DE1818054C2 - Process for the production of cytobiotic globulins - Google Patents

Description

(a) All of the immunoglobulins are brought into solution by adding to that by dialysis Sodium chloride and sodium hydroxide were added to the suspension obtained compared to pure water to obtain a pH of 7.3 ± 03 and an ionic strength of 0.06, whereupon activated charcoal is added to the solution and, after one hour, the supernatant liquid is separated by filtration,

(b) the separated liquid comes into contact with the insoluble residue of the homogenate of the organ or tissue that was used in the production of the antigenic extract, which served to immunize the blood donor animal was obtained, which previously was initially suspended in a 0.15 M citrate solution (pH 3.1 ± 0.1) and then with one on one pH 7.5 ± 0.3 buffered 0.15 M NaCl solution has been carefully washed was

(c) after separating the globulins which did not adhere to the homogenate and which remained in solution the adsorbed cytobiotic globulins are treated with a 0.15 M citrate solution (pH 3, l) eluted,

(d) the eluate is neutralized to a pH of 7.5 ± 0.3, the insoluble components become separated, and

(e) the solution is against a 0.15 M sodium chloride solution! dialyzed, sterile-filtered and freeze-dried

The invention relates to a method for obtaining cytobiotic globulins, in which an antigenic Extract is made from animal organs or tissues with which an animal of another species (blood donor animal) is immunized, and total immunoglobulins from the blood of the immunized animal be separated.

In particular, the invention relates to a method for obtaining cytobiotic globulins, wherein a Selection of the cytobiotic globulins is made possible by choosing from the totality of the fractions with immunological Properties the most stable fractions are separated, which have an optimal cytobiotic activity exhibit.

The invention also relates to the use of the cytobiotic globulins as a biological stimulant and Activator of cell metabolism.

In DE-PS 9 40 728 regenerative organ-specific sera are already described, which are produced by immunizing an animal of a different species with aniigcncn extracts from animal organs.

It was generally known that the actual immunological active ingredients in sera are globulins. A method for obtaining such stable, highly purified ^ -Globulin preparations is z. B. in DE-PS 9 17 209 described. In this process, the bulk of the /? - globulins are separated from the raw globulin fraction of human or animal serum or plasma, which has been freed from albumins, at a certain ionic strength and a certain pH value and at a protein concentration of 3 to 4% and then in a second Stage the main amount of -globulins by precipitation and then separates, with the addition of a certain amount of aluminum hydroxide gel, remaining small residual amounts of α- and γ-globulin as insoluble aluminum compounds.

The object of the invention is to create a method for obtaining cytobiotic globulins of high purity and high activity, in which an antigenic extract from animal organs or tissues with which an animal of another species is immunized and from the blood of the immunized All immunoglobulins are separated from the animal.

This object is achieved according to the invention by providing a method of the above specified type, which is characterized in that the cytobiotic globulins from the whole of immunoglobulins isolated according to the following stages:

2. Use of the cytobiotic globulins obtained according to claim 1 as a biological stimulant and activator of cell metabolism.

(a) All of the immunoglobulins are brought into solution by opposed to that by dialysis Sodium chloride and sodium hydroxide are added to the suspension obtained in pure water, to obtain a pH of 7.3 ± 0.3 and an ionic strength of 0.06, whereupon the solution Activated charcoal is added and, after one hour, the supernatant liquid is separated off by filtration will,

(b) the separated liquid comes into contact with the insoluble residue of the homogenate of that Organ or tissue that is involved in the production of the antigenic extract that is used for the Immunize the blood donor animal, which was previously received in a 0.15 M citrate solution (pH 3.1 ± 0.1) suspended and then buffered with a to a pH of 7.5 ± 0.3 0.15 M NaCl solution had been carefully washed,

(c) after separating those that are not adhering to the homogenate and globulins remaining in solution become the adsorbed cytobiotic globulins eluted with a 0.15 M citrate solution (pH 3.1),

(d) the eluate is neutralized to a pH value of 7.5 ± 0.3, the insoluble components are separated off, and

(e) the solution is dialyzed against a 0.15 M sodium chloride solution, sterile-filtered and freeze-dried.

The cytobiotic globulins have the following

Features on:

(a) they have no aggressive or toxic activity towards the human organism;

(b) they have a specific tropism for the homologous tissues of animal tissues that make up they have been separated or isolated;

(c) they are able to exert a regenerative effect on damaged tissues leading to restoration normal biological cycle.

1. Separation of the immune globulins

washed with a concentration of 1.56M.

After the precipitate has been absorbed in the form of a paste in purified or distilled water, it becomes subjected to dialysis against purified water until the complete disappearance of the ammonium salt. Dialysis must be carried out in such a way that a correct concentration of protein is available for it to be carried out the second phase of the procedure, d. H. the selection the globulins, is maintained.

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The method according to the invention is explained in more detail below

The organ selected for the production of specific cytobiotic globulins, which for example can be the kidney for globulins with kidney action or the heart for globulins with heart action used immediately after receipt from the slaughterhouse or, if this is not possible, after freezing, which is immediately after removal of the organ from the animal.

After a rapid chopping, the organ is subjected to an initial wash with 0.9% sodium chloride solution to remove most of the remaining blood. Thereafter, the minced organ is crushed in a mixer or similar device and suspended in the same saline solution in an amount equal to ten times its weight. After a one-hour stirring and Inberührunghalten with the salt solution, the insoluble part is separated and at a temperature below - frozen at -20 ⁰ C, in order to cause rupture of the cells.

After thawing, this insoluble part will again be ten times its weight Suspend amount of saline solution, secrete and wash again until washing liquid is dip no longer carries a soluble element.

An antigenic extract is obtained from the animal organ or animal tissue obtained in the manner described above produced, with which a blood donor (e.g. a horse) is immunized.

However, the method can also be applied to human organs or tissues.

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The blood of the immunized animal is collected on an anticoagulant, the final Concentration of the anticoagulant is 0.02 M.

After the blood cells have been separated, the plasma obtained can be processed according to one of the following two methods known working methods are dealt with:

(a) Operation using
Ammonium sulfate

A saturated aqueous solution of ammonium sulfate is added to the plasma so that a final concentration of 1.025 M of the ammonium salt. The resulting precipitate is removed by centrifugation severed.

The ammonium sulfate concentration is then increased by adding a suitable amount of saturated aqueous Solution brought to 1.56 M. The precipitate is collected and washed with an ammonium sulfate solution

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eo

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(b) Operation using a
Metal salt

The plasma is first diluted in three volumes of an aqueous sodium chloride solution, using the salt concentration is a function of the species from which the treated blood is derived

A zinc salt is then added to give a concentration of 10 millimoles. The one at room temperature The resulting precipitate is separated off by centrifugation. The liquid is filtered and the same zinc salt is added to the filtrate to increase the concentration to 18.6 millimoles, as well as one basic / amino acid added to a concentration of 0.43 millimoles.

After centrifugation, the precipitate is washed in purified water (one volume to three Volumes of starting plasma). After centrifuging again and pouring away the washing water, the precipitate was brought to a paste-like consistency with purified water and then for 24 hours dialyzed against purified water

2. Selection of the cytobiotic globulins

(a) The total immune globulins obtained according to procedure 1. (a) or 1. (b) above are brought into solution by purifying the suspension resulting from the dialysis Water, sodium chloride and sodium hydroxide are added so that the pH is close to the neutrality point and an ionic strength is obtained which is suitable for eliminating various globulins is.

The optimal conditions are:

Concentration of proteinaceous

Ingredients 5%

pH 7.3 ± 0.3

Ionic strength 0.06

After mixing and dissolving the soluble proteins under the conditions given above, 1 to 2% activated carbon added. The mixture is left to stand for an hour, and after decanting or centrifugation, the supernatant liquid is filtered with a clarifying filter.

In step (b) the globulins which have an optimal cytobiotic activity are separated off in that the solution of the globulins in contact with the insoluble residue of the homogenate des Organ or tissue used in the manufacture of the antigen used to immunize of the blood donor animal, e.g. B. Horse, has served. Then the globulins separated in this way subjected to the further treatment steps (c) to (e) according to claim 1.

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The invention is explained below with reference to Example 1. Effect on embryonic organs

len explained in more detail in a culture of organs

Example 1 The embryonic organ in organ culture is a selected

5th material to determine the biological activity and degree

(a) Separation of the immune globulins to make the cytotoxicity of a product recognizable,

from the totality d-ίΓ globulins The sensitivity of the cultural tissue is extraordinary

very high and enables very fine determinations.

61 blood of an immunized horse are mixed with the culture medium were eight drops of a 1% strength 100 ml of a 33% trisodium citrate solution together with a solution of the examined globulin per ml of the medium quantity added

A volume of a saturated ammonium sulfate solution in the case of the embryonic shinbone (tibia) was made

Three volumes of the plasma are added to the solution. After the daily growth recorded photographically, Separation of the precipitate, 220 ml become saturated and the comparison with a control culture showed a The ammonium sulfate solution per liter of the supernatant is accelerated and more intensive growth in the presence Liquid added. That after the second precipitation of the globulin as well as a considerable decrease collected precipitate is means of a mixture of deformities. These deformities contributed from 38 parts of saturated ammonium sulfate solution and 62 the control samples 70% and in the case of the speci parts washed with purified water. He will then fish for globulin-treated shin samples only resuspended in purified water and a 20 20%.

-sDialysis under the conditions mentioned above One subjected to the bodies of these shin samples, guided autoradiography showed a much more intense inclusion of radioactive sulfur S35 (the one from (b) Selection of the globulins of the marked methio added to the nutrient medium

25 nin) than the control samples.
The dialysate is reduced to a protein concentration of

50/0 brought 35 g of sodium per liter of suspension 2. The exerted on the tissue

umchloride added. The pH is set to 7.5 ± 03 renewing effect

brought by means of the necessary and sufficient Amount of normal sodium hydroxide solution, 30 The specific globulins called the cure of which has previously been determined on an aliquot, experimental wounds in a markedly shorter one

The working process is then shown in the above-mentioned time as for the control samples,
This phenomenon is due to bone damage

made visible to a rat. The healing rate was on average twice as high as Example 2 for the control samples.

(a) Separation of the immune globulins (a) Effect on the kidney
from the entirety of the globulins

40 The generation of kidney damage of the amylose species

One volume of equine blood plasma is given three volumes by injection of sodium caseinate through the

A 0.9% sodium chloride solution was added at the same time as the specific globulin was administered

Then 30 ml per liter of the dilution solution is slowed down or prevented. The protection of the kidney was

ner 9.6% aqueous zinc sulfate solution was added. such that the treated animals survived while

After the precipitate has been separated off, 45 control animals per liter will die.
Filtrate 21 ml of 9.6% aqueous zinc sulfate solution and

5 ml of 0.65% aqueous glycine solution were added. (b) Effects on the liver

(b) Selection of the globulins _c ^D _f ^ie tissue renewal moved to an operative

50 Removal of a large part of the liver much faster after the administration of liver globulins

The procedure is as in the previous example 1,
went.

The invention further relates to the use of the (c) effect on the skin
cytobiotic globulins in a medicine in the form of 55

a pharmaceutical composition, which in particular also in this case was tissue renewal

special as a biological stimulant and activator of the accelerates when the experimental wound with a

Cell metabolism is suitable is solution of the specific skin globulin treated

The de; compared to the control samples, the

The cytobiotic globulins obtained are doubled in the form of 60 wound healing

A very light powder, the one a little gray on the basis of various tests carried out

white color and in water and in physio- could also be determined that the cytobioti-

logical serum is soluble. These cytobiotic GIo- globulins see the following properties in animals

buline were subjected to a pharmacological test:
that made their biological activity recognizable 65

which leads to a metabolic stimulation of the protection of the mouse against acute poisoning by ammo

Tissue leads. nium chloride;

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Accelerated metabolism in the rat exposed to an unbalanced and malnourished diet;
Doping effect in malnourished animals subjected to physical activity test when normal diet began;

Protido-anabolic effect, in which any androgenic effect was absent;

Delay the onset of muscle fatigue; Restrained effect on arterial pressure of the Rabbit;

Accelerating the healing of experimental wounds from burns in rabbits.

The toxicological investigation of the cytobiotic globulins has shown:

that they do not cause acute poisoning in mice and guinea pigs;

that they do not cause chronic poisoning in the rabbit and guinea pig;
that they do not contain antibodies which can be found by the diffusion method in gelose against various human, amniotic and porcine cells;

that they do not contain antibodies which, by means of fixation of the complement, against ground human, amniotic Cells and grated pig kidneys are detectable;

that they do not cause teratogenic activity in the mouse, rat and rabbit.

For use in human therapy, the pharmaceuticals mentioned below can be used as examples Compositions are used:

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Solution for parenteral injection

Lyophilisate of specific

Globulins (kidney, liver, skin) 10 mg

physiological serum

2 ml per ampoule

Solution for local applications

Lyophilisate of specific

Multiple tissue globulins 0.60 g
Solvent (distilled water

or physiological serum) q.s.p. 100 ml

It can also be other forms, such as. B. suppositories can be used.

The cytobiotic globulins are used in human therapy for disorders in nutrition, Asthenia and emaciation symptoms have been investigated.

The drug was given in the form of an injectable solution. Intramuscular administration was also found to be free from drawbacks.

The assumed rhythm of administration consisted of two to three injections per week. The duration of the Treatment was three weeks.

The results obtained were generally good.

In geriatrics, the results were even more pronounced. The rate of improvement was in the order of magnitude of 80% of the cases.

The repetition of the injections did not result in any impairment of sensitivity.

Claims (1)

Patent claims: 1. Process for the production of cytobiotic globulins, in which an antigenic extract is made from animal organs or tissues with which an animal of another species (blood donor animal) is immunized, and the entire immunoglobulins are separated from the blood of the immunized animal are, characterized in that the cytobiotic globulins from the whole of immunoglobulins isolated according to the following stages: