PT87923B - The present invention relates to a method for the preparation and manufacture of pharmaceutical compositions useful for the preparation of non-nucleoside resistant cells - Google Patents

Abstract

The invention relates to a process for the preparation of a dsRNA (double stranded RNA) which comprises ligating complex oligomers, polymer complexes or both, preferably oligonucleotide peptides, which hybridize to single-stranded polymer RNA complementary to form the duplex dsRNA.

Description

EPIGRAPH: .. PROCESS FOR THE PREPARATION OF RNAdC AND

PHARMACEUTICAL COMPOSITIONS THAT CONTAIN USEFUL FOR THE MODULATION OF LYMPHOKIN-RESISTANT CELLULAR STATES

INVENTORS: Dr. William A. Carter.

Claim of the right of priority under Article 4 of the Paris Convention of 20 March 1883.

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Description related to HEM Research, Inc., North American, industrial and co-inertial patent, domiciled at 12220 Wilkins Avenue, Rockville, Maryland 20852, United States of America (inventor; Dr. William A. Carter, resident in the USA), for, PROCESS FOR THE PREPARATION OF RNAdC AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN USEFUL FOR THE MODULATION OF CELLULAR STATES

LYMPHOKIN RESISTANT.

description

Domain of the invention

The present invention relates to a process for the preparation of double-stranded ribonucleic acid (hereinafter referred to as dRNA) for use alone or in combination with lymphokines to inhibit the proliferation of neonatal cells in the body of an animal , including humans, for the purpose of correcting defects that occur in non-tumor or cancer cells; the body of an animal, including humans, and to treat disorders of the immune system linked to the predisposition of certain individuals to be susceptible to tumoral or cancerous conditions, whether this predisposition is of genetic (hereditary) origin or of environmental causes. The invention relates to a process for the preparation of pharmaceutical make up ¹ BAD ORIGINAL sions containing dsRNA in combination with ¹ s, I and other chemical biological agents for the treatment of tumor or cancerous conditions.

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BACKGROUND OF THE INVENTION [!

I. IFN and ARNdc's functions as IFN inducers

Interferons (hereinafter referred to as MFIs) constitute a class of proteins capable of inhibiting virus replication in animal cells. These proteins are cellular in origin, can be produced in vitro or in vivo, and are stimulated by a wide variety of materials (The Encyclopedia of Bioche mistry, edited by Roger Williams and Edwin Lansford, King. Nhold Publishing Company, 1967). IFN is known to exert antiproliferative effects as well as antiviral effects (Nature 262, 300, 1978), to reestablish contact inhibition (J. Cell Eiol., 56 846, 1973) and to inhibit the growth of neoplastic cells on agar semi-solid (nature, 231, 20, 1971).

oor laughs. _ η n of interferon, po in induction of rFN, enzymes induced by IFN,

DRNAs are inducers of different molecular forms of human IFN including alpha (leukocytes), beta (fibroblasts) and gamma (immune) types (J. Reticuloendothelial Society, 2 3, 299, 1978), with the possibility of IFN production be stimulated by both natural and synthetic dsRNAs. It is known to use synthetic dsRNA in the form of complexes of polyriboinosinic and polyribocytidylic acids (hereinafter referred to as rC or poly IC) strictly as an example, ca. U.S. Patent no. 3,666,646 on behalf of Lampson et al .. In addition to its role, dRNA is also an activator of two a protein kinase and a 2'-5'-oligoadenylate synthase (Proc. Natl. Acad. Sci. 75 5893 , 1978). The molecular basis for the effects

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However, the antiproliferative and antineophasic effects of interferon and its relationship to dDNA are, however, unknown and prior to the present invention, any antiproliferative effects of ddNA were also unknown in addition to its role as an interferon inducer.

II. Unpaired synthetic analogs of dRNA

More recently it has been found that IFN can also be induced by unpaired analogues of rl _n . rC _n> as described in U.S. Patents N ^e . 4 130 641 and 4 024 222 on behalf of Ts ¹ o and Carter.

These analogs are formed by modifying the ri. rC in order η n to incorporate non-corresponding bases (uracil and guanine) along the polyribocytiaylate (rC) chain or to n modify the ribosyl backbone of polyribinosinic acid (rl ^). Unpaired analogues, particularly those modified in the rC chain, retain the ability to induce interferon on the condition that certain structural requirements are met. As the figures illustrate, long regions of: dRNA are not necessary with perfectly matched bases for the induction of interferon. In fact, the structural prerequisite for triggering the synthesis of interferon appears to be the conservation of 1/2 to 1 turn of the dsRNA helix with perfectly matched bases.

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There was an accelerated speed;

hydrolysis when exposed to disassembled polymers to nucleases (J. Molec. Biol., 70, 567, 1972), although these cDNAs ¹ were almost as active as kidney. rC in terms of interferon induction. It was projected that the prolonged conservation of a structure in double he-! The intact lymphoma could be responsible for the numerous other responses to dsRNA commonly related to its toxicity. Studies carried out on the analog (unpaired Rj, ri. R (C .. ..), Ampligen ^, a brand

ORIGINAL BAD registered in S. U. A. by HEM researcn, Inc., Rockville,

Md., 10852, USA, in various animal systems have shown that ri. r (C. ~ U) is less toxic than ri .rC ri x rL f η on (see Foly IC witn mismatched bases, prospects for cancer therapy, in Augmenting Agents in Cancer Therapy, edited by Ξ.Μ. Hersh et al, P.aven Press, New York, 177, 1981).

In particular, repeated administration of laugh (C

12,

U) to rats results in much less toxicity to critical sensitive organs including elements of the bone marrow / bone, liver cells, thymocytes and splenocytes (J. Mole. Biol., 70, 567, 1972), while preserving the effectiveness in protecting against various lethal viral threats (Mole. Pharm., 12, 299, 1976).

Also, laughs. r (C _ni .. U) n 11-14, n has reduced pyrogenicity (100-fold reduction) compared to ri. rC in rabbits, a lower mitogenicity (5 to 10 fold reduction) with rat and human cells in vitro and a decreased formation of dsRNA antibody formation (5 to 10 fold reduction) in rabbits and rats. Against this picture of reduced cellular toxicity, ri .r (C .. „U) retains its ability to induce interferon, stimulate the function of natural destructive cells (natural killer = NK) (J. Immuno, 124, 1852 , 1980) and activate the aforementioned intracellular mediators, 2'-5 ¹ -oligo-adenylate synthase and a specific protein kinase. It is clear, ¹ therefore, that in a variety of assay systems (rabbits, rats and humans) and under different dosing schedules measuring various toxicities and therapeutic effects, the unpaired analogue laughs (Ampli- ¹ gene) has a therapeutic relationship enhanced. The preparation and formulation of this mismatched inducer / activator has already been described (J. Molec. Bio. 70, 567,

1972).

The use of analogues of laugh

BAl) ORIGINAL

. rC _n modified for the specific purpose of inducing the production of interferon in animal cells to fight the virus attack is already known, for example from U.S. Patents ^Nos HIS. 4 13G 641 and 4,024,222 mentioned. However, with regard to their use as antiproliferative agents, there is an impediment to IFN therapy (as well as other lymphokines when administered alone) because these lymphokines have only a marginal effect on many individuals. As a result, relatively large amounts of lymphokines are required for administration, resulting in abnormally high levels of lymphokines in the body that would not be generated in the course of the normal human response and an ordinary viral infection, immune cell differentiation or natural mechanisms of immunovigilance, antivirals and host defense. At these high levels the body can treat lymphokine like IFN as if it were a foreign substance and treated individuals have the ability to generate antibodies against various lymphokines including IFN. These resulting antibodies generally disrupt any residual therapeutic benefits.

In addition, neoplastic cells in particular have the ability to acquire resistance to the therapeutic properties of various lymphokines including IFN and the different members of the interleukin family. As shown below, this ability of neoplastic cell lines to develop resistance against IFN is acquired in the form of a non-random phenotype and therefore constitutes a serious inconvenience to any therapeutic method designed to inhibit proliferation based on the use of lymphochia. in isolation. The present invention represents a solution to this deficiency in the prior art by introducing new antiproliferative agents and compositions that stop or dramatically retard cell growth

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cancerous cells, whether or not the cells have developed resistance to lymphokines. Furthermore, the present invention provides processes for the preparation of compositions containing interferon in such a way that the body levels of interferon, although lower by a factor of 20 or 50 than the levels reached by the prior art, still result in an even greater therapeutic efficacy. Since the compositions of the present invention that contain dsRNA act synergistically, the therapeutic benefits of dsRNA are likewise far superior to those described in the prior art and undoubtedly provide a therapeutic attack on tumor nuclei in bone matrix sanctuaries, sanctuaries These tumors are notoriously resistant to the attack of lymphokines applied exogenously when they are administered as simple therapy.

III. Natural destructive cells (NK) as an immunovigilance mechanism against tumor cells

Numerous studies have been published that support a central role for populations of NK cells in the mechanisms of immunovigilance against neoplastic cells. Rats with high NK cell activity have been shown to be more resistant to growth of NK-sensitive tumor cells than rats with low NK cell activity (Immunol. Rev., 44, 105, 1979). It was also reported that mice brown O573L / 6 quej have a selective deficiency in NK cells, are more sus- ¹ ceptíveis to tumor growth than with normal syngeneic mice NK cell activity (Nature (London),

84, 622, 1980; Nature, 2 34, 624, 1980). A similar condition exists in human patients with Chediak-Higashi syndrome and it has been suggested that deficiency in NK cells in this disease may be causally related to the subsequent development of lymphomas in these patients (J. Exp. Iled., 151, 1039, 1980 J. Exp. Fie d.,

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151, 1049, 1980); Nature (London), 284, 553, 1980).

Patients with chronic lymphocytic leukemia (CLL) are known to have a high incidence of secondary malignancies. Ziegler and Zarling (Int. J. Cancer, 27, 321, 1981) evaluated the activity of NK cells in lymphocytes of chronic leukemia patients after elimination. nation of leukemic cells. Significantly, most CLL patients have minimal or undetectable NK cell activity, despite the presence of lymphocytes that bind to NK sensitive targets. In addition, kidney transplant recipients, who are treated with high doses of immunosuppressive products, have a risk of developing malignancies approximately 100 times greater and have extremely reduced or abolished NK cell activities (Transplantation, 29, 214, 1980). In contrast, antibody-dependent cell-mediated cytotoxicity (CODA) is not as low in these receptors. This fact suggests that the deficiency in the activity of immune cells is specific, and, when present, is associated with a profound propensity for the development or recurrence of malignant diseases.

SUMMARY OF THE INVENTION

The present invention is based on multiple findings including the observation that even if tumor cells are resistant to IFN, the use of specific lincocuins in combination with unpaired dsRNA results in a synergistic or enhanced effect with respect to their respective antiproliferative action. This means that the use of AxNdc alone or in combination with lymphochipines in individuals who exhibit a marginal response to simple modality therapy results in an increased antiproliferative / immunomodular effect of cDNA compared to that resulting from use-based therapy.

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In addition, for many individuals therapy with lymphokines, including IFN, is a viable method of treatment, that is, these individuals do not need abnormally high dosage levels of IFN and, as a result, are at a much lower risk of to develop antibodies against lymphokines or to have other side effects of the lymphokine administered, since these side effects are directly related to the dosage of lymphokine. In these individuals, dsRNAs enhance the action of lymphokines and thus make IFN therapy a more effective therapeutic method. Therefore, the therapeutic methods and compositions described in the present invention improve existing treatment methods based on isolated lymphokines and, at the same time, provide access to these treatment methods to those individuals who would otherwise be excluded from effective treatment by one or more of the prayers pointed out.

A preferred embodiment of the present invention relates to a dsRNA that is an unpaired analog of ri. rC _n . As previously mentioned, these unpaired analogs result from the interruption of any of the chains with non-corresponding bases (i.e., that do not form Watson-Crick base pairs) such as uracil and guanine. These analogues of-! semi-matched represent preferred embodiments due to the fact that they retain the anti-proliferative effects of the unchanged ri rC while the pressure does not have a considerably reduced tendency to trigger undesirable side effects, as they are, non-specific cell death (ie that is, the death of normal body cells) and antigenicity

In addition, I also found

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L

than the specific unpaired analog rl ^. ^r ( ^c qq_q4 '

U) has drastic effects even superior to those previously seen in connection with the antiproliferative capabilities of a typical dsRNA, such as ri. ^rC n * Specifically, it enables the ability in some cases to correct deficiencies in tumor cells in such a way that these cells are reprogrammed and functionally convert to normal cells. In fact, I isolated cancer cells from patients and repeatedly observed this phenomenon. In addition, I have found that Ampligen can actually function as a cancer cell normalizing agent in situations in which lymphokines really pose a complete obstacle to that treatment. Certain chemical and biological substances are known to have some ability to normalize cancer cells independently. DRNAs ^{1 are} unpaired, notably. r (C ^ - _L _ ^ ₄ , U), is capable of amplifying this type of tumor cell normalization activity, while certain lymphokines, especially lymphokines, often have the opposite effect, that is, they reduce or cancel this normalization process .

In addition, the present invention also allows for the correction of immunological disorders in individuals predisposed to the development of cancer in a rude form of family history (i.e., for here dictated reasons) or defects in the immune system. This ability is apparently a foreign effect of IFN and lymphokines and evidences the fact that the dsRNA can affect ¹ are qualitatively cancer cells, which are isolated unable lymphokines.

Therapeutic compositions and therapeutic methods allow the treatment of cancer, tumors and neoplastic cells. In this context, treatment is a generic term covering cancer prevention, stopping tumors (whether partial or complete),

BAD ORIGINAL inhibition of tumor resurgence in patients previously treated by established therapy, the conversion of tumor cells into normal cells, the correction of cancer-related immune deficiencies and the improvement of host defenses necessary to provide protection against viral infections as well as against various autoimmune and inflammatory states. It is crucial to understand that the present invention is applicable not only to the treatment of cancer or tumors since they have already manifested clinically but also to the prevention or abolition of these malignant states while they are incipient or sub-clinical. This means that cancerous conditions can be sub-clinical for decades before they become openly evident, and then become the subject of treatment with existing therapeutic methods. The present invention, on the other hand, makes it possible to warn of a cancerous condition even before it reaches the clinically evident condition, for example in individuals who have a history that indicates a high predisposition to cancer. Seen from a different perspective, cancer is

simply part of a biological continuum (possibly lasting decades) in which the early states of the continuum may simply be a predisposition. The present invention not only enables the correction of openly evident cancer, but also allows the prevention of cancer if it is treated during the early stages of this continuum, possibly decades before its actual appearance. ί

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It is therefore an object of the present invention to provide processes for the preparation of compositions useful for the treatment of cancer in animations including man, the term treatment (when used in conjunction with cancer, tumor, etc.) being a generic term covering the prevention of cancer, the stopping of a tumor (whether partial or complete), the inhibition of reappearance; of tumors in patients previously treated by 10 BAD ORIGINAL

established cell, the conversion of tumor cells to normal cells or the correction of cancer-related immune deficiencies and the improvement of host defenses necessary to provide protection against viral infections as well as against various autoimmune / inflammatory states.

Compositions prepared according to the process of the present invention are useful for use in therapeutic methods for inhibiting the proliferation of neoplastic cells in animals, including humans, which contain unpaired dsRNA in a form suitable for administration to the animal having need for this therapeutic treatment.

Additionally, compositions prepared according to the process of the present invention are useful for use in improved therapeutic methods for inhibiting the proliferation of IFN-resistant neoplastic cells in a form suitable for administration to the animal in need of this therapeutic treatment. .

It is also an object of the present invention to provide a process for the preparation of pharmaceutical compositions useful for potentiating the antiproliferative effects of dsRNA on neoplastic cells in the body of an animal, including a human being, characterized in that an unpaired dsRNA is incorporated as an active ingredient. combination with a lymphokine.

It is yet another object of the present invention to provide a process for the preparation of pharmaceutical compositions useful for the treatment of shifts, including tumors resistant to isolated IFN, in the body of an animal, including a human, characterized by being incorporated as an active ingredient a dRNA

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isolated or in combination with a lymphochemistry The pharmaceutical compositions subject to the process of the present invention are to functionally convert cancer cells into the body of an animal, including a normal human being.

It is yet another objective to provide a process for the preparation of pharmaceuticals useful for the conversion of cancerous or tumoral functions into the body of an abnormal one characterized by incorporating an unpaired dsRNA into chemical or biological combinations that it has unpaired in.

According to the usefulness for tumors or cells, in cells there is invention of cell compositions badly in cells in the ingredient with another independently some capacity for normalization of neoplastic cells.

The pharmaceutical compositions obtained in accordance with the process of the present invention are also used to improve or eliminate immunological deficiencies.

or chronic viral, j i

BRIEF DESCRIPTION OF THE DRAWINGS;

The present invention is illustrated;

attached drawings in which: i ί

FIGURE 1a is a graph showing the indirect measurement of induction of cAMP levels by measuring the activity catalyzed by adenylate in i cells

human glioma treated with unpaired dsRNA (two concentrations) for 30 minutes;

FIGURE 1B is a similar graph! similar to that of Figure 1a, which presents similar results

- 12 ORIGINAL BAD tei s gic as sit hair

over a 24-hour period;

FIGURE 2a is a graph showing a direct measurement of the induction of cAMP levels by RIA measurement in human glioma cells treated with unpaired dsRNA (two concentrations) for 30 minutes;

FIGURE 23 is a graph similar to that of Figure 2a showing similar results over a 24 hour period;

FIGURE 3 is a bar graph showing protein synthesis in human glioma cells (untreated, treated with IFN, treated with dsRNA and treated with IFN and dsRNA simultaneously) at 24, and 72 hours with progressive differentiation and loss of malignant cell phenotype, a property that is not from linauauins, for cells treated with dsRNA;

FIGURE 4 is a graph of the results!

I tates of a 31-day experiment with dimension measurement! tumors (length times width) of tumors grafted on athymic rats among untreated rats (comparison) and treated with IFN and dRNA; i

FIGURE 5 is a graph of the results of colonogenic analysis in melonorn cells for increasing amounts of IFN-alpha, IFN-beta and dsRNA;

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FIGURE 2 is a graph of the results of a collogenic analysis in human renal carcinoma cells for isolated IFN-alpha and two combinations of IFN-alpha at zero and 100, 30C and 1GC0 units with 50 and 100 pg / ml of the dsRNA demonstrating effects synergistic antipro;

liferatives for the combinations; I i

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J

FIGURE 7 shows the response already a patient with a malignant melanoma in terms CS tumor volume and synthetase activity ¹ 2 ' , 5'-ademilato before and after over 75 days of treatment with dsRNA;

FIGURE 8 is a series of four HPLC plots that measure the presence of several oligomers, especially p ^ A ^ z before and after the initiation of dsRNA therapy in the patient in Figure 7;

FIGURE 9 is an illustration of elevated LAK activity in a stabilized carcinoid cancer following more than 30 months of dsRNA therapy compared to LAK activity in patients with solid tumors who did not receive dsRNA therapy and normal patients for comparison, and is used to verify the amounts of maintenance therapy for dRNA needed to maintain a favorable clinical effect over an extended period of time; Hey

FIGURE 10 shows the answer! of a patient with an adenocarcinoma in the lung in terms of NK cell activity and the mediastinal dimension of the tumor before and almost 400 days after unpaired dsRNA therapy.

!

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SUMMARY OF IN72NÇÃ0;

Processes for preparing useful therapeutic compositions for modulating the growth of tumor cells towards cell normalization and for the loss of the malignant cellular phenotype containing exogenous unpaired dsRNA by administration are described. these compositions to a patient thereby increasing the patient's intracellular dsRNA level. Tumor cells that did not respond or that only responded poorly to lymphokine thus became capable of

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give answer. Therapeutic compositions can contain i

a lymphokine for joint administration with dsRNA. Particularly drastic responses are achieved by making tumor cells indolent capable of responding to the antiproliferative effects of interions.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As already stated, many individuals may not respond satisfactorily to anti-cancer therapy based on the use of IFN alone. As a consequence, high dosage levels are required which may result in these individuals becoming resistant to their natural IFN. In addition, the fact that a resistance of another type can also be developed by neoplastic cells in an acquired phenotype! non-randomly constitutes a consideration of extra ordinary importance in understanding the drastic improvement that the pharmaceutical compositions of the present invention provide in relation to any method that seeks to inhibit

I proliferation through the use of isolated IFN.

The administration of IFN and any dRNA, that is, perfectly matched or mismatched dRNA, “in combination includes embodiments in which the two agents are administered together 'in the form of a therapeutic mixture and also when the two agents are administered separately but simultaneously, for example, by means of separate intravenous injections in the same individual. Combined administration further includes separate administration of the two products in such a way that one of the products is given first followed shortly after the second. All of these forms of administration have inherent advantages. separate but simultaneous administration, for example, allows for independent regulation of each agent and therefore enables therapeutic optimization on an individual basis 15 BAD ORIGINAL '

dual. Separate but simultaneous administration, for example, allows for independent regulation of each agent and therefore enables therapeutic optimization on an individual basis. Separate administration allows the IFN given alone to establish an effect on cells, albeit marginal, which is then amplified through the use of dsRNA. The preparation of IFN and RNA-γ dc in the form of a mixture allows, of course, instant access to this therapeutic composition in cases where the optimum levels have been previously established. The foregoing comments apply to all cases presented in this specification, including! the claims, when a therapeutic agent is administered in combination with another.

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Likewise, the therapeutic agents and compositions of the present invention can be administered by any of the body routes currently used as is known to medical science. It was ί

Intravenous administration is mentioned but other routes can be used, including intramuscular, intrathecal, intracranial and intraperitoneal.

Lymphokines include interferons (alpha, beta and gamma), preferably alpha interferon, interleukins, specifically interieukuins 1, 2 or 3 and recombinant interleukin 2 (rlL-2), and tumor necrosis factor (tumor necrosis factor = TNF). Lymphokine activated killer cells (LAK) formed in animals in response to exposure to lymphokine are also considered to be included.

When the lymphokine used is the interferon (alpha), the amount administered should be such that it provides O, Cl at 10C 000 IRU per milliliter of fluid; patient's body. When the lymphokine is IL-2, I

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preferably rlL-2, the amount administered is 2 in a range from about 10 units of iL-2 per kg of the patient's body weight to a value that approaches the unacceptable levels of toxicity in that patient, which can be as elevated as 10θ units of IL-2. However, a dosage in the range from about 3.7 to about 10 units of IL-2 per kg body weight is preferred.

When the two agents, dsRNA and lymphokine, are administered as described above, they can be administered as a mixture, or administered separately but simultaneously or sequentially.

When lymphokine and any dsRNA are administered in combination, it can be administered in such an amount as to result in a level of 1 to 1 000 µg of dsRNA / ml of body fluid, that is, 3 serum solution, salts, vitamins , etc., which circulates inside the body and bathes the tissues. For example, administration of a composition containing 10 mg of dsRNA to an individual weighing 160 lb (72.5 kg) will result in an dsRNA level of approximately 1 yug / ml. By hand:

similarly, the amount of IFN present when administering a combination of dRNA-IFN RNA is the amount that will result in a level of 1 to 100,000 IRU / ml in the body. The mode of administration of the combination may be as previously described, that is, I am in the form of a mixture, by separate but simultaneous administration or sequentially. What matters in this case is that the administration is functionally synergistic, whatever the physical form of implementation.

By unpaired APXdc (?) Is meant that dsRNA in which the hydrogen bonds (base stacking) between the corresponding strands

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they are relatively intact, that is, they are interrupted on average by less than one base pair in every 29 consecutive base residues. The unpaired ASKdc term must be understood accordingly. The dRNA can be a complex of a polyinosinate with a polycytidylate containing a proportion of uracil or guanidine bases, for example, from 1 in 5 to 1 in 30 of these bases (poly I. Poly (C. - C _o ^x or G) Alternatively, appropriate oligonucleotides (small nucleotide fragments) can be complexed with the appropriate complementary polynucleotides or with oligonucleotides in certain circumstances.

DRNA can be poly I. poly C, U, in which case the ratio of C to U is about 13 to 1 and the sedimentation coefficients of poly I and poly C, U are both less than 9 and at a distance of 2 units from each other , both preferably 6.5 to 7.5.

DRNA can be of the general formula ri. r (C _ni U) and specifically laughs. r (C _nn , U).

n 11-14 n ‘n 12 n

Next, other examples of adequately | From.

The preferred mismatched dsRNAs in the present invention are based on copolinucleotides selected from poly (C, U) and poly (C, G) where η n n is an integer with a value from 4 to 29 and are i

unpaired analogs of polyribocytidylamide (C) complexes, for example by including 2 'n - C-methyl-ribosyl residues. These unpaired analogues of j rl. rC, of which the preferred ones have the general formula I η η [

Π. r (C ^ 2 ' ^u ) _n > were described by Carter and Ts'o nas>

US patents 4,130,641 and 4,024,222. The dsRNA's described in these patents are in general suitable for use according to the present invention.

- IS BAD ΟΓ-C! P -

unmatched in:

for use action in the present invention poly (I). poly (c ₄ , u) poly (I). poly (C. ₇ , U) poly (I). poly ^(C 13 ' ^U) poly (I). poly (c ₂₂ , u) poly (I). poly (C _2nd , G) poly (I). poly (C _2g , G) and poly (I). poly (C) 23 G> P D

The amount of dsRNA mismatches. of the administered is preferably sufficient to cause a peak blood concentration from 0.01 microgroups per milliliter of dsRNA to 1000 micrograms per milliliter of dsRNA in the systemic blood circulation immediately following distal administration in relation to the melting point.

As previously described, a preferred form of the present invention consists of the process of preparing unpaired dsRNA of formula r1. rC (C _n . U) and pharmaceutical compositions η n 11-14 'n that contain it useful to inhibit the proliferation of neoplastic cells. We verified that the unRNAs are mismatched | cause milder pharmacological effects compared to i sRNAs ¹ s that present a perfect base pairing.

The activities of IFN types other than human fibroblast IFN were also examined, including the natural IFN varieties of human leukocytes and the IFNs produced in bacteria using recombinant DNA technology (rIFN). ;

In all cases, IFNs had a lesser effect on tumor cell growth when injected alone than when injected together with or

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followed by dsRNA, whether it was paired dsRNA (ri rC) or unpaired ri ^{r (C} ll-14 ' ^U) n'

The maximum tumor cell growth response achieved by all IFNs administered alone was approximately 33% across a wide range of injected IFN concentrations (10% _to 3 x 10 10 IRU per day).

In contrast, animals treated with quantities as small as 5

IRU per day of IFN in combination with a dsRNA showed a reduction in the growth of human tumor cells of 65% or higher,

Thus, the dsRNA used in conjunction with any form of IFN results in a quantitatively superior effect. In addition, human tumors generally need an individualized treatment regimen in order to generate the optimal therapeutic effect. Additional efforts to individualize the treatment program should result in therapeutic efficacy 10 times greater or more whenever unpaired dsRNA is used in conjunction with IFN.

The data clearly demonstrate that dsRNA amplifies the therapeutic effectiveness of all forms of IFN, including natural, synthetic and hybrid forms, such as those derived partly from alpha IFNs and partly from beta or gamma IFNs, all of which are sbrengi. within the scope of the present invention. Thus, the start of unpaired dsRNA, in particular the formula laughs. r (C _n . U), is an ootent formulation with n 11 -14 '- against cancer, far exceeding the capacity of the isolated IFN. In addition, it is known that the studied human tumor material contains various types of viral genetic information that positively contributes to the malignant process and / or the pathological process in human tissues, causing disease in the tissues and their destruction.

The data therefore demonstrate the important fact that a combination of RNAdc - IFN is not only effective as an anti-cancer agent, but also as an anti-cancer agent, but also as an agent against the components and diseases.

viral diseases. In fact, the therapeutic compositions of the present invention act against any disease in which IFN treatment can be indicated, including viral diseases manifested by acute, subacute, latent or chronic phases and also including the dysimmune human diseases in which viral activity serves. to start loyia duck but it can also disappear as the human autoimmune disease becomes self-perpetuating.

It should be noted that the role of any dsRNA in synergistic reinforcement of any combined treatment of dsRNA-IFN is not that of an IFN inducer, as in this case no synergism would be observed.

On the contrary, the dRNA is a unique agent that contributes to supplement and complement the action of the IFN, thus providing a degree of flexibility and effectiveness that could not be achieved by either the dRNA or the IFN when isolated in the treatment of human viral diseases. nas and cancer.

I observed low levels of NK cell activity in some members of a study group including patients with lung cancer and individuals with a family history of a high incidence of cancer. These observations are detailed below. I investigated whether the low activity of NK cells in patients with a family history of a high incidence of cancer could be restored to normal levels by adding various types of interferon and / or with unpaired dsRNA. Recent reports demonstrate a difference in the efficiency of increasing NK cells (cells derived from normal individuals) both between natural types (Brit. J. Kaem., 50, 85, 1982) and cloned subtypes (Can, Res, 42 , 1312, 1982) with interferon. I noticed that the unpaired dsRNA has the most pronounced ability to restore normal levels of increased NK cells in individuals at risk of developing cancer.

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The present invention is explained in more detail by means of the following examples in which all parts and percentages are expressed by weight. The dsRNA (unpaired) used in the following examples was of formula ri. r (C _in U), sometimes n 11-14 n is not represented by ri. r (C. „, U).

n 12 n

A. New mechanism of dRNA in growth modulation The new mechanism of growth modulation presented by unpaired RNAs is not shared by prototype lymphokines. This demons tra broad - based utility of dsRNA are ¹ in the modulation of human aberrant cell growth by a mechanism different from IFN induction. Specifically, induction by cyclic adenosine monophosphate (cyclic adenosine monophosphate = cAMP) by dsRNA was found in cells that were known to be insensitive to alpha interferon. These studies were conducted with two cAMP kinase inhibitors called fi-7 and HA 1004 (known metabolic inhibitors of protein kinase C and CAMP kinase) in order to assist in the assessment of the new anti-growth modulation activity of Unpaired dsRNA in the face of resistance to single-mode lymphokine therapy using alpha interferon for prototype lymphokine.

In order to demonstrate that the mismatched dsRNA does not work by inducing interferon propagation in IFN-insensitive cells, human glioraa cells (brain tumor) (A 1235) have been treated with the dsRNA for several periods of time since 0 to 8 hours. The dRNA was eliminated and fresh medium was added for 24 hours, determining the interferon titers (IRU / ml) then by analyzing the cytopathic effect (Finter, NG, J. Gen Virol. 5: 419- 427, 1969). The results are presented in the following table in which the limit

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detection interior was 5 IRU / ml.

TABLE 1

Induction of Interferon in Human Glioma cells treated with dsRNA

Indicator cell

HFF

WI3H

Duration of treatment (n) 0 2 4 6 8

5 5 5 5

5 5 5 5

From these results it was concluded that the dsRNA does not induce interferon in these IFN insensitive cells.

Next, identical cells were subjected to a confirmatory assay in order to determine whether interferons play any role in the anti-proliferative effects caused by dsRNA on the cells in question. This assay was done by using interferon antibodies that, if present, will bind to the antibody and reduce the antiproliferative effect inuuzieo by the dsRNA. Three different interferon antibodies (alpha, beta and gamma) at 240 neutralization units / '/ ml and one blank or comparison sample were used. Cell growth inhibition assays were carried out as described by iiubbell et al, Cancer Res. 44: 3252-3257 (1984). The percentage of comparison growth for the antibody was calculated as follows:

treated cells (24 h) - comparison cells (0 n) comparison cells (24 h) - comparison cells (0 h)

ORIGINAL BAD

J

The

I • Η t— (ο

Μ a

• Η

-μ d

Φ

π) ε

φ

Already • Η Ç • Η CM 0 {φ Ο The d φ Q 3 * 4 The Ρ Η 31 φ 0 Ρ φ

ο fi μ

Ο ο

Η

-Ρ

Ç

Φ results are

I o

— R — l cn φ

Ρ φ (0 Γ — 1 ο

t — d 'φ ο

Ε φ

υ ρ

<

ο ο

a φ

ρ • Η

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Ρ d

• Ο Ο d ο φ ίΦ ε σ 3 φ η μ φ φ η ε ο

a μ

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-μ d

<

> - =>

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H

I

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P d

(0 «ο_ a

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-μ

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presented in the following table

ι — 4 ο 3 r-1 • • • « ΓΟ Γ \ 1 m + 1 + 1 + 1 + Ί C0 η σ 1 "4 • « ♦ « Γ * π Q Q Ρ σ \ C0 LO 3 3 ο σ> ί — Ί ι — 4

Γ ~ • « • ι ^- 1 ro + 1 + 1 + 1 C0 Ρ σ • • • ο ο Q Ίΐ · σ ρ 3 00

σ ι — 1 ο • • • Ρ CN ro + 1 + 1 + 1 ω Ρ • • • ι — 1 ιΓ) Γ — 1 Q Ω Ω σ \ ι_Ω σ \ 3

'3 3 RO ω « • • • CN ΓΟ + 1 + 1 + 1 + 1 σ σ (Ν • • • • σ> ι — 1 ο ο m and Γ-

r_j ε σ <Η ε \ Ρ LOL Ηi γ-4 ε \ Ρ Γν * Η ί — I Ρ \ Ρ a ι ο Ο Ο Ρ ο ο ο CN CN > —1 ι — 1

d φ

0 Ρ ".You • Η Κ <41 pi d <“Η I I 1 φ α s 3 3 £ Ç Ω The The Φ ΓΟ Ο < Η H The H Ρ cxl φ 1-1 -tf μ Η Η The

ORIGINAL BAD 1

- - —d

The results shown are in!

is the percentage of growth of the comparison sample in 24 hours. ND = Not available. These data again confirm that dsRNA does not induce IFN in the cells studied.

The dRNA was found to induce cyclic AI4P in human glioma cells which was directly attributable to the dRNA and not by inducing IFN in these cells. Three known metabolic inhibitors, H-7, KA 1004 and pertussis toxin, inhibit or antagonize the antitumor effect of dRNA, as shown in the following tables. Percentage cell growth was again determined, as in Table 2 above, with the aim of obtaining a low percentage of growth in relation to the comparison sample. The isolated unpaired dsRNA causes a reduction in the percentage growth of the comparison sample; however, the presence of a metabolic inhibitor increases cell growth.

Table 3, below, shows the antagonism of the anti-tumor effect of dRNA by the metabolic inhibitors H-7 and AN 1004 in human glioma cells, again the values presented as a percentage of the growth of the comparison sample for dsRNA in concentrations 0 (blank or comparison sample), 25 and 200 µg / ml.

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TABLE 3

DsRNA (^ ug / ml)

0 25 2 00 1 H-7 (^ àM) 0 66.7-12.6 ! 48.2-5.4 5 103.3-13.6 108.3-15.5 75.2 ± 4.5 25 87.5- 6.7 89.7-11.0 72.8-7.9 HA1004 (yUE) 5 112.7-16.2 113.4— 3.2 94.1-13.4 25 113.2-14.3 117.2-16.7 10.7 ± 6.8

Similarly, a pretreatment of the same human glioma cells with

I pertussis inhibited dsRNA-induced antiproliferation, I as shown below. In Table 4 the percentage 1 of the growth of the comparison sample is reported at 24 hours in cells pretreated for 4 hours with pertussis toxin before the addition of dsRNA. It turns out that!

the known metabolic inhibitors H-7 and HA 1C04 have one!

i minimal effect on growth inhibition in cells treated with alpha interferon, while co-i, toxin queluche appears to have an oizasic effect - - does not cause any change in growth inhibition induced by: alpha interferon and high doses and increases the antiproliferative effect at low doses.

I

I

- 26 - '1

BAD ORIGINAL I

TABLE 4

DsRNA (yug / ml)

Treatment 0 25 200 0 88.4-2, I ^a 71.7 ± 5.7 3 µg / ml 98.7-7.1 102.5-7.3 99.9-6.3 1 µg / ml 94.0-5.2 93.8-7.1 + 1 93.2-5.1 C, 3 µg / ml 99.4-7.2 95.6-4.1 102.7-5.7 Per other hand, the IFN-alpha not au

increases the levels of intracellular cAMP in the same cells over a period of 24 hours, as shown in the following table. In this procedure, human gioma cells (A 1235) were exposed to zero (comparison sample), 25 ° and 1000 IRU / ml of IFN-alpha for periods from the beginning of the experiment to 24 hours.

TABLE 5

Duration of _ (IRU / ml) _ treatment O 250 1000 <0.02

1 min <0.02 <0.02 5 min <0.02 <0.02 10 min <0.02 <0.02 30 min <0.02 <0.02 1 H <0.02 <0.02 2 H <0.02 <0.02 4 H <0.02 <0.02 O H <0.02 <0.02 24 H <0.02 <0.02

- 27 ORIGINAL RAD ^

In this table, the values are shown in cAMP / µg protein picamoles; the detection limit was 0.02 pmol cAMP / pg of protein.

Levels of intracellular cAMP were then measured indirectly in human gland cells treated with dRNA (A 1235) by measuring adenylate catalysis activity (pmol cAMP produced in 15 minutes) immediately following antiproliferative doses of dRNA (25 and 200 µg / ml) in 30 minutes, as shown in Figure 1a, and after 24 hours in Figure 1B. In this procedure, the cells were treated with unpaired dsRNA, 25 (filled triangle) or 200 (filled squares) µg / ml of unpaired dsRNA. At the appropriate times (Fig. 1a; 0.5 to 30 min and Fig. 1B:

0.5 to 25 h) the medium was removed and the cells were quickly frozen on dry ice. Adenylate cyclase activity in cells treated by ultrasound was measured by the method of Young et al., Kolec. Endocrinol. 1: 884-988, 1987. Cell counts were performed at 24 hrs in order to verify antiproliferative activity.

l £ _.

The induction of cAKP in human glioma cells (A 1235) treated with dsRNA was measured directly in CAMP picamoles per microgram of protein and the results are shown in Figure 2a for 30 minutes and in Figure 23 for 24 hours. In this procedure, A 1235 cells were treated with 25 (triangles) or 20C (solid squares) jug / ml of the unpaired dsRNA. We

At appropriate times, the medium was eliminated and the intra-cellular CAI.P was solubilized in 0.1 N HCl. The cAKPi levels were determined using an RIA method, as described by Reisíne et al., J. Cell. Biol. 102: 1630-1637, 1986. cAKP levels in comparison cells and æ 1 µg / ml of dsRNA were below the cell counts at 24 h to verify antiproliferative activity- 28 cells treated with limit of detection .

ORIGINAL BAD ___. Uncle

t iva

These four graphs show a dramatic increase in cAMP after just 30 seconds of exposure to dsRNA (Figs. 1a and 2a) and a maintenance level after 30 minutes. The measurement at 24 hours, as in Figs. 1B and 2B, shows the effect of dsRNA over time.

i

These studies confirm that the increase in cAMP levels is directly attributable to ARBdc since ddRNA does not induce IFN in the cells used. Antiproliferative doses of dRNA induce adenylate cyclase activity within 30 seconds after the start of treatment. Similarly, levels of intracellular cAMP increased in an antiproliferative dose-dependent manner within 30 seconds. Pretreatment of cells with pertussis toxin, also an intracellular cAMP inhibitor, followed by treatment with dsRNA inhibits dsRNA-induced growth inhibition. In contrast, antiproliferative doses of natural human IFN-alpha do not increase intracellular cAMP levels during a 24-hour treatment period. In addition, the known metabolic inhibitors H-7 and HA 1004 have a minimal effect on growth inhibition in cells treated with IFN-alpha, whereas pertussis toxiin is found to have a biphasic effect, as noted above.

In studies of the cAl-P system in IFN-induced antiproliferation (Panniers et al., J.

Cell Eci., 48: 259, 1981; Banerjee et al. , Virology 129:

230, 1983; Ebsworth et al., J. Cell Physiol. 120: 14th,

1934), these authors found that cAitP was not associated with the degree of antiproliteration status. As far as I know, there was no study, before the ones reported here, on the RNAdc ¹ s in the same system. These studies support the conclusions that (a)

ORIGINAL BAD

DRNAs and lymphokines, of which xFN is a prototype, have different mechanisms of action and (b) anti-proliferation;

dRNA-induced interaction is associated with the cAMP system (while lymphokines are not).

Since caAP has an imbiquity nature, that is, most, if not all, cells have the genetic information necessary to synthesize a cAMP under the appropriate stimulus, these experiments indicate the occurrence of a broad utility base of unpaired dsRNA in growth modulation of aberrant human cells, these cells being relatively or completely resistant to IFN or other lymphokines when administered alone. These results suggest that the dsRNA acts through two sequential mechanisms - the first mediated by IP-kinase kinase, followed by the modulation of protein kinase C.

B. Synthesis of protein j

I í

I

Intracellular protein changes in cells treated with dsRNA reflect the differentiation and loss of the malignant cell phenotype, a property that is not characteristic of lymphokines. Cell normalization after treatment with dsRNA was studied according to

I with the method of Soslau et al. , Biochemical and Biophysi-;

i cal Research Communications, 119: 941,943, 1984. In this procedure, human brain tumor cells (A 1235) treated with antiproliferative doses of unbound ddNA (200 yum / ml) or natural human IFN alpha (100 pm /, / ml) showed significantly different patterns of protein synthesis over a 72-hour period. Protein synthesis is an important parameter since it indicates that the treated abnormal cells are in the process of normalization and indicates the extent or duration of the therapeutic effect. The results of this study are represented in the bar graph in Figure 3. Protein synthesis was

- 30 ORIGINAL BAD ______J

assessed by determining the counts per minute of cells at 1235 marked by radioactive isotopes for a pre-established number of cells. These cells were untreated cells (comparison sample, blanks blank), cells treated only with IFN-alpha at a concentration of ICO µm / ml (dashed dotted down from right to left), with A.RNdc (vertical dashes) at 200 yum / ml and together with IFN-alpha in the indicated amounts (dashed dashes rising from right to left). Measurements had been taken at 24 and 72 hours. A. The line above each bar represents a unit of standard deviation.

Although nominal changes were observed at 24 hours with both agents compared to the comparison samples, at 48 hours, both dsRNA and IFN showed significant increases in protein synthesis, with protein synthesis stimulated by dsRNA being significantly higher than at of cells treated by IFN. Surprisingly, there was a 3-fold increase in protein synthesis in cells treated with dsRNA compared to the comparison samples and the cells treated with IFN, in which the protein synthesis dropped to the levels of the comparison samples. The continuous increase in protein synthesis in cells?

I treated with dsRNA apparently reflects changes si- [j

gnificatives in the types of specific proteins produced as a result of cellular changes induced by: dsRNA for a more differentiated or standardized cell phenotype. ;

Protein phosphorylation studies; bladder carcinoma cells treated with interferon-betha showed significant changes in the phosphorylation of various proteins compared to untreated cells (Soslau et al., Biochem. Biophy. Ac ta, Vol. 119 , 1934, p. 941). In contrast my

- 31 - -j

ORIGINAL BAD

stuual statues (polyacrylamide gels not shown) with ri. r (C ,, .., U) demonstrate entirely different changes in phosphorylation patterns compared to cells treated with interferon. These data reinforce even more the molecular differences between dsRNAs and lympho -ouines in terms of mechanisms of tumor cell modulation, return to more normal cell phenotypes and fundamental differences in the pathways used in the modulation of immunodifferentiation and immunocompetence.

For a specialist in oncological and immunological techniques, in the practice of the basic invention described here in various ways, a specific protein characteristic of the malignant process for a cell of interest or a characteristic of the recovery of the malignant cell of interest will be selected and selected. changes in the selected protein or protein group will be monitored in the light of the invention described herein. Factors that vary from individual to individual can affect the net need for unpaired dsRNA over a period of time, however, not diminishing the usefulness of the present invention. Note - the presence of extraordinarily high levels of nucleases or phosphodiesterases can alter or cause the doctor to modify the concentration of the necessary dsRNA therapy as opposed to those presented in the examples and experiments mentioned here. Variations from patient to patient are to be expected, as in many aspects of clinical medicine.

Protein synthesis studies allow the underlying mechanisms to be followed from beginning to end;

I determine the kinetics of triggering the action of the product; the greatness of the product's action thus makes it possible to evaluate the number of cells in normalization and the duration of action j of the product and therefore the duration of the therapeutic effect, not yet anticipated by the numerous results of I

- 32 b ORIGINAL Menu j

investigation reported in this field by the applicant. | i

C. Intrinsic need for dsRNA in tumor cells for IFN anti-growth activity

I have determined that the anti-growth activity of interferon in tumor cells occurs only rarely and especially when IFN-sensitive tumor cells have pre-existing intrinsic dsRNA in a sufficient amount residing in the cells. anti-growth activity in cells that do not have intrinsic dsRNA or that have only an insufficient amount can be caused by application of dsRNA (by administration) to create conditions of dsRNA sufficiency.

Interferons are known to induce the 2 ', 5'-oligo-A RNA-dependent synthase RNA-dependent enzyme, which is implicated in inhibiting cell growth. The results of my research indicate that double-stranded RNAs essentially function as cofactors for enzyme activation, a distinct phase from interferon induction of this enzyme. ΐ

Historically, these two actions have been wrongly grouped, resulting in a drastic decrease in the potential of the therapeutic benefits of the two agents, either alone or in combination. Now demonstrate that ARMdc ¹ s occur naturally in cells mononucle! ares of peripheral blood (Ci-iSPs) of some individuals, 'notably in patients with filamentous cell leukemia, although they do not occur in the CNSPs of normal individuals. Undoubtedly, the presence of ARMdc's in patients i _r!

with filamentous cell leukemia now apparently explains for the first time the well-known efficacy of in-; terferon in cases where interferon is given alone and provides an additional critical dichotomy between IFN and dsRNA, which can be explored therapeutically.

ORIGINAL BAD

Namely, the clinical efficacy of interferon is due to the induction of an enzyme (the 2 ¹ , 5'-oligo-a synthase) in j!

presence of pre-existing intracellular natural RNA, while exogenous RNA acts mainly by activating pre-existing enzymes in this particular growth regulation pathway.

In order to verify the validity of my original hypothesis, I isolated nuclear RNAs from normal individuals for comparison and rIFN-c <A of leukemia cells (HeLa) from filamentary cells, I fractionated this material in sucrose gradients and investigated its activation capacity of 2 ', 5'-A purified high molecular weight synthetase. None of the RNA fractions from untreated HeLa cells showed synthase activation capacity. However, the fraction of the heterogeneous nuclear RNA (nRNA) of the IFN-treated cells activated the synthetase in a dose-dependent manner. High pressure liquid chromatography (HPLC) analysis of the enzyme products showed that active 2 ', 5'-A trimers and tetramers were formed. I also fractionated nuclear RNA from mononuclear cells from patients with filamentous cell leukemia, which are extremely sensitive to IFN therapy, and demonstrated the occurrence of high levels of dsRNA ¹ s activating the 2 ', 5'-A synthase in the ARNnH. HPLC analysis revealed the occurrence of trimer, tetramer, pentamer and he- | 2 ', 5 ¹ -A biologically active shamans (approximate ratios 53: 15: 4: 1). Nuclear RNAs from normal mononuclear cells did not cause activation. These results indicate that there are natural nuclear RNAs that quan- ¹ present in relevant cells, neoplastic or other, can participate in the regulation of growth by IFN. In fact, there is a critical need for an interferon response so far undetected. These facts allow me to deduce that deficiencies in these NRAs can lead to an ineffectiveness of the IFN, which can, however, - 34 -

BAD ORIG ^tM AL!

be overtaken by exogenous dsRNA, a point that I was able to investigate and confirm through clinical trials as I have described elsewhere in the present specification.

In order to be effective, IFN activity requires the presence of dRNA in the cell where anti-growth activity is sought, which is rare. This presence is apparently necessary in order for the tumor cell to be receptive / sensitive to IFN therapy. . Some researchers wrongly group the IFNs and the ARNdc's together. From these and related investigations it appears that ¹ s dRNAs are cofactors that activate the 2 ', 5'-oligo-A synthase enzyme. IFN-insensitive tumor cells, when exogenous dsRNA are supplied, become susceptible to successful dsRNA therapy. In cells that respond to treatment with IFN is thought that dsRNA or dsRNA's intrace lulares have a three dimensional structure very similar to the mismatched dsRNA s ^1, particularly the ^ rl. r (C ^^ U) of the present invention, based on the respective biological and catalytic properties and lack of toxicity, on the enhanced immune function when compared to the cytotoxic function as well as on other data, such as the parameters of cAKP and shift cells.

Cells that do not respond to lymphokines in general and IFNs in particular become able to respond if they are first provided with an effective amount of exogenous dsRNA in order to induce the synthesis of intracellular dsRNA intended to participate in the regulation of cell growth in association with an interfering, and therapeutic, surround the dc RNA, then supplying the IFN, now made while optionally continuing the required formulation.

ORIGINAL BAD

D. Human kidney tumor graft responses

The responses of human kidney grafts to IFN and dRNA are fundamentally different in terms of long-term survival and magnitude of the effect, as explained in the investigation that follows.

I used several original animal systems using grafts of human tumors in athymic rats in order to show an unexpected and differential response to interferons and dsRNA. For example, human renal cell carcinoma demonstrated progressive tumor growth and there was no increase in survival during treatment with alpha interferon, while I observed significant decreases in tumor size and dramatic increases in survival with dsRNA treatment. In fact, after death aged 2 to 4 years due to natural causes, 95% of rats treated with dsRNA did not show the histological presence of tumors at autopsy. This result is completely unexpected, since cancer remissions caused by lymphokines (for example, by IFN) are notoriously short-lived and there are few or no examples of animals without a tumor after several months. Thus, the observation of a 95% cure rate by histological criteria and survival data is completely unexpected. Furthermore, the activity of natural destruieoras cells ⁽¹ in = tural killer NK) spleen was increased in animals treated with dsRNA while not noticed increased activity of NK cells in mice treated with interferon.

Similar differences were noted in the growth inhibition between gamma interferon and dsRNA in two different graft models and I also observed pronounced differences in human bladder carcinoma RI'4 and in human brain glioma A 1235, as shown

- 36 ORIGINAL BAD

above. In both cases, there was a statistically significant inhibition of tumor growth in animals treated with dsRNA while minimal inhibition of growth was observed with isolated gamma interferon. Significant differences were also observed in the activity of splenic NK cells with increased NX activity by dsRNA but no increase in NK activity was observed by gamma interferon.

Similar effects have been observed in terms of increased immune activity; Table 6 below shows the results with brain tumors. Dosages were 20,000 IRU of interferon per day and 2 to 3 weekly doses (200 to 500 yjg per dose) of ri. r (C .., ,,, U) · A careful study of an 11—14 n samples of blood (obtained serially at regular intervals from the caudal vein) showed that the chemical cDNA RNA described elsewhere in this specification could be administered indefinitely without any renal effect, on bone marrow or on inconvenient immune function ', it being noted that these organs have already been hysterically considered to express toxicity.

In Table 6, shown below, immune cell activity is shown in spleen cells of rats treated with INN-gamma and / or with dRNA.

The study gathers effecry cells (oothiomatic immune cells by surgery from spleens of animals with human tumor grafts) to targeted cells (in this case, human tumors or cherry cells) in incicaca proportion. The effector cell is an immune or immune cell and the target cell is a specific cancer cell of interest. The results are presented as a percentage! cytotoxicity (cell death) as a result of treatment (IFN, dsRNA or both). A higher percentage of toxicity than the comparison sample indicates an improvement in the eradication of tumor cells.

- 37 BAD ORIGINAL frame

Effector cell treatment

Comparation

200: 1

100: 1: 1

IFN range

200: 1

100: 1: 1

ARNdc

00: 1

100: 1: 1

IFN gamma + dsRNA

200; 1

100: 1: 1

Comparation

16.3

14.2

7.5

6.4

2.9

2.8

2.3

22.8

13.2

28.7

22.8

17.2

Interestingly, in this assay the ipq-gamma killed fewer qo cells than in the comparison group (without any treatment), while the isolated dsRNA gave the best results with the combined treatment immediately afterwards,

- 3 3 - ORIGINAL BAD 'ι

The effectiveness of dsRNA, specifically ri. r (C ,, U), against brain tumors hu π jl i “JL - n humans grafted onto athymic rats compared to IFN-gamma is illustrated in Figure 4 of the drawings. This figure is a graph that compares the two-dimensional dimension of the tumor, calculated in length times width, in n; ^z .

The line joining the circumferences refers to the comparison group (without any therapy); the line joining the white triangles refers to the isolated IFN-gamma (p>0.5); the line that joins the squares in bold refers to the RNA dl ^. ^r ( ^c j_2_q4 ' ^u ) _n (p> 0.50). Tumors are measured to IO ^{9 9} 31 days following the start of the experiment.

DRNA was effective in reducing tumor size when compared to IFN-gamma which was only slightly more effective than the comparison sample.

E. Clinical examples of lymphoguin-resistant tumors that respond to dsRNA

A methodology was developed. ternative from an animal model to assess tumors susceptible or resistant to IFN therapy and to identify cases where a potential clinical synergy is likely for the combination therapy of IFN and dRNA. One technique as mentioned is the technique of collogenic analysis of human tumors developed by Hamburger and Salmon (primary Bioassay of Numan Tumor Stem Cells, Science, 197: 461-463, 1977), which allows to safely predict the clinical response or resistance to anti-cancer products in. individual patients with a degree of accuracy of 90 to 95 p at the cellular level with a degree of confidence for inactive products and d 'for active products.

7 '5 o c

Colonogenic analysis is carried out according to. the Hamburger and Salmon procedures as follows. Tumor cells or cells

- 39 ORIGINAL BAD

Fresh leukemics are obtained from the patient and prepared in the form of a single-cell suspension. It is applied to an agar or methyl cellulose plate or to cells after culturing a predetermined concentration of the candidate therapeutic agent, calculated from the literature and other dosage standards. the cells are washed and inoculated on agar or methyl cellulose and incubated in Petri dishes at 37 ° C to allow the growth of cell colonies. Following a predetermined period of cell growth, colonies are inspected visually with a microscope and the number of colonies on each Petri dish is counted. Colony biology and cell composition are then studied in more detail, including cell morphology, cariology, auto-renewal and immunology and the effects of cells in rats up to now. (naked). The cells are analyzed using monoclonal antibodies and nucleic acid probes. colonogenic analyzes represent a reliable approach to the assay of tumor cell chemosensitivity in vitro.

i j

Colonogenic analysis assays have been carried out on various tumor specimens in the inventor's lab. unlike the absence of activi. expected antitumor activity, as described in several post-cancer treatment sims (Compilation of Phase II Results with Single Antineoplastic Agents, US Department of Health and Human Services, Public Health Service, National Institutes of Health, Volume 4, 19S5) that report that about 1G0 individuals received poly I.poli C without any or all or partial responses, the inventor noted) a predicted response rate of 42 using an AxHdc de- ^! unmatched in a wide variety of human tumors histologically insensitive to polynucleotide therapy. '!

ί l

For the purpose of clinical evaluation

- 40 BAD ORIf? '& ξξ§ $ &# ft * C £ aayv ?. -v ^ - _

preliminary, tumors considered sensitive showed a cell decrease of over 60% in the number of tumor cell clones.

In the vast majority of examples, competing trials were carried out with individuals both in the laboratory and in the clinic in order to determine their tumor sensitivity to various generic IFN classes and / or to various types of interleukins. Specifically, individuals who participated in the dc RNA studies had previously undergone various unsuccessful clinical treatments with interferences or interleukins at doses deemed effective based on published medical data. In all cases, the methodology with the specific lymphokine was not successful in preventing the continuation of the growth of the tumors and in a majority of cases the profiles of the immune cells remained unchanged or in many cases they even decreased (worsened) in relation to to the values verified before the infusion of the isolated specific lymphokine.

clinical laboratory in which the investigations of the present invention were carried out established at least three unexpected results:!

. the preferred unpaired dsRNA gives good results while poly I. poly C does not;

. the preferred unpaired dsRNA is effective as long as the isolated IFN is ineffective;

j

I. synergism exists when the preferred unpaired dna RNA and one are used in comination. lymphokine.

The relative sensitivity of various histological types of human tumors to unpaired dsRNA was studied (see Table Ί below). The inbadorigijy experience .L '

hint that more than 42% of a wide variety of different tumors, especially solid tumors, although resistant to exposure to lymphokines when isolated, are extremely sensitive to the antiproliferative effects of the specific molecular configuration of dsRNA's.

Logical histo type

TABLE 7

Quantity Quantity of resistant sensitive

Endometrial carcinoma 2

Glioblastoma 4

Lung carcinoma 3

Renal cell carcinoma 33

Carcinoid 3

Practical carcinoma 1

Breast carcinoma 4

Kelanoma 5

Colorectal carcinoma 5

Sarcoma 1

Carcinoma of the esophagus O

Kesothelioma 0

Pancretic carcinoma O

Gastric carcinoma 0

Meduloblastorna 0

7

2

Percentage of sensitive

100 80 60 52 50 50 36 36 3 õ 13 ⁰ i 0 O

TOTAL 61 85 42% 1

This observation is unexpected, however, for two reasons - the biomeric history of the dsRNAs, ~ 1 in general and the lack of clinical utility in the 1nroquinas.

The effects of IFN-alpha, IFN-beta, and the preferred unpaired dsRNA in the colonogenic analysis described above are shown graphically in cells

-T-J

BAD ORIG / N

of melanoma in Figure 5. The percentage of colonies in j relative to the comparison sample is plotted against the dose of ri. r (C _ln .., U) in micrograms oor ml and in IRU / ml for interferons; the baseline corresponds to 100% of the colonies in the comparison sample. Note that both IFNs are above the baseline (unregulated) except when in cytotoxic quantities. j í

!

I

Figure 6 shows the synergistic antiproliferative effect of IFN-alpha and r1 ^ r (C ₁₂ , U) on human renal cell carcinoma, also as a percentage of the number of colonies in the comparison sample. Two amounts of ri were used. r (C _n _, U) at 50 and 100 n 12 n micrograms per ml in each combination. the concentration of IFN-alpha was 0 to 3000 IRU / ml, which is well above the normal therapeutic contractions of IFN-alpha.

There is an interruption in the line following the initial readings (indicating no effect on the IFN comparison colonies, about 62 / para; 50 and about 50% Dara ICO ri. R (C \ „, U) and I then read a reading for 100 IRU / ml of IFN-alpha. As these data show, IFN-alpha alone was not effective in the clinically acceptable dosage range, it was only effective for IFN-alpha above these dosages but at levels cititóxicos, demasiaaamente high levels for clinical use. the results of this analysis for a inoicam therapy effective amount of IFN i must be reduced to a clinically acceptable level, and preferably ^should! anriproliferativo be supplemented with another agent.

Specific cases of synergistic effects have been found in 63% of melanomas, 50% of breast cancers, 80% of ovarian carcinomas and 65 / of renal c & r for combined IFN-alpha + rl ^ / therapy

I. r (C. „, U). In this way it can be determined from n

of these examples that (a) the application of dsRNA's is unmatched

- 43 ORIGINAL BAD

those specific to lymphokine-resistant states have clinical utility; (b) in cases of resistance to isolated dsRNA, a judicious combination with lymphokines should result in a significant therapeutic benefit in most cases.

F. Below are three clinical examples that confirm the effectiveness of the present invention

Patient No. 1 - Malignant melanoma

--- - ----- - - - - - - - Patient η® 1, male and middle aged, had a malignant melanoma that does not respond clinically to either IFN or IL-2. The administration of dsRNA (300 mg twice a week) resulted in the complete elimination of the tumor (according to the determination of the tumor mass) after 80 days of therapy; see Figure 7. This favorable response continues and approaches □ approximately 2.5 years after initial treatment. The current maintenance dose is between 50 and 100 mg twice a week. Dosage is determined by considering the clinical response in the light of laboratory parameters !, i

including the relative level of the 2 ', 5'-a synthase enzyme and the ability to detect biochemical intermediates active in the effect induced by the dsRNA, such as the cyclic ARP levels described above or the presence of specific classes of 2', 5'- molecules The ones that can harm the tumor's stopping situation as well as an improved profile of the immune cells. These circumstances are shown in Figure 7, in which the inventor determined approximately contemporary increases in bioactive 2 ', 5 ^{* 1} -a, cAKP and 2 ¹ , 5'-a oligomers! none of which was present while the patient received lymphocyte therapy or in his Nash-out period without treatment. 2 ', 5 synthase activity (-ademylate (pmol ATP / µg protein) is indicated by

- 44 ORIGINAL BAD

points represented by solid circles in relation to the volume of the tumor mass (indicated by points represented by solid triangles) for several days before the start of therapy, with the start of unpaired dsRNA therapy and at the end of therapy when a complete response (virtually cancellation of tumor volume).

See also Figure 8 for the same patient, in which the oligomers designated by P3A3 and higher (determined by high pressure liquid chromatography) are shown, verifying that these oligomers do not appear until after administration of the unpaired dsRNA, and note the correlation between the presence of these oligomers and the clinical response. The various oligomers were measured at intervals of 25 days before therapy with unpaired dsRNA and 1, 23 and 55 days after the start of therapy. It has been found, both in the inventor's laboratory and elsewhere, that low molecular weight oligomers (dimers) are especially bioinert in terms of anti-growth (antiproliferation), antiviral and immune-enhancing properties.

Similar biochemical phenomena impair the prolonged increase in the activity of lymphokine-activated destructive cells (lymphokine-acti. Vated killer = LAK) and the stable and / or drastic regression of the resulting tumor, as shown in Figure 9, for which it appears the measure of the elevation of LAK cells (as% Cr specific release) in a patient with a stabilized disease state (carcinoid cancer) following more than 30 months of therapy with dsRNA (left column) in comparison with patients with solid tumors who did not receive dsRNA therapy (center) and normal patients or individuals for comparison (right column). These data were used to check the relative amount of therapeutic agent

- 45 BAD ORIGINAL tic needed to maintain a favorable clinical effect for long periods of time, as in maintenance therapy following the initial tumor domain, even in the presence of lymphokine resistance.

Patient # 2

Of particular significance is the recognition by the inventor that the spread of cancer to the bones can be reduced dramatically by combining dsRNA with IFN. For example, patient no. 2, with renal cell cancer and abundant bone metastases, showed a rapid and prolonged response after receiving a daily dose of just 1.5 m units of IFN-alpha and 300 mg of dsRNA administered twice a week. In one case of chronic leukemia, a similar regimen induced prolonged remissions in a high percentage of subjects, while patients who received IFN-alpha alone did not show any significant clinical response.

Patient # 3, rd 3, a middle-aged male patient with an IFN-resistant kidney tumor, experienced a rapid onset of tumor reduction at the same time as a change in the profile of the 2 ', 5'-A olicomers, a rapid onset modification of the cANP pathway and a dramatic increase in immunovigilance activity. These significant clinical and laboratory checks persisted for 36 consecutive months and were not accompanied by any therapy-related side effects.

I

Similar clinical observations have been obtained with clinical success in most lymphokine-resistant tumors, including the most lethal and

- 46 BAD ORIGIN /.

lung cancer, with the respective data being presented sumptuously in Figure 10. the comparison of NK cell activity (measured in lytic units and in mediastinal dimension of tumor size (in 2 mm)) was measured by a CT scrutinizer. Results for the start and end of about 400 days of unpaired dsRNA therapy are shown.

patient had a complete clinical response to therapy based on measurements of tumor size.

Claims (1)

Process for the preparation of an unpaired dsRNA (double-stranded RNA) characterized by oligomer complexes, polymeric complexes or both, preferably oligonucleotides, that hybridize with complementary single-stranded polymeric RNA to form the duplex RNA. 23. The method according to claim 1, characterized in that the dRNA contains regions of breakage of binding and has the favorable therapeutic proportion properties of ri, r (C _ni , „, U). n 11-14 n Process according to either of Claims 1 and 2, characterized in that the dRNA has a three-dimensional structure identical to that of the dRNA in which the tumor cells to be treated are deficient. - 4 - i J ΐ J Process for the preparation of a pharmaceutical composition useful for modulating the growth of tumor cells and for the loss of the malignant cell phenotype characterized by incorporating an dDNA as an active ingredient when prepared according to any of claims 1 to 3. - 5th according to claim ORIGINAL BAD Process 48 cation 4, characterized in that the pharmaceutical composition also contains lymphokine in an amount effective for inhibiting tumor growth. 6. A process according to claim 5, characterized in that the lymphokine is an interleukin or an interferon. Lisbon, 6 July 1988. ORIGINAL BAD PROCESS FOR THE PREPARATION DS ARNdc 5 DE PHARMACEUTICAL COMPOSITIONS THAT CONTAIN IT USEFUL FOR THE MODULATION OF CELLULAR STATES LYMPHOKIN RESISTANT The invention relates to a process for the preparation of an unpaired dsRNA (double-stranded RNA) which comprises linking oligomer complexes, polymeric complexes or both, preferably optimal oligonucleus, which hybridize to complementary single-stranded polymeric RNA. to form the duplex dsRNA.