MODULATION AND DIAGNOSIS OF CYTOKINE DYSFUNCTIONS

Abstract: Cytokines, the natural macromolecular cellular products which enhance normal cell functions, when disregulated can cause or aggravate various human diseases. dsRNAs, when administered in specific scheduled doses, biologically neutralize potentially damaging effects of dysregulated cytokine and lymphokine states and encourage host recovery of diseases.

Description: MODULATION AND DIAGNOSIS OF CYTOKINE DYSFUNCTIONS Cytokines are natural macromolecular cellular products which can enhance normal cell functions, including those of lymphocytes which, in turn, convey desirable properties termed "immune surveillance." However, dysregulation of cytokines, characterized by aberrancies in production and/or distribution in bodily tissues, can actually cause or aggravate various human diseases. This invention describes a facile way of biologically neutralizing the potential damaging effects of dysregulated cytokine and lymphokine states. By judicious use of certain dsRNAs in specific scheduled dosages, further organ damage is minimized and host recovery from.disease is encouraged. Cytokine and lymphokine (produced by, or targeted to, lymphocytes) dysfunctions are being progressively linked to serious human diseases. No effective remedies have been proposed until those I dislcose herein. Even though needed in normal function of bodily cells (e.g., bone marrow, immune system, joint function, pulmonary function, etc.), aberrancies in cytokine production are associated with disease acceleration and morbidity. Aberrancies in production can consist of: (a) inappropriate production by cells not normally producing certain cytokines, (b) overproduction by cells naturally producing these macromolecules, (c) aberrant polypeptide production, such as effector cytokine molecules with subtle errors in their amino acid sequence causing alteration in activity or cellular target range, and (d) abnormalities in tissue localization, or concentration within cells, which cannot tolerate their presence without undergoing cellular injury. SUBSTITUTESHEET SPECIFIC EXAMPLES OF ABERRANT LYMPHOKINES AND HUMAN DISEASES (A) Tumor Necrosis Factor (TNF). TNF is a macrophage/monocyte derived immune mediator, which has been implicated in septic shock, cachexia, graft vs. host disease (Piguet et al Journal of Experimental Medicine, 1987 vol. 166, pp 1280-1289), parasitic infections, vascular disease and neoplastic diseases. Specifically, elevated levels are associated with a poor clinical outcome. TNF can also stimulate cells to produce other cytotoxic factors which can in turn cause additional cell inflammation, or cell death, even in brain cells (certain cells called oligodendrocytes). Patients with HIV diseases showing elevated TNF (greater than 50 picograms per milliliter) have progressive loss of developmental milestones and intellectual abilities (Mintz et al Am. Journal Diseases of Children, 1989, vol.143, pp. 771-774). Interleukins (especially IL-1) may promote TNF production, such that diseased human hosts often exist in a milieu of not one, but a group, of aberrant (as defined above) lymphokines—induced through a type of biological cascade effect. The therapeutic goals, which are the subjects of the present invention, are to biologically neutralize, or convey various levels of control, over a range of aberrant lymphokines which may co-exist in the same individual. TNF levels are increased in persons with malignancies above the upper limit of normal which, in children, is approximately 40 nanograms per liter (Saarinen et al, Cancer Research, 1990, vol. 50, pp 592-595). SUBSTITUTESHEET (B) INTERFERONS Enhanced production of interferon gamma is characteristic of HIV infected thymus-derived cells (Arya et al, Proc. Natl. Acad. Sci., U.S. 1985, vol. i - 82, pp 8691-8695). HIV infected diseased lymph nodes with follicle lysis show high levels of interferon-gamma production (Emile et al, Journal of Clinical Investigation vol 86, pp 148-159). Individuals with high interferon levels have a poorer clinical prognosis than matched cohort groups who have undetectable, or lower, lymphokine levels. Aberrant lymphokine production (as defined above) thus contributes to the very disease pathology they were intended in nature to correct. (C) Interleukins. IL-1 beta levels are elevated in infants with severe perinatal complications (280 ± 116 picograms per ml; Miller et al, Journal of Pediatrics, 1990, vol. 117, pp 961-965). These studies indicate IL-1, IL-6 and TNF in spinal cord plasma relate to clinical complications in the perinatal period. Concentrations of IL-1 are also related to severity of malaria; see Kwitetkowski et al, Lancet, vol. 336 (November 17, 1990, pp 1201-1204) . Transiently increased TNF production is a normal response to malaria infection, but excessive levels of production predispose the patient to cerebral malaria and a atal outcome. (D) Other Metabolic or Cellular Manifestations of Dysregulated Lymphokine Production. A convenient biochemical method to detect derangement is through measurement of a lymphokine-sensitive intracellular pathway, namely the 2'-5' oligoadenylate pathway (sometimes called the 2'-5'-oligoaldenylate system or 2-5 OAS for short). This pathway can be tested in different cell types which, in turn, may be fractionated by different methods such as 2 or 3 color flow cytometry, gradient SUBSTITUTESHEET centrifugation, etc. Specific derangements may concurrently exist in T4 h lper/inducer cells, T8 suppressor/cytotoxic cells, NK (natural killer) cells, B cells (involved in antibody production), CT (cytotoxic T cells), or ADCC (antibody-dependent cytotoxic cells). These changes may be effected by extracellular TNF, interferons (alpha, beta, gamma) or IL1, 2, 3, 4, 5, 6, 7, 8—each acting alone or in concert or through a multiplying "cascade" effect. Patients with rheumatoid disorders (arthritis, systemic lupus) have typically measurable interferonemi (greater than 6 IU by bio assay per ml or equal or greater than 1 IU by RIA) . When patients develop, by contrast, an interferon-specific antibody they may have inactive rheumatoid disease without visceral involvement (von Wussow et al, Rheumatology International, 1988, vol. 8, pp 225-30). One objective of my invention is to develop the functional or operational equivalent of an antibody, i.e., to "fine tune" the level of extracellular cytokines to those which are relatively non-toxic to the host and may be medically beneficial. Correlative measuring cytokines in synovial fluid, especially TNF and IFN, in patients with arthritis support 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 can even lead to vascular inflammation associated with elevated IL-2 receptors and TNF. This may be particularly accentuated in Kawasakai disease involving coronary artery lesions as recently described in Japanese Journal of Allergology (vol. 39, pp 118-123, 1990, Feb.) SUBSTITUTESHEET Excessively activated immunocompetent cells (e.g., T cell-mediated cytotoxicity) can also invade muscle cells causing a myositis and inflammatory disease of muscle groups. Elsewhere in this specification I show that biochemical derangement in 2-5 OAS pathway functions correlate with inflammatory disease of muscle and associated neurosensory pain and that the symptoms can be effectively controlled by biologically neutralizing the offending cytokines. The dsRNA used in the procedures described herein may be a complex of a polyinosinate and a polycytidylate containing a proportion of uracil bases or guanidine bases, e.g., from 1 in 5 to 1 in 30 such bases (poly I.» poly(C 4 2g x>U or G) ) . The dsRNA may be of the general formula rI n «r(C 11 _- 4 ,U) or rl •r(C 12 ,U) . Other suitable examples of dsRNA are discussed below. By "mismatched dsRNA" are meant those in which hydrogen bonding (base stacking) between the counterpart strands is relatively intact, i.e., is interrupted on average less than one base pair in every 29 consecutive base pair residues. The term "mismatched dsRNA" should be understood accordingly. The mismatched dsRNAs preferred for use in the present invention are based on copolynucleotides selected from poly (C ,U) and poly (C ,G) in which n is an integer having a value of from 4 to 29 and are mismatched analogs of complexes of polyriboinosinic and polyribocytidilic acids, formed by modifying rl TC to incorporate unpaired bases (uracil or guanidine) along the polyribocytidylate (rC ) strand. Alternatively, the dsRNA may be derived from SUBSTITUTESHEET poly(I) ' poly(C) dsRNA by modifying the ribosyl backbone of polyriboinosinic acid (rl ), e.g., by including 2'-O-methyl ribosyl residues. The mismatched complexes may be complexed with an RNA-stabilizing polymer such as lysine and cellulose. These mismatched analogs of rln-rCn, preferred ones of which are of the general formula r l • (c n_i ' u) n or rI n" r C 29' G ^ ' are described bv Carter and Ts'o in U.S. Patents 4,130,641 and 4,024,222. The dsRNAs described therein generally are suitable for use according to the present invention. The preferred mismatched dsRNA is rln•(C.11..-,14„,U)n or AMPLIGEN® of HEM Pharmaceuticals Corporation of Rockville, MD, USA, available as a lyophilized powder. Other examples of mismatched dsRNA for use in the invention include: - poly (I) • poly (C 4 U) poly (I) • poly (C ? ,U) poly (I) • poly (C 13 ,U) poly (I) • poly (C 22# U) poly (I) • poly (C 20 ,G) poly (I) • poly (C 2g ,G) and poly (I) • poly C p23 G>p Another class of dsRNAs suited to the practice of this invention are short dsRNAs of defined structure, for example oligonucleotides of the formula: where m and n are each more than 5 and less than 100, I is inosine monophosphate, C is cytidine monophosphate, and where the locks in one strand are complementary to locks in SUBSTITUTESHEET the opposite strand, or an oligonucleotide of the structure: 5'lock-t (I) χ A] .-lock 3' 3'lock-t (C) U], -lock 3' y κ where x and y are each more than 5 and less than 25, j and k each at least 1 and less than 10, I and C are as identified above, A is a nucleotide which is not I, and U is a nucleotide which base pairs with A. Alternatively, the short oligonucleotide may have the structure: 5'(I) n -hinge-(C) m 3* where n, m, I and C are as defined above. These oligonucleotides may have substitutions in one strand not complementary to nucleotides in the opposite strand. Preferably these oligonucleotides are stabilized by internal registers of complementary heteropolymer and desirably the lock or hinge or both contain regions of complementary heteropolymer. These oligonucleotides desirably have single-stranded tails. These oligonucleotides are described in more detail in PCT/US89/02172. EMBODIMENTS OF THE INVENTION ILs, TNF-α and IFNS are immuno regulatory proteins but their dysregulation actually causes tissue pathology. IL-1 and TNF share activities including pyrogenicity, activation of T cells, and neutrophil activation. High levels are SUBSTITUTE SHEET found in patients suffering from rheumatoid arthritis and other inflammatory diseases. I observed in male Sprague-Dawley, rats (300-400 grams in weight), that E. coli liposaccharide (2 mg per kilogram) produced, as expected, the classical peak levels of TNF (from 0 to >50,000 units/ml) in 60 minutes or less. Shortly thereafter, a significant percentage of the animals developed an illness with lassitude, fever, dehydration and death. However, I discovered that if animals were pretreated or post treated with mismatched dsRNA (1-20 mg/kg), which resulted in blood levels from 1-200 micro grams/ml, they survived and clinically had only a minimal, transitory, illness. Thus, I had discovered that dsRNA, in certain levels and given in certain schedules, had a novel and unexpected ability to biologically neutralize certain lymphokine actions associated with host morbidity. Then I proceeded to identify a group of individuals suffering from recurrent fevers and lymph node enlargement. Some also gave evidence of viral infection as I describe below. I detected evidence of elevated circulating IL-1 and TNF proteins in their blood by ELISA measurements and in addition parallel evidence of hyperactivity of the lymphokine activated intracellular biochemical pathway (2'-5 oligo adenylate synthetase) which has as its terminal mediator the protein RNase L. The 2'-5'-oligoadenylate/RNase L pathway is illustrated in Fig. 2 of my European application 0285 263 A3 and was assessed using the procedures described in Carter et al The Lancet, 1286-1292 (June 6, 1987). This study related to the metabolic hyperactivity in cytokine-dependent pathways associated with musculo-joint inflammation. RNase L activity was studied in Charlotte, SUBSTITUTESHEET North Carolina for sixteen evaluable individuals who were distributed as follows with respect to RNase L activity: 10 of 16: elevated RNase L activity (range: 110-500). 3 of 16: normal RNase L activity (62, 67, 94). 3 of 16: low RNase L activity (7, 44, 58). For purposes of classification, elevation is defined as greater than 100, normal in the range of 60 to 100 and low less than 60. Clinical observation reveals that those with the highest RNase L levels experienced the most severe neurosensory pain whereas mid-range elevated RNase L levels produced mixed symptomatology. Those having the lowest RNase L levels were encephalopathic, that is evidencing derangement in the central nervous system function characterized by cognition deficiencies. Those individuals with normal RNase L levels presented a relatively good clinical status with mild chronic fatigue and were able to work or return to school if teenagers. Figure 1 shows a giant cell/HHV-6 assay in which peripheral blood cells from several patients were cultured for 10 days and stained with monoclonoal antibodies against HHV-6. The percentage of HHV-6 positive cells was recorded as a function of time of dsRNA treatment. The reduction of HHV-6 positive cells was statistically significant by the paired t-test (p<0.01, 2 sided). SUBSTITUTESHEET Measurements were conducted over a period of some 80 weeks while initial evaluations were taken prior to dsRNA therapy. Figure 1, attached, shows that the pathway was excessively elevated in blood cells (lymphocytes) from the patients with severe neurosensory, muscle pain as well as recurrent fever. When the pathway was more normal in peripheral blood, the individuals suffered more in terms of cerebral function, suggesting that the aberrant cytokine production was localizing their effects to the central areas (brain) more than the molecules were damaging peripheral body tissues in these particular individuals. By achieving blood levels with mismatched dsRNA similar to those I found to be efficacious in animals, more than 50% of the individuals improved clinically and the intracellular abnormality in the 2-5A biochemical pathway normalized as in the animal studies. A high percentage of the individuals examined were concurrently infected with a human herpes virus (HHV-6) which interacts intimately with cells of the immune system (Levy et al. Virology, 1990, vol. 178, pp 113-121). I discovered (as shown in Fig. 1) that individuals receiving dsRNA also showed a concurrent reduction in circulating HHV-6 titers as well as in levels of a retrovirus with limited structural homology to HIV (the AIDS virus). In companion laboratory experiments, I discovered that HHV-6 could in fact cause elaboration of various cytokines from human blood SUBSTITUTESHEET cells, including cytokines designated TNF and IL-1, in amounts comparable to those which I discovered dsRNA would biologically neutralize in animals or man. Two panels were analyzed for cytokine derangement. The first, panel A, was analyzed for IL-1-beta protein release after HHV-6 infection of human cells. Amount of IL-1 Produced Inducer (picograms/ml) A. HHV-6 3,400 B. Lipopolysaccharide 5,300 (positive control) C. Unstimulated 725 (negative control) In the second panel analysis of TNF alpha protein release was studied after HHV-6 infection of human cells. Amount of TNF Inducer (picograms/ml) A. HHV-6 1,825 B. LPS 810 C. Unstimulated 190 The above tables indicate that cytokine levels, IL-1 and TNF alpha, respectively were measured in supernatant fluids using ELISA kits according to the manufacturer's guidelines (R and D Systems, Minneapolis, MN) . HHV-6 (10 2 TCID β0 /ml) was added to PBMC cultures and SUBSTITUTE SHEET lymphokine/cytokine level determined after 2 to 6 days of culture. Their action at the target cell level at which they were incurring ongoing damage to vital cell functions. Importantly, the viral intermediary,y by producing albeit "normal" lymphokines in cells and tissues not typically associated with cytokine production, in fact brings about a cytokine dysfunctional state since the cells bathed in the newly released cytokines cannot withstand their noxious effects and undergo morphological and functional changes leading to disease acceleration and morbidity. Thus, I concluded that the novel mechanisms of neutralizing these noxious macromolecules could result from either stemming their production, in the first instance or alternatively, neutralizing their action at a target cell level at which they were incurring ongoing damage to vital cell functions. SUBSTITUTESHEET

Claims: WHAT IS CLAIMED IS: 1. A method of modulating disease pathology associated with aberrant cytokine production, comprising administering to a patient having a cytokine dysfunction a homeostatic effective amount of a dsRNA sufficient to restore aberrant, cytokine release or to neutralize the cytokines harmful effects on target tissues. 2. The method according to claim 1, wherein the cytokine is an interleukin, an interferon or tumor necrosis factor. 3. The method according to claim 1, wherein the dsRNA is a mismatched dsRNA. 4. The method of claim 3 in which the mismatched dsRNA is a polyadenylic acid complexed with polyuridylic acid. 5. The method of claim 4, in which the mismatched dsRNA is a complex of polyinosinate and polycytidylate containing from 1 in 5 to 1 in 30 uracil quanidine bases. 6. The method of claim 5 in which the mismatched dsRNA is rl •r(C 11 _ 1 », U)n or the mismatched dsRNA contains regions of bond breakage and exhibits the favorable therapeutic ratio property of rl •r(C 11 _ ll> U) . SUBSTITUTESHEET 7. The method of claim 8 in which the amount of mismatched dsRNA administered results in a level of from 2 to 1,000 micrograms of the mismatched dsRNA per milliliter of the patient's systemic blood circulation. 8. The method of claim 3, in which the dsRNA is a short oliglnucleotide of defined structure of the formula: where m and n are each more than 5 and less than 100, I is inosine monophosphate, C is cytidine monophosphate, or 5'lock-[(I) A 3 j 'lock 3 ' 3'lock-[(C) U] k -lock 3' where x and y are each more than 5 and less than 25, j and k each at least 1 and less than 10, I and C are as identified above, A is a nucleotide which is not I, and U is a nucleotide which base pairs with A, or (I) n -hinge-(C) m 3 where n, m I and C are as defined above, provided that the locks in one strand are complementary to locks in the opposite strand. SUBSTITUTESHEET 9. The method according to claim 8, in which the oligonucleotide is stabilized by internal registers of complementary heteropolymer and the lock or hinge or both contain regions of complementary heteropolymer. SUBSTITUTESHEET

Inventors: CARTER WILLIAM, CARTER, WILLIAM
Applicants: HEM PHARMA CORP, HEM PHARMACEUTICALS CORP.
Application year: 1992
Main classification: A01N43
IPC codes: A01N43, A61K31, A61K45, A61P37