PT100688A - PROCESS FOR THE MODULATION OF PATHOLOGIES ASSOCIATED WITH CYTOKINE DISORDERS BY DUAL CHAIN RNA - Google Patents

Description

Description of the patent application of HEM Pharmaceuticals Corp., North American, industrial and commercial, established in One Penn Center Suite 660, Philadelphia, Pennsylvania 19103, United States of America, (inventor: Willi-am Carter, United States of America), to " PROCESS FOR THE MOVEMENT OF PATHOLOGIES ASSOCIATED WITH DYSFUNCTIONS OF CYTOKINES BY DU PLA CHAIN RNA ".

DESCRIPTION

Cytokines are natural macromolecular cell products that have the ability to enhance normal cell functions, including those of lymphocytes, which in turn have useful properties called " immune surveillance ". However, cytokine dysregulations characterized by aberrations in production and / or distribution in body tissues may cause or aggravate a number of human diseases. The present invention describes an easy way to biologically neutralize the potential undesirable effects of conditions characterized by cytokine and lymphokine dysregulations. By judicious use of certain ARNs in specific programmed dosages it is possible to minimize adjuvant organ damage and to encourage host recovery from disease states.

Dysfunctions of cytokines and lymphokines (produced, by or directed to lymphocytes) have been progressively associated with severe human diseases. No effective remedies have yet been proposed,

in the present invention.

Although normal functions of body cells (eg, bone marrow, immune system, connective tissues, lung function, etc.) require cytokines, aberrations in their production are associated with disease acceleration and morbidity.

The aberrations in the production of cytokines may consist of: (a) insufficient production by cells which normally produce certain cytokines, (b) overproduction by cells which naturally produce such macromolecules, (c) production of aberrant polypeptides such as active cytokine molecules with minor errors in the respective amino acid sequences that cause changes in the activity or range of target cells, and (d) abnormalities in tissue localization or concentration in cells that can not tolerate the presence of these cytokines without injury cell phones.

SPECIFIC EXAMPLES OF ABERRANT LYMPHOQUINS AND HUMAN DISEASES (A) Tumor Necrosis Factor (TNF). TNF and an immune mediator derived from macrophages / monocytes, which has been implicated as implicated in septic shock, cachexia, host disease versus grafts (Piguet et al., Journal of Experimental Medicine, 1987 vol 166, pp. 1280-1289 ), parasite infections, vascular diseases and neoplastic diseases. In particular, elevated TNF levels are associated with unfavorable clinical outcomes. TNF can also stimulate cells to produce other cytotoxic factors which may in turn cause additional cellular inflammation or death of cells, even in brain cells (certain cells called oligodendrocytes). Patients with HIV disease who have a high level of TNF (greater than 50 picograms per milliliter) have a progressive loss of developmental patterns and intellectual abilities (Mintz et al., Am. Journal of Diseases of Children, 1989, vol. 143, pp. 771-774). Interleukins (in particular IL-1) may facilitate the production of TNF, such that affected human hosts often exist in an absence of lymphokine environment except for a group of lymphocytes. aberrant foci (as defined above) - induced by -

a biological cascade effect.

The therapeutic objectives to be attained with the present invention are intended to neutralize by biological means or to provide various levels of control of several aberrant lymphokines which may coexist in the same individual.

Levels are elevated in people with malignant tumors beyond the normal upper limit, which in children is about 40 nanograms per liter (Saarinen et al., Cancer Research, 1990, vol 50, pp. 592 -595). (B) INTERFERES High production of interferon gamma and characteristic of HIV-infected thymus derived cells (Arya et al., Proc. Natl Acad Sci USA 1985, 82, pp. 8691-8695). Lymphatic nodules infected with HIV in the follicles show elevated levels of interferon gamma production (Emile et al., Journal of Clinical Investigation, vol 86, pp. 148-159). Individuals with high interferi levels) have more unfavorable clinical prognoses than comparison groups having lower or undetectable levels of lymphokines. The production of aberrant lymphokines (as defined above) thus contributes to the disease pathology itself which are by nature intended to correct. (C) INTERLEUQUINES. IL-1 beta levels are elevated in children with severe perinatal complications (280 ± 116 picograms per ml; Mille et al., Journal of Pediatrics, 1990, vol 117, pp. 961-965). These investigations indicated that IL-1, IL-6 and TNF in spinal cord plasma are related to clinical complications in the perinatal period. IL-1 concentrations are also related to the severity of malaria; see Kwitetkowski et al., Lancet, vol. 336 (November 17, 1990, pp. 1201-1204). Transiently elevated TNF production is a normal response to malaria infection, but excessive levels of production predispose the patient to cerebral malaria and a fatal outcome. (D) Other Metabolic or Cellular Manifestations of Pro-. duction of lymphokines. A convenient biochemical method for detecting deregulation may be based on the measurement of an intra-

(eg sometimes referred to as the 2, 5'-oligoadenylate system or shortly 2-5-DAS). This metabolic pathway may be assayed in different cell types, which in turn may be fractionated by different methods such as 2 or 3-color flow cytometry, gradient centrifugation, etc. Simultaneous conditions may be present at the same time. T4 helper / inducer T4 cells in γ-cytotoxic suppressor T8 cells, natural killer (NK) cells, B cells (involved in the production of antibodies), CT (cytotoxic T cells) or ADCC cells ( antibody-dependent cytotoxic cells). These alterations can be effected by extracellular TNF, by interferons (alpha, beta or gamma) or by IL1, 2, 3, 4, 5, 6, 7 or 8 - each acting alone or together or through a multiplier effect in " cascade ".

Patients with rheumatoid conditions (arthritis or systemic lupus) typically have a measurable interferometry (greater than 6 IU per bioanalysis per ml or equal to or greater than 1 IU per RIA). When, on the contrary, patients develop a specific antibody to an interferon they may have inactive rheumatoid diseases without visceral involvement (von Wussow et al., Rheumatology International, 1988, vol.8, pp. 225-30). It is an object of my invention to provide the functional or operational equivalent of an antibody, i.e., to perform " fine tuning " from the level of extracellular cytokines to the cytokines which are relatively non-toxic to the host and which can be physically beneficial.

Correlating measurements of cytokines in synovial fluid, especially TNF and IFN, in patients with arthritis are consistent with the existence of a relationship between tissue damage and lymphokine activity (Hopkins et al., Clinical and Experimental Immunology, 1988 , vol 73, pp. 88-92). Aggressive activation of immunocompetent cells may even cause vascular inflammation associated with a high level of IL-2 and TNF receptors. This situation may be particularly marked in Kawasakai disease involving coronary artery lesions, as recently described in the Japanese Journal of Allergology (vol.39, pp. 118-123, 1990, Feb.). • Excessively activated immunocompetent cells. may also invade the muscle cells causing myositis and a disease-

in groups of muscles. In the present specification also show how a biochemical condition in the functions of the metabolic pathway 2-5-OAS is related to inflammatory diseases of the muscles and associated neurosensory pain and that symptoms can be effectively controlled by biological neutralization of the causative cytokines. The dsRNA used in the procedures described herein may be a complex of a polyinosinate and a polyglycidylate containing a ratio of uracil or guanine bases, for example from 1 in 5 to 1 in 30 of these bases (poly I, poly <C ^ _2gX> U or G)).

The dsRNAs may be of the general formula (IX) or (C19-U). Other suitable examples of dsRNA are discussed below.

&Quot; Unpaired dRNA &quot; is meant a dsRNA in which the hydrogen bonds (stacking of bases) between the corresponding chains are relatively intact, i.e., are halved on average less than one base pair in every 29 consecutive base pair residues. The term &quot; unpaired dsRNA &quot; must be understood in agreement.

Preferred dsRNAs for use in the present invention are based on copolynucleotides selected from poly [0U) and II 9

poly (Cn, G) in which n is an integer having a value of from 4 to 29 and are mismatched analogues of polyriboinosine and polyribotidylic acid complexes formed by modification of R1C in order to incorporate non-corresponding bases ( uracil or guanine) along the polyribo-citi dilate (rCn) chain. Alternatively, dsRNA can be derived from poly (I) poly (C) dsRNA by modifying the ribosyl backbone of polyribo-nosinic acid (rIn), for example by inclusion of 2'-O-methyl ribosyl residues. The mismatched complexes can be complexed with an RNA stabilizing polymer such as for example cellulose and cellulose. These mismatched analogues of R1, R2, R3, R6, R6, R6, R6, R6, R6 and R6 are as defined above. are described by Carter and Ts'o in U.S. Patents "14 Π Π Cà Π 4 130 641 and 4 024 222." The dsRNAs described in the said patents are generally suitable for use in accordance with the present invention. The preferred mismatched dsRNA or AMPLIGEN ™ from HEM Pharmaceuticals Corporation of Rockville, MD, USA, is commercially available in the form of a lyophilized powder.

Other examples of unpaired dsRNA for use in the present invention include:

poly (I). . poly (C4, u) poly (I). . poly (C7, U) poly (I). . poly (C 13 U) poly (I). . poly (c22, u) poly (I). poly (C 20, S) poly (I). poly (CggsG) and poly (I). poly Cp23 G &gt; P

Another class of dsRNAs suitable for the present invention are the short dsRNAs of defined structure, for example oligonucleotides of the formula: (i) -N- both greater than 5 and less than 100, inosine monophosphate, C and cytidine monophosphate, and wherein the closures in a chain are complementary to the closures in the opposite chain, or an oligonucleotide having the structure: ) vA_7 .-- closure 3 '

(C) y1_7k-closure 5 'where x and y are both greater than 5 and less than 25, j and k are both greater than 1 and less than 10, I and C are as defined above, A and a nucleotide other than I, and U is a nucleotide that forms a base pair with A.

Alternatively, the short oligonucleotide may have the structure: wherein: n, m, I and C have the meanings defined above.

These oligonucleotides may have substitutions on a chain which are not complementary to nucleotides on the opposite strand. Preferably such oligonucleotides are stabilized by internal registers of complementary heteropolymers and it is desirable that the closures and flaps of the non-

both oligonucleotides contain regions of complementary heterolpolymer. These oligonucleotides preferably have single stranded extensions. These oligonucleotides are described in more detail in PCT / US89 / 02172.

METHODS OF CONCRETIZATION OF THE PRESENT INVENTION

ILs, TNF-β and IFNS are immunoregulatory proteins but the deregulation of these proteins ends up causing pathological phenomena in the tissues. IL-1 and TNF have common activities, including pyrogenicity, T-cell activation, and neutrophil activation. Elevated levels are found in patients suffering from rheumatoid arthritis and other inflammatory diseases.

I observed in male Sprague-Dawley rats (300 to 400 grams in weight) that the E. coli liposaccharide (2 mg per kilogram) produced, according to expectations, the usual peak TNF levels (from 0 to &gt; 50,000 units / ml) in 60 minutes or less. Shortly afterwards, a significant percentage of the animals developed a disease with lassitude, fever, dehydration and death. However, I have found that if animals with unpaired dsRNA were treated previously (1 to 20 mg / kg), resulting in blood levels of 1 to 200 micrograms / ml, they survived and, from a clinical standpoint, they had only a minimal transient disease . Thus, it concludes that dsRNA at certain levels and administered according to specific programs had a new and unexpected ability to biochemically neutralize certain actions of the lymphokines associated with a high morbidity.

Next, I identified a group of individuals suffering from recurrent fevers and swelling of the lymph nodes. Some also showed signs of a viral infection, as I describe later. I detected evidence of elevated levels of IL-1 and TNF proteins circulating in the blood of these subjects by ELISA and also simultaneously detected signs of hyperactivity of the intracellular biochemical metabolic pathway associated with lymphokines (2'-5-oligo-adenylate synthetase ) whose terminal mediator is the RNase L protein. The 2'-5'-oligoadenol / RNase L metabolic pathway is shown in Fig. 2 of my European patent application 285,263 and was studied using procedures described in Carter | et al., The Lancet, 1286-1292 (June 6, 1987). - 7 -

This study refers to metabolic hyperactivity in metabolic pathways dependent on associated cytokines and inflammation of muscles and joints. The activity of RNase L was studied in Charlotte, North Carolina, for sixteen subjects under analysis, which were distributed as follows with respect to RNase L activity:

10 of 16: high RNase L activity (range: 110 to 500) 3 of 16: normal RNase L activity (62, 67 and 94) 3 of 16: reduced RNase L activity (7, 44 and 58)

For classification purposes, high levels are considered to be higher than 100, normal in the 60 to 100 range, and reduced to less than 60. Clinical observation reveals that individuals with higher RNase L levels suffer from more severe sensorineural pain while that intermediate RNase L levels cause mixed symptomatology. Individuals with the lowest RNase L levels were encephalopathic, ie, they had signs of central nervous system disorders characterized by cognitive deficits. Individuals with normal RNase L levels had a relatively good clinical status with mild chronic fatigue and were able to work or attend classes if they were collared. Figure 1 shows a giant cell / HHV-6 analysis in which peripheral blood cells from several patients were cultured for 10 days and stained with monoclonal antibodies against HHV-6. The percentage of cells positive for HHV-6 was recorded as a function of the time of treatment with dsRNA. The reduction of HHV-6 positive cells was statistically significant by means of the double t-test (p <0.01, 2 sides). The determinations were conducted over a period of about 80 weeks, while initial evaluations were performed prior to dsRNA therapy. Figure 1, attached, shows that the metabolic pathway was found to be excessively elevated in the blood cells (lymphocytes) of patients with severe neurosensitive muscle pain as well as recurrent fever. When the metabolic pathway was closer to normal,

peripheral blood, individuals suffered more in terms of brain function, leading to the conclusion that aberrant cytokine production localized to &gt; effects on the central (brain) areas instead of observing that the molecules caused lesions in the peripheral tissues of the body in these individuals.

By reaching levels of blood-dysregulated dsRNA similar to those I had determined to be effective in animals, more than 50% of the individuals showed clinical improvements and the abnormality in the biochemical metabolic pathway 2-5A was abrogated, as in animal experiments.

A high percentage of the subjects examined were simultaneously infected with a human herpes virus (HHV-6) that interacts intimately with the cells of the immune system (Levy et al., Viriol, 1990, vol 178, pp. 113 -121). (As shown in Fig. 1) that individuals given dsRNA showed a simultaneous reduction of circulating HHV-6 titers as well as levels of a retrovirus with limited structural homology to HIV AIDS).

In laboratory experiments performed simultaneously, I have found that HHV-6 can in fact cause a number of cytokines to be elaborated from human blood cells, including the cytokines designated TNF and IL-1, in amounts comparable to those found to be neutralized by by dsRNA in animals or man.

Two panels were analyzed for cytokine dysfunction. The first, panel A, was analyzed for IL-1-beta protein release following infection with HHV-6 in human cells.

Inductor IL-1 amount produced (picograms / ml) A. HHV-6 3 400 B. Lipopolysaccharide 5 300 (positive control) C. No stimulation 725 (negative control)

In the second panel the analysis of TNF-alpha protein release after HHV-6 infection in human cells was studied. - 9 -

Claims (1)

Inducer TNF (peak / ml) A. HHV-6 1 825 B. LPS 810 C. No est. 190 The tables shown indicate that the levels of cytokines, IL-1 and TNF-alpha, respectively, were measured in supernatant fluids using ELISA assay kits according to the general instructions of the supplier (R and D Systems, Minneapolis, MN). HHV-6 (10 TCID50 / ml) was added to PBMC cultures and the level of the cytokines was determined after 2 to 6 days of culture. The action occurs at the level of target cells, in which lesions impairing vital cell functions occur. It should be noted that the viral intermediate y, by causing the production of lymphokines which nevertheless are &quot; normal &quot; in cells and tissues not typically associated with the production of cytokines actually causes a state of cytokine production dysfunction since cells bathed in the cytokines thus released can not withstand the detrimental effects thereof and undergo morphological and functional changes which lead to an acceleration of disease and morbidity. Thus, I have come to the conclusion that the mechanisms now studied for the first time to neutralize these harmful macromolecules may result from a curtailment of the production of these, in a first hypothesis, or alternatively, the neutralization of their action at the level of the target cells in which would injure the vital functions of cells. A method for modulating the pathology of diseases associated with an aberrant cytokine production characterized in that it comprises administering to a patient suffering from a cytokine dysfunction of a non- An effective homeostatic amount of a double-stranded ribonucleic acid (dsRNA) sufficient to restore aberrant cytokine release or to neutralize the deleterious effects of cytokines on target tissues. The method of claim 1 wherein the cytokine is an interleukin, an interferon or a necrosis factor or tumors. A process according to claim 1 wherein dsRNA is an unpaired dsRNA. A process according to claim 3 wherein the mismatched dsRNA is a polyadenylic acid complexed with polyhydric acid. A process according to claim 4 wherein the mismatched dsRNA is a polyinosinate and polycytidylate complex containing from 1 to 5 to 1 to 30 bases of uracil or guanine. A method according to claim 5 wherein the mismatched dsRNA is either DnRNA or DnRNA dsRNA contains regions of cleavage of binding and exhibits the property of the favorable therapeutic production of rIn (Ci- . A method according to claim 6 wherein the amount of mismatched dsRNA administered results in a level of 2 to 1000 micrograms of mismatched dsRNA per milliliter of blood from the patient's systemic circulation. The method of claim 3 wherein the dsRNA is a short oligonucleotide of structure defined by the formula: - (I) n-closure 3 '3' - (C) m-closure 5 in which males are both greater than 5 and less than 100, and inosine monophosphate, C and cytidine monophosphate, or 5'fecho- / (I) A3 / 3 ' ), Wherein x and y are both greater than 5 and less than 25, and both are greater than 1 and less than 10, I and C are as defined above, A is a nucleotide which is not and I and U are a nucleotide that forms a base pair with A, or 5 '(I) n-hinge - (C) ni3' wherein n, m and I are as defined above, provided that the latches in a chain are complementary to the latches of the opposite chain. A method according to claim 8 characterized in that the oligonucleocyte is stabilized by internal complementary heteropolymer registers and the closure or hinge or both contain complementary heteropoiele regions. The applicant claims the priority of the U.S. application filed on July 16, 1991, under serial number USSN 07/730, 229. Lisbon, July 15, 1992. - 12 -