DE1212678B - Process for the production of medicinal preparations - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. Cl .:

A61k

German KL: 30 h -2/04

Number: 1212 678

File number: C 20232IV a / 30 h

Filing date: November 24, 1959

Opened on: March 17, 1966

The invention relates to a method for obtaining medicinal preparations with therapeutic Animal tissue activity. The active ingredients contained in these medicinal preparations of proteolytic Activity have interesting therapeutic properties, especially a strong effect against leprosy and post-poliomyelitic paralysis.

A method for the production of a hemostatic agent is known, in which by lOgenöss Maceration of a paranchymatic organ an alcohol-soluble phosphatide with hemostatic properties is obtained. This phosphatide has no enzymatic activity comparable to that of the active ingredients according to the invention, and also otherwise shows completely different behavior.

The method according to the invention is characterized in that comminuted animal tissue is used under sterile conditions in physiological saline solution for at least 2 days, but for that long Macerates until the Abderhalden's microreaction to organ-specific ferments at a maximum number different organ substrates is positive, then either

a) the maceration liquid obtained after filtration with a water-miscible organic Solvent, preferably acetone, falls, separates the precipitate and from the precipitate the active ingredients are eluted with physiological NaCl solution or

b) the macerate in the autoclave is ^{heated to 120 °} C. for about 1 hour and then filtered,

whereupon the eluate obtained under a) or that according to b) recovered filtrate is gently dried.

In obtaining the new preparations according to the invention, it was found that their fermentative Activity undergoes major changes during maceration. It is therefore advisable to control the maceration liquid in order to try to obtain the To determine moments of greatest effectiveness. One leads to this goal at regular intervals the Abderhalden's microreaction, as z. B. is described in "Results of Enzyme Research", Volume 11, Leipzig 1950, pp. 20ff. According to this reaction, the fermentation-containing liquid with different brought into contact with pure organ substrates (protein). If organ-specific ferments are present are, a digestion of the corresponding organ substrate occurs, the z. B. by presence of amino acid is detectable. The fermentative activity is determined by the number of organs which the digestive effect of these ferments proves method for the production of medicinal preparations

Applicant:

Dr. Henri Joseph Marie Clement,
Ettelbruck (Luxembourg)

Representative:

Dr.-Ing. A. van der Werth, patent attorney,
ίο Munich 8, Lucile-Grahn-Str. 22nd

Named as inventor:

Dr. Henri Joseph Marie Clement, Ettelbruck
(Luxembourg)

Claimed priority:

Luxembourg of November 26, 1958 (36 616) - -

put, expressed. Among these particular organs used for this purpose are the liver, spleen, hypothalmus, Pituitary, adrenal, thyroid, quadriceps, ovaries, testes and conjunctival tissue to name a few. This list is not limiting. The organs used can come from humans or animals.

It has been found according to the invention that the therapeutic activity of the preparations is proportional their fermentative activity is.

The maceration time necessary to achieve optimal activity depends on the working conditions and especially on the type of tissue macerated and the temperature away. The duration is generally between 2 and 30 days. This number is sometimes exceeded however, in most cases the maximum occurs between the 10th and the 20th day. Man assumes that the best or almost the best activity is achieved when the ferments present in the maceration water are at least ten organ substrates seven fermentative columns.

The maceration water separated from the animal raw material on the 9th day enables, for example, if it comes from a goat quadriceps, the digestion of three or four substrates. These Activity reaches seven substrates when separated on day 8 and becomes zero after 22 days.

The new water-soluble active ingredients have to maintain their fermentative activity from the Maceration solution are separated. This separation is possible in two ways.

609 538/356

3 4

With the one possibility of separation under the milieus in which they are located. It's almost zero

if the maceration liquid is thrown to a warming in a markedly acidic environment (pH 3.3),

in an autoclave at 12O ⁰ C for one hour. becomes essential at a pH of 6 and reaches her

As a result of this autoclave treatment, there was an over-maximum in neutral water (pH T), then, surprisingly, an increase in fermentative and '5 decreases rapidly and is only very weak in light

the therapeutic activity of the active ingredients in solid alkaline solutions (pH 8.5).

sets, although when cooking in an open vessel. The preparations of the invention are used, for. B. for loading

Loss of activity occurs. The macerated leprosy treated in this way. Numerous clinical results

tion liquid is then filtered sterile. This confirms their amazing effectiveness. Lepers

The filtrate, dried at 37.degree. C., gives a residue which has ulcers, spots and even holes

in a sealed bottle and with the exclusion of her diseases after a few injections

is kept by light. It has been found that it heals. Muscle contractures as a result of an old one

the powder obtained in this way, its fermentative activity leprosy also disappear. During several

for several months even at temperatures years of the usual type, d. H. with injections of

of about 30 ° C. On dissolving this powder 15 cups without any improvement in their condition

in sterile water one finds the initial enzyme-treated patients with the invention

table activity again. This remains to be eventually cured during several appropriate remedies. Which

Days constant and then suffers a gradual, from the treatment of the maceration fluids with

but slower reduction than that which would result in a water-miscible solvent-

is observed in the maceration liquid. 20 the substances (precipitation) have a stronger one

The second way of obtaining the active ingredients therapeutic effect against paralysis, for example

it says that the muscular contractures evident in animal tissue are considered to be those which are evident

obtained separated maceration liquid with a water-treated liquid in an autoclave

miscible organic solvents, for example.

with acetone, alcohols or mixtures thereof, 25 The active ingredients according to the invention can also

treated. A precipitate forms, which can be used successfully in the treatment of poliomyelitic

separates and from which the active ingredients are applied with physio-paralysis,

logical NaCl solution eluted, whereupon this extract- In the following examples, the recovery

tion solution under the same conditions as those of the active substances described according to the invention,

The liquid treated in the autoclave is evaporated. 30 starting material can be from the goat

The powders obtained can also be used without a muscle, lungs, cubital nerve, kidney, brain,

Keep changes and are easily soluble in the spinal cord, skin, thyroid, liver, heart; from the

Water. Billy goat of testicles; a muscle from the ox,

Although the adrenal gland, liver, heart; pork side

Preparations in powder form are well preserved, subject to 35 kidney gland; a placenta from humans. That invented.

they in solution a gradual loss of activity. Proper method is by no means based on this

For example, the list of fabrics can be limited by certain additives or certain treatments

this loss of activity can be prevented. the tissues of other animals can be taken.

Short-term treatment with Die is recommended to investigate fluctuations in activity

X-rays (distance 7 cm, 60 kV, 20 mA, 40 microreaction used during maceration

60 seconds) or with ultraviolet rays (Ab- von Abderhalden could in principle by others

stood 10 cm, 500 W, 90 seconds). Of course, tests for ferments will be replaced,
the preceding numbers are just examples and

can be changed. For example

The preparations 45 obtained in accordance with the invention

The existing new active ingredients must not be used with the Under sterile conditions, a quadriceps biological irritant from Filatov is removed like a freshly slaughtered goat. The muscle represents which apparently no fermentation activity is cut into small cubes and placed in a sterile one (Filatov, La Therapeutique tissulaire, container. 400 ecm physiological NaCl-Edit. Franc. Moscou, 1955, p. 56). The defense ferments 50 solution and 50,000 potassium peniculine units, in 1 ecm of Abder heaps on the other hand, which are only dissolved in water, are added. The bottle is shaken to a living, non-cachectic and not clogged organism, and brought to a place where an organism deprived of a pancreatic gland occurs at a temperature of about 5 ° C.
(R. Abderhalden and RW Martin, Fer- The quantity ment research necessary for the activity experiments, 16 [1940], 245), do not withstand the 55 are taken on the 1st, 4th, 6th, 9th and 12th day. For warmth (R. Ab der hai den and S. Buadze, the Adberhalden reaction one chooses the ten ferment research, 11 [1931], p. 361). These sub-following organs: liver, spleen, hypothalamus, different show indisputably that the new active substances pituitary, adrenal gland, thyroid gland, bovine derivation. 60 fabrics. Positive reactions result from the

After all, the already known proteoly samples each have two, eight, three, four

enzymes such as pepsin and trypsin do not affect the own organs or eight organs. After the last result will be

like properties of the products of the invention, 200 ecm maceration liquid and about half

namely the organ-specific activity, resistance of the quadriceps pieces taken,

in the warmth and the activity at pH 7 65 The maceration is carried out under the same conditions

maximum. continued as before. There are test samples

The enzymatic activity of the new preparations taken on the 15th, 18th, 24th, 31st, 48th and 73rd day. the

according to the invention depends on the acidity of the number of digested organs according to the microreaction

von Abderhalden is 5, 8, 0 (24th day), 2.5 and 0 (73rd day). These results clearly show the fluctuations in activity during maceration.

If you z. B. liver, one finds a zero reaction on the 1st, 9th, 24th, 31st and 73rd day. A positive one Result will be on the 6th day, a fairly strong effect on the 4th and 18th days, and finally a substantial one Effective on the 12th and 48th day.

To obtain the preparations according to the invention, 100 ecm of the liquid withdrawn on the 12th day is taken and an approximately corresponding amount is added to the removed quadriceps pieces, ie the fourth part of the original pieces. The mixture is heated in an autoclave at 12O ⁰ C for one hour. It is then allowed to cool down to about 50 ° C. and filtered in a sterile manner at this temperature. The filtrate is distributed in five petri dishes, which are brought in an oven at about 37 ⁰ C. After it has completely dried out, the residue is taken up with the aid of sterile instruments and placed in a well-sealed bottle. Keep it out of light and, if possible, in a cool place. This latter condition is not absolutely necessary.

The other 100 ecm of the liquid removed on the 12th day is filtered through sterile paper and 150 ecm of a mixture of equal parts of acetone and ethanol is added. The precipitate is then left to stand for 12 hours. The supernatant liquid is carefully separated off and the precipitate is eluted with 20 ml of sterile physiological NaCl solution for 20 hours with occasional stirring with the aid of a sterile glass rod. Filter through sterile paper. The filtrate is placed in petri dishes and dried at 37 ⁰ C. The powder obtained is stored as described above. The same results are obtained if the acetone-ethanol mixture is replaced by acetone.

Example 2

Under sterile conditions, a muscle of a freshly slaughtered ox is cut into small cubes and placed in a sterile container. 400 ecm physiological NaCl solution and 50,000 units of potassium penicillin, dissolved in 1 ecm water, are added. The bottle is closed, shaken and brought to a place with a temperature of about 5 ^° C. The quantities required for the activity tests are taken on the days indicated in the table below. For the reaction after Ab derhalden, the organs mentioned in Example 1 are used. The positive responses vary as indicated in the table.

Maceration days Number of organs with
positive reaction 1 2 4th 8th 6th 3 9 4th 12th 8th 15th 5 18th 5 24 0 29 1 47 5 73 0

In order to obtain the active substances, the maceration must be interrupted on the 12th day, if out of ten tested substrates eight give a positive reaction.

The active substances are therefore extracted from the maceration liquid on the 12th day according to one of the im Example 1 extracted procedure.

Example 3

Under sterile conditions, a lung lobe from a freshly slaughtered goat is cut into small cubes and placed in a sterile container. 300 ecm of physiological NaCl solution and 50,000 units of kahumpenicillin, dissolved in 1 liter of water, are added. The bottle is closed, shaken and brought to a location of about 5 ^° C. Then the necessary quantities for the activity tests are taken. These are carried out on the 14th, extra 15th and 16th day. The following 10 organs are selected for the Abderhalden reaction: hypothalamus, pituitary gland, thyroid gland, liver, spleen, adrenal gland, testes, ovaries, muscle connective tissue and muscles. There are positive reactions eras with two, two or seven organs. At this last result the maceration is interrupted. The active substances are extracted from the maceration liquor removed on the 16th day according to one of the methods described in Example 1.

Example 4

A cubital nerve of a goat is macerated by the method described in Example 3. the Maceration is interrupted on the 14th day. For the reaction according to Abderhalden one has that in Example 5 specified organs taken. On the 14th day there were positive reactions with eight organs. The active ones Substances are extracted from the maceration liquid taken on the 14th day according to one of the methods in Example 1 described procedure extracted.

Example 5

Following the procedure described in Example 3 a goat kidney is macerated. The maceration is stopped on the 16th day. For the response after Abderhalden, the organs listed in Example 3 are used. Surrendered on the 16th day positive reactions with six organs. The active substances are taken from the on the 16th day Maceration liquid extracted by one of the methods described in Example 1.

Claims (2)

Patent claims: 1. Process for the production of medicinal preparations with therapeutic activity from animal Tissue, characterized in that one shreds animal tissue under sterile Conditions in physiological saline solution for at least 2 days, but macerated until the Abderhalden's microreaction to organ-specific ferments with a maximum number of different Organ substrates is positive, then either a) the maceration liquid obtained after filtration with a water-miscible organic solvent, preferably acetone, falls, the precipitate is separated and from the Precipitate the active ingredients with physiological NaCl solution or eluted b) the macerate is heated in the autoclave to 120 ° C for about 1 hour and then filtered, whereupon the eluate obtained according to a) or the filtrate obtained according to b) is gently dried. 2. The method according to claim 1, characterized in that that the solution produced by redissolving the powder of the short-term exposure to X-rays or ultraviolet rays. IO Publications considered: German Patent No. 820,787;
Swiss patent specification No. 276 050. 609 538/356 3.66 © Bundesdruckerei Berlin