Note: Descriptions are shown in the official language in which they were submitted.
36~17~
A FIBRINOLYTICALLY ACT~VE AGENT AND A METHOD F3R
THE PREPARATION THEREOF
BACKGROUND OF THE IMVENTION
The present invention relates to a novel fibrinoly-
tically active agent and a method for the preparationthereof. ~ore particularly, the invention ~elates to a
novel fibrinolytically active agent e~tracted from earth-
worms as well as the method for the preparation thereof by
the extraction of earthworms with an aqueous extractant.
1~ In recent years, attention is directed ~y the practi-
tioners of medicine and pharmaceutics to various types of
the deseases due -to -the coagulation of blood occurring in
many cases of prime and aged adults from the standpoint of
geria-trics. Several of the well known deseases of such a
type are, for example, myocardina] infarction, cerebral
thrombosis, syndrome of disseminated intravascular coagu-
lation and the like and, as i5 well known, urokinase of
man origin and streptokinase are used as a therapeu-tic
medicament therefor.
These medicaments are, however, not quite satisfactory
in several respects. For example, urokinase of man origin
is prepared from human urine as the starting material so
that the supply thereof is limited by the availability of
this starting material in large volumes. Streptokinase is
defective due to the antigenicity. In addition, both of
these medicaments must be used by instillation so that the
patient under treatment suffers great pains unavoidably.
Therefore, it has been long desired to develop a novel
fibrinolytically active agent usable as a therapeutic
medicament of the above mentioned deseases without the
problems in the conventional medicam~nts therefor. That
is, one of the recent problems in the pharmaceutics has
been to develop a novel fibrinolytically active agent free
from the limitation by the availability o-E the starting
material in large quantities and capable of being ~mi ni s-
trated to the patient not by the instillation but by other
72
.
means, desirably orally~ without giving pains to the
patient.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to
provide a novel fibrinolytically active agent useful as a
therapeutic medicament for th~ diseases due to the coagula-
tion of blood free from the limi~ation in the availability
of the starting material in large quantities and suitable
for administration not by instillation. With the above
object, the inventors have conducted extensive investi-
gations with a variety of natural resources for the desired
effective ingredient and arrived at a discovery that
tissues of earthworms contain such a substance in a good
content leading to the completion of the present inventionO
Accordins to the invention there is provided a method
for the preparation of a fibrinolytically active agent
which comprises: (a) extracting the tissues of earthworms
belonging to the family of Lumbricidae with an aqueous
extractant having a pH in the range of 5 to 10 to
give an aqueous extract solution containing the active
ingredients, and (b~ concentrating or dehydrating the
aqueous extract solution,
Thus, the fibrinolytically active agent of the
present invention is an extracted material from
earthworms belonging to the family of Lumbricidae
with an aqueous extractant having a p~ in the range
of 5 to 10, preferably followed by purification,
and then concentration and dehydration and the
method of th~ present invention for the preparation of such
a fibrinoly~ically active agent preferably comprises dis-
persing finely ground tissues of earthworms in an aqueous
extractant to extract the effective ingredient into the
extractant, separating the extract solution containing the
effective ingredient from the insoluble matter and
concentrating or dehydrating the effective ingredient
contained in the extract solution.
~198G72
- 2a -
The above obtained concentrate or dehydrated material
is, although it is still in a crude state, effective as such
but it is preferable that the crude product is further
purified by a suitable means such as adsorption, fractional
S precipitation with a polar organic solvent, salting-out,
ultrafiltration, ion-exchange chromatography, gel filtration,
affinity chromatography, hydrophobic chromatography and the
like.
~ .
,. . .
BRIEF DESCRIPTION OF THE DRAWING
FIGURE 1 is a graph illustrating the condition of
separation of the inventive fibrinolytically active
in~redient in the isoelectric focusing.
FIGURE 2 is a graph illustrating the increases in
the fibrinolytic activity of the peripheral bloods of three
patients of hypertension.
FIGURE 3 shows the fibrinolytic activity and the
optical density of the fractions obtained in the column-
chromatographic fractionation of the earthworm extract
in Example 14.
FIGU~ES 4a and 4b show the euglobulin dissolving time
and the fibrinolytic activity, respectively, of the
peripheral blood of men orally administrated with the
fibrinolytically active agents obtained in Example 14 in
the lapse of time (see Example 16).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
As is known, earthworms belonging to the family of
Lumbricidae have been utilized from time imm~m~rlal in
the oriental coun-tries as a kind of legendary medicine for
an anodyne, antipyretic, diuretic and the like. However,
nothing is disclosed or suggested in the prior art liter-
atures on the possibility that the tissues of earthworms
may contain certain fibrinolytically active ingredient
capable of exhibiting the desired effect of increasing the
fibrinolytic activity of the peripheral blood of man when
it is administrated to a patient, in particular, orally.
Therefore, it was quite unexpected that the extracted
material rom earthworms with an aqueous extractant may
exhibit such a fibrinolytic activity.
In th ollowing, the procedure for the preparation
of the fibrinolytically active agent from earthworms is
described in detail.
The starting material is obtained from the tissues of
earthworms and fresh bodies of living earthworms and the
bodies of earthworms from which the entrails have been
removed as well as the entrails themselves are all suitable
f
-- 4 --
as the starting material. The zoological kind of -the
earthworms is not particularly limitative within the
family of Lumbricidae and earthworms of any kind are
substantially equally suitable including, for example,
Lumbricus rubellus, Lumbricus terrestris, Eisenia foetida
and the like. The earthworm bodies are used as a homoge-
nate by finely grinding. It is a convenient way that tne
tissues of earthworms are dried in advance by heating,
vacuum-drying or freeze-drying and pulverized into a fine
powder which may be used as such or after defatting. The
most preferable starting material is a freeze-dried powder
or fresh earthworm tissues with or without defatting.
The aqueous extractant, with which the finely ground
tissues of earthworms are extracted, should have a p~ in
the range from 5 to 10 or, preferably, from 6 to 8. The
aqueous ex-tractant suitable for the purpose is not limited
to pure water but may include physiological saline solu-
tions, buffer solutions and undermentioned prepared salt
solutions which may further contain a small amount of a
polar organic solvent miscible with water such as methyl
alcohol, ethyl alcohol, propyl alcohol, acetone, diethyl
ether, dio~ane and the like. The most preferable aqueous
extractants are the physiological saline solutions and
buffer solutions having a pH of 5 to 10 or, preferably,
from 6 to 8. Phosphate, acetate, borate, citrate and
tris/hydrochloric acid buffer solutions are equally
suitable. The above-mentioned prepared salt solution is
a dilute aqueous solution prepared with admixture of a
water-soluble organic or inorganic acid, such as hydro-
chloric f sulfuric, phosphoric, acetic, lactic, citric andsuccinic acids, and an alkali, such as hydroxides and
carbonates of an alkali metal and ammonia, to have a pH of
5 to 10 or, preferably, 6 to 8. The suitable proportion
of the aqueous extractant to the starting material is in
the range from 1 to 100 times by weight or, preferably,
from 5 to 30 times by weight based on the dry weight of
the starting material. The extraction is performed at a
temperature not higher than 60 C or, preferably, in the
i7;~
-- 5
range from 5 to 40 C Eor a sufficient time up to 500 days
or, preferably, from 30 minutes to about 30 days.
The extraction of the finely ground tissues of earth-
worms with the aqueous extractant is performed by agitating
or shaking the slurried mixture or by passing the aqueous
extractant -through a bed of the powdered starting material.
It is preferable that the ear-thworm tissues are homogenized
by use of a homogenizer, blender, ultrasonic disintegrator,
pressurizing ce]l destroyer, grinder or the like machine
into a homogenate, i.e. an aqueous suspension of the cell
constituents, in order to destroy the cells of the earth-
worm tissues before the slurried aqueous mixture is
incubated to effect extraction.
When the extraction of the effective ingredients in
the earthworm tissues is completed, the slurried aqueous
mixture is filtered and the clear filtrate, i.e. the
extract solution, is, optionally as combined ~ith the
washings of the residue in the above filtration and kept
for a suitable time at a suitable temperature, concentrated
by a suitable known method such as evaporation with heating
or under reduced pressure and/or ultrafiltration or
dehydrated by evaporation of the aqueous solvent under
reduced pressure or by freeze-drying into a solid material.
It is preferable that a small amount of an antiseptic or
~5 preservative is added to the aqueous slurried mixture of
the earthworm tissues under incubation or the aqueous
extract solution under the processing for concentration
or dehydration~ In this respect of preventing denatura-
tion, the addition of the above mentioned polar organic
solvent to the aqueous extractant is eEfective in addi-tion
to the effect of enhancing or accelerating the extraction
of the effective ingredients thereby. The concentration
of the organic solvent in the aqueous extractant should be
determined depending on the kind of the solvent and other
parameters although the concentration of the organic
solvent should not exceed 50% by volume in the aqueous
extractant.
6~7~:
-- 6
The above obtained aqueous extract solution followed
by concentration or dehydration contains the desired
fibrinolytically active ingredients in a crude state so
that purification thereof should preferably follow. The
purificatlon or fractionation of the effective ingredients
is undertaken with the concentrated aqueous extra~t solu-
tion as such or with the dehydrated material as dissolved
in a small volume of water. The method of purification
may be a conventional purification method for high poly-
meric substances in gèneral. Following is a descriptionof several methods for the purification or fractionation
of the fibrinolytically active ingredients obtained in
the above.
The methods applicable to the purification or frac-
tionation of the effective ingredients in a crude stateas in an aqueous solution include treatments by use of an
adsorbent or polar organic solvent, salting-out, ultra-
filtration, ion-exchange chromatography, gel filtration,
affinity chromatography, hydrophobic chromatography and
the like. Either one of the above mentioned methods may
be sufficient in some cases but it is usual that two or
more of the above methods are utilized in combina~ion in
a suitable order to remove the undesirable impurities.
The adsorbent utilizable here is exemplified by active
charcoal, acid clay, activated clay and synthetic resin-
based adsorbent, for example, sold under a tradename of
Amberlite XAD. The polar organic solvent used for the
fractional precipitation of the effective ingredients is
exemplified by methyl alcohol, ethyl alcohol, propyl
alcohol, acetone, diethyl ether, dioxane and the like,
of which ethyl alcohol, acetone and propyl alcohol are
preferred.
The salt suîtable for the salting-out is exemplified
by ammonium sulfate, sodium sulfate, magnesium sulfate,
potassium phosphate, sodium chloride, potassium chloride,
sodium citrate and the like, of which ammonium sulfate
is preferred. The ion exchanger suitable for the ion
exchange chromatography is exemplified by those based on
'7~
-- 7
the hydrophilic polysaccharides such as cellulose, dextran,
agarose and the like, of which diethylaminoethylcellulose
(hereinafter referred to as DEA~-cellulose), triethylamino-
ethylcellulose (hereinafter referred to as TEAE-cellulose),
aminoethylcellulose (hereinafter referred to as AE-cellu-
lose), carboxymethylcellulose (hereinafter referred to as
CM-cellulose), phosphocellulose (hereinafter referred to as
P-celiulose), phosphomethylcellulose (hereinafter referred
to as PPM-cellulose), diethylaminoethylcellulofine (here-
inafter referred to as DEAE-cellulofine) and the like are
preferred.
The ion exchange chromatography may be carried out
also by use of a conventional ion exchange resin including
weakly acidic cation exchange resins such as those sold
under trade~arks of Amberlite IRC-50, Amberlite IRC-75,
Amberlite IRC-84, Dowex CCR-2 and the like and weakly
basic anion exchange resins such as those under trademarks
Amberlite IR-4B, Amberlite IR-45, Amberlite IRA-40Q,
Dowex 3 and the like.
The gel filtration may be performed by use of those
sold under trademarks of Sephadex, Sephalose, Biogel,
Toyopearl Ultragel, Cellulofine and the like. The station-
ary phase used in the affinity chromatography may be formed
with various adsorbents such as Sephalose and Toyopearl
as the carrier. Further, the hydrophobic chromatography
can be carried out by use of an adsorbent such as those
with a hydrophilic polysaccharide, e.g. agarose and cellu-
lose as the base to which hydrophobic groups have been
introduced ~y use of an aliphatic, alicyclic or aromatic
compound having 2 to 20 carbon atoms in a molecule and
having an amino group.
Following schemes 1 to 16 are several preferable
examples of the processes of the inventive method given
in the form of a flow chart.
8 --
Scheme 1~
¦earthworm tissues ¦
aqueous extractant
~ with pH 6-8
¦incubation ¦
filtration or
¦washout ¦ ~~ centrifugal separation
.,
aqueous
extractant ~, residue
with pH 6-8 l extract
solution
~washingS ¦
combination of extract
solution and washings
~_ ultrafiltration or
vacuum evaporation
~r
¦concentrated solution¦
vacuum-, freeze-
~ or flash-drying
crude fibrinolytic
substance
- 9 -
Scheme 2
¦earthworm tissues ¦
aqueous extractant
~ with pH 6-8
¦extraction and/or incubation ¦
fil-tration or
¦washout¦ c centrifugal separation
aqueous
ex-tractant ~ residue ¦
with pH 6-8
extract
solution
¦washings ¦ l
combination of extract
solution and washings
I
¦incubation ¦
_ ultrafiltration or
vacuum evaporation
¦concentrated solution ¦
c vacuum-, freeze~
or flash-drying
crude fibrinolytic
substance
-- 10 --
Scheme 3. Scheme 4.
concentrated concen-trated
solution in solution in
Scheme 1 or 2 Scheme 1 or 2
polar polar
-- organic ~- - organic
solvent solvent
¦ precipitates ¦ ¦ precipltates ¦
¦ dissolution ¦ ¦ dissolution ¦
¦ ion exchange ¦ ¦ gel filtration ¦
fractionated ¦ ion exchange
material
`1/ purified
¦ precipitates ¦ fibrinolytic
substance
gel filtration
or affinity
chromatography
purified
fibrinolytic
substance
I~L 1~ ~ ~ IY ~d
- 11
Scheme~5. Scheme 6.
concentrated concentrated
solution in solution in
Scheme 1 or 2 Scheme 1 or 2
~ salting-out ¦ ~ salting-out ¦
v v
¦precipitates ¦ ¦ precipitates ¦
¦ dialysis ¦ ¦dialysis ¦
¦ion exchange ¦ ¦ gel filtration ¦
¦gel filtration ¦ affinity
I chromatography
~/ I
purified
fibrinolytic purified
substance fibrinolytic
substance
- 12 -
Scheme 7. Scheme 8.
dehydrated crude dehydrated crude
fibrinolytic - fibrinolytic
substance in substance in
Scheme 1 or 2 Scheme 1 or 2
c aqueous aqueous
solvent ~~ solvent
¦solution ¦ ~solution ¦
polar ~ salting-out ¦
- organic
solvent
V ¦ precipitates ¦
¦precipikates ¦
1, ¦ solution ¦
¦solution ¦
`I'
dialysis and/or
gel filtration
ion exchange
and/or
gel filtra-tion
¦ion exchange ¦
.
fractionated
material fractionated
I material
~b i
purified
fibrinolytic purified
substance fibrinolytic
substance
72
Scheme 9. Scheme 10.
learthworm tissues ¦
fracti.onated
material in
Scheme 7 or ~
aqueous
~ solvent
v ~/
affinity homogenization
chromatographyand incubation
u filtration or
c centrifugal
purified separation
fibrinolytic
substance
~I
¦extract solution¦
adsorption
treatment
~'
hydrophobic
chromatography
purified
fibrinolytic
substance
72
14 -
Scheme 11.Scheme 12.
conce~trated concen~rated
solution in solution in
Scheme l or 2 Scheme l or 2
decolorization decolorization
~, \,
adsorption with ion exchange
synthetic
adsorbent
purified
fibrinolytic
purified substance
fibrinolytic
substance
'7~2
- 15 --
Scheme 13. Scheme 14.
dehydrated crude dehydrated crude
fibrinolytic fibrinolytic
substance in substance in
Scheme 1 or 2 Scheme 1 or 2
aqueous ~ aqueous
~ solvent solvent
decolor- ~ .decolor-
ization ization
ultrafiltration ultrafiltration
purified dïalysis or
fibrinoly-tic gel filtr.ation
substance
purified
fibrinolytic
substance
7~
~6
Scheme 15. Scheme 1~
concen-trated concentrated
solution in solution in
Scheme 1 or 2 Scheme 1 or 2
¦ decolorization ¦adsorpt~on with
synthetic
adsorbent
polar
organic
solvent6~ salting-out ¦
¦ precipltates ¦¦ precipitates ¦
¦ dissolution ¦¦ dissolution ¦
¦ ultrafiltration ¦¦ ultrafiltration ¦
hydrophobic ¦ ion exchange ¦
chromatography
purified
purified fibrinolytic
fibrinolytic substance
substance
~L¢~7~
- 17 -
Following is a description of a preferred embodiment
of the inventive method for the preparation of a fibrino-
lytically active agent from earthworms given in further
detail as well as the characterization of the thus obtained
products~
I. Preparation of the fibrinolytically active agents
A defatted powder of freeze-dried earthworms is
dispersed in lQ times by weight of a 50 mM phosphate buffer
solution having a pH of 7.0 and subjected to incubation
at 37 C for 200 hours to extract the fibrinolytically
active ingredients into the aqueous phase. The aqueous
extract solution is concentrated by ultrafiltration and the
concentrated extract solution is admixed with a sufficient
volume of ethyl alcohol to fractionally precipitate the
effective ingredients. The precipitates are dissolved
in distilled water and fractionated by use of a DEAE-
cellulose into three fibrinolytically active fractions
called hereinafter F-I, F-II and F-III, respectively.
Each of the fractions F-I and F-II is subjected to
salting-out with ammonium sulfate followed by the trea-tment
with Sephadex G-75 and freeze-drying of the active frac-
tion. The third fraction F-III is subjected to desalting
as such ollowed by freeze-drying to give a purified
product.
II. Characteristic properties common to fractions F-
~to F-~III and the mPthod for activity determination
(l) Activity. each of them has an activity to solubilize
fibrin.
(2) Substrate specificity: each oE them has a strong
activity to decompose ~ibrin~
(3) Optimum pH and stabilizing pH: the optimum pH of the
fibrinolytic substance is about 8-10 while the stabilizing
pH is about 5-10.
(4) Activity determination fibrinogen is dissolved in
a 0.17M borate buffer solution having a pH of 7.8 and
containing 0.01M sodium chloride in such a concentration
7~
that the concentration of the coagulable protein is 0.15%
by weight and 10 ml of the solution are taken in a steril-
ized glass dish of 80 mm diameter with admixture of 0.5 ml
of a 20 units/ml solution of thrombin followed by standing
for 1 hour at room temperature with the dish covered.
The standard fibrin plate test is performed by
dropping 0.03 ml of the above prepaSed test solution on
a standard fibrin plate prepared with 10 ml of a 0.15~
fibrinogen solution and, after keeping for 10 minutes with
a filter paper inserted under the glass cover, placing it
in a thermostat at 37 C to be kept there for 18 hours
to effect the reaction. The fibrinolytic activity is
expressed by the product in mm2 of the lengths of the
major and minor axes of the area on the standard fibrin
plate formed by dissolving.
(5) Stability: at least 92% of the residual activity is
obtained with the fibrinolytically active ingredients of
the invention in an aqueous solution having a pH of 7.5
or 9.0 ater 30 minutes at 50 C.
t6) Inhibitors: the activity of the fibrinolytic substance
is inhibited by aprotinin (Trasylol, a ~rademark of
Baeyer Co.), tranexamic acid (Transamine, a trademark of
Dai~ichi Seiyaku Co.) and soybean trypsin inhibitor
~available from Miles Laboratories, Inc.) and serum.
(7) Fibrinolytic activity: the fibrinolytically active
agent of the invention has an activity of plasminogen
activation so that fibrin is solubilized indirectly in
addition to the direct reaction therewith to effect solu-
bilization. Further, similar activity is also exhibited
by an extract solution prepared by the incubation at 37
C for 20 days of a homogenate of 300 g of a freeze-dried
powder of earthworm tissues in 3 liters of a physiological
saline solution or the freeze-dried material of the extract
solutio~. ~
In an isoelectric focusing undertaken with an extract
solution obtained in the above described manner after
filtration, the pH gradient curve and the optical density
curve at 750 nm by use of the copper-Folin reagent of the
,,
~.~
-
-- 19 --
fractions each in a 2.5 ml volume were as shown in FIGURE
1 by the curves I and II, respectively. These results
indicate the presence of proteinous or quasi-proteinous
substances having the isoelectric points at pH values of
1.5, 3.4 and 5.6.
FIGURE 1 also inc~udes -the results of the determina-
tion of the fibrinolytic activity of the fractions in the
isoelectric focusing of the above obtained extract solution
as filtered or after dialysis by the curves III and IV,
respectively. These results indicate the presence of a
highly fibrinolytic substance in the fractions of the
extract solution ~ithout dialysis obtained at and near the
isoelectric point of pH 3.4 and the presence of fibrinolyt-
ically active substances having somewhat lower activity
than above in the fractions obtained at and near the
isoelectric points of pH 1.5 and pH 4Ø In contrast
thereto, the fractions obtained at and near the isoelectric
point of pH 5.6 have no fibrinolytic activity. On the
other hand, the dialysis of the extract solution has an
cO effect to remove the fibrinolytically active substance
appearing in the fractions of the extract solution before
the dialysis obtained at and near the isoelectric point
of pH 1.5 while the activity of the fractions obtained
at and near the isoelectric point of pH 3.4 is retained
even after the dialysis. Thus it is concluded from the
results shown in FIGURE 1 that the active ingredient has
an isoelectric point at about pH 3.4 while the substance
having an isoelectric point of about pH 5.6 is irrelevant
to the fibrinolytic activity of the extracted material
from earthworms.
III. In vivo activity test of the inventive fibrinolytic
substance
In the use of the inventive fibrinolytically active
agent as a therapeutic medicament for human patient by
oral admirlistration, the crude or partially purified
products at any intermediate stages of purification in
the above given schemes for purification may be used
672
- 20 -
although, needless to say, the highly purified final
product is the most preferable form from the standpoint
of exhibiting excellent effectiveness as a therapeutic
medicamen-t by oral administration.
The purified product obtained in the following
Example l was orally administrated to three male patients
of 60, 73 and 59 years old suffering from arteriosclerosis
each in a dose of 600 mg as freeze-dried and the peripheral
blood of each pa-tient was taken periodically at 1 hour
intervals, for which the fibrinolytic activity in mm2 was
examined by the euglobulin fractionation to give the
results shown in FIGURE 2 by the curves I, II and III for
the above three patients, respectively. The white circles
and black circles on each of the curves correspond to
complete and incomplete solubilization of fibrin, respec-
tively. The conclusion derived from these results is that
the fibrinolytic activity in the peripheral blood begins
to increase 2 hours after administration and reaches the
maximum value 4 to 6 hours after administration of the
inventive fibrinolytically active agent.
Example l.
An aqueous slurry of 1 kg of a finely powdered,
freeze-dried tissues of earthworms of the species
Lumbricus rubellus after defatting with acetone dispersed
in lO liters of a 50 mM phosphate buffer solution having
a pH of 7~0 was agitated for lO0 hours at 37 C to effect
extraction of the water-soluble ingredients followed by
filtration. The residue from the filtration was washed
with 3 liters of the same buffer solution and the washings
were combined with the filtrate to give a total volume of
12.8 liters of a clear extract solution. The fibrinolytic
activity o~ this extract solution was 490 mm2/ml after
lO times dilution with distilled water.
The above obtained extract solution was concentrated
by ultrafiltration to give a volume of 1.75 liters of the
concentrated extract solution having a fibrinolytic
6~
- 21 -
activity of 550 mm2/ml after 60 times dilution. The
effective ingredients contained in this concentrated
extract solution was fractionally precipitated by first
adding 1.75 liters of e-thyl alcohol to the extract solution
and then by adding 7.0 liters of ethyl alcohol to the
filtrate from the first step precipitation. The precipi-
tates collected from the above two-step precipitation were
combined and dissolved in l.l liters of the same buffer
solution. The thus obtained solution had a fibrinolytic
activity of 694 mm /ml after 60 times dilution. The
solution was further subjected to chromatographic frac-
tionation by use of DEAE-Sephalose (a product by Pharmacia
Co.) into 3 fractions of F-I, F-II and F-III. Each of
the ~ractions F-I and F-II was subjected to salting out
by 60% saturation with ammonium sulfate ~ollowed by the
treatment with Sephadex G-75 and freeze-drying to give 625
mg of a dehydrated powdery material having a fibrinolytic
activity of 12,300 mm2/mg and 665 mg of a powdery material
having a fibrinolytic activity of 10,700 mm2/mg from the
fractions F-I and F-II, respectively. The third fraction
F-III was, after desalting and concentration, freeze-dried
to give 1200 mg of a powdery material ~aving a fibrinolytic
activity of 11,500 mm2/mg.
Example 2.
Live earthworms of the same species as in Example 1
weighing 84 g were added to a physiological saline solution
to give a total volume of 300 ml and ground into a homoge-
neous suspension, which was incubated for 100 hours at
37 C followed by centrifugal separation into an extract
solution and insoluble residue~ The residue was washed
with 150 ml of the physiological saline solution and the
washings were combined with the extract solu-tion to give
a total volume of 400 ml of the combined solution having
a fibrinolytic activity of 375 mm2/ml after lO times
dilution. The dehydrated material obtained from this
combined solution by freeze-drying had a fibrinolytic
activity o~ 142 mm2/mg.
- 22 -
Example 3.
Live earthworms weighing g4 g were added -to 500 ml of
distilled water containing 0.3 g of phenol and ground into
a homogeneous suspension which was incubated for 76 hours
at 30 C followed by filtration to remove the insoluble
residue from the aqueous extract solution. The residue
was washed with 200 ml of distilled water and the washings
were combined with the extract solution to give a total
volume of 650 ml of the combined solution ha~ing a fibrino-
lytic activity of 220 mm2/ml after 10 times dilution~
Example 4.
~ive earthworms weighing 84 g were added to an aqueous
mixture of 400 ml of distilled water and 30 ml of ethyl
alcohol and ground into a homogeneous suspension, which
was incuba~ed for 240 hours at 25 C followed by centrif-
ugal separation to give a clear extract solution having
a fibrinolytic activity of 350 mm2/ml a~ter 10 times
dilution.
Example 5.
An aqueous suspension was prepared by a~mixi ng 50 g
of a powder of vacuum-dried earthworms with 400 ml of a
physiological saline solution and 100 ml of a salt solution
having a pH of 6.5 as pr~pared with a 1.8% aqueous solution
of phosphoric acid and a 1.8% ammonia water and the sus-
pension was incubated for 100 hours at 38 C followed by
iltration to give a clear extract solution having a
fibrinolytic activity of 725 mm2/ml after 10 times dilution
corresponding to an activity of 72.5 mm2/mg of the dry
powder of earthworms.
Example 6.
~ n aqueous suspension was prepared by dispersing 50 g
of a powder of freeze~dried earthworms in a mixture of 250
ml of a dilute salt solution having a pH of 6.3 as prepared
with a 2% aqueous a~etic acid solution and a 2% aqueous
sodium hydroxide solution, 200 ml of a physiological saline
~9~
- 23 -
solution and 50 ml of~ distilled water with admixture of
0.5 g of sodium azide and the suspension was incubated
for 72 hours at 37 C followed by filtration to give a
clear extract solution having a fibrinolytic activity
of 460 mm2/ml after 10 times dilution.
Example 7.
An aqueous suspension was prepar~d by dispersing 50
g of a defatted powder of freeze-dried earthworms into a
mixture of 200 ml of an acetate buffer solution having a
pH of 7.0, 200 ml of a borate buffer solution having the
same value of p~ as above, 100 ml of distilled water, 10
ml of propyl alcohol and 10 ml of dioxane and the suspen-
sion was incubated for 2~0 hours at 32 C followed by
filtration to give a clear extract solution having a
fibrinolytic acitivity of 772 mm2/ml after 10 times
dilution.
Example 8.
An aqueous suspension was prepared by dispersing 50
g of a defatted powder of freeze-dried earthworm bodies
with the entrails removed into an aqueous mixture of 250
ml of a dilute salt solution having a pH of 6.8 as prepared
with an aqueous acid mixture containing 1.8% of phosphoric
acid and 3.5% of hydrogen chloride and a 2N aqueous
solution of potassium hydroxide, 225 ml of a physiological
saline solution and 25 ml of acetone and the suspension
was incubated for 96 hours at 25 C followed by filtration
with suction to give a clear extract solution having a
fibrinolytic activity of 600 mm2/ml after lG times
~ilution.
Example 9.
An aqueous suspension was prepared by dispersing 10
g of a defatted powder of earthworms dehydrated by high-
temperature flash drying into an a~ueous mixture of 50 ml
of a phosphate buffer solution having a pH of 6.~ and 50
ml of a citrate buffer solution having a pH of 6.5 and
the suspension was incubated for 7 hours at 37 C ~ollowed
by filtration to give a clear extract solution. The
residue was washed with a physiological saline solution
and the washings were combinecL with the above extract
solution -to give a total volume of 120 ml of the combined
solution. This combined extract solution was admixed with
0.1 g of sodium azide and further incubated for 10 hours
at 37 C. The resultant solution had a fibrinolytic
activity of 280 mm2/ml after 10 times dilution.
Example 10.
An aqueous suspension was prepared by dispersing 10
g of a defatted powder of freeze-dried earthworms into an
aqueous mixture of 50 ml of a phosphate buffer solution
having a p~I of 7.4 and 50 ml of a physiological saline
solution and the suspension was agitated for ~ hours at
22 C to effect extraction of the water-soluble ingredients
into the aqueous solution followed by centrifugal separa-
tion to give a clear extract solution. The fibrinolytic
activity of this extract solution was, after admixture of
0.07 g o~ sodium azide and incubation for 5 hours at 37
C, 190 mm~/ml after 10 times dilution.
Example 11.
Live earthworms weighiny 10 g were added to an aqueous
mixture of 70 ml of an aceta~e buffer solution having a
pH of 7.0 and 30 ml of distilled water and ground into a
homogeneous aqueous suspension which was agitated for 2
hours at 20 C to e~fect extraction of the water-soluble
ingredients into the aqueous solution followed by centrif-
ugal separation to give a clear extract solution. The
residue was washed with water and the washings were
combined with the extract solution to give a total volume
of 180 ~1 of -the combined solution having a ~ibrinolytic
activity of, after admixture of 0.1 g of sodium azide
and incuba-tion ~or 7 hours at 37 C, 40 mm2/ml after 10
times dilution.
367~
~ 25 -
Example 12.
An aqueous suspension prepared by dispersing 1 kg
of a freeze-dried powder of earthworms in 10 liters of
an aqueous solution containing 0.1% by weight of sodium
benzoate and 0.9% by weight of sodium chloride was agitated
for 96 hours at 32 C to extract the water-soluble ingre-
dients followed by filtration. The residue from the above
filtration was washed with 3 liters of the same aqueous
solution of sodium benzoate and sodium chloride as above
and the washings were combined with the filtrate to give
12.5 liters of a clear extract solution having a fibrino-
lytic activity of 490 mm2/ml after 10 times dilution.
The thus obtained extract solution was concentrated
to a total volume of 0.5 liter by ultrafiltratio.n and the
concentrated solu~ion was subjected to fractional precipi-
tation by first adding O.S liter of ethyl alcohol thereto
to obtain precipitates and then by adding a furt~er volume
o~ ethyl alcohol to the filtrate from the first precipita-
tion to give a final ethyl alcohol concentration of 80%
so that an additional amount of precipitates was obtained.
The precipitates obtained in the above two-step precipita-
tion were combined and washed with ethyl alcohol fol.iowed
by vacuum-drying to give 40.5 g of a dry powder having
a fibrinolytic activity of 1285 mm2/mg.
The above obtained powder was dissol~ed in 1 liter
of a lOmM phosphate buffer solution having a pH of 8.0
and the solution was passed through a column filled with
a hexyl-Sepharose prepared by the reaction of hexylamine with
agarose ac-t.ivate~ with epichl.orohydrin (Sepharose, trademark,
a product by Pharmacia Fine Chemicals Co.) to have the
active ingredients adsorbed thereon. After washing with
-the same buffer solution as above, elution of the column
was undertaken with the same buffer solution as above but
containing sodium chloride in a ~oncentration of 0.25M
as the eluant to give 1 liter of an eluate solution.
The eluate solution was, after dialysis, dehydrated
by freeze-dryin~ to give 5.75 g of a dehydrated material
having a fibrinolytic activity of 7241 mm2/mg.
,
~ c~
6~7~
- 26 -
Example 13.
The procedure down to the fractional precipi-tation
with ethyl alcohol followed by vacuum-drying was substan-
tially the same as in Example 12 above and 47 g of the
dried powder having a fibrinolytic activity of 1100 mm2/mg
were dissolved in 1 liter of a 20mM phosphate buffer
solution having a pH of 7Ø This solu-tion was passed
through a column filled with an ETI (albumen trypsin
inhibitor)-Sepharose prepared by combining an albumen
trypsin inhibi-tor (a product b~ Sigma Co.) to agarose
activated with epichlorohydrin to have the active ingre-
dients adsorbed thereon. After washing first with the
same buffer solution as above and then with a O.lM acetate
bufer solution having a pH of 5.0, elution of the column
was undertaken with the same acetate buffer solution as
above but containing sodium chloride and alginine in
concantrations oE lM and 0.5M, respectively, as the eluant
to give 0~8 liter of an eluate solution.
The eluate solution was, af-ter dialysis, dehydrated
by freeze-drying to give 255 mg of a dehydrated material
haviny a fibrinolytic activity of 70,960 mm2/mg.
Example 14.
An aqueous dispersion of 1 kg of a powdex of freeze-
dxied earthworms in 10 liters of an aqueous salt ~olutioncontaining 0.1% of sodium benzoate and 0.9% of sodium
chloride was agita-ted for 72 hours at 30 C to effect
extraction of the water-soluble ingredients into the
aqueous solu~ion followed by filtration to give a clear
e~tract solution. The residue was washed with 3 liters
of the same salt solution as above and the washings were
combined wlth the extract solution to give a total volume
of 13 liters of the clear combined solution having a
fibrinolytic activity of 450 mm2/ml after 10 times
dilution.
The above obtained aqueous solution was concentrated
by ul~rafiltxation into a liquid volume of 0.71 litex
and then admixed with equal volume of ethyl alcohol to
- 27 -
precipitate solid material which was collected by filtra-
tion. The filtrate was further admixed with ethyl alcohol
to give a final concentration of e-thyl alcohol of 80% to
give further precipitates which were collected and washed
with ethyl alcohol followed by vacuum-drying into a dry
powder. The total yield of the dry powdery products in
the above two-step precipitation was 42 g and the fihrino-
lytic activity thereof was 1322 mm2/mg.
The above obtained powdery product was dissolved in
1000 ml of distilled water and subjected to column-
chromatographic fractionation by use of an adsorbent of
DEAE-Cellulofine (a product by Chisso Co.) to give three
fractions F-I, F-II and F-III. FIGURE 3 gives the results
of the fibrinolytic activity in mm2 and the optical density
at 280 nm of the eluate fractions each in a 20 ml volume
obtained in the above mentioned column chromatography by
the curves I and II, respectively. The broken line in
FIGURE 3 indicates the concentration of sodium chloride
in the eluate fractions given by the electric conductivity
in m mho.
Each of the fractions F-I to F-III was subjected to
a treatment of salting-out by ~0~ saturation with ammonium
sulfate and the precipitates were dissolved in a small
volume of a 10 mM phosphate buffer solution having a pH
of 8Ø The solution was successively subjected to gel
filtration with Sephacryl S-200 and desalting concentration
by ultrafiltration followed by freeze-drying ~o give 629
mg, 879 mg or 1070 mg of the dehydrated product having a
fibrinolytic activity of 13,780 mm2/mg, 9,290 mm2/mg or
17,620 mm2/mg rom the fractions F-I, F-II and F-III,
respectively.
The plasminogen activator activity was examined for
each of the above obtained dehydrated products. Thus,
the dehydrated product was dissolved in water in a con-
centration o~ 0.1 mg/ml and 20 ~1 of this solution wereadmixed with 10 ~1 of ~he plasminogen having an activity
of 5 units/ml ~a product by Si~ma Co.~ and 30 ~1 of a 0.17M
borate buf~ex solution having a pH of 7.8 and containing
16i~
- 28 -
0.01M sodium chloride and, after standing for 10 minutes
at 37 C, 0.03 ml of the mixed solution was dropped
vertically on to a fibrin plate free of plasminogen (a
product by Miles Laboratories, Inc.). The area in mm2
of the dissolved portion was determined on the plate
after 18 hours of the reaction at 37 C. When the above
obtained area was taken as A and the corresponding area
in mm2 obtained by use of 10 ~1 of the 0.17~ borate buffer
solution in place of -the plasminogen was taken as B, then
the plasminogen activator activity is expressed by A-B.
The values of the thus determined plasminogen activator
activity were 2025 mm2/mg, 1721 mm2/mg and 12~3 mm2/mg
for the dehydra-ted products obtained from the fractions
F-I, F-II and F-III, respectively.
Example 15.
The fibrinolytically active products obtained in
Example 14 were examined for the reactivity with fibrin
and fibrinogen. Thus, 0.18 ml of blood plasma of man,
0.02 ml of a 250 mM aqueous solution of calcium chloride
and 0.02 ml of an aqueous solution of one of the fibrino-
lytically active products in a varied concentration were
mixed and the FDP (fibrin-decomposition peptide) produced
by the reaction for 30 minutes at 37 C in the above
mixture was determined by the latex coagulation test
using a kit for the thrombo-wellco test (manufactured by
Wellcome Co.). The results are shown in Table 1 in the
columns under the heading of CaC12 (+) for 5 different
concentratlons of 10~ to 10 1 ng/ml. The marks (+), (+~)
and (~++) in the table in~icate the formation of FDP from
fibrin, the increase of th~ number of the + marks corre-
sponding to the increase in the formation of FDP, while
the mark (-) indicates the absence~ OL formation of FDP
from fibrin for each concentration.
On the other hand, the sam~ test as above was repeated
în the absence of calcium chloride, i.e. by the use of
a physiological saline solution in place of the aqueous
solution of calcium chloride, to find that no FDP was
- 29 -
formed irrespective of the fraction of the fibrinolytically
active products and the concentration thereof as is shown
in the columns of Table 1 under the heading of CaCl2 (~).
These results support the conclu ion that the fibrinolyti-
cally ac-tive agent of the invention has reactivity with
fibrin but not with fibrinogen.
T a b l e
~ F - I F - II F - III
Concentration \
of fibrinolytic \ CaCl2 (+) ( ) (+) ( ) (+
substance
104 ng/ml - - + - ++~ -
103 _ _ +++ _ +++
2 _ _ +++ _ ~++
1 0 + _ +++ _ +
+ _ ++~ _ +
+ _ ++ _ +
Example 16.
Each of the freeze-dried purified products of the
fractions F-I to F-III in Exampoe 14 was orally adminis-
-trated to healthy men in a dose of l ~g/kg body weight and
the peripheral blood of the subjects was taken periodically
to give the euglobulin fractions, with which measurements
were undertaken for the time of complete dissolution of
euglobulin in hours and the fibrin dissolving activity in
mm2 by the s andard fibrin plate test. The results are
shown in FIGURES 4a and 4b, respectively.
As is shown in FIGRURE 4a, the time for the solubi-
lization of euglobulin was remarkably decreased about 2
7~:
- 30 -
hours after the oral administration of the purified
fibrinolytically active agents obtained from the fractions
F-I (curve I) and F-III (curve III) and the decrease in the
time of euglobulin solubilization continued sustainedly.
On the other hand, the time for the euglobulin solubili-
zation began to gradually decrease about 6 hours after
the oral administration of the fibrinolytically active
agent obtained from the fraction F-II (curve II). These
results support the conclusion that each of the purified
fibrinolytically active agents obtained from the fractions
F-I to F-III has an effect to enhance the fibrinolytic
activity of ~he peripheral blood of man when administrated
orally.
Further, FIGURE 4b indicates that, though with consid-
1~ erable differences between individuals, the fibrinolytic
activity of the euglobulin fractions obtained from the
peripheral blood was maximum at 2 to 7 hours after the
oral administration of the active agents obtained from
the fractions F-I to F-III (curves I to II~, respec-tively)
to the subjects and the fibrinolytic activity thereof was
kept sustainedly even 10 hours after administration.
