U.S. patent application number 10/410203 was filed with the patent office on 2003-11-13 for liposome.
This patent application is currently assigned to Terumo Kabushiki Kaisha. Invention is credited to Kasukawa, Hiroaki, Kawahara, Kazuo, Kimura, Junji, Uchiyama, Hideki, Ushijima, Hideto.
Application Number | 20030211142 10/410203 |
Document ID | / |
Family ID | 26488987 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211142 |
Kind Code |
A1 |
Kawahara, Kazuo ; et
al. |
November 13, 2003 |
Liposome
Abstract
This invention provides a liposome having a drug included
therein, which has active targeting property and ensured stability
in blood and can be used effectively in the diagnosis and/or
treatment of diseases, particularly renal diseases, that accompany
production of proteoglycan, comprising (1) a basic compound which
takes positive charge within a physiological pH range, (2) a lipid
derivative of a hydrophilic polymer and (3) a lipid which
constitutes the liposome, as its membrane constituting components,
wherein their constituting ratios are from 1 to 20 mol % of (1)
based on (3) and from 0.2 to 5 mol % of (2) based on the total of
(1) and (3).
Inventors: |
Kawahara, Kazuo; (Kanagawa,
JP) ; Ushijima, Hideto; (Kanagawa, JP) ;
Uchiyama, Hideki; (Kanagawa, JP) ; Kimura, Junji;
(Kanagawa, JP) ; Kasukawa, Hiroaki; (Kanagawa,
JP) |
Correspondence
Address: |
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Assignee: |
Terumo Kabushiki Kaisha
|
Family ID: |
26488987 |
Appl. No.: |
10/410203 |
Filed: |
April 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10410203 |
Apr 10, 2003 |
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09582710 |
Sep 21, 2000 |
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6562371 |
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09582710 |
Sep 21, 2000 |
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PCT/JP99/06108 |
Nov 2, 1999 |
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Current U.S.
Class: |
424/450 |
Current CPC
Class: |
Y10T 428/2984 20150115;
A61K 9/1271 20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 009/127 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 2, 1998 |
JP |
10-311620 |
Jun 10, 1999 |
JP |
11-163595 |
Claims
1. A liposome in which a drug is included, which comprises (1) a
basic compound which takes positive charge within a physiological
pH range, (2) a lipid derivative of a hydrophilic polymer and (3) a
lipid which constitutes the liposome, as its membrane constituting
components, wherein their constituting ratios are from 1 to 20 mol
% of (1) based on (3) and from 0.2 to 5 mol % of (2) based on the
total of (1) and (3).
2. A liposome in which a drug is included, which comprises (1) a
basic compound which takes positive charge within a physiological
pH range, (2) a lipid derivative of a hydrophilic polymer and (3) a
lipid which constitutes the liposome, as its membrane constituting
components, wherein their constituting ratios are from 5 to 15 mol
% of (1) based on (3) and from 0.2 to 5 mol % of (2) based on the
total of (1) and (3).
3. The liposome according to claim 1 or 2, wherein the basic
compound which takes positive charge within a physiological pH
range is a basic compound which has an amidino group, a basic
compound which has two or more amino groups, a basic compound which
has a piperidine ring or a basic compound which has a quaternary
amine.
4. The liposome according to claim 3, wherein the basic compound
which takes positive charge within a physiological pH range is any
one of those represented by the following formulae 1 to 4; 14(in
the formula 1, A represents an aromatic ring, each of R.sup.1 and
R.sup.2 represents an alkyl or alkenyl group having from 10 to 25
carbon atoms, wherein R.sup.1 and R.sup.2 may be the same or
different from each other, each of X.sup.1 and X.sup.2 represents
--O--, --S--, --COO--, --OCO--, --CONH-- or --NHCO--, wherein
X.sup.1 and X.sup.2 may be the same or different from each other, m
is 0 or 1 and n is 0 or an integer of from 1 to 6), 15(in the
formula 2, R.sup.3 represents hydrogen or an alkyl or alkenyl group
having from 1 to 8 carbon atoms, R.sup.4 represents hydrogen or an
alkyl or alkenyl group having from 1 to 8 carbon atoms, each of
R.sup.5 and R.sup.6 represents hydrogen or an alkyl or alkenyl
group having from 1 to 25 carbon atoms (excluding a case in which
R.sup.5 and R.sup.6 are both hydrogen), wherein R.sup.5 and R.sup.6
may be the same or different from each other, X.sup.3 represents
--O-- or --S--, p is 0 or 1 and q is 0 or an integer of from 1 to
10), 16(in the formula 3, each of R.sup.7 and R.sup.8 represents an
alkyl or alkenyl group having from 1 to 8 carbon atoms, wherein
R.sup.7 and R.sup.8 may be the same or different from each other,
R.sup.9 represents hydrogen or an alkyl or alkenyl group having
from 1 to 8 carbon atoms, each of R.sup.10 and R.sup.11 represents
hydrogen or an alkyl or alkenyl group having from 1 to 25 carbon
atoms (excluding a case in which R.sup.10 and R.sup.11 are both
hydrogen), wherein R.sup.10 and R.sup.11 may be the same or
different from each other, X.sup.4 represents --O-- or --S--, r is
0 or 1 and s is 0 or an integer of from 1 to 10), and 17
5. The liposome according to any one of claims 1 to 4, wherein the
lipid derivative of a hydrophilic polymer is a lipid derivative of
polyethylene glycol.
6. The liposome according to claim 5, wherein the aforementioned
lipid derivative of polyethylene glycol is a compound which
contains a polyethylene glycol chain and a diacyl glycerol in one
molecule.
7. The liposome according to any one of claims 1 to 6, wherein the
lipid derivative of a hydrophilic polymer has a molecular weight of
from 1,000 to 7,000.
8. The liposome according to any one of claims 1 to 7, wherein the
aforementioned drug to be included in the liposome is a drug for
use in diagnosing and/or treating renal diseases.
9. The liposome according to claim 8, wherein the aforementioned
drug for use in diagnosing and/or treating renal diseases is an
adrenocortical steroid and/or a derivative thereof.
10. The liposome according to any one of claims 1 to 9, wherein the
aforementioned drug to be included in the liposome is a drug for
use in diagnosing and/or treating diseases which accompany
production of proteoglycan in injured portions of tissues and/or
organs.
11. The liposome according to any one of claims 1 to 10, wherein
the major particle size range of the aforementioned liposome is
from 90 to 200 nm.
12. The liposome according to any one of claims 1 to 11, wherein
the lipid which constitutes the aforementioned liposome is a
phospholipid or a hydrogenated product of a phospholipid.
13. The liposome according to any one of claims 1 to 12, wherein it
further contains a stabilizing agent or an antioxidant.
14. Use of the liposome of claim 1 or 2 as a drug for treating
inflamed kidney.
15. Use of the liposome of claim 1 or 2 in the production of a
therapeutic drug for inflamed kidney.
16. A method for treating inflamed kidney, which comprises
administering the liposome of claim 1 or 2 to mammals including
human.
Description
TECHNICAL FIELD
[0001] This invention relates to a liposome having active targeting
property and ensured stability in blood, which can be used in the
diagnosis and/or treatment of diseases, particularly renal
diseases, that accompany production of various types of
proteoglycan in injured portions of tissues and/or organs.
BACKGROUND ART
[0002] In recent years, there have been increased studies on a drug
delivery system (DDS) based on a so-called targeting technique in
which a drug is efficiently distributed in an organ of interest.
Liposomes are one of the most extensively studied means as such a
DDS because of their ability to include, or trap, drugs
therein.
[0003] However, there are various problems to be resolved in
putting these liposomes into practical use, particularly, the
difficulties in escaping from the mechanism of living body for
recognizing foreign bodies and in controlling intracorporeal
kinetics. That is, liposomes cause aggregation in blood by their
mutual reaction with various blood plasma proteins, including
opsonin, and are captured by a reticuloendothelial system (RES)
such as in the liver or spleen, so that it has been difficult to
deliver liposomes selectively to the target tissues or cells.
[0004] In recent years, it became possible to prevent aggregation
of liposomes in blood and avoid being captured by RES, by coating
the surface of liposome with a hydrophilic polymer such as
polyethylene glycol (PEG) and thereby preventing adsorption of
various blood plasma proteins, including opsonin, to the liposome
surface (U.S. Pat. No. 5,013,556, U.S. Pat. No. 5,676,971).
[0005] There are a number of studies based on these techniques with
regard to the targeting at tissues of interest, such as tumor
tissues, regions of enhanced vascular permeability, inflamed
tissues, the liver, the brain and lymph tissues, but all of these
cases are based on the effect of so-called passive targeting
property, which is induced as a result of the improvement of the
stability in blood by avoiding the capture by RES (Advanced Drug
Delivery Reviews, 24 (1997), 337-334), so that concern has been
directed toward the development of a liposome having the ability to
bind to the tissues of interest, in which the targeting function is
reinforced by so-called active targeting techniques.
[0006] Regarding the active targeting techniques, methods for the
modification of the liposome surface with target factors such as
antibodies, antibody fragments, amino acids, peptides or
saccharides have been studied, as well as techniques for making the
liposome surface into cationic nature. Particularly, the cation
formation can be cited as one of the desirable modification
techniques, because it has the ability to deliver genes and the
like into cells, as a gene introducing technique, and it improves
accumulation of liposome on, for example, an injured portion of
vascular endothelium (JP-A-7-89874; the term "JP-A" as used herein
means an "unexamined published Japanese patent application").
However, strong aggregation and the like phenomena mediated by the
binding of blood protein to liposome were observed by the cationic
liposome when compared with a neutral or anionic liposome
(Biochimica et Biophysica Acta, 1280 (1996), 149-154), so that its
sufficient active targeting property in the living body was not
obtained as such.
[0007] On the other hand, proteoglycans which keep the cell surface
anionic are known as the components which their interactions to
cationic liposome are expected. It has been reported that
overproduction of various types of proteoglycans occurs in tissues
of fibrosis in a large number of organs (the liver, the lungs, the
heart, the pancreas, the bone marrow and arteries) of tumors such
as large bowel cancer, of cell proliferative nephritis and of other
inflammatory diseases (Acta Pathol. Jpn., 36 (6): 827 (1986), FEBS
Lett., 244: 315 (1988), J. Rheumatol., 18 (10): 1466, (1991), J.
Dent. Res., 71 (9): 1587, (1992). This reaction in the living body
is considered to be a result of excess repairing reaction which
occurs during the step of wound healing or of excess cell
proliferation in a tumor or the like tissue, so that it is used as
a pathological marker of the foci of the aforementioned
diseases.
[0008] However, very little is known about reports which aim at
achieving active targeting for tissues and/or organs in which such
proteoglycan is overproduced. For example, JP-A-8-27030 describes
that a drug carrier whose surface is modified with a basic compound
which takes positive charge within a physiological pH range is
accumulated on a cortical portion of the kidney, but there are no
reports which disclose a targeting technique for the specific
accumulation of a carrier on renal glomerulus, particularly renal
glomerulus which caused inflammation by an injury, and proteoglycan
producing tissues and/or organs typified thereby.
[0009] Glomerulonephritis is one of the diseases so far reported in
which production of various types of proteoglycan is accelerated in
injured portions (Clin. Exp. Immunol., 108: 69, (1997), Kidney
International, 49: supple. 54, s 55 (1996), J. Am. Soc. Nephrol.,
2: s 88 (1992)). Importance of the targeting of a drug for a tissue
and/or organ overproducing proteoglycan can easily be understood by
merely citing this case of glomerulonephritis.
[0010] According to the materials produced in 1996 by The Japanese
Society of Dialysis Medicine, it is said that about 27,000 patients
are subjected to dialysis due to renal insufficiency, and about 50%
of the cases is caused by chronic nephropathy, and about 30%
thereof by diabetic nephropathy. Since the primary disease in both
of the chronic nephropathy and diabetic nephropathy is
glomerulonephritis, these patients will be released from the
struggling against dialysis when an effective method for the
treatment of glomerulonephritis is established. Up to now, however,
no effective glomerulonephritis treating method has been found.
[0011] For example, a glomerulonephritis, called IgA nephritis, is
a glomerulonephritis most frequently found in the Japanese people,
and it is said that its patients are estimated to be close to
300,000 in Japan alone and it occupies around 40% of the
glomerulonephritis in adults and close to 30% of that in children.
As a result of a follow up study carried out in Japan for recent 20
years, it was found that about 40% of IgA nephritis cases,
estimated to be 5,000 to 6,000 cases per year (1995), reached
terminal renal insufficiency and were subjected to dialysis, so
that it is considered that the development of a therapeutic method
specific for IgA nephritis is a world-wide pressing need. However,
close to 5,000 patients have been subjected to dialysis every year
because of the absence of sufficient means for its treatment.
[0012] It has been reported that, as a result of 10 years of
clinical efficacy evaluation on IgA nephritis, the steroid therapy
which is now actively used in Japan for nephrotic syndrome and
rapidly progressive glomerulonephritis, and its efficacy is said to
be established, also has a possibility of keeping renal functions
for a prolonged period of time by its 2 to 3 years of continuous
use at an early stage of progressive IgA nephritis.
[0013] On the other hand, however, side effects become a serious
problem when it is continuously administered for a certain period
for such a chronic disease, because problems such as
arteriosclerosis, osteoporosis and immunity reduction in adults,
slow development in children and the like cannot be avoided. As a
result, it becomes a problem that its sufficient efficacy cannot be
obtained, because not only QOL (quality of life) of the patient is
reduced but also its administration cannot be continued due to side
effects and, particularly, its administration for a prolonged
period of time is impossible.
[0014] In order to resolve these problems, it is necessary to think
out a means for expressing its drug effect alone, by a dose which
does not express the aforementioned side effects, and it is
desirable for this purpose to design a system for efficiently
delivering a drug to the glomerulus which caused inflammation by an
injury.
[0015] The same requirement is also applied to other diseases in
which proteoglycan is excessively produced in injured portions of
tissues and/or organs, so that it is expected to develop a method
which can exert sufficient therapeutic effect without inducing side
effects, by actively targeting the organ of interest with a drug
which shows a strong drug effect but cannot be used due to its
toxicity.
[0016] In consequence, this problem is resolved by including a drug
into a liposome which is efficiently accumulated on tissues and/or
organs that accompany production of proteoglycan, typified by renal
glomerulus which caused inflammation by an injury, and by
delivering the drug efficiently to such tissues and allowing it to
perform continuous action. This purpose is achieved by realizing a
liposome having active targeting property and ensured stability in
blood, which also has the ability to be accumulated on injured
portions of tissues and/or organs that accompany production of
various types of proteoglycan.
DISCLOSURE OF THE INVENTION
[0017] Accordingly, this invention contemplates providing a
liposome having active targeting property and ensured stability in
blood, which can be used in the diagnosis and/or treatment of
diseases, particularly renal diseases, that accompany production of
various types of proteoglycan in injured portions of tissues and/or
organs.
[0018] In the process of carrying out studies on factors which
control intracorporeal kinetics of liposome, the present inventors
have found surprisingly that, when a liposome is prepared by
incorporating a basic compound which takes positive charge within a
physiological pH range, within a certain range of mixing ratio, and
combining a hydrophilic polymer to its surface in an amount
controlled within a certain range, it shows such properties that
not only its stability in blood is ensured, its capture by the
liver and the like can be avoided and its retention in blood is
improved, but also its ability to recognize proteoglycan in injured
portions is sharply improved.
[0019] It was found also that, when its major particle size range
is from 90 to 200 nm, its accumulation on and selectivity and
persistency of the effects are improved for tissues and/or organs
in the living body, where proteoglycan is produced in excess
amount, particularly for renal glomeruli. In this case, the term
"major particle size range" means a range of particle size in which
70% or more of particles are included when the particle size
distribution of liposomes is defined by scattering intensity
distribution in the particle size measurement based on the laser
light scattering methods.
[0020] The present inventors have accomplished this invention by
conducting intensive studies based on the aforementioned new
knowledge.
[0021] Accordingly, the present invention provides a liposome in
which a drug is included, which comprises (1) a basic compound
which takes positive charge within a physiological pH range, (2) a
lipid derivative of a hydrophilic polymer and (3) a lipid which
constitutes the liposome, as its membrane constituting components,
wherein their constituting ratios are from 1 to 20 mol % of (1)
based on (3) and from 0.2 to 5 mol % of (2) based on the total of
(1) and (3).
[0022] More preferred is a liposome which comprises (1) a basic
compound which takes positive charge within a physiological pH
range, (2) a lipid derivative of a hydrophilic polymer and (3) a
lipid which constitutes the liposome, as its membrane constituting
components, wherein their constituting ratios are from 5 to 15 mol
% of (1) based on (3) and from 0.2 to 5 mol % of (2) based on the
total of (1) and (3).
[0023] The aforementioned basic compound which takes positive
charge within a physiological pH range is preferably a basic
compound which has an amidino group, a basic compound which has two
or more amino groups, a basic compound which has a piperidine ring
or a basic compound which has a quaternary amine, more preferably
any one of those represented by the following formulae 1 to 4.
1
[0024] In this connection, the compound of formula 1 is described
in WO 97/42166, the compounds of formulae 2 and 3 are described in
JP-A-9-263579 and the compound of formula 4 is described in Chem.
Papers, 39 (1), 125-134 (1985), but these documents describe
nothing about a liposome having active targeting property and
ensured safety in blood, which can be used in the diagnosis and/or
treatment of diseases, particularly renal diseases, that accompany
production of various types of proteoglycan in injured portions of
tissues and/or organs. 2
[0025] (In the formula 1, A represents an aromatic ring, each of
R.sup.1 and R.sup.2 represents an alkyl or alkenyl group having
from 10 to 25 carbon atoms, wherein R.sup.1 and R.sup.2 may be the
same or different from each other, each of X.sup.1 and X.sup.2
represents --O--, --S--, --COO--, --OCO--, --CONH-- or --NHCO--,
wherein X.sup.1 and X.sup.2 may be the same or different from each
other, m is 0 or 1 and n is 0 or an integer of from 1 to 6.) 3
[0026] (In the formula 2, R.sup.3 represents hydrogen or an alkyl
or alkenyl group having from 1 to 8 carbon atoms, R.sup.4
represents hydrogen or an alkyl or alkenyl group having from 1 to 8
carbon atoms, each of R.sup.5 and R.sup.6 represents hydrogen or an
alkyl or alkenyl group having from 1 to 25 carbon atoms (excluding
a case in which R.sup.5 and R.sup.6 are both hydrogen), wherein
R.sup.5 and R.sup.6 may be the same or different from each other,
X.sup.3 represents --O-- or --S--, p is 0 or 1 and q is 0 or an
integer of from 1 to 10.) 4
[0027] (In the formula 3, each of R.sup.7 and R.sup.8 represents an
alkyl or alkenyl group having from 1 to 8 carbon atoms, wherein
R.sup.7 and R.sup.8 may be the same or different from each other,
R.sup.9 represents hydrogen or an alkyl or alkenyl group having
from 1 to 8 carbon atoms, each of R.sup.10 and R.sup.11 represents
hydrogen or an alkyl or alkenyl group having from 1 to 25 carbon
atoms (excluding a case in which R.sup.10 and R.sup.11 are both
hydrogen), wherein R.sup.10 and R.sup.11 may be the same or
different from each other, X.sup.4 represents --O-- or --S--, r is
0 or 1 and s is 0 or an integer of from 1 to 10.) 5
[0028] The aforementioned lipid derivative of a hydrophilic polymer
is preferably a lipid derivative of polyethylene glycol, more
preferably, said lipid derivative of polyethylene glycol is a
compound which contains a polyethylene glycol chain and a diacyl
glycerol in one molecule.
[0029] It is desirable that the aforementioned hydrophilic polymer
has a molecular weight of from 1,000 to 7,000.
[0030] Regarding the aforementioned liposome, it is desirable that
its major particle size range is from 90 to 200 nm.
[0031] It is desirable that the aforementioned drug to be included
in the liposome is a drug for use in the diagnosis and/or treatment
of renal diseases or a drug for use in the diagnosis and/or
treatment of diseases which accompany overproduction of
proteoglycan in injured portions of tissues and/or organs.
[0032] It is desirable that the aforementioned drug for use in the
diagnosis and/or treatment of renal diseases is an adrenocortical
steroid and/or a derivative thereof.
[0033] It is desirable to use the aforementioned liposome as a drug
for the treatment of inflamed kidney, and it is desirable to
produce a medicament for the treatment of inflamed kidney making
use of such liposomes.
[0034] The invention also provides a method for the treatment of
inflamed kidney, which comprises administering these liposomes to
mammals including human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a graph showing a relationship between liposomes
modified by a basic compound which takes positive charge within a
physiological pH range and a hydrophilic polymer lipid derivative
and their affinity for rat mesangial cells as proteoglycan
producing cells.
[0036] FIG. 2 is a graph showing participation of proteoglycan in
the affinity of liposomes modified by a basic compound which takes
positive charge within a physiological pH range and a hydrophilic
polymer lipid derivative for rat mesangial cells as proteoglycan
producing cells.
[0037] FIG. 3 is a graph showing a relationship between the amount
of a basic compound which takes positive charge within a
physiological pH range contained in the liposome constituting lipid
and the affinity for rat mesangial cells.
[0038] FIG. 4 is a graph showing a relationship between the amount
of a basic compound which takes positive charge within a
physiological pH range contained in the liposome constituting lipid
and the affinity for human vascular endothelial cells.
[0039] FIG. 5 is a graph showing a relationship between the amount
of a basic compound which takes positive charge within a
physiological pH range contained in the liposome constituting lipid
and the affinity for rat mesangial cells.
[0040] FIG. 6 is a graph showing a relationship between the amount
of a basic compound which takes positive charge within a
physiological pH range contained in the liposome constituting lipid
and the affinity for human vascular endothelial cells.
[0041] FIG. 7 is a graph showing a relationship between the amount
of a basic compound which takes positive charge within a
physiological pH range contained in the liposome constituting lipid
and the stability of liposome in serum.
[0042] FIG. 8 is a graph showing a relationship between the
modification by a hydrophilic polymer lipid derivative and the
affinity for proteoglycan in a liposome modified by a basic
compound which takes positive charge within a physiological pH
range.
[0043] FIG. 9 is a graph showing a relationship between the
modified amount of a hydrophilic polymer lipid derivative and the
stability in serum in a liposome modified by a basic compound which
takes positive charge within a physiological pH range.
[0044] FIG. 10 is a schematic illustration showing accumulation of
liposomes modified by a basic compound which takes positive charge
within a physiological pH range on injured kidney.
[0045] FIG. 11 is a schematic illustration showing that liposomes
having neutral surfaces do not accumulate on injured kidney.
[0046] FIG. 12 is a graph showing a relationship between average
particle size of liposomes and their accumulation on injured
kidney.
[0047] FIG. 13 is a graph showing a relationship between kinds of
basic compound to be used in the surface modification of liposomes
and their accumulation on injured kidney.
[0048] FIG. 14 is a graph showing pharmacological effects of the
liposome of the invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0049] The following describes the invention in detail.
[0050] The membrane constituting components of the liposome of the
invention are comprised of a basic compound which takes positive
charge within a physiological pH range, a hydrophilic polymer lipid
derivative and a liposome constituting lipid, and it is possible to
use a stabilizing agent, antioxidant and the like as occasion
demands.
[0051] The liposome constituting lipid is not particularly limited,
with the proviso that it can form liposome, but a phospholipid or a
derivative thereof or a lipid other than phospholipid or a
derivative thereof can be used suitably from the viewpoint of
providing liposomes which are stable in the living body.
[0052] Examples of the aforementioned phospholipid include natural
or synthetic phospholipids such as phosphatidylcholine (lecithin),
phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, sphingomyelin,
cardiolipin and the like, or their hydrogenated products obtained
in the usual way.
[0053] Examples of the stabilizing agent include a sterol which
reduces membrane fluidity, such as cholesterol; and saccharides
such as glycerol and sucrose.
[0054] Examples of the antioxidant include a tocopherol homologue,
namely vitamin E. Tocopherol exists in 4 isomer forms of .alpha.,
.beta., .gamma., and .delta., all of them can be used in the
invention.
[0055] The liposome of the invention contains a basic compound
which takes positive charge within a physiological pH range
(sometimes to be referred to as basic compound hereinafter) and a
hydrophilic polymer lipid derivative as membrane constituting
components. The physiological pH range cannot be defined, because
it varies depending on the condition of the living body, but it is
a range of from 7.0 to 7.5 for example.
[0056] The basic compound which takes positive charge within a
physiological pH range on the liposome surface is not particularly
limited, with the proviso that it does not spoil structural
stability of the liposome, and its examples include lipid
derivatives in which a basic compound having an aliphatic primary
amino group, an aliphatic secondary amino group, an aliphatic
tertiary amino group, an aliphatic quaternary amino group; an
amidino group; a guanidino group; an aromatic primary amino group,
an aromatic secondary amino group, an aromatic tertiary amino group
or an aromatic quaternary amino group is linked, directly through
these groups or via an appropriate spacer, to a hydrophobic
compound such as a long chain aliphatic alcohol, a sterol, a
polyoxypropylene alkyl or a glycerol fatty acid ester. When linked
to a hydrophobic compound, the portion which takes positive charge
within a physiological pH range can easily be positioned on the
surface of liposome. Among these compounds, a basic compound which
has an amidino group, a basic compound which has two or more amino
groups, a basic compound which has a piperidine ring or a basic
compound which has a quaternary amine is preferable in terms of the
strong mutual reaction.
[0057] In this connection, when these basic compounds are linked to
hydrophobic compounds via a phosphate group or the like substituent
group which takes negative charge within a physiological pH range
(e.g., diacylglycerophosphatidylcholine,
diacylglycerophosphatidylethanolamine and a derivative of
diacylglycerophosphatidylethanolamine in which a neutral or
negative substituent group is linked to the ethanolamine
substituent group via an amido bond), the resulting compounds are
neutrally or negatively charged within the physiological pH range
due to such negative charge so that, as a matter of course, they
are not included in the basic compound which takes positive charge
within a physiological pH range.
[0058] These basic compounds may be used alone or as a mixture of
two or more. Illustrative examples of the aforementioned compounds
include DOTMA (U.S. Pat. No. 61,161,246), DOTAP (U.S. Pat. No.
5,508,626), Transfectam (U.S. Pat. No. 2,292,246), TMAG (U.S. Pat.
No. 4,108,391), DOSPA, Tfx.TM.-50, DODAC, DC-CHOL, DMRIE and the
like known compounds. As more preferred examples, those in which
the basic compound is any one of the aforementioned formulae 1 to 4
can be cited.
[0059] Amount of the basic compound which takes positive charge
within a physiological pH range is preferably from 1 to 20 mol %
based on the liposome constituting lipid, namely other membrane
constituting component excluding said basic compound and the
hydrophilic polymer. The amount if less than 1 mol % would entail
poor accumulation on proteoglycan producing tissues and/or organs
and if exceeding 20 mol % would spoil physical stability of
liposomes in the living body.
[0060] Also, amount of the basic compound which takes positive
charge within a physiological pH range is more preferably from 5 to
15 mol % based on the liposome constituting lipid, namely other
membrane constituting component excluding said basic compound and
the hydrophilic polymer. Within this range, selectivity for
proteoglycan, particularly proteoglycan chondroitin sulfate,
becomes excellent, and accumulation on the tissues and/or organs
which produce proteoglycan chondroitin sulfate becomes particularly
excellent.
[0061] Since the surface of the liposome of the invention takes
positive charge within a physiological pH range, its accumulation
on the tissues and/or organs which produce proteoglycan is
expressed thereby.
[0062] Also, since the liposome of the invention is modified by a
hydrophilic polymer, its stability in blood and RES avoiding effect
are thereby obtained.
[0063] Various methods can be cited without particular limitation
for modifying the liposome surface by a hydrophilic polymer, but it
is desirable to employ a method in which said hydrophilic polymer
is linked to a hydrophobic compound such as a long chain aliphatic
alcohol, a sterol, a polyoxypropylene alkyl, a glycerol fatty acid
ester or a phospholipid, and the portion of said hydrophobic
compound is inserted into the liposome membrane.
[0064] The lipid derivative of hydrophilic polymer is not
particularly limited, with the proviso that it does not spoil
structural stability of the liposome, and its examples include
lipid derivatives of polyethylene glycol, dextran, pullulan,
Ficoll, polyvinyl alcohol, a styrene-maleic anhydride alternating
copolymer, a divinyl ether-maleic anhydride alternating copolymer,
a synthetic polyamino acid, amylose, amylopectin, chitosan, mannan,
cyclodextrin, pectin, carrageenan and the like, of which
polyethylene glycol and a lipid derivative of polyethylene glycol
are particularly preferable.
[0065] The lipid derivative of polyethylene glycol is not
particularly limited, but it is preferably a compound which
contains a polyethylene glycol chain and a diacyl glycerol in one
molecule.
[0066] It is desirable that the hydrophilic polymer has a molecular
weight of from 1,000 to 7,000.
[0067] Particularly, polyethylene glycol and a lipid derivative of
polyethylene glycol having a molecular weight of from 1,000 to
7,000 are desirable because of their significant effect to improve
the retention in blood circulation.
[0068] Blending amount of the hydrophilic polymer lipid derivative
is preferably from 0.2 to 5 mol % based on the total amount of the
basic compound which takes positive charge within a physiological
pH range and the liposome constituting lipid, though it varies
depending on its molecular weight and adding method. Within this
range, accumulation on the tissues and/or organs where proteoglycan
is produced.
[0069] The reason for this is as follows. When blending amount of
the hydrophilic polymer lipid derivative is within the above range,
interaction between plasma protein and liposome is inhibited by
hydrophilic polymer on the liposome surface, so that the stability
in blood becomes excellent. On the other hand, when blending amount
of the hydrophilic polymer lipid derivative is within the above
range, binding of liposome with certain biological polymer
components is not inhibited. Chondroitin sulfate, dermatan sulfate
and the like as constituting components of proteoglycan belong to
galactosaminoglycan (GAG) which is a fibrous anionic biological
polymer component, but binding of such a fibrous anionic biological
polymer with the liposome is not inhibited by the specified
blending amount of the hydrophilic polymer lipid derivative of the
invention. The liposome of the invention selectively recognizes a
tissue and/or organ where proteoglycan having such GAG is
produced.
[0070] An effect has been well known that concentration of
liposomes in blood is maintained for a certain period of time by
such modification by a hydrophilic polymer. On the other hand, the
hydrophilic polymer lipid derivative has an aspect that it makes
the surface of liposome hydrophilic and thereby diminishes its
accumulation on organs. However, it has not been known that
characteristics of the liposome surface are not diminished when
modified by a hydrophilic polymer and that its characteristics can
rather be improved through modification by the hydrophilic polymer
within a certain range of concentration.
[0071] For example, a liposome in which the other lipid, a basic
compound which takes positive charge within a physiological pH
range and a hydrophilic polymer lipid derivative are blended within
the range of 20:1:1 is disclosed in Biochemistry, 37: 12875,
(1998). It is described in this report that, when about 5 mol % of
a hydrophilic polymer which itself is anion and the same amount of
a basic compound are blended, binding of cationic liposome to cells
is inhibited by the hydrophilic polymer, but it does not describe
that a liposome which has excellent stability in blood and binds to
cells via its interaction with proteoglycan can be prepared by the
blending ratio of the present invention.
[0072] Also, it has been reported in Biochimica et Biophysica Acta,
1326: 236, (1997) that interaction between a liposome which is not
cationic and cells is inhibited by the blending of 1 to 5 mol % of
a hydrophilic polymer lipid derivative. However, as shown in Test
Example 1, the interaction between a cationic liposome and cells is
completely different from the case of a neutral or anionic
liposome, and binding strength of the cationic liposome to cells
increases as the blending ratio of a basic compound which takes
positive charge within a physiological pH range is increased (Test
Examples 3 and 4), so that the just cited report in which such
points are not examined is of no help with regard to the blending
ratio of the hydrophilic polymer most suited for the interaction
between the cationic liposome and cells.
[0073] A cationic liposome in which a PEG ceramide is blended as a
hydrophilic polymer lipid derivative is disclosed in WO 96/10391.
In this document, it is disclosed that the stability in blood is
maintained by the blending of 10 mol % or more of the PEG ceramide
in the case of a liposome which contains a basic compound that
takes positive charge within a physiological pH range in an amount
of about 17.5 mol % based on other lipids (about 15 mol % of the
total lipids), and that its interaction with cells is maintained
(retained) by the blending of 5 mol % of the PEG ceramide. However,
it does not disclose that a liposome in which its stability in
blood is maintained and its interaction with cells via proteoglycan
is optimized can be prepared by the blending ratio of the present
invention (blending of from 1 to 20 mol %, preferably from 5 to 15
mol %, of a basic compound with from 0.2 to 5 mol % of a
hydrophilic polymer derivative, based on other lipids).
[0074] WO 98/51285 discloses a liposome which contains a basic
compound which takes positive charge within a physiological pH
range and a combined product of PEG and
dilauroylphosphatidylethanolamine or a liposome which contains
diphthanoylphosphatidylethanolamine in addition to the basic
compound and PEG derivative. According to the basic compound of
this liposome, its content of 50% based on other lipids is
disclosed, but nothing is disclosed about the blending ratio within
the range of the present invention.
[0075] According to this invention, the blending ratio of the basic
compound which takes positive charge within a physiological pH
range and the hydrophilic polymer lipid derivative is a definitely
important factor. Regarding the stability of liposomes in blood in
particular, their interaction with plasma protein and incorporation
into the RES system via the interaction have been regarded as
important but, in addition to these factors, their interaction with
fibrous anionic biological polymer components such as GAGs and the
like which are constituting components of proteoglycan on the cell
surface is important in the case of cationic liposomes.
[0076] In the case of glomerulonephritis, mesangial cells of the
glomeruli generate a proliferative change and thereby cause the
progression of disease. In Test Example 1, mesangial cell-binding
ability of a liposome containing 8.7 mol % of a basic compound
(3,5-dipentadecyloxybenzamidine hydrochloride) based on the other
lipids is shown in comparison with a liposome which does not
contain the basic compound. The liposome binds to mesangial cells
only in the presence of the basic compound. Also, as shown in Test
Example 2, this binding does not occur when GAGs linked to
proteoglycan on the cell surface are cut out, thus showing that
this binding is effected via the GAGs of proteoglycan. In addition,
it is shown in Test Example 1 that, since the binding ability
decreases as the amount of the hydrophilic polymer to be added is
increased, it is desirable that the blending amount of the
hydrophilic polymer does not exceed mol %.
[0077] On the other hand, it is shown in Test Example 4 that, when
the hydrophilic polymer is 5 mol % or less, aggregation in blood
cannot sufficiently be inhibited if the basic compound which takes
positive charge within a physiological pH range is blended in an
amount exceeding 20 mol % based on other lipids. That is, it is
desirable that blending ratio of the basic compound is set to 20
mol % or less based on other lipids.
[0078] Certain types of proteoglycan exist also in endothelial
cells which cover the surface of blood vessels. In consequence,
when interaction with such endothelial cells is strong, physical
stability of liposomes in blood circulation is spoiled by this
interaction with endothelial cells, thus arising a possibility of
the inhibition of efficient delivery to an injured portion. Thus,
it is more desirable that the liposome of interest can bind to
proteoglycan in the injured portion but does not interact with
proteoglycan on vascular endothelial cells. Surprisingly, it was
revealed by some studies carried out by the present inventors based
on Test Examples 3 and 4 that such a liposome can be realized when
the basic compound is used at a blending ratio of between 1 mol %
or more and 20 mol % or less, preferably 5 mol % or more and 15 mol
% or less, based on other lipids.
[0079] As described above, the liposome which can bind to injured
portions of tissues and/or organs where proteoglycan is produced,
while maintaining its stability in blood, can be realized for the
first time by the extremely limited ranges of blending ratios of a
cationic lipid and a hydrophilic polymer, so that the liposome of
the invention is clearly different from already known liposomes in
terms of the construction and purpose.
[0080] According to the invention, a surface modifying agent other
than the hydrophilic polymer lipid derivative can be used jointly.
Examples of the surface modifying agent other than the hydrophilic
polymer include water-soluble polysaccharides such as glucuronic
acid, sialic acid and the like.
[0081] As the drug to be included in the liposome, pharmaceutically
acceptable pharmacologically active substances, physiologically
active substances and/or substances for diagnosis use can be used
depending on each purpose for the diagnosis and/or treatment of
renal diseases and other diseases that accompany overproduction of
proteoglycan in injured portions of tissues and/or organs.
[0082] Properties of the drug to be included in the liposome are
not particularly limited, but electrically neutral or anionic
property is desirable, because the surface of liposome is modified
by a substituent group which takes positive charge within a
physiological pH range, and high inclusion ration can therefore be
obtained.
[0083] Types of the drug to be included in the liposome are not
particularly limited so far as the formation of liposome is not
spoiled. Their examples include adrenocortical steroids such as
prednisolone, methylprednisolone, dexamethasone and the like;
non-steroidal anti-inflammatory drugs such as aspirin, indometacin,
ibuprofen, mefenamic acid, phenylbutazone and the like; mesangial
cell growth inhibitors such as heparin, low molecular weight
heparin and the like; immunosuppressants such as cyclosporin and
the like; angiotensin converting enzyme (ACE) inhibitors such as
captopril and the like; AGE (advanced glycation endoproduct)
inhibitors such as methylguanidine and the like; TGF-.beta.
antagonists such as biglycan, decorin and the like; PKC (protein
kinase C) inhibitors; prostaglandin preparations such as PGE.sub.1,
PGI.sub.2 and the like; peripheral vasodilators such as papaverine
drugs, nicotinic acid drugs, tocopherol drugs, Ca antagonist and
the like; phosphodiesterase inhibitors; anti-thrombus agents such
as ticlopidine, aspirin and the like; anticoagulants such as
warfarin, heparin, anti-thrombin agent and the like; thrombolytic
agents such as urokinase and the like; chemical mediator release
inhibitors; antibiotics; antioxidants; enzyme preparations; lipid
incorporation inhibitors; hormone preparations; vitamin C; vitamin
E; radical scavengers such as SOD and the like; antisense
oligonucleotides which have the action to inhibit growth of
mesangial cells; carcinostatic agents; decoys or genes; X-ray
contrast media; radioisotope-labeled nuclear medicinal diagnostic
agents; and MRI contrast media. Particularly, an adrenocortical
steroid and/or a derivative thereof and/or a mixed preparation
thereof containing other components can be suitably used by this
invention. Because, despite their strong efficacy, they cause
considerably strong systemic side effects. The continuous or
long-term administration by this invention can minimize the side
effect.
[0084] Examples of the diseases in which proteoglycan is
excessively produced in the injured portions of tissues and/or
organs include renal diseases in which inflammation is formed on
glomeruli, as well as diseases in which fibrosis occurs in the
liver, the lungs, the heart, the pancreas, the bone marrow,
arteries and the like, the tumors such as large bowel cancer and
the like and inflammatory diseases.
[0085] Examples of the aforementioned renal diseases include
minimal change type focal glomerular sclerosis, IgA nephritis,
mesangial proliferative glomerulonephritis, membranous
glomerulonephritis, membranoproliferative glomerulonephritis types
I, II and III, intracanalicular proliferative glomerulonephritis
and crescentic glomerulonephritis (=extracapillary proliferative
glomerulonephritis) as primary glomerulonephritis; and lupus
nephritis, Goodpasture syndrome, diabetic nephropathy, systemic
vascular inflammation, thrombotic microvascular inflammation,
intraglomerular thrombosis, benign nephrosclerosis, malignant
nephrosclerosis, progressive systemic sclerosis (scleroderma),
glomerular disorder accompanied by infection and drug induced renal
disorder as tonic glomerulonephritis.
[0086] Most suitable size of the liposome of the invention is from
90 to 200 nm as the major particle size range. As will be shown
later in Test Example 8, the liposome if within this range can be
easily transferred into inflammed tissues and/or organs accompanied
by vascular damage and production of proteoglycans including renal
glomeruli. The major particle size range if less than 90 nm would
facilitate its transfer into normal tissues and/or organs too and
if exceeding 200 nm would hinder smooth transfer into injured
tissues. On the other hand, average particle size is an average
value of total particle size measured by the light scattering
method in the same manner, and this value is a value within the
major particle size range and its range is preferably from 90 to
200 nm as a matter of course.
[0087] The liposome of the invention can be obtained easily in the
usual way. An example thereof is shown in the following.
[0088] A basic compound which takes positive charge within a
physiological pH range and other membrane constituting components
such as a phospholipid, a stabilizing agent, an antioxidant and the
like are mixed in organic solvent such as chloroform or the like in
a flask, the solvent is evaporated and then the resulting residue
is dried in vacuo to effect formation of a thin film on the inner
wall of the flask. Next, a drug is put into the flask and
vigorously stirred, thereby obtaining a liposome dispersion liquid.
The thus obtained liposome dispersion liquid is centrifuged, and
the supernatant is subjected to decantation to remove the
unstrapped drug. Thereafter, the liposome of the invention is
obtained by adding a hydrophilic polymer lipid derivative solution
and heating the mixture. In this connection, the liposome of the
invention can also be obtained by adding the hydrophilic polymer
lipid derivative solution at the time of the mixing of membrane
constituting components. Though both of these methods for adding
the hydrophilic polymer lipid derivative solution do not have
particular problems, the adding method at the time of the mixing of
membrane constituting components entails inclusion of the
hydrophilic polymer inside the liposome too, so that it will
sometimes cause substantial reduction of the surface modification
ratio and reduction of inclusion volume.
[0089] Alternatively, the liposome of the invention can also be
obtained by mixing the aforementioned respective constituting
components and effecting high pressure discharge of the mixture
using a high pressure discharge type emulsifier.
[0090] The liposome of the invention specifically accumulates on
proteoglycan producing tissues and/or organs, particularly on renal
glomeruli which are caused inflammation by injury. The ratio of the
accumulated amount of the liposome of the invention on injured
tissues and/or organs to its accumulated amount on normal tissues
and/or organs is approximately from 2 to 10 times when compared
after its administration or after a certain lapse of time. In
consequence, the use of the liposome of the invention renders
possible efficient delivery of a drug to injured
proteoglycan-overproducing tissues and/or organs, particularly to
injured renal glomeruli, and its continuous action therein. In
addition, even if a drug having large side effects is used as the
drug, its side effects can be reduced to the minimum, so that when
an adrenocortical steroid and/or a derivative thereof is used for
example, the liposome can be used suitably for IgA nephritis or the
like glomerulonephritis.
EXAMPLES
[0091] The following illustratively describes the invention with
reference to examples, but the invention is not limited
thereto.
[0092] 1. Preparation of Various Types of Liposomes
Preparation Example 1
Preparation of a Liposome Which Contains
3,5-Dipentadecyloxybenzamidine Hydrochloride and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative (PEG-PE) as Membrane
Constituting Components
[0093] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0094] Phosphatidylcholine (concentration, 100 mM): 3.0 ml
[0095] Cholesterol (concentration, 100 mM): 2.54 ml
[0096] 3,5-Dipentadecyloxybenzamidine hydrochloride represented by
the following formula (100 mM): 4.6 ml 6
[0097] After evaporation of chloroform, the resulting residue was
dried overnight in vacuo to effect formation of a lipid thin film
on the inner wall of the flask. Next, 10 ml of 150 mM sodium
chloride solution was put into the flask and treated with a bath
type sonicator to obtain a liposome (MLV) dispersion liquid. This
was compressed using a polycarbonate film of 0.4 .mu.m in pore size
and then compressed using a polycarbonate film of 0.1 .mu.m to
carry out dressing of grain.
[0098] The same volume of a solution prepared by dissolving a
polyethylene glycol-phosphatidylethanolamine derivative represented
by the following formula in physiological saline (concentration,
0.0125, 0.025, 0.05, 0.1, 0.2 or 0.4 w/v %) was added to the
liposome dispersion liquid, and the mixture was heated at
60.degree. C. for 30 minutes to obtain a dispersion liquid of the
title liposome whose surface was modified by the hydrophilic
polymer lipid derivative. 7
[0099] When a 100 .mu.l portion of the thus obtained liposome
dispersion liquid was dispersed in 3 ml of 150 mM sodium chloride
aqueous solution and the particle size was measured using a
particle size distribution analyzer Zetamaster-S manufactured by
Malvern, the average particle size was from 127.1 to 132.5 nm and
the major particle size range was from 90 to 200 nm.
[0100] Also, when the phosphatidylcholine content was measured by a
choline oxidase method, and the cholesterol content by a
cholesterol oxidase-phenol method and the
3,5-dipentadecyloxybenzamidine hydrochloride content by a liquid
chromatography, the 3,5-dipentadecyloxybenzamidine hydrochloride
content was 8.7 mol % based on other lipids.
[0101] Separately, the thus obtained liposome dispersion liquid was
centrifuged at 100,000 g, and the supernatant was separated by
decantation. The polyethylene glycol-phosphatidylethanolamine
derivative (PEG-PE) content of the supernatant was measured by a
picric acid reagent color developing method, and modification ratio
of the PEG-PE derivative was calculated by subtracting the measured
value from its added amount. The PEG-PE derivative modification
ratio of the thus obtained liposomes were 0.11, 0.23, 0.45, 0.91,
1.8 and 3.8 mol %, respectively.
Preparation Example 2
Preparation of a Liposome Which Contains
N,N-dioctadecyl-2-(piperidin-4-yl- -oxy)acetamide and a
Polyethylene Glycol-Phosphatidylethanolamine Derivative as Membrane
Constituting Components
[0102] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0103] Phosphatidylcholine (concentration, 100 mM): 3.0 ml
[0104] Cholesterol (concentration, 100 mM): 2.54 ml
[0105] N,N-Dioctadecyl-2-(piperidin-4-yl-oxy)acetamide represented
by the following formula (10 mM): 4.6 ml 8
[0106] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0107] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 112.4 to 127.8 nm, the major particle size range was
from 90 to 200 nm, the
N,N-dioctadecyl-2-(piperidin-4-yl-oxy)acetamide content was 8.7 mol
% based on other lipids, and the PEG-PE derivative modification
ratios were 0.11, 0.23, 0.45, 0.91, 1.8 and 3.8 mol %,
respectively.
Preparation Example 3
Preparation of a Liposome Which Contains
N'-pentadecyldiethylenetriamine and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0108] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0109] Phosphatidylcholine (concentration, 100 mM): 3.0 ml
[0110] Cholesterol (concentration, 100 mM): 2.54 ml
[0111] N'-Pentadecyldiethylenetriamine represented by the following
formula (10 mM): 4.6 ml 9
[0112] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0113] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 111.7 to 125.8 nm, the major particle size range was
from 90 to 200 nm, the N'-pentadecyldiethylenetriamine content was
8.7 mol % based on other lipids, and the PEG-PE derivative
modification ratios were 0.11, 0.23, 0.45, 0.91, 1.8 and 3.8 mol %,
respectively.
Preparation Example 4
Preparation of a Liposome Which Contains Glucamine Palmitate and a
Polyethylene Glycol-Phosphatidylethanolamine Derivative as Membrane
Constituting Components
[0114] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0115] Phosphatidylcholine (concentration, 100 mM): 3.0 ml
[0116] Cholesterol (concentration, 100 mM): 2.54 ml
[0117] Glucamine palmitate represented by the following formula
10
[0118] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0119] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 112.5 to 124.5 nm, the major particle size range was
from 90 to 200 nm, the glucamine palmitate content was 8.7 mol %
based on other lipids, and the PEG-PE derivative modification
ratios were 0.11, 0.23, 0.45, 0.91, 1.8 and 3.8 mol %,
respectively.
Preparation Example 5
Preparation of a Liposome Which Contains
1,2-dipalmitoyl-3-trimethylammoni- umpropane and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0120] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0121] Phosphatidylcholine (concentration, 100 mM): 3.0 ml
[0122] Cholesterol (concentration, 100 mM): 2.54 ml
[0123] 1,2-Dipalmitoyl-3-trimethylammoniumpropane represented by
the following formula (10 mM): 4.6 ml 11
[0124] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0125] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 113.4 to 130.1 nm, the major particle size range was
from 90 to 200 nm, the 1,2-dipalmitoyl-3-trimethylammoniumpropane
content was 8.7 mol % based on other lipids, and the PEG-PE
derivative modification ratios were 0.11, 0.23, 0.45, 0.91, 1.8 and
3.8 mol %, respectively.
Preparation Example 6
Preparation of a Liposome Which Contains
1,2-dipalmitoyl-3-dimethylammoniu- mpropane and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0126] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0127] Phosphatidylcholine (concentration, 100 mM): 3.0 ml
[0128] Cholesterol (concentration, 100 mM): 2.54 ml
[0129] 1,2-Dipalmitoyl-3-dimethylammoniumpropane represented by the
following formula (10 mM): 4.6 ml 12
[0130] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0131] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 113.4 to 130.1 nm, the major particle size range was
from 90 to 200 nm, the 1,2-dipalmitoyl-3-dimethylammoniumpropane
content was 8.7 mol % based on other lipids, and the PEG-PE
derivative modification ratios were 0.11, 0.23, 0.45, 0.91, 1.8 and
3.8 mol %, respectively.
Preparation Example 7
Preparation of a Neutral Liposome Preparation
[0132] Each of the following 2 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0133] Phosphatidylcholine (concentration, 100 mM): 3.48 ml
[0134] Cholesterol (concentration, 100 mM): 2.54 ml
[0135] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0136] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 111.4 to 126.5 nm, the major particle size range was
from 90 to 200 nm, and the PEG-PE derivative modification ratios
were 0.11, 0.23, 0.45, 0.91, 1.8 and 3.8 mol %, respectively.
Preparation Example 8
Preparation of a Liposome Which Contains
3,5-dipentadecyloxybenzamidine Hydrochloride and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0137] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0138] Phosphatidylcholine (concentration, 100 mM): 3.12 ml
[0139] Cholesterol (concentration, 100 mM): 2.66 ml
[0140] 3,5-Dipentadecyloxybenzamidine hydrochloride (10 mM): 2.3
ml
[0141] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0142] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 124.5 to 132.4 nm, the major particle size range was
from 90 to 200 nm, the 3,5-dipentadecyloxybenzamidine hydrochloride
content was 4.2 mol % based on other lipids, and the PEG-PE
derivative modification ratios were 0.11, 0.23, 0.45, 0.91, 1.8 and
3.8 mol %, respectively.
Preparation Example 9
Preparation of a Liposome Which Contains
3,5-dipentadecyloxybenzamidine Hydrochloride and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0143] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0144] Phosphatidylcholine (concentration, 100 mM): 2.76 ml
[0145] Cholesterol (concentration, 100 mM): 2.36 ml
[0146] 3,5-Dipentadecyloxybenzamidine hydrochloride (100 mM): 8.63
ml
[0147] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0148] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 113.3 to 123.2 nm, the major particle size range was
from 90 to 200 nm, the 3,5-dipentadecyloxybenzamidine hydrochloride
content was 17.6 mol % based on other lipids, and the PEG-PE
derivative modification ratios were 0.11, 0.23, 0.45, 0.91, 1.8 and
3.8 mol %, respectively.
Preparation Example 10
Preparation of a Liposome Which Contains
3,5-dipentadecyloxybenzamidine Hydrochloride and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0149] Each of the following 3 membrane constituting components was
added as chloroform solution to a 50 ml capacity eggplant type
flask and mixed.
[0150] Phosphatidylcholine (concentration, 100 mM): 2.28 ml
[0151] Cholesterol (concentration, 100 mM): 1.94 ml
[0152] 3,5-Dipentadecyloxybenzamidine hydrochloride (100 mM): 1.73
ml
[0153] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0154] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 125.3 to 133.3 nm, the major particle size range was
from 90 to 200 nm, the 3,5-dipentadecyloxybenzamidine hydrochloride
content was 42.9 mol % based on other lipids, and the PEG-PE
derivative modification ratios were 0.11, 0.23, 0.91, 1.8 and 3.8
mol %, respectively.
Preparation Example 11
Preparation of a Liposome Which Contains 1,
2-dipalmitoyl-3-trimethylammon- iumpropane and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0155] Each of the following 3 membrane constituting components was
added as chloroform solution to a 0.150 ml capacity eggplant type
flask and mixed.
[0156] Phosphatidylcholine (concentration, 100 mM): 3.24 ml
[0157] Cholesterol (concentration, 100 mM): 2.75 ml
[0158] 1,2-Dipalmitoyl-3-trimethylammoniumpropane (10 mM): 0.29
ml
[0159] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0160] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 115.4 to 126.3 nm, the major particle size range was
from 90 to 200 nm, the 1,2-dipalmitoyl-3-dimethylammoniumpropane
content was 0.5 mol % based on other lipids, and the PEG-PE
derivative modification ratios were 0.11, 0.23, 0.91, 1.8 and 3.8
mol %, respectively.
Preparation Example 12
Preparation of a Liposome Which Contains
1,2-dipalmitoyl-3-trimethylammoni- umpropane and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0161] Each of the following 3 membrane constituting components was
added as chloroform solution to a 0.150 ml capacity eggplant type
flask and mixed.
[0162] Phosphatidylcholine (concentration, 100 mM): 3.06 ml
[0163] Cholesterol (concentration, 100 mM): 2.66 ml
[0164] 1,2-Dipalmitoyl-3-trimethylammoniumpropane (10 mM): 2.88
ml
[0165] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0166] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 114.5 to 128.1 nm, the major particle size range was
from 90 to 200 nm, the 1,2-dipalmitoyl-3-dimethylammoniumpropane
content was 5.3 mol % based on other lipids, and the PEG-PE
derivative modification ratios were 0.11, 0.23, 0.91, 1.8 and 3.8
mol %, respectively.
Preparation Example 13
Preparation of a Liposome Which Contains
N',N"-dipentadecyltriethyltetrami- ne and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0167] Each of the following 3 membrane constituting components was
added as chloroform solution to a 0.150 ml capacity eggplant type
flask and mixed.
[0168] Phosphatidylcholine (concentration, 100 mM): 3.24 ml
[0169] Cholesterol (concentration, 100 mM): 2.75 ml
[0170] N',N"-Dipentadecyltriethyltetramine represented by the
following formula (10 mM): 0.29 ml 13
[0171] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0172] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 111.4 to 123.1 nm, the major particle size range was
from 90 to 200 nm, the N',N"-dipentadecyltriethyltetramine content
was 0.5 mol % based on other lipids, and the PEG-PE derivative
modification ratios were 0.11, 0.23, 0.91, 1.8 and 3.8 mol %,
respectively.
Preparation Example 14
Preparation of a Liposome Which Contains
N',N"-dipentadecyltriethyltetrami- ne and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components
[0173] Each of the following 3 membrane constituting components was
added as chloroform solution to a 0.150 ml capacity eggplant type
flask and mixed.
[0174] Phosphatidylcholine (concentration, 100 mM): 3.06 ml
[0175] Cholesterol (concentration, 100 mM): 2.66 ml
[0176] N',N"-Dipentadecyltriethyltetramine (10 mM): 2.88 ml
[0177] Thereafter, a dispersion liquid of the title liposome was
obtained by the same method of Preparation Example 1.
[0178] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was from 122.5 to 130.5 nm, the major particle size range was
from 90 to 200 nm, the N',N"-dipentadecyltriethyltetramine content
was 5.3 mol % based on other lipids, and the PEG modification
ratios were 0.11, 0.23, 0.91, 1.8 and 3.8 mol %, respectively.
Preparation Example 15
Preparation of a Liposome Which Contains
3,5-dipentadecyloxybenzamidine and a Polyethylene
Glycol-Phosphatidylethanolamine Derivative as Membrane Constituting
Components and Includes Prednisolone Phosphate Sodium Therein
[0179] Each of the following 4 membrane constituting components was
added as chloroform solution to a 0.150 ml capacity eggplant type
flask and mixed.
[0180] Phosphatidylcholine (concentration, 100 mM): 4.2 ml
[0181] Cholesterol (concentration, 100 mM): 1.20 ml
[0182] 3,5-Dipentadecyloxybenzamidine hydrochloride (10 mM): 5.75
ml
[0183] Polyethylene glycol-phosphatidylethanolamine derivative (1
mM): 12 ml
[0184] After evaporation of chloroform, the resulting residue was
dried overnight in vacuo to effect formation of a lipid thin film
on the inner wall of the flask. Next, 10 ml of 10 mM sodium
chloride aqueous solution containing 140 mM prednisolone phosphate
sodium was put into the flask and treated with a bath type
sonicator to obtain a liposome (MLV) dispersion liquid. This was
compressed using a polycarbonate film of 0.4 .mu.m in pore size and
then compressed using a polycarbonate film of 0.1 .mu.m to carry
out dressing of grain, thereby obtaining a dispersion liquid of the
title liposome.
[0185] When the thus obtained liposome was measured in the same
manner as described in Preparation Example 1, the average particle
size was 133.1 nm and the major particle size range was from 90 to
200 nm.
Comparative Example 1
[0186] A liposome was prepared in the same manner as described in
Preparation Example 15, except that physiological saline was used
instead of the 10 mM sodium chloride aqueous solution containing
140 mM prednisolone phosphate sodium used in Preparation Example
15. The average particle size was 103.4 nm and the major particle
size range was from 90 to 200 nm.
Preparation Example 16
Preparation of Liposome Having an Average Particle Size of
Exceeding 200 nm
[0187] A dispersion liquid of the title liposome was obtained in
the same manner as described in Preparation Example 1, except that
the polycarbonate film of 0.4 .mu.m in pore size alone was used for
the dressing of grain and the polycarbonate film of 0.1 .mu.m in
pore size was not used.
[0188] When particle size of the thus obtained liposome was
measured in the same manner as described in Preparation Example 1,
the average particle size was 273 nm and the major particle size
range was from 180 to 350 nm.
Preparation Example 17
Preparation of Liposome Having an Average Particle Size of Less
than 90 nm
[0189] A dispersion liquid of the title liposome was obtained in
the same manner as described in Preparation Example 1, except that
the dressing of grain was carried out using a polycarbonate film of
0.4 .mu.m in pore size, a polycarbonate film of 0.1 .mu.m in pore
size and then a polycarbonate film of 0.05 .mu.m in pore size.
[0190] When particle size of the thus obtained liposome was
measured in the same manner as described in Preparation Example 1,
the average particle size was 79.6 nm and the major particle size
range was from 40 to 90 nm.
[0191] 2. Test Examples on the Performance of Various Liposomes
[0192] Performances of the thus obtained various liposomes were
tested.
Test Example 1
Affinity for Proteoglycan Producing Cells (1)
[0193] The purpose of this test is to know the effect of the
surface modification by a basic compound which takes positive
charge within a physiological pH range on the adhesion and
incorporation of liposomes to and into proteoglycan producing
cells, and the effect of a hydrophilic polymer lipid derivative on
this case.
[0194] <Method>
[0195] Rat mesangial cells were cultured using a 12 well microplate
until their proliferation reached about 4.times.10.sup.4/well as
the number of cells. Each of the Rhodamine-labeled liposomes
prepared in accordance with Preparation Examples 1 and 7, in which
the liposome membranes were labeled with Rhodamine-PE, was added to
each well in an amount of 50 .mu.g as lipid. After 24 hours of
incubation at 37.degree. C., the plate was washed twice with
physiological saline. The cells were lysed by adding 0.1% SDS
solution, and the fluorescence intensity was measured to calculate
the amount of liposomes adhered to and incorporated into cells.
[0196] <Results>
[0197] The results are shown in FIG. 1.
[0198] When liposomes whose surfaces were modified by a basic
compound which takes positive charge within a physiological pH
range were further modified by various concentrations of a
polyethylene glycol-phosphatidylethanolamine derivative,
aggregation occurred during the test when they were not modified by
the polyethylene glycol-phosphatidylethanolamine derivative. Also,
adhesion to and incorporation into cells did not occur in the case
of liposomes having no basic compound which takes positive charge
within a physiological pH range on their surfaces.
[0199] That is, in order to ensure stable adhesion to and
incorporation into proteoglycan-producing cells, it is necessary
that their surfaces are modified by the basic compound which takes
positive charge within a physiological pH range, and it is
necessary for more stable incorporation that the modifying amount
of the hydrophilic polymer lipid derivative is 5 mol % or less.
Test Example 2
Affinity for Proteoglycan Producing Cells (2)
[0200] The purpose of this test is to know importance of
proteoglycan for the adhesion of liposomes, in which their surfaces
were modified by a basic compound which takes positive charge
within a physiological pH range, to proteoglycan producing
cells.
[0201] <Method>
[0202] Rat mesangial cells were cultured using a 12 well microplate
until their proliferation reached about 4.times.10.sup.4/well as
the number of cells. Thereafter, they were enzymatically treated
with chondroitinase ABC to remove proteoglycan from the cell
surface.
[0203] The Rhodamine-labeled liposome prepared in accordance with
Preparation Examples 1 having a polyethylene
glycol-phosphatidylethanolam- ine derivative modification ratio of
0.45 mol %, in which the liposome membrane was labeled with
Rhodamine-PE, was added to each well in an amount of 50 .mu.g as
lipid. After 3 hours of incubation at 37.degree. C., the plate was
washed twice with physiological saline. The cells were lysed by
adding 0.1% SDS solution, and the fluorescence intensity was
measured to calculate the amount of liposomes adhered to and
incorporated into cells.
[0204] <Results>
[0205] The results are shown in FIG. 2.
[0206] The liposomes whose surfaces were modified by a basic
compound which takes positive charge within a physiological pH
range adhered to the cells whose surfaces were covered with
proteoglycan, but their adhesion to the treated cells in which
chondroitin sulfate was cut out from proteoglycan by chondroitinase
ABC was considerably reduced.
[0207] That is, a liposome whose surface is modified by a basic
compound which takes positive charge within a physiological pH
range adheres by binding to proteoglycan on the cell surface via
chondroitin sulfate or the like GAG in the molecule.
Test Example 3
Affinity for Proteoglycan Producing Cells (3)
[0208] The purpose of this test is to know the effect of the
surface-modifying amount of a basic compound which takes positive
charge within a physiological pH range on the affinity for
proteoglycan producing cells.
[0209] <Methods>
[0210] Rat mesangial cells were cultured using a 12 well microplate
until their proliferation reached about 4.times.10.sup.4/well as
the number of cells. Each of the Rhodamine-labeled liposomes
prepared in accordance with Preparation Examples 8, 1, 9 and 10,
and 11 to 14, in which the liposome membranes were labeled with
Rhodamine-PE, was added to each well in an amount of 50 .mu.g as
lipid. After 24 hours of incubation at 37.degree. C., the plate was
washed twice with physiological saline. The cells were lysed by
adding 0.1% SDS solution, and the fluorescence intensity was
measured to calculate the amount of liposomes adhered to and
incorporated into cells.
[0211] In the same manner, human vascular endothelial cells were
cultured using a 12 well microplate until their proliferation
reached about 4.times.10.sup.4/well as the number of cells, and
each of the Rhodamine-labeled liposomes prepared in accordance with
Preparation Examples 8, 1, 9 and 10, and 11 to 14, in which the
liposome membranes were labeled with Rhodamine-PE, was added to
each well in an amount of 50 .mu.g as lipid. Thereafter, the amount
of liposomes adhered to and incorporated into cells was calculated
by repeating the same procedure of the case of rat mesangial
cells.
[0212] <Results>
[0213] The results are shown in FIGS. 3 to 6.
[0214] Their adhesion to the cells varies depending on the amount
of the basic compound which takes positive charge within a
physiological pH range on the cell surface.
[0215] Their adhesion to rat mesangial cells and human vascular
endothelial cells did not occur when the basic compound content was
0.5 mol % or less, but the adhesion to rat mesangial cells capable
of producing a large amount of chondroitin sulfate proteoglycan was
improved when the content was 1 mol % or more (FIG. 3 and FIG. 5).
Also, the adhesion to rat mesangial cells increases as the basic
compound content was increased. However, when the content was
increased to 20 mol % or more, the adhesion to human vascular
endothelial cells capable of producing a large amount of heparan
sulfate proteoglycan also increased (FIG. 4 and FIG. 5). Also,
strength of the adhesion to mesangial cells varied depending on the
kind of basic compound, and the adhesion was ensured generally at 5
mol % or more in each case of the membrane materials and the
adhesion to mesangial cells was observed even within the range of
from 1 to 5 mol % depending on the membrane material.
[0216] That is, the adhesion to proteoglycan producing cells occurs
at a cationic membrane material content of 1 mol % or more. It is
preferably 5 mol % or more. Also, when it is within the range of
from 5 to 15 mol %, a liposome which does not bind to vascular
endothelial cells but shows adhesion selectively for proteoglycan
produced by the target cells on an injured portion can be prepared,
so that such a range is particularly desirable.
Test Example 4
Stability of Various Liposomes in Serum
[0217] The object of this test is to know effect of the amount of
liposome surface basic compound which takes positive charge within
a physiological pH range on the stability of liposomes in
serum.
[0218] <Method>
[0219] A 100 .mu.l portion of rat serum was mixed with 100 .mu.l of
a dispersion liquid prepared by diluting each of the liposomes
obtained in Preparation Examples 8, 1, 9 and 10 with physiological
saline to 10 mM as the total lipid, and the mixture was incubated
at 37.degree. C. for 1 hour. After cooling, this was diluted by
adding 2 ml of physiological saline, and its turbidity was measured
at an absorbance of 450 nm.
[0220] <Results>
[0221] The results are shown in FIG. 7.
[0222] Aggregation of liposomes in serum varies depending on the
amount of the basic compound which takes positive charge within a
physiological pH range.
[0223] No problems were observed regarding their aggregation in
serum when the basic compound content was 15 mol % or less. In the
case of from 15 to 20 mol %, it was able to prevent their
aggregation when the PEG-PE derivative-PE modification ratio was
0.5 mol % or more. When the basic compound content was increased to
20 mol % or more, aggregation of liposomes occurred and their
stability was spoiled even by the addition of-2 mol % of the PEG-PE
derivative-PE.
[0224] That is, in order to prevent aggregation of liposomes in
blood and ensure their stability, it is most desirable to set the
basic compound content to 20 mol % or less.
Test Example 5
Affinity of Various Liposomes for Proteoglycan
[0225] The object of this test is to know influence of a
hydrophilic polymer lipid derivative upon the adhesion of
proteoglycan to liposomes whose surfaces were modified by a basic
compound which takes positive charge within a physiological pH
range.
[0226] <Method>
[0227] Chondroitin sulfate C was dissolved in physiological saline
to a concentration of 0.1 mg/ml. A 0.5 ml portion of this solution
was mixed with 0.5 ml of each of the liposomes obtained in
Preparation Example 1, which had been diluted with physiological
saline to a total lipid concentration of 10 mM, and the mixture was
incubated at 37.degree. C. for 1 hour. After cooling, its turbidity
was measured at an absorbance of 550 nm to examine influence of the
polyethylene glycol-phosphatidylethano- lamine derivative
modification ratio upon the adhesion of chondroitin sulfate C based
on the correlation between the turbidity and the polyethylene
glycol-phosphatidylethanolamine derivative modification ratio.
[0228] <Results>
[0229] The results are shown in FIG. 8.
[0230] When modified by various concentration of the polyethylene
glycol-phosphatidylethanolamine derivative, the adhered amount of
proteoglycan increased as the modification concentration is
reduced. Also, the adhesion of proteoglycan was effective when the
modifying amount was 5 mol % or less.
[0231] That is, adhesion of proteoglycan to liposomes whose
surfaces were modified by a basic compound which takes positive
charge within a physiological pH range can be controlled by
modifying the liposome surface by a hydrophilic polymer lipid
derivative, and 5 mol % or less is most suitable for this
purpose.
Test Example 6
Stabilization in Blood by Hydrophilic Polymer Lipid Derivative
Modification
[0232] The object of this test is to know influence of the
modification by a polyethylene glycol-phosphatidylethanolamine
derivative upon the stability of liposomes in blood.
[0233] <Method>
[0234] A citrated human blood sample was centrifuged at 3,500 rpm
for 15 minutes to obtain human blood plasma. A 1.95 ml portion of
this human plasma was mixed with 0.05 ml of the liposome obtained
in Preparation Example 4, and the mixture was incubated at
37.degree. C. for 1 hour. After cooling, turbidity of the mixture
was measured at an absorbance of 550 nm to calculate polyethylene
glycol-phosphatidylethanolamine derivative modification ratio of
the aggregated liposomes.
[0235] <Results>
[0236] The results are shown in FIG. 9.
[0237] When modified by various concentrations of the polyethylene
glycol-phosphatidylethanolamine derivative, aggregation occurred at
a low concentration modification range, and the modification range
was 0.2 mol % or less.
[0238] That is, in order to prevent aggregation of liposomes in
blood and ensure their stability, it is most desirable to set the
modification ratio of hydrophilic polymer lipid derivative to 0.2
mol % or more.
Test Example 7
Examination of Accumulation on the Kidney (1)
[0239] The object of this test is to know influence of the surface
modification by a basic compound which takes positive charge within
a physiological pH range upon accumulation on proteoglycan
producing tissues typified by the injured kidney.
[0240] <Method>
[0241] Anti-Thy-1 antibody nephritis rats were prepared by
intravenously administering an anti-Thy-1 antibody solution through
the caudal vein of each CD (SD) male rat. On the fifth day after
administration of the anti-Thy-1 antibody, a Rhodamine-labeled
liposome having a polyethylene glycol-phosphatidylethanolamine
derivative modification ratio of 0.45 mol %, in which the liposome
membrane was labeled with Rhodamine-PE, which had been prepared in
accordance with the method of Preparation Example 1 or 7, was
administered by intravenous injection to each rat at a dose of 500
.mu.l through the caudal vein. The kidney was excised from each rat
24 hours thereafter. Unfixed frozen sections were prepared from the
excised kidney, and the distribution of liposomes therein was
observed under a fluorescence microscopy.
[0242] For the sake of comparison, distribution of liposomes in the
kidney of normal CD (SD) male rat was observed by the same
method.
[0243] <Results>
[0244] The results are shown in FIGS. 10 and 11. FIGS. 10 and 11
are schematic illustrations prepared based on about 400.times.
magnification fluorescence microphotographs. In FIGS. 10 and 11,
liposomes of Preparation Examples 1 and 7 were used
respectively.
[0245] When the liposomes prepared using an amidino
group-containing compound (3,5-dipentadecyloxybenzamidine
hydrochloride) as the basic compound which takes positive charge
within a physiological pH range (Preparation Example 1) was
compared with the liposomes prepared without using the compound
(Preparation Example 7), it was confirmed that the former are
accumulated specifically on the kidney which caused inflammation,
particularly on the glomerulus. (In FIG. 10, white objects are
Rhodamine-labeled liposomes.) Though not shown in the drawings, it
was confirmed that the liposomes of Preparation Example 1 do not
accumulate on the normal kidney.
[0246] That is, the liposomes whose surfaces are modified by a
basic compound which takes positive charge within a physiological
pH range have an excellent property to be accumulation on tissues
and/or organs which excessively produce proteoglycan, typified by
the injured kidney, particularly the glomerulus which was injured
and thereby caused inflammation.
Test Example 8
Examination of Accumulation on the Kidney (2)
[0247] The object of this test is to know influence of the average
particle size of liposomes upon their accumulation on proteoglycan
producing tissues typified by the injured kidney.
[0248] <Method>
[0249] Anti-Thy-1 antibody nephritis rats were prepared by
intravenously administering an anti-Thy-1 antibody solution through
the caudal vein of each CD (SD) male rat. On the fifth day after
administration of the anti-Thy-1 antibody, a Rhodamine-labeled
liposome having a polyethylene glycol-phosphatidylethanolamine
derivative modification ratio of 0.45 mol %, in which the liposome
membrane was labeled with Rhodamine-PE, which had been prepared in
accordance with the method of Preparation Example 1, 16 or 17, was
administered by intravenous injection to each rat at a dose of 500
.mu.l through the caudal vein. The kidney was excised from each rat
24 hours thereafter. Rhodamine-PE was extracted from the excised
kidney and its fluorescence intensity was measured to calculate the
distributed amount of liposomes in the kidney.
[0250] For the sake of comparison, distributed amount of liposomes
in the kidney of normal CD (SD) male rat was observed by the same
method.
[0251] <Results>
[0252] The results are shown in FIG. 12.
[0253] It was confirmed that the liposomes having an average
particle size of 122.2 nm and a major particle size range of from
90 to 200 nm (Preparation Example 1) have excellent property to
accumulate on the kidney of a morbid state (injured kidney). It was
confirmed also that, in the case of the liposomes having an average
particle size of 273 nm and a major particle size range of from 180
to 350 nm (Preparation Example 16), their accumulated amount on the
kidney of a morbid state is smaller than that of the case of
Preparation Example 1, namely 2/3 or less, and their accumulated
amount on the normal kidney is almost the same as the case of
Preparation Example 1, and, in the case of the liposomes having an
average particle size of 79.6 nm and a major particle size range of
from 40 to 90 nm (Preparation Example 17), their accumulated amount
on the kidney of a morbid state is smaller than that of the case of
Preparation Example 1, namely about 2/3, and their accumulated
amount on the normal kidney is about 2 times larger than the case
of Preparation Example 1.
[0254] That is, in order to inhibit their accumulation on normal
tissues and/or organs and to simultaneously effect their selective
accumulation on proteoglycan overproducing tissues and/or organs
typified by the kidney of a morbid state, it is most suitable to
control the major particle size range and average particle size at
a level of from 90 to 200 nm.
Test Example 9
Examination of Accumulation on the Kidney (3)
[0255] The object of this test is to know, in liposomes whose
surfaces are modified by a basic compound which takes positive
charge within a physiological pH range, influence of the kind of
basic compound upon their accumulation on proteoglycan
overproducing tissues and/or organs typified by the injured
kidney.
[0256] <Method>
[0257] Anti-Thy-1 antibody nephritis rats were prepared by
intravenously administering an anti-Thy-1 antibody solution through
the caudal vein of each CD (SD) male rat. On the fifth day after
administration of the anti-Thy-1 antibody, a Rhodamine-labeled
liposome having a polyethylene glycol-phosphatidylethanolamine
derivative modification ratio of 0.45 mol %, in which the liposome
membrane was labeled with Rhodamine-PE, which had been prepared in
accordance with the method of Preparation Examples 1, 2 or 3, was
administered by intravenous injection to each rat at a dose of 500
.mu.l through the caudal vein. The kidney was excised from each rat
24 hours thereafter. Rhodamine-PE was extracted from the excised
kidney and its fluorescence intensity was measured to calculate the
distributed amount of liposomes in the kidney.
[0258] For the sake of comparison, distributed amount of liposomes
in the kidney of normal CD (SD) male rat was observed by the same
method.
[0259] <Results>
[0260] The results are shown in FIG. 13.
[0261] In the liposomes having any one of the basic compounds,
their accumulation on the kidney of a morbid state was improved in
comparison with the normal kidney, so that it was confirmed that
modification of the surface by the basic compound which takes
positive charge within a physiological pH range is important for
positive targeting at the kidney of a morbid state. Also, the
accumulation property varied depending on the kind of basic
compound, and it was confirmed that particularly excellent
accumulation property is obtained when 3,5-dipentadecyloxybenza-
midine hydrochloride (Preparation Example 1) or
N,N-dioctadecyl-2-(piperid- in-4-yl-oxy)acetamide (Preparation
Example 2) is used.
Test Example 10
Pharmacological Effect of the Liposomes of the Invention in Which a
Drug is Included
[0262] The object of this test is know the degree of practical
pharmacological effect exerted by the liposomes of the invention
confirmed by the above Test Examples 1 to 9.
[0263] <Method>
[0264] Anti-Thy-1 antibody nephritis rats were prepared by
intravenously administering an anti-Thy-1 antibody solution through
the caudal vein of each CD (SD) male rat. On the fifth day after
administration of the anti-Thy-1 antibody, the liposome dispersion
liquid obtained in Preparation Example 15 (1 mg as prednisolone
phosphate sodium) was administered by intravenous injection. On the
tenth day after administration of the anti-Thy-1 antibody, urine
was collected to measure the total protein content.
[0265] For the sake of comparison, the preparation of Comparative
Example 1 (a liposome dispersion liquid having no included drug) or
physiological saline was administered by the same method, and urine
was collected on the tenth day after administration of the
anti-Thy-1 antibody to measure the total protein content.
[0266] Also, a prednisolone phosphate sodium solution (1 mg as
prednisolone phosphate sodium) which was not made into liposomes
was administered once on the fifth day after administration of the
anti-Thy-1 antibody, or five times (once a day for 5 days) started
on the fifth day after administration of the anti-Thy-1 antibody,
and urine was collected on the tenth day after administration of
the anti-Thy-1 antibody to measure the total protein content.
[0267] Urine was also collected from un-treated normal rat to
measure the total protein content.
[0268] <Results>
[0269] The results are shown in FIG. 14.
[0270] Marks in FIG. 14 are (1): a group in which the prednisolone
phosphate sodium solution was administered once, (2): a group in
which the prednisolone phosphate sodium solution was administered
five times, (3): a group in which the liposome dispersion liquid
containing no drug was administered once (Comparative Example 1)
and (4): a group in which physiological saline was
administered.
[0271] When the liposome of Preparation Example 15 as a liposome of
the examples of the invention was administered, remarkable effect
to inhibit excretion of protein into urine was found. This effect
was even superior to a case in which prednisolone phosphate sodium
equivalent to its amount included in the liposome of the invention
was administered continuously for 5 days, namely five times larger
amount as the total dose of prednisolone phosphate sodium, without
making it into liposome.
[0272] Also, when the liposome with no drug included therein was
administered or prednisolone phosphate sodium was administered once
without making it into liposome in the same amount included in the
liposome of the invention, the excreted amount of protein in urine
was almost the same as the case of the administration of
physiological saline, so that no effect was found.
[0273] That is, by only one administration, the liposome of the
invention can exert a pharmacological effect similar to or larger
than the case of a continuous administration of a drug which is not
made into liposome. In consequence, it was confirmed that the use
of the liposome of the invention renders possible insurance of the
efficacy of a drug and reduction of the total dose of the drug.
Test Example 11
Acute Toxicity
[0274] The object of this test is to know the degree of toxicity of
the liposome of the invention when compared with the toxicity of a
conventional liposome. For this purpose, a liposome similar to the
invention but having no drug included therein and a conventional
liposome (also having no drug included therein) are respectively
subjected to a rat lethal toxicity test.
[0275] <Preparation of Liquids to be Tested>
[0276] (1) A Liposome Dispersion Liquid of the Invention Which
Contains 3,5-dipentadecyloxybenzamidine Hydrochloride and a
Polyethylene Glycol-Phosphatidylethanolamine Derivative
[0277] The liposome dispersion liquid of the invention having a
polyethylene glycol-phosphatidylethanolamine derivative
modification ratio of 0.45 mol % obtained in Preparation Example 1
was used as a liquid to be tested, after its concentration using an
ultrafiltration membrane and subsequent dilution with physiological
saline as occasion demands.
[0278] (2) Conventional Liposome Dispersion Liquid
[0279] The liposome dispersion liquid of the invention having a
polyethylene glycol-phosphatidylethanolamine derivative
modification ratio of 0.45 mol % obtained in Preparation Example 7
was used as a liquid to be tested, after its concentration using an
ultrafiltration membrane and subsequent dilution with physiological
saline as occasion demands.
[0280] <Method>
[0281] Quarantined mice of five weeks of age were divided into
groups of 5 animals per group, and each of the aforementioned
liquid to be tested was administered through the caudal vein of
each animal at a dose of 100 ml/kg. On the other hand,
physiological saline was administered at a dose of 100 ml/kg in the
solvent control group.
[0282] After administration of the liquid to be tested, general
conditions were carefully observed at least once a day for 7 days,
and toxicity signs and death cases were recorded. Also, pathologic
autopsy was carried out after 7 days and each organ was excised.
Pathologic sections were prepared from each organ and observed.
[0283] <Results>
[0284] Similar to the case of the conventional liposome (liposome
dispersion liquid of (2)), no death case was found during the
observation period in the liposome similar to that of the invention
except that a drug was not included therein (liposome dispersion
liquid of (1)). Also, no problematic pathological findings were
obtained by the pathologic observation of each organ excised after
7 days.
[0285] That is, it was confirmed that the liposome of the invention
has markedly low toxicity and high safety.
INDUSTRIAL APPLICABILITY
[0286] Thus, as has been described in the foregoing, in comparison
with the conventional liposomes, the liposome of the invention has
high targeting ability for tissues and/or organs where proteoglycan
is excessively produced typified by injured kidney, particularly
the glomerulus which caused inflammation by injury, and also has
high safety.
[0287] In consequence, the liposome of the invention is markedly
effective for the purpose of diagnosing and treating diseases which
accompany overproduction of proteoglycan in injured portions of
tissues and/or organs, such as the case of glomerulonephritis.
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