Note: Descriptions are shown in the official language in which they were submitted.
- 10~73~16
1 This invention relates to medicinal preparations
having a pharmacodynamic action on the bio-protective
mechanisms in living organisms, said preparations being
composed primarily of cholesterol derivatives expressed ~ -
by the following general formula (I):
~'
; R
H
(where R represents -OH or =0). ;
Among the compounds expressed by the above-
shown general formula (I), the most preferred examples
for use in this invention are 7-hydroxycholesterol and
7-ketocholesterol.
These compounds are already known in the art
and mostly acknowledged as catabolic products of
cholesterol in living organisms. This invention is a
discovery of novel use of such bio-metabolic products as
a medlcament. This invention also features finding of the
~` active center of the immunoregulatory substance in living
organisms known as IRA.
The present inventors and their co-workers have
previously discovered a method for preparation of a
peptide-like material having an immunoregulatory activity
in serum and placental blood (Japanese Patent Application
l No. ~421t76). The study on this immunoregulatory
l substance has been rapid progress even since such
.i ~ .,:
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1~173~
substance was isolated from Cohn IV-l paste of human serum and identified
as a peptide-like material with molecular weight of approximately 5,000 by
Occhino et al., in 1973 ~The Journal of ImmunologyJ 110, (3), 685, (1973~).
This peptide-like material is presently called IRA (immunoregulatory a-
globulin~. The present inventors have also pursued their research into the
entity of this immunoregulatory substance acting in the living organisms.
The study by the present inventors has been aimed principally at
elucidating the active center of said immunoregulatory substance existing
in living organisms and realizing chemical synthesis of the substance which,
as compared with the conventional extracts, can be used for a wide variety of
remedial purposes such as for immunoregulatory treatment, can be refined to
a desired purity and can be also determined both physically and by way of
analytical chemistry and which can therefore be administered as a remedial
medicament at a correct dosage to the relevant patients.
The present inventors have employed a method in which the slightly
water soluble material obtained from Cohn IV-l paste by organic solvent
extraction was subjected to column chromatography to batch off the immunoregu-
latory active substance, and found out that this substance is a cholesterol
derivative~ The present inventors also disclosed the fact tha~ this
substance has an antiphlogistic activity.
According to the pTeSent invention, there is provided a medicament
having an immunoregulatory action B~ which comprises a cholesterol derivative
oxpressed by the following general formula: -
O ~
H
,:
-- 2 --
' . , , . .: :
107381~
where R represents -OH or -O, and physiologically acceptable carrier, the
cholesterol derivative being present in an amount of 1.0 to 60% by weight
of the medicament.
Typical examples of the compounds expressed by the general formula
(I) are 7-hydroxycholesterol and 7-ketocholesterol. For obtaining either of
these substances, Cohn IV-l paste of human serum fraction is extracted with
a chloroform-methanol system and the obtained extract is subjected to A1203
and SiO2 column chromatography and further to cellulose and liquid
chroma~ograph~ to batch off the cholesterol derivative fraction. The
desired substance is isolated in the form of crystals. They may be also
chemically synthesized by merely oxidizing cholesterol according to a known
method.
The compounds of the general formula (I) can be used as an immuno-
regulatory agent, particularly as a cytoplasmic immunoregulatory medicinal
base and also prove useful as an antiphlogistic remedial medicament. Now,
the general pharmacodynamic activities, method of administration, effective
dosage and toxicity of the compounds of the general formula ~I) are described.
. . .
1073~16
1 (1) Immunoregulatory action (in vitro experi~ents)
The PHA reaetion 50!~ inhibition concentration
of the compounds of the general formula (I) ~ere examlned
by using the PHA method by Cooperband, S.R., et al., (The
Journal of Imr,lunology, 109, (1), 154, (1972)). ~he
results are shown in the following table. For sake of
eomparison, the effect of IRA obtained according to the
Oechino et al., method (The Journal of Immunology, 110,
(3), 685, (1973)) was also shown.
7-hydroxycholesterol 5 ~g/ml
7-ketocholesterol 8 ~g/ml
IR~ 20 ~g/ml
(2) Immunoregulatory action (in vivo experiments)
~ he following animal tests were conducted by
using the compounds of the general formula (I) (7-hydroxy-
eholesterol and 7-~etocholesterol).
Of the total 15 mice used for the tests, 5
mice were given 7-hydroxycholesterol (at the dose of
8 mg/kg/day) and another 5 mice were given 7-keto-
eholesterol (at the dose of 10 mg/kg/day), both through
intravenous administration continuously for 20 days, and
the effect on skin grafting was examined. For admini-
stration, each compour.d was emulsified with a surfaceaetive agent and prepared into a physiological isotonic
solution with end concentration of 3.0 ~!eight %, and this
solution l7as previously administered to eaeh mouse of the
~ .
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lQ'738~6
. . ..
1 above-said two test groups 24 hours before start of
grafting, and thereafter the solution was adrninistered
once a day for the total period of 20 days.
The surface observation after 20-day test
showed that the skin-grafted ratio of 80 to 9~0 in said
both tested groups in contrast with 0~ in the control.
This is indicative of the excellent pharmacodynamic
effect of the compounds of this invention as an immuno-
regulatory medicament.
(~) Antiphlogistic action
20 Mice were divided into four groups of five
and the mice of two test groups were administered intra-
abdominally with 7-hydroxycholesterol and 7-
ketocholesterol, respectively, with the dose of 200 mg/kg
(physiologically isotonic 2.0 weight % aqueous solution),
and 1 hour later, 0.05 ml of l~~o carrageenin was admini-
stered as a phlogogenic agent subcutaneously to the hind
legs of these mice.
The sizes of the edemata grO~^Jn on the mice legs
.
with time after carrageenin administration were measured
by a volume differential meter and compared with those
of the control mice to which an isotonic sodium chloride
solution was administered.
; There was observed 82~o growth of edema in four
hours after carrageenin administration in the mice of the
control group and 70~0 growth of edema in the cholesterol
group (this group was administered with cholesterol for
. reference data). In the 7-hydroxycholesterol group, on
the other hand, the growth of edema was limitcd to about
... ~, .. . .
f 10'~
1 40~ in five hours after carrageenin administration,
signifying an excellent antiphlogistic effect of this
compound. In the 7-l~etocholesterol group, there was
seen only about 50~0 gro~th of edema, attesting to a
notable antiphlogistic action of this compound, too.
The test results are shown in the graph of Fig. 1 of the
accompanying drawing. In the graph, percent of growth
of edema is given on the vertical Y-axis and time (hour)
after carrageenin administration is given on the
horizontal X-axis. The numerals for the respective curves
represent the respective specimens used in the test, that
is, 1 represents isotonic sodium chloride solution, 2
cholesterol, 3 7-ketocholesterol, and 4 7-hydroxy-
cholesterol.
(4) Dosage and method of administration
The 50 percent effective doses of 7-hyd;^oxy-
cholesterol and 7-ketocholesterol for the immunoregulatory
and antiphlogistic activities were examined by way of
animal experiments. It was acertained that the most
~ 20 preferred form of administration for remedial use as a
; medication is injection, and the recommended dosage is
10 to 1,000 mg/kg in gross volume. The compounds are also
effective when administered externally or orally, but the
dosage in such cases is usually 40 to 2,000 mg/l~g in
gross volume. In the case of external administration,
a small dosage will do as locallzed application is
- possible.
. ~or use as an injection, it is recommended to
prepare the material of this inventlon into a
- 6 -
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:1073~16
1 physiologically isotonic aqueous solution by emulsifying
the material with a suitable emulsifier as the material
of this invention is insoluble in water. The emulsifier
used for this purpose may be selected from the kno~n
types of compounds commonly used for medicinal prepara-
tions. The loadings of such emulsifier should be 2 to
lO~o by weight. The compounds of this invention can be
contained in an amount of 1.0 to 60~o by weight in the
medicament. It is also possible to contain 10 to 1,000 -
mg of active principle in the composition.
When the compounds of this invention are used -
in the form of an aqueous solution, it is highly advan-
tageous to add a stabilizer of the kno~n types of steroid
such as albumin. Such stabilizer may be added in loadings
of 0.1 to 5 weight % for obtaining due effect.
, When used for oral administration, the compounds
of this invention may be prepared into suitable tablets
or liquids according to a pertinent method known in the
art. The cholesterol derivatives used in the medicaments
~ 20 according to this invention are scarcely disintegrated in
] ~ the stomach, and there are even available some data
suggestive of absorption from the stomach and intestinal
tracts. Thus, it seems that no specific consideration
is needed in preparation of the medicaments for oral
administration.
The compounds of this invention can be also
prepared into an oitment for external use according to a
known method. In this case, an immediate effect is
obtained as the preparation can be acted directly to the
affected part.
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1 (5) Acute toxicity test
An acute toxicity test of 7-hydroxycholesterol
and 7-ketocholesterol was conducted on one group of rats
(5 rats) with average body weight of about 200 gr. ~ach
compound was suspended in a physiological solution of
sodium chloride and made into a uniform suspension by
a supersonic treatment, and then such suspension ~7as
administered intro-abdominally to each rat at the doses
of 500 mg/kg, 1,000 mg/kg, 1,500 mg/kg and 2,000 mg/kg,
respectively. No death was seen in 5-day observation of
the tested rats.
As understood from the foregoing description,
the medicaments acting on the bio-protective mechanisms
aecording to this invention have little toxicity and no
antigenic properties as they contain as ac-tive principle
a substance identical ~ith the active center of IRA which
derives itself from the living organisms.
It is to be also noted that the in vitro
experiments show an extremely high specific activity of
the compounds of this invention as compared with the
eompounds which have been known heretofore as IRA.
~'urther, as the medicaments of this invention were
eonfirmed as composed of a sing]e compound, it is possible
to adjust the dosage to avoid any excess administration.
~5 ~urther, as these compounds can be obtained from a
chemical synthesis, their medicinal preparations can be
provided at extremely low cost.
Sho~m in the following are some examples of
the method for production of the compounds according to
this invention.
< ~0~38~6
.
1 Production Example 1
25 Grams of co~mercially available guaranteed
cholesterol (a product by Kishida Chemicals) w~s
dissolved in 1,000 ml of hot ethanol, and the entire
amount of the mixture was added portionwise into a solu-
tion prepared by dissolving 5 grams of sodium stearate
(a product by Kishida Chemicals) in 5 litres of distilled
; water of about 70C, with pH of the solution being
adjusted to 8.5 + 0.1.
Then a cooling pipe was connected to the top of
the reactor and the external portion of the reaction
solution was heated while preventing evaporation of the
- solution, and when the solution temperature reached
85 + 1C, the reaction solution was subjected to aeration
- 15 for 5 to 7 hours.
After the reaction, a small quantity of
hydrochloric acid was added to make the solution acidic
(pH: 6 - 4) and then, by adding an equivalent quantity of
chloroform and agitating the mixture vigorously, the
object material was extracted while collec+ing the
; chloroform layer. Such extracting operation was repeated
twice and the collected chloroform layer was concentrated
by heating it to around 45C under vacuum. This concent-
rated extract was then dissolved in a small amount of
, .
chloroform and poured into a column of chloroform-
equilibrated silica gel (a product by Melc Inc.), whereby
the object material was adsorbed in the silica gel.
Washing with chloroform caused elution of
~ .:
first the unreacted cholesterol and then 7-ketocholesterol
detected by an ultraviolet light irradiator, and further
.
_ 9 _
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. ' - ,. . . . . .
, - : . . :
~073816
1 pourin~ of a solvent prepared by addin~ methanol in the
ratio of 5~0 to chloroform resulted in elution of 7-hydroxy-
cholesterol.
The eluted 7-ketocholesterol portion and 7-
hydroxycholesterol portion were collected respectivelyand subjected to a similar treatment wi-th a column of
benzene-equilibrated silica gel (product by Melc Inc.)
for increasing purity.
The melting points of thus obtained 7-keto-
cholesterol and 7-hydroxycholesterol agreed with those
shown in the literature (The Journal of ~iological
Chemistry, 141, 597, (1941)). The above-mentioned
literature, secondary decomposition products were by-
produced due to use of Girard's reagent for separating
the two compounds, but in the chromatography method used
by the present inventors, it was possible to prevent such
secondary reaction.
Example 2
1 Kg of paste of IV-l fraction obtained according
to Cohn's alcohol fractionation method was suspended and
extracted in 4 liters of CHC13-MeOH (1 : 1) mixed solution,
and the obtained extract was developed in a silica gel
column and the adsorbed material was eluted out with
chloroform containing a sma]l amount of methanol. The
eluate was concentrated and subjected to cellulose column
chromatography and two fractions having the inhibitory
activities were rcmovcd by means of thin-layer chromato-
graphy aeve]oped with n-hexane - ethyl acetate (1 : 1)
mixed solution and the PHA method and then conccntrat;cd
-- 10 -
~' .
1 under vacuum to isolate them in the form of crystals.
Thin-layer chromatography and elemental analysis
confirmed that these materials are 7-ketocholcsterol and
7-hydroxycholesterol.
Medicinal Preparation Example 1 (Injection)
300 Grams of sterilized 7-hydroxycholesterol
was dissolved in 9 litres of distilled water with 300
grams of a polyoxyethylene-polyoxypropylene copolymer
with average molecular weight of 8,350, and the mixture
was emulsified and then made isotonic physiologically
by adding 1 litre of lactated Ringer's solution to obtain
an aqueous solution with final concentration of 3.0~
This solution was distributed into vials such that each
vial would eontain 100 mg of 7-hydroxychole~terol, thereby
preparing injeetions.
Medieinal Preparation Example 2 (Oral tablets)
The following substances:
7-hydroxycholesterol 300 grams
Calcium phosphate 50 grams
Starch - 600 grams
~iquid gelatin 150 grams
were mixed and kneaded by adding water~ and the mixture
~; was granulated into globular grains and the latter were
dried under vacuum to obtain grains with diametcr of
about 5 mm. These grains were compressed by a presser
and prepared into tablets each weighing 500 mg.
107~8~i
1 Medicinal Preparation Example 3
10 Grams of a mixture of 7-ketocholesterol and
7-hydroxycholesterol and 10 grams of phospholipid were
suspended in 100 ml of water, and the suspension was
uniformalized by a supersonic treatment and prepared into
aseptic injections.
Medicinal Preparation Example 4
10 Grams of phospholipid was added in 100 grams
of soya-bean oil, to which were further added 4 grams of
7-hydroxycholesterol and 400 ml of water, and the mixture
was prepared into a uniform and steril injection by using
a supersonic treating method.
