IE52794B1 - Insoluble crosslinked cytotoxic oxidase-peroxidase system - Google Patents

Abstract

A fluorescent-activated spatially quantitative light detector for sensing light reflected from an information bearing surface, said light detector comprising a generally cylindrical rod containing a fluorescent dye dispersed throughout a generally transparent medium and a photosensor at one end of, and in operative association therewith. The reflected light incident on the light detector is absorbed by said dye and reradiated at the fluorescent wavelength, a portion of this reradiated light reaching the photosensor causing an output signal to be generated.

Description

This invention relates to biochemistry and more particularly to an enzymatic preparation for the control of tumour growth and infectious diseases and a process of inserting said preparation into mammals, Ample evidence has shown that the combination of certain peroxidases with hydrogen peroxide and a halide ion produces a system with strong cytotoxic properties. The myeloperoxidase-hydrogen peroxiderchloride system forms a potent cytotoxic system effective against bacteria, fungi, viruses, mycoplasma, and various mammalian cells. Similarly, the lactoperoxidase-hydrogen peroxide-thiocyanate system and the horeseradish peroxidase-hydrogen peroxide-chloride system have been shown to have potent cytotoxic activities.

An equally cytotoxic system is obtained when instead of hydrogen peroxide, a hydrogen peroxide generating system is. used. Thus,, the glucose oxidase-horseradish peroxidasechloride combination yields a potent cytotoxic system upon the addition of glucose. Galactose oxidase and xanthine oxidase have also been shown to be effective in this respect.

Furthermore, we showed that the endogenous NADH oxidase activity of the horseradish peroxidase is also capable of promoting the cytotoxic activity of the enzyme in the presence of chloride ions.

A large body of evidence indicates that cytotoxic systems such as those described above may be operative .in polymorphonuclear leukocytes, eosinophils, macrophages, and other cell types with cytotoxic properties. Such cells in general apper to utilize an NADH or NADPH oxidase as the peroxide-generating enzyme.

Macrophages are a necessary component in the augmentation of natural killer cell activity by Bacillus Calmette-Guerin (BCG) in mice. BCG also 'increases the peroxide and superoxide production by macrophages. The possibility thus exists that the peroxidase system of the macrophages-plays a role in the augmentation of the natural killer cell activity. Similarly, 53794 peripheral lymphocytes, which are predominantly T-cells, contain a cytotoxic peroxidase. Chemiluminescence resulting from peroxide generating oxidative metabolism is observed when T lymphocytes are stimulated by Concanavalin A. •Furthermore, immunization of mice with either soluble or particulate antigens' causes an increase in peroxidase activity in the spleen which precedes the generation of specific antibody. These observations suggest that oxidase and/or peroxidase activity is in some way involved in developing specific immune responses.

) Thus far none of the cytotoxic system described above have been used in any in vivo experiments. However, some relevant experiments were done some time ago by Schultz and his colleagues. Schultz, Snyder, Wer, Berger and Bonner; Chemical Nature and Biological Activity of Myeloperoxidase: Molecular i Basis of Electron Transport, Academic Press, New York, N.Y., pp. 301-321 (1972); and Schultz, Baker and Tucker; Myeloperoxidase, Enzyme-Therapy of Rat Mammary Tumors; Cancer Enzymology, Academic Press, New York, N.Y. pp. 319-334 (1976). Using mice bearing 20-methylcholanthrene induced tumours, these authors injected myeloperoxidase in combination with thio-TEPA, an antitumour drug. They observed a significant reduction.in tumour growth in the treated mice, but no complete remissions. Neither myeloperoxidase nor thio-TEPA alone were effective in reducing tumour growth. The inhibition of tumour growth lasted as long as the treatment with myeloperoxidase and thio-TEPA was continued.

These results indicated that the activity of myeloperoxidase could play a role in the control of tumour growth, either directly or indirectly. Definite conclusions are difficult to D obtain with such experiments, however, because the biological half-life of myeloperoxidase is only about twenty-four hours.

It is noteworthy, that the toxic activity appeared to be specifically directed to the tumour issue.

According to the present invention there is provided a process for preparing a composition of matter possessing cytolytic activity toward viruses and prokaryotic and eukaroyotic cells comprising: dissolving a first oxidase enzyme with a second' peroxidase enzyme capable of chemically cross-linking with said first enzyme and with a protein in a buffer solution; and adding a polymerizing agent to said solution and allowing 5 said solution to polymerize, wherein both enzymes remain active.

According to a further aspect of the present invention there is provided a composition of matter possessing cytolytic activity toward viruses and prokaryotic and eukaryotic cells formed by dissolving a first oxidase' enzyme with a second peroxidase enzyme capable of chemically cross linking with said first enzyme and with a protein in a buffer solution and polymerizing said solution by adding a polymerizing agent to said solution, wherein both enzymes remain active.

According to yet another aspect of the present invention there is provided a composition of matter comprising a first oxidase enzyme, a second peroxidase enzyme and a protein, said first and second enzymes and said protein being chemically cross-linked, and which composition possesses cytolytic activity toward viruses and prokaryotic and eukaryotic cells.

According to yet another aspect of the present invention there is provided a process for cytolysis of viruses and prokaryotic and eukaryotic cells in non-human living organisms comprising the preparation of a mixture of a carrier medium and a composition of matter possessing cytolytic activity toward prokaryotic and eukaryotic cells, said composition of matter being formed by dissolving a first oxidase enzyme, a second peroxidase enzyme, and a protein in a buffer solution and polymerizing said composition of matter by adding a polymeriz30 ing agent thereto and inserting said mixture into a. living organism.

It is an object of this invention to provide a preparation and a process for the control of tumour growths and infectious diseases in .non-human mammals.

It has been discovered that several peroxidases, when used in combination with a hydrogen peroxide and/or superoxide generating system, possess a potent cytotoxic activity when 52784 administered to tumour-bearing animals. It has further been discovered that this cytotoxic activity appears to be exclusively directed toward the neoplastic tissues.

In order- to obtain the tumouricidal activity, it is necessary that the two enzymes be kept in close proximity to each other and that the enzymes be stabilized such as to significantly increase their in viv.o half-life. Both these requirements can be met .by immobilizing the enzymes, either separately or in combination, onto an insoluble support. This immobilization can be done by either chemically attaching the enzyme molecules onto the insoluble support or by.entrapment of the enzyme molecules within the molecular matrix of the support.

For the purpose of this Patent Application, immobilization is defined as the association of active protein molecules with an insoluble macromolecule‘by any means that prevents the protein molecules from moving away from the insoluble support.

An insoluble compound is defined as a compound that does not form a true solution in an aqueous medium at around physiological pH values; aqueous media being aqueous buffer niedia as well as any bodily fluids. Compounds that form colloidal solutions in the above-mentioned aqueous media are considered to be insoluble compounds.

One or more injections of the immobilized enzyme conjugate described above into tumour-bearing animals results in a partial or total regression of the tumour tissue. It is preferable that the material be injected into the tumour or proximal to the tumour; however, primary tumours as well as metastasized tumours are subject to the cytotoxic action of the Immobilized enzymes.

It has further been discovered by virtue of the present invention that the immobilized enzyme systems act as potent activators of the specific immune response in tumour-bearing animals. This activation of the specific immune response therefore may all or in part be responsible for the regression of the tumours. Similarly, injection of potent inhibitors of the cytotoxic activity of the immobilized enzyme system results 53794 β in a depression of the specific immune response and makes animals more susceptible to develop tumour growth.

The foregoing indicate that the immobilized enzyme system augments and/or activates the natural mechanism(s) that the body uses to eliminate transformed or foreign cells present in the body, whereas inhibitors of the cytotoxic system are able to inhibit this natural defence mechanism of the body against the growth of abnormal cells.

As a preliminary matter, it is to be noted that as a 10 matter of convenience to the reader, the insoluble crosslinked cytotoxic oxidase-peroxidase system of the present invention will be referred to hereinafter as ICCOPS. In addition, it. is also to be noted that three to four-week-old Sprague-Dawley 'rats were each injected intraperitoneally with 0.5 or 1.0 ml - of Novikoff hepatoma suspension. The tumours were allowed to develop for five days or until ascites was evident. At this time some animals were sacrificed to establish the presence of tumour. The remaining tumour-bearing animals were then divided into test and control groups and treatment was initiated.

Preparation of ICCOPS The crosslinked enzyme was prepared by dissolving 10 mg of horseradish peroxidase and 300 mg of bovine serum albumin or human serum albumin in a solution containing 2 ml glucose oxidase (2400 units) and 2.5 ml of 0.1M phosphate buffer, pH 6.9. Polymerization was initated by adding 75 μΐ of 25% glutaraldehyde to the enzyme solution. The mixture was incubated for at least 24 hours at 4°C or until a gel was formed. The gel was soaked in 100 times its volume of distilled water. The water was decanted off and the gel was further washed with 100 more volumes of distilled water over a sintered glass filter. The gel was then frozen and lyophilized. The lyophilized gel was ground in a mortar with a pestle to a size that would pass through the 18 gauge hypodermic needle when · swollen in phosphate buffered saline (PBS), pH 7.4. The powdered ICCOPS was stored at -20°C until used.

Peroxidase activity of immooilized enzyme Oxidase and peroxidase activity were qualitatively determined by adding 1 mg of the immobilized enzyme system to 0,1 V, glucose in PBS, pH 7.4, containing 1.5 mg ABTS per ml.

If the green solution turned blue within an hour, the oxidase and peroxidase were considered active.

Procedures for treatments of tumour-bearing rats The basic procedure for treatment with ICCOPS required three consecutive days of treatment with 5 mg of the preparation suspended in 1 ml PBS, pH 7.4, containing 0.1% glucose. The suspension was injection into the abdominal cavity of rats bearing 5-day old tumours. Variations in the treatment regimens were used as indicated in the results which are described later herein.

Assay of the Cellular Immunological Response in Rats A single-cell suspension was produced from the spleens by passage through a stainless-steel screen (Collector, Bellco Glass Co., Vineland, New Jersey). The cells were washed once in Hank’s Balanced Salt Solution (IIBS^, Colorado Serum Co., Denver, Colorado), and then suspended in a Tris-ammonium chloride solution. The Tris-ammonium chloride solution was prepared from 10 ml 0.17 M Tris and 90 ml 0.16 H ammonium chloride and the solution was adjusted, to pH 7.2. The suspension was incubated in a water bath at 37°C to lyse the erythrocytes. The cells were centrifuged at 1000 rpm in a CRC 5000 DAMON/IEC Division centrifuge for ten minutes and the supernatant was discarded. The cells were washed in HBSS and again centrifuged. The pellet was suspended in Modified Eagles Medium (Gibco, Grand Island, New York) supplemented with 10% (V/V) normal rabbit serum (KC Biologicals, Linexa, Kansas) , Hepes buffer 10 mM (Gibco), non-essential amino acids (Gibco) and penicillin-streptomycin. A 100 μΐ aliquot of cells was withdrawn and mixed with 100 μΐ 0.4% trypan blue. After three minutes, the cells and dye mixture were diluted with 19.8 ml physiological saline solution resulting in a 1:200 dilution of cells. The cells were counted in a hemocytometer and their concentration adjusted 52784 to 2 x 106 viable cells/ml. Viability was routinely greater than 90%.

The various antigens and mitogens were stored sterile at 4°C dissolved in PBS, pH 7.2, at concentrations of 1· mg/ml, 100 ug/ml, and 10 p.g/ml. Cultures were performed in standard 96-well microtiter plates constructed from tissueculture quality plastic (Falcon Plastics, Los Angeles, California). To the wells requiring antigen at 1 jig/ml, ΙΟ ·μ·1 of antigen at 10 pg/ml was added. To achieve final concentrations of 10 pg/ml or 100 p.g/ml, for example, 10 μ 1 of 100 pg/ml or 1 mg/ml were added, respectively. Other concentrations of various mitogens were prepared in a similar manner. The specific antigen utilized was Keyhole Limpet Hemocyanin (KLH, Schwartz/Mann Laboratories, Orangeburg, New York) with which the rats were inoculated subcutaneously 14 days prior to the introduction of tumour cells. Each rat received 200 pg of KLH in 0.1 ml PBS, pH 7.6. Concanavalin A (ConA) from Boehringer Mannheim (Indianopolis, Indiana) was the mitogen utilized in the assays.

The negative control consisted of the addition of 10 μΐ of PBS at pH 7.'2 to each control well. After the addition of antigen or mitogen, each well received 100 μΐ-of the cell suspension. The plates were incubated at 37°C in a humid atmosphere containing 5% C02 for 24 hours. Next, the wells were pulsed with· 100 pi of MEM containing tritiated-thymidine at 5 pCi/ml, specific activity 2Ci/mmol. After 48 hours of culture, the cells were harvested onto glass-fibre filters using a titerte.ch cell harvester (Flow Laboratories, McLean, Virginia). After drying, the filters were counted in a scintillation fluid consisting of toluene, PPO, and POPOP.

TEST RESULTS ATTAINED BY USE OF THE PREFERRED EMBODIMENT AND THE PROCESS Regression of Novikoff Hepatomas in Rats Young female Sprague-Dawley rats were intraperitoneally inoculated each with 0.5 ml of a Novikoff hepatoma cell suspension. Five days later the tumours had developed and in most cases ascites fluid was present in the peritoneal cavity.

At this point each rat was injected intraperitoneally with 5 mg ICCOPS.suspended in a 0.1% glucose solution.

The following day the rats were again given 5 mg ICCOPS, and this was .repeated on the third day. Control experiments were done by injecting ICCOPS from which either the glucose oxidase or the peroxidase was omitted or by injecting only the glucose solution: Five days after the last treatment four of the seven animals treated with the complete ICCOPS system were sacrificed together with all the control animals that were still alive. All control animals had developed massive abdominal tumours amounting to as much as 25 grams of tumour tissue. The animals treated with the complete . ICCOPS system, on the other hand, showed the presence of some yellow fibrous connective tissue, but no signs of active tumour growth. The three animals that were saved here remained alive ‘for eight months with no apparent ill effects. (See Table I).

Thus far a total of 24 Novikoff hepatoma bearing rats have been treated with the complete ICCOPS system. A complete regression of the tumour tissue occurred in each one of the animals, and in all cases no apparent ill effects were present following the treatment.

Table I Remission of Novikoff Hepatoma tumours in Inoculated Rats Treatment Remission HRP-GOD-BSA (3 days) 15/17 HRP-BSA (3 days) 0/2 GOD-BSA (3 days) 0/3 LPO-GOD-BSA (5 days) 4/4 None 0/5 Effect on ICCOPS on Hepatoma Tumours A young female rat was inoculated intraperitoneally with 0.5 ml of Novikoff hepatoma cell suspension and the tumour was allowed to develop for five days. At this point, the abdominal cavity was opened under anesthesia. Typical tumour development was observed accompanied by ascites. 1θ The abdominal cavity was subsequently closed, and 5 mg 1CCOPS was injected i.p. The rat was subsequently given two more doses of 5 mg ICCOPS on two consecutive days following the first administration, and the abdominal cavity was opened again on the tenth day after the last treatment. At this point, the tumour was converted to a hard, nodular, yellow fibrous mass. The rat subsequently bred two months later and produced a normal litter. This experiment illustrated that the peroxidase promoted tumour regression occurs by means of tumour cell lysis. Most of' the ICCOPS particles were found in the omental tissue. Apparently there is little or no direct contact between the ICCOPS particles and the tumour cells, suggesting that the cytotoxic activity of the ICCOPS particles is transferred to the tumour cells via one or more soluble intermediates. Histopathological Analysis A hematoxylin and eosin preparation of the actively growing Novikoff hepatoma was studied. The tumour cells appeared to be of epithelial origin and little connective tissue stroma or encapsulation was evident. The cells were poorly differentiated. The tumour consisted of a continuum of. cells forming occasional small cystic appearing structures. The tissue showed an occasional limited differentiation towards pseudorosettes of the cells around the vasculature, and in some areas attempted differentiation towards a papillary pattern. Mitotic figures were common. Vascularity was limited and there were numerous areas of necrosis present in the tissue. The tumour was therefore designated an undifferentiated carcinoma with some characteristics of a papillary cystic adenocarcinoma.

The same preparation of the yellow remains of the tumour taken ten days after the final treatment with ICCOPS was studied. The tissue consisted of islands of necrotic cells encapsulated by an active fibroblastic response. There was a marked collapse of the neoplastic tissue and necrosis appeared to be total. The tissue thus demonstrated tissue necrosis with secondary fibroplasia. 03794 The mesenteric lymph nodes of the same .animal showed disruption by an infiltration of neoplastic cells into the nodes. The neoplastic cells had started the formation of discrete, islands of tissue, which is indicative of active neoplastic metastasis. Thus, this animal had already suffered metastasis when the primary tumour was being destroyed. If this observation is representative of the other animals, one can only conclude that any metastasized tumour was subsequently destroyed, since all remaining rats fully recovered.

Effect of Peroxidase Inhibitors In order to obtain additional evidence that the observed tumour regression was indeed promoted by the ICCOPS system, an experiment was carried out in which some animals were injected with a potent peroxidase inhibitor. The DOPA analog 3-aminotyrosine is -a potent inhibitor of horseradish peroxidase with of 5 μΜ.

In this experiment ICCOPS was administered to eight hepatoma-bearing rats. Four of the animals were also injected' with 100 pg of '3-aminotyrosine.

The results indicated that the 3-aminotyrosine inhibited the action of ICCOPS and all four rats that received 3aminotyrosine eventually died of the tumour, whereas the four rats receiving ICCOPS only showed complete remission. This confirms that the regression of the hepatomas is indeed promoted by the ICCOPS. ' Effect of’ICCOPS on Normal Tissue Two of the male rats with regressed Novikoff hepatomas were sacrificed on the tenth day following the treatment with ICCOPS in order to evaluate the effects of the treatment on fast proliferating normal .cells. For this purpose, the testes of the animals were examined by electron microscopy and found that the sertoli cells and spermatogenesis appeared completely normal. 100 mg. of ICCOPS were also injected into three normal healthy animals on three consecutive days. On the fourth day one of the animals was sacrificed. Microscopic 5279 4 examination showed that the ICCOPS particles were again embedded in the omental tissue. The other two rats were kept alive for another six months,. During this time, nothing was observed that distinguished them from any other normal healthy animal.

Effect of ICCOPS on other Tumours In order to determine whether or not the cytotoxic action of ICCOPS in vivo is specific for rat adenocarcinomas, the effect of ICCOPS was tested on several types of tumours.

A malignant mouse melanoma (B16-F1) was grown in mice, and after an appreciable amount of tumour cells were present in the animals, treatment with ICCOPS was initiated. The results showed that tumour necrosis occurs as a result of the ICCOPS treatment. However, treatment for four to five consecutive days was necessary to obtain complete remission.

Several spontaneous rat breast tumours were also treated.

In these cases, 5 mg ICCOPS was injected in the proximity of the tumour for,three to four consecutive days. A biopsy, taken ten days after the last day of treatment showed that complete remission of the tumour had occurred. Pathological examination revealed that both a spontaneous breast carcinoma and a breast sarcoma could successfully be treated with ICCOPS.

Furthermore, a dog with advanced Hod^tin' s disease (a malignant lymphoma) was treated. ICCOPS was injected directly into one of the diseased lymph nodes for three days. At that point, the dog died. However, severe necrosis of the lymphoma was observed in the treated lymph nodes, indicating that this lymphoma is also sensitive to the action’ of ICCOPS.

Finally, in a similar manner, a complete remission of a benign tumour (a fibroadenoma) in a rat was achieved.

These data indicate that at least several types of tumours are affected by the cytotoxic activity of ICCOPS, and animals bearing such tumours can successfully be treated in this manner. Such data indicate, however, that not all tumours respond equally well to ICCOPS treatment; a different 53794 treatment schedule is required for each type of tumour in order to achieve complete remission. ΛιιI 11 muon r Activity, of Other Peroxidases Substitution of lactoperoxidase for horseradish petoxidase in the ICCOPS preparation yields a product that is also effective in promoting tumour regression in vivo.

Its in vivo activity toward rat hepatomas is somewhat less than that of the horseradish peroxidase system; administration had to be carried out for four consecutive days to achieve the same results as with three days of ICCOPS administration.

These results show that the tumouricidal activity is not specific for the horseradish peroxidase, but can also be promoted by certain other mammalian peroxidases. Enchancement of Tumour Growth by Aminotyrosine In order to evaluate whether or not endogenous peroxidases play a role in the natural resistance to neoplastic growth, some rats were injected with a potent inhibitor of the cytotoxic activity of peroxidases and the animals were subsequently challenged with a suspension •of hepatoma cells.

Four adult male rats were injected i.p. with 1 mg of 3-aminotyrosine in 1 ml of water. Four control rats were injected with 1 ml of water. The following day, all rats were injected with 1 ml of a dilute Novikoff hepatoma cell suspension; the number of cells being normally insufficient to produce tumour growth. On the same day, an additional 1 mg aminotyrosine was administered to the rats that received the inhibitor the previous day and this was repeated for three more days. The four control rats similarly received water injections.

On the ninth day following the inoculation with tumour, all rats were sacrificed and examined for the presence of tumour. No tumour was present in any of the control animals. On the other hand, each of the animals that were treated with aminotyrosine had developed tumour growth. One rat had a large central omental tumour, whereas the other three rats 53794 contained a number of small tumours scattered through the mesenteric omentum and over the surface of the small intestines.

This dramatic difference indicates that 3aminotyrosine inhibits the natural mechanism by which the animal protects itself against neoplasia. As such its action is similar to that of various immune suppressant compounds.

Effects of ICCOPS on the Immune System Table II illustrates the change in response to Concanavalin A stimulation of spleen cells obtained from tumour-bearing rats and from rats with regressed tumours as compared to normal healthy animals. The data show that spleen cells from tumour-bearing animals are highly active in incorporating thymidine and are insensitive to further stimulation by Concanavalin A. In fact, even at low concentrations, the mitogen acts as an inhibitor. Spleen cells obtained from animals with regressed tumours, on the other hand, can be stimulated by the mitogen, even though they are more active than the cells from healthy rats.

When spleen cells from animals with actively regressing tumours were tested, a pattern was observed similar to that of animals with completely regressed tumours. These results suggest that the changes in the immune response occur soon after the administration of ICCOPS. Similar results were obtained with spleen cells of rats previously immunized with KLH. (Table III). Again, the cells of animals with actively regressing tumours behaved more like cells from normal animals in their response to stimulation with KLH than like cells from tumour-bearing animals.

These data indicate that changes in the immune system occur concomitantly · with the tumour regression as a result of the action of the peroxidase system. Whether these changes occur as a result of the regressing tumour or are directly caused by the presence of the peroxidase system cannot be ascertained at the present time.

TABLE II Responses of Rat Spleen Cells to Concanavalin Λ In Tumour-Bearing Rats and Rats with Regressing Tumours (5 days after Treatment) Con A Normal Rats Tumour-Regressing Tumour-Bearing Rats Rats CPMa Slb CPM SI CPM SI 0 1165+34 1.0 6341+1266 1.0 23343+284 1.0 1 Ug 1409+34. 1.2 10062+946 1.6 25357+817 1.0 10 2627+127 2.-2 12854+1203 2.0 23084+482 0.98 50 dig 3130+860 2.7 3028+214 0.48 17970+3855 0.77 100 1346+36 1.2 1833+832 0.29 2150+813 0 a) CPM of tritiated-thymidine taken up by 2 x 10 cells + the standard deviation for three samples. b) SI = Stimulation index: mitogen induced response divided by the control response without mitogen.

TABLE III The Specific Immune response (Secondary) to KLI1 by Spleen Cells From Normal, Tumour-Regressing, and Tumour-Bearing Immunized Rats.

KLH Normal Rats Tumour-Regressing Tumour-Bearing Rats Rats CPM SI* CPM* SI CPM SI 0 386+62 1.0 3864+61 1.0 18295+122 1.0 1 ./ig 557+57 1.4 3967+688 1.0 10288+387 0.6 10 M<3 1973+407 5.1 6705+600 1.7 10712+115 0.6 100 Mg 598+118 1.5 G501+359 1.7 13442+226 0.7 * The stimulating index (SI) and the mean CPM + .0, were, determined as in Table II.

CONCLUSION The data presented in this Application shows that 5 ICCOPS, an immobilized system with .glucose oxidase and horseradish peroxidase as its functional components, rapidly and selectively destroys a very malignant and advanced adenocarcinoma in rats. In addition, other malignant tumours, including a melanoma, a sarcoma and a lymphoma, are subject to selective destruction by ICCOPS. Previous in vitro studies by others using soluble glucose oxidase and horseradish peroxidase showed no specificity for tumour cells as compared with normal cells. A similar lack of specificity was observed with the myeloperoxidase and lactoperoxidase systems.

In an in vivo study, on the other hand, a very high degree of selectivity was obtained as illustrated by two facts. Firstly, ho obvious damage was found to normal tissues, including rapidly proliferating testicular cells; and secondly,. the histopathological tissue slices show that fibroblasts located adjacent ,to necrosing tumour cells are essentially normal.

It was also demonstrated that peroxidases other than horseradish peroxidase can be used in ICCOPS. Tumour regression has been obtained using the lactoperoxidase and cytotoxic activity in vitro was demonstrated using myeloperoxidase and snake skin peroxidase.

The function of the glucose oxidase in the ICCOPS system is merely to provide the hydrogen peroxide and/or superoxide needed to convertthe peroxidase into the cytotoxic form. Glucose oxidase was chosen for reasons of convenience, since its substrates (glucose and oxygen) are readily available for its in vivo use. However, its function can be fulfilled by any enzyme that produces either hydrogen peroxide or superoxide.

It was also found that the peroxidase system activates a secondary antitumour mechanism. This secondary system SS 7 r* φ appears to be the cell-mediated portion of the immune system. The spontaneous blastogenic activity of spleen cells of rats with actively growing tumour indicates a maximally stimulated immune system that fails to produce an effective response. Introduction of ICCOPS, however, rapidly modifies the immune system by reducing the ineffective response and restoring the mitogenic and specific immune response. These results indicate that ICCOPS acts as an immunostimulant in animals whose normal response is insufficient. The increased sensitivity of animals toward a challenge with tumour cells as a result of 3-aminotyrosine administration, thus acting as an immunosuppressant, also supports this conclusion.

Hence, ICCOPS as well as peroxidase inhibitors could also be useful as therapeutic agents in diseases affecting the immune system, in regulating the 'immune system following organ transplants, and other tissue implantations, as well as in any therapy that requires either a suppression or activation of the endogeneous immune response.

There are, of course, many variations to the specific steps, techniques, dosage, materials, and the like, used in the work described herein.

Claims (20)

1. CLAIMS;

1. A process for preparing a composition of matter possessing cytolytic activity toward viruses and prokaryotic and eukaryotic cells comprising: dissolving a first oxidase enzyme with a second 5 peroxidase enzyme capable of chemically cross-linking with said first enzyme and with a protein in a buffer solution; and adding a polymerizing agent to said solution and allowing said solution to polymerize, wherein both enzymes 10 remain active.·

2. A process as claimed in Claim 1, wherein said polymerized solution is cooled until a gel is formed and any soluble material in said gel is removed thereby resulting in an insoluble gel; and 15 said insoluble gel is dried.

3. A process 'as claimed in Claim 2, wherein said insoluble gel is frozen and lyophilized.

4. A process as claimed in Claim 2 or 3, wherein said step of removing any soluble material in said gel comprises 20 soaking said gel in a liquid water to remove any soluble material in said gel, thereby resulting in a mixture of an insoluble gel in a liquid medium; and filtering said mixture resulting from said soaking to separate said insoluble gel.from said liquid medium. 25 5. A process as claimed in Claim 3 or 4, further comprising grinding said lyophilized insoluble gel; and mixing said ground lyophilized insoluble gel in a saline solution. 6. A process as claimed in any preceding claim, wherein 30 said first enzyme comprises a glucose oxidase. 7. A process as claimed in any preceding claim, wherein said second enzyme comprises horseradish peroxidase. 8. A process as claimed in any of claims 1 to 6, wherein said second enzyme comprises lacto peroxidase. 9. A process as claimed in any preceding claim, wherein said protein comprises bovine serum albumin. 10. A process as claimed in any of claims 1 to 8, wherein said protein comprises human serum albumin. 11. Ά composition of matter possessing cytolytic activity toward viruses and prokaryotic and eukaryotic cells formed by dissolving a first oxidase enzyme with a second peroxidase enzyme capable of chemically cross linking with said first enzyme and with a protein in a buffer solution and polymerizing said solution by adding a polymerizing agent to said solution, wherein both enzymes remain active. 12. A composition of matter as claimed in Claim 11, wherein the polymerized solution is cooled to form a gel and the gel is dried and soluble material is removed therefrom. 13. A composition of matter as claimed in Claim 11 or 12, wherein said first enzyme comprises a glucose oxidase. 14. A composition of matter as claimed in any of claims 11 to 13, wherein said second enzyme comprises horseradish peroxidase. 15. A composition of matter as claimed in any of claims 11 to 13, wherein said second enzyme comprises lacto peroxidase. 53794 16. A composition of matter as claimed in any of claims 11 to 15, wherein said protein comprises bovine serum albumin. 17. A composition of matter as claimed in any of claims·

5. 11 td 15, wherein said protein comprises human serum albumin. 18. A composition of matter comprising: a first oxidase enzyme, a second peroxidase enzyme and a protein, said first and second enzymes and said protein

6. 10 being chemically cross-linked, and which composition. possesses cytolitic activity toward viruses and prokaryotic and eukaryotic cells. 19. A composition of matter as claimed in Claim 18, wherein said composition comprises a solid polymer.

7. 15 20. A composition of matter as claimed in Claim 18 or 19, wherein said first enzyme comprises glucose oxidase. 21. A composition of matter as claimed in any of claims 18 to 20, wherein said peroxidase comprises horseradish peroxidase.

8. 20 22. A composition of matter as claimed in any of claims 18 to 20, wherein said second enzyme comprises lacto peroxidase.

9. 23. A composition of matter as claimed in any of claims 18 to 22, wherein said protein comprises bovine serum albumin.

10. 24. A composition of matter as claimed in any of claims 18

11. 25 to 22, wherein said protein comprises human serum albumin. 25. A process for cytolysis of viruses and prokaryotic and eukaryotic cells in non-human living organisms comprising the preparation of a mixture of a carrier medium and a composition of matter possessing cytolytic activity toward prokaryotic and eukaryotic cells, said composition of matter being formed by dissolving a first oxidase enzyme, a second peroxidase enzyme, and a protein in a buffer solution and polymerizing said composition of matter by adding a polymerizing agent 5 thereto and inserting said mixture into a living organism. '

12. 26. The process of Claim 25, wherein the composition of matter is cooled to form a gel which is then dried and all soluble material is removed therefrom before insertion into a living organism. 10

13. 27. A process for cytolysis of prokaryotic and eukaryotic cells in living organisms as set forth in Claim 25, wherein said living organism is from the group of animals known as mammals, but excluding humans.

14. 28. A process for cytolysis of prokaryotic and eukaryotic 15 cells in living organisms as set forth in Claim 25, wherein said cytolytic activity is directed against neoplasia.

15. 29. A process for cytolysis of prokaryotic and eukaryotic cells in non-human living organisms as set forth in .Claim 25, wherein infusion of said mixture comprising said medium and said 20 composition of matter stimulates the activity of the endogenous immune system of the living organism.

16. 30. A process according to Claim 1 for preparing a composition of matter possessing cytolytic activity toward viruses . and prokaryotic and eukaryotic cells, substantially 25 as hereinbefore described.

17. 31. A composition of matter possessing cytolytic activity toward viruses and prokaryotic and eukaryotic cells, whenever prepared by a process claimed in any one of Claims 1-10 or 30.

18. 32. A composition of matter according to Claim 11, substantially as hereinbefore described. 5

19. 33. A composition of matter according to Claim 18, substantially as hereinbefore described.

20. 34. a process according to Claim 25 for cytolysis of viruses and prokaryotic and eukaryotic cells in non-human living organisms, substantially as hereinbefore described.