WO2014169043A1 - Agents anti-inflammatoires et leurs procédés d'utilisation - Google Patents

Agents anti-inflammatoires et leurs procédés d'utilisation Download PDF

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WO2014169043A1
WO2014169043A1 PCT/US2014/033509 US2014033509W WO2014169043A1 WO 2014169043 A1 WO2014169043 A1 WO 2014169043A1 US 2014033509 W US2014033509 W US 2014033509W WO 2014169043 A1 WO2014169043 A1 WO 2014169043A1
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cationic polymer
nucleic acid
polymers
cationic
polymer
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PCT/US2014/033509
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English (en)
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Bruce A. Sullenger
Eda HOLL
Hemraj JUWARKER
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Duke University
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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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • the present invention relates, in general, to methods of neutralizing the effects of proinflammatory nucleic acids in a subject (e.g., a human) or in cells and to cationic polymers suitable for use in such methods.
  • the invention further relates to methods of identifying antiinflammatory cationic polymers, e.g., by screening combinatorial libraries for nucleic acid- binding polymers,
  • Nucleic acids are released from dead and dying cells. These extracellular nucleic acids (RNAs and DNAs) can be taken up by inflainmatory cells and can activate multiple nucleic acid- sensing Pattern Recognition Receptors (PRR) such as the Toll-Like Receptors (TLRs 3 S 7, 8 and 9 in particular), which are localized in endosomes (Kawai and Akira, Nat. ' .Immunol. 1 1 (5):373- 84 (2010)).
  • PRR Pattern Recognition Receptors
  • TLRs 3 S 7, 8 and 9 the Toll-Like Receptors
  • the inappropriate activation of these TLRs can elicit a variety of inflammatory and autoimmune diseases, for example, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. (Lee et al, Proc. Natl Acad. Sci. USA 108(34): 14055-60 (2011).) It has been previously reported that certain
  • PAMAM-G3, COP, HDMBr can inhibit activation of nucleic acid-sensing PRRs, irrespective of whether they recognize ss A, dsRNA or hypomeihy!ated DMA (Lee et al, Proc. Natl. Acad. Sci. USA 108(34): 14055-60 (201 .1 )).
  • Systemic administration of such polymers can prevent fatal liver injury caused by pro-inflammatory nucleic acids in a murine acute toxic shock model.
  • the present invention results, at least in part, from studies designed to generate a library of cationic polymers that can be screened for polymers having the ability to neutralize the effects of pro-inflammatory nucleic acids, for example, in TLR 9 activation, These studies have resulted in the identification of certain cationic polymers that can neutralize the proinflammatory effecis of any nucleic acid, regardless of the sequence, structure or chemistry of the nucleic acid.
  • the present invention relates to identifying polymers capable of blocking the pro-inflammatory effects of various extracellular nucleic acids, for example, nucleic acids released from dead and dying cells. More specifically, the invention relates to methods of neutralizing the effecis of such nucleic acids in a subject (e.g., a human) and to cationic polymers suitable for use in such methods. The invention further relates to methods of identifying antiinflammatory polymers, e.g., by screening combinatorial libraries of nucleic acid-binding polymers.
  • methods of inhibiting nucleic acid-induced activation of a PRR in particular an endosomai TLR
  • the methods may include contacting a cell with a cationic polymer, e.g. by adding the cationic polymer to the extracellular space or media of the ceils, in an amount effective to inhibit activation of a PRR (TLR activation) by the nucleic acid.
  • the methods also include administering a cationic polymer to a subject in an amount effective to inhibit TLR. activation by the nucleic acid.
  • the administration may include systemic or localized administration of the cationic polymer compositions.
  • the cationic poly mers suitable for use in the methods are disclosed herein but are al!
  • cationic polymers capable of binding to a nucleic acid and inhibiting the ability of the nucleic acid to activate a PRR, such as an endosomal TLR, are provided. Some of these cationic polymers are provided in Figure 10 wherein n is between 1 and 500, 5 to 250, 10-200, 20-150, 30-100 or any combination thereof.
  • a polymer library can be screened for anti -inflammatory polymers by selecting the cationic polymers that: I) are capable of inhibiting IL-6 production by cells in response to treatment with a nucleic acid stimulator of a TLR; 2) have minimal to no effect on the stimulation of the cell through TLR2, TLR4, or TL 5 in response to a stimulator of these T LRs; 3) have minimal cytotoxicity to the cells; 4) are capable of binding nucleic acids; and optionally 5) are not taken up by the cells, but remain extracellular.
  • Figure 1 is a set of synthetic schemes for generation of the combinatorial nucleic acid- binding cationic polymer library, Michael Addition of primary or secondary amines to acryiate/acryi amide or epoxide ring opening of giycidyl ethers by primary or secondary amines was used to generate the polymers in the library.
  • n is, for example, 1 to 500.
  • Figure 2 shows the monomer structures used to generate the nucleic acid-binding polymer combinatorial library.
  • the lettered structures (A-K and AA-CC) represent the backbone monomers.
  • the numbered structures (1-34) represent the R side chain amine linkers, linking the monomers to form the cationic polymers.
  • Combinatorial synthesis affords 196 polymers. The names of each of the monomers used is indicated under each structure.
  • Figure 3 is a graph and array depiction showing the results of the ethidium bromide displacement assay for some of the polymers demonstrating the ability of the cationic polymers to bind the CpG 1668 and displace the ethidiurn bromide.
  • Poiyethylenimine ( ⁇ ) was included as a positive control. Lighter shades indicate more displacement and a higher binding affinity.
  • Figure 4 is a set of arrays depicting the results of the ethidiurn bromide displacement assay and the (3 ⁇ 4o analysis (competitive exclusion 50) which is the amount of the polymer necessary to decrease the ethidiurn bromide fluorescence by 50%.
  • the dashed lines represent three sidechain linkers showing the best ability to displace the ethidiurn bromide (9, 10 ad 13). Lighter shades are indicative of higher affinity of the polymers.
  • Figure 5A-5D is a set of bar graphs showing the ability of selected nucleic acid-binding polymers to inhibit TLR-9 activation by pro-inflammatory DNA (CpG 1668) and the resulting reduction of IL-6 cytokine production at varying doses in primary dendritic cells.
  • the effect of the polymers on TLR4 activation in response to lipopofysacchari.de (LPS) was also assessed.
  • the polymer was administered for the first 10 minutes then the CpG or LPS were added for 18 hr incubation prior to measuring IL-6 production by the cells.
  • These polymers are selective for nucleic acids that do not inhibit synthetic, non-nucieic acid agonist LPS.
  • Figure 6A and 6C is a set of graphs showing the ability of selected nucleic acid-binding polymers to inhibit TLR9 activation by CpG1 68 and the resulting reduction of IL-6 production at lower doses of the indicated polymers in primary dendritic cells.
  • Figure 6B and 6D are graphs showing the effect of similar doses of the nucleic acid-binding polymers on TLR.4 activation by LPS in primary dendritic cells.
  • Figure 7A and 7B are graphs showing the combined results from the in vitro assays for the polymer backbones (A-K and AA--CC in Fig. 7 A) and for the monomer side chain linkers ( 1 - 14 in Fig. 7B).
  • Polymer backbones A, B, H and K performed better than the other backbones tested.
  • Polymer backbones C, Dminister E, F, and AA each produced at least one suitable polymer.
  • Monomer side chains 1, 6, 8, 9, 13 and 14 performed better than the other monomer side chains tested.
  • Monomer side chains 3 and 4 each produced at least one suitable polymer as well.
  • Figure 8 is a set of FACS analyses showing that the polymers are capable of reducing cellular uptake of a fluoreseently labeled nucleic acid by cells and that the cells were ceils of the macrophage/dendritic lineage (CD1 lb * and GDI le ⁇ ).
  • Several of the polymers were better than PAMAM at reducing cellular uptake of the labeled CpGl 668,
  • Figure 9 is a bar graph showing the percent viability of cells by an MT ' T assay after incubation with each of the indicated polymers.
  • the backbone monomer of the polymer is indicated above the bar graph and the monomer side chain is indicated along the bottom of the graph.
  • a star (*) indicates that 100% of the cells were non-viable (100% cell death).
  • Figure 10 shows the structures of the lead candidate polymers (n is 1 to 500).
  • nucleic acid agonists include any nucleic .acid capable of activating a PRR and inducing a cell to produce cytokines such as 1L-6.
  • Nucleic acid agonists include dsRNA, ssRNA, un- or hypo-methylated DNA or ssDNA and the agonists may be completed with proteins.
  • the present invention relates, at least in part, to a combinatorial library of nucleic acid-binding cationic polymers and to methods of identifying catio ic polymers suitable for use as anti -inflammatory therapeutics in a subject (e.g., a mammal, preferably, a human) comprising screening the library for polymers that neu tralize or Inhibit the effects of pro-inflammatory nucleic-acids.
  • a subject e.g., a mammal, preferably, a human
  • the invention further relates to anti-inflammatory polymers so identified and to methods of using same.
  • a combinatorial librar of 1.96 polymers (140 poly(P-amux) esierjs, 1 disulfide containing poly(p-amido amine)s and 42 poiy(p-hydroxyi amine)s) were synthesized from the reactions of primary or secondary amines with either bisacrylates, bisacrylamides or diglyeidylethers (Fig. 1) (poly($-amino ester )s: Akinc et al, BiocoTijugate Chemistry 14(b): 979- 988 (2003); poly(p -hydroxy!
  • amines Barua et al, Molecular Pharmaceutics 6(1): 86-97 (2009); and ⁇ ! ⁇ ( ⁇ -amido amine)s: Lin et al, Bioconjugate Chemistry 18(1): 138-145 (2007)).
  • These cationic polymer classes have been studied for the purpose of non-viral gene delivery (e.g., siRNA) (Pack et at, Nat. Rev. Drug Discov. 4:581 -593 (2005)), however, they have not been studied in other therapeutic areas, such as inflammation and thrombosis. While these polymers were developed to bind to and deliver nucleic acids into the nuclei of cells, they can be chemically tuned (e.g., by the selection of backbone monomer functionalities) to prevent cellular uptake and improve anti -inflammatory function.
  • these nucleic acid-binding polymers were tested for their ability to inhibit TLR-9 activation in the presence of pro-inflammatory ssD A.
  • Primary bone marrow-derived plasmacytoid dendritic cells were incubated with varying doses of nucleic acid-binding polymers and a subsequent dose of a known TLR-9 agonist.
  • Functional polymers were selected based on their ability to decrease l.L-6 production after an overnight incubation without suppressing cytokine production in the presence of a non-nucleic acid T ' LR agonist, LPS (Fig, 5-6). This demonstrated the selectivity of these cationic polymers for nucleic acids.
  • the polymers suitabl have low or no cytotoxicity.
  • the cationic polymers provided herein are not cytotoxic or have low cytotoxicity as compared to untreated cel ls. Low
  • the cationic polymers provided herein do not allow or inhibit cellular uptake of the nucleic acid agonists.
  • the cationic polymers provided herein work at least partially by inhibiting cellular uptake of the nucleic acid agonists of the PRRs and thus inhibit interaction o f the nucleic acid agonists with the receptors.
  • the cationic polymers may inhibit uptake of the nucleic acid by 1.0%, 20%, 30%, 40%, 50% or even 60% or more as compared to control cells provided a distinct polymer or no polymer at all
  • the present invention relates, in one embodiment, to methods of inhibiting nucleic acid- induced activation of PRRs, such as endoso.mal TLRs (e.g., TLR 9).
  • the methods include contacting a cell with a cationic polymer (e.g., by adding the polymer to the extracellular space or media) or administering to a subject (e.g., a human) in need thereof the cationic polymers described herein.
  • the cationic polymer is capable of binding nucleic acids responsible for induction of PRR (TLR) activation in an amount and under conditions such tha inhibition of that activation is effected.
  • the agent binds the nucleic acids in a manner that is independent of the nucleotide sequence, the chemistry (e.g., DNA or RNA, with or without base or sugar modifications) and/or the structure (e.g., double-stranded or single-stranded, comp!exed or uncomplexed with, for example, a protein) of the nucleic acids responsible for inducing nucleic acid receptor (TLR) activation.
  • TLR nucleic acid receptor
  • the present method can he used to treat inflammatory and/or autoimmune responses resulting from inappropriate activation of nucleic acid receptors on or in cells.
  • Administration or addition of the cationic polymers inhibits activation of the nucleic- acid receptor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more in a dose-dependent manor such that addition of small amounts of the cationic polymer axe not or only slightly capable of snliibiting receptor activation and addition of higher amounts of the cationic polymers results in additional inhibition up to full inhibition of activation of the receptor by the nucleic acid.
  • the percentage inhibition of the receptor may refer to the percentage inhibition or reduction in cytokine production (e.g. l ' L-6) in response to the nucleic acid agonist in
  • Nucleic acid-binding polymers of the invention include pharmaceutically acceptable cationic polymers that can bind pro-inflammatory nucleic- acids in, for example, biologic fluids, prevent cellular uptake and thereby inhibit TLR. activation.
  • Such cationic polymers include poly(P-aramo ester)s, disulfide containing poly$-arnido amine)s and poly(
  • Preferred polymers include those in Tig. 10, particularly preferred are AA9, H3, H4, H8, HI 3 and 1114 where "n" is, for example, I to 500, preferably, 5 to 250, more preferably, 10-200, 20- 150 or 30- 100.
  • polystyrene resin examples include A 1 , A2, A6, A9, ALT A 1.4, B5, B6, 88, B9, B13, E13, F6, F8, F9, H2, H3, H4, H6, H7, H8, B9, H13, HI 4, II , ⁇ 3.. ⁇ 4, K6, K.9, K14, AA1 , AA9, and BBl.
  • the backbone is the structure listed as A- or AA-CC as shown in Figure 2 and the monomer side chain has the structure indicated as 1-14 in Figure 2.
  • the polymers are made from the monomers shown in Figure 2 using the reactions shown in Figure 1 to generate the polymers listed.
  • Cationic polymers of the invention include biodegradable and non-biodegradable polymers and blends or copolymers thereof.
  • the binding affinity of a nucleic acid-binding cationic polymer of the invention for a nucleic acid is in the pM to mM range, preferably, less than or equal to 50 nM; expressed in terms of binding constant ( ), the binding affinity is advantageously equal to or greater than 10 S M " ', preferably, I0 5 M " ⁇ to 1G S M ' ⁇ more preferably, equal to or greater than 10 6 M "! .
  • the binding affinity of the sequence-independent nucleic acid-binding cationic polymers can be, for example, about 1 x 10' M "1 , 5 x 10 s M *1 , 1 x 10 6 M ' !
  • ITC Isothermal Calorimetry
  • FRET Forster Resonance Energy Transfer
  • surface piasmon resonance a real time binding assay such as Biacore.
  • nucleic acid-binding cationic polymers of the invention simultaneously limit the activation of multiple nucleic acid binding PRRs (endosoraa! TLRs, e.g., TLR3, TLR7, TLRS and TLR9 and possibly cytosolic nucleic acid sensors such as RIO-I) by binding to a wide array of different nucleic acids including ssRNA, ssDNA, dsRNA and dsDNA and of which may be presented in a complex with, protein such as viral proteins, histones, HMGB3 or R1G-L
  • the nucleic acid-binding polymers do not inhibit activation of non-nucleic acid binding TLRs such as TLR 2, TLR.4, TLR5, or TLR6.
  • the cationic polymers do not inhibit activation by LPS, lipoproteins, or flagellin.
  • the cationic polymers are not taken up by the cells and are not cytotoxic.
  • the present invention provides a method of controlling (inhibiting or preventing) autoimmune and/or inflammatory responses associated with activation of PRRs by nucleic acids (e.g., endosoraa! TLRs, such as TLR9).
  • nucleic acids e.g., endosoraa! TLRs, such as TLR9.
  • Such responses play a role in the pathogenesis of diseases/disorders that are associated with presence in the circulation of the subject of free nucleic acids, either pathogen-derived (e.g., viral- or bacterial-derived) nucleic acids or nucleic acids released from dead or damaged host cells.
  • nucleic acid-binding polymers of the invention include infectious diseases, cardiovascul r disease, cancer, bacterial sepsis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, COPD, obesity, psoriasis, atherosclerosis, diabetes, wound healing, burns, infectious diseases, reperfussion injury, renal failure/dialysis, organ transplantation, neurodegenerative disease and traumatic brain inj ury. (See also, infectious diseases, cardiovascul r disease, cancer, bacterial sepsis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, COPD, obesity, psoriasis, atherosclerosis, diabetes, wound healing, burns, infectious diseases, reperfussion injury, renal failure/dialysis, organ transplantation, neurodegenerative disease and traumatic brain inj ury. (See also
  • the cationic polymers can also be used in combination with other treatments.
  • the cationic polymers may be used in conjunction with another therapeutic, such as a cancer therapeutic, known to result in a robust inflammatory response by releasing nucleic acids.
  • Such treatments may be treatments known to induce cell death or nucleic acid based inflammation.
  • the cationic polymers are administered to ceils or a subject which previously received or were exposed to a nucleic acid-based pharmaceutical composition, such as an siRNA, DNA vaccine or aptamer based therapy.
  • a nucleic acid-based pharmaceutical composition such as an siRNA, DNA vaccine or aptamer based therapy.
  • the polymers described herein may be useful to limit inflammatory side effects associated with administration of such therapeutics.
  • nucleic acid-binding polymers described herein Another application of nucleic acid-binding polymers described herein is to counteract the effects of D A, RNA or polyphosphate molecules thai are released from cells and subsequently induce thrombosis (Kannemeier et al, Proc. Natl. Acad. Sci.. 104:6388-6393 (2007); Fuchs et al. Proc. Natl. Acad. Sci. Published Online before Print August 23, 2010). It has been observed that RNA and DNA molecules can activate the coagulation pathway as well as platelets and thereby engender blood clotting (Kannemeier et al, Proc. Natl. Acad. Sci. 104:6388-6393 (2007); Fuchs et al, Proc. Natl. Acad. Sci.. Published Online before Print August 23, 2010).
  • nucleic acid-binding cationic polymers described herein can bind RNA and DNA molecules and shield them from other potential binding partners, such agents can be employed to inhibit the ability of DNA and RNA molecules to bind and acti vate coagulation factors and platelets. In so doing, these RNA/DNA -binding polymers can be utilized to limit nucleic acid- induced pathological blood coagulation.
  • nucleic acid-binding cationic polymers described herein represent novel entities for preventing the induction and progression of a variety of thrombotic disorders, including myocardial infarction, stroke and deep vein thrombosis.
  • cells may be contacted with the polymers directly or indirectly in vivo, in vitro, or ex vivo.
  • Contacting encompasses administration to a cell, tissue, mammal, subject, patient, or human.
  • contacting a ceil includes adding the polymers to a ceil culture, such as by including or adding the polymer to the media in which the cell is incubating or providing the polymer to the extracellular space.
  • Other suitable methods may include introducing or administering the polymers descri bed herein to a cell, tissue, mammal, or patient using appropriate procedures and routes of administration as defined below.
  • nucleic acid-binding polymers of the invention can be administered to the subject via any route such that effective levels are achieved in, for example, the bloodstream.
  • the compounds described herein may be administered by any means including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, transdermal,
  • the compounds may be formulated as an intestable, injectable, topical or suppository formulation.
  • the compounds may also be delivered with in a liposomal or time-release vehicle.
  • the nucleic acid-binding polymer can also be administered, for example, directly to a target site, for example, directly lo a joint when arthritis is the disease to be treated.
  • the nucleic acid-binding polymer is administered as soon as clinical symptoms appear and administration is repeated as needed.
  • Administration of the compounds to a subject in accordance with the invention appears to exhibit beneficial effects in a dose-dependent manner.
  • the optimum, dosing regimen will depend, for example, on the nucleic acid-binding polymer, the subject, the condition being treated and the effect sought.
  • administration of larger quantities of the compounds is expected, to achieve increased beneficial biological effects than administration of a smaller amount.
  • efficacy is also contemplated at dosages below the level at which toxicity is seen.
  • the specific dosage administered in any given case will be adjusted in accordance with the compound or compounds being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activit of the compound or the response of the subject, as is well known by those skilled in the art.
  • the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied.
  • Dosages for a given patient can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the compound of the invention and of a known agent, such as by means of an appropriate conventional, pharmacological or prophylactic protocol.
  • the maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects.
  • the number of variables in regard to an individual prophylactic or treatment regimen is large, and a considerable range of doses is expected.
  • the route of administration will also impact the dosage requirements, it is anticipated that dosages of the compound will reduce symptoms of the condition at least 10%, 20%, 30%, 40%, 50%, 60%, 70%. 80%, 90% or 100% compared to pre-treatment symptoms or symptoms is left untreated. It is specifically contemplated thai pharmaceutical preparations and compositions may palliate or alleviate symptoms of the disease without providing a cure, or, in some embodiments, may be used to reverse the disease or disorder, such as an autoimmune or inflammatory disease.
  • Suitable effective dosage amounts for administering the compounds may be determined by those of skill in the art, but typically range from about 1 microgram to about 500,000 micrograms per kilogram of body weight weekly, although they are typically about 100 milligrams or less per kilogram of body weight weekly.
  • the effective dosage amounts described herein refer to total amounts administered, that is, if more than one compound is administered, the effective dosage amounts correspond to the total amount administered.
  • the compound can be administered as a single dose or as divided doses.
  • the composition may be administered two or more times separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks, if added to cellular media, the cationic polymers may be added such that the concentration of the polymer is between ⁇ and lOmM, 50nM and 5mM, ⁇ and 3mM, or 500nM and 2mM.
  • the nucleic acid-binding cationic polymers, or pharmaceutically acceptable salts thereof, can be formulated with a carrier, diluent or excipient to yield a pharmaceutical composition.
  • the compounds may be used to make pharmaceutical compositions.
  • Pharmaceutical compositions comprising the compound of formula ( ⁇ ) or any of the compounds described above and a pharmaceutically acceptable carrier are provided.
  • a pharmaceutically acceptable carrier is any carrier suitable for in vivo administration. Examples of pharmaceutically acceptable carriers suitable for use in the composition include, but are not limited to, water, buffered solutions, glucose solutions, oil-based or bacterial culture fluids. Additional components of the
  • compositions may suitably include, for example, exclpients such as stabilizers, preservatives, diluents, emulsifiers and lubricants.
  • pharmaceutically acceptable carriers or diluents include stabilizers such as carbohydrates (e.g., sorbitol, raannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing agents such as bovine serum or skimmed milk and buffers (e.g., phosphate buffer).
  • the composiiion is suitable for freeze-drying or spray-drying.
  • the composition may also be emulsified.
  • the patient (subject) to be treated can be a mammal, preferably a human.
  • the subject can be, for example, a farm animal such as a cow, pig, horse, goat or sheep, or a companion animal such as a dog or a cat.
  • the cationic polymers described herein may be administered in combination with each other or in combination with other therapeutics such as an antimicrobial, cancer therapeutic, nucleic acid based therapeutic or inflammatory mediator.
  • the compositions may be administered in combination with each other or in combination with other therapeutics such as an antimicrobial, cancer therapeutic, nucleic acid based therapeutic or inflammatory mediator.
  • the compositions may be administered in combination with each other or in combination with other therapeutics such as an antimicrobial, cancer therapeutic, nucleic acid based therapeutic or inflammatory mediator.
  • the compositions may be any suitable therapeutics such as an antimicrobial, cancer therapeutic, nucleic acid based therapeutic or inflammatory mediator.
  • the two therapeutics may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 1.2 hours, 16 hours, 20 hours, .1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more.
  • an effective amount or a therapeutically effective amount as used herein means the amount of a composition that, when administered to a subject for treating a state, disorder or condition is sufficient to effect a treatment.
  • the therapeutically effective amount will vary depending on the compound, formulation or composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated as described for dosing above,
  • endosomal TLR- containing ceils preferably, mammalian cells (e.g.. mammalian plasmacytoid dendritic cells)
  • a first PRR agonist such as an endosoraal TLR agonist (e.g., CpG DNA or single or double stranded RNA or nucleic acid-containing particles)
  • a test agent e.g., a cationic polymer selected from the combinatorial library described above and in the Example that follows.
  • a culture supernatant sample can be taken and analyzed for the presence of a product of an intracellular signaling event initiated by activation of the nucleic acid responsive receptor (TLR9), for example, one or more cytokines (e.g., it-6).
  • TLR9 nucleic acid responsive receptor 9
  • cytokines e.g., it-6
  • PRR agonist or an endosoma! TLR agonist having a sequence, chemistry and/or structure different from that of the first agonist, e.g.
  • a test agent that inhibits activation of the nucleic acid responsive receptor preferably, in a manner independent of the sequence, chemistry and/or structure of the nucleic acid agonist used (that inhibition of activation being evidenced by inhibition of production of a product of an intracellular signaling event initiated by PRR activation (e.g., cytokine production) (e.g., in a dose dependent manner)) can then be tested in vivo, for example, in mice, to further assess its suitability for use in the methods described herein.
  • PRR activation e.g., cytokine production
  • the polymers are also tested for the inability to block activation and cytokine production by cells in response to non-nucleic acid binding PRRs (TLRs) such as LPS activation of TLR4; Pam3CS 4 activation of TLR2; endogenous DAMP or heparan sulfate activation of TLR4.
  • TLRs non-nucleic acid binding PRRs
  • the polymers should also be tested for cytotoxicity to cells after incubation and for lack of toxicity when administered to subjects such as mice. Cytotoxic or toxic polymers should not be selected whereas those having no or little toxicity may be selected for further evaluation.
  • the polymers are also selected for the ability to bind to the nucleic acids with high affinity and to not be taken up by cells into an intracellular space.
  • Reactions employed to generate a combinatorial library 1 for functional screening is set forth in Fig. ⁇ and the monomer building blocks used to synthesize the polymer library are set forth in Fig. 2.
  • Amine monomers 1 -14 were reacted with backbone monomers (A-K, AA-CC) in. 1 :1 stoichiometry at 1.6 M in DMSO for 5 days at 56°C in multi-well polypropylene plates.
  • the library consists of 3 main polymer classes, namely, poly(j5-amino ester)s, disulfide containing poly( -amido amine)s and poly(p-bydroxyi arnine)s>
  • the monomer building blocks were selected to include multiple functionalities in both the backbone and side-chains of the polymer to enhance biodegradability and bioreducibiiity and to lower toxicity and reduce cellular uptake (Fig. 2).
  • poly( -ammo ester )s, and poly( -amido amine)s were synthesized via Michael- type additions of amines to bisacrylates or bisacrylamides to generate (pofy(p-amino ester )s (see Akinc et ai, Bioconjugate Chemistry 14(5): 979-988 (2003)); and ⁇ ! ⁇ ( ⁇ -amido amine)s (see Lin et ah Bioconjugate Chemistry 18(1): 138-145 (2007)).
  • the po.!y(f3 ⁇ hydrox i amine)s were synthesized via epoxide ring opening of digiycidyl ethers by either primary or secondary amines (see Barua et al, Molecular Pharmaceutics 6(1): 86-97 (2009)). These reactions are robust, efficient and offer a modular strategy by which a large number of chemical functionalities can be introduced onto a polymeric backbone in a combinatorial fashion.
  • CpG1 68 oligodeoxynucleotides ODN
  • ethidium bromide displacement assay as described by (Tse and Soger (2005) Current Protocols in Nucleic Acid Chemistry. 20:8.5, 1 8,5.1 1.), Briefly, CpG oligodeoxynucleotides (CpGl 668 ODN) were incubated with ethidium bromide to allow intercalation ([Pj j EtBr] ⁇ : 4/1 ) in a 96 well plate.
  • the 196 polymers of the Library were added to the CpG:EtBr complexes.
  • the graph shows a representative set of polymers. Polymer A4 (diamonds) was not able to bind the CpG ODN and did not result in decreased fluorescence of the CpG:EtBr complex, in contrast, polymers B9 (upright triangles), A13 (downward triangles), 813 (squares) and positive control PEf
  • the 196 nucleic acid-binding polymers were also tested for their ability to inhibit TLR.-9 activation in the presence of a pro-inflammatory ssDNA (CpG).
  • CpG pro-inflammatory ssDNA
  • Primary bone marrow-derived plasmacytoid dendritic cells were incubated with varying doses of the nucleic acid-binding polymers for 10 minutes and a subsequent dose of a known TLR-9 agonist, CpG 3668 was added for 18 hours.
  • Functional polymers were selected based on their ability to decrease IL-6 production in response to the CpG ODN after a period of 18 hours without suppressing IL-6 production in the presence of LPS - a synthetic, non-nucleic acid TLR.4 agonist (Fig. 5).
  • Fig. 6A-D Further dose curves for the ability of the polymers to inhibit IL-6 production by the cells in response to CpG, but not inhibit the response to LPS are shown in Fig. 6A-D.
  • Fig. 7A and Fig. 7B compile the data from these experiments into a structural analysis for the backbone and side chain monomers, respectively,
  • the polymer candidates were also tested for cellular uptake using an Alexa488-CpG uptake FACS assay (Lee et al, Proc. Natl. Acad. Sci. USA 108(34): 14055- 4060 (2011)).
  • Potential candidates were then subjected to further screening for cytotoxicity in a murine macrophage cell line, RAW264.7.
  • the polymers were added to the cells at a concentration of 0.5 mg/fnL for 24 hours prior to being washed with PBS and the MT ' F assa reagent added to the cell for 2 hours.
  • An MTT assay was performed to assess the effect of the polymers on cell viability. The results are shown in Figure 9. The percentage of viable ceils is by comparison of the cells incubated with the polymer as compared to similarly treated ceils incubated in the absence of polymer. Many of the polymers tested had little or no effect on cellular viability.
  • the refined candidates consist of those displaying a high inhibition of TLR-9 activation in primary cells, low cytotoxicity as determined by MTT assays, low inhibition of LPS mediated activation and low cellular uptake of Alexa488-CpG.
  • the lead candidate polymers are shown in Figure 10.

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Abstract

La présente invention concerne, d'une manière générale, les effets pro-inflammatoires de divers acides nucléiques extracellulaires et, en particulier, des procédés de neutralisation des effets de tels acides nucléiques chez un sujet (par exemple, un être humain) ou dans une culture cellulaire et des polymères cationiques appropriés pour être utilisés dans de tels procédés. L'invention concerne en outre des procédés d'identification de polymères cationiques anti-inflammatoires, par exemple, par criblage de bibliothèques combinatoires de polymères de liaison d'acide nucléique.
PCT/US2014/033509 2013-04-09 2014-04-09 Agents anti-inflammatoires et leurs procédés d'utilisation WO2014169043A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20090298710A1 (en) * 2005-12-15 2009-12-03 Farokhzad Omid C System for Screening Particles
US20120128782A1 (en) * 2009-05-15 2012-05-24 The Johns Hopkins University Multicomponent Degradable Cationic Polymers
US20120183564A1 (en) * 2009-09-16 2012-07-19 Sullenger Bruce A Inhibition of endosomal toll-like receptor activation
USRE43612E1 (en) * 2000-10-10 2012-08-28 Massachusetts Institute Of Technology Biodegradable poly(β-amino esters) and uses thereof
WO2013040552A2 (fr) * 2011-09-16 2013-03-21 Georgia Health Sciences University Procédés permettant de favoriser la tolérance immunitaire

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
USRE43612E1 (en) * 2000-10-10 2012-08-28 Massachusetts Institute Of Technology Biodegradable poly(β-amino esters) and uses thereof
US20090298710A1 (en) * 2005-12-15 2009-12-03 Farokhzad Omid C System for Screening Particles
US20120128782A1 (en) * 2009-05-15 2012-05-24 The Johns Hopkins University Multicomponent Degradable Cationic Polymers
US20120183564A1 (en) * 2009-09-16 2012-07-19 Sullenger Bruce A Inhibition of endosomal toll-like receptor activation
WO2013040552A2 (fr) * 2011-09-16 2013-03-21 Georgia Health Sciences University Procédés permettant de favoriser la tolérance immunitaire

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