CA2113573A1 - Modulation and diagnosis of cytokine dysfunctions - Google Patents

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

WO ~3/()1717 2 1 ~ 3 5 7 3 P~ JS92tl~5286 M DU~TI~lN ~ DIA~IOSI S OF
~X~ ~

Cytokines are natural macromolecular cellular pre:duc~s which can en~ance normal cell ~Eunctions, including those of lymphocytes which, in turn, coT~vey desirable ~properti~
termed "immune urveillance. " ~Iowever, dy~regulation of cyltokines, characteri~d by aberrancies in production andjor ~ tribution in bodily ti~ues, can ac~ually cause or ag~ravate ~Taric>us human di~eases. This invention describ~s facile way of biologically neutralizing the potential damaging effects of dysregulated cytokin~ and lymphokine state~. ~y judic:iou~ use of eertain d~RNA in sp~cific scheduled dosages, furt:her organ dama~ is ~.
minimized and host recovery from disea~;e is encouraged.
:~`

Cytokine and lymphokine ( produ ::~d by, or ~arqeted to, ~ - -lymphocytes ) dysf~ tion~ ar~ b~ing progressively linked to ~''i!
. erious human di~ea~es. Mo effectis~e remedi~ ha~e been proposed until those I di~3lcos~ ~herein. Even though needed in normal function ~of ~odily cells ~e.g., bone marrow~ -..
immurle s~ t~m, joint function, pulmonary f~nction, eltc. ), :::
aberrancies in cytokine production are ~associat~d with di~ea~e acceleration and mor~idity. Aberrancie~ in production can consist of: (a,) inappropriat~ production by cell~ not normally producing certain cytokine~, 6b) overproductioni by cell~ ~naturally producing the~e macromolecules, (c) abe~rrant polypepticle produ~tion, ~uch as effector cytvkine molecul~s with subtle errors in: their amino acid setIuence cau~ing alteratiorl in activîty or ::
cellular target range, and (d) abnormalities in ti~ue localization, or concentration within cells, which cannot tolerat~ their pr~sence without undergoing cellular injury.

SUBSrITUTE SltlEET

2113a~3 WO 93/~l717 PCr/VS92/~528' SPE;C:IFIC ~X~L.ES OF ABER~aT L~HOKI~æS
AND ~N DI 53~SE:S

(A) Tu~Dr Necrosi~3 Factor (~NF~ . TNF is a macrophage/monocyte derived i~unune mediator, whi ::h has been i~nplicated in ~;eptic æhock, cachexia, graft ~. ho~t disease (Piguet et al Journal of Experimental Medicine, 1987 vol. 166, pp l28(:)-128g), para~itic infection~, vascular disease and n~oplasti c di;ease~3. Specifically, ~-elevated level~ are as~;ociated with a poor clinical outcome.

TNF c:an alsc~ stimulate cells to produce ot~er cytotoxic fac::tor~; which :can in :~rn cau~;e additional cell ---inflammatic>rl, or cell deat~, sv~en in br in cells (certain cell~; calledl oligod~ndrocyt~s~. Patient~; ~with HIV di~aases ~how ng ele~ated TNF ( ~reater than 50 picoyrams per milliliter) have: progressive Ios~ of ~d~elopm~3nt~1 ~milestones and intellectual abilities (Mintz et al ~Tn.
Jc~urnal Dig;eases of C:hildren, 1989, vol.l43, pp. 771-774). ~:
Interleukin~ (especially Il,-l) may promote TNF production, ~;uch t:hat diseased human host:s often exigt in ~ milleu of not one, bu~ a group, of :aberrant ~a~ defihed above) lymphokines-~induced through a type of biological cascade efect.

The kherapeutic goals, which are the :subjects of~ the present inventior~, are to biologically neutrallze, ~r convey variou~ levels OI control, ovex. a range of aberrant lylTphokines which may co-exi~t in the same inclividual.

TNF levels are increased in persons with malignanci~s ~;
above the upper limit of normal which, in children, is ~pproxim~tely 40 nanogram~ per liter (Saarinen et al, Cancer Res~arch, lg90, vol. 5û, pp 592-595).

SUBSTITUTE SHEET

WC) 93J01717 2113 :~ 7 3 PCl/IIS92/052B6 (B) INTEREERONS Enhanced production of interferon gamma i~ characteristic of HIV infected thymus-derived cell~ (Arya e~ al, Prc~c. Natl. Acad. Sci., U.S. 1985, vol. . ~:-82, pp 8691~86~5). HIV infected diE;ea~:ed lymph nodes with follicle lysis show high levels of interf~3ron-gamma production (~:mile et al, Journal o~ C:linical Investigation vol 86, pp 148 159 ) . Individuals wi~h high in~erferon l~vels have a poorer clinical progno~3iE; than matched cohort group~ who have undetectable, or lower, lymphokine levels.
Aberrant lymphokine produ~:ltion ( a~; defined above ) thus cs:srltributes to the very di seas~ pathology they were intendLed in nature to correc:t.
,,"_. ;~
~ C) Int~rleuki~s~ l beta levels are elevated in inIanlts with ~evere perinatal complica~ions ~280 :t 116 picos~ram~ per ml; Miller et al, Journal of Pediatric~, 1990, vol. 117, pp 96ï-965). The~;e studies indic:ate I~
IL-6 and TNF in spinal cord pla~ma relate to clinical c:omplications in the perinatal perio~l. Concentrativns of IL-l are al~o r~lated to severity of malaria; ~ee Kwitetkow~ki ~t al, Lancet, vol. 336 (Novem3~er 17, 1990, pp ;:
1201-1204). Transiently increased TNF production i8 a :~:
normal re~ponse to malaria infection, bu~ ex::essi~e levels of production predi pose th~ patient to cerebral m~laria and a fatal outcome.

(D) Other M~tabolic or Cellular Mani~e~tation~ of Dysregulated L~phokine ~roduetion. A conv~nient biochemical method to detect derangement is through measurement of a lymphokine-sensitive intracellular pathway, namely ~he 2'-5' oligoadenylate pathway (sometimes called the 2'-5'-oligoaldenylate system or 2-~ OAS for short). This pathway can be tested in different cell types which, in turn, may b~ fractionated by different methods such as 2 or 3 color flow cytometry, gradient SlJBSlrlTUTE SHEET

WO93/~1717 2 1 1 3 S 7 .3 PC~/US92/~528~

centrifugation, etc. Specific derangement~ may concurrently exist in T4 helper/inducer cells, T8 suppressor/cytotoxic cells, NK tnatural killer) cells, B
cells (involved in antibody production), CT (cy~otoxic T
cells), or ADCC ~antibody-dependent cytotoxic cells). These ~hanges may be @ffec~d by ex~racellular TNF, in~erferons (alpha, beta, gamma) or ILl, 2, 3, 4, 5, 6, 7, 8--each ~;
acting alone ~r in concert or through a multiplying "cascade" effect.

Patients with rheumatoi~ di~order~ (arthritis, ~ystemic lupus) have typically measurable i~t~rferonemia .
(greater than 6 IU by bio as~ay per ml or equal or greater -~
than 1 IU by RIA). Whe~ patien~s de~elop, by contrast, an interferon-~pecific a~ti~ody they may have i~aetivé ~-~
rheumatoid dis~ase without visceral involvement ~von Wussow et al, Rheumatology International, 1988, vol. 8, pp 225-30). One objective of ~y invention is to develop the . ~:
functional or operational equivalent of an antibody, i.e., to "fine tune" the level ~f e~tracellular cytokines to those which are relativ~ly non-toxic to the host and may be medically ben~ficial.

Correlative measuring cytokines in æynovial fluid, especially ~NF and IFN, in patients with arthritis sup*ort a relationship between tissue damage and lymphokine ~.
activity, (Hopkins et al, Clinical and Experimental Immunology, l988, vol 73, pp. 88-92).

Aggressive activation of immunocompetent c~lls ~an :~
even lead to vascular inflammation associated with elevated IL-2 receptors and TNF. This may be particularly accentuated in Kawasakai di~ease involvin~ coronary artery lesions a~ re~ently des~ribed in Japanese Journal of Allergology tvol. 39, pp 118-123, 1990, Feb.) SUBSTITUTE SHEET

W093/0~717 2 ~ i 3 ~ 7 3 PCT/US92/05286 ~ xcessi~ely activated immunocompetent cell~ (e.g., T
cell-mediated ~ytotoxicity) can also invade muscle cells causing a my~sitis and inflammatory disease of mu~cle groups. Elsewhere in this ~peciication I show that biochemical derangement in 2-5 OAS pathway functiorls correlate with inflammatory disea~e of mu~cle and associated ~euro~enEsory pain and ~hat the ~ymptoms can be effectively controlled by biologically neutralizi~g the offending ~ytokines.

The dsRNA u~ed in the procedures d~cribed her~in may be a comple~x of a polyinosinate and a polycytidylate contairling a proportion of uracil bases s: r guanidine ba~es, e . g., from 1 in 5 to 1 in 30 ~uch bases ~poly I
poly5C4_29X~U or G) ?

The d~ may be of ~he general formula :.
rIn-r(C11-14~U)n or r~ r(C12,U3n. Other ~uitabl~
exampIes of d~RNA are di~cu sed below.

By "mismatched ds~NA" are meant those in which :~
hydrogen bonding (ba~e stacking~ between the counterpart strands is relatively intact, i.e., is in~errupted on aver~ge l~ss than one base pair in every 29 consecutive base pair residues. The term "mi~matched dsRNA" should be under~tood accordingly.

The mismatched dsRNAs preferred for use in the pre~ant invention are~based on copolynucleot.ides selected from poly (Cn,U) and poly (C~,~) in which n is an integer having a value of from 4 to 29 and are mismatched analoys of complexe~ of polyriboinosinic and polyribocytidilic acids, formed by modifying rIn~rCn to incorporate unpaired bases (uracil or guanidine) along the polyribocytidylate (rCn) strand. Alt rnati~ely, the dsRNA may be derived from SUBS~lTlJTE~ SHEET

2 1 ~ 3 !~ 7 ~ P~/~'S92/0528f poly( I ) p~ly~C) dsRNA by modifying the ribosyl backbone of polyriloinosinic acid ( rIn) , e . g ., by including 2 ' -0-methyl ribosyl residues. The mi~;matc:hed complexes may be -~
complexed with an RNA-stabiliz;ing polymer f;uch as lysine .
and c:ellulose . These mismatched analogs of rIn~ rCn, preferred ones o which are of the general formula rIn (C~ 4tu)n or rIn-r(C29,G3n, are described by Car~er and Ts'o in U.S. Patents 4,130,641 and 4,024,222. The d~RNAs clescribad therein gener~lly are suitable for u~e according tc~ the present inverltion. The pr~ferred mismatchecl d~;RNA is rIn- IC~ 4,U~n or ~M~LIGE~ of HEM
Pharmaceuticals Corporation of Rockville, MD, U~;A, avai lable a~; a lyophi lixed powder . ~;

C)ther examples of mismatched clsRNA for u~e in the inventi on inc lude: ~

poly ( I ) o poly (~ U) pol~ p~ly ( C7, U ) poly ~ poly ( C13, U ) poly (I) poly (C22,U) poly ( I ) poly (.C~, G ) poly ~ poly (C29,G~ and poly ( I ) poly Cp23 G>p :
Another class of dsRNAs suited to the practice cf ~his invention are short d~RNAs o defined structllre, for example oligonucleotides of the foxmula:

5 ' lock- ( I ) n~ locX 3 ' 3 ' locX- (C)m-lock 5 ' where m ancl n are each more than 5 and less than 100, I is inosine monophc~sphate, C i ~ cytidine monophosphate, and where ~he locks in one strand are complementary to los::ks in SUBSTITUTE SHEET

WO93/01717 ~ 1 ~ 3 ~ ~ ~ PCT/US9~05~8~ ;

the opposîte strand, or an oligonucleotide of the structure: ~.

5'lock-[(I)xA]j-lock 3' 3'l~ck-l(c)y~]k-lo~ 3 where x and y are each more than 5 and less than 25i j and ~`~
k each at least l and l~ss than l0, I and C are a~ ~
iden~î~ied above, A is a nucleotide which i~ not I, and U ~.
is a nucleotide which ba~ pairs with A. ::~

Alternati~ely, the ~hort oli~onucleotid~ may have the .~`
~~structure S (I)~-hing~-(C~m3 wh~re n, m, I and C are aæ defin~d above.

The~e oligonucleotides may have su~stitutions in o~e ~trand not compl~mentary to nucl~eotides in the opposi~e 6trand. Pref~rably the~e oligonucleotide~ are ~tabilized by inter~al re~isters of complem~n~ary het~ropoIymer and :~de~irably the lock or hinge or b~oth co~tain reglons of~
~c~mplementary heteropolymer. These oligo~ucleo~ides ~.
desirably have ~ingle-~tran~ed tails. These vlîgonu~leotide~ are:described in more detail in ; ~ : PCT/US89/02172. ~ ;~

EMBODIMENTS OF T~E INVENTION

ILs, TNF-a and IFNS are i~muno regulatory proteins but their ~ysregulation actually causes ti~sue pathslo~y. IL-l and TNF share activitie~ including pyrog~nicity, ~ctivation of T cell~, and neutrophil activation. High levels ar~

.

SlJBSTlTVTE S~1EIET

WO93/01717 2 1 1 3 5 7 ~3 P~r/US92/052X~ ; :

found in patients suffering from rheumatoid arthriti~ and other inflammatory di~ea~es.

I observed in male Sprague-Dawley, rats ~300-400 grams in weight), that E. coli liposaccharide (2 mg per kilogram) produced, as expected, ~he classical peak levels of ~NF (from 0 to >50,000 unit~/ml ) in 60 mi~utes or le~s.
Shortly thereafter, a ~igniicant p~rcentage of the animals developed an ill~es~ with la~itude, f2ver, dehydration and daath. How~ver, I di#cover~d that if animal~ were pretreated or po~t treated with mi~atched d~RNA (1-20 mg/kg), which resulted in blood levels from 1-200 micro grams/ml, ~hey survived and clinically had only a minimal, transitory, illness. T~u~, I had discovered that d~NAt in certain le~el~ and gi~en in certain ~chedules, had a novel and unexpected ability to biologically neutralize certain lymphokine action~ o~iated with ho~t morbidity.

Then I proceeded to identify a group of individuals ~uffering from recurrent ~ever~ and lymph node ~nlargem~nt. Some also gave evidence of ~iral infection as I describe below. I det~cted eviden~e of el~vated circulating IL-l 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 ad~nylate synthetase~ which has as it5 terminal mediator the protein RNase L. The 2t-5'-oligoadenylate/RNase L pathway is illustrated in Fig.
2 of my Europea~ application 0 285 263 A3 and was assessed using the procedur~s de~cribed in Carl:er et al The Lancet, ~:
12~6-1292 (June 6, 1987). :.

~ his study rela~ed to the metabolic hyperactivity in cytokine-dependent pathways associated with mus~ulo-joint ~:
inflammation. ~Nase L activity wa~ studied in Charlotte, SUlBSTlTlJTE SHEET

~ W~3/~1717 2 1 ~ 3 S 7 3 I'CT/US~2/OS~S6 North Carolina for sixteen evaluable individuals wh~ were distributed as follows with reEpect to RNase L activity;

10 ~f 16: elevated RNa~e L acti~ity ~range: 110-500 3 of 16: normal ~Na~e h activity (6~, 67, 9 3 of 16: low RNase L activity (7, ~, 58).
' .:
Eor purposes of clas~ification, elevation i~ defined as gr~ater than 100, normal in the range of 60 to 100 and lc)w le~s than 60. C~ ic:al ob~ervation ~-reveals that tho~e with ths hig~e~t RNase L leve~s e~perienced the moæt severe neuro~ensory pain whereas mid-range ele~ated~RNase L levels produ~ed :mixed symptomatology. Those hav:ing the l~west RNa~e L level~;were encephalopathic, that i~ evid~nci~g dera~gemen~ in the c~nkral nervous rystem unction ~haracterized by cognition deficiencies. Those indi~iduals with normal RNa~e L levels preæented a ~ :.
relatively good clinical statu~ with mil~ chronic fatigue and were able to work or r~turn to 6~hool if teenager~.

Figure 1 ~hows a giant cell/H~-6 aB~ay in which peripheral blood cells from ~everal patien~s were cultured for 10 day~ and tained with monoclonoal antibodie~ against HHV-6. The pereent~ge of H~V-6 positive cells was recorded as a function of time of dsRNA tre tment. The ~eduction of H~V-6 posikive cells was ~tatistically significant by th~ paired t-test (p<O.O1, 2 sided). -~
,'.

.
SUE~STITUTE SHEET

~1 i 357~
W~93fO1717 P~r~l,'S92/0528~f:

Measurements were conducted over a period of ~:ome 80 weeks whil~ initial evaluationE; were taken prior to d~RNA therapy.
Figure 1~ attach~d, ~hows that the pathway was excessiv~ly elevated in blc>od cell~
( lymphocytes) from the patients with ~evere neuroæensory, muscle pain a~ w~ll a~ recurrent feY~r. When the pathway wa~; more normal in peripheral }~ od, ~he individual~ ~uf~E~red more in terms of cerebr 1 function, ~ugg~stirlg that th0 aberra~t cyto}cine production was localizing th~i r effects to the central areas (brain~ more than the rnolecul~s were damaging peripheral body tis~ues in these particular individ~lal~ .

By a~hieving blood level~ with mi~atched d~RNA ~imilar to tho~e I found to be efficacious in animals, :more than 50~ of the individua}s improved clinically and the intracellular abnormality in the 2-5A bioch~mical pathway normalized as in the animal ætudie~ .
':~
A hi~ percentage of the indi~idual~
- examined were coDcurrently infected with a human herp~s virus ~ 6) w~i~h interact~ in~imately with ~;
cells of the imrnune ~y~tem ~Le~y et al, Virology, 1990, vol. 178, pp 113-121). I di covered (as shown in Fig. 1 ) that individuals rec~ivirlg dsRNA al~o ~ihowed a concurrent reduction in circul~ating HHV-6 titer~; as well as in level~; of a retroviru~; with limited struc:tural homology to HIV (the AIDS viru~3).

In compani~n laboratory experimellts, I
di~covered that ~IV-6 could in fact cau~e elaboration of ~arious cytokines from human blood SUBSTITUTE SHEE~T

WO 93/0l717 2 1 ~ 3 5 7 ~ PCI/US92/052~6 cells/ including cytokines designate!d TNF and IL-l, in amounts compa~able to those w~ich I di ~;covered d~RNA woul ;l biologically neutralize in animal~ or man.

Two panels were analyzed for cytokine derangement. The first, pan~l A, was analyzed for IL-l-beta protein releas~ after H~1-6 infection of human c:e 11~ .
Amount of IL-l Produced Induc~r (pico~rams/ml ) ~A. HHV-6 3,400 B. Lipopc~ly6acc:haride5, 300 (positiv~3 control ~
C. Unsti~ulal:ed 725 ( negati~e control ) In the second panel analy~is of TNF
alpha protein release was studied after HHV-6 inection of human cells.
: ., ~ .Amount of TNF -;:: Inducer~picos~rams/rnl ) A. ~IV-6 1,82S
8. ~PS 810 ~:
C. Un~timulated 190 '.-The abo~e tables: indicate that cytokine levels, IL 1 and TNF alpha, respectively were -~
mea~;ured in ~up~rnatant 1uids using ELISP~ kits - according to the manufacturer' s guidelines ~R and D
~;ystems, MinrleapGlis, ~) . ~IV-6 ( 1 o2 TCXDs n/ml) was added to PBMC cultures and ~:

SUBSTITUTE SHEET

~113~,73 WO 93~017l7 PCr/lLJS92J0528' lympho}~ine/cytokine level determined after 2 to 6 days of culture.

Their action at the target cell level at whic:h they were inc~lrring ongoing damage to vital cell function~ . Importantly, the viral intermediary, y by producing alb~it "normal" lymphokirles in ~ells and tissues not typically ~ssociated with cytokine production, in fact bring~ about a cytokine dy~unctional ~tate ~ince the c:~lls bathed in the n~wly relea~ed cytokine~ cannot withstand their nt:~xious effQcts and undç~rgo mo~phologic~l and ~-~functional c:hanges leading to di~ea~e acceleration and morbidity. . ~.
Thu~, I concluded that the novel mechani ~ms of neutralizing theRe noxious ::-macromolec:ule~ could re ult from either ~temming their prodllc~ion, in th~ firs~ instanc~ .:
or alternatively, neutralizing their action at a target c~ll level at which they were incurring ongoing damage to vital ct311 functions.
.::
:~ .

SUBSTITUTE SHEET

Claims (9)

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 complexes 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 rIn?r(C11-14, U)n or the mismatched dsRNA contains regions of bond breakage and exhibits the favorable therapeutic ratio property of rIn?r(C11-14, 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 5'(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 loclc or hinge or both contain regions of complementary heteropolymer.