WO2008047067A1 - Traitement d'une maladie inflammatoire - Google Patents

Traitement d'une maladie inflammatoire Download PDF

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WO2008047067A1
WO2008047067A1 PCT/GB2006/050345 GB2006050345W WO2008047067A1 WO 2008047067 A1 WO2008047067 A1 WO 2008047067A1 GB 2006050345 W GB2006050345 W GB 2006050345W WO 2008047067 A1 WO2008047067 A1 WO 2008047067A1
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erg
modulator
cells
inflammatory
cell
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PCT/GB2006/050345
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Simon Brown
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University Court Of The University Of Edinburgh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/314—Phosphoramidates
    • C12N2310/3145—Phosphoramidates with the nitrogen in 3' or 5'-position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring

Definitions

  • the present invention relates to methods of modulating inflammatory responses.
  • it relates to methods of modulating recruitment of leukocytes and phagocytosis of apoptotic cells, and the use of such methods in the treatment of inflammatory diseases.
  • CD31 also known as the cell adhesion molecule PECAM-1 , is an important regulator of leukocyte transmigration across both the endothelium (Muller et al., 1993; Muller, 2003; Nourshargh & Marelli-Berg, 2005) and perivascular basement membrane (Liao et al., 1995;
  • Antibody ligation of leukocyte CD31 in vitro is known to activate ⁇ 1 , ⁇ 2, and ⁇ 3 integrins to promote adhesion (Newman and Newman, 2003) in which the small GTPase Rap1 has been implicated (Reedquist et al., 2000). More recently, induced oligomerization of CD31 independent of homophilic or antibody binding was found to promote ⁇ 5 ⁇ 1 -dependent adhesion of transfected epithelial cells to immobilized fibronectin (Fn) whereas dimerization led to homophilic cell-cell interactions (Zhao and Newman, 2001 ).
  • CD31 is also known to interact with ⁇ v ⁇ 3 and although this was originally reported to occur in trans (Buckley et al., 1996) it now appears that it is a cis interaction (Wong et al., 2000). Thus CD31 can associate with integrins and regulate integrin activation.
  • CD31 interactions have analogous consequences in macrophage discrimination of living from dying cells (Brown et al., 2002; Dogusan et al., 2004). Healthy CD31 -positive leukocytes receive a propulsive "move away" signal when they encounter macrophage CD31 , whereas apoptotic cells bearing CD31 are tightly bound and phagocytosis is then promoted. The firm binding and engulfment of apoptotic cells by macrophages appears to be mediated by CD31 -directed integrin adhesion (Vernon-Wilson et al., 2006) in keeping with leukocyte transmigration. Nevertheless, it remains a paradox that CD31 can function on one hand in both transmigration and engulfment to promote integrin-dependent adhesion (attachment) while on the other motility and migration (detachment).
  • Inflammation is a defence mechanism used by the body to deal with noxious endogenous or exogenous stimuli. It is associated with tissue injury, characterized by capillary dilation and leukocyte infiltration. Efferocytosis is the phagocytic recognition and clearance of apoptotic cells (deCathelineau & Henson, 2003). In normal inflammatory responses, delayed apoptosis of inflammatory cells contributes to their accumulation in order to enhance the inflammatory response to a pathogen. However inappropriate inflammatory responses contribute and cause many acute and chronic diseases. In such diseases, excessive leukocyte recruitment to a site of infection or inflammation may exacerbate the inflammation by increased release of pro-inflammatory cytokines, oxidative agents etc. Further, inflammatory responses may result in disregulation of apoptosis of cells at the site of inflammation. The disregulation of the apoptosis of inflammatory cells may result in exacerbation of the inflammatory response.
  • Kav100 cells bind and ingest fibronectin- coated LatexTM beads (Fn-beads) in a ⁇ 1 integrin-dependent manner (Blystone et al., 1994).
  • Kav100 are K562 cells stably transfected with the integrin ⁇ v ⁇ 3 in which ⁇ v ⁇ 1 is the only other integrin expressed.
  • K562 cells are devoid of many receptors implicated in the phagocytic clearance of apoptotic cells, including CD31 , and therefore represent a reductionist, but genetically tractable system, to model phagocytosis.
  • the inventors expressed CD31 ectopically in Kav100 cells.
  • ERG ether-a go-go related gene potassium channel
  • ERG ether-a go-go related gene potassium channel
  • ERG ether-a go-go related gene potassium channel
  • ERG current was shown to be essential for CD31 -dependent regulation of apoptotic thymocyte uptake.
  • a method of modulating phagocytosis of apoptotic cells by phagocytic cells comprising administering to said cells a modulator of ERG current.
  • CD31 is known to play a role in leukocyte recruitment.
  • ERG ERG-dependent uptake of apoptotic thymocytes
  • the inventors further examined the effect of ERG inhibition on leukocyte migration in a zebrafish wound model of inflammation. The inventors demonstrated that inhibitors of ERG significantly reduce leukocyte recruitment to the site of a wound.
  • a method of modulating leukocyte recruitment comprising administering to said leukocytes a modulator of ERG.
  • ERG inhibitors not only reduces the number of inflammatory cells in peritoneal lavages but also reduces the levels of certain pro-inflammatory cytokines. It is believed that the reduced concentration of pro-inflammatory cytokines is a result of inhibition of cytokine release from macrophages.
  • a method of modulating the release of pro-inflammatory cytokines from macrophages in a cell population comprising administering to said cell population a modulator of ERG.
  • the release of proinflammatory cytokines from macrophages in a cell population is inhibited, with said modulator being an inhibitor of ERG.
  • the release of pro-inflammatory cytokines from macrophages in a cell population is increased, with said modulator being an activator or agonist of ERG.
  • the present invention provides a method of treatment of an inflammatory disease in an individual, wherein said method comprises administration to said individual of an effective dose of a modulator of ERG.
  • an ERG modulator in the preparation of a medicament for the treatment of an inflammatory disease.
  • the invention may be used in the treatment of any inflammatory disease or condition.
  • the inflammatory disease is a chronic inflammatory disease.
  • the disease is peritonitis, arthritis, pleurisy, COPD or asthma.
  • inflammatory diseases should be understood to also encompass a disease or condition associated with excessive or inappropriate inflammation.
  • excessive or inappropriate inflammation means an inflammatory response which contributes to or prolongs a pathological state.
  • the inflammatory disease is a chronic wound condition.
  • Chronic wound conditions include, but are not limited to, venous ulcers, diabetic ulcers and pressure ulcers.
  • ERG inhibitors may be of particular use in the treatment of such conditions.
  • the present invention may also be used to enhance leukocyte recruitment to a site of injury.
  • This may be of particular use in the to promote wound repair and or healing, in particular acute wounds, in particular with respect to patients manifesting a defect in leukocyte responses, for example as a result of a neutropenia or Leukocyte Adhesion Deficiency type I or II.
  • the administration of agents which enhance ERG current should contribute to the healing of wounds.
  • the ERG modulator is an ERG agonist.
  • an ERG modulator in the preparation of a medicament for the treatment of wound condition.
  • ERG modulator in the preparation of a medicament for the promotion of wound healing.
  • compositions for the treatment of a wound comprising an ERG modulator.
  • a wound dressing comprising an ERG modulator.
  • ERG modulators for use in promoting wound healing may be provided alone or in combination with other wound healing agents.
  • a pharmaceutical product comprising an ERG modulator and a further wound healing agent for the simultaneous, separate or sequential use to promote wound healing.
  • the further wound healing agent may be any other agent known to promote wound healing. Such agents are well known to the skilled person and may include .
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF fibroblast growth factor
  • IGF- 1 insulin-like growth factor
  • any suitable modulator of ERG may be used in the present invention.
  • the modulator is a selective modulator of ERG.
  • the modulator is an inhibitor of ERG.
  • the modulator is a Class III antiarrhythmic compound
  • ERG inhibitor Any suitable ERG inhibitor may be used in the methods of the invention.
  • selective inhibitors of ERG which may be used in the present invention are methanesulfonanilides.
  • Suitable methanesulfonanilides which may be used include, but are not limited to E-4031 , WA Y- 123,398, dofetilide, d-sotalol, MK-499, ibutilide, and sematilide.
  • modulators of ERG which may be used in the present invention include agents which promote the association of CD31 with ERG proteins. Such agents may be small molecules, antibodies or antibody fragments Further agents which may be used to modulate ERG channel current include antisense molecules, for example, morpholino antisense molecules, RNAi, viral delivery of shRNA directed against ERG.
  • the ERG modulators may be ERG agonists.
  • ERG agonists include but are not limited to the ERG channel activating urea derivatives described in e. g. WO 94/22807, WO 96/25157, WO97/45400, WO97/451 1 1 , WO 98/47879, WO 2000/24707 and WO 2004/022529.
  • Other agonists include mallotoxin, and NS1643.
  • the present invention is based on the surprising demonstration that ERG inhibition promotes phagocytosis of apoptotic cells and inhibits recruitment of leukocytes to sites of inflammation.
  • the two effects of ERG modulation may be exploited in the treatment of inflammatory diseases.
  • treatment includes any regime that can benefit a human or non-human animal.
  • the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment).
  • Treatment may include curative, alleviation and/or prophylactic effects.
  • the present invention may be used to treat any disease in which modulation of leukocyte recruitment and/or the phagocytosis of apoptotic cells is therapeutically useful.
  • any disease in which modulation of leukocyte recruitment and/or the phagocytosis of apoptotic cells is therapeutically useful.
  • it will generally be desirable to accelerate phagocytosis and inhibit leukocyte recruitment.
  • it will generally be preferred to employ an agent which specifically inhibits ERG.
  • Inflammatory conditions for which the present invention may find use include, but are not limited to, inflammatory conditions such as peritonitis, arthritis, pleurisy, lung fibrosis, systemic sclerosis and chronic obstructive pulmonary disease (COPD).
  • inflammatory conditions such as peritonitis, arthritis, pleurisy, lung fibrosis, systemic sclerosis and chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • inflammatory lung disease atopic dermatitis
  • NERDS nodules eosinophilia, rheumatism, dermatitis and swelling
  • pulmonary fibrosis pulmonary fibrosis
  • IBD inflammatory bowel disease
  • vasculitic granulomatous diseases including polyarteritis and Wegeners granulomatosis
  • sarcoiditis idiopathic pulmonary fibrosis
  • auto-immune diseases eosinophilic pneumonia, reperfusion damage caused by myocardial infarction, atheroma
  • glomerular nephritis rheumatoid arthritis
  • ERG modulators may be used to aid wound healing.
  • ERG modulators may be used to enhance leukocyte infiltration to a site, for example, to aid wound healing .
  • ERG modulators which inhibit leukocyte infiltration to a site may be used.
  • any suitable ERG modulator may be used.
  • the ERG modulator is an inhibitor of ERG.
  • Such inhibitors include methanesulfonanilides such as E-4031 , WA Y- 123,398 , d-sotalol, MK-499, ibutilide, and sematilide.
  • the ERG modulator is an interference RNA that downregulates the expression of the ERG channel, for example a morpholino siRNA molecule.
  • Other modulators which may be used are those which act to inhibit the association of CD31 with ERG.
  • the ERG modulators may act to increase current through the ERG channel.
  • ERG channel openers which may be used in this aspect of the invention include the ERG channel activating urea derivatives such as those described in e. g. WO 94/22807, WO 96/25157, WO97/45400, WO97/451 1 1 , WO 98/47879, WO 2000/24707 and WO 2004/022529, mallotoxin, and NS 1643.
  • treatment with combinations of the ERG modulators described herein with other agents useful for treating particular disorders is provided.
  • the choice of other agent will depend on the particular condition being treated and will be at the discretion of the physician.
  • ERG inhibitors in the invention include but are not limited to NSAIDs, cyclooxygenase-2 (COX-2) inhibitors, glucocorticosteroids, disease- modifying antirheumatic drugs (DMARDs- e.g, intramuscular gold, hydroxychloroquine, sulphasalazine and methotrexate- for arthritis) and anti-TNF therapy with biologies.
  • NSAIDs cyclooxygenase-2
  • COX-2 cyclooxygenase-2
  • DMARDs- e.g, intramuscular gold, hydroxychloroquine, sulphasalazine and methotrexate- for arthritis e.g, intramuscular gold, hydroxychloroquine, sulphasalazine and methotrex
  • ERG inhibitors also include molecules which inhibit or reduce the level or activity of an expression product of a gene encoding an ERG protein.
  • Such molecules may include antisense molecules, ribozymes and RNAi molecules.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC.
  • Antisense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • Antisense oligonucleotides can be derived from any nucleic acid molecule that encodes an ERG receptor. Generally, antisense molecules comprise from about 8 to about 30 bases and typically comprise from about 12 to about 25 bases.
  • Antisense molecules which may be used include phosphorothioates, siRNA, PNAs, LNAs and morpholinos.
  • PNA(peptide nucleic acid) differ from DNA and RNA in the composition of its "backbone.” which is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • Locked nucleic acids also known as inaccessible RNA, is an RNA nucleotide, in which the ribose moiety of LNA nucleotide is modified with an extra bridge connecting 2' and 4' carbons. The bridge "locks" the ribose in 3'-endo structural conformation.
  • Morpholinos are synthetic molecules typically 25 bases in length, and bind to complementary sequences of RNA by standard nucleic acid base- pairing. Morpholinos differ from RNA in that in Morpholinos, the nucleic acid bases are bound to morpholine rings instead of deoxyribose rings and are linked through phosphorodiamidate groups instead of phosphates. They do not degrade their target RNA molecules, unlike many antisense structural types (e.g. phosphorothioates, siRNA). Instead, Morpholinos act by "steric blocking", binding to a target sequence within an RNA and simply getting in the way of molecules which might otherwise interact with the RNA.
  • Morpholino antisense RNA is a chemically stable antisense RNA strategy for the knock-down of mRNA for genes of interest based on a complementary strand binding to mRNA and blocking the activity of RNA Pol to inhibit protein translation.
  • Antisense may be delivered by any cell transfection method, for example by micro-injection.
  • RNAi makes use of short-interfering (19-25bp) RNA (siRNA), designed to hybridise with the mRNA of the target gene and prevent its expression.
  • siRNA short-interfering (19-25bp) RNA
  • RISC enzyme complex
  • shRNA is short hairpin RNA that is encoded within the genetic material of a modified viral vector which when expressed drives double stranded RNA within target cells. This shRNA is then recognized allowing a RISC complex to form to target mRNA of interest.
  • Any suitable vector may be used to deleiver antisense, siRNA molecules etc.
  • a replication defective HIV- based vector may be used.
  • the ERG modulator for use in the invention may be administered as a pharmaceutical composition.
  • Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention may comprise, in addition to active ingredients, a pharmaceutically acceptable excipient, a carrier, buffer stabiliser or other materials well known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, 21 st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.).
  • Such materials may include buffers such as acetate, Tris, phosphate, citrate, and other organic acids ; antioxidants; preservatives; proteins, such as serum albumin, gelatin, or immunoglobulins ; hydrophilic polymers such as polyvinylpyrrolidone ; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine ; carbohydrates; chelating agents; tonicifiers; and surfactants.
  • the pharmaceutical compositions may also contain one or more further active compounds selected as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect the activity of the ERG modulator. For example, in the treatment of inflammatory disease such as arthritis, in addition to ERG modulator, other anti-inflammatory agents such as cyclooxygenase-2 (COX-2) inhibitors, may be used.
  • COX-2 cyclooxygenase-2
  • the active ingredients may be administered via microspheres, microcapsules, liposomes, other microparticulate delivery systems.
  • active ingredients may be entrapped within microcapsules which may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-
  • microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for further details, see Remington: the Science and Practice of Pharmacy, 21 st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.
  • Sustained-release preparations may be used for delivery of active agents.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, suppositories or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl- methacrylate), or poly (vinylalcohol)), polylactides (U. S. Pat. No.
  • compositions of and for use in the invention are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
  • the actual dosage regimen will depend on a number of factors including the condition being treated, its severity, the patient being treated, the agent(s) being used, and will be at the discretion of the physician.
  • the optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient(s) being administered and the route of administration.
  • Figure 1 illustrates that CD31 and membrane depolarization promote ⁇ 1 integrin-dependent binding of Fn-coated LatexTM beads: a,b,c,d, May- Grunwald staining of cytospin preparations of Kav31 WT following trypsin- EDTA treatment (a,b) or SEM of adherent cells before trypsin-EDTA treatment (c,d) revealing phagocytic cup formation, but never the internalization of Fn-beads.
  • Kav100 lines stably expressing cytoplasmic tail truncations in which the proximal 1 , 30, 50, and 71 amino acids are present, f, Fn-bead binding by Kav31WT and Kav2YF is reduced at low cell density (LCD, ⁇ 10% confluence), g, Fn-bead binding at HCD was sensitive to ⁇ 1 and ⁇ v ⁇ 3 integrin blockade with P5D2 and LM609 mAbs (10 ⁇ g/mL) respectively or RGDS (1 mM). All histograms represent the mean ⁇ S. D. of at least three experiments, each performed in duplicate. * and ** denotes p ⁇ 0.05 and ⁇ 0.01 respectively by Anova and Tukey's post hoc test compared with KavHygro (e) or untreated cell line (g).
  • Figure 2 illustrates that the voltage-gated potassium channel ERG co- associates with CD31.
  • a A Coomassie blue stained gel of a Fab fragment of YRI31.8 which recognizes Ig domain 4 of CD31 , coupled with a biotin label transfer reagent, sulfo-SBED.
  • b Immunofluorescence labelling of Kav31 WT with the Fab reagent and an Alexa-488 conjugated streptavidin revealed a typical staining pattern in which CD31 concentrates at the lateral border/junctions of Kav31 WT at HCD.
  • FIG. 3 CD31 and ERG show evidence of colocalization.
  • (a) Kav31WT cells immunostained punctate for ERG (green)(upper left panel) and CD31 (red)(upper middle panel). The upper right panel shows a merged image of the upper left and middle panels,
  • (b) Human monocyte-derived macrophages (HMDM) immunostained punctate for ERG (green) and CD31 (red) in which an apoptotic cell (AC) is bound.
  • HMDM Human monocyte-derived macrophages
  • AC apoptotic cell
  • (c) A region of a lamellopodia is expanded to reveal co-clustering of CD31 and ERG (c) in which an overlap image is shown (d).
  • (e) Two histograms are shown for horizontal transactions of (c) taken at positions i and ii.
  • ERG affects membrane polarization in which the depolarized state is permissive for Fn-bead binding.
  • Figure 5 illustrates the effect of depolarisation on human monocyte- derived macrophages (HMDM) binding of apoptotic neutrophils
  • HMDM human monocyte- derived macrophages
  • a Human monocyte-derived macrophages exhibit an increased capacity to bind apoptotic neutrophils when depolarized with 54.5 mM KCI (DEPOL) which is lost if HMDMs are allowed to repolarize upon return to normal saline conditions (REPOL).
  • the effect of repolarization can be blocked in the presence of 10 ⁇ M DOF.
  • b representative images of HMDMs binding apoptotic PMNs under untreated and depolarized conditions
  • c Overall bead binding indices for (a) are computed. All error bars represent the 95% c.i.
  • Figure 6 illustrates that 1) there is a high degree of heterogeneity within the macrophage population with respect to resting membrane potential, 2) for those macrophages with a low membrane potential [ ⁇ 50 RFI (relative fluorescence intensity)] we see that they are readily depolarized by apoptotic cells and 3) repolarization is inhibited by CD31.
  • a 5-7 day old HMDMs exhibit significant heterogeneity in relative membrane potential as observed with FLI PR-blue
  • b Viable PMNs essentially exclude FLI PR- blue whereas apoptotic cells do not in which FLI PR-blue loading is identical to that for HMDMs (a).
  • c d Representative time-lapse images of an HMDM (2) binding an apoptotic cell (white arrows). Quantitation of
  • n represents the number of individual HMDMs monitored and error bars represent the 95% c.i.
  • Figure 7 illustrates the effect of dofetilide and P2B1 F(ab') 2 on macrophage repolarization following depolarization with 54.5 mM KCI.
  • HMDMs were depolarized with 54.5 mM K + for 5 min before returning cells to saline media conditions containg either 10 ⁇ M DOF, P2B1 F(ab') 2 , or an irrelevant IgG F(ab') 2 in which the relative membrane potential is reported as a fold-increase in RFI over that seen for HMDMs left untreated
  • b Representative time-lapse traces for individual HMDMs repolarizing in which HMDMs have been grouped according to whether they started out with a low or high RFI.
  • c Representative low magnification images of HMDMs under depolarized and repolarized settings.
  • CONTROL normal saline
  • All histograms represent the mean ⁇ S. D. of between 3 and 8 experiments, each performed in sextuplets where %Phagocytosis represents the percentage of macrophages engulfing apoptotic cells.
  • Figure 9 illustrates how a ventral tail fin nick in Zebrafish is repaired following an initial contraction followed by an influx of neutrophils before reopening and the neutrophils reverse migrating
  • a shows a photograph at magnification x3.5 of a zebrafish embryo
  • b shows a section of the zebrafish at magnification 11.5, showing the applied nick
  • c infiltrating leukocytes are stained with MPO and appear as brown stellate bodies, d, contractive phase of the wound response seen at 0, 2, 4 and 6 minutes post tail-nick.
  • Figure 10 illustrates the results of time-lapse DIC light microscopic analysis on point of entry or exit of leukocytes from the caudal vein (a) as well as the associated path length (b) and migration velocity (c) of each transmigrated leukocyte as it migrates to the damaged site. Also shown in (c) is the effect of 10 ⁇ M dofetilide on migration velocity forthose leukocytes that transmigrated ( ⁇ ). In all cases recruitment was promoted in the presence of Pseudomona aruginosa and only wound sizes greater than 500 ⁇ m 2 and less than 2500 ⁇ m 2 are considered
  • Figure 11 illustrates that Class III antiarrhythmic methanesulfonanilides block leukocyte recruitment at 2 h post tail fin injury when the leukocyte response is found to be maximal.
  • Figure 12 illustrates that antisense RNA knockdown of zERG significantly attenuates the inflammatory response of 3dpf embryos to a tail fin nick in the presence of P. aruginosa. zERG knockdown was confirmed by its effect on the ventricular heartbeat.
  • Match is an exact complementary strand for ERG whereas mismatch is a 5 base-pair mismatch to act as a control
  • Figure 13 illustrates the anti-inflammatory effects of dofetilide on both leukocyte recruitment and pro-inflammatory cytokine production when administered intraperitoneal ⁇ on mice subsequently challenged with a thioglycollate model of "sterile" peritonitis, a, Illustrates total cell counts from peritoneal lavages after 4 hours exposure to 30OuI of 10% TG.
  • A Positive control (TG, no dofetilide)
  • B 1 mg/kg dofetilide
  • C 5mg/kg dofetilide
  • D Negative control (No TG, no dofetilide).
  • b Illustrates cytokine levels from peritoneal lavage fluid after 4 hours;
  • A Positive control (TG, no dofetilide),
  • B 1 mg/kg dofetilide,
  • C 5mg/kg dofetilide,
  • D Negative control (No TG, no dofetilide).
  • Figure 14 illustrates the anti-inflammatory effect of dofetilide when administered in the drinking water on mice challenged with sterile peritonitis and illustrates total cell counts from peritoneal lavages after 4 hours (50OuI 6% TG; 10mg/kg dofetilide).
  • Figure 15 illustrates the anti-inflammatory effect of dofetilide on mouse peritoneal macrophages challenged with TG ex vivo by measuring cytokine levels from cell media 24 hours following TG challenge (1% TG)
  • K562 stably expressing ⁇ v ⁇ 3 (Kav100) (a gift from S. D. Blystone) were maintained in DMEM-F12 +Glutamax supplemented with 10% FBS.
  • Stable lines expressing variant CD31 forms were generated by transfection following electroporation (750V, 25 ⁇ F; BIORAD GENE PULSE).
  • 5x10 6 cells were resupended in HBSS, pH 6.7, containing 10 mM HEPES, 1 nM ATP, 2 mM glutathione, with 20 ⁇ g of plasmid DNA. All CD31 plasmid constructs were subcloned from pCDNA3 (gifts from CD.
  • Kav100 lines were typically used after removal from antibiotic selection for at least two passages.
  • Jurkat T cells were maintained in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and routinely selected for CD31 expression using 9G11 immobilized to DynabeadsTM.
  • Murine bone-marrow-derived and resident peritoneal macrophage preparations as well as in vitro phagocytosis of apoptotic thymocytes was as described elsewhere (Brown et al, 2002; Taylor et al, 2000).
  • Fn-coated LatexTM beads were prepared with 3.0 ⁇ m Polybead Amino Microspheres (Polysciences Inc) modified with glutaraldehyde and reacted with human plasma Fn as described (Blystone et al, 1994). Kav100 cells were typically washed with DMEM (no serum) and approximately 4x10 7 beads were added per 48-well assay. Fn-beads were allowed to settle and bind for 30 min before gently removing all media and adding trypsin- EDTA for a further 15 min to remove loosely bound Fn-beads. Fn-bead ingestion was scored by light microscopy and expressed as the number bound into phagocytic cups per 100 cells (phagocytic index).
  • RGDS (1 mM) and all antibodies, including control IgG mAb, were added at 10 ⁇ g/ml for 15 min at room temperature prior to the addition of Fn-beads except for Fig. 4f where P2B1 and Fn-beads were added simultaneously.
  • Fab fragments of anti-CD31 mAbs (P2B1 , 9G1 1 , YRI31.2, YRI31.8, YRI31.9, YRI31.12) were generated with immobilized Ficin before modifying with sulfosuccinimidyl[2-6-(biotinamido)-2-(p-azidobenzamido)- hexanoamido]-ethyl-1 , 3 ' -dithiopropionate (Sulfo-SBED) (1 ⁇ g Sulfo- SBED/10 ⁇ g Fab), both according to the supplier's instructions (Perbio Sciences, UK).
  • FLIPR-Blue reagent is a membrane potential reporting dye which shows good correlation with patch clamping and is amenable to fluorescence imaging (Molecular Devices).
  • Kav100 cell lines and murine macrophages were incubated with FLIPR-Blue according to the manufacturer's instructions in which the reconstituted dye was added direct to the culture medium at a 1 :5 dilution in which cells were maintained at room temperature. Following a minimum 5 min loading period, cells were visualized by fluorescence microscopy.
  • the FLIPR-Blue dye was reconstituted in DMEM/10% FBS and used at a 1 :1 dilution as this was found to give a superior signal to noise ratio.
  • Kav31 WT either at HCD or LCD, were incubated with WM59 or P2B1 for 15 min at 37°C before fixing with 4% freshly-made paraformaldehyde. Cells were then immunolabelled for ERG using a rabbit pAb (Abeam) before washing and applying Alexa-488 and Alexa-568 secondary antibodies.
  • Kav100 cell lines and resident peritoneal macrophages cultured in 48-well plates, were loaded with FLIPR-Blue dye according to manufacturers instructions. Fluorescence imaging was performed with Improvision OpenLab software using an ORCA camera attached to an Axioskop Il MOT microscope. In the case of FLIPR-Blue, whole field images were collected with x5 objective and the low fluorescence signal subtracted from the mean. To correct for inter-experimental variations, the mean corrected fluorescence for Kav31 WT cells was expressed as the fold-increase over that seen for untreated Kav100.
  • the patch pipette (intracellular) contained in mM: 140 KCI, 2 MgCI 2 , 10 HEPES, 30 Glucose, 1 BAPTA (1 ,2-bis-o-aminophenoxy ethane-N,N,N',N'-tetraacetic acid, tetrasodium salt), 1 ATP, pH 7.3 with intracellular free calcium [Ca 2+ ]j buffered to 100 nM.
  • Cells were voltage clamped at -50 mV and depolarized to the respective potentials for 100 ms. Steady-state outward current was determined 80 ms into the pulse and was stable for >30 min under these conditions.
  • Vrest Resting membrane potential was determined in the conventional whole-cell current clamp mode under the same ionic conditions. Vrest was monitored for 5-10min following establishment of the whole-cell configuration to ensure stability and the Vrest averaged over 5min. All data acquisition and voltage protocols were controlled by an Axopatch 200B (or 200A) amplifier and pCLAMP9 software (Axon Instruments Inc., Foster City, CA). All data were sampled at 10 kHz and filtered at 2 kHz. Pipettes were manufactured from Garner no. 7052 glass, with resistances of 1 - 3 M ⁇ in physiological saline after fire polishing. DOF (10 ⁇ M), mAb P2B (10 ⁇ g/mL) or KCI (54.5 mM) were applied in bath solution by gravity-driven perfusion at a flow of 3-5 ml/min or by direct application to the bath.
  • the zebrafish embryos were incubaed at 28.5°C and samples collected as a time series before staining for MPO.
  • the zebrafish embryos were photographed, at minute intervals, whilst under mild anaesthetic (MS222), to follow leukocyte migration. Images of timed-leukocyte migration were collated and then compiled into a QuickTime movie.
  • the zebrafish embryos were pre-nick incubated (30 mins) with anti-arrhythmic agents (5), tail nicked and then time series sampled for MPO staining.
  • Antisense Knockdown of zERG Approximately 5 ng of FITC-labelled antisense phosphoramidate morpholinos targeting the 5' region of zERG were micro-injected into the yolk of eight cell-stage Zebrafish embryos. Embryos at 2 or 3 dpf (days post fertilization) were selected based on fluorescense incorporation throughout the embryo body.
  • Example 1 CD31 ligation promotes ⁇ 1 integr in-dependent binding of Fibronectin -beads (Fn-beads).
  • Fibronectin is an important opsonin in wound repair and host defences.
  • Fibronectin coated beads may be used to study the phagocytic behaviour of cells in vitro. Stable expression of CD31 by Kav100 cells (Kav31 WT), grown at high cell density (HCD), promoted the binding of Fn-beads into distinct phagocytic cups (Fig. 1 a-d) at levels greater than that seen for Kav100, or a hygromycin vector control, KavHygro (Fig. 1 e).
  • CD31 ability of CD31 to promote Fn-bead binding at HCD was independent of an ITIM motif found within the cytoplasmic tail of CD31 (Newman, 1999) but was dependent on homophilic interactions that are abrogated by a lysine to alanine point mutation (K89A, Newton et al., 1997, Fig. 1 e). In contrast, increased Fn-bead binding was not observed at low cell density (LCD) (Fig. 1f).
  • Example 2 CD31 inducibly associates with the voltage-gated potassium channel ERG
  • CD31 -directed ⁇ 1 integrin function was independent of the ITIM motif within the cytoplasmic tail of CD31 , and therefore independent of either SHP-1 or SHP-2 recruitment, the inventors sought novel protein partners for CD31 in Kav31 WT cells grown at HCD where bead binding was constitutively high.
  • Protein crosslinking with a biotin transfer reagent, Sulfo-SBED (Pierce Biotechnology), conjugated to a Fab antibody recognizing Ig domain 4 of CD31 (YRI31.8), but not Fabs directed against Ig domains 1 & 2 (data not presented) was found to incorporate biotin into neighbouring proteins distinct from CD31 or Ig heavy or light chains (Fig. 2c).
  • MS/MS analysis unequivocally identified a biotinylated protein at 140 kDa as the pore-forming ⁇ -subunit of the voltage-gated potassium channel, ERGI a (Fig. 2d).
  • Example 3 ERG does not function in K562 cells to set resting membrane potential (Vrest)
  • DOF dofetilide
  • E4031 two highly selective inhibitors of ERG with no known alternative activity
  • Neither DOF nor E4031 had any significant effect on bead binding by either cell line, even after 24 h of pretreatment (data not shown).
  • the inability of DOF to affect Vrest was confirmed with a fluorescent membrane potential dye (FLIPR-Blue) (Fig. 4a) and by whole- cell current clamp recordings where DOF alone at 10 ⁇ M had no significant effect on a resting membrane potential of -33.6 ⁇ 3.5 mV.
  • FLIPR-Blue fluorescent membrane potential dye
  • Example 4 ERG current is activated following cell depolarization.
  • ERG current is activated by membrane depolarization (Mitcheson & Sanguinetti, 1999).
  • Kav31WT cells repolarized to varying degrees in a concentration-dependent manner (Fig. 4a) that correlated strongly with the number of Fn-beads that bound over a subsequent 30 min period (Fig. 4b).
  • P2B1 is an anti-CD31 mAb that we have previously shown inhibits the phagocytosis of apoptotic cells by macrophages (Brown et al., 2002; Vemon-Wilson et al., 2006).
  • P2B1 whole IgG and a F(ab') 2 but not a Fab fragment inhibited the rapid repolarization of Kav31WT cells following a brief exposure to 54.5 mM K + (Fig. 4c).
  • the inhibitory effect of P2B1 on repolarization closely paralleled that for dofetilide.
  • Figure 5 illustrates that deliberate depolarization of human monocyte- derived macrophages augments the binding of apoptotic neutrophils.
  • 54.5 mM KCI was used to depolarize macrophages (B).
  • the data reveal that dofetilide inhibits repolarization to maintain high levels of apoptotic cell binding.
  • depolarization appears to recruit new macrophages into binding apoptotic cells while also increasing the number of apoptotic cells bound.
  • Figure 6 illustrates that 1 ) there is a high degree of heterogeneity within the macrophage population with respect to resting membrane potential (Panel a), 2) for those macrophages with a low membrane potential [ ⁇ 50 RFI (relative Fluorescence Intensity)] we see that they are readily depolarized by apoptotic cells and 3) repolarization is inhibited by CD31.
  • HMDMs with an initial RFI of less than 60 before contact with an apoptotic cell. HMDMs were then followed for at least 20 min after contact in which the maximum and subsequent minimum RFI was determined. HMDMs were pretreated with either nothing (f), P2B1 Fab (g), IgG Fab (h), or P2B1 Fab + 10 ⁇ M DOF (i). Dofetilide and P2B1 F(ab') 2 inhibit macrophage repolarization following depolarization with 54.5 mM KCI ( Figure 7).
  • Example 6 functions to repolarize depolarized murine macrophages.
  • Example 7 ERG inhibition is required for CD31 -dependent engulfment of apoptotic cells.
  • Fig. 8a The efficacy of wild-type macrophages to phagocytose apoptotic thymocytes was significantly reduced when apototic thymocytes were derived from CD31 knockout mice compared to wild-type controls. As depolarization was permissive for Fn-bead binding the inventors asked whether K + -induced depolarisation of macrophages could rescue the reduced phagocytosis of CD31 knockout thymocytes.
  • CD31 negative macrophages may also explain why phagocytosis of CD31 negative thymocytes was significantly lower than that observed with CD31 positive thymocytes (Fig. 8c) which hitherto had always been inexplicable. Taken together these data indicate that 1 ) apoptotic thymocytes depolarise murine macrophages (see discussion) and 2) ERG and CD31 are functionally linked in that inhibition of ERG current was required to specifically promote CD31 - directed apoptotic cell engulfment.
  • macrophages from CD31 -/- mice exhibit a reduced phagocytic capacity that is not modulated by ERG inhibition with 10 ⁇ M dofetilide.
  • Example 8 Class III antiarrhythmics inhibit leukocyte recuitment Zebrafish (Danio rerio) represents a genetically tractable and optically transparent organisms with which to model and visualize human disease. During very early zebrafish embryonic development the innate immune system is functional, although it takes longer to acquire an adaptive immune response. The inventors have developed an infected wound model of inflammation to ascertain the importance of ERG in leukocyte recruitment based on the demonstration as described above of the functional relationship between ERG and CD31. As CD31 is a cell adhesion molecule that is a key mediator of leukocyte transmigration and interstitial migration the inventors assessed whether or not ERG is involved in the regulation of leukocyte migration. The inventors assessed the efficacy of class III anti-arrhythmic methanesulfonanilides which are highly selective for ERG on leukocyte recruitment and, as described below, confirm that ERG is an important regulator of leukocyte function.
  • FIG. 10 illustrates the analysis of time-lapse Differential Interference Contrast (Normarski) light microscopic images for leukocyte transmigration across the caudal vein (point of entry) and subsequent path length and time taken (velocity) to migrate to the wound.
  • average leukocyte migration velocity is relatively constant regardless of time and entry. Early transmigrating leukocytes are more likely to wander before finding the wound.
  • Dofetilide (A) causes transmigrated leukocytes to stop and stall more often and for longer than untreated ( ⁇ ) controls explaining the apparent reduction in migration velocity.
  • Figure 11 illustrates the results of a study in which zebrafish embryos were pre-incubated with no drug (panel A), dofetilide 1 ⁇ M or dofetilide 10 ⁇ M, tail nicked and then time series sampled for MPO staining.
  • Dofetilide at 10 ⁇ M was found to inhibit leukocyte recruitment (P ⁇ 0.08) Leukocytes were identified following MPO Staining of paraformaldehyde-fixed 3dpf embryos
  • Example 9 Dofetilide reduces inflammatory cell counts in peritoneal lavages in an in vivo model of peritonitis.
  • the inventors have identified a novel mechanism for regulation of phagocytosis dependent upon CD31 -directed inhibition of the membrane repolarization function of mammalian ERG. They first identified a role for ERG in CD-31 mediated regulation in a reductionist model system. Importantly, this regulatory mechanism was also involved in macrophage clearance of apoptotic cells demonstrating the importance, and conservation, of this mechanism in native cells. In addition, the observations of Fn-bead binding indicate that membrane depolarization directly enhanced ⁇ 1 integrin adhesive function. Importantly, CD31 promoted ⁇ 1 integrin adhesive function and phagocytosis in an ITIM- independent manner.
  • CD31 is considered a member of the inhibitory receptor family that includes SIRP ⁇ and is thought to function through the recruitment of SH2 domain containing protein tyrosine phosphatases to the ITIM (Newman, 1999). In each case calcium influx is a key second messenger and would be favoured by membrane depolarization. It is therefore plausible to speculate that the inhibitory function of CD31 in these cells is not by direct negative regulation of an ITIM bearing receptor as proposed but rather by regulating membrane potential.
  • apoptotic cells may also provide a stimulus for depolarization independent of CD31 reminds us of early descriptions of surface charge in not only defining apoptotic cells (Morris et al, 1984) but also their clearance mechanism (Savill et al, 1989) in which K + leakage (Vu et al, 2001) would be expected to depolarize macrophages upon cell- cell contact. It is plausible to speculate further that depolarization would then allow formation of a phagocyte "synapse" in which the tight binding and recognition of apoptotic cells promotes phagocytic clearance (Gregory & Devitt, 2004).
  • Fc-mediated phagocytosis which has been implicated in apoptotic cell clearance (Hart et al., 2004) and is morphologically very similar, is also associated with membrane depolarization (Young et al, 1983; Holevinsky & Nelson, 1995; Floto et al, 1997; Campo et al, 2003).

Abstract

La présente invention concerne des procédés permettant de moduler la phagocytose de cellules apoptotiques et le recrutement de leucocytes sur un site d'inflammation. Les procédés impliquent la modulation de l'activité du canal ERG. Les procédés permettent l'utilisation de modulateurs ERG pour le traitement de maladies inflammatoires.
PCT/GB2006/050345 2006-10-20 2006-10-20 Traitement d'une maladie inflammatoire WO2008047067A1 (fr)

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