WO1999001154A1 - Method for inhibiting immunostimulatory dna associated responses - Google Patents

Method for inhibiting immunostimulatory dna associated responses Download PDF

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Publication number
WO1999001154A1
WO1999001154A1 PCT/US1998/013820 US9813820W WO9901154A1 WO 1999001154 A1 WO1999001154 A1 WO 1999001154A1 US 9813820 W US9813820 W US 9813820W WO 9901154 A1 WO9901154 A1 WO 9901154A1
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group
hydrogen atom
immunostimulatory dna
lower alkyl
butylamine
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PCT/US1998/013820
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French (fr)
Inventor
Donald E. Macfarlane
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University Of Iowa Research Foundation
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Application filed by University Of Iowa Research Foundation filed Critical University Of Iowa Research Foundation
Priority to EP98931779A priority Critical patent/EP1009440B1/en
Priority to DE69840025T priority patent/DE69840025D1/en
Publication of WO1999001154A1 publication Critical patent/WO1999001154A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention relates to a method of inhibiting cellular and humoral responses to immunostimulatory DNA with compositions including 4-aminoquinolines, 9- aminoacridines and derivatives thereof.
  • CpG-ODN CpG-ODN mimic many of the actions of bacterial DNA (Krieg et al, 1996. Antisense & Nucleic Acid Drug Deve 6: 133-9). and powerfully inhibit the induction of apoptosis in mouse WEHI 231 B-cells by anti-surface IgM (Yi et al , 1996. J. Immunol. 157:4918-25; Macfarlane et al, 1997. Immunology 91 :586). This system is a convenient and reproduc- ible model to study responses to immunostimulatory DNA.
  • DNA and oligodeoxynucleotides are endocvtosed into acidic vesicles, and are then transported to the cytoplasm and nucleus of cells.
  • Chloroquine, hydroxychloroquine and quinacrine are known to induce remissions of systemic lupus erythematosus and rheumatoid arthritis by an unknown mechanism.
  • These drugs bind to DNA by intercalation. They are weak bases and partition into acidic vesicles At high concentrations, chloroquine can collapse the pH gradient and disrupt the action of endosomal hydrolytic enzymes and the trafficking of receptors
  • the invention is based on the discovery that qumac ⁇ ne, chloroquine and selected 9- aminoac ⁇ dine and 4-am ⁇ noqu ⁇ nol ⁇ ne compounds unexpectedly block the action of immunostimulatory DNA. in cells at concentrations much below those needed for the other immunomodulatory effects of these compounds in vitro
  • the 9-am ⁇ noac ⁇ dme and 4- aminoquinohne compounds utilized in the methods of the invention inhibit the anti- apoptotic effect of CpG-ODN and the CpG-ODN-induced secretion of IL-6 On the other hand, these compounds do not influence the anti-apoptotic effects of other agents, for example, hpopolysaccha ⁇ des
  • the effects of the compounds are highly specific to immunostimulatory DN ⁇ These agents are useful for inducing remissions of autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, leading to generally useful remittive and anti-inflammatory agents
  • the methods of the invention are useful for inhibiting immunostimulatory DNA- associated responses in a subject, and involve the administration of a composition to a subject exhibiting responses initiated or exacerbated by immunostimulatory DNA
  • the compositions include 4-am ⁇ noqu ⁇ nohnes, 9-am ⁇ noac ⁇ d ⁇ nes and derivatives thereof These compounds may be linked to each other by linkers
  • the responses initiated or exacerbated by immunostimulatory DNA include those which result in the initiation or exacerbation of various disease states
  • the disease states include septic shock, inflammatory bowel diseases, asthma, sinusitis, various autoimmune disorders such as hemolytic anemia, and others
  • the responses to be inhibited include the production of immunomodulatory proteins such as cytokines, interleukins, mterferons, cell growth factors and chemokines
  • IL-6 IL-6
  • responses to be inhibited by the methods of the invention are high levels of erythrocyte sedimentation, and high levels of immunoglobuhn
  • the invention also features a method of screening compounds useful for inhibiting immunostimulatory DNA-associated responses This method involves contacting a ceil with immunostimulatory DN A., thereby inducing a measurable immune response, and a
  • the cells can be hyb ⁇ doma plasma cells, including 7TD 1.
  • nucleic acid or “DNA” means multiple nucleotides (i.e. , molecules comprising a sugar (e.g , ⁇ bose or deoxy ⁇ bose)) linked to a phosphate group and to an exchangeable organic base, which is either a substituted py ⁇ midine (e.g., cytosme (C). thymine (T) or uracil (U)) or a substituted purine (e.g.. adenine (A) or guanine (G))
  • a substituted py ⁇ midine e.g., cytosme (C). thymine (T) or uracil (U)
  • purine e.g.. adenine (A) or guanine (G)
  • the term refers to ⁇ bonucleotides as well as oligodeoxynucleotides
  • polynucleosides i.e...
  • Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g. , genomic or cDNA), but are preferably synthetic (e.g. , produced by o gonucleotide synthesis)
  • a "stabilized nucleic acid molecule” or “nuclease-resistant nucleic acid molecule” means a nucleic acid molecule that is relatively resistant to in degradation (e.g , via an exo- or endo-nuclease).
  • Stabilization can be a function of length or secondary structure
  • Unmethvlated CpG-containing nucleic acid molecules that are tens to hundreds of kilobases long are relatively resistant to in vivo degradation.
  • secondary structure can stabilize and increase their effect.
  • the nucleic acid molecule becomes stabilized and therefore exhibits more activity
  • Preferred stabilized or nuclease-resistant nucleic acid molecules referred to in the instant invention have a modified backbone.
  • nucleic acid molecules are phosphorothioate-modified nucleic acid molecules (i.e. , at least one of the phosphate oxygens of the nucleic acid molecule is replaced by sulfur).
  • the phosphate modification occurs at or near the 5' and or 3 ' end of the nucleic acid molecule.
  • phosphorothioate-modified nucleic acid molecules can increase the extent of immune stimulation of the nucleic acid molecule, which contains an unmethvlated CpG dmucleotide
  • nucleic acid molecules include non-tonic DNA analogs, such as alkyl- and ary 1- phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or ary l group), phosphodiester and alkylphosphot ⁇ esters, in which the charged oxygen moiety is alkylated Nucleic acid molecules that contain a diol, such as tetraethy leneglycol or hexaethyleneglycol, at either or both termini have also been shown substantially resistant to nuc lease degradation
  • immunostimulatory DNA refers to bacterial DNA, viral DNA, other DNA. synthetic double- or single-stranded DNA, DNA synthesized with a nuclease-resistant backbone or at least a partially nuclease resistant backbone, (such as a phosphorothioate-backboned DNA) which stimulates (e.g , has a mitogenic effect on, or induces or increases cytokine expression by vertebrate lymphocytes).
  • immunostimulatory DNA includes that which contains an unmethylated cytosine, guanine (CpG) dinucleotide sequence.
  • CpG or “CpG motif refers to a nucleic acid having cytosine followed by a guanine linked by link containing phosphorus
  • methylated CpG refers to the methylation of the cytosine on the py ⁇ midme ring, usually occurring at the 5'-pos ⁇ t ⁇ on of the py ⁇ midme ring
  • unmeth lated CpG refers to the absence of methy lation of the cytosine on the py ⁇ midme ring Methylation. partial removal, or removal of an unmethylated CpG motif in an ohgonucleotide of the invention is believed to reduce its effect.
  • Methy lation or removal of all unmethylated CpG motifs in an ohgonucleotide substantially reduces its effect
  • the effect of methy lation or remov l of a CpG motif is "substantial" if the effect is similar to that of an ohgonucleotide that does not contain a CpG motif
  • airw ay obstruction includes respiratory ailments such as asthma, sinusitis, as well as airway obstructions caused by an infection, or exacerbated by an infection
  • lower alkyl and lower alkoxy refer to alkyl- and alkoxy groups comprising up to 7 carbon atoms The terms include straight and branched chain groups
  • ahc hc reters to a non-aromatic carbon chain system forming a ring, and hav ing from 4 to 10 members
  • polyalicychc refers to a non-aromatic carbon chain system forming more than one ring, each ring having from 4 to 10 members
  • aryl group refers to aromatic hydrocarbons having from 6 to 26 carbons
  • Fig 1 is a graph showing the effect of various concentrations (log scale) of selected compounds (quinac ⁇ ne, amodiaqume, chloroquine, hydroxychloroquine, quinidine, quinine, and p ⁇ maquine) on ODN 1760 protection against the -sIgM-mduced inhibition of [ ⁇ ] thymidine uptake in WEHI 231 cells
  • Fig 2a is a graph showing the effect of various cell medium additives (ODN 1 ⁇ 60. ⁇ - slgM. ODN 1760/ ⁇ -sIgM, quinac ⁇ ne, quinac ⁇ neODN 1760, quinac ⁇ ne c -sIgM. and quinac ⁇ ne/ODN l "60/ ⁇ -sIgM) on the W ⁇ HI 231 cell count measured at different time periods after incubation
  • Fig 2b is a bar graph showing the effect of various cell medium additiv es (ODN 1 ⁇ 60. ⁇ - slgM. ODN 1760/ ⁇ -sIgM, quinac ⁇ ne, quinac ⁇ neODN 1760, quinac ⁇ ne c -sIgM. and quinac ⁇ ne/ODN l "60/ ⁇ -sIgM) on the W ⁇ HI 231 cell count measured at different time periods after incubation
  • Fig 2b is a bar graph showing the effect of various cell
  • Fig 3 is a g Dh showing the effect of various concentrations of ODN ⁇ ⁇ 60 on W amount of [ H] thymidine uptake in the presence of various concentrations ot quinacne in W EHI 2 1 cells treated with ⁇ -sIgVl
  • Fig 4 is a bar graph showing the effect of additions of quinac ⁇ ne at va ⁇ ous times after incubation on the extent of [ ⁇ ] thymidine uptake m WEHI 231 cells treated with ⁇ - slgM Also displayed is a comparison of the effect on [ H] thymidine uptake of incubation of ⁇ -sIgM-treated WEHI 231 cells with ODN 1760 and a phosphodiester ODN with the same sequence as ODN 1760
  • Fig 5 is a bar graph showing the effect of quinac ⁇ ne on the protection by hpopolysaccha ⁇ des against -sIgM-induced inhibition of [ ⁇ ] thymidine uptake by W EHI 231 cells
  • Fig 6 is a bar graph showing the effect of qumac ⁇ ne on the protection by ODN 1 " 60 against C-ceramide-induced inhibition of [ H] thymidine uptake in WEHI 231 cells
  • Fig 7 is a bar graph showing the effect of quinac ⁇ ne on the protection by ODN against thapsigargm-induced DNA fragmentation in WEHI 231 cells
  • Fig 8 is a graph showing ODN 1760-mduced effects on percentage of [ H] thymidine uptake in human mononuclear cells
  • Fig 9 is a graph showing ODN 1760- ⁇ nduced effects on percentage of IL-6 production in human mononuclear cells
  • chloroquine Being a diprotic weak base, chloroquine (and many of the other compounds which w ere used) partitions into the acidified cellular vesicles, such as Ivsosomes At high concentration, chloroquine can collapse the pH gradient of Ivsosomes and induce their swelling
  • the compounds chloroquine, hydroxychloroqume and qumac ⁇ ne induce remissions of rheumatoid arthritis and systemic lupus erythematosus The beneficial effect of these compounds becomes apparent ov er the course of sev eral weeks To account for this valuable therapeutic action, a number of investigators have explored the effects of the compounds on immune and inflammatory responses in xitro.
  • the invention features methods for inhibiting immunostimulatory DNA-induced responses in a subject.
  • This method includes the administration of a therapeutically effective amount of a composition to the subject.
  • the composition contains a compound that is effective in inhibiting immunostimulatory DNA-induced responses.
  • R ⁇ can be a hydrogen atom, a lower alkyl group, or can be further linked to R B by an alkyl chain. If R A is a lower alkyl group, it is preferred to be methyl or ethyl. If R A is further linked to R B by an alkyl chain (in addition to the depicted linkage by the nitrogen atom), the alkyl chain can be -(CH ) n -, where n is from 4 to 7, and is preferably 4 or 5.
  • the substituent R B can be a hydrogen atom, an alicyclic group (such as a cyclopentyl. cyclohexyl or cycloheptyl group), an alkyl secondary, tertiary or quaternary amino group or alkenyl secondary, tertiary or quaternary amino group.
  • R B can be an unsubstituted alkyl amine, such as 4-[pentyl-N,N-dialkyl amine], 4-[pentyl-N-alkyl amine], 4-[ ⁇ entyl amine], 4-[butyl-N,N-dialkyl amine], 4-[butyl-N-alkyl amine], 4-[butyl amine].
  • N- substituents are generally lower alkyl. but can also include hydroxy-substituted lower alkyl, such as 2-hydroxyethyl.
  • Unsaturated chains include 4-[pent-2-enyl-N,N-dialkyl aminej and 4-[pent-2-enyl-N-alkyl amine]. Longer amine-containing alkyl chains can also be utilized, and the nitrogen need not be located at the terminus of the substituent group
  • Cyclic amines can be included in the alkyl chain
  • the alkyl chain can terminate in. or be interrupted by. a pyrrole ring, a piperazidyl ring, a pipe ⁇ dyl ring, or a morpholinyl ring, any of which may be further substituted with lower alkyl groups
  • R B is an unsubstituted amine-containmg group
  • the preferred variants of R B can be 2-[ethyl-N,N-d ⁇ methyl amine], 2-[ethyl-N-methyl amine], 4-[pent ⁇ l- N.N-diethyl amine], and 4-[butyl-N.N-d ⁇ ethyl amine]
  • R B amine-contammg chains described above can be variously substituted Substituents include lower alkyl groups, such as methyl, ethyl and propyl
  • Other useful R B substituents include substituted or unsubstituted aryl groups, such as pheny l. anisyl. hydroxyphenyl, chlorophenyl, dichlorophenyl, fluorophenyl, naphth l. thiophenyl. which can be substituted at o-, m- or p-positions, or at 1- or 2-pos ⁇ t ⁇ ons in case of naphthy l.
  • heterocychc groups such as py ⁇ dyl, pyrrolyl, pipe ⁇ dyl, and piperazidy l, or halogen, such as chloro, bromo, and fluoro, and other substituents such as hydroxyl. and alkoxyl
  • halogen such as chloro, bromo, and fluoro
  • hydroxyl such as hydroxyl. and alkoxyl
  • Further substitution of this amine-containing alkyl chain can comprise, for example, amide or ester linkages, ether or thioether linkages
  • the amine-containing alkyl chain can terminate with a substituent group such as a primary, secondary or tertiary amine, a hydroxy group, a thiol. a carboxyhc acid, or an amide
  • R B can also be a ahpolycyclic group, such as bicycloheptyl, bicyclooctyl. or adamantyl, and can be linked to any position of these groups R B can also be linked to R ⁇ by an alkyl chain
  • the substituent R can be a hydrogen atom, a lower alkyl group, or an aryl group R : can also be a heteroaromatic group, such as 2-, 3-, or 4-py ⁇ dyl. 1-, 2-, or 3- pyrroly l. or an aryl substituted lower alkenyl group, such as trans- ⁇ -styryl and trans- ⁇ -[ ⁇ , ⁇ -trans- dimethyl-p-chlorostyryl] It is believed that bulky groups at R : contribute to the efficacv of the compounds of the invention Thus, many of the embodiments include bulky groups at this position Among the unsubstituted aryl groups useful as R, substituents include phenyl, 1- and 2-naphthyl.
  • substituted aromatic hydrocarbons include the above-mentioned aryl groups, substituted with lower alkyi groups such as methyl and ethyl, halogens, such as chlorine, fiuorne and bromine, perfluoroalkyl groups such as t ⁇ fluoromethyl and pentafluoroethyl.
  • alkoxy groups such as methoxy; aryloxy groups such as phenoxy; amine-containmg substituents such as N-[ N,N-d ⁇ methyl ethylenediamine], or l-[4-methylp ⁇ peraz ⁇ ne]
  • substituent R can be a hydrogen atom, a lower alkyl group such as methyl or ethyl, or an aromatic group such as phenyl
  • the substituent R is hydrogen or methyl
  • the substituent R ⁇ can be a hydrogen atom, a lower alkyl group such as methyl or ethyl, or a halogen atom, such as chlorine, bromine or fluorine
  • the substituent R () can be a hydrogen atom, a lower alkyl group such as methy l or ethyl, a lower alkoxy group such as methoxy or ethoxv, an aryloxy group such as phenoxy, an aryl group such as phenyl, an amine group such as N,N-d ⁇ methy l amino or N,N-d ⁇ ethylam ⁇ no, or a thioether group such as phenylthioether or benzylthioether It is found that bulky substituents on position 6 of the quinohne ring tend to enhance activity, so that many of the preferred embodiments include bulky groups at this position
  • the substituent R- can be a hydrogen atom, a lower alkyl group such as methyl or ethyl, a lower alkoxy group such as methoxy or ethoxy, an aryloxy group such as phenoxy, a halogen atom such as chlorine, bromine or fluorine, or a lower haloalkyl group, namely a lower perfluoroalkyl group, such as t ⁇ fiuromethyl or pentafluoroethyl.
  • the substituent R 3 can be a hydrogen atom or a lower alkoxy group such as methoxy or ethoxy
  • Pharmaceutically acceptable salts of any of these compounds are also included in the invention These salts include protonated or deprotonated atoms on the 4- aminoquinolme and counte ⁇ ons including potassium, sodium, chlo ⁇ ne, bromine, acetate and many other commonly recognized counte ⁇ ons If R- is a halogen, then at least one of R 2 .
  • R : , R,, R 6 , or R s is not a hydrogen atom and R B is not 4-[N,N-d ⁇ alkyl-n-pentylamme] or 4-[N-alkyl-N-hydroxyalky l-n-penty lamme]
  • the 4-am ⁇ noqumolme compounds which inhibit immunostimulatory DNA-associated responses can be linked together via a linker
  • the linker can be connected to each molecule at either the same position on the compound, or at different positions Preferred linked 4-am ⁇ noqu ⁇ nohne compounds are linked at the same position on each compound.
  • the prefe ⁇ ed position for linkage between the individual 4-aminoquinoline compounds is at the 4-amino position, shown as R ⁇ or R B in the structure.
  • the linker can be of a number of types. Preferred linker types are alkyl chains, alkyl chains interrupted with nitrogen atoms, or alkyl chains interrupted with amide linkages.
  • the alkyl chains are preferred to be at least two carbons in length and not more than twelve carbons in length. If the alkyl chain is interrupted by nitrogen atoms, it is preferred that the overall length of the chain be between two and twelve atoms.
  • Prefe ⁇ ed linker molecules include those of the formula -[(CH 2 ) nl N(R)(CH ) ,J , r where nl, n2 and n3 are independently between I and 5, and R is a hydrogen atom, a carbonyl group or a lower alkyl group.
  • the linker can further include substituents to vary the hydrophobicity or hydrophilicity of the linked compound as a whole.
  • R B can be a hydrogen atom or an alkyl secondary, tertiary or quaternary amino group.
  • R B can be an unsubstituted alkyl amine, such as 4- [pentyl-N,N-dialkyl amine], 4-[pentyl-N-alkyl amine], 4-[pentyl amine], 4-[butyl-N,N- dialkyl amine], 4-[butyl-N-alkyl amine], 4-[butyl amine], 2-[ethyl-N,N-dialkyl amine], 2- [ethyl-N-alkyl amine], 2-[ethyl amine], 3-[propyl-N,N-dialkyl amine], 3-[propyl-N-alkyl amine], and 7-[hepta-4-methyl-4-azaamine].
  • the N-substituents are generally lower alkyl. but can also include hvdroxv-substituted lower alkyl, such as 2-hydroxyethy .
  • Unsaturated chains include 4-[pent-2-enyl-N,N-dialkyl amine] and 4-[pent-2-enyl-N-alkyl amine]. Longer amine-containing alkyl chains are also possible, and the nitrogen need not be located at the terminus of the substituent group.
  • Cyclic amines can be included in the alkyl chain.
  • the alkyl chain can terminate in, or be interrupted by, a pyrrole ring, a piperazidyl ring, a piperidyl ring, or a morpholinyl ring, any of which may be
  • R B is an unsubstituted amine-containing group
  • the preferred va ⁇ ants of R B are 2-[ N.N-dimethy l ethylamine], 2-[N-methyl ethylamine], 4-[N,N-d ⁇ ethyl pentylamine], and 4-[N,N-d ⁇ ethyl butylamine]
  • Preferred embodiments of the 9-am ⁇ noac ⁇ d ⁇ nes useful in the invention hav e R,j as 4-[4-aryl-N,N-d ⁇ alkyl butylamine], 4-[4-heteroaromat ⁇ c-N.N-d ⁇ alkyl butylamine], 4-
  • R B amine-containmg chains described above can be variously subst'tuted Substituents include lower alkyl groups, such as methyl, ethyl and propyl Other useful
  • R B substituents include substituted or unsubstituted aryl groups, such as phenyl. amsyl hydroxyphenyl, chlorophenyl. dichlorophenyl, fluorophenyl, naphthyl, thiophenyl. which can be substituted at o-, m- or p-positions, or at 1- or 2-pos ⁇ t ⁇ ons in case of naphthyl heterocychc groups, such as pyridyl, pyrrolyl, pipe ⁇ dyl, and piperazidyl, or halogen, such as chloro, bromo and fluoro. and other substituents such as hydroxyl, and alkoxy 1
  • this amine-containmg alkyl chain can comprise, for example, amide or ester linkages, ether or thioether linkages
  • the amine-containmg alkyl chain can terminate with a substituent group such as a primary, secondary or tertiary amine, an hydroxy group, a thiol.
  • a carboxylic acid, or an amide Preferred embodiments have 4-[ N,N-d ⁇ alkyl pentylamine] , 4-[4-aryl-N,N-d ⁇ alkyl butylamine] or 4-[4-heteroaromat ⁇ c-N,N- dialkyl butylamine] groups at this position, in which the aryl or heteroaromatic group is unsubstituted or substituted bv halogen or alkoxy, and the N,N-d ⁇ alkyl groups are N N- diethyl groups
  • the substituent R- is a lower alkyl group, such as methyl or ethyl Preferred embodi- ments have methyl at this position
  • the substituent R can be a hydrogen atom, or a lower alkoxy group
  • the substituent X is a halogen atom, such as chlorine, bromine or fluor ne Pre ⁇ e ⁇ e ⁇ embodiments have chlorine at this position
  • the 9-am ⁇ noac ⁇ dme compounds which inhibit immunostimulatory DN ⁇ -associated responses can be linked together via a linker Tne linker can be connected to each molecule at either the same position on the compound or on different positions Preferred linked 9-ammoac ⁇ d ⁇ ne compounds are linked at the same position on each compound
  • the preferred position for linkage between the individual 9- aminoac ⁇ dine compounds is at the 9-am ⁇ no position, shown as R or R B in the structure
  • the linker can be of a number of types Preferred linker types are alkyl chains, alkyl chains interrupted with nitrogen atoms, or alkyl chains interrupted with amide linkages
  • the alkyl chains are preferred to be at least two carbons m length and not more than twelve carbons in length If the alkyl chain is interrupted by nitrogen atoms, it is preferred that the overall length of the chain be between two and twelv e atoms
  • Preferred linker molecules include those of the formula -[(CH ; ) nl N(R)(CH : ) n2 ] n - where nl , n2 and n3 are independently between 1 and 5, and R is a hydrogen atom, a carbonyl group or a lower alkyl group
  • Further useful compounds for inhibiting immunostimulatory DNA-associated responses include 4-am ⁇ noqu ⁇ nol ⁇ ne compounds and 9-am ⁇ noac ⁇ d ⁇ ne compounds linked together by a linker Preferred linked 4-am ⁇ noqu ⁇ nohne/9-ammoac ⁇ d ⁇ ne compounds are linked at analogous positions on each compound The preferred position for linkage for the 9-ammoac ⁇ d ⁇ ne compounds is at the 9-am ⁇ no position, shown as R x or R B in the structure and the linker is further linked to the 4-am ⁇ no position of the 4-am ⁇ noqu ⁇ nohne compound
  • the linker can be of a number of types Preferred linker types are those discussed above in connection with the 4-am ⁇ noqu ⁇ nolme and 9-ammoac ⁇ d ⁇ ne compounds
  • the compositions used in the methods of the present invention are useful to inhibit cellular and humoral immune responses Such immune responses can be associated with infections These immune responses are associated with immunostimulatory DNA, and
  • the immune responses which are inhibited by the present methods include those associated with septic shock, inflamma- tory responses such as inflammatory bowel disease, respiratory tract infections such as asthma and sinusitis, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythemetosus, and autoimmune diseases involving humoral immune responses, such as hemolytic anemia
  • the immunostimulatory DN A-mduced responses to be inhibited by the compounds discussed herein can lead to production of cytokines such as IL-6
  • the methods of the present invention are intended to inhibit production of IL-6 in cells If subjects have circulating levels of IL-6 which exceed 20 picograms/milhhter, the subject can be a candidate for treatment with the methods of the invention
  • Normal circulating levels of IL-6 are approximately 10 picograms/ milhhter, while circulating levels of subjects with systemic lupus erythemetosus (50 picograms, milhliter), cancer ( 70 picograms milhliter . and septic shock ( 100 picograms/ milhliter) are generally much higher ( Papadapoulos 1995
  • the methods of the present invention will be useful for subjects exhibiting immunostimulatory DNA-induced disorders, and having circulating levels of IL-6 of at least 20 picograms milhliter
  • the subjects w ill have at least 40 picograms/ milhliter of circulating IL-6
  • concentration of compounds included in compositions used in the methods of the invention can range from about 10 ru ⁇ I to about 10 ⁇ M, preferably about I n nM to about 1 ⁇ M Most preferably, the compounds are found m concentrations of from about 10 nM to about l OOnM
  • the immunostimulatory DNA-induced responses to be inhibited by the compounds discussed herein can lead to an increase in gammaglobuhn over the normal v alues found in subjects Immunoglobuhns can be measured by the nephelomet ⁇ c method, or by electrophoresis (both described in "Protein Abnormalities", by Stephan Ritzman, Alan Riss, 1982)
  • the normal gammaglobuhn levels according to the nephelomet- ⁇ c method are IgG, 700-1500 milligrams/ 100 milhhters, IgA 70-400 milligrams/ 100 milhhters, IgM 30-300 milligrams/ 100 milhhters, and IgD 0-40 milligrams- 100 milhhters When measured by electrophoresis.
  • the normal value for gammaglobuhn is 0 7 to 1 milligrams/ 100 milhhters
  • the immunostimulatory DNA-induced responses to be inhibited by the compounds discussed herein can lead to an increase in the erythrocvte sedimentation rate, as measured by the Westergr ⁇ n sedimentation rate determination (Internal Medicine vol 1 Jay H Stein, ed , 1983 Little Brown Co p 162) Normal sedimentation rates are up to 20 mm/hour, so that the methods of the invention can be used w ith subjects ⁇ ' ibiting ' ⁇ estergren erythrocvte sedimentation rates of more than 24 mm, hour
  • Administering the pharmaceutical composition of the present inv ention can be accomplished by various means known to the skilled artisan
  • the pnarmaceutical compositions according to the invention can be administered locally or svstemically
  • the term "therapeutically effective amount” means the quantity of a compound according to the invention necessary to inhibit a symptom in a subject
  • a "subject” is any vertebrate animal, preferably a mammal, including humans
  • Amounts effective for therapeutic use will, of course, depend on the severity of the disease and the weight and general state of the subject
  • dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition
  • Animal models w ell known to those of skill in the art can be used to determine effective dosages for treatment of particular disorders Various considerations are described, e g , in Gilman et al .
  • compositions according to the invention are in general administered topically, intravenously, orally, or parenterally, or as implants, and ev en rectal use is possible in principle
  • suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (m ⁇ cro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and, or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavo ⁇ ngs, sweeteners or solubhzers are customarily used as described above
  • the pharmaceutical compositions are suitable for use m a variety of drug delivery systems
  • Langer, 1990, Science 249 1527-33 which is incorporated herein by reference
  • the pharmaceutical preparations are preferably prepared and administered in dose units
  • Solid dose units are tablets, capsules or suppositories
  • different daily doses are necessary Under certain circumstances, however, higher or lower daily doses may be approp ⁇ ate
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and ⁇ !so by multiple administration of subdivided doses at specific intervals
  • the invention provides a method for screening compounds for their efficacy in inhibiting the immune response triggered by immunostimulatory DNA Anv compounds which are suitable for administration to subjects can be screened this way
  • the compounds are selected from those having st ctures A and B. as described above
  • This method initially involves a determination of the simulation of the immune response which is triggered by immunostimulatory DN A
  • immunostimulatory DN A means either a humoral (antibody) or cellular (T lymphocyte) immune response
  • the "stimulation index" is a measure of a immunostimulatory DNA to affect an immune response which can be tested in various immune cell assays
  • the stimulation of the immune response can be assayed by measuring various immune parameters, e.g , measuring the antibody- forming capacity, number of lymphocyte subpopulations, mixed leukocvte response assay, and lymphocyte proliferation assay
  • the stimulation of the immune response can also be measured in an assay to measure resistance to infection or tumor growth
  • Methods for measuring a stimulation index are well known to those skilled in the art
  • one assay is the incorporation of radioactive precursors, such as 3 H labeled undine or thymidine in a B-cell culture
  • the induction of secretion of a particular cytokine can also be used
  • One particularly useful cell line for the assay of immunostimulatory DNA action is the mouse-mouse hyb ⁇ doma plasma cell line 7TD1 (ATTC ⁇ CRL 1851 ), which requires exogenous IL-6 for growth, and has been used to characterize and assay IL-6 (Papadopolous et al 1995 J Clin Lab Anal.
  • cell culture conditions were as follows WEHI 231 cells (ATTC #CRL 1702) were grown in log phase in a medium of RPMI 1640 supplemented with 10% fetal bovine serum (heat inactivated at 65°C for one hour), 0 3 grams/Liter of L-glutamine, 50 milligrams/Liter gentamycin, and 0 05 mM 2-mercaptoethanol in a 3 "?
  • the 7TD1 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (heat inactivated at 65°C, 1 hour). 0 3 grams/Liter L-glutamine, 50 milligrams/Liter gentamycin, 0 05 mM 2-mercaptoethanol, and 10 units per milhliter mu ⁇ ne IL-6 in a 3 7 °C humid atmosphere with 5% C0 2 Viable 7TD1 cells were counted in a hemocytometer chamber in duplicate after dilution with 0 4% trypan blue Cell counts were within 10%) or 100 microhters of each other
  • IL-6 enzyme-linked immunosorbent assay ELISA
  • cells were incubated as indicated. At 48 hours, the cell culture supernatant was harvested and assayed for mu ⁇ ne IL-6 by ELISA according to manufacturer's instructions (PharMingen, San Diego CA)
  • CpG-ODN 1760 has the sequence 5'-A ⁇ AATCGACGTTCAAGCAAG-3 ' synthesized on a phosphorothioate or (when indicated) on a phosphodiester backbone
  • the oligonucleotides were purchased from Genosys (The Woodlands TX) Mu ⁇ ne IL-6, mu ⁇ ne neutralizing antibody to IL-6 and ELIS A antibodies were purchased from PharMingen (San Diego CA)
  • the following compounds were all purchased from Sigma Chemical Co , St Louis
  • MO goat anti-mouse IgM ( ⁇ -sIgM. Cat £M8644).
  • Canton MA Verapamil was from American Reagents Laboratory, Shirley NY C -ceram ⁇ de was purchased from Matreya, Inc .
  • PA Ac ⁇ dine homodimer (b ⁇ s-(6-chloro-2-methoxy-9-ac ⁇ d ⁇ nyl)spermme) and 9-ammo-6- chloro-2-methoxyac ⁇ d ⁇ ne were purchased from Molecular Probes, Eugene OR
  • the assay of [ H] Thymidine incorporation was carried out as follows WEHI 231 cells (200 ⁇ L at 2 x 10 'milhliter, in duplicate or triplicate) were incubated in a 96-well plate (Costar, Cambridge MA) Additions were made as indicated After incubating for 16 hours, 0 5 ⁇ Ci [methyl- H] thymidine (2 Ci/milhmole, DuPont NEN, Boston ⁇ LA) w as added to each well, and the cells were incubated for another 4 hours Ceilular DNA was then captured onto glass fiber filters (Whatman 934AH) using a cell harvester (Brandei.
  • WEHI 231 cells (ca. 2 x 10 per milhliter) were incubated for 16 hours at 37°C with 10 ⁇ grams/milhhter ⁇ -sIgM, 6 ⁇ grams/milhhter ODN 1760. and the indicated concentration of the compound After 16 hours, 0 5 ⁇ Ci [ H] thymidine was added After 4 more hours incubation, the cellular DNA was harvested onto glass fiber filters The dry filters were counted in a liquid scintillation counter
  • Fig 1 shows the ratio of the uptake of cells incubated with ⁇ -sIgM divided by the uptake of cells incubated with ⁇ -sIgM with CpG-ODN. each measured w ith the indicated concentration of test compound Each experimental point was the mean of at least duplicate measurements
  • the DNA fragmentation assay was carried out generally as follows WEHI 23 1 cells were incubated with 0 5 ⁇ Ci/ milhliter [ H] thymidine for 3 hours, centrifuged. resus- pended in 2 milhhters of fresh medium at 2 x I0 ⁇ m ⁇ ll ⁇ hter. and incubated in a 24-weH plate with additions as indicated After incubation for 16- 18 hours. 1 milhliter a quots of the cells were centrifuged, washed with 200 ⁇ hters PBS This suspension was drawn into a pipette tip containing 25 ⁇ hters of 2% SDS, and the mixture was immediately ejected
  • the gel was electrophoresed for 1 hour at 65 volts, destained in water, and photographed under ultraviolet light
  • the lanes of the gel were cut into three approximately equal portions
  • the first portion included the gel well and the first 2 millimeters of the running lane
  • the second and third pieces were the remainder of the lane
  • the gel portions w ere melted in glass scintillation vials and mixed with a 20 milhliter of scintillation cocktail and counted in a scintillation counter
  • the amount of fragmented DNA (DN A in the second and third gel pieces) was expressed as a percentage of the total counts recov ered m the lane Referring to the experiments summarized in Fig 2a, WEHI 231 cells (2 x 10" per milhliter) were incubated with or without 6 ⁇ grams/milhhter ODN 1760.
  • CpG-ODN protects cells against -sIgM-induced apoptosis Quinac ⁇ ne rev ersed this protection by ODN 1760, revealed by measurement of both cell count ov er a period of three days (Fig 2a) and percentage of fragmented DNA (Fig. 2b) Similar results were obtained with chloroquine These data show that the effect of certain compounds on thymidine uptake is a true reflection of their effect on cell growth and protection from apoptosis
  • the assay of IL-6 was generally carried out as follows WEHI cells ( 2 x 10' mill liter) were incubated with or without ODN 1760 (6 ⁇ gram/milhhter) and with or without chloroquine or quinac ⁇ ne After 24 hours, the IL-6 content of the supernatant was assayed by enzyme-linked immunoassay using two monoclonal antibodies in a sandwich technique as described by the manufacturer , using standards supplied (PharVIingen. San Diego CA)
  • CpG-ODN stimulated the synthesis of cytoLines.
  • cytoLines include- g IL-6
  • WEHI 231 cell elaborated about 700 picograms IL-6 per milhliter ODN 1760 increased this elaboration to over 10,000 picograms/milhhter
  • quinac ⁇ ne ( 10 nM) or chloroquine (30 nM) completely reversed the effect of ODN 1760
  • the B-cell repertoire is regulated by a number of factors that modulate clonal deletion, including hpopolvsaccha ⁇ des (endotoxm, LPS, derived from gram-negativ e bacteria) w hich promote B-cell growth and inhibit B-cell apoptosis Chloroquine and other compounds have been reported to block LPS-induced responses in mononuclear cells
  • CpG-ODN has been previously shown to block apoptosis in WEHI 23 1 cells which is induced by stimuli other than ⁇ -sIgM (Macfarlane et al , 1997 Immunolog ⁇ 91 586)
  • the effect of quinac ⁇ ne on the protection by ODN 1760 from cell death induced by agents other than ⁇ -sIgM was examined, to determine if the action of the studied compounds w as restricted to ⁇ -sIgM-mduced apoptosis C 2 -ceram ⁇ de mediates receptor-induced cell death (Hannun, 1996 Science 2 ⁇ 4 1855-9) and inhibits thymidine incorporation
  • Thapsigargin induces an increase in intracellular calcium, resulting in massive DN A fragmentation Fig 6 shows that the effect of C 2 -ceram ⁇ de was reduced by ODN 1 7 60.
  • WEHI 231 cells were incubated with C 2 -ceram ⁇ de as indicated with or without 6 ⁇ grams/milhhter ODN and 0 1 ⁇ M quinac ⁇ ne and thymidine incorporation was estimated Referring to the experiments summarized in Fig 7, WEHI 231 cells were prelabeled with 0 5 ⁇ Cv milhliter [ 3 H] thymidine for 3 hours, and resuspended in fresh medium containing 10 nM thapsigargin, 10 ⁇ grams/milhhter ⁇ -sIgM, 6 ⁇ grams/milhhter ODN, and/or 0 1 ⁇ M quinacrme as indicated Cells were lysed 24 hours later, and the DNA was electrophoresed as described above in Example 2 to estimate the percentage fragmentation of DNA
  • Fig 8 shows H uptake as a percentage of ODN-treated cells
  • the H uptake per donor for control samples was as follows donor 1 , 4 31% donor 2 2 76%, donor 3 6 39% Thymidine incorporation for untreated samples was less than 0 05% Samples from three donors were averaged, and error bars indicate 1 standard deviation
  • PBMC peripheral blood mononu- clear cells

Abstract

The invention features methods for inhibiting immunostimulatory DNA-associated immune responses, through the administration of specific compounds of the general class of 9-aminoacridines and 4-aminoquinolines. The invention also features methods of screening compounds useful in inhibiting immunostimulatory DNA-induced immune responses.

Description

METHODS FOR INHIBITING
IMMUNOSTIMULATORY
DNA ASSOCIATED RESPONSES
This application claims priority of provisional U.S. Patent Application No. 60/05 1.066. filed July 3, 1997.
Background of the Invention
The invention relates to a method of inhibiting cellular and humoral responses to immunostimulatory DNA with compositions including 4-aminoquinolines, 9- aminoacridines and derivatives thereof.
Bacterial DNA is increasingly recognized as a powerful modulator of immunity (Krieg,
1997. Trends in Microbiol. 4:73-6), stimulating the polyclonal proliferation of B-cells and the production of cytokines by monocytes and other cells (Ballas et al , 1996. J. Immunol. 157:1840-5). This activity is attributed to unmethylated CpG sequences in bacterial DNA, which are uncommon in vertebrate DNA (Krieg et al, 1995. Nature 374:546-9). Single stranded oligodeoxynucleotides which have the motif PuPuCGPyPy
(CpG-ODN) mimic many of the actions of bacterial DNA (Krieg et al, 1996. Antisense & Nucleic Acid Drug Deve 6: 133-9). and powerfully inhibit the induction of apoptosis in mouse WEHI 231 B-cells by anti-surface IgM (Yi et al , 1996. J. Immunol. 157:4918-25; Macfarlane et al, 1997. Immunology 91 :586). This system is a convenient and reproduc- ible model to study responses to immunostimulatory DNA.
Bacterial DNA immobilized on beads does not stimulate immune responses, suggesting that internalization of the DNA is required for activity. DNA and oligodeoxynucleotides are endocvtosed into acidic vesicles, and are then transported to the cytoplasm and nucleus of cells. Chloroquine, hydroxychloroquine and quinacrine are known to induce remissions of systemic lupus erythematosus and rheumatoid arthritis by an unknown mechanism. These drugs bind to DNA by intercalation. They are weak bases and partition into acidic vesicles At high concentrations, chloroquine can collapse the pH gradient and disrupt the action of endosomal hydrolytic enzymes and the trafficking of receptors
Summary of the Invention The invention is based on the discovery that qumacπne, chloroquine and selected 9- aminoacπdine and 4-amιnoquιnolιne compounds unexpectedly block the action of immunostimulatory DNA. in cells at concentrations much below those needed for the other immunomodulatory effects of these compounds in vitro The 9-amιnoacπdme and 4- aminoquinohne compounds utilized in the methods of the invention inhibit the anti- apoptotic effect of CpG-ODN and the CpG-ODN-induced secretion of IL-6 On the other hand, these compounds do not influence the anti-apoptotic effects of other agents, for example, hpopolysacchaπdes The effects of the compounds are highly specific to immunostimulatory DN Λ These agents are useful for inducing remissions of autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, leading to generally useful remittive and anti-inflammatory agents
The methods of the invention are useful for inhibiting immunostimulatory DNA- associated responses in a subject, and involve the administration of a composition to a subject exhibiting responses initiated or exacerbated by immunostimulatory DNA The compositions include 4-amιnoquιnohnes, 9-amιnoacπdιnes and derivatives thereof These compounds may be linked to each other by linkers The responses initiated or exacerbated by immunostimulatory DNA include those which result in the initiation or exacerbation of various disease states The disease states include septic shock, inflammatory bowel diseases, asthma, sinusitis, various autoimmune disorders such as hemolytic anemia, and others The responses to be inhibited include the production of immunomodulatory proteins such as cytokines, interleukins, mterferons, cell growth factors and chemokines
One such protein is IL-6 Other responses to be inhibited by the methods of the invention are high levels of erythrocyte sedimentation, and high levels of immunoglobuhn
The invention also features a method of screening compounds useful for inhibiting immunostimulatory DNA-associated responses This method involves contacting a ceil with immunostimulatory DN A., thereby inducing a measurable immune response, and a
_τ_ test compound. Any inhibition of the immune response is detected. The cells can be hybπdoma plasma cells, including 7TD 1.
A. "'nucleic acid" or "DNA" means multiple nucleotides (i.e. , molecules comprising a sugar (e.g , πbose or deoxyπbose)) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyπmidine (e.g., cytosme (C). thymine (T) or uracil (U)) or a substituted purine (e.g.. adenine (A) or guanine (G)) As used herein, the term refers to πbonucleotides as well as oligodeoxynucleotides The term also includes polynucleosides (i.e.. a polynucleotide minus the phosphate) and any other organic base- containing polymer Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g. , genomic or cDNA), but are preferably synthetic (e.g. , produced by o gonucleotide synthesis) A "stabilized nucleic acid molecule" or "nuclease-resistant nucleic acid molecule" means a nucleic acid molecule that is relatively resistant to in
Figure imgf000005_0001
degradation (e.g , via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure Unmethvlated CpG-containing nucleic acid molecules that are tens to hundreds of kilobases long are relatively resistant to in vivo degradation. For shorter immunostimulatory nucleic acid molecules, secondary structure can stabilize and increase their effect. For example, if the 3' end of a nucleic acid molecule is self-complementary to an upstream region, so that it can fold back and form a stem loop structure, then the nucleic acid molecule becomes stabilized and therefore exhibits more activity Preferred stabilized or nuclease-resistant nucleic acid molecules referred to in the instant invention have a modified backbone. For use in immune stimulation, especially preferred stabilized nucleic acid molecules are phosphorothioate-modified nucleic acid molecules (i.e. , at least one of the phosphate oxygens of the nucleic acid molecule is replaced by sulfur). Preferably the phosphate modification occurs at or near the 5' and or 3 ' end of the nucleic acid molecule. In addition to stabilizing nucleic acid molecules, phosphorothioate-modified nucleic acid molecules (including phosphorodithioate-modified) can increase the extent of immune stimulation of the nucleic acid molecule, which contains an unmethvlated CpG dmucleotide
International Patent Application Number WO 95/26204 entitled "Immune Stimulation by Phosphorothioate Ohgonucleotide .Analogs" also reports on the non-specific immunostimulatory effect of phosphorothioate modified oligonucleotides International Patent Application WO 96/02555 discloses the ability of unmethylated CpG-contaming oligonucleotides to activate lymphocytes. Unmethylated CpG-containing nucleic acid molecules having a phosphorothioate backbone have been found to preferentially activ ate B-cell activity, while unmethylated CpG-containing nucleic acid molecules having a phosphodiester backbone have been found to preferentially activate monocytic
(macrophages, dendritic cells and monoc tes) and NK (natural killer) cells Other stabilized nucleic acid molecules include non-tonic DNA analogs, such as alkyl- and ary 1- phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or ary l group), phosphodiester and alkylphosphotπesters, in which the charged oxygen moiety is alkylated Nucleic acid molecules that contain a diol, such as tetraethy leneglycol or hexaethyleneglycol, at either or both termini have also been shown substantially resistant to nuc lease degradation
As used herein, the term "immunostimulatory DNA" refers to bacterial DNA, viral DNA, other DNA. synthetic double- or single-stranded DNA, DNA synthesized with a nuclease-resistant backbone or at least a partially nuclease resistant backbone, (such as a phosphorothioate-backboned DNA) which stimulates (e.g , has a mitogenic effect on, or induces or increases cytokine expression by vertebrate lymphocytes Such immunostimulatory DNA includes that which contains an unmethylated cytosine, guanine (CpG) dinucleotide sequence. A "CpG" or "CpG motif refers to a nucleic acid having cytosine followed by a guanine linked by link containing phosphorus The term "methylated CpG" refers to the methylation of the cytosine on the pyπmidme ring, usually occurring at the 5'-posιtιon of the pyπmidme ring The term "unmeth lated CpG" refers to the absence of methy lation of the cytosine on the pyπmidme ring Methylation. partial removal, or removal of an unmethylated CpG motif in an ohgonucleotide of the invention is believed to reduce its effect. Methy lation or removal of all unmethylated CpG motifs in an ohgonucleotide substantially reduces its effect The effect of methy lation or remov l of a CpG motif is "substantial" if the effect is similar to that of an ohgonucleotide that does not contain a CpG motif
As used herein, the term "airw ay obstruction" includes respiratory ailments such as asthma, sinusitis, as well as airway obstructions caused by an infection, or exacerbated by an infection The terms "lower alkyl" and "lower alkoxy" refer to alkyl- and alkoxy groups comprising up to 7 carbon atoms The terms include straight and branched chain groups The term "ahc hc" reters to a non-aromatic carbon chain system forming a ring, and hav ing from 4 to 10 members The term "polyalicychc" refers to a non-aromatic carbon chain system forming more than one ring, each ring having from 4 to 10 members The term "aryl group" refers to aromatic hydrocarbons having from 6 to 26 carbons
L nless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety In case of conflict, the present specification, including definitions, will control In addition the materials, methods and examples are illustrative only and not intended to be limiting
Brief Descπption of the Drawings Fig 1 is a graph showing the effect of various concentrations (log scale) of selected compounds (quinacπne, amodiaqume, chloroquine, hydroxychloroquine, quinidine, quinine, and pπmaquine) on ODN 1760 protection against the -sIgM-mduced inhibition of [Η] thymidine uptake in WEHI 231 cells
Fig 2a is a graph showing the effect of various cell medium additives (ODN 1 ~60. α- slgM. ODN 1760/α-sIgM, quinacπne, quinacπneODN 1760, quinacπne c -sIgM. and quinacπne/ODN l "60/α-sIgM) on the WΕHI 231 cell count measured at different time periods after incubation Fig 2b is a bar graph showing the effect of various cell medium additiv es (ODN
1760 α-sIgM. ODN 1760 -sIgM, and quinacπne ODN 1760/α-sIgM) on me percentage of DN A fragmentation detected from WEHI 231 cells
Fig 3 is a g Dh showing the effect of various concentrations of ODN ι ~60 on W amount of [ H] thymidine uptake in the presence of various concentrations ot quinacne in W EHI 2 1 cells treated with α-sIgVl Fig 4 is a bar graph showing the effect of additions of quinacπne at vaπous times after incubation on the extent of [Η] thymidine uptake m WEHI 231 cells treated with α- slgM Also displayed is a comparison of the effect on [ H] thymidine uptake of incubation of α-sIgM-treated WEHI 231 cells with ODN 1760 and a phosphodiester ODN with the same sequence as ODN 1760
Fig 5 is a bar graph showing the effect of quinacπne on the protection by hpopolysacchaπdes against -sIgM-induced inhibition of [Η] thymidine uptake by W EHI 231 cells
Fig 6 is a bar graph showing the effect of qumacπne on the protection by ODN 1 "60 against C-ceramide-induced inhibition of [ H] thymidine uptake in WEHI 231 cells
Fig 7 is a bar graph showing the effect of quinacπne on the protection by ODN against thapsigargm-induced DNA fragmentation in WEHI 231 cells
Fig 8 is a graph showing ODN 1760-mduced effects on percentage of [ H] thymidine uptake in human mononuclear cells Fig 9 is a graph showing ODN 1760-ιnduced effects on percentage of IL-6 production in human mononuclear cells
Detailed Description Unexpectedly we find that compounds quinacπne, chloroquine, and other structurally related 4-amιnoquιnolιnes and 9-amιnoacπdιnes are extremely potent inhibitors of the action of immunostimulatory DNA These compounds added in low nanomolar concentrations, completely block the ability of immunostimulatory DNA to protect W EHI 23 1 cells from growth arrest, DNA fragmentation and inhibition of thymidine incorporation induced by the engagement of its surface IgM, as well as apoptosis induced by other agents such as hpopolysacchaπdes The inhibitory effect is non-competitive with respect to CpG- ODN, and it occurs even when quinacπne is added after the immunostimulatory DN A The potency of chloroquine, qumacπne and derivatives is magnified by inhibition of the multi-drug resistance pump, suggesting that their site of action is intracεllular The specificity of the effect toward CpG-ODN is illustrated by the fact that quiracπne does not influence the ability of hpopolysacchaπdes (LPS) to inhibit apoptosis WEHI 23 1 cells These results can be reviewed in light of the known biological effects in vitro of quinacπne and chloroquine
Being a diprotic weak base, chloroquine (and many of the other compounds which w ere used) partitions into the acidified cellular vesicles, such as Ivsosomes At high concentration, chloroquine can collapse the pH gradient of Ivsosomes and induce their swelling The compounds chloroquine, hydroxychloroqume and qumacπne induce remissions of rheumatoid arthritis and systemic lupus erythematosus The beneficial effect of these compounds becomes apparent ov er the course of sev eral weeks To account for this valuable therapeutic action, a number of investigators have explored the effects of the compounds on immune and inflammatory responses in xitro. rev ealing a wide range of inhibitory actions Quinacπne inhibits phosphohpase A2 (Roberts et al , 1996 British J Haematologv 92 804- 14. blocks ion channels (Sakuta et al , 1994 Eur J Pharm 252 LI 7-21 ), binds to receptors (Tamamizu et al , 1995 Cell & Mol Neurobiol 15 42"- 38), and inhibits fMLP-induced superoxide production and enzyme release by granulocytes ( Foldes-Filep et al , 1992 / Leuk Biol 52 545-50) Chloroquine and qumacπne ha e little influence on other functional assays of neutrophils (Feπante et al , 1986 Immunol 58 125-30) Chloroquine inhibits cytokine release from mononuclear cells induced by endo- and exo-toxms (Mattson et al, 1996 J Infect Dis 173 212-8) Each of these described effects requires concentrations of chloroquine or quinacπne markedly higher than the concentrations used in the present invention to inhibit CpG-ODN-dnven responses in WEHI 231 cells, and none of them satisfactorily explains the remittive effect of these drugs on autoimmune disorders Chloroquine delays the recycling of proteins to the cell surface from lysosomes. resulting in altered trafficking of lysosomal enzymes and receptors (Chou et al , 1974 in Antibiotics III Mechanism of action of antimicrobial and antuumor agents (Corcoran. J , and Hahn, F , eds ). pp 281-92, Springer Verlag, Berlin) These effects on lysosomal functions are seen only when cells are exposed to higher concentrations of chloroquine than used here, so it is improbable that the suppression ov the compounds of CpG-ODN responses can be attributed solely to these relatively gross actions, even though experiments wth ammonia plus bafilomvcin A suggest that the immunostimulatory DNA-associated response requires vesicular acidification Methods of Inhibiting Immunostimulatory DNA-induced Responses
The invention features methods for inhibiting immunostimulatory DNA-induced responses in a subject. This method includes the administration of a therapeutically effective amount of a composition to the subject. The composition contains a compound that is effective in inhibiting immunostimulatory DNA-induced responses.
Compounds found to be useful for inhibiting immunostimulatory DNA-associated responses include 4-aminoquinoline compounds of the formula
Figure imgf000010_0001
RΛ can be a hydrogen atom, a lower alkyl group, or can be further linked to RB by an alkyl chain. If RA is a lower alkyl group, it is preferred to be methyl or ethyl. If RA is further linked to RB by an alkyl chain (in addition to the depicted linkage by the nitrogen atom), the alkyl chain can be -(CH )n-, where n is from 4 to 7, and is preferably 4 or 5.
The substituent RB can be a hydrogen atom, an alicyclic group (such as a cyclopentyl. cyclohexyl or cycloheptyl group), an alkyl secondary, tertiary or quaternary amino group or alkenyl secondary, tertiary or quaternary amino group. For example, RB can be an unsubstituted alkyl amine, such as 4-[pentyl-N,N-dialkyl amine], 4-[pentyl-N-alkyl amine], 4-[ρentyl amine], 4-[butyl-N,N-dialkyl amine], 4-[butyl-N-alkyl amine], 4-[butyl amine]. 2-[ethyl-N,N-dialkyl amine], 2-[ethyl-N-alkyl amine], 2-[ethyl amine], 3-[propyl-N.N- dialkyl amine], 3-[propyl-N-alkyl amine], and 7-[hepta-4-methyl-4-azaamine]. The N- substituents are generally lower alkyl. but can also include hydroxy-substituted lower alkyl, such as 2-hydroxyethyl. Unsaturated chains include 4-[pent-2-enyl-N,N-dialkyl aminej and 4-[pent-2-enyl-N-alkyl amine]. Longer amine-containing alkyl chains can also be utilized, and the nitrogen need not be located at the terminus of the substituent group
Cyclic amines can be included in the alkyl chain For example, the alkyl chain can terminate in. or be interrupted by. a pyrrole ring, a piperazidyl ring, a pipeπdyl ring, or a morpholinyl ring, any of which may be further substituted with lower alkyl groups In embodiments in w hich RB is an unsubstituted amine-containmg group, the preferred variants of RB can be 2-[ethyl-N,N-dιmethyl amine], 2-[ethyl-N-methyl amine], 4-[pent\ l- N.N-diethyl amine], and 4-[butyl-N.N-dιethyl amine]
The RB amine-contammg chains described above can be variously substituted Substituents include lower alkyl groups, such as methyl, ethyl and propyl Other useful RB substituents include substituted or unsubstituted aryl groups, such as pheny l. anisyl. hydroxyphenyl, chlorophenyl, dichlorophenyl, fluorophenyl, naphth l. thiophenyl. which can be substituted at o-, m- or p-positions, or at 1- or 2-posιtιons in case of naphthy l. heterocychc groups, such as pyπdyl, pyrrolyl, pipeπdyl, and piperazidy l, or halogen, such as chloro, bromo, and fluoro, and other substituents such as hydroxyl. and alkoxyl Further substitution of this amine-containing alkyl chain can comprise, for example, amide or ester linkages, ether or thioether linkages The amine-containing alkyl chain can terminate with a substituent group such as a primary, secondary or tertiary amine, a hydroxy group, a thiol. a carboxyhc acid, or an amide
RB can also be a ahpolycyclic group, such as bicycloheptyl, bicyclooctyl. or adamantyl, and can be linked to any position of these groups RB can also be linked to Rκ by an alkyl chain
The substituent R, can be a hydrogen atom, a lower alkyl group, or an aryl group R: can also be a heteroaromatic group, such as 2-, 3-, or 4-pyπdyl. 1-, 2-, or 3- pyrroly l. or an aryl substituted lower alkenyl group, such as trans-β-styryl and trans-β-[α,β-trans- dimethyl-p-chlorostyryl] It is believed that bulky groups at R: contribute to the efficacv of the compounds of the invention Thus, many of the embodiments include bulky groups at this position Among the unsubstituted aryl groups useful as R, substituents include phenyl, 1- and 2-naphthyl. 1-, 2-. 3-, 4-, or 9-phenanthryl and the like Among the substituted aromatic hydrocarbons include the above-mentioned aryl groups, substituted with lower alkyi groups such as methyl and ethyl, halogens, such as chlorine, fiuorne and bromine, perfluoroalkyl groups such as tπfluoromethyl and pentafluoroethyl. alkoxy groups, such as methoxy; aryloxy groups such as phenoxy; amine-containmg substituents such as N-[ N,N-dιmethyl ethylenediamine], or l-[4-methylpιperazιne] Any of the above groups can be present at o-, m-, or p-positions on a phenyl ring, or at any syntheticallv feasible position on another aryl system Multiply substituted aryl rings are also possible The substituent R, can be a hydrogen atom, a lower alkyl group such as methyl or ethyl, or an aromatic group such as phenyl In certain preferred embodiments, the substituent R, is hydrogen or methyl
The substituent R^ can be a hydrogen atom, a lower alkyl group such as methyl or ethyl, or a halogen atom, such as chlorine, bromine or fluorine The substituent R() can be a hydrogen atom, a lower alkyl group such as methy l or ethyl, a lower alkoxy group such as methoxy or ethoxv, an aryloxy group such as phenoxy, an aryl group such as phenyl, an amine group such as N,N-dιmethy l amino or N,N-dιethylamιno, or a thioether group such as phenylthioether or benzylthioether It is found that bulky substituents on position 6 of the quinohne ring tend to enhance activity, so that many of the preferred embodiments include bulky groups at this position
The substituent R- can be a hydrogen atom, a lower alkyl group such as methyl or ethyl, a lower alkoxy group such as methoxy or ethoxy, an aryloxy group such as phenoxy, a halogen atom such as chlorine, bromine or fluorine, or a lower haloalkyl group, namely a lower perfluoroalkyl group, such as tπfiuromethyl or pentafluoroethyl. The substituent R3 can be a hydrogen atom or a lower alkoxy group such as methoxy or ethoxy Pharmaceutically acceptable salts of any of these compounds are also included in the invention These salts include protonated or deprotonated atoms on the 4- aminoquinolme and counteπons including potassium, sodium, chloπne, bromine, acetate and many other commonly recognized counteπons If R- is a halogen, then at least one of R2. R:, R,, R6, or Rs is not a hydrogen atom and RB is not 4-[N,N-dιalkyl-n-pentylamme] or 4-[N-alkyl-N-hydroxyalky l-n-penty lamme]
It is further found that the 4-amιnoqumolme compounds which inhibit immunostimulatory DNA-associated responses can be linked together via a linker The linker can be connected to each molecule at either the same position on the compound, or at different positions Preferred linked 4-amιnoquιnohne compounds are linked at the same position on each compound. The prefeπed position for linkage between the individual 4-aminoquinoline compounds is at the 4-amino position, shown as RΛ or RB in the structure. The linker can be of a number of types. Preferred linker types are alkyl chains, alkyl chains interrupted with nitrogen atoms, or alkyl chains interrupted with amide linkages. The alkyl chains are preferred to be at least two carbons in length and not more than twelve carbons in length. If the alkyl chain is interrupted by nitrogen atoms, it is preferred that the overall length of the chain be between two and twelve atoms. Prefeπed linker molecules include those of the formula -[(CH2)nlN(R)(CH ) ,J ,r where nl, n2 and n3 are independently between I and 5, and R is a hydrogen atom, a carbonyl group or a lower alkyl group. The linker can further include substituents to vary the hydrophobicity or hydrophilicity of the linked compound as a whole.
Further compounds found to be useful for inhibiting immunostimulatory DNA- associated responses include 9-aminoacridine compounds of the formula
Figure imgf000013_0001
The substituent RB can be a hydrogen atom or an alkyl secondary, tertiary or quaternary amino group. For example, RB can be an unsubstituted alkyl amine, such as 4- [pentyl-N,N-dialkyl amine], 4-[pentyl-N-alkyl amine], 4-[pentyl amine], 4-[butyl-N,N- dialkyl amine], 4-[butyl-N-alkyl amine], 4-[butyl amine], 2-[ethyl-N,N-dialkyl amine], 2- [ethyl-N-alkyl amine], 2-[ethyl amine], 3-[propyl-N,N-dialkyl amine], 3-[propyl-N-alkyl amine], and 7-[hepta-4-methyl-4-azaamine]. The N-substituents are generally lower alkyl. but can also include hvdroxv-substituted lower alkyl, such as 2-hydroxyethy . Unsaturated chains include 4-[pent-2-enyl-N,N-dialkyl amine] and 4-[pent-2-enyl-N-alkyl amine]. Longer amine-containing alkyl chains are also possible, and the nitrogen need not be located at the terminus of the substituent group. Cyclic amines can be included in the alkyl chain. For example, the alkyl chain can terminate in, or be interrupted by, a pyrrole ring, a piperazidyl ring, a piperidyl ring, or a morpholinyl ring, any of which may be
- I I - further substituted with lower alkyl groups In embodiments in which RB is an unsubstituted amine-containing group, the preferred vaπants of RB are 2-[ N.N-dimethy l ethylamine], 2-[N-methyl ethylamine], 4-[N,N-dιethyl pentylamine], and 4-[N,N-dιethyl butylamine] Preferred embodiments of the 9-amιnoacπdιnes useful in the invention hav e R,j as 4-[4-aryl-N,N-dιalkyl butylamine], 4-[4-heteroaromatιc-N.N-dιalkyl butylamine], 4-
[4-aryl-N-alkyl butylamine], and 4-[4-heteroaromatιc-N-alkyl butylamine] Quaternary nitrogen-containing variants of these residues are also envisioned as useful m the inven¬
The RB amine-containmg chains described above can be variously subst'tuted Substituents include lower alkyl groups, such as methyl, ethyl and propyl Other useful
RB substituents include substituted or unsubstituted aryl groups, such as phenyl. amsyl hydroxyphenyl, chlorophenyl. dichlorophenyl, fluorophenyl, naphthyl, thiophenyl. which can be substituted at o-, m- or p-positions, or at 1- or 2-posιtιons in case of naphthyl heterocychc groups, such as pyridyl, pyrrolyl, pipeπdyl, and piperazidyl, or halogen, such as chloro, bromo and fluoro. and other substituents such as hydroxyl, and alkoxy 1
Further substitution of this amine-containmg alkyl chain can comprise, for example, amide or ester linkages, ether or thioether linkages The amine-containmg alkyl chain can terminate with a substituent group such as a primary, secondary or tertiary amine, an hydroxy group, a thiol. a carboxylic acid, or an amide Preferred embodiments have 4-[ N,N-dιalkyl pentylamine] , 4-[4-aryl-N,N-dιalkyl butylamine] or 4-[4-heteroaromatιc-N,N- dialkyl butylamine] groups at this position, in which the aryl or heteroaromatic group is unsubstituted or substituted bv halogen or alkoxy, and the N,N-dιalkyl groups are N N- diethyl groups
The substituent R-, is a lower alkyl group, such as methyl or ethyl Preferred embodi- ments have methyl at this position
The substituent R, can be a hydrogen atom, or a lower alkoxy group
The substituent X is a halogen atom, such as chlorine, bromine or fluor ne Preιeτeα embodiments have chlorine at this position
It is further found that the 9-amιnoacπdme compounds which inhibit immunostimulatory DN Λ-associated responses can be linked together via a linker Tne linker can be connected to each molecule at either the same position on the compound or on different positions Preferred linked 9-ammoacπdιne compounds are linked at the same position on each compound The preferred position for linkage between the individual 9- aminoacπdine compounds is at the 9-amιno position, shown as R or RB in the structure The linker can be of a number of types Preferred linker types are alkyl chains, alkyl chains interrupted with nitrogen atoms, or alkyl chains interrupted with amide linkages
The alkyl chains are preferred to be at least two carbons m length and not more than twelve carbons in length If the alkyl chain is interrupted by nitrogen atoms, it is preferred that the overall length of the chain be between two and twelv e atoms Preferred linker molecules include those of the formula -[(CH;)nlN(R)(CH:)n2]n - where nl , n2 and n3 are independently between 1 and 5, and R is a hydrogen atom, a carbonyl group or a lower alkyl group
Further useful compounds for inhibiting immunostimulatory DNA-associated responses include 4-amιnoquιnolιne compounds and 9-amιnoacπdιne compounds linked together by a linker Preferred linked 4-amιnoquιnohne/9-ammoacπdιne compounds are linked at analogous positions on each compound The preferred position for linkage for the 9-ammoacπdιne compounds is at the 9-amιno position, shown as Rx or RB in the structure and the linker is further linked to the 4-amιno position of the 4-amιnoquιnohne compound The linker can be of a number of types Preferred linker types are those discussed above in connection with the 4-amιnoquιnolme and 9-ammoacπdιne compounds The compositions used in the methods of the present invention are useful to inhibit cellular and humoral immune responses Such immune responses can be associated with infections These immune responses are associated with immunostimulatory DNA, and can result in disease states or can exacerbate disease states. The immune responses which are inhibited by the present methods include those associated with septic shock, inflamma- tory responses such as inflammatory bowel disease, respiratory tract infections such as asthma and sinusitis, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythemetosus, and autoimmune diseases involving humoral immune responses, such as hemolytic anemia
The immunostimulatory DN A-mduced responses to be inhibited by the compounds discussed herein can lead to production of cytokines such as IL-6 Thus, the methods of the present invention are intended to inhibit production of IL-6 in cells If subjects have circulating levels of IL-6 which exceed 20 picograms/milhhter, the subject can be a candidate for treatment with the methods of the invention Normal circulating levels of IL-6 are approximately 10 picograms/ milhhter, while circulating levels of subjects with systemic lupus erythemetosus (50 picograms, milhliter), cancer ( 70 picograms milhliter . and septic shock ( 100 picograms/ milhliter) are generally much higher ( Papadapoulos 1995
J Clin Lab Anal 9 234-7) Thus the methods of the present invention will be useful for subjects exhibiting immunostimulatory DNA-induced disorders, and having circulating levels of IL-6 of at least 20 picograms milhliter Preferably, the subjects w ill have at least 40 picograms/ milhliter of circulating IL-6 The concentration of compounds included in compositions used in the methods of the invention can range from about 10 ru\I to about 10 μM, preferably about I n nM to about 1 μM Most preferably, the compounds are found m concentrations of from about 10 nM to about l OOnM
The immunostimulatory DNA-induced responses to be inhibited by the compounds discussed herein can lead to an increase in gammaglobuhn over the normal v alues found in subjects Immunoglobuhns can be measured by the nephelometπc method, or by electrophoresis (both described in "Protein Abnormalities", by Stephan Ritzman, Alan Riss, 1982) For example, the normal gammaglobuhn levels according to the nephelomet- πc method are IgG, 700-1500 milligrams/ 100 milhhters, IgA 70-400 milligrams/ 100 milhhters, IgM 30-300 milligrams/ 100 milhhters, and IgD 0-40 milligrams- 100 milhhters When measured by electrophoresis. the normal value for gammaglobuhn is 0 7 to 1 milligrams/ 100 milhhters
The immunostimulatory DNA-induced responses to be inhibited by the compounds discussed herein can lead to an increase in the erythrocvte sedimentation rate, as measured by the Westergrεn sedimentation rate determination (Internal Medicine vol 1 Jay H Stein, ed , 1983 Little Brown Co p 162) Normal sedimentation rates are up to 20 mm/hour, so that the methods of the invention can be used w ith subiects ≤ ' ibiting 'Λestergren erythrocvte sedimentation rates of more than 24 mm, hour
" Administering" the pharmaceutical composition of the present inv ention can be accomplished by various means known to the skilled artisan The pnarmaceutical compositions according to the invention can be administered locally or svstemically The term "therapeutically effective amount" means the quantity of a compound according to the invention necessary to inhibit a symptom in a subject A "subject" is any vertebrate animal, preferably a mammal, including humans Amounts effective for therapeutic use will, of course, depend on the severity of the disease and the weight and general state of the subject Typically, dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition Animal models w ell known to those of skill in the art can be used to determine effective dosages for treatment of particular disorders Various considerations are described, e g , in Gilman et al . eds . Goodman and Gilman's The Pharmacological Bases of Therapeutics 8<h ed , Pergamon Press, 1990, and Remington s Pharmaceutical Sciences. 17th ed . Mack Publishing Co . Easton, Pa , 1990, each of which is herein incorporated by reference
The pharmaceutical compositions according to the invention are in general administered topically, intravenously, orally, or parenterally, or as implants, and ev en rectal use is possible in principle Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (mιcro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and, or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoπngs, sweeteners or solubhzers are customarily used as described above The pharmaceutical compositions are suitable for use m a variety of drug delivery systems For a brief review of present methods for d g delivery, see Langer, 1990, Science 249 1527-33 which is incorporated herein by reference
The pharmaceutical preparations are preferably prepared and administered in dose units Solid dose units are tablets, capsules or suppositories For treatment of a patient, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and bodv weight of the patient, different daily doses are necessary Under certain circumstances, however, higher or lower daily doses may be appropπate The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and ι!so by multiple administration of subdivided doses at specific intervals
I D- Method of Screening Antagonists of Immunostimulatorv DNA
In another aspect, the invention provides a method for screening compounds for their efficacy in inhibiting the immune response triggered by immunostimulatory DNA Anv compounds which are suitable for administration to subjects can be screened this way In preferred embodiments, the compounds are selected from those having st ctures A and B. as described above
This method initially involves a determination of the simulation of the immune response which is triggered by immunostimulatory DN A The term "immune response * means either a humoral (antibody) or cellular (T lymphocyte) immune response The "stimulation index" is a measure of a immunostimulatory DNA to affect an immune response which can be tested in various immune cell assays The stimulation of the immune response can be assayed by measuring various immune parameters, e.g , measuring the antibody- forming capacity, number of lymphocyte subpopulations, mixed leukocvte response assay, and lymphocyte proliferation assay The stimulation of the immune response can also be measured in an assay to measure resistance to infection or tumor growth Methods for measuring a stimulation index are well known to those skilled in the art For example, one assay is the incorporation of radioactive precursors, such as 3H labeled undine or thymidine in a B-cell culture The induction of secretion of a particular cytokine can also be used to assess the stimulation index In general, methods known to those skilled in the art for detection of cell proliferation or viability of cells can be used in the present invention Immunostimulatory DNA is contacted with a cell, thereby stimulating a measurable immune response The inhibition of this immune response by the addition of a compound is then determined
One particularly useful cell line for the assay of immunostimulatory DNA action is the mouse-mouse hybπdoma plasma cell line 7TD1 (ATTC ≠ CRL 1851 ), which requires exogenous IL-6 for growth, and has been used to characterize and assay IL-6 (Papadopolous et al 1995 J Clin Lab Anal. 9 234-7, Krakauer, 1993 J
Figure imgf000018_0001
14 267-77) Growth of this cell line was not known to be supported by agents other \ιan IL-6 Unexpectedly it is found that immunostimulatorv DN A can substitute for IL-6 n this cell line (Manzei et al , 1998 Life Science 62 23-7) Thus, the cell line 7TD 1 ι3 the first established cell line shown to be immunostimulatory DNA-dependent for growth An additional useful assay of antagonists of immunostimulatory DNA action is the protection by immunostimulatory DNA from the killing effect of antibody to cell surface immunoglobuhn-M on WEHI 23 1 muπne B-cells Inhibition of this protection by the addition of a compound is then determined The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims
Examples The following examples illustrate specific embodiments of the compounds of the invention, and demonstrate their efficacy m some aspects of the claimed methods
General procedures were employed as described here
Unless otherwise noted, cell culture conditions were as follows WEHI 231 cells (ATTC #CRL 1702) were grown in log phase in a medium of RPMI 1640 supplemented with 10% fetal bovine serum (heat inactivated at 65°C for one hour), 0 3 grams/Liter of L-glutamine, 50 milligrams/Liter gentamycin, and 0 05 mM 2-mercaptoethanol in a 3"?°C humid atmosphere with 5% C02 WEHI 231 cells were diluted with 0 4% trypan blue in phosphate-buffered saline (PBS) and live cells were counted in a hemocytometer chamber Cells were resuspended in medium at 2 x lOVmillihter in 24-well tissue culture plates (Costar, Cambridge MA) Appropriate additions were made, and the cells were incubated and counted daily WEHI 231 cells were diluted with fresh medium if the concentration exceeded 7 x 10 /milhliter
The 7TD1 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (heat inactivated at 65°C, 1 hour). 0 3 grams/Liter L-glutamine, 50 milligrams/Liter gentamycin, 0 05 mM 2-mercaptoethanol, and 10 units per milhliter muπne IL-6 in a 37°C humid atmosphere with 5% C02 Viable 7TD1 cells were counted in a hemocytometer chamber in duplicate after dilution with 0 4% trypan blue Cell counts were within 10%) or 100 microhters of each other
Preparation and sources of materials are as follows For the production of IL-6 enzyme-linked immunosorbent assay (ELISA). cells were incubated as indicated. At 48 hours, the cell culture supernatant was harvested and assayed for muπne IL-6 by ELISA according to manufacturer's instructions (PharMingen, San Diego CA) CpG-ODN 1760 has the sequence 5'-AΓAATCGACGTTCAAGCAAG-3 ' synthesized on a phosphorothioate or (when indicated) on a phosphodiester backbone The oligonucleotides were purchased from Genosys (The Woodlands TX) Muπne IL-6, muπne neutralizing antibody to IL-6 and ELIS A antibodies were purchased from PharMingen (San Diego CA) The following compounds were all purchased from Sigma Chemical Co , St Louis
MO goat anti-mouse IgM (α-sIgM. Cat £M8644). hpopolysacchaπde ( E coh sεrotype 0127 B8 Cat τ=L3129). amodiaquine, chloroquine, qumacπne, pπmaquine, quinidine. quinine, coralyne chloride, and thapsigargm Hydroxychloroquine w as purchased from Copley Pharmaceutical. Inc . Canton MA Verapamil was from American Reagents Laboratory, Shirley NY C -ceramιde was purchased from Matreya, Inc . Pleasant Gap
PA Acπdine homodimer (bιs-(6-chloro-2-methoxy-9-acπdιnyl)spermme) and 9-ammo-6- chloro-2-methoxyacπdιne were purchased from Molecular Probes, Eugene OR
The other compounds listed in Tables 2 and 3 were generous gifts from Dr Jill Johnson of the National Cancer Institute These compounds were synthesized generallv according to known methods of synthesis (Elslager et al , 1969 J Med Chem 12 970-4,
Elslager et al , 1969 J Med Chem 12 600-7, Marquez et al , 1972 J Med Chem 36- 9) The compounds are available from Developmental Therapeutics Program, National Cancer Institute. Executive Plaza North, Bethesda MD, 20892
Example 1 Simultaneous Addition of Immunostimulatorv DNA and Compounds
The assay of [ H] Thymidine incorporation was carried out as follows WEHI 231 cells (200 μL at 2 x 10 'milhliter, in duplicate or triplicate) were incubated in a 96-well plate (Costar, Cambridge MA) Additions were made as indicated After incubating for 16 hours, 0 5 μCi [methyl- H] thymidine (2 Ci/milhmole, DuPont NEN, Boston λLA) w as added to each well, and the cells were incubated for another 4 hours Ceilular DNA was then captured onto glass fiber filters (Whatman 934AH) using a cell harvester (Brandei. Gaithersberg MD) The filters were dried, placed into plastic scintillation lals with 10 milhhters of scintillation cocktail (EconoSafe. Research Products International. Mount Prospect IL ) and counted in a liquid scintillation counter (Beckman LS-3 13 T) The H recovered on the filter was expressed as a traction of the H added In a t pical experiment, WEHI 23 1 cells incorporate about 15% of added JH
- 1 3- Referring to experiments summarized in Fig. 1, WEHI 231 cells (ca. 2 x 10 per milhliter) were incubated for 16 hours at 37°C with 10 μgrams/milhhter α-sIgM, 6 μgrams/milhhter ODN 1760. and the indicated concentration of the compound After 16 hours, 0 5 μCi [ H] thymidine was added After 4 more hours incubation, the cellular DNA was harvested onto glass fiber filters The dry filters were counted in a liquid scintillation counter
In the absence of other additions, cells incorporated about 20% of the added radioactivity This was reduced to about 1 % by the addition of α-sIgM In the absence of a compound, ODN 1760 restored the uptake to about 15% of added radioactivity The vertical axis of Fig 1 shows the ratio of the uptake of cells incubated with α-sIgM divided by the uptake of cells incubated with α-sIgM with CpG-ODN. each measured w ith the indicated concentration of test compound Each experimental point was the mean of at least duplicate measurements
In the absence of other additions, α-sIgM powerfully inhibited [JH] incorporation This inhibition is almost completely relieved by ODN 1760 Fig 1 shows that quinacπne, hydroxychloroquine, chloroquine and amodiaquine (substituted 4-amιnochloroquιnolιnes) completely inhibited the CpG-ODN effect at nanomolar concentrations The compounds themselves did not inhibit [3H] thymidine incorporation unless added at concentrations well in excess of 30 μM Quimdine and quinine (substituted 4-methylquιnohnes) and pπmaquine (a substituted 8-ammoqumohne) had little effect on CpG-ODN action at concentrations less than 10 μM Qumacπne and chloroquine showed promise as representative members of structural families useful in inhibiting immunostimulatory DNA-induced responses
Example 2 Effect on Growth and Apoptosis
The DNA fragmentation assay was carried out generally as follows WEHI 23 1 cells were incubated with 0 5 μCi/ milhliter [ H] thymidine for 3 hours, centrifuged. resus- pended in 2 milhhters of fresh medium at 2 x I0\mιllιhter. and incubated in a 24-weH plate with additions as indicated After incubation for 16- 18 hours. 1 milhliter a quots of the cells were centrifuged, washed with 200 μhters PBS This suspension was drawn into a pipette tip containing 25 μhters of 2% SDS, and the mixture was immediately ejected
- 1 - into a well of a 0 8% agarose TBE gel containing 0.5 μhter/milhhter ethidium bromide
The gel was electrophoresed for 1 hour at 65 volts, destained in water, and photographed under ultraviolet light The lanes of the gel were cut into three approximately equal portions The first portion included the gel well and the first 2 millimeters of the running lane The second and third pieces were the remainder of the lane The gel portions w ere melted in glass scintillation vials and mixed with a 20 milhliter of scintillation cocktail and counted in a scintillation counter The amount of fragmented DNA (DN A in the second and third gel pieces) was expressed as a percentage of the total counts recov ered m the lane Referring to the experiments summarized in Fig 2a, WEHI 231 cells (2 x 10" per milhliter) were incubated with or without 6 μgrams/milhhter ODN 1760. 10 μgrams, milhliter α-sIgM, or 0 1 μM quinacπne as indicated Live cells were counted daily The cells were diluted in medium if their concentration exceeded 7 x 10" per milhliter Referring to the experiments summarized in Fig 2b, [Η]-labeled WEHI 23 1 cells were incubated with no addition, or simultaneous additions as indicated (α-sIgM 10 μgrams/milhhter, ODN 1760 6 μgrams/ milhliter, quinacπne 0 1 μM) After 16 hours, cells were loaded into wells of 0 8% agarose gel by lysing with 1% SDS, and DNA electrophoresed 1 hour at 65 volts The percentage fragmentation was estimated as described above In this and other figures, error bars indicate 1 standard deviation Quinacπne alone influenced neither cell growth nor cell death induced by α-sIgM
CpG-ODN protects cells against -sIgM-induced apoptosis Quinacπne rev ersed this protection by ODN 1760, revealed by measurement of both cell count ov er a period of three days (Fig 2a) and percentage of fragmented DNA (Fig. 2b) Similar results were obtained with chloroquine These data show that the effect of certain compounds on thymidine uptake is a true reflection of their effect on cell growth and protection from apoptosis
Example 3 Effect on IL-6 Synthesis
The assay of IL-6 was generally carried out as follows WEHI cells ( 2 x 10' mill liter) were incubated with or without ODN 1760 (6 μgram/milhhter) and with or without chloroquine or quinacπne After 24 hours, the IL-6 content of the supernatant was assayed by enzyme-linked immunoassay using two monoclonal antibodies in a sandwich technique as described by the manufacturer , using standards supplied (PharVIingen. San Diego CA)
Besides inhibiting apoptosis, CpG-ODN stimulated the synthesis of cytoLines. includ- g IL-6 In the absence of other additions, WEHI 231 cell elaborated about 700 picograms IL-6 per milhliter ODN 1760 increased this elaboration to over 10,000 picograms/milhhter The addition of quinacπne ( 10 nM) or chloroquine (30 nM) completely reversed the effect of ODN 1760
Example 4 Non-competitive Kinetics
Refemng to the experiments summarized in Fig 3, 10 μgrams/ milhliter α-sIgM and the indicated concentrations of ODN 1760 and qumacπne were added simultaneously to WEHI 231 cells Thymidine incorporation was estimated at 16 hours as described for Example 1 In the experiments summarized in Fig 3, the inhibition by α-sIgM of [ H] thymidine incorporation in the presence of a range of concentrations of ODN 1760 and quinacπne was measured The data show that increasing the concentration of ODN 1760 did not overcome the inhibitory effect of quinacπne, suggesting that the compounds did not compete with immunostimulatory DNA for a common site, such as a receptor, an enzyme active site or a transport protein.
Example 5 Subsequent Addition
Referring to the experiments summaπzed in Fig 4, WEHI 231 cells were incubated with α-sIgM and ODN 1760 At times thereafter 0 1 μM qumacπne was added (data shown in the left three bars) Cells were incubated with 10 μgrams, milhliter α-sIgM and ODN 1760 (6 μgrams, milhliter, phosphorothioate or a phosphodiester ODN with the same sequence as 1760 (PD ODN) (30 μM at time 0 hours and 8 hours, data represented in right six bars in Fig 4j [3H] thymidine incorporation was estimated at 16 hours
It could be hypothesized that the compounds interfere with the transport of ODN to their intracellular site of action, in which case the compounds would prevent, but not reverse, the effect of nuclease-resistant CpG-ODN This prediction did not appear to be correct Fig 4 shows that quinacπne blocked the action of CpG-ODN even when added 8 hours after the CpG-ODN, suggesting that quinacπne interfered with the mechanism the cell uses to recognize CpG-ODN (or with subsequent signal-response coupling), rather than transport CpG-ODN used in typical experiments was synthesized on a nuclease-resistant phosphorothioate backbone The phosphodiester ODN with the same base sequence as ODN P60 is considerably less potent than ODN 1760 Fig 4 shows that quinacπne w as effectiv e in blocking the action of phosphodiester ODN, although the innibitory effect w as less complete than with phosphorothioate ODN
Example 6 Effect of Ouinacπne on Protection bv Lipopolvsacchaπde
The B-cell repertoire is regulated by a number of factors that modulate clonal deletion, including hpopolvsacchaπdes (endotoxm, LPS, derived from gram-negativ e bacteria) w hich promote B-cell growth and inhibit B-cell apoptosis Chloroquine and other compounds have been reported to block LPS-induced responses in mononuclear cells
(Jacobsen et al , 1995 Trap Med Parasitol 46 8-92, Hannun, 1996 Science 274 1855-
9)
In the experiments summarized in Fig 5, the influence of LPS on α-sIgM-mduced suppression of thymidine incorporation was examined Even at high concentrations, LPS has much less ability than ODN 1760 to reverse α-sIgM-induced growth inhibition m W EHI 231 cells Qumacπne at a concentration that completely reversed the effect of ODN 1760 had no effect on the action of LPS Thus (at the concentration used) quma- cπne did not block the detection of LPS and subsequent signal-response coupling in the same ay that it did with CpG-ODN Lipopolysacchaπde (LPS) was added at the indicated concentration witn or without 10 μgrams, milhliter α-sIgM and 0 1 μM qumacπne The small degree of protection against c-sIgM-induced inhibition of [Η] thymidine uptake afforded by LPS is not influenced by quinacπne Example 7 Immunostimulatorv DNA blockade of apoptosis induced bv other agents
CpG-ODN has been previously shown to block apoptosis in WEHI 23 1 cells which is induced by stimuli other than α-sIgM (Macfarlane et al , 1997 Immunolog\ 91 586) The effect of quinacπne on the protection by ODN 1760 from cell death induced by agents other than α-sIgM was examined, to determine if the action of the studied compounds w as restricted to α-sIgM-mduced apoptosis C2-ceramιde mediates receptor-induced cell death (Hannun, 1996 Science 2~4 1855-9) and inhibits thymidine incorporation Thapsigargin induces an increase in intracellular calcium, resulting in massive DN A fragmentation Fig 6 shows that the effect of C2-ceramιde was reduced by ODN 1760. and that quinacπne blocked this protective effect Fig 7 shows that quinacπne also blocked the protective effect of ODN 1 "60 against thapsigargin-induced DN A fragmentation Thus, quinacπne appeared to block the protectiv e effect of immunostimulatory DN A generally not just the protection against α-sIgM-induced apoptosis
Referring to the experiments summarized in Fig 6, WEHI 231 cells were incubated with C2-ceramιde as indicated with or without 6 μgrams/milhhter ODN and 0 1 μM quinacπne and thymidine incorporation was estimated Referring to the experiments summarized in Fig 7, WEHI 231 cells were prelabeled with 0 5 μCv milhliter [3H] thymidine for 3 hours, and resuspended in fresh medium containing 10 nM thapsigargin, 10 μgrams/milhhter α-sIgM, 6 μgrams/milhhter ODN, and/or 0 1 μM quinacrme as indicated Cells were lysed 24 hours later, and the DNA was electrophoresed as described above in Example 2 to estimate the percentage fragmentation of DNA
Example 8 Inhibition of ~ D1 cell growth bv compounds
Little growth occurs when 7TD1 cells are incubated in RPMI 1640 supplemented with
"> fetal calf serum without added IL-6 or CpG-ODN The addition of IL-6 supported vigorous growth with half-maximal effect at about 10 microhters/ milhliter The phosphorothioate CpG-ODN 1760 supported the growth of the 7TD 1 cells n the absence of added IL-6 Growth stimulation by CpG-ODN was blocked by chlorouume and quinacπne. but these 4-amιnoquιnolme compounds had little effect on lL-6-ιnduced 30 growth (Table 1) The 7TD1 cells were incubated for five days and were washed twice with medium to remove IL-6, and resuspended at 2 x 104 per milhliter in medium. Cell counts were performed and the percent inhibition of growth was computed. The term "anti" refers to 40 nanograms/milliliter neutralizing antibody to IL-6.
Table 1. Inhibition of 7TD1 cell growth
Addition 7TD1 cells/μL % Growth Inhibition
Figure imgf000027_0001
Example 9 Immunostimulatorv Effects on Human Mononuclear Cells
The effects of the compounds is not restricted to immortal mouse cells Lnfractionated human peripheral blood cells incorporated thymidine and produced IL-6 when exposed to ODN 1760 as displayed in Fig 8 and 9, respectivelv Both effects w ere inhibited by quinacrine and chloroquine
Referring to the experiments summarized in Fig 8, human mononucleai cells (2 x 10"/ milhliter) from three different donors were incubated with or without ODN 1760 and the indicated concentration of quinacrine or chloroquine for four davs [ H] thvmidine incorporation ( 18 hour pulse) was then measured Fig 8 shows H uptake as a percentage of ODN-treated cells The H uptake per donor for control samples was as follows donor 1 , 4 31% donor 2 2 76%, donor 3 6 39% Thymidine incorporation for untreated samples was less than 0 05% Samples from three donors were averaged, and error bars indicate 1 standard deviation
Referring to the experiments summarized in Fig 9, human peripheral blood mononu- clear cells (PBMC) from three donors were incubated with or without ODN 1760 and the indicated concentration of test compound After 24 hours, the concentration of IL-6 w as determined in the supernatant The figure indicates the percentage of IL-6 in reference to ODN-treated cells The IL-6 amounts for control sample per donor were as follows donor 1, 203 picograms/milhhter, donor 2, 71 5 picograms/milhhter, donor 3, 465 picograms/milhhter IL-6 measured from untreated cells was 0-21 picograms/milhhter
Results from the three donors ere averaged, and error bars indicate 1 standard deviation
Example 10 9- Ammoacπdines and 4-Amιnoquιnohne Deπvatives
Some specific embodiments of 9-amιnoacndιnes useful in the methods of the invention are included in Table 2 Some specific embodiments of 4-amιnoquιnohnes useful in the methods of the vention are included m Table 3
The structures are listed as changes from quinacπne or chloroquine The symbol - indicates no change Vie and Et indicate -CH and - CH2CH respectivelv Aromatic substituents are in the para position except as indicated The activity is -ιog M) of the concentration (M) required tor half maximal inhibition of CpG-ODN effect on [ H] thymidine uptake in the presence of α-sIgM. The last column is the activity expressed as a fraction of activity of chloroquine (fold).
17-
Figure imgf000030_0001
Figure imgf000031_0002
Figure imgf000031_0001
Figure imgf000031_0003
Figure imgf000031_0004

Claims

CLAIMSWhat is claimed is:
1. A method of inhibiting immunostimulatory DNA-associated responses in a subject, the method comprising administering a therapeutically effective amount of a composition to a subject exhibiting responses initiated or exacerbated by immunostimulatory DNA, the composition comprising a compound having the structural formula A
Figure imgf000032_0001
wherein R v is a hydrogen atom, a lower alkyl group, or linked to RB by a substituted or unsubstituted alkyl chain; RB is a hydrogen atom, an alicyclic group, an alkyl secondary, tertiary or quater- nary amine, or an alkenyl secondary, tertiary or quaternary amine; R2 is a hydrogen atom, a lower alkyl group, an aryl group, a heteroaromatic group, or a lower alkenyl group substituted with an aryl group; R3 is a hydrogen atom, a lower alkyl group, or an aromatic group; R, is a hydrogen atom, a lower alkyl group, or a halogen atom; RΓÇ₧ is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group, an aryl group, an amino group or a thioether group; R- is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group, a haloalkyl group, or a halogen atom; and RΓÇ₧ is a hydrogen atom, or a lower alkoxy group, and pharmaceutically acceptable salts thereof, with the proviso that if R- is a halogen. then at least one of R2, RJ; R5, R6 or R are non-hydrogen and RB is not 4-[N,N- dialkyl-n-pentylamine] or 4-[N-alkyl-N-hydroxyalkyl-n-pentylamine].
2. The method of claim 1, wherein RB is selected from the group consisting of a 4-[2- substituted butylamine], a 4-[3-substituted butylamine], a 4-[4-substituted butylamine], a 4-[5-substituted pentylamine], and a 2-[2-substituted ethylamine].
3. The method of claim 2, wherein RB is an aryl-substituted butylamine.
4. The method of claim 1, wherein the compound inhibits immunostimulatory DNA- associated responses at concentrations of less than 10 ╬╝M.
5. The method of claim 1, wherein the immunostimulatory DNA-associated response results in a disease state.
6. The method of claim 5, wherein the disease state is rheumatoid arthritis.
7. The method of claim 5, wherein the disease state is systemic lupus erythematosus.
8. The method of claim 1, wherein the immunostimulatory DNA-associated response results in exacerbation of a disease state.
9. The method of claim 8, wherein the immunostimulatory DNA associated response exacerbates inflammatory bowel disease.
10. The method of claim 5, wherein the immunostimulatory DNA-associated response initiates an autoimmune disorder.
11. The method of claim 5, wherein the immunostimulatory DNA-associated response exacerbates an autoimmune disorder.
12. The method of claim 11, wherein the autoimmune disorder is hemolytic anemia.
13. The method of claim 1, wherein the immunostimulatory DNA-associated response is the production of an immunomodulatory protein.
14. The method of claim 13, wherein the immunomodulatory protein is IL-6.
15. The method of claim 1, wherein the immunostimulatory DNA-associated response is an erythrocvte sedimentation rate of at least 24 mm/hour.
16. The method of claim 1, wherein the immunostimulatory DNA-associated response is an increase in immunoglobulin level.
17. A method of inhibiting immunostimulatory DNA-associated responses in a subject, the method comprising administering a therapeutically effective amount of a composition to a subject exhibiting immunostimulatory DNA-associated responses, the composition comprising a compound having the structural formula B
Figure imgf000035_0001
wherein, RB is a hydrogen atom, or an alkyl secondary or tertiary amino substituent selected from the group consisting of 4-[4-aryl-N,N-dialkyl butylamine], 4-[4-heteroaromatic- N,N-dialkyl butylamine], 4-[4-aryl-N-alkyl butylamine] and 4-[4-heteroaromatic-N- alkyl butylamine]; R2 is a lower alkyl group; and R3 is a hydrogen atom, or a lower alkoxy group; X is a halogen atom, and pharmaceutically acceptable salts thereof.
18. The method of claim 17, wherein RB in structure B is selected from the group consisting of 4-[4-p-halophenyl-N,N-dialkyl butylamine], 4-[4-ρhenyl-N,N-dialkyl butylamine], 4-[4-naphthyl-N,N-dialkyl butylamine], 4-[4-p-alkoxyphenyl-N,N-dialkyl butylamine], 4-[4-(2-thienyl)-N,N-dialkyl butylamine], and 4-[4-(3-pyridyl)-N,N- dialkyl butylamine].
19. The method of claim 18, wherein the compound inhibits immunostimulatory DNA-associated responses at concentrations of less than 10 ╬╝M.
20. The method of claim 17, wherein the immunostimulatory DNA-associated response results in a disease state.
21. The method of claim 17, wherein the immunostimulatory DNA-associated response results in exacerbation of a disease state.
22. The method of claim 21. wherein the immunostimulatory DNA associated response exacerbates inflammatory bowel disease.
23. The method of claim 20, wherein the immunostimulatory DNA-associated response initiates an autoimmune disorder.
24. The method of claim 20, wherein the immunostimulatory DNA-associated response exacerbates an autoimmune disorder.
25. The method of claim 24, wherein the autoimmune disorder is hemolytic anemia.
26. The method of claim 17, wherein the immunostimulatory DNA-associated response is the production of an immunomodulatory protein.
27. The method of claim 26, wherein the immunomodulatory protein is IL-6.
28. The method of claim 17, wherein the immunostimulatory DNA-associated response is an erythrocvte sedimentation rate of at least 24 mm/hour.
29. The method of claim 17, wherein the immunostimulatory DNA-associated response is an increase in immunoglobulin level.
30. A method of inhibiting immunostimulatory DNA-associated responses in a subject, the method comprising administering a therapeutically effective amount of a composition to a subject exhibiting immunostimulatory DNA-associated responses, the composition comprising a linked compound formed by linking two compounds having the structure A,
Figure imgf000037_0001
wherein
RA is a hydrogen atom, a lower alkyl group, or linked to RB by a substituted or unsubstituted alkyl chain;
RB is a hydrogen atom, an alicyclic group, an alkyl secondary, tertiary or quaternary amine, or an alkenyl secondary, tertiary or quaternary amine;
R2 is a hydrogen atom, a lower alkyl group, an aryl group, a heteroaromatic group, or a lower alkenyl group substituted with an aryl group;
R3 is a hydrogen atom, a lower alkyl group, or an aromatic group;
R5 is a hydrogen atom, a lower alkyl group, or a halogen atom;
R<5 is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group, an aryl group, an amino group or a thioether group;
R7 is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group, a haloalkyl group, or a halogen atom; and
Rs is a hydrogen atom, or a lower alkoxy group, and pharmaceutically acceptable salts thereof, with the proviso that if R- is a halogen, then at least one of R2. R3, R5, RΓÇ₧ or R3 is non-hydrogen and RB is not 4-[N.N-dialkyl-n- pentylamine] or 4-[N-alkyl-N-hydroxyalkyl-n-pentylamine], wherein the linking is accomplished by a linking group.
3 1. The method of claim 30, wherein the linking group links positions RB in the two compounds of structure A.
32. The method of claim 31. wherein the linking group comprises an alkyl chain, or an alkyl chain interrupted by nitrogen atoms.
33. A method of inhibiting immunostimulatory DNA-associated responses in a subject, the method comprising administering a therapeutically effective amount of a composition to a subject exhibiting immunostimulatory DNA-associated responses, the composition comprising a linked compound formed by linking two compounds having the structure B
Figure imgf000038_0001
wherein,
RB is a hydrogen atom, or an alkyl secondary or tertiary amino substituent selected from the group consisting of 4-[4-aryl-N.N-dialkyl butylamine], 4-[4-heteroaromatic- N.N-dialkyl butylamine], 4-[4-aryl-N-alkyl butylamine] and 4-[4-heteroaromatic-N-alk l butylamine];
R2 is a lower alkyl group: and
R, is a hydrogen atom, or a lower alkoxy group:
X is a halogen atom, and pharmaceutically acceptable salts thereof, wherein the linking is accomplished by a linking group.
34. The method of claim 33. wherein the linking group links positions RB in the two compounds.
35. The method of claim 34. wherein the linking group comprises an alkyl chain, or an alkyl chain interrupted by nitrogen atoms.
36. A method of inhibiting immunostimulatory DNA-associated responses in a subject. the method comprising administering a therapeutically effective amount of a composition to a subject exhibiting immunostimulatory DNA-associated responses, the composition comprising a linked compound formed by linking one compound of structure A
Figure imgf000039_0001
wherein RA is a hydrogen atom, a lower alkyl group, or linked to RB by a substituted or unsubstituted alkyl chain; RB is a hydrogen atom, an alicyclic group, an alkyl secondary, tertiary or quaternary amine. or an alkenyl secondary, tertiary or quaternary amine; R2 is a hydrogen atom, a lower alkyl group, an aryl group, a heteroaromatic group, or a lower alkenyl group substituted with an aryl group; R3 is a hydrogen atom, a lower alkyl group, or an aromatic group: R is a hydrogen atom, a lower alkyl group, or a halogen atom; RΓÇ₧ is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group. an aryl group, an amino group or a thioether group; R7 is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an
Figure imgf000039_0002
loxy group, a haloalkyl group, or a halogen atom; and Rs is a hydrogen atom, or a lower alkoxy group, and pharmaceutically acceptable salts thereof, with the proviso that if R7 is a halogen, then at least one of R2, R3, R5. R^ or Rs is non-hydrogen and RB is not 4-[N,N-dialkyl-n- pentylamine] or 4-[N-alkyl-N-hydroxyalkyl-n-pentylamine], and a compound of structure B,
Figure imgf000040_0001
wherein, RB is a hydrogen atom, or an alkyl secondary or tertiary amino substituent selected from the group consisting of 4-[4-aryl-N,N-dialkyl butylamine], 4-[4-heteroaromatic- N.N-dialkyl butylamine], 4-[4-aryl-N-alkyl butylamine] and 4-[4-heteroaromatic-N-alkyl butylamine]; R2 is a lower alkyl group; and R3 is a hydrogen atom, or a lower alkoxy group; X is a halogen atom, and pharmaceutically acceptable salts thereof. wherein the linking is accomplished by a linking group.
37. The method of claim 36, wherein the linking group links positions RB in the two compounds.
38. The method of claim 37, wherein the linking group comprises an alkyl chain, or an l chain interrupted by nitrogen atoms.
39. A method of reducing immune response to infection, the method comprising adminis- tering a therapeutically effective amount of a composition to a subject exhibiting responses associated with an infection, the composition comprising a compound having the structural formula A
Figure imgf000041_0001
wherein RA is a hydrogen atom, a lower alkyl group, or linked to RB by a substituted or unsubstituted alkyl chain; RB is a hydrogen atom, an alicyclic group, an alkyl secondary, tertiary or quaternary amine, or an alkenyl secondary, tertiary or quaternary amine; R2 is a hydrogen atom, a lower alkyl group, an aryl group, a heteroaromatic group, or a lower alkenyl group substituted with an aryl group; R3 is a hydrogen atom, a lower alkyl group, or an aromatic group; R5 is a hydrogen atom, a lower alkyl group, or a halogen atom; Rή is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group. an aryl group, an amino group or a thioether group; R- is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group, a haloalkyl group, or a halogen atom; and Rs is a hydrogen atom, or a lower alkoxy group, and pharmaceutically acceptable salts thereof, with the proviso that if R7 is a halogen. then R2. R3, R5, R,, or Rs must be non-hydrogen and RB is not 4-[N.N-dialkyl-n- pentylamine] or 4-[N-alkyl-N-hydroxyalkyl-n-pentylamine] .
40. The method of claim 39. wherein the infection results in septic shock.
41. The method of claim 39. wherein the infection results in airway obstruction.
42. A method of identifying a compound that inhibits an immunostimulatory DNA- induced response, the method comprising contacting a cell with an immunostimulatory DNA. thereby inducing a measurable immune response, and a test compound and detecting inhibition of the immunostimulatory effect of the immunostimulatory DNA.
43. The method of claim 42. wherein the cell is a hybridoma plasma cell.
44. The method of claim 42. wherein the test compound is selected from the group consisting of compounds with structure A, and compounds with structure B.
45. The method of claim 42, wherein the detecting is by measuring IL-6 production.
46. The method of claim 43, wherein the hybridoma plasma cell is 7TD 1.
47. A method of inhibiting immunostimulatory DNA-associated responses in a subject, the method comprising administering a therapeutically effective amount of a composition to a subject exhibiting responses initiated or exacerbated by immunostimulatory DNA, the composition comprising a compound having the structural formula A
Figure imgf000043_0001
wherein RA is a hydrogen atom, a lower alkyl group, or linked to RB by a substituted or unsubstituted alkyl chain; RB is a hydrogen atom, an alicyclic group, an alkyl secondary, tertiary or quaternary amine, or an alkenyl secondary, tertiary or quaternary amine; R2 is a hydrogen atom, a lower alkyl group, an aryl group, a heteroaromatic group, or a lower alkenyl group substituted with an aryl group; R3 is a hydrogen atom, a lower alkyl group, or an aromatic group; R5 is a hydrogen atom, a lower alkyl group, or a halogen atom; R^, is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group. an aryl group, an amino group or a thioether group; R7 is a hydrogen atom, a lower alkyl group, a lower alkoxy group, an aryloxy group, a haloalkyl group, or a halogen atom; and RΓÇ₧ is a hydrogen atom, or a lower alkoxy group, and pharmaceutically acceptable salts thereof, wherein the immunostimulatory DNA-associated response results in a disease state selected from the group consisting of septic shock, inflammatory bowel disease, asthma. sinusitis, and hemolytic anemia.
48. A method of inhibiting immunostimulatory DNA-associated responses in a subject, the method comprising administering a therapeutically effective amount of a composition to a subject exhibiting responses initiated or exacerbated by immunostimulatory DNA, the composition comprising a compound having the structural formula B
Figure imgf000044_0001
wherein. Rg is a hydrogen atom, or an alkyl secondary or tertiary amino substituent; R2 is a lower alkyl group; and R3 is a hydrogen atom, or a lower alkoxy group; X is a halogen atom, and pharmaceutically acceptable salts thereof, wherein the immunostimulatory DNA-associated response results in a disease state selected from the group consisting of septic shock, inflammatory bowel disease, asthma, sinusitis, and hemolytic anemia.
49. The method of claim 1, wherein the immunostimulatory DNA-associated response is the inhibition of the induction of B-cell death.
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