RU2003334C1 - Method for treatment of patients having tumors - Google Patents

Description

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The invention relates to the use of double-stranded ribonucleic acid RNA) alone or in combination with lymphokines to suppress the proliferation of tumor cells in animals and humans, 5 correction of disorders in tumor and cancer cells in animals and humans, and the effects on immune system disorders arising from due to the predisposition of certain organisms to tumor or cancer, regardless of whether they arise under the influence of genetic or environmental factors. The invention relates to the use of RNA in combination with other chemical and biological agents to suppress the effects of these factors.

1. Interferons (IFNs) constitute a class of proteins capable of inhibiting 20 replication of viruses in animal cells. They are of cellular nature, can be produced in vitro or fn vivo and are stimulated by a large number of substances (The Encyclopedia of Biochemistry, ed. By 25 Roger WHIIams and Edwin Lansford, Relnhold Publishing Company, 1967). INF is known for its both antitumor and antiviral effects (Nature 262, 300, 1978), its ability to restore contact inhibition and suppression of growth in the semi-fluid agar of tumor cells.

Dual-filament RNAs are inducers of various molecular forms of human INF, of three types: alpha 35 (white blood cell), beta (fibroblast) and gamma (immune), which can be stimulated by both natural and synthetic dnitium RNA. The use of synthetic double-stranded RNA to form 40 complexes of polyriboinosine and polyribocytidylic acids (hereinafter referred to as rln hSp or ioli-IC) as an interferon inducer is known, for example, from US patent N; 3,666,646 issued by Lampsonet 45 at. In addition to its role as an IFN inducer, dvnithium RNA is an activator of two IFN-induced enzymes: protein kinases and 2-5-oligoadenylate synthetase (Proc. Natl. Acad. Scl., 75, 5893, 1978). The molecular bases for the antiproliferative and antitumor effects of interferon and their association with dvuhnitievoy RNA were not known before the present invention, as well as antiproliferative properties 55 of two witnium RNA, resulting from its role as an IFN inducer.

2. Synthetic incompatible analogues of double-stranded RNA.

More recently, it was found that IFN can also be induced by incompatible analogues of hSp. as described in patents Mg 4130641 and No. 4024222 issued by T 8O and Carter. These analogues are formed by modified rln-hSp to incorporate unpaired bases (uracil or guanine) along the polyribocytidylate (hSp) chain or by modified vertebral ribosyl of polyriboinosinic acid (rln). Incompatible analogues, especially those modified in the hCp chain, retain the ability to induce IFN for as long as it meets the structural requirements of

Long sections of the paired bases of dvnithium RNA are not necessary for inducing interferon. Indeed, a necessary condition for the synthesis of rapidly acting interferons is the preservation of the 1/2 - 1-helical turn of the twin bases of dvuhnitievoy RNA (dnRNA).

An increase in hydrolysis rate was observed when incompatible polymers were exposed to nuclease (J. Mole-c. Blol. 70,567,1972), however, these double-stranded RNAs were almost as active as unmodified rln rCn, starting from the induction of interferon. It has been suggested that long-term maintenance of an intact double helical structure may be responsive to many other biological reactions to ILV, commonly referred to as drug toxicity. Studies of incompatible analogues: rln4 r (Cii-i4) n Ampllgen®, a registered trademark of HEM Research, Inc., Rochvllte, 10852, SIGA, in various animal systems have shown that rln t (Cu, U) n is less toxic than rln - rCn (see Poly-1C with Incompatible Bases, Cancer Therapy Studies In augmenting agents in Canar Therapy eo teo EMH g e a1, Pa e P e, New York, 177, 1981). In particular, repeated administration of rln r (C 12. U) n mice showed even less toxicity to such sensitive organs as bone marrow of the spine, liver cells, thymocytes, splenocytes (. Blol., 70, 567, 1972), since the effectiveness of the protection against infection with a variant of the lethal virus has been maintained (Molec. Pharm, 12,299. 1976).

In addition, rln g (Cn-14, U) n shows a decrease (100-fold decrease) in pyrogenesis relative to rln hSp in rabbits, a decrease in mitogenesis (5-19-fold decrease) in mouse and human cells in vitro, and a decrease education

dsRNA antibodies (5-10-fold decrease) in rabbits and mice. Against the background of this decrease in cellular toxicity, rln r (Cn-i4, U) n retains its ability to induce interferon, stimulating the function of natural killer cells of OH (J. Immunology, 124 1852.1980) and activating these intracellular mediators 2 - 5-oligoadenylate - synthetase and specific protein kinase. Therefore, it is obvious that in various control systems (rabbit, mouse and human) and with different dosing regimens, measurements of toxic and therapeutic effects show that the incompatible analog rln r (Ci 1-14, U) n (Ampllgen) has enhanced tera- song index. The preparation and preparation of this inducer / activator is described in J.Molec. Biol .. 70, 567.1972).

The use of modified rln rCn analogs for the sole purpose of inducing interferon in animal cells for antiviral action is described in US Pat. Nos. 4,130,641 and 4,024,2222. However, with regard to their use as an antitumor agent, the disadvantage of IFN therapy (as well as other lymphokines administered in its pure form) is that it has only a marginal effect in many organisms. As a result, the relatively large amount of lymphokines required for introduction into the body leads to an unnaturally high level of accumulation of lymphokines in it, which could not be generated by the body itself during the normal response of a person to a viral infection, immune cell differentiation or the natural mechanism of immunoprotection against cancer , viral and other lesions. At such high levels of lymphokines, the body reacts to them as foreign bodies and has the ability to generate anti-IFN antibodies against various lymphokines. The resulting antibodies destroy the residual therapeutic benefit. In addition, neoplastic cells, in particular, are capable of developing resistance to therapeutic properties of various lymphokines such as IFN and other members of the lymphokine family. As will be shown hereinafter, this ability of neoplastic cell lines to develop IFN resistance is achieved by a non-random phenotype, therefore this serves as some obstacle to therapeutic methods designed to inhibit the polymerization based on the use of pure lymphokines.

The present invention eliminates the drawbacks of using these therapeutic agents by discovering new antiprofilative agents and their compositions that stop or significantly slow down the growth of cancer cells, regardless of whether these cells develop resistance to lymphokines. In addition, the proposed method provides compositions containing interferon, in which the degree of content is at least 20-50 times less than in the described applications, the therapeutic efficacy achieved by the means described in the invention is not less, but even more. Since the compositions described in the proposed method contain synergistically active dnRNAs, the therapeutic effect of dnRNAs is correspondingly higher than that described in the known inventions, and indeed had a therapeutic effect on the tumor bodies of the bone matrix, the tumor of which is known for its resistance to the effects of exogenous lymphokines when they are used in monomodal therapy.

3. Cells - natural killers as an immunoprotective antitumor mechanism.

Many articles have been published in support of the main role of the cell population - natural killers (NKs) in the immunoprotective mechanism against neoplastic cells. Mice with high NU cell activity are more resistant to tumors with NK-susceptible tumor cells than mice with low NU cell activity (Immunol. Rev., 44. 165, 1979). C57B1 / 6 beige mice that were selected for NU cell deficiency showed a higher susceptibility to tumor growth than isogenic mice with normal NU cell activity {Nature Lond. 284, 622.1980; Nature. 284, 624.1980) A similar situation was observed in people with Chediak-Higashi syndrome, it was assumed that NU-cell deficiency in this disease could cause further development in these patients with lymphoma (J. Exp. Med, 151.1039.1980; J. Exp . Med., 151. 1049, 1980; Nature Lond, 284.553. 1980). Patients with chronic lymphocytic leukemia (CLL) have a higher metastasis rate. Zlegler and Zarltnf (int. J, Cancpr, 27, 321, 1981) evaluated NU cell activity in leukocytes taken from a patient with chronic leukemia after removal of leukemia cells. It is important to note that the majority of patients with CLL have minimal or non-destructive NU-cell activity, despite the presence of lymphocytes associated with NU-susceptible cell objects - 5 and. In addition, recipients with renal lantograft who were prescribed high-dose immunosuppressive drugs had a 100-fold higher risk of malignant development of 10 diseases, they also had an extremely reduced or complete loss of NU-accurate activity (Transplantation, 29, 214 , 1980). In contrast, antibody-mediated cell cytotoxicity (ACCC) of these 15 recipients was not inhibited. It follows that the deficiency of immunocellular activity is specific, and if it exists, it entails a serious predisposition to malignant 20 development / relapse of the disease.

The present invention contains many discoveries, one of which is that even if tumor cells are IFN-resistant, the use of specific lymphokines in combination with incompatible dcRIC results in a synergistic and potentiating effect with respect to antiproliferative effects. For example, the use of mismatched 30 dsRNA in pure form or in combination with lymphokines in the body, which show only a marginal response to the monomodal effect of lymphokines, results in an increase in the antiproliferative / immunomodulating effect of dsRNA in addition to that observed in therapy pure dsRNA. In addition, for many individuals, lymphokine therapy, such as IFN, 40 is an acceptable treatment method, i.e. these patients do not need an excessively high dosage of IFN and, therefore, they have too little risk of developing antibodies against 45 lymphokines or because they have side effects on the administration of lymphokines, since these side effects are directly dependent on the dosage of lymphokines. In this sense, dsRNA enhances the effect of lymphokines 50 and at the same time gives IFN therapy methods greater efficiency. Therefore, the therapeutic methods and formulations contemplated by this invention improve existing methods based only on lymphokines, 55 and also make it possible to apply these methods to those patients who, for one reason or another, cannot use the known ones.

In a preferred embodiment of this invention, the dsRNA is an incompatible rhn analog of hcp. As indicated, this analogue is the result of rupture of a chain by unpaired bases, i.e. they are not Watson-Crick paired bases such as uracil and guanine. These incompatible analogs are a preferred embodiment of the present invention due to the fact that they retain the antiproliferative effects of unmodified rln hSp and at the same time exhibit a significant reduction in the tendency to the rapid occurrence of side effects such as fever, death of non-specific cells, etc. e. death of normal body cells, and antigenicity. In addition, it was found that the specific incompatible analogue of nV g (Cc-14, U) n gives a sensational effect, even more than that already described, in connection with the antiproliferative abilities of a typical dsRNA like rln hSp. In particular, this allows in some cases to correct defects in the tumor cells so that they are reprogrammed and functionally converted to normal. The applicant actually separated the cancer cells from the patient and repeatedly observed this phenomenon. In addition, it has been found that AmpHgen can effectively act as a cancer cell normalizing agent in situations where there are obstacles to the use of lymphokines. It is known that some chemical and biological substances have the ability to independently normalize cancer cells. Incompatible dsRNA, namely rln r (Cii-i4, U), expands these possibilities in the normalization of cancer cells, while lymphokines often have the opposite effect, i.e. reduce or destroy the normalization process.

In addition, the present invention makes it possible to correct immune disorders expressed in a predisposition to cancer due to hereditary factors or disorders in the immune system. These properties, obviously, are not related to the action of IFN, lymphokine, most likely they can be attributed to the action of incompatible dsRNA on cancer cells in a qualitatively different way than lymphokine does.

These therapeutic compositions and methods are used to treat cancer, tumors, and neoplastic cells. Treatment in this context is a term meaning cancer prevention, developmental delay.

tumors (partially or completely), elimination of tumor relapses, degeneration of tumor cells as normal, correction of cancer-related immune defects and providing the necessary protection against the effects of viral infections, as well as various autoimmune reactions to inflammatory conditions.

It is important to note that the present invention is used not only to treat a cancer tumor after it has been clinically detected, but also preventively to prevent malignancy at an early stage of the onset or subclinical course of the disease, i.e. the precancerous condition can last quite a long time before visible signs of a disease requiring therapeutic treatments appear. The present invention is also intended to prevent cancer before it reaches a clinical stage, for example, in organisms having a high degree of hereditary predisposition to cancer. In other words, cancer can be thought of as part of a biological continuum that can exist for quite some time, the early stages of which can be seen simply as a predisposition. The invention is intended not only for the treatment of clinical cancer, but also allows its preventive use long before the onset of visible signs of the disease.

The aim of the invention is to increase the effectiveness of treatment.

The method uses therapeutic agents and methods to modulate tumor cells towards normalization and loss of their malignant phenotype by introducing exogenous incompatible + dcRN K into tumor cells. Such methods, for example, are the administration of incompatible dsRNA and an increase in the intracellular level of dsRNA. Tumor cells that are not responsive or have little responsiveness to lymphokines thus become reactive. The administration of dsRNA can be carried out in conjunction with lymphokines. A particularly striking effect can be achieved in transferring to cells of slowly developing tumors the ability to respond to the antiproliferative effect of interferon on them.

As noted, many organisms do not have a satisfactory response to carcinotherapy. based on the use of IFN in its purest form. Consequently, the high dosage level required for the effective effect

for such patients, it may develop the body's resistance to production within itself of natural IFN. In addition, the fact that another type of resistance can also be developed by neoplastic cells in the form of a randomly acquired phenotype attaches paramount importance in understanding the significant improvement in therapeutic methods and agents obtained in

0 of the present invention, in comparison with those methods that were used to suppress tumor proliferation in patients by using only one IFN. The introduction of IFN and some dsRNA,

5 i.e. with paired bases or incompatible, in the implementation of this invention is carried out in combination in which both agents are administered either together as a therapeutic mixture or separately.

0 not simultaneously, as if by separate systems for intravenous infusion into one organism. Combined use also includes the administration of these drugs separately, in which one is given first from the bottom and the second shortly after the first. All three of these embodiments of the invention have advantages.

Separate but simultaneous administration of them, for example, allows for independent control of each agent and obtains the optimal remedy for each patient.

Separate administration allows for a clear IFN to restore the marginal effect on cells, which is enhanced by the use of dsRNA. The preparation of IFN and dsRNA as a mixture provides quick access to this therapeutic agent

0 provided that if you determine in advance its optimal composition. Comments have previously been given on all cases contemplated by the present invention, including the claims, wherein one therapeutic agent is administered in combination with another.

Similarly, the therapeutic agents and mixtures thereof contemplated by the method can be administered by the route, usually

0 used and known in medical practice. One of them is intravenous, but there are others: intramuscular, subshell, intracranial and intraperitoneal,

5 Lymphokines incorporate interferons (alpha, beta, gamma), preferably alpha, interleukins, specific interleukins (1. 2 or 3) and recombinant interleukin-2 (rlL-2), tumor necrotic factor (TNF ) This also includes

lymphokine-activated killer cells (LAK), which are formed in the body as a result of a response to the action of fokin.

If interferon (alpha) is used 5 as imfokine, then the required amount is from 0.01 to 10,000 units. Since IR per ml of human body fluid, white IL-2, and preferably rlL-2, is a lymphokine, the amount administered should be from 10 units, 10 IL-2 per kg of human body weight to a value approaching an unacceptable level for the patient toxicity, up to 106 units. IL-2. However, these limits are from TO3 to 104 units. IL-2 per kg of human body weight. fifteen

If both dsRNA agents and lymphokine are administered in combination, as described, they can be introduced into the mixture, separately, but simultaneously or one after the other. "

If lymphokines and dsRNA are administered in a combination of 20, then dsRNA can be introduced in an amount that leads to a level of 1-1000 mg of dsRNA per ml of fluid in the body, i.e. a solution of serum, salts, vitamins, etc., circulating in the body. 25 For example, the administration of a composition containing 10 mg of dsRNA to an organism weighing 160 stones (72.4 kg) results in an RNA content of about 1 mg / ml. In a similar way, the amount of interferon with 30 injections of dsRNA - IFN will be in the range of 1-100000 units. IFN per ml of fluid in the body. The route of administration of this combination, as described, can be performed as follows; as a mixture, 35 separately, but simultaneously or one after another. The thing is that the introduction of these agents gives a synergistic effect, no matter in the practical embodiment of this combination, to take them.40

By incompatible dsRNAs are meant dsRNAs in which the hydrogen bonds (base packing) between the double helices are relatively intact, i.e. they are interrupted on average by less than 45 one base pair in every 29 consecutive basic residues. In accordance with this, the term incompatible dsRNA should be understood. dsRNA can be a complex of polyinosinate and half-50 iticidylate, which make up the proportion of uracil and gunadine bases, for example, from (1-5) to (1-30) (poly I poly (C4-C29 to x U or G). on the other hand, the corresponding oligonucleotides (small nucleotide fragments 55) may be coupled to the corresponding complementary polynucleotides or oligonucleotides under certain circumstances.

dsRNA can be represented by the basic formula rln r (Cn-i4, U) n and, in particular, rl (Cia, U) n. Other suitable examples of dsRNA are discussed below.

Incompatible dsRNAs that are most suitable for use in the present invention are based on copolyinucleotides selected from poly (Cn. U) and poly (Cn. G), where n is an integer of 4-29 and is incompatible analogues of the complex of polyribibocytidylate (hSp) compounds, for example, formed by the inclusion of 2-0-methylribosyl residues. These incompatible rln rCn analogues, the most preferred of which is rln (Ci2. U) n, are described by Carter and in U.S. Patent Nos. 4,130,641 and 4,024,222. The dsRNA described therein is generally the most suitable for use in the invention.

Specific examples of inconsistent dsRNAs for use in the present invention include:

poly (I) poly (C4, U) poly (I) poly (C, U) poly (I) poly (Cia. U)

POLY (ABOUT POLY (C22. U) POLY (I) POLY (C20, G)

poly (I) poly (Cp) 23 O r.

An amount of incompatible dsRNA is introduced sufficiently to achieve a peak blood concentration of 0.01 fi g per ml dsRNA to 1000fi t per ml dsRNA in the circulatory system shortly after administration.

It has been found that in the proposed method, it is best to use dsRNAs of the rln rC (Cn-i4, U) n type for inhibition of neoplastic cells. Inconsistent dsRNAs have been observed to have a weaker pharmaceutical effect compared to paired base dsLNAs. The activity of other types of IFN, in addition to IFN fibroblast, was also investigated, including natural human leukocytes and IFN produced in bacteria using recombinant DNA technology (rIFN). In all cases, IFNs have a weaker effect on human tumor cells when they are poured in pure form than when phni are poured together or sequentially in normal or mismatched RNA. The maximum reduction of the human tumor was 33% in width when injected with an IFN concentration of 105-3 106 units. daily. In contrast, in animals subjected to the introduction of IFN in combination with dsRNA in an amount of 10 units. IFN daily, there was a decrease in the tumor by 65% more than the decrease

According to this, dsrnA, used in combination with any one type of IFN, results in a qualitatively higher effect. In addition, “with the tumor, the tumors require an indipidual treatment regimen in order to obtain an optimal therapeutic effect. treatment regimens lead to the expected 10-fold increase in therapeutic effect where incompatible dsRNA is used in combination with IFN. The data convincingly show that dnRNA extends the therapeutic efficacy of all forms of IFN, including natural, synthetic and hybrid forms, such as those derived in part from alpha, beta and gamma-IFN. In accordance with this combination of dnRNA. especially inconsistent dsRNA, namely r (Cu-i4, U) n, with IFN is included in the preparation of a potent anti-cancer drug that far exceeds the capabilities of pure ION. In addition, when studying the structure of the cheloretic tumor, it becomes apparent that the content of various types of viral genetic information in it certainly contributes to the malignant and / or pathological processes in human tissues that are the cause of their parasites and diseases. Therefore, it is important that the dsRNA-IFN combination is not only an antitumor agent, but also an effective anti-viral agent. Indeed, the therapeutic mixtures described in this invention will be effective against diseases for which IFN treatment is indicated, including viral diseases in the acute, subacute, latent and chronic stages, as well as diseases associated with impaired immune spasis, but which may disappear when autoimmune defense is restored.

It is important to emphasize that the role of dsRNA in the spe- cistically enhanced treatment effect when using the dsRNA-IFN compound is not LJ. that it stimulates IFN, otherwise this synergism would not be observed. Most likely, incompatible dsRNA introduces a specific effect that supplements the action of IFN, making it more plastic and effective, which could not be achieved using in the treatment of cancer or viral diseases

only IFN or dsRNA alone.

Some patients with lung cancer and patients with a mild hereditary predisposition to cancer were observed.

low activity of NU cells. These observations are described below. It was tested whether f-southerly in patients with a high hereditary risk factor restore NU-cell activity to a normal level using additional types of IFN and / or incompatible dsRNAs. Recent studies show a discrepancy in the effective increase in NU cells (cells were taken from normal organisms) among natural types (Brit. J. May., 50, 85, 1982) and cloned interferon subtypes (Can. Res , 42, 1312. 1982), It was noted that incompatible RNA has a more pronounced ability to stabilize the normal level of NU cells in patients with a high degree of hereditary predisposition to cancer.

The following are examples illustrating the present mnFr ete "iie. all proportions and percentages in which are taken by weight. The dsRNA (incompatible) used in the examples has the formula rin g (Cc-14, U) n, sometimes known as rin (Ci2, U).

A. dsRNA - a new mechanism for modulating tumors,

A new mechanism for modulating tumors by incompatible dsRNA is not only part of the basic mechanism of action of lymphokines. This study demonstrates the broad reasons for using abnormal human tumor cells to modulate, the mechanism of this modulation being different from that of IFN induction. In particular, dsRNA induction of cyclic adenosine monophosph (sLMR) was also detected in cells known for their insensitivity to alpha-interferon. These studies were performed with two c-LMR kinase inhibitors designated as H-7 and HA 1004 (known metabolic inhibitors of protein kinase C and cAMPA) to evaluate the new antitumor modulating activity of incompatible dcRPC as resistance to unimodal limfokine-interfering, aka prototype lymphokine. To demonstrate incompatible dsRNA, no interferon was induced in IFN-insensitive cells; for this, human glioma cells (brain tumors) (A1235) were treated with dsRNA for various periods of time from 0 to 8 hours. The dsRNA was removed and fresh was added medium for 24 hours, then tigers of interferon (units, IFN / ml)

tested for cytopathic effect (Pinter. N.G., J., Gen virol. 5: 419-427. 1969). The results are shown in table. 1, where the lower detection limit was 5 units. IFN / ml

From this it follows that dsRNA did not induce interferon in these IFN-insensitive cells.

Then, such cells were subjected to a supporting check in order to determine whether interferon plays the role of antiproliferative effect in these cells caused by exposure to dsrnA. This was done by using antibodies to interferon, which, if present in the cell, would be bound to the antibody, the antiproliferative dsrnA-induced effect would be reduced.

Three types of antibodies to interferons (alpha, beta, gamma) in 240 neutral, units / ml and control cells were used. Tumor growth inhibition assay was performed as described by Hubbell et

ab. Cancer Res. 44: 3252-3257 (1984). The percentage of control antibody culture was calculated as follows:

treated cells (24 h) - control cells (Pts) 100 control cells (24 h) - control cells (and h)

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The results are presented in table. 2. These data also confirm that dsRNA did not induce IFN in the studied cells.

It has been found that dsRNA induces cyclic AMP in human glioma cells, which can be directly attributed to dsRNA rather than IFN induction in these cells. Three well-known metabolic inhibitors: H-7, HA 004 and pertussis toxin - inhibit or counter the antitumor effect of dsRNA, as shown in the table. 3. The percentage of cell cultures was again evaluated to obtain a low percentage of control cultures, as in Table. 2. Incompatible dcrn K in its pure form works to decrease the percentage of control cells, the presence of an inhibitor increases cell culture. Figure 3 shows the counteraction by metabolic inhibitors of H-7 and HA 1004 to the antitumor effect of incompatible dsRNA in human glioma cells. Values are again given as the percentage of control cells for dsRNA at a concentration of 0 (blind sample or control), 25 and 200 mg / ml.

Similarly, it is shown below how pretreatment of human glioma cells with pertussis toxin mngibs dsRNA-induced antiproliferation. 8 tab. Figure 4 shows the percentage of control culture for 24 hours in cells pretreated with pertussis toxin for 4 hours before dsRNA was added. This shows that irro H-7 and HA 1004, two known inhibitors, have minimal effect on tumor inhibition in cells treated with alpha interferon, while pertussis toxin, which apparently has a two-phase effect, does not change the inhibition tumor cells with alpha interferon at high doses and enhances the antiproliferative effect in small doses.

IFN-alpha does not increase the intracellular cAMP level in the same cells for 24 hours, as shown in table. 5. In this technique, human glioma cells (A-1235) were exposed to interferon-alpha in an amount of 0 (control.), 250 and 1000 units. IFN / ml within 24 hours from the start of the experiment.

In this table, pm & l cAMP / mg protein are given; the accuracy limit is 0.02 pmol cAMP / mg protein.

The intracellular cAMP level was then determined indirectly by measuring the catalytic activity of andenylate (pmol cAMP formed in 15 min) in human glioma cells immediately after treatment with an antiproliferative dose of dsRNA (25 and 200 g / ml) for 30 min and 24 h In this procedure, cells were treated with incompatible dsRNA, 25 (solid triangle) or 200 (solid square) / l g / ml of incompatible dsRNA. At the appropriate point in the treatment time (0.5-30 min and 0.5-25 h), the medium was removed and the cells were quickly frozen with dry ice. The activity of adenylate cyclase in homogenates obtained from cells upon exposure to ultrasound was studied by the Jonng method. et al Molec. Enfocrinol. 1: 884-888, 1987. Cells were counted for 24 hours to check for antiproliferatin activity. Induction of cAMP in dsRNA-treated human glioma cells (A 1235) was measured directly as picomole

cAMP on a microgram of protein, results of more than 30 minutes and more than 24 hours. In this method A, 1235 cells were treated with 25 (triangles) or 200 (solid squares) / g / ml of incompatible dsRNA. At appropriate time points, the medium was removed and intracellular cAMP was cured at 0.1 HCI. CAMP levels were determined using the RIA method as described by Relslne et al, J. Cell. Biol. 102: 1630-1637, 1986. cAMP levels in control cells and cells treated with IjU g / ml dnRNA. were below the limit of detection accuracy. Cells were counted 24 hours to test antiproliferative activity.

A pronounced increase in cAMP was observed after only 30 s of dsRNA. this level was maintained for 30 minutes.

Studies confirm an increase in the cAMP level, which is directly related to the action of dsRNA, so dsRNA was not an IFN inducer in the cells used. Antiproliferative doses of dnRNA induced edenylate cyclase activity 30 seconds after the start of the test. Similarly, intracellular cAMP levels increased depending on the antiproliferative dose after 30 seconds. Pretreatment of the cells with pertussis toxin as well as an intracellular cAMP inhibitor followed by dsRNA treatment inhibits the dsRNA-induced tumor inhibitor. On the contrary, antiproliferative doses of natural human IFN-alpha do not increase intracellular cAM P-levels for 24 hours of the experiment. In addition, two known inhibitors of H-7 and HA 1004 have minimal effect on tumor inhibition in IFN-alpha treated cells, while pertussis toxin appears to have a biphasic effect, as indicated above.

Investigation of a cAMP system with IFN-induced proliferation (Panniers et al, J. Celt Sci., 48: 259, 1981, Banerjee et al, Virology 129: 230.1983, Ebsworth et al. J. Celt Physio. 120: 146, 1984), it was found that cAMP is not related to the degree of antiproliferative state. As for dsRNA in the same systems, these studies have not been previously conducted. The studies conducted in the present invention confirm the conclusion that a) dNRNAs and lymphokines, the prototype of which is IFN, have different mechanisms of action and B) dsRNA-induced antiproliferation is associated with the cAMP system (while lymphokines are not) .

In this sense, cAMP is essentially a ubiquitous system, i.e. more or 5 less all cells possess the genetic information necessary for the synthesis of cAMP with appropriate stimulants. These experiments show ample potential for using mismatched dsRNA to modulate abnormal cells that are partially or completely resistant to IFN or other lymphokines used in pure form. In addition, these results suggest that inconsistent dsrnA acts by two sequential mechanisms: the first via cAMP kinase, and the second through modulation of pro-kinase C.

0 B. Protein synthesis.

Intracellular protein changes in dsRNA-treated cells, reflective progressive differentiation and loss of the phenotype of malignant cells,

5 anti-lymphokine properties.

The normalization of cells after dnRNA treatment was, in a sense, studied by Soslau et aK Biochemical and Biophysical Research Communications. 119: 941-948, 1984.

0 The method in which brain tumor cells (A 1235) were treated with an antiproliferative dose of incompatible dsRNA (100 / g m / ml) or natural human IFN-alpha (100 4 m / ml) showed significant differences in the nature of protein synthesis during period 72 hours. Protein synthesis is an important parameter, as it shows the normalization of the treated atypical cells and the duration of the therapeutic effect. Protein synthesis was assessed by counting the radioactive isotope-labeled A 1235 cells with respect to a predetermined number of cells. These cells were not processed; only IFN-alpha was processed in

concentration of 100 / g m / ml, dsRNA in conc.

200 / i m / ml together with IFN-alpha with dsRNA.

The measurements were carried out in 24, 48 and 72 hours.

Over 24 hours of measurement, changes in synthesis

0 protein in comparison with the control sample was insignificant; measurements within 48 h showed a significant increase in protein synthesis stimulated by both dsRNA and IFN agents,

5 dsRNA-processing, this increase is significantly higher than with IFN-processing. It is striking that after 72 h of measurement, protein synthesis in cells treated with dsRNA increases by 3 times compared with the control, while s cells treated with IFN, this level drops to the control, maintaining the increase in protein synthesis in cells treated with dsRNA , apparently, reflects significant changes in the types of certain proteins obtained as a result of dsRIC-induced changes in cells to a more differentiated or normalized cellular phenotype.

A study of protein phosphorylation in human IFN-alpha-treated bladder carcinoma showed significant changes in protein phosphorylation compared to untreated cells (Soslau et at, Blochem, Biophy, Acta, vol. 119.1984, p. 941). The studies of the present invention (giulacrylamide gels not shown) with rln r (CiM4i U) n showed changes completely different from the results obtained for interferon-treated cells. These data, moreover, more vividly show the molecular differences between dsrnAs and lymphokines based on the modulation mechanism of tumor cells, their return to a more normal phenotype, and fundamental differences in the way immunodifferentiation and immune activity are used in the modulation.

For oncologists / immunologists, in the practical part of the invention, a number of methods have been described for selecting specific protein characteristics of a malignant process for a given cell type or regeneration characteristics of malignant cells of a given hype. and control changes for the selected protein or group of proteins in light of the descriptions provided by the present invention. Factors varying from person to person may be able to make their own requirements for dsRNA over time, but this does not diminish the advantages of the invention. It should be noted that the presence of an unusually high level of nuclease or phosphodiesterase may become a reason for the clinician to change the concentration of dsRNA in the required therapy, as opposed to that indicated in the examples and experiments.

Studies of protein synthesis make it possible to trace the mechanisms underlying it, the dynamics of drugs, to determine the degree of this action, by establishing, for example, the number of normalized cells, the period of action of the drug and from what time the positive effect began. until now known works in this field,

C. Own dsRNA necessary for antitumor IFN activity in

tumor cells.

It was determined that the antitumor activity of IFN in tumor cells is quite rare and mainly when IFN-sensitive cells

have their own early dsRNA inherent in these cells. Lack of antitumor activity or a sufficient amount of intrinsic dsRNA in cells can be compensated by the introduction of dsRNA to create the conditions for its necessary presence in sufficient quantity.

Interferons are known to induce dsRNA-linked enzyme 2.5-oligo-A synthetase, which is involved in

cell growth inhibitor. The discovery made in the present invention shows that double-stranded RNAs work primarily as cofactors for enzyme activation, which is clearly different from that

claims that interferons induce this enzyme. These actions erroneously combine, significantly reducing the potential for success in treating with each agent alone or in

their combination. It has been shown that dsRNA is naturally found in the peripheral roofs of mononuclear cells (PMMK) of some individuals with leukemic reticuloendotheliosis, although they were not found in the PCMK of normal individuals. Indeed, the presence of dsRNA in the hair cells (HeLa) of patients with leukemia seems to explain, to a first approximation, the known efficacy of interferon in cases where interferon is used alone and provides a decisive additional dichotomy between IFN and dsRNA that can be used therapeutically. The clinical efficacy of interferon is due to the induction of the enzyme (2, 5 oligo A synthetase) in the presence of early early intracellular natural dsRNA, while exogenous dsRNA works in significant

degrees by activating enzymes, thereby controlling cell growth.

To confirm this new hypothesis, sod RNA was isolated from control and treated with IFN-a A hairy cells (HeL a), fractionated by sucrose gradients and tested for the ability to activate purified high molecular weight 2, 5 A syntheses. None of the fractions of untreated RNA HeLa cells could activate synthetase. However, the heterogeneous sod of the RNA fraction of the IFN-treated cells activated the synthetase in a dose-dependent manner. Hot denaturation of g RNAcustran triggered this activation. Analysis of the enzyme product by high pressure liquid chromatography (HPLC) showed that biologically active 2, 5-A trimers and tetramers were obtained. The mononuclear cell turf of RNA was also fractionated from hairy cells with free leukemia, which were very sensitive to IFN therapy and showed a high level of 2, 5 A synthetase activating dsRNA in g of the RNA fraction. HPLC analysis showed the formation of biologically active 2), 5 A trimers, tetramer, pentamer and hexamer (their ratio is 53: 15: 4: 1, respectively). The normal mononuclear cell sod of RNA did not show any activity. These results show that natural RNA sod exists only in relevant cells, neoplastic or, in other words, when it can participate in the regulation of growth by IFN. Until now, there is a latent and urgent need for an interferon response. This allowed us to conclude that the lack of dsRNA / lodge leads to inefficiency of interferon, which can be compensated by exogenous dsRNA. This statement can well be verified and confirmed in clinical conditions.

To be more convincing, it can be noted once again that for the IFN activity, the presence of a rare dsRNA, an inhabitant, in cells requiring antitumor activity is necessary. Here, apparently, tumor cells require susceptibility / sensitivity to IFN therapy. Some authors mistakenly group IFN and dsRNA together. From the present studies, it is evident that dsRNA is a cofactor which activates the enzyme 2,5-oligo A synthetase. Tumor cells Insensitive to IFN. When exogenous dsRNA is added, they become susceptible to dsRNA therapy. In cells responding to IFN, dsRNA obviously has a three-dimensional structure like incompatible dsRNA, namely, eln g (Sc-14. U) n, based on their biological and catalytic similarities, lack of toxic properties, enhanced immune functions, comparable to cytotoxic functions, as well as other characteristics such as cAMP and

parameters of their antitumor effect.

Cells that are mainly immune to lymphokines and to interferons in particular restore their susceptibility by first adding an effective amount of exogenous dsRNA to induce the synthesis of the necessary intracellular dcRK K, which is involved in the regulation of cell growth in combination with interferon, and then when the now therapeutically active MFN, as already containing the necessary dsRNA supplement.

D. Reactivity of the human renal tumor xenograft.

The susceptibility of the human kidney xenograft to IFN and dsRNA is completely different, as the studies described below, based on observations of long-term

LIVING ANIMALS.

The use of the human tumor xenograft in atimic mice yielded unexpected and different reactions to interferon and dsRNA. For example, human renal carcinoma showed progressive tumor growth and did not increase survival time during interferon-alpha treatment, while dcRC treatment showed a significant decrease in size and an extremely long survival of the organism. Indeed, after death at 2–4 years of age due to natural causes, 95% of all dsRNA-treated mice were not found in histology after autopsy, tumor lalichi. This was very unexpected, since lymphokines (for example, IFN) cause a very short remission of cancer and there are only a few, if any, specimens of animals in which no tumors were found even after several months. Obtaining 95% of the recovered by histological criteria and survival time was very unexpected. In addition, the activity of spleen cells of natural killers (NUs) increased with dsRNA treatment of animals, while the activity of mice with IFN did not increase NU activity.

A similar difference in the inhibition of tumor growth was observed between interferon-gamma and dsRNA in the other two xenotranspantant models; a noticeable difference was also observed in experiments with human PT4 bladder carcinoma and human tumor head molum A 1235. As described, in all cases, statistically significant inhibition of tumor growth was observed in animals treated with dsRNA, and minimal in the treatment of interferon-gamma, used in pure form. A significant difference was also observed in a ciency splenic NU-klvtok with an increase in the processing of the dsRNA NU-activity and lack of increase in the processing of interferon gamma,

A similar effect was observed due to an increase in immunoactivity, the results of experiments with glioma are presented in Table 6. Dosage: 20,000 units, IFN / interferon daily. etc dn -, L) n no 2 or 3 times a week (200-500 fi g per dose). A thorough examination of blood samples obtained sequentially throughout the entire time of testing from the tail vein showed that dnRNA, the chemical class of which is described elsewhere in the present invention, can be introduced indefinitely, with any adverse effect. on the functions of the kidneys, brain and immune functions, i.e., on the functions of organs most susceptible to toxic agents.

In the table. 6, which is presented below, provides immunological activity data for spleen cells from mice treated with IFI gamma and / or dsRNA. Table 6 shows the ratios of the components of the studied mixture of cells: effector cells (immune cells obtained surgically from the spleen of animals that have been injected with a human tumor xenograft) and target cells (in this case, a brain tumor of a person). Effector cells are immune cells of the immune system, and target cells are cells of the studied tumors. The results are presented as percent cytotoxicity resulting from treatment with IFN, dsRNA, or both agents together. A higher percentage of toxicity indicates an increase in eliminated tumor cells.

It is curious that in this experiment the killed cells after treatment with IFN gamma are even less than the control ones (untreated), and the effect after processing separately taken dsRNA is greater than when processing combinations of dsRNA + IFN gamma.

The efficacy of dsRNA, especially dn g (C11-14, U) n, against a human brain tumor inoculated in atimic mice is compared with that of IFN-gamma. Tumors were measured on day 10 and

on the 31st day after the start of the experiment. dsRNA was more effective in reducing the size of the tumor compared with the efficiency of IFN, which turned out to be almost less than the control one,

E. Clinical examples of lymphokine-resistant tumors that are susceptible to dnRNA.

An alternative technique has been developed, different from that for animal models, to evaluate tumor susceptibility or resistance, which presents a possible synergy clinic for a combination of IFN and dsRNA therapy. One such technique is the method of cologenic analysis developed by Hamburber and Salmon (Primary Bioassay of Human Tumor Strm Cells Science

197: 461-463, 1977), which can really predict the susceptibility or resistance of the patient's body to anticancer drugs with a high accuracy of 90-95% reliability for inactive drugs and 75-80% for active ones.

In accordance with a technique developed by Hamburger and Salmon, a halogen analysis is carried out as follows. A monocellular mixture is prepared from fresh tumor cells or cells taken from leukemia patients. The pre-determined concentration of the selected therapeutic agent, calculated from literature or methodological

recommendations are added to a plate with agar or methyl cellulose medium or in the lungs after plating. Cells purified and seeded in agar or methyl cellulose medium are incubated in a Petri dish at 37 ° C.

which allows to obtain a colony of cell cultures. In accordance with a predetermined period of cell growth, cell colonies are visually observed with a microscope and the number in each Petri dish is calculated. Biologically and cell mixtures of colonies were studied very thoroughly, including cell morphology, karyology, immunology, and cell behavior in atimic mice. Cells were investigated

using monoclonal antibodies / probes with nucleic acids. Cologne analysis is a reliable approximation to in vitro analysis of tumor cells for chemical sensitivity.

Cologic analysis of ne multiple tumor samples can be performed in the laboratory. In contrast to the expected lack of antitumor activity noted on various works

on cancer research (Compilation of Phase D. Results with single Antlneoplastlc Agents, US Departmen of Health and Human Services, Public Health Service, National Institutes of Health, volume 4, 1985), which describe about 100 animal samples obtained from poly I poly With C-treatment and being completely insensitive or partially sensitive to the agent used, this study observed 42% of the predicted responses to incompatible dsRNA treatment in a wide range of solid human tumor species that are known to be insensitive to polynucleotide therapy.

According to preliminary clinical estimates, tumors, considered sensitive, produce more than 60% reduction in the number of tumor clones.

A large number of such tests, both in laboratories and clinics, have been performed on individuals to determine their tumor sensitivity to different genetically IFN classes and / or different types of interleukins. In particular, patients participating in dsRNA experiments. before that, they were subjected to unsuccessful clinical treatment with multferon or interleukins in doses reasonably taken from published medical data. In all these examples, the use of certain lymphokines was unsuccessful to prevent tumor growth, in most examples the immune cells remained unchanged, in many examples there was an actual decrease in the size of the tumor compared to its size before treatment with pure lymphokines.

Laboratory studies of the present invention have established at least three unexpected results:

The selected incompatible dsRNA is effective when the poly I -field C is ineffective,

The selected incompatible dsRNA is effective when non-effective IFN is used alone.

Synergism has when selected incompatible dsRNA and lymphokine are used in combination.

In the table. Figure 7 shows the relative susceptibility values of various histological types of human tumors to incompatible dsRNAs. Experiments show more than 40% susceptibility to incompatible dsRNA, i.e. dsRNAs with a specific molecular structure, of a different class of tumors, especially solid tumors, while pure lymphokine-resistant tumors are also susceptible to incompatible dsRNAs in the same era.

These results are unexpected for two reasons: the known bioinertness of dsRNA in general and the clinical lack of benefit in lymphokines.

The effects of IFN-beta, IFN-beta, and incompatible dnRNA were studied in a cologenic analysis for melanoma. The percentage of control cells is derived with respect to the dose r1n r (6ti-i4, U) n in micrograms per ml and in units. IFN / ml for interferons.

The synergistic antiproliferative effect of IFN-alpha and rln KS 12 was also considered. U) n in human kidney carcinoma in the same units. Combinations were used for the amount of rln rfViz, i) at 50 and 100 / d7 ml. The concentration of IFN-alpha ranged from 0 to 3000 units. IFN / ml, which is significantly higher than normal IFN-alpha therapeutic concentration.

At the beginning of the reference, there is a line break, indicating no effect on the counter, colony for O IFN, about 62% for 50 and 50% for 100 rln rfCi2. U) n and then for 100 units. IFN / ml IFN-alpha.

As these data show, IFN-alpha in its pure form is not effective at clinically acceptable doses, its effectiveness is observed at a higher dosage at a cytotoxic level, a level too high for clinical use. The results of this study showed that for the effectiveness of therapy, the amount of IFN should be reduced to a clinically acceptable level. probably supplemented or replaced by other antiproliferative agents.

Certain examples of synergistic action were observed in 63% of melanoma, 50% of breast cancer. 80% of ovarian cancer and 65% of kidney cancer for combined IFN-alpha + rln r (Ct2. UJn-therapy. Thus, from these results we can conclude that a) the use of a certain incompatible dsRNA in imfokine-resistant states will be beneficial in clinical therapy, c) in the case of resistance of the body to dsRNA in its pure form, an acceptable combination with lymphokines would have a beneficial therapeutic effect in most cases.

D. Three clinical examples confirming the effectiveness of the present invention

Patient 1 - malignant melanoma. Patient 1 is a middle-aged man with malignant melanoma, clinically immune to IFN and 1 L 2 therapy. The introduction of dnRNA (300 mg twice a week) as a result led to the complete resorption of the tumor (the amount of tumor mass was calculated) during the 80-day treatment period. This beneficial effect persists for approximately 2.5 years from the start of treatment. Currently, a dose of between 50 and 100 mg 2 times a week is maintained. The dosage was determined by taking into account clinical susceptibility in the light of laboratory larametroa, including a relative level of 2, 5 A synthetase and the ability to detect active biochemical intermediaries in the dsRNA-induced effect, such as the cyclic AMR levels described above, or the existence of certain classes 2, 5 -A molecules, as well as increased immune cell activity. An almost simultaneous increase in 2, 5-A synthetase, cAPM and bioactive 2 "5-A oligomers was observed, while none of them was detected with lymphocyte therapy or during the period of ineffective treatment. Various oligomers were measured in the range of 25 days before therapy and 1, 23 and 55 days after the start of dlRNA teralia. The low molecular weight of the 2, 5-A oligomers (dimers), as determined in the laboratory and elsewhere, makes it especially bioinert with respect to antiproliferative, antiviral and immuno-enhancing properties.

A similar biochemical phenomenon is explained by a long-term increase in lymphokine-activated activity of Iubian cells (I AK), resulting in a stable state of the disease and / or significant regression of the tumor, which was measured by increased I AK activity (as% 51Cg cleavage) in patients in a stable state of the disease (carcinoid cancer), after 30 months of dsRNA therapy, and for comparison in patients who did not receive dsRNA therapy and normal or control patients. These data were used to control the relative amount of therapeutic agents required for a beneficial clinical effect over

for a long period of time, for example, to maintain therapy after the start of control, even with lymphokine resistance.

Patient 2.

Particularly noteworthy is the fact that bone metastases in bones can be significantly reduced by treatment with dsRNA and IFN,

taken in combination with each other. For example, patient 2 with kidney cancer and extensive bone metastases showed a quick and long-term susceptibility to this therapy after he received a daily dose of just 1.5m units. IFN and 300 mg dsRNA administered twice a week. Similar to the case of chronic leukemia, this treatment regimen caused prolonged remission in a large number (in percentage) of patients, while in patients treated with IFN-alpha alone, this effect was not observed.

Patient 3.

Patient 3 is a middle-aged man with an IFN-resistant kidney tumor, which at the very beginning of treatment showed a rapid decrease in the tumor simultaneously with a change in the profile of the .5-A oligomers, a rapid disruption of the metabolic pathway to cAMP and a sharp increase in immunoactivity. These important clinical and laboratory data were stored for 36 months; no side effects were detected by this therapy.

A similar beneficial clinical effect of the described therapy was observed in most lymphokine-resistant tumors, including the most lethal and inactive types of lung cancer. The dependences of NU-cell activity (in litical units) and the corresponding sizes of the median tumor (in mm) measured by CT scanning are shown. results

were obtained at the beginning and after completion of about 400 days of therapy with incompatible dsRNA. The patient showed complete susceptibility to therapy, as confirmed by measurements of tumor size

(56) Patent EP No. 0113162, class A 61 K 31/70, 1984.

29200333430

dsRNA can be poly I poly C, U in Table

Induction of interferon in dsRNA-treated human glioma cells

wherein the ratio of C to U is about

Antibodies to interferons do not inhibit dsRNA-induced antiproliferation in human glioma cells

Note. The results are presented as a percentage of the control culture for 24 hours. ND was not done.

table 2

Table 3

Table 4

Table 5

Table 6

Claims (1)

The claims METHOD FOR TREATING PATIENTS WITH TUMORS by correction of immune disorders with double-stranded ribonucleic acid, characterized in that, with Continuation of the table. 6 Table 7 in order to increase the effectiveness of treatment, Ampligen is used as ribonucleic acid in an amount of 50 to 300 mg per 1 ml of blood immediately after administration of the drug or after its administration in combination with interferon,