GB2346884A - Methods for obtaining protein mixtures and control factors from wound granulation epithelialization or determined blastocyst tissue - Google Patents

Abstract

The invention relates to methods of obtaining control factors for the start and end phases of wound granulation and epithelialization from animal wound tissue or human or animal blood, whereby the granulation tissue and/or epithelialization tissue is removed from the wound of an animal or from the blood of a human being or an animal undergoing a healing process or from the blastocyst system of a human being or an animal; the lipids are separated from the above mentioned tissue or blood or serum thereof; solids and high molecular protein are removed from the first product obtained and the desired control factors are isolated from the second product obtained. The control factors are suitable for the production of medicaments.

Description

2346884

Descriptio

The invention concerns different methods, especially a) for the production of low-molecular-weight protein mixtures from wound tissues of animals, b) for the production of control factors for start phases and termination phases of wound granulation and epithelization from wound tissues from animals or from blood of human beings or animals (the said control factors are substances which control the granulation and epithelization processes in wound tissues; that is, they activate or terminate the individual phases of these processes), f or the production of low-molecular weight protein mixtures from blastocyst tissue in the gastrulation stage and of control factors for the termination of proliferation' of blastocyst tissue in the stage of gastrulation, d) for the production of low-molecular-weight protein mixtures from blood or blood serum from human beings or animals which has been stimulated by wound healing processes, and e) for the production of lipid extracts from wound healing or blastocyst tissues.

These methods represent a unified method complex because they all remove animal granulation or epithelization tissue, human or animal blastocyst tissue, or blood or blood serum as their starting point and serve to provide substances for the treatment of certain diseases.

From DE-PS 862942, a method for the production of a tissue extract and a serum stimulated by this extract is known. With this method, simple incised wounds are made in mammals that are closedwith sutures.

After a wound healing process of 5 to 8 days, the granulation tissue formed in the.wound is removed. This is then freed from general protein and lipids. The dry powder obtained is processed into an aqueous extraction. This extract can be, injected into animals, and blood ftom which the said stimulated serum can be produced is withdrawn from them 6 to 8 days later.

The known method was further developed in accordance with DE-PS 885127 wherein wound granulation tissue removed from animals was cultured in the presence of a nutrient medium, the culture carefully injured after visible growth, and a tissue extract was produced from the culture after a certain additional growth time.

The wound healing process is a convincing natural classic example of how tissue damage is first repaired by a juvenile undifferentiated tissue, and how this undifferentiated tissue then comes under the control of central circulation and central metabolism, is differentiated again, and is integrated into the whole organism.

2 At first, juvenile cells sprout out ftom the still intact tissue in the periphery of the wound, and these are then stimulated by proliferation promoting factors to enormous cell division (proliferation) for the purpose of bridging-over and filling in the wound. As soon as this objective has been achieved, the body produces factors which abruptly stop cell division as well as cause the transformation of the juvenile wound tissue into differentiated tissue structures and their integration into the whole organism.

Within the context of the invention, these factors are termed "control factors". They are also contained in the previously known extract from wound granulation tissue.

When in the embryonic, undifferentiated cellular tissue aggregate of the morula during the phase in which the blastocyst develops and, during gastrulation when the transition into the final anatomically and functionally differentiated germ layers occurs, a process which is biologically identical to the wound healing processes takes place, At this time, therefore, the regulatory mechanisms which inhibit the further proliferation of undifferentiated embryonic tissue and stimulate differentiation must also become effective.

Therefore, the blastocyst in its determination stage can also be used as starting material for the extraction of effective substances analogous to those of the wound tissue (see example In addition, it is know that healing wounds themselves are practically irmnune even against severe pathogenic infections (e.g. anthrax).

Studies showed that the parenteral adn-dnistration of the known extract from wound granulation tissue causes marked stimulation of the reticuloendothelial system and activation of the immunologic system, erythropoietic and leukopoletic systems, and cellular phagocytosis.

Therefore, as clinical studies demonstrate, the known extract from wound granulation -tissue and the known serum that has been stimulated by wound healing processes can be successfully used for the treatment of infectious diseases (caused by bacteria and/or viruses and/or fungi). Thereby, a short-term or immediate arresting of the disease among other things is achieved, that is, the often dangerous symptoms Re poor general condition, fever, chills, vomiting, coughing, dyspnea, circulatory insufficiency, and generalization of the infection disappear in a considerably shorter time than with the application of other forms of treatment.

Other clinical studies have demonstrated that the known extract from wound granulation tissue and the known serum that has been stimulated by wound healing processes can also be successfully used for the treatment of malignant tumors, slowhealing wounds, ulcer diseases, cirrhosis of the liver, atrophic kidney, degenerative diseases, and lack of immunity.

I I 3 Moreover, the question of a vaccination prophylaxis for patients at risk due to these diseases or for those predisposed to these diseases must be considered.

In addition, it can be assumed that for endemic and/or epidemic infectious diseases, the known extract &om wound healing tissue offers a generally effective vaccination method.

In this connection, reference is made, for example, to the following literature:

J. Hammerschmid, "MUnchener Medizinische Wochenschrift", Vol. 95 (1953), No. 39 A. Kressner, "Wrichener Medizinische Wochenschrift", Vol. 96 (1954), No. 30 V. di Carlo, "La Riforma Medica", 1954, No. 45 J. Hammerschmid, "Mijnchener Medizinische Wochenschrift"., Vol. 97 (1955), No. 39 J. Hammerschrnid, "Medizinische Klinik", Vol. 50 (1955), No. 27 S. Nitschkoff, "Munchener Medizinische Wochenschrift", Vol. 97 (1955), No. 46 A. Kirchdorfer, "Rassegna Internationale di Clinica e Terapia", Vol.)= (195 5), No. U Therefore, there is a great need to provide the protein mixtures and control factors 4contained in wound tissues and/or blastocyst tissues in sufficient quantities to be able to extensively use them to treat the aforesaid diseases and defects.

The invention describes methods by which 1. the said protein mixtures can be obtained in larger quantities, in higher - -;concentrations, and with lower costs than previously (the therapeutic use of these more highly concentrated protein mixtures should ultimately achieve a crreater curative effect) and, 2. from the said protein mixtures,- those control factors can be extracted in isolated form which regulate the prol iferation, integration, and epithelization processes in wound tissues or the differentiation of the blastocyst tissue in the gastrulation stage. --- I Ultimatel. y, the isolated control factors have a considerably greater curative effect on cancer than the protein mixtures.

4 In accordance with the invention, these aims are achieved by methods which, compared to the methods described in DE-PS 862942 and DE-PS 885127, represent new methods and are described below as well as in the patent claims.

The decisive factor of the invention is the explanation of previously unknown findings-, namely, in which tissues and at what times therapeutically efficacious substances are formed, and how they are usable as medicinal agents.

The steps of the method carried out in accordance with the invention are explained below:

In accordance with a first form of the invention, new surgical methods qompared to the known methods are applied for production of the starting material (ground substance):

(aa) For production of wound granulation tissue, wounds were made in marnmals using special surgical methods (see example I and Fig. 1).

(ab) Previously, only wound granulation tissue was used as the starting material.

However, since carcinomas, for examp16, develop from ectodermal cells, it can be assumed that the active substances which are formed in epithelization tissue analogously to those in wound granulation tissue have a greater, affinity-induced curative effect with diseases of tissue systems of ectodermal origin (skin, mucosa). Therefore, methods analogous to the methods described in (aa) were also developed for the production of epithelization tissue (see example 2 and Fig. 2 and 3).

I I (ac) As a starting material, blastocyst tissue in the determination condition is used for the extraction of effective substances analogous to those of the wound tissue.

The invention is explained in an exemplary fashion in the accompanying figures.

Fig. I shows a flow diagram which schematically depicts the processing tracks G, S, and L in accordance with the different forms of the invention.

Fig. 2 shows a schematic sectional view of the implantation of a fish trap-like object in an artificial wound.

Fig 3. shows a schematic sectional view similar to Fig. I but for the. case of the implantation of a netlike object in an artificial wound.

From the starting material, the following therapeutically efficacious end products are produced by different procedural tracks:

Procedural track G = low-molecular-weight total proteins in a concentrated form of administration Procedural track S = control factors for proliferation termination and proliferation stimulation Procedural track L = lipid fraction for the production of medicaments for skin diseases -,The procedural tracks ja, S, and L are explained in Fig. I In Fig. 1, the new procedural steps compared to DE-PS 862942 and DE-PS 885127 are darkly marked.

With the aforementioned surgical procedures, the starting material is obtained either as C) ____ 1.), wound granulation tissue, described in Fig. I as "procedural step aa", as epithelization tissue, described in Fig. I as "procedural step ab", or as blastocyst tissuej described in Fig. I as "procedural step ac".

In Fig. 1, the sequence of events of procedural track S (production of the control factors) is presented from top to bottom in the left half, and the sequence of events of procedural track G (production of the lowmolecular-weight total proteins) is presented from top to bottom in the right half In addition, procedural track L (production of a specific lipid fraction) is shown in Fig. 1, and depicted there as a step branching off from right to left from procedural step bg of procedural track G.

Reference is explicitly made to the terms used in Fig. I and the sequence of procedural steps that are clearly shown there.

As previously mentioned, the aforesaid diseases can be successfully treated with the known protein mixtures. The treatment of cancer, however, represents a special case which will be discussed later.

With the known methods, only the protein mixtures, and these only in relatively low concentrations, could be extracted previously. Moreover, the starting material could only be previously produced in small quantities. This led to extremely high production costs.

Procedural track G The product of this procedural track is a low-molecular-weight protein n- dxture.

For the production of the low-molecular-weight protein mixture in larger quantities, in higher concentrations, and at lower costs, procedural track 0 is applied in accordance with a special form of the invention, the end product is a pure low-molecular-weight total protein mixture.

The starting material is produced by means of the aforesaid new, economical methods aa, ab, and ac, and it is processed further as follows (see also Fig. 1):

(bg) Separation of the lipids from the granulation tissue, epithelization tissue, blastocyst tissue, blood, or serum to produce a fat-free albun-inprotein mixture product.

(cg) Separation of solid particles, albumins, and high-molecular-weight protein fractions from the albumin-protein mixture product to produce an aqueous and low-molecular-weight protein mixture product, which can be employed for the -1 treatment of the diseases mentioned at the beginning.

(dg) Concentration of the proteins dissolved in an aqueous solution by evaporation in a vacuum, or production of a IyopHized dry preparation by freeze-drying.

The procedural steps (bg) with (dg) of procedural track G are described in example 6.

Treatment of malignant tumors (cancers) with substances from wound healing tissues According to the current state of knowledge, malignant tumors develop specific proteins which can trigger off an enormous proliferation phase and make independent metabolism, infiltrative growth, and formation of metastases possible.

7 From recent publications, extracts from those will be discussed below which point to a certain relevance with regard to possible applications of the control factors which can be extracted from wound tissues.

American Association for Cancer Research San Diego, April 97, J. W. Shay, Southwestern Medical Center, University of Dallas, and D. S. Coffey, John Hopkins University, Houston Telomerase is a biochemical active substance which stimulates the cell division of ova (probably also spermatozoa) and thereby guarantees the continued existence ("immortality") of the "body" (i.e. whole organism) of the individual. Telomerase is also formed in the embryonic cell aggregate. It disappears when the undifferentiated embryonic cell aggregate develops into the differentiated -fetus in the womb (see here also example 3).

Therefore, there must be natural control mechanisms which inactivate the telomerase and thereby terminate the further "endless" proliferation of embryonic tissue. It has been proven that in biologically determined organisms, only cancer cefls can routinely produce telomerase. In this way, cancer cells can continuously and indefinitely divide without being subject to the normal aging p I rocess and cell death. Throughout the world, the substance is being sought which can inactivate telomerase and, thereby, stop the continuous proliferation of cancer ceUs (i.e. "starve the cancer").

In- this connection, reference is made to the following literature:

Journal of Clnical Investigation, April 1997 C. Malbon, Stoney Brook State University, New York, and L. Norton, Sloan-Kettering Institute, New York Malbon et al. have identified an active substance ("chemical switch") which stimulates breast cancer ceHs to proliferate extremely rapidly. They believe, therefore, that this discovery opens up very promising possibilities for combating this disease. Tfie substance was called "" kinase". It normally causes cell divisions with information that is kept remarkably brief Studies showed that cells taken from breast cancer tissue contain 5 to 20 times more MAP kinase than cells from normal tissue. With such an excessive amount of MAP kinase in cancer cells, it seems logical that this is the reason this tissue is stimulated to continuous cell division. In the view of the scientists, this in turn leads, depending on the concentration of MAP kinase, to the different forms of cancer (degree of malignancy; intensity of growth, infiltration, and formation of metastases).

In laboratory experiments, it was demonstrated that after an inactivation of the MAP kinase, the breast cancer ceUs stop dividing and die. It was found that small particles of deoxyribonucleic acid (DNA), which were implemented in the cancer tissue culture, inactivated those chemical messenger substances that trigger off the production of MAP kinase. However, it is not yet known whether and how a corresponding treatment strategy for human beings can be derived ftom these findings, particularly since corresponding biological inactivators are as yet unknown.

Larry Norton from the Sloan-Kettering Institute in New York reported that diverse laboratory investigations have indicated for quite some time that MAP kinase plays an important role in the growth of cancers. The recent studies by Malbon et al. would have strengthened these suspicions, above all based on experiments with human breast caAcer cells. Norton commented: "These experiments provide very important evidence which indicates that our views are on the right track and that, in addition to other substances, MAP, kinase plays an important role in regulating the ceH division in cancer tissues. Somewhere, the optimal way will be found to biologically inactivate the substances that are continuously stimulating cell division in cancer tissues. It wiU then be possible to specifically influence this cell division, so that one will also be able to biologically kill cancer cells in this way."

In this connection, reference is made to the following literature Nature Medicine, Vol. 1, No. 12, December 1995 "Unrestricted cell cycling and cancer" M. Strauss et al., Max Delbrack Center for Molecular Medicine, Max Planck Gesellschaft, Humboldt University, Berlin Knowledge concerning the genesis and development of cancer has progressed so far today that a number of typical changes are known which correiate with the responsiveness of the tumor to the different treatments like chemotherapy, hormonotherapy, and/or radiotherapy. An important marker in this connection is a -protein classified as p53.

It is present in approx. 50% of malignant tumors. As soon as p53 can no longer be detected in malignant tumors, the responsiveness of malignant tumors to chemotherapy, hormonotherapy, and radiotherapy rapidly decreases, and the course of tumor growth then becomes extremely rapid and fatal.

I All multicellular organisms (including human beings) have developed a very closely interlocked control mechanism which regulates cell proliferation. This mechanism represents the precondition for the survival of these organisms, and it functions as a kind of switch between the alternative routes in the direction of cell division and temporal arresting of the division cycle which ultimately results in either cell differentiation or cell death. In view of the important role played with regard to the control of cell proliferation, this switch mechanism was called the "restriction point (R point) in mammalian cells". If this "R point" is damaged or destroyed during the primary phases of gene replication of cells, this leads to unregulated cell growth with disastrous consequences for the entire organism.

With the protein mixtures which are produced in accordance with methods described in DE-PS 862942 and DE-PS 885127, it could be demonstrated in clinical studies that in addition to the treatment of the aforementioned diseases, exceptional therapeutic successes were achieved with these protein mixtures particularly with the treatment of canceroses. Depending on the stage of the disease, this treatment led to a regression of the tumor up to the point of its disappearance, the disappearance of the paratumorous infiltrates, an arresting and/or disappearance of the metastases, an extensive stabilization or normalization of the general condition, and therefore the achieving of a normal quality of life and a significant prolongation of life expectancy (see above).

In this connection, as a consequence of the procedural techniques employed at that time, particularly with regard to the production of the starting material, these protein mixtures did not contain anything like the high concentrations of efficacious substances that are possible with the method described in the invention. Moreover, the,extraction and prodution methods were so elaborate and costly, that under the conditions prevaiIing at that time, meeting the demand on the large-scale basis was impossible. But above a1l, there was as yet no techniques which could be applied to produce those substances (i.e. control factors) in isolated form which could arrest the growth of malignant tumors and their metastases.

I In the biologically determined organism, a tissue aggregate develops if necessary whick like an embryonic tissue aggregate (which develops from the fertilized ovum containing a diploid set of chromosomes), also shows all of the characteristics of an undifferentiated proliferative juvenile tissue; namely, the wound tissue. In this connection, nature provides a classic example of how biological substances are produced during the wound healing process which first abruptly stop the highly stimulated cell divisions of the juveni.le tissue aggregate and then integrate its metabolism and blood supply into that of the entire organism.

By means of a therapeutic application of control factors regulating the wound healing processes, a therapeutically efficacious method would be available with which cancer can be specifically, selectively, and above all biologically treated.

The observations of Hammerschniid, Nitschkoff, and Kressner among others (see references) prove that precanceroses (significant precursors of the transition into cancer tissue) completely disappear under treatment with the known low-molecularweight protein mixture from wound tissues.

It can therefore be assumed that with the application of the control factors, a successful preventive method for avoidance of the development of cancer with diagnosed or suspected precanceroses is available. In addition, the question of a vaccination prophylaxis for patients at risk of or predisposed to cancer must be taken into consideration.

To economically produce the isolated control factors in sufficient quantities, it is best to use the genetic engineering method described in procedural track S (see Fig. 1):

(bs) Genetic engineering processing of the control factors and cloning of cells with the genetic characteristics of these factors (see example 7); (cs) Culturing of the cloned cells in vitro (see example 8); (ds) Processing of the tissue culture in accordance with the "total proteins procedural track" (see example 9).

Procedural track L The end products of this procedural track are pure lipid fractions from wound tissues.

Since the effect of ointments whose medicament-free base was enriched with lipid fractions separated with solvents from granulation and/or epithelization tissues was tested in clinical studies, which showed surprising successes for the treatment of skin diseases, among others with degenerative, chronic, infected, and superinfected skin -diseases and slow-healing lesions (ulcus cruris etc.), the lipid fractions, which were separated from granulation and/or epithelization tissue in accordance-with a special form of the invention, were freed from the solvents to produce them as therapeutically usable ground substance for dermatologic treatment. This form of procedural track L is explained in example 10.

The aforesaid procedural steps are explained based on the following examples:

Example I Implantation of a fish trap-like object in a wound Example 2: Net implantation over an artificial skin lesion Example 3: Use of blastocyst tissue as starting material Example 4: Determination of the best time for removing the tissue Example 5 i Removal of granulation, epithelization, or blastocyst tissue I I Example 6: Processing of the starting material into low-molecular-weight total proteins Example 7: Genetic engineering method for the production of genetic characteristics of the control factors and cell cloning Example 8: Culturing of the cloned cells Example 9: Processing of the cloned tissue culture analogous to the procedural steps bg with dg Example 10: Production of lipid extracts from wound healing tissues or blastocyst tissues Example I

Implantation of a fish trap-likeoiect in a wound Procedural step aa, Fig. I and 2 With mammals, predominately the pig (due to chromosomes which are, apart from the primates, most closely related to human tissues), a wound as large as possible in terms of length and depth is made under sterile conditions using a surgical procedure under local anesthesia. Most suitable in this regard is the neck region because no.severe stress reactions take place there. In the wound, a distortion-stable, fish traplike object made ftom a neutral material (e.g. plastic, titanium, silicone etc.) is implanted under retraction (see Fig. 2), and the wound is closed over it layer by layer with sutures.

With the previously known procedure for creating the wound (simple separation via incision with subsequent joining together of the edges of the wound with sutures), only a very narrow space was necessary for filling the wound, and so the yield of granulation tissue was relatively small. The advantage of the new method is that due to the implant, a hollow space is created between the wound margins that must be filled by proliferating granulation tissue. In this way, a much greater amount of granulation tissue is formed, which is a great economic advantage for the production of the -star-dng material.

Experiments:

In 4 laboratory rats, a 2 cm. long, 12 to 14 cm deep wound was made in the region of the neck-thoracic girdle. A fish trap-like object consisting of two fine-meshed silicone lamellae (12 mrn x 16 mm) was inserted in this wound, which formed an average cavity of 8 mm. The wound was closed with layered sutures, and the wound tissue was removed after 6 days.

In all animals, a massive amount of granulation tissue had proliferated into the implant.

12 Example 2

Net implantation over an artificial skin lesio Procedural step ab, Fig. I and 2 Analogous to the aforementioned procedure (aa), the total epithelium is resected over as large an area as possible to produce epithelization tissue, possible underlying fasciae are removed, and then the muscle tissue is superficially damaged. Directly over the muscle wound bed, a tightly-meshed net made of silicone etc. is inserted, attached to the skin edges with sutures, and the lesion is covered over with special gauzes under sterile conditions. In this way, a many times greater amount of epithelization tissue develops compared to the previous method.

Example 3

Use of blastocyst- tissue as starting material Procedural step ac, Fig. I Scientific basis:

After implantation, the fertilized ovum first forms a solid, globular, undifferentiated cell aggregate via divisions, the so-called morula. Around the 4th day after fertilization, the blastocyst develops from it, which forms a blastula (blastodermic cavity) with outer (thrpphoblast) and inner (embryoblast) cell layers.

As a result of a cleavage, the invagination of the blastula and, thereby, the development of the anatomically and functionally differentiated and finally determined blastodermic layers of the ectoderm, endodem-4 and mesoderm takes place. This process is called "gastrulation".

With the beginning of gastrulation, the exact time of the differentiation of tle embryonic tissue is therefore determined. Thus at this time, the regulatory mechanisms which inhibit the proliferation (cell divisions) of undifferentiated embryonic tissue and cause it to start differentiating must become effective. These regulatory mechanisms have therapeutic effects that are identical to the effects of the substances from granulation and epithelization tissues.

To obtain blastocyst tissue, the blastocyst is surgically removed fibm an animal on the 4th day after fertilization, or after slaughtering.

I I 13 To determine the optimal time (in terms of hours) for removal as precisely as possible, it is recommended that it be determined, by means of prior experiments using histological examination, at what time the invagination of blastula (via cleavage) and, therefore, the development of the anatomically and functionally differentiated and finally determined blastodermic layers, ectoderm., endoderm, and mesoderm, take place. In different animal species, the timing of this process can differ by a matter of hours.

In addition, it is possible with abortions that are carried out around the 4th day after fertilization to obtain human blastocyst tissue writh the informed consent of the patient. Here too, it must be clarified byhistological examination whether the tissue can be used.

Example 4

Determination of the best time for removing the tissue The control factor PA (proliferation termination), which is to be used above all for ending proliferation of cancer tissues, can be extracted from all starting materials. To extract the control factor PA from epithelization tissue, this must be removed at the end of epithelization; that is, at the conclusion bf wound covering.

The control factor PF (proliferation stimulating), which promotes the proliferation of cancer tissues and normal tissues, and serves as an initial treatment for cancer and for the treatment of retarded healing, can only be extracted from juvenile and proliferating,tissue.

Both factors are produced in the aforesaid starting materials and, furthermore, are produced by different animal species at different times, and so establishing the specific time of production is important for optimal extraction. For doing this, three methods are available:

a) Visual examinations These examinations are made considerably easier by applying a fenestrated bandage.

The beginning of wound granulation, which as a rule is on the 3)rd to 5th postoperative day, is easily ascertainable macroscopically from the appearance of the typical granulation tissue.- - - Like,,Nrise, the beginning of epithelization is also easily ascertainable macroscopically from the marginal proliferation of skin into the wound bed, and end of epithelization by the complete wound closure with skin. These processes take place between the 8th and 40th postoperative day.

14 b) Histological tissue examinations The distinctions between proliferating juvenile or determined tissues are easy for histologists to make. For this purpose, a biopsy needle, for example, is used to take investigational material from the wound granulation, epithelization, or blastocyst tissue at the presumed points in time.

Example 5

Removal of wound tissue and blastocyst tissue The. wound tissue obtained in accordance with procedural steps (aa) or (ab), or the blastocyst tissue obtained in accordance with (ac), can either be surgically removed from living animals under sterile conditions and local anesthesia, or from dead animals after slaughtering.

Of course, the most economical method differs from country to country. Animal protection regulations must also be considered.

In any case, the tissue removed must be immediately covered with a layer of toluene.

With the surgical removaL the animal can confinue to be kept in the normal way; and then with later slaughtering, the common utilization obligation in accordance with sanitary measures (slaughterhouse sale as lower-grade meat, loss of value of meat) involved in the removal of tissues after slaughtering does not apply. However, the technical measures required are more expensive than when removing tissues after -. 4slaughtering. The effeqt of time/effort vs. benefit must be considered.

If the wound tissue is removed from a slaughtered animal, then there is no need for repeated aseptic operations because the tissue is completely sterilized by the immediate immersion in toluene.

The-entire wound tissue is first removed by making an excision far into the healthy tissue. Then the pure granulation tissue or epithelization tissue is detached by severing the connection to the surrounding tissue (muscles, sldn) at the visible edges of the fish trap-like implant or over the net. Then the granulation tissue formed in the hollow implant, or the epithelization tissue formed over the net, is removed and processed fiirther as described in example 6.

This offers another advantage compared to the known method: whereas most of the tissue to be processed consisted previously of connective tissue substances, almost exclusively pure granulation or epithelization tissue is obtained with the present method. This means that the starting material and therapeutically efficacious substances are available in a decisively higher degree of purity and, consequently, also with a higher degree of efficacy than was previously the case.

I I Example

Processing of the starting material into lQw-molecular-weight total proteins Total proteins procedural track, procedural steps bg=dg Step bg:

In this step of the method carried out in accordance with the invention, the granulation tissue, epithelization tissue, blastocyst tissue, or serum obtained from an animal or human being is freed from lipids. By lipids is meant both the true fats and the fatfike substances.

To do this, the tissue together with toluene is first ground up and homogenized in the sterilized processing apparatus (e.g. in a mincer). Then the toluene is drawn off and mixed with four times as much acetone by weight, and extraction is carried out in a sterilized agitator for 2 to 3 hours-at an environmental temperature of O'C. Then the acetone is drawn off.

The tissue residue is then mixed with the same quantity by volume of a mixture of 1: 1 acetone and diethyl ether, again extracted for 30 minutes in the agitator, and again aspirated. Then the same volume of diethyl ether is added, and extraction is again carried out for 30 minutes. The ether is then drawn off in a vacuum. After separation of the last diethyl ether solution, a first protein product is obtained in the form of a sterile, lipid-free dry powder.

All available lipid extraction substances are kept for extraction and further use of the lipids, and they are processed further in accordance with the lipid procedural track.

Step cg:

In this step of the method carried out in accordance %kith the invention, the lipid-free dry powder is mixed with a __ hundredfold quantity by weight of sterilized, distilled water and with a I '/:?-fold quantity by weight of sterilized Ringer's solution, covered with a layer of toluene, and subjected to sterile aqueous extraction in a cold storage room for 24 hours at a temperature of +2'C.

The toluene is then drawn off from the present mixture in aqueous solution, and the solid particles are then separated by_centrnging with at least 5000 rpm. In this way, the first lipid-free total protein product in aqueous solution is obtained; it is processed as follows:

To remove the high-molecular-weight, parenterally intolerable protein fractions, the protein full extract is processed in a tubular spiral dialyser in which the low-molecularweight fractions diffuse into the outer liquid medium.

The outer dialysate obtained in this way contains a mixture of lowmolecular-weight proteins with which one can achieve the aforementioned therapeutic effects.

This protein mixture also contains the aforesaid control factors which are especially suitable for the treatment of canceroses (see treatment of malignant tumors). if necessary, it can also be used for the direct extraction of isolated control factors.

Step dg The water of the outer dialysate is evaporated in a vacuum evaporator at a maximum temperature of 360C (because higher temperatures decisively diminish the efficacy of the proteins) until a 70- to 100-fold concentrated aqueous solution is obtained, or until a water-soluble dry powder is formed (1yophilization). Both products can be processed into parenterally administrable therapeutic preparations in'the usual way.

As an alternative to the above-described methods for obtaining the starting material, blood can also be withdrawn from human beings (with their informed consent) when they are going through a wound healirig process. For the further processing, the serum obtained from the blood in the normal way can be used and processed like the wound tissue obtained in the aforesaid manner.

Example 7

Genetic engineering method for the production of a control factor Jhe expert is familiar with the genetic engineering procedures referred to in the following examples. Therefore, their description will be limited to the essentials. In the event that companies are cominissioned to carry out such procedures, reference will only be made to this without explaining these procedures. If prefabricated tools are available, the use of these will on]y be explained to the extent required to understand how the method is carried out in accordance with the patent.

The control factors present in both the low-molecular-weight total proteins and in the prepared starting materials (steps aa, ab, and ac) can be, if required, relatively easily identified analytically. For this purpose, the CLONTECH Inc. company, for example, provides reliable tools. Various kits as well as the Northern Blot make it possible to carry out simple investigations of genes with differentiated significance even with small quantities.

The low-molecular-weight total proteins obtained in the "total proteins procedural step" contain those proteins that can be therapeutically used as control factors.

To identify the specific protein (control factor) which is being sought, comparative analyses with the protein mixtures produced from other tissues (e.g. with muscle tissue from the wound area) using the same procedure are carried out.

I I 17 One first chromatographically isolates those proteins that first appear in the comparison test in comparison to other tissues. One of the desired proteins (control factor) is isolated using the normal separation method (e.g. using preparative column chromatography, including BPLC and isoelectric focusing) and demonstrated in pure form.

It is then tested whether the isolated protein is identical to the control factor PA being sought. For this purpose, experimental animals, for example, are injected with fuhninantly growing cancerous ulcers, or in vitro cultures of profusely proliferating cancer tissue are sprayed with it. If the protein used is identical to the control factor PA, the proliferation of the cancer tissues will be stopped.

On the other hand, if it is to be tested whether the control factor PF is identical to the isolated protein, it is injected into animals with a freshly created wound that is left open and the wound covered with a fenestrated bandage. If the protein is identical to the PF control factor, the granulation (proliferation) in the wound 1AU be significantly stimulated.

From the proteins identified in this way, the amino acid sequences can be determined by a commissioned companyfmstitute (e.g. by BACBEM Inc.). The amino acid code serves as among other things to check whether during the further procedural steps, the original protein or its original secondary products are still in evidence.

The relevant protein is expressed from the cells of the starting material (wound tissue (aa) and (ab) or blastocyst tissue (ac)), and the corresponding mRNA is extracted 'frorn it.

The mRNA must contain the code of the amino acid sequences of the original protein (sequence test).

This mRNA can (e.g. via SMART technology) be transferred via a singlestranded cDNA with primers to the double-stranded DNA.

The bland-end DNA fragment is inserted with restriction enzymes into a bland-end cut vector and bound with ligase. After the transfection with, for example, E. coil, the plasrrlid created is cloned. The (round) plasnU created is tranduced into suitable mammalian cells with, for example, adenoviruses. The transduced cells are then cultured in vitro.

A special therapeutic method consists of using, for example, cancer cells of a patient obtained during operations or from biopsies as recipient cells. For this purpose, they are transduced with a plasmid which contains the genetic characteristics of the control factors. If these transduced cells are replanted in the patient, it is possible that a biological chain reaction could be triggered off w1-:lich leads to the termination of proliferation and, thereby, to the natural death of the cancer tissue.

18 Example 8

Culturing of the cloned substances, procedural step es The genetically engineered transduced cells are now cultured in vitro in tissue cultures, wherein the traced genetic characteristics of the control factor also multiply per cellular proliferation in the ratio of 1: 1.

As a nutrient mediun- one can use, for example, the manufactured product, DMEM solution. The solution is filled into culture flasks (50-500 ml), whose valve must remain open so that the C02 necessary for culture combustion w1iich is added to the incubator air can penetrate into the flask. Then the genetically manipulated cell material is introduced. For stimulation of the greatest possible mitotic activity, the control factor PF can be added if necessary. Incubation -is carried out in the normal way in a heated, thermostatically-controlled, sterilized incubator.

After the specific, optimal growth time, the culture is removed and used as starting material. From it, a low-molecular-weight protein which contains a control factor in concentrated form is produced in the normal way (analogous to the total proteins procedural step).

This product is processed into a therapeuticaUy applicable preparation using the wellknown methods.

Example 9

Processing of the cloned cell cultures In principle, the cell cultures cultured in accordance with example 8 can be processed according to the procedural steps of the "total proteins procedural track" (example 6). The removal of lipids can be ornitted.

-After the centrifugation and precipitation of protein substances, the protein mixture is dialyzed, and the outer dialysate is separated and then concentrated and lyophilized in the usual way.

Example I

Production of lipid extracts from wound healing tissue The extraction media (e.g. acetone, diethyl ether, toluene) which are used to separate the lipids from the wound healing tissues in procedural step (bg) are drawn off and/or evaporated using the pressure reduction method. If necessary, the residue is elutriated out with ethanol.

The lipid fractions extracted in this way are mixed into an ointment, cream, or lotion vehicle, or in other solution vehicles, and used as dermatologic medicaments.

I (9

Claims (19)

1. ?Itent Claims aun Xethod tor Producing control Fac-mrs 5cc the sart.

   phases and te ation ohases oE wound granulation and eioitheli=tion Erom wound tissues of animals or IL-lom the blood of'numan, be;mas or animals using the procedural stelps, (a) Removal, of ganulation and/or elpithelization tissue from the wound of an animal, or withdrawina blood E-orri a. human be'Uria or an animal which is croina thrcuzh a wound healing process, -(b) separaition of the dulds from the granulation tissue, epithelizxtion tissue, blood, or serum, to oroduce a. -trr, protvin produ cz, and (c) secLaration of sodds and --hr protein =aczons tom:he -O;s-, protem oroduc: to produce a second protein oroducz, wherein (d) the desire-d control 'Lactors are isolated E-om the second protein or'oducz.
2. 2 Method in accordance with claim 1, wherein in step (a) after -%.he withdrawing of blood-the serum is is'olated from this blood.
3. 3. Method for producing control factors for the start phases and termination phases of wound granulation and epithelization ffom a tissue of an animal or human being, Characterized by the procedural steps (a) Removal of a blastocyst tissue in the detem-uriation stage fr an being or an animal, om a hum (b) separation of the I;Plds from the blasto-cyst tissue to obtain a first protein product, (c) separation of solids and high-molecular-weight protein fraczions firom he first protein product to produce a second protein product, and (d) isolation of the desired control factors firom the second protein product.
4. 4. Niethod For producing control factors for the start phases and te 'nation phases of wound granulation and epithelization from wound tissues of animals or from the blood of human beings or animals using the procedural steps (a) removal of granulation and/or epithelization tissue from the wound of an animal, or the withdrawal of blood from a human being or an animal which is Just clOinZ throuah a wound healing process, % (b) separation of the lipids from the granulation tissue, epithelization tissue, blood, or serum to produce a first protein product and (c) separation of solids and high-molecular-weight protein fractions from the first product to produce a second protein product, characterized by (d) isolation of the desired control factors from the second protein product, (e) genetic engineering processing of the control factors by means of demonstration of the coded RNA-s and DNAs and the vectors for the purpose of cloning suitable recipient cells with one or more DNA ffagments, which code for the control factors present in the products obtained in step (d), (f) culturing of the cloned tissue in a nutrient medium and 7 (a) isolation of the desired control factors from the tissue culture of step (f).
5. 1 Method in accordance with claim 4, wherein in step (a) after the withdrawing of blood.-the serum is isolated from this blood.
6. 6. Method for producing control factors for the start phases and terrmination phases of wound granulation and epithelization from wound tissues of animals, or from a tissue of an ard"mal or a human being characterized by the procedural steps (a) removal of blastocyst tissue in the determination stage &orn a human bein-a or an animal, (0 separation of the lipids from the blastocyst tissue to produce a first lipid free protein product, (c) separation of solids and high-molecular-weight protein &actions from the -
7. 7 first protein product to produce a second protein product and (d) isolation of the desired control factors ftorn the second protein product, (e) genetic engineering processing of the control factors by means of demonstration of the coded RNAs and DNAs and the vectors for the purpose of cloning of suitable recipient cells with one or more DNA fragments, which code for the control factors present in the products. obtained in step (d), (f) culturing, of the cloned tissue in a nutn'ent medium and (8) isolation of the desired control factors from the tissue culture of step 7. Method in accordance to one of the claims I to 6, wherein in step (d) the second protein product is chromatographically fractionated, and the'fractions which contain the desired control factors are isolated.
8. 8 Method in accordance to one of the claims I to 7, wherein in step (a) the gumulation or epithelization tissue is removed from the animal, or the blastocyst tissue in the deten-nination condition is removea from the human be;ng or the animal, or blood is withdrawn from the human being or the animal at the time when the maximum quantity of the control factors to be extracted has been formed.
9. 9 Method in accordance with one of the claims I to 8, wherein in step (a) the granulation or epithelization tissue is removed from the animal during the4th to --I Oth postoperative day, the epithelization tissue during the 6th and 44th postoperative day, and the blas-tocyst tissue around the 4th day after fertilization.
10. Method in accordance with one of the claims I to 9, wherein in step (a) obtained granulation, epithelization, or blastocyst time is inoculated into a nutrient medium and allowed to profferate until the formation of the maximum quantirl of the control factors to be extracted.
11. 11 Me,-hod in accordance with one of the claims 1,4, and 7 to 10, wherein in step (a) the granulation tissue or epithelization tissue is removed fTorn the animal in an advancea sta-ae of wound healina.
12. 12 Method in accordance with one of the claims 2 and 6 to 10, where in in step (a) the blastocyst, tissue is obtained from a human being or animal during the staae of aastrulation.

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13. 13 Method in accordance with one of the claims 1. 4, and 7 to 11, where! n [or the- production of granulation tissue, in step (a) a wound is created in the animal, an object is inserted in the wound which shows at least one hollow space and, on its surface, lattice-like openings leading into the hollow space, and the wound is then sutured; after maximum granulation or after the termination of granulation, the said object alona, with the anulation tissue formed are gr removed from the wound and the granulation tissue is detached from the object.
14. 14. 'i'viethod in accordance with one of the claims 1, 4, and 7 to 11, where i n for the production of epithelization tissue, in step (a) a skin lesion is made in an animal, the exposed musculature is superficially damaged, then a netlike abiect is inserted in the lesion, the wound is covered by fenestrated banclage which makes it possible to observe the beEoining or completion of epithe'lization, and then the desired tissue formed is removed after a sufficient amount ottime.
15. 15. Method in accordance with one of the claims I to 14, wherein in step (a) the tissue removed from the animal or human being is processed using zenetic engineering methods in order to produce cells with the genetic C; characteristics of the control factors to be obtained, and then these cells are cloned.
16. 16. Methodin accordance -,vith claim 15, wherein the cloned cells for producing the genetic characteristics of the control factors are cultured in vitro.
17. 17. Use of one of the control factors or their aenetic active substances obtained using, a method in accordance with one of the claims I to 16 for the production of a medicament.
18. 18. Use of the second protein product obtained in step (c) of one of the methods in accordance with one of the claims I to 16 tor the production of a medicament.
19. 19. Use of the lipids obtained 'in step (b) of one of the methods in accordance with one of the claims I to 16 for the production of a medicament.

    23 Summa Methods are described for producing control factors for the start phases and termination phases of wound granulation and epithelization ftom wound tissues from animals or ftom blood from human beings or animals, wherein granulation and/or epithelization tissue from the wound in an animal, or blood from a human being or an animal just going through a wound healing process, or the blastocyst tissue from a human being or animal are removed, the lipids are separated from the said tissues or blood or its serum, solids and hich-molecular-weight protein fractions are removed from the first product obtained, and the desired control factors are isolated from the second product obtained. The control factors are suitable for the production of medicaments.

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