenient
and efficient coating of fine particles with lipid membrane is now
possible.
* * * * *