AU672449B2 - Process for production of cell cultures with elevated content of endogenous cytokines - Google Patents

Abstract

The process entails exposing cultures of patient's autologous blood to an ozone/oxygen mixture and fractionating. The cell cultures are cultivated in a manner known per se and lysates are added as stimulants. The lysates are obtained by ozonolysis and proteolysis of blood or blood fractions and/or urine.

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION For a Standard Patent

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: Actual inventor: Address for Service: HORST KIEF HORST KIEF 4 44,4 4,44 44 *4 4 #9 44,.

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4 4' *4 4 44 4 444444 4 S 4 44 WRAY ASSOCIATES, Primary Industry Terrace, Perth, Western Australia, 6000.

House, 239 Adelaide Attorney code: WR Invention Title: "Process, for Production of Cell Cultures with Elevated Content of Endogenous Cytokines" I roinJ The following statement is a full description invention, including the best method of performing it us:of this known to -2- THIS INVENTION relates to a process for the production of cell cultures with an elevated content of endogenous cytokines.

Cytokines are maeawgatr subntaneon that relay signals between lzmunocompetent cells and either cause the recipient ceall to assume certain functions and/or stimulate it to divide. The increased growth of certain immuanocompetent cell types in the blood should be considered the most important task of cytokines. At present, there is only one way of producing cytokines in rather large amounts at a reasonable market price; gone technology. Although the technology has matured and mass production is now under way, the process is no expensive that the cytokinos are presently among the most costly medications on the m rket. Although their structure is known and they can be produced in basically pure form, the use of cytokines still involves serious side effects.

Moreover, the administration of cytokines produced bY gone technology shown that, no matter how pure the production, foreign reactions still cannot be avoided, as in shown by the sometimes high rate of side oeffects of the res.pective products on the market. Furthermore, these suffer from one fundamental disadvantage; since they usually have a single cell type or group as the target cells and do not make use of the remainder of the immune system, the effeativeness of 4144 cytokines that are technologically produced and individually administered is not physiologically natural.

3. 0 In one form the invention resides in a process for the production of cell cultures with increajed content of ondogenous cytokines, comprising treating a patient's own blood cultures with an ozone-oxygen mixture and fractionation, wherein the cell cultures are cultivated in usual fashion and lysates are added as stimulants, the lysates having been obtained by ozonolysis and ft fractionation from blood or blood fractions and/or urine.

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-3- The process of the invention makes it possible to obtain endogenous cytokines which, because of their origin, once incorporated again in their own organism, are much more tolerable, and since they are left in the body's ewn medium, neither do they exhibit the above-mentioned defet of unphysiological action.

The possibility of producing immunomodulatory substances was first set forth in EP No. 0 265 548 Bl. There, lysates were obtained after deaggregation, by ozonolysis and proteolysise from individual blood fractions, as well as urine. Since then, the clinical effectiveness of these in autoimmune diseases has been established. This patent already describes the exposure of cell cultures to ozone gas and the use of the resulting lysates as immunomoculatory substances. Meanwhile (1990), the production of gamma-interferon from leukocyte cultures exposed to ozone gas has been demonstrated by Bocci. Bocci was also able to show that the information of Kief 20 regarding the concentration of 02/03 mixture ensures an optimal yield of gamma-interferon from whole blood ecultures. As can be seen from this work, the maximum yield in this procedure amounts to roughly 140 picograms per milliliter. If one uses whole blood or other fractions, such as leukocytes and lymphocytes together for a shortterm culture, it is also possible to obtain other cytokines .il. by EP No. 0 265 548 Bl, such as interleukin 2, which is present at 40 times more than the initial values in the I supernatant solution (the layer of nutrient solution VO x -4 I I2 -4 located above the calls that sink onto the bottom of the culture vessel) of such d culture, processed according to this technique. But in the process of the invention, much higher yields can be obtained, and various cytokine S mixtures can also be produced. Thus, the pro-mss of the invention starts with use of the process according to EP No. 0 265 548 B1. Its advanta is that it uses the lysates obtained by EP No. 0265 548 El for stimulation of mitosis in leukocyte and lymphocyte cultures that are first treated according to EP No. 0265 548 El. According to this technique, a variable spectrum of cytokines can be produced, depending on the choice of lysates obtained from serum, leukocytes, lymphocytes, erythrocytes and/or urine.

The primary procedure of the process for production of endogenous cytokines is as follows: First, whole blood is ozonized and incubated. After this, one obtains leukocytes by extraction of the buffycoat or lymphocytes by means of separating agents and zone centrifugation. These populations have already experienced 20 a selection through the buildup of oxygen radicals in the whole blood, since radical-sensitive cells are destroyed during this treatment and only the resistant cells survive.

These surviving cells are then cultivated in the usual way and lysates are added as stimulants. These lysates may be obtained, for example, from erythrocytes, granulocytes, or lymphocytes alone, or also from serum of the patient according to EP No. 0265 548 El.

A major advantage of the invented process is the exclusive use of endogenous material. This becomes very evident when compared with the usual nutrient solutions that are employed in liquid cell cultures and that almost always 4 4 .9*4 *4 *4 4, 4 4 4* 4 44 *4 4.

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44 4 4 .j :i i.l I 4 I t* 5 contain calf serum. The method according to the invention precludes a sensitization to animal protein or transmission of animal-borne disease.

Depending on the choice of the starting material, different cytokine mixtures can be induced; for example, when using a lysate obtained from serum according to EP No. 0265 548 B1, an especially abundant amount of gamma-interferon and interleukin 2 is obtained. After this, the cell cultures are lysed in the way already described by EP No. 0265 548 B1 and an injectable solution is prepared according to pharmaceutical procedures. Such lysates, even in dilutions of 1:1,000,000 and administered subcutaneously in amounts of 0.1 ml, induce an endogenous production of gammainterferon in the organism itself, which at first screening are found to be seven times higher than the starting value.

The interleukin 2 levels were on average six times above the norm. On the other hand, the soluble IL-2 receptors can be reduced to h to 1/3 in the first-described technique, which is an important criterion in the treatment 20 of autoimmune diseases.

A further increase is possible when blood stimulated in this way is again cultivated in usual fashion and exposed to lysates obtained according to EP No. 0265 548 Bl, which is a kind of culture cascade with even higher cytokine levels. The use of the lysates obtained per EP No. 0265 548 B1 as a stimulant for endogenous blood cultures, however, not only results in specific cytokines according to the choice of lysate and culture cell, but also increased budding of macrophages and killer cells, which are suitable for special application in malignant diseases.

The ozone treatment of the blood cultures has the further advantage that malignant cells from the corresponding donor I Ir I$ I

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r 4 IL 'M M -6blood are selectively inhibited in their mitosis by ozone (cf. Sweet, Ming-Shien Kao, Song-Chien, D Lee, Science Vol 209, 932), or even selectively destroyed, given an appropriate dose and overall mass. The special expense that is required in the preparation of endogenous stem cell cultures from the blood of cytostatically treated cancer patients is largely eliminated thanks to the ozone treatment. Urine contains, besides alpha 2 glycoprotein, transferrin, hemosiderin, immunoglobulin A and immunoplobulin G. Furthermore, it is possible to detect varying amounts of chains or chain fragments of immunoglobulins, in addition to ceruloplasmin, haptoglobulin solution fragments, and traces of alpha 2 macroglobulin. These components are an expression of both passive and active filtration of the kidneys. Thus, urine or even a protein mixture that has been concentrated according to the state of the art presents a possibility of stimulation of such cells, which could hardly be selected from the blood by laboratory techniques. This is shown by 20 the content of cytokines that can be detected spontaneously in the urine, and also when kidney disease is present (cf.

S. Sweet, Ming-Shien Kao, Song-Chien D. Lee, Science, Vol.

209 932).

If urine is processed according to EP No. 0265 548 B1 and 25 added as antigen to such a culture, the yield of interleukins 3, 4, 5 and 6 is substantially increased, Swhile at the same time the harvest of eosinophils is much I larger in number.

One can easily see that endogenous cytokines produced in 30 this way are much cheaper, along with the above mentioned advantage that the endogenous substances offer by virtue of their tolerance. It is not even necessary to prepare the cytokines in pure form, since the interplay of cytokine mixtures, endogenous proteins of the culture medium, cell nucleotides, and plasma component evidently exert a stronger, immunomodulatory effect than the cytokine alone.

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Claims (9)

1. A process for the production of cell cultures with Increased content of endogenous cytokines comprising treating a patient's blood cultures with an ozone-oxygen mixture and fractionatlon, wherein the cell cultures are cultivated in usual fashion and lysates are added as stimulants, the lysates having been obtained by ozonolysis and fractionatlon from blood or blood fractions and/or urine.

2. A process according to claim 1 wherein repeated doses of lysates are added to the cell cultures.

3. A process according to claim 2 wherein one further dose of lysates is added to the cell culture.

4. A process according to any one of the preceding claims wherein the lysates *are obtained from serum. t

5. A process according to any one of the preceding claims wherein the cell 15 cultures are whole blood, leukocyte, lymphocyte, or mixed cultures. so S6. A process according to claim 1 substantially as herein described,

7. A lysate obtained from the cell culture obtained from the process according I to any one of the preceding claims.

8. A pharmaceutical preparation comprising a lysate of claim 7 and a S 20 p;harmaceutically acceptable carrier. Ri 'i a I iL u i_.

9. A process for Influencing the Immune system of a recipient comprising administering to the recipient an effective amount of the pharmaceutical preparation of claim 3. Dated this Seventh day of August 1996. DR HORST KIEF Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant ftftfftftJ ft ft ,IftA

111.1W 1-1 ABSTRACT Process for production of cell cultures with increased content of endogenous cytokines, by treatment of the patient's own blood cultures with an ozone-oxygen mixture .and fractionation, wherein the cell cultu,,res are cultivated in usual fashion and lysates are added as stimulants, the lysates having been obtained by ozonolysis and proteolysis from blood or blood fractions and/or urine. 044 0