Note: Descriptions are shown in the official language in which they were submitted.
~ 36S6
ACRGROUND OF THE INVENTION:
The present invention relates to liposomes including
an active substance, particularly a physiologically active
substance.
Hitherto, in cases where a physiologically active
substance such as a medicine is directly administered into a
living body, there have been many occasions causing (1) immunolo-
gical problems of proliferation of the antibody to the medicine
within the body, (2) problems of augumented side effects-of the
medicine resulting from the uptake of the medicine by the
tissues other than the target of the medicine, or inversely, (3)
problems caused by the inability of the medicine of passing
through the target tissue, and further (4) problems caused by
the inability of the medicine of maintaining i~s activity owing
to its degradation, inactivation, etc. by enzymes within the
living body.
However, it is considered that the above-mentioned
problems will be resolved by administering the physiologically
active substance such as a medicine Ioaded on a carrier which is
able to transfer the active substance directly into the target
tissue within the livinq body while protecting the active
substance.
From the above-mentioned viewpoint, Japanese Patent
Application Laying Open ~o. 118826/74 (DE-OS - 2:2 49 552) has
recently proposed a liposome comprising a vesicle having a
closed lamellar structure (micelle) composed of at least one
~ 5~
bimolecular layer formed by a compound, represented by the general
formula, X-Y, wherein X represents a polar and hydrophilic group
such as phosphate, carboxyl, amino, hydroxyl or choline and Y
represents a non-polar and hydrophobic group such as alkyl,
alkenyl or alkynyl, for example, a purified phospholipid such as
lecithin, phosphatidylethanolamine, phosphatidylserine, etc. as
a material for forming the membrane of the above-mentioned layer
and containing a physiologically active substance dissolved in
an aqueous solution within the vesicle of the liposome. Since
the wall membrane which forms the liposome protects the active
substance in the aqueous solution within the vesicle even under
severe conditions, for instance, those in gastrointestinal
tracts, the activity of the active substance is not spoiled even
. when the liposome is ora1ly administeEed.` In addition, since
.the permeability of the liposome to a tissue of living body
changes depending on its particle size (diameter), it is possible
to raise the permeability of the liposome to the tissue by
adjusting the diameter of the liposome. Accordingly, the above-
: mentioned liposome has been given attention as a possible means
of selectively supplying a physiologically active substance
contained in the liposome into a specified tissue of a living
body.
~owever, the above mentioned wall membrane which forms
the above-proposed liposome and is consisted of pure:phospholipid
has defects of lack of pliability and of insufficient mechanical
strength. In addition, owing to the excessively large effluent
~ 56
velocity of the physiologically active substance contained in
the vesicle to the outside, the liposome is not necessarily satis-
factory concerning its property of releasing the above-mentioned
physiologically active substance slowly within the living body,
that is, so-called the slow-releasing property. Particularly,
the above-mentioned liposome has a defect that the effluent
velocity of the above-mentioned active substance is highly raised
at a temperature higher than the transition temperature of the
wall membrane which forms the above-mentioned liposome.
In order to improve the strength of the wall membrane
of the above-mentioned liposome, a method is well-known in which
a steroid lipid such as cholesterol is admixed with the above-
mentioned phospholipid as the material for forming the membraneous
layer of the liposome. Although the strength of the wall membrane
forming the liposome is somewhat improved by the proposed method,
the so-called slow-releasing property is not so much improved.
In consideration of the above-mentioned situation,
the inventor of the present invention, after studying the methods
for supplying the liposome having a strong wall membrane and
having a favorable slow releasing property of the physiologically
active substance into the living body, has found that the wall
membrane of the liposome which was formed by a phospholipid
containing molecules of oily substance such as "crude lecithin"
has a higher pliability and is more excellent-in the slow-
releasing property of the physiologically active substance into
the living body as compared to the wall membrane of the
~3~56
conventional and publicly known liposome formed by using purified
phospholipid. Accordingly, it is surprising to see that the
liposome formed by using a phospholipid containing molecules of
oily substance has the above-mentioned specific properties.
Accordingly, the purpose of the present invention is
to provide a liposome containing physiologically active substance,
of which the wall membrane is strong enough and which has a
favorable slow-releasing property of the above-mentioned active
substance within the living body.
The other purposes of the present invention will be
made clear from the following description:
BRIEF EXPLANATION OF THE DRAWINGS:
FIGURE 1 shows the schematic illustration of the lipo-
some of the present invention and FIGURE 2 shows a relationship
between the amount of molecules of oily substance contained in
the membraneous material forming the liposome according to the
present invention containing glucose in the aqueous solution
within the vesicle of the liposome and the percentage of the
amount of glucose entrapped within the vesicle to the total
amount of glucose used for forming the above-mentioned liposome
loading glucose (briefly~e~pressed hereinafter as the rate of
entrapment). FIGURE 3 shows a comparison of permeability of the
above-mentioned glucose from the liposome of the pres-ent
invention and from the liposome shown in Comparati-ve Example.
FIGURE 4 shows a comparison of the persistency of hypoglycemic
~3~5~i
action of insulin in a living body, which is supplied by the
liposome of the present invention containing insulin therewithin
to that by the liposome of Comparative Example also containing
insulin therewithin. FIGURES 5 to 8 show the slow-releasing
property to an active substances included within the respective
liposomes of the present invention.
Among them, FIGURE 5 shows the change of the amount of
radio-isotope in blood with the passage of time after the
subcutaneous injection of the liposome prepared by using the
above-mentioned substance, however, radio-labelled, as the active
substance, and FIGURE 6 shows the change of the amount of radio-
isotope in urine with the passage of tlme after the same treatment
as above. FIGURE 7 shows the change of the residual amount of
radio-isotope, at the site where the free radio-labelled active
substance-was directly injected subcutaneously as it is, with
the passage of time, and FIGURE 8 shows the change of the residual
amount of radio-isotope, at the site where the liposome including
the radio-labelled active substance within its vesicle was
injected subcutaneously, with the passage of time.
DETAILED DESCRIPTION OF T~E INVENTION:
.
The characteristic feature of the present invention is,
on the formation of a liposome including an active substance
within its vesicle surrounded by a micellar membraneous layer,
the use a material comprising micellar membraneous layer of
phospholipid, in which molecules of a fatty substance are present
ti56
as the wall membrane of the above-mentioned liposome.
Another characteristic feature of the liposome, includ-
ing an active subs-tance, formed by using the above-mentioned
material according to the present invention is its W/0/W-type
complex emulsified state as shown in FIGURE 1 and its excellent
property of slowly releasing the above-mentioned active substance
included within its vesicle to -the outside when taken into a living
body. In FIGURE 1, X represents a hydrophilic group, Y a hydro-
phobic group, P a vesicle containing an aqueous solution.
A molecules of fatty substance and Q an aqueous solution outside
of liposome, respectively.
The material for forming the liposome of the present
invention, as has been described above, comprises phospholipid
with which molecules of a fatty substance are mixed or.to which
they are bonded. .~he phospholipid used herein is not particularly
limited provided it has been used as the material for a membra-
neous layer of the conventional liposome, for instance, a single
compound of lecithin, phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylserin, phosphatidyl-
inositol, sphingomyelin, cardiolipin, etc. or its mixture, and
according to the necessity, a sterol compound such as cholesterol
may be contained.
The fatty substance present in the above-mentioned
phospholipid is triglyceride, wax or mineral oil or the mixture
thereof, for instance, those selected from vegetable oil such as
soy-bean oil, cotton seed oil and sesame oil, and also from
mineral oils of coal-origin or petroleum-origin.
Particularly preferable is the single use of crude
lecithin or the use of a mixture of crude lecithin and other oily
substance mentioned above.
The term "crude lecithin" herein used means a fraction
which eluates with chloroform or a mixture of chloroform and
methanol in a ratio of 100 :1 to 3 :2 when a component rich in
phospholipid derived from substance such as yolk and soybean oil
is fractioned by column chromatography using alumina as a column
material, the fraction consisting of 97 to 80% by weight of pure
lecithin and 3 to 20% by weight of oily substance, for instance,
triglyceride and carotenoid.
In cases where the liposome of the present invention is
prepared by using the above-mentioned crude lecithin as a
material for membraneous layer, the content of the oily substance
in crude lecithin is adjusted so that the content of the fatty
substance in the thus formed wall membrane becomes to 3 to 20%
by weight, preferably 5 to 15% by weight. In addition, care
should be taken to keep the content of the fatty substance in
the above-mentioned wall membrane not over 20% by weight or the
formation of the wall membrane is impaired to have reduced yield
of the liposome. On the other hand, in the case where the
content of the fatty substance is less than 3% by weight, the
above-mentioned object of the present invention cannot be attained.
By the way, on the formation of the liposome according
to the present invention, sterols such as cholesterol and
~ 656
ergosterol and a substance capable of changing the electrically
loaded state of the surface of the liposome, for instance,
phosphatidic acid, dicetyl phosphate or ganglioside of bovine
brain for giving negative charge, or stearylamine for giving
positive charge may be admixed with the material for membraneous
layer as the third component. The amount of addition of the
third component may be determined adequately according to the
property of the phospholipid suitably, and usually is 0 to 10%
by weight of the material for membraneous layer.
lo In the case of forming the liposome of the present
invention while using a mixture of the above-mentioned phospho-
lipid and the oily substance as a material for membraneous layer,
the conventional method for preparing a liposome is possibly
applied. For instance, a method in which a thi~ film is formed
from the above-menti~ned material and after bringing the thus
formed membrane in*o contact with a continuous phase containing
an active substance to form a dispersion by agitation, a super-
sonic vibration i5 applied on the dispersed system, a method
in which after mixing a solution of the above-mentioned material
for membraneous layer in a solvent insoluble in water with an
aqueous solution containing the above-mentioned (water-soluble)
active substance, the mixture is subjected to a supersonic
treatment for forming a precursor of liposome and then the
solution containing the precursor is subjected to ultracentrifugal
treatment in the co-presence of an aqueous medium, or a method
in which after coating the surface of glass-beads and the like
11~3~56
with the above-mentioned material for membraneous layer, the
coated beads are mixed with an aqueous solution containing the
above-mentioned active substance to disperse the coated beads
into the solution is applicable.
The amount of the above-mentioned material for membra-
neous layer on forming the above-mentioned liposome is 1 to 500
mg/ml of the liquid in which the liposome is suspended.
The membraneous layer of the liposome obtained by the
above-mentioned method is formed as a result of mutual action
lo between the hydrophobic group possessed by the phospholipid in
the material for membraneous layer which forms the liposome and
the molecules of oily substance which are present in the material
for membrane layer. Accordingly, the morphological state of
the liposome of the present invention is to be said quite dif-
ferent substantially from the state of the conventional liposome
comprising the micelle of purified phospholipid.
In addition, the oily substance as a constituent of
the wall membrane of the liposome according to the present
invention, on the formation of the liposome from the material
for membraneous layer, brings about the improvement of the
liposome-yield, and after forming the liposome, brings about the
easiness in the process of separating the liposome, the uniformity
of the particle size of the liposome and the improvement of
pliability, the strength of the wall membrane of the liposome and
the slow-releasing property of the active substance included in
the vesicle of the liposome when administered within the living
body.
~3f~56
The active substance, particularly the physiologically
active substance used on the formation of the liposome of the
invention is a medicine of which the side effect is to be
reduced or a medicine of which the persistency within the living
body is to be improved, and is exemplified by peptide hormons
such as insuline, oxytocin, vasopression, adrenal-cortex stimulat-
ing hormon (ACTH), luteinizing hormon-releasing hormon (LH-RH),
carcitonin, somatostatin, steroid hormons such as progesteron,
follicular hormon, adrenal cortex hormon and other hormons such
lo as prostaglandin, adenosine-3',5'-cyclicmonophosphate, anti-tumor
agents such as chlorambutyl, streptozocin, methothorexate, 5-
fluorouracil, sitosin arabinoside, mitomycin C, breomycin,
polysaccharide derivative, antibiotics such as penicillin,
cepharospolin, streptomycin, enzymatic preparations such as
aminoglucosidase; invertase, etc.
The liposome according to the present invention com-
prises particles of 0.01 to 10 microns in average diameter with
a narrow range of distribution of particle diameter, and those
particles of 0.5 to 5 microns in average diameter are obtainable
easily with a uniform diameter. The particles of liposome of
the present invention are excellent in pliability, and they can
be concentraied by a low speed-centrifugal treatment at 2,000 to
4,000 r.p.m. Moreover, the re-dispersion after concentration can
be carried out only by a short time-shaking without necessitating
an ultrasonic treatment to be recovered to the original state.
The liposome according to the present invention, as is
~3~5~
understood from its specific properties described above, is
excellent in its capability of maintaining the active substance
included within its vesicle as compared to the conventional
liposome of the type of pure phospholipid micelle, and also its
slow-releasing prop.erty to the active substance included there-
within is favorable. Accordingly, the liposome of the present
invention is particularly suitable as a protecting agent for an
unstable medicine within the living body, and also for a medicine
which causes side effects by an inevitable excessive administra-
]0 tion. By these reasons, the liposome according to the present
invention is possibly utilizable as a medicine.
For instance, in the case where the liposome of the
present invention is applied as an insulin injection, an intra-
muscular administration of the liposome of the present invention
once two to seven days at a dose of 0.2 - 20 mg as insulin to
an adult gives the same effect as that of the direct intramuscular
administration of free insulin three times a day at a single
dose of one third of 0.1 to 4.0 mg.
In cases where the liposome of the present invention is
utilized as a medicine, its administration is possible via
several routes such as percutaneous, subcutaneous, intramuscular,
intraperitoneal, intravenal, intrarectal and topical, preferably
by subcutaneous or topical administration. The amount of admini-
stration depends naturally to the method and the route of
administration, the kind of active substance and the extent of
treatment, however, usually it is 0.1 to 1 times as large as
l~ S~
the amount of the active substance directly administerea per day,
and further the interval of administrations can be extended.
In addition, the liposome of the present invention is
possibly administered as an injection after dispersing the
liposome into an aqueous physiological saline solution.
Particularly, the liposome of the present invention containing
a peptidic physiologically active substance including peptide
hormons exhibits a remarkable effect when applied as a subcuta-
neous or intramuscular injection. Since peptidic physiologically
lo active substances are generally degraded promptly in the living
body, it is necessary to carry out frequent administration
(injection) for the maintenance of the effect of the substance
resulting not only in causing the heavier load on the patient
but also causing the large fluctuation-of the concentration of
the active substance in blood. These situations are apt to reduce
the effect of administration of the active substance and to
cause side effects of the active substance.
Whereas, the liposome of the present invention, as are
shown later in Examples, shows an excellent slow release of the
included active substance on subcutaneous or intramuscular
injection, and accordingly, the times of administration are
possibly reduced materially with a maintenance of the uniform
concentration of the peptidic physiologically active substance
in blood, and accordingly, the effect of the active substance is
fully exhlbited with the suppression of its side effects.
According to the acute toxicity test of the above-
~1~3~5~;
mentioned materials for forming the above-mentioned liposome of
the present invention using rats via two routes of subcutaneous
and intravenous injections, no toxic sign was observed on the
treated rats until the dose rate reached 1,000 mg/kg concerning
every material, the fact showing that the liposome of the present
invention is safely applicable as a medicine from the viewpoint
of the wall membrane of the liposome.
The followings are the concrete explanation of the
present invention while referring to Examples.
Example 1:
A solution containing 100 mg of a commercial crude
yolk lecithin (manufactured by Merck Company), 11.6 mg of
cholesterol and 2.7 mg of stearylamine in 10 ml of chloroform
was placed in a 25-ml round-bottomed flas]c set on a rotatory
evapora~or, and by distilling choloroform from the solution while
rotating the evaporator at a temperature of 38C under reduced
pressure, a film was formed on the inner wall of the flask.
Then, one milliliter of an aqueous 1% by weight solution of
adenosine 3',5'-cyclic-monophosphate (hereinafter abbreviated
as C-AMP) was added to the flask, and by shaking the flask for
30 minutes the film was exfoliated from the inner wall of the
flask and the film was dispersed into the solution. By treating
the dispersion with a supersonic treating machine (manufactured
by Nippon Seiki Company, Model NS 200-2) for 20 min, a suspension-
dispersion of particles of 1 to 2 microns in average particle
,
~ 3~5~
diameter was obtained. In the next step, an aqueous physiological
saline solution of 6 times by volume of the above-mentioned
suspension-dispersion was added to the suspension-dispersion,
and the mixture was trea-ted 3 times with centrifugal separation
at 3,000 r.p.m. for each 10 min to separate completely the thus
formed liposome, 1 - 1 and the residual solution of C-AMP which
had not uptaken into the liposome. For comparison, four kinds
of liposomes were prepared by using the materials for membraneous
layer shown in Table 1, as Comparative Examples, l - 2, 1 - 3,
lo 1 - 4 and 1 - 5 in the same procedures as in 1 - 1.
The rate of entrapment of C-AMP, that isl the percen-
tage by weight of C-AMP collected within the vesicle of the
liposome to the amount of C-AMP used, and the extent of release
of C-AMP from the liposome after 24 hours~of malntaining the
liposome at a temperature of 37C, that is, the residual percen-
tage in the liposome, are shown in Table 1. As is seen in Table
1, by the use of the crude lecithin as the material for the
membrane layer, the liposome of improved rate of entrapment and
of the improved residual amount in the vesicle of the liposome
as compared to those of the liposome prepared by using the
conventional material for membraneous layer.
1143~5ti
~ ~ rl~^ 7
~ .~ ~ ~ ~ ~ ~
.~ b " c~ oo oo oo co a
a~ ~o ~ _ Q~
a # a) o ~ . ~ P' e
aJ h ~~ oO ,1 ~ O ~i tn ,i
JJ c~l ~n
a ~ e e a
`D ~, ~ I` ~ . ~ ~a o
G' ~,1 O ,i ~ o ,i c~l O N O ~1 O ~rl
~1 O ~1 O O --1 O rl ~d
~ ~ +
e e ~ o ~ h r-
P. 4~ ~ ~ ~ ~. ~ C~
O + ~ ~ .,1 .~ O
. ~ ~ ~ R
,~ ~ . ~ ~ ~ ,1 ~,~
JJ ~ ~ ~ ~ C ~ ~ ~ ~ al ~
~r1 0 ~ ~1 0 ~rl ~1 ~ ~ O ~ a) ~ o
o- eaJ ~ ~~ ~ ta ~ ~ ~ ~ ~.~ P~ O
~ ~ ~ a~ u ,~ 2) ~ a~
~3 8 0 ~ ~ rl ~ ~ a) ,1
P; C~ 4~ ~ ~1 ~ .,~ a ." ~ ~1 ~ ~ .,~
~ O ~ h O O ~ ~ :1 ~ 4
,~ u o u~ ~ u ~n _ ~ u ~ e
a~ _ ~ ~ .
e
E~ e ~ ~ ~ ~ u~ ~
. , U ~
Q OJtl~ ~_ K
u R . ,_~ O
R ~ ~ X S~;
td.,1 ~Qf~
C~.P~ , C~
~3ti~6
In addi-tion, commercial crude lecithin (manufactured
by Merc~ Co.) and commercial purified lecithin (manufactured by
Sigma Co.) had the following compositions, respectively:
unit: % by weight
Specimen PhopholipidCholesterolOily substance
Crude lecithin( ) 93.8 1.1 5.1
Purified lecithin )99.5 0.3 0.2
. Example 2:
Liposomes were prepared by the same procedures as in
10 - Example 1 except for using one milliliter of an aqueous 20% by
weight of glucose solution instead of an aqueous solution of
C-AMP in Example 1 using.each of the following materials for
membraneous layer of the liposome shown in Table 2.
The amount of cotton seed oil as a component of the
materials for the membraneous layer and the rate of collecting
glucose in each liposome are correlated in Fig. 1. In addition,
the residual amount of glucose in the liposome as compared to
. the lnitial amount of glucose in the liposome by percentage
after maintaining for 24 hours at a temperature of 37C was
shown in Fig. 3, the determination of the above-mentioned glucose
being carried out on Specimens of 2 - 7, 2 - 8 and 2 - 9.
From these figures, the superiority of the liposome
of the present invention to the conventional liposomes is clearly
recognized.
~ 3~56
Table 2
Composition of Materials for Membraneous Layer
Composition of Materials for
ClassificationSpecimenMembrane
Base material Cotton seed
(mg) oil (mg)
Present invention 2 - 1 Crude lecithin 100 5o
2 - 3 ,, . 15
2 - 5 " 20
Comparative example 2 - 6 (++) 40
.2 - 7 Purified lecithin~ 100 0
Present invention 2 - 8 . 150
2 -10 l ll 15
2 -11 ~ 20
Comparative example 2 -12 40
Note: ~+) commercially available
.++)icommercially available
- 17 -
1~3~:i5~
Example 3:
By using the materials for membraneous layer having
compositions shown in Table 3, while using the procedures
described in Example l, each film was formed on the inner wall
of a round-bottom flask. ~fter adding one milliliter of an
aqueous citric acid-buffer solution containing lO mg of insulin
per lO ml of the solution (pH of 2.3) to the flask, a suspension-
dlspersion of liposome particles of l to 2 microns in diameter
was prepared by the same procedures as in Exampl-e l. After
leaving the suspension-dispersion at room temperature for 24
hours, it was treated one with 6 ml of an aqueous physiological
solution and two times with a 6 :l mixture ~by volume) of a
physiological saline solution and an aqueous citric acid-buffer
solution and subjected to centrifugal treatment to obtain a
liposome. After adding an aqueous citric acid buffer solution
to the thus prepared liposome to adjust the concentration of
insulin in the mixture to 40 IU/ml (IU means an International
standard unit), the mixture was subjected to the following
experiment:
18 -
3~56
~ ~`Y
~365~
Experiment: Persistency test of insulin liposome within living
bodies
Four groups of SD female rats artificially attacked
by diabetes with the administration of streptozocine were re-
spectively given subcutaneous injection of each liposome prepared
as above, and their blood sugar was determined before and after
the injection of the liposome. Fig. 4 shows the values of blood
sugar with the lapse of time, the ordinate showing the percentage
of the concentration of glucose in the blood after injection to
the concentration of glucose in the blood before injection, and
the abscissa showing the days after injection.
As is seen in Fig. 4, the persistency of insulin in the
rat body (in other words, the fact that the reduced value of
glucose was maintained for a long period of time) due to the
application of the liposome of the present invention prepared
by using crude lecithin inherently containing oily substance is
far superior to that due to the application of the conventional
liposome prepared by using purified lecithin not containing the
oily substance.
Example 4:
The present example shows the transition of the active
substance whlch has been included in the vesicle of the liposome
after administration into the living body. For that purpose,
the following experiments were carried out using a liposome
including the tritium-labelled luteinizing hormon-releasing
5~
horrnon (LH-RH) as the ac~ive substance. The liposome was pre-
pared by the following procedures:
Prepara-tion of liposome:
The same film consisting of crude yolk-leclthin,
chlos-terol and stearylamine was prepared on the inner wall of a
round-bo-ttomed flask as in the first parayraph of Example 1.
To the flask, 1 ml of an aqueous physiological saline solution
containlng tritium-labelled LH-RH (hereinafter referred to as
H-LH-RI~) (250 ~ Ci/7.3 ~g, prepared by New England Nuclear
lo Company) at a rate of 1 ~y/ml was added, and after exfoliating
and dispersing the film from the inner wall of the flask by
shaking -the flask for 30 min, the thus formed dispersion was
treated by the supersonic treating machine (refer to Example 1)
for 15 min to prepare a supension-dispersion of particles of 1
to 2 ~ in average particle diameter. Six times by volume of an
aqueous physiological saline solution as much as the suspension-
dispersion was added to the suspension-dispersion and by
subjecting the mixture to 2 times of centrifugal separation at
3,000 r.p.m. for each 10 min, the thus prepared liposome was
completely separated from the solution of 3H~LH-RH which has not
been taken into the vesicle of the liposome.
The ra-te of collecting 3H-LH-RH was 10~ by weight.
After adjusting the concentration of 3H-LH-RH to 3.42 ~Ci/ml by
the addition of an aqueous physiological saline solution, the
solution was used for the following experiments.
~ 6
E periment 1:
The thus prepared liposome and the free 3H-L'I-RH not
included in liposome were respectively injected subcutaneously
into each ICR male mouse of body weight of 30 to 32 g of each
group consisting of 3 animals at a dose rate of 0.34 ~Ci/animal,
and the content of 3H-LH-RH (expressed by the amount of the
radio-isotope, RI) in the animal's blood was traced with the
passage of time by collecting each 0.25 ml of blood specimen from
each animal at a predetermined time interval after treating the
specimen with a sample oxidizer (made by Packard Company) and
determining the radioactivity by a liquid scintillation counter.
The results of determination were shown in Fig. 5, by
taking the content of 3H-LH-RH in the blood specimen after 15
min of the administration as 100.
As is seen in Fig. 5, the hormon included in the
vesicle of the liposome prepared according to the present
invention is slowly released within the living body.
Experimént 2:
The above-mentioned liposome and the free 3H-L-~-RH
were respectively injected subcutaneously to each SD male rat of
each group consisting three animals, and the excreted amount
of the radio-isotope in urine and feces was tracsd with the
passage of time. The rate of administration was 0.68 ~Ci/animal,
and the excreted amount of the radio-isotope was determined by
scintillation counting the specimen after diluting the urine
~ ~3~
specimen with distilled water to 100 ml, or drying the feces and
oxidizing it.
The results are shown in Fig. 6 by taking the admini-
stered amount as 100, and integrating the excreted amount to the
time of determination. No radio-isotope was detected in the faces.
From Fig. 6, it will be recognized that the hormon
included in the liposome prepared according to the present
invention is slowiy released from the liposome within the living
body.
Experiment 3:
The same liposome as used in Experiments 1 and 2, and
the free 3H-LH-RH were respectively injected subcutaneously into
each ICR male mouse weighing 30 to 32 g of each group consisting
2 mice, and the residual amount of the radio-isotope at the site
of injection was traced with the passage of time after the
administration of 0.165 ~Ci. The determination of the residual
amount of the radio-isotope was carried out by taking out the
region of injection and after dissolving the region into SOLUENE
(supplied by Packard Company) using scintillation counting.
The results are: (1) a~ is shown in Fig. 7, after injecting the
free 3H-LH-RH, the residual amount of RI decreased remarkably in
a short time after 1njection, whereas (2) as is shown in Fig. 8,
it showed an extremely slow reduction when administered in the
state of inclusion in the vesicle of the liposome.
According to the results of the above-mentioned
d~ p1ar~
3~
experiments 1 to 3, the slow releasing property of the active
s~bstance included in the vesicle oE the liposome according to
the present invention has been verified.
