WO1993001717A1

WO1993001717A1 - Modulation and diagnosis of cytokine dysfunctions

Info

Publication number: WO1993001717A1
Application number: PCT/US1992/005286
Authority: WO
Inventors: William Carter
Original assignee: Hem Pharmaceuticals Corp.
Priority date: 1991-07-16
Filing date: 1992-06-23
Publication date: 1993-02-04
Also published as: AU2268892A; IE922145A1; EP0596912A4; EP0596912A1; TW271398B; ZA925263B; PT100688A; JPH07501787A; CA2113573A1; CN1068738A; MX9204164A

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. 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). (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. 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 fatal 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 centrifugation, etc. Specific derangements may

concurrently exist in T4 helper/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 interferonemia (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.) 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 29}x>U or G)).

The dsRNA may be of the general formula

rI_n·r(C_11-14,U)_n or rl_n·r(C₁₂,U)_n. 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_n,U) and poly (C_n,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_n·rC_n to incorporate unpaired bases (uracil or guanidine) along the polyribocytidylate (rC_n) strand. Alternatively, the dsRNA may be derived from poly(I)·poly(C) dsRNA by modifying the ribosyl backbone of polyriboinosinic acid (rl_n), 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 rl_n·rC_n, preferred ones of which are of the general formula

rl_n·(C_11-14'U)_n or rI_n·r(C_29'G)_n, 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 rl_n·(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 (C₄,U)

poly (I) ● poly (C₇,U)

poly (I) ● poly (C₁₃,U)

poly (I) ● poly (C₂₂,U)

poly (I) ● poly (C₂₀,G)

poly (I) ● poly (C₂₉,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:

5'lock-(l)_n-lock 3'

3'lock-(C)_m-lock 5' 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 the opposite strand, or an oligonucleotide of the

structure:

5'lock-[(I)_xA]_j-lock 3'

3'lock-[(C)_yU]_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.

Alternatively, the short oligonucleotide may have the structure:

5'(I)_n-hinge-(C)_m3' 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 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, 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). 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 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 (10²

TCID₅₀/ml) was added to PBMC cultures and 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.

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_n●r(C_11-14,

U)n or the mismatched dsRNA contains regions of bond breakage and exhibits the favorable therapeutic ratio property of rl_n●r(C_11-14, U)_n.

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:

5'lock-(I)_n-lock 3'

3'lock-(C)_m-lock 5' 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)_xA^]j^-lock 3' 3'lock-[(C)_yU]_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)_m3' 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.

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.