WO2007039151A1 - Blockers of transforming growth factor beta and its receptors for the treatment of infectious diseases - Google Patents

Blockers of transforming growth factor beta and its receptors for the treatment of infectious diseases Download PDF

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WO2007039151A1
WO2007039151A1 PCT/EP2006/009245 EP2006009245W WO2007039151A1 WO 2007039151 A1 WO2007039151 A1 WO 2007039151A1 EP 2006009245 W EP2006009245 W EP 2006009245W WO 2007039151 A1 WO2007039151 A1 WO 2007039151A1
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tgfβ
receptor
blocker
infections
inhibits
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PCT/EP2006/009245
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French (fr)
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Adriano Fontana
Ursula Malipiero
Uwe Koedel
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Universität Zürich
Ludwig-Maximilians-Universität München
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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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents

Definitions

  • This invention relates to the treatment of infectious diseases of the central nervous system (CNS) and the skin with particular focus on bacterial meningitis and infected leg using blockers of transforming growth factor beta (TGF ⁇ )
  • TGF ⁇ Transforming growth factor beta
  • TGF ⁇ is a multifunctional cytokine involved in the regulation of proliferation, differentiation, migration and other functions of many different cell types Three isoforms are present in mammals, i.e. TGFp 1 , TGF ⁇ 2 and TGF ⁇ 3 , which, to a great extent, show overlapping functions.
  • TGF ⁇ is secreted in a latent form, which needs an activation step mediated by proteases or thrombospondins before it can bind to its specific type I and type Il serin/threonin kinase receptors TGF ⁇ mediates the association of TGF ⁇ receptor type I (TGF ⁇ RI) and TGF ⁇ RII, which triggers TGF ⁇ RII to phosphorylate and activate TGF ⁇ RI.
  • TGF ⁇ RI TGF ⁇ receptor type I
  • TGF ⁇ RII TGF ⁇ RII
  • SMAD2 and SMAD3 downstream signaling involving receptor-activated SMAD transcription factors (SMAD2 and SMAD3) become phosphorylated, associate with co-SMADs (SMAD4), and the formed complex moves into the nucleus and associates with transcriptional co- activators or co-repressors Inhibitory SMADs, including SMAD7, induced by TGF ⁇ family ligands and other cytokines function in a negative feedback loop to reduce TGF ⁇ induced signaling
  • SMAD7 transcriptional co- activators or co-repressors
  • TGF ⁇ family ligands and other cytokines function in a negative feedback loop to reduce TGF ⁇ induced signaling
  • TGF ⁇ TGF ⁇ RII
  • SAD3 downstream signaling molecules
  • TGF ⁇ plays a pivotal role in the propagation of wound healing and in the development of excessive fibrosis, as seen in patients with scleroderma, in glomerular scarring in glomerulonephritis, or in the fibrosis of the lung as seen in idiopathic pulmonary fibrosis or secondary conditions due to side-effects of drugs such as methotrexate or exposure to inhaled antigens.
  • TGF ⁇ inhibitors are considered for treating such diseases. Indeed, experimental strategies aimed at interfering with the activity of TGF ⁇ have proved therapeutically successful in cancer models and in fibrosis of the lung and kidney. The specific use of TGF ⁇ inhibitors in the treatment of infections in the CNS or in infections of skin ulcers has not been investigated yet.
  • This invention focuses on the treatment of infections of the CNS including bacterial meningitis, a disease occurring worldwide (approximately 1.2 million cases a year) with a high mortality rate and incidence of neurologic sequelae. Even in industrial countries, and despite early and intensive treatment with antibacterial antibiotics and intensive care, death occurs in 25% of adults.
  • the major causes of bacterial meningitis in Western Europe and the United States are Streptococcus pneumoniae and Neisseria meningitis.
  • the introduction of Haemophilus influenzae conjugated vaccines provoked a declining incidence of invasive H. influenzae infections.
  • PMN polymorphonuclear leukocytes
  • PMN and macrophages are also essential in the resolution of fungal infections and protozoan diseases of the CNS.
  • Examples of frequently seen fungal CNS diseases are infections with aspergillus and Candida; and Toxoplasma gondii serves as an example of protozoan CNS infections.
  • These non-bacterial infections of the CNS may typically be present in a subacute fashion with focal neurologic signs such as seizures or altered mental status due to meningoencephalitis. Since the clearance of protozoan or fungal infectious agents depends on the activation of macrophages by T-lymphocytes, infections of the CNS with these pathogens are most frequently seen in T-cell deficiency such as HIV-infections.
  • Skin ulcers are common and cause a substantial burden to the patient
  • the major causes of skin ulcers comprise arteriosclerosis, postthrombotic venous insufficiency, diabetes mellitus, and chronic renal insufficiency
  • arteriosclerosis arteriosclerosis
  • postthrombotic venous insufficiency venous insufficiency
  • diabetes mellitus venous insufficiency
  • chronic renal insufficiency In a given patient a combination of these risk factors is seen
  • the chronicity of the aforementioned diseases also the skin ulcers are very often chronic, and their complete healing difficult to achieve
  • the major complication of skin ulcers is their infection Despite intensive antibiotic treatment and adequate debridement, amputation of the affected foot is often required Local immunosuppression which is due to poor invasion of PMN into the skin ulcers and defective bacterial clearance of PMN in patients with diabetes provide the basis for their impaired elimination of bacteria and the risk of chronicity of the infection, which counteracts the healing process
  • Elimination of pathogens require leukocytes to migrate to the site of infection and eliminate the pathogen This process is hindered in the CNS because the blood brain barrier (BBB) prevents the easy travel from the blood to the CNS parenchyma In the skin of patients with arteriosclerosis, venous insufficiency, diabetes mellitus and renal insufficiency, migration of leukocytes into chronic ulcerations are hindered because of angiopathy and scarring of the lesions
  • BBB blood brain barrier
  • the present invention relates to a method of treating infectious CNS diseases and infections of skin ulcers comprising administering a TGF ⁇ blocker, and the use of such blockers in said treatment and in the manufacture of medicaments for treating infectious CNS diseases and infections of ulcers of the skin.
  • the invention further relates to a method of screening for a compound effective in the treatment of infectious CNS diseases and/or infections of ulcers of the skin comprising contacting a candidate compound with a TGF ⁇ or a TGF ⁇ receptor, and choosing candidate compounds which selectively reduce activity of TGF ⁇ or the TGF ⁇ receptor .
  • the invention further relates to compounds selected by these methods of screening.
  • Fig. 1 Generation of mice with phagocyte-specific deletion of TGF ⁇ receptor II.
  • Fig. 2 Deletion of TGF ⁇ RII in phagocytes determined by Taqman real-time PCR.
  • TGF ⁇ inhibits the LPS induced TNF ⁇ secretion in macrophages of TGF ⁇ Rllflox/flox mice, but not in macrophages of phag-TGF ⁇ RII-/- mice. Macrophages were pre-stimulated for 2 h with TGF ⁇ followed by a 6 h incubation period with 0.01 ng LPS/ml. TNF ⁇ content was determined by ELISA.
  • Fig. 3 Deletion of TGF ⁇ RII in phagocytes results in increased numbers of leukocytes in the CNS and more efficient bacterial killing.
  • Lane a uninfected TGF ⁇ Rllflox/flox mice
  • Lane b infected TGF ⁇ Rllflox/flox mice
  • Lane c infected phag-TGF ⁇ RII-/- mice
  • CSF cerebrospinal fluid
  • Meningitis-associated intracranial complications including a rise in intracranial pressure (A, y-axis intracranial pressure, mm Hg) and vasogenic brain edema (B, brain albumin content, ng per ⁇ g protein), as assessed by albumin content in the CNS, are less pronounced in infected ceftriaxone-treated phag-TGF ⁇ RII-/- mice than in the corresponding TGF ⁇ Rllflox/flox
  • TG F ⁇ is a chemoattractant for neutrophils
  • pretreatment of the cells with the cytokine reduces their chemotaxis
  • the present invention relates to a method of treating infectious CNS diseases and infections of ulcers comprising administering a TGF ⁇ blocker, and the use of such blockers in said treatment and in the manufacture of medicaments for treating infectious CNS diseases and infections of ulcers of the skin.
  • TGF ⁇ blockers diminish or prevent TGF ⁇ receptor signaling in PMN and macrophages.
  • PMN and monocytes-macrophages are recruited more efficiently from the blood to the CNS or the skin, and become more potent phagocytic cells.
  • Infectious CNS diseases considered are, for example, bacterial meningitis and CNS infections with fungus or protozoan pathogens such as infections with aspergillus, Candida and Toxoplasma gondii.
  • Infections of ulcers considered are, for example, such infections in patients suffering of arteriosclerosis, postthrombotic venous insufficiency, diabetes mellitus, and chronic renal insufficiency.
  • TGF ⁇ blockers are compounds which inhibit the production of TGF ⁇ or the activation of TGF ⁇ from its latent form, inhibit the production or the activation of TGF ⁇ receptors, partly or entirely inactivate active TGF ⁇ , inhibit binding of TGF ⁇ to its receptors, or inhibit the TGF ⁇ mediated receptor downstream signaling pathway.
  • TGF ⁇ production can be inhibited by anti-sense oligodeoxynucleotides or siRNAs to the three TGF ⁇ isoforms.
  • Prevention of the formation of active TGF ⁇ can be achieved by targeting thrombospondin (TSP1 and/or TSP2) with anti-thrombospondin antibodies or with inhibitors which interfere with the synthesis of thrombospondins.
  • thrombospondins are matricellular glycoproteins, which activate latent TGF ⁇ .
  • the active form of TGF ⁇ can also be inhibited by polypeptides and fragments thereof, which are related to the human TGF ⁇ latent binding protein family, and thereby bind to the active TGF ⁇ protein.
  • TGF ⁇ blockers examples include TGF ⁇ blockers according to the invention.
  • a further example of a compound considered as TGF ⁇ blocker according to the invention is a binding composition which specifically and/or selectively binds TGF ⁇ i, TGF ⁇ 2 and/or TGF ⁇ 3, and which neutralizes TGF ⁇ i , i.e. a compound comprising antigen binding sites of anti-TGF ⁇ Fabs as described in WO 2005/010049 (Eh LiIIy)
  • TGF ⁇ blockers a monoclonal antibody to TGF ⁇ i , which inhibits the function of TGF ⁇ i (from Cambridge Antibody Technology); GC-1008, a humanized monoclonal antibodies to TGF ⁇ i, TGF ⁇ 2 and TGF ⁇ 3 (from Cambridge Antibody Technology), Lerdelimumat, a monoclonal antibody to TGF ⁇ i (from Cambridge Antibody Technology, Cordeiro M F , Curr Opin MoI Ther 5 199-203, 2003) Antibodies to TGF ⁇ have been used successfully in animal studies to lower scarring in experimental glomerulonephritis (W.A Border et al , Nature 360 361-364, 1992) Alternatively, peptides comprising parts
  • TGF ⁇ i amino acids 41-65 of TGF ⁇ 3, since they are capable of blocking TGF ⁇ effects (US 6'500'920, Univ of St Louis)
  • TGF ⁇ blockers according to the invention are compounds which by binding to TGF ⁇ interfere with TGF ⁇ receptor activation, such as proteins of the family of small leuci ⁇ e- ⁇ ch proteoglycans, which have a high affinity for TGF ⁇
  • One small leucine-rich proteoglycan is decorin which is composed of repeated motifs with leucine residues flanked by cysteine-rich regions.
  • TGF ⁇ induced pulmonary fibrosis Small leucine-rich proteoglycans which can be used to bind and neutralize TGF ⁇ also include other proteins such as biglycan, fibromoduhn and lumican Compositions of these proteins are provided by Alcon Lab lnc , and are examples of TGF ⁇ blockers according to the invention
  • TGF ⁇ blockers On the level of the membrane receptor, TGF ⁇ binding can be inhibited with soluble TGF ⁇ receptors
  • TGF ⁇ blockers are the soluble TGF ⁇ type III receptor fusion proteins comprising all or a portion of the Fc tail of human IgG covalently linked to all or an active portion of a splice variant of the extracellular domain of human TGF ⁇ or activin type Il or type Il B-receptor (WO 2005/028517, Gen Hospital Corp.)
  • Interference with TGF ⁇ mediated signaling is achieved with (1) the overexpression of SMAD7 by virus-mediated gene transfer, which results in the inhibition of the TGF ⁇ receptor mediated phosphorylation of SMAD transcription factors required for the transcription of TGF ⁇ regulated genes, and (2) small organic compounds, which block TGF ⁇ receptor kinases, and thereby activate of SMAD proteins.
  • TGF ⁇ blockers comprise novel pyrazole compounds and related dihydropyrrolo pyrazoles, and are highly potent inhibitors of the TGF ⁇ receptor kinase by binding to the ATP-binding pocket of the kinase.
  • TGF ⁇ blocker a further TGF ⁇ receptor kinase blocker
  • Ly 2157299 from EIi Lilly & Company, which modulates TGF ⁇ Rl function by inhibiting SMAD2 phosphorylation.
  • Other compounds considered as TGF ⁇ blockers according to the invention, which interfere with TGF ⁇ receptor signaling include
  • Gene therapy can make use of vectors harboring cDNA, which encode for proteins encoding for TGF ⁇ or TGF ⁇ receptor genes or for genes involved in the activation or inactivation of TGF ⁇ or of TGF ⁇ receptors or which encode genes mediating TGF ⁇ receptor signaling
  • vectors which lead to overexpression of inhibitory SMAD proteins such as SMAD7
  • TGF ⁇ blockers according to the invention are also widely known TGF ⁇ inhibitors, for example, as disclosed in Patent Applications WO 92/00330, WO 99/48904, WO 99/50296, WO 00/12551, WO 02/26935, WO 02/62793, WO 2004/010929, WO 2004/056352, WO 2004/081009, WO 2004/112710, WO 2005/010049, WO 2005/019422, WO 2005/023248, WO 2005/028517, WO 2005/039570, US 6'500'920
  • TGF ⁇ blockers are:
  • Soluble TGF ⁇ type III receptor fusion proteins comprising all or a portion of the Fc tail of human IgG covalently linked to all or an active portion of a splice variant of the extracellular domain of human TGF ⁇ or activin type Il or type Il B-receptor (WO 2005/028517, Gen Hospital Corp )
  • SB-4314542 a small molecular inhibitor of the TGF ⁇ receptor type I which blocks phosphorylation and nuclear translocation of SMAD proteins (J. Rich, Duke University
  • HTS466284 a small molecule inhibitor of the TGF ⁇ receptor type I which inhibits downstream signaling events (ET. Akporiaye and M. Rausch, Department of Microbiology and Immunology, University of Arizona); and
  • Ac-SDKP N-acetyl-seryl-aspartyl-lysyl-proline
  • Nitrogen-containing heterocyclic compounds and other aromatic organic compounds (WO 2004/056352, Scios Inc.)
  • siRNA Small interfering ribonucleic acid
  • TGF ⁇ i and TGF ⁇ 2 antisense constructs from Antisense Pharma GmbH
  • RNA ligand TGF ⁇ i selected from a group of 137 sequences identified by the SELEX
  • TGF ⁇ inhibitory proteins and of proteins which interfere with the intracellular TGF ⁇ signaling cascade SMAD6 and SMAD7 nucleic acids and polypeptides (WO 99/50296, EIi Lilly); and vectors comprising nucleic acids which encode the hLTBP-3 polypeptide (WO 00/12551 , EIi Lilly & Company).
  • TGF ⁇ blockers are: - antibodies to TGF ⁇ and TGF ⁇ receptors;
  • cytoxazon derivate which inhibits the TGF ⁇ signal transduction pathway (WO 2005/039570, RIKEN KK); and - nitrogen-containing heterocyclic compounds and other aromatic organic compounds (WO 2004/056352, Scios Inc.).
  • TGF ⁇ blockers are:
  • One aspect of the invention relates to a method of treating infectious diseases of the CNS or infected skin ulcers comprising administering TGF ⁇ blockers as defined hereinbefore in a quantity effective against the CNS or skin disease to a mammal in need thereof, for example to a human requiring such treatment.
  • the treatment may be for prophylactic or therapeutic purposes.
  • the TGF ⁇ blocker is preferably in the form of a pharmaceutical preparation comprising the TGF ⁇ blocker in chemically pure form and optionally a pharmaceutically acceptable carrier and optionally adjuvants.
  • the TGF ⁇ blocker is used in an amount effective against the infectious CNS disease or the infected skin ulcer.
  • the dosage of the active ingredient depends upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, the mode of administration, and whether the administration is for prophylactic or therapeutic purposes.
  • the daily dose administered is from approximately 1 mg to approximately 500 mg, preferably from approximately 10 mg to approximately 100 mg, of a TGF ⁇ inhibitor or TGF ⁇ receptor inhibitor
  • compositions for enteral administration such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as subcutaneous, intravenous, intramuscular or injections into the cerebrospinal fluid (CSF) compartment are especially preferred.
  • the pharmaceutical compositions comprise from approximately 1% to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient
  • compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known perse, for example by means of conventional dissolving and lyophilizing processes.
  • suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, and also binders, such as starches, cellulose derivatives and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, flow conditioners and lubricants, for example stearic acid or salts thereof and/or polyethylene glycol Tablet cores can be provided with suitable, optionally enteric, coatings Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient
  • Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the capsules may contain the active ingredient in the form of granules, or dissolved or suspended in suitable liquid excipients, such as in oils
  • Transdermal application is also considered, for example using a transdermal patch, which allows administration over an extended period of time, e g. from one to twenty days.
  • Another aspect of the invention relates to the use of TGF ⁇ blockers as described hereinbefore in the treatment of infectious CNS diseases and infected skin ulcers and in the manufacture of medicaments for treating these diseases.
  • Such medicaments are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing.
  • the TGF ⁇ blocker can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of fixed combinations of a TGF ⁇ blocker and one or more other therapeutic agents known in the treatment of infectious CNS diseases and infected skin ulcers, the administration being staggered or given independently of one another, or being in the form of a fixed combination.
  • the invention further relates to a method of screening for a compound effective in the treatment of infectious CNS diseases and infected skin ulcers comprising contacting a candidate compound with a TGF ⁇ or a TGF ⁇ receptor and choosing candidate compounds which selectively reduce the activity of TGF ⁇ .
  • the invention further relates to compounds selected by these methods of screening.
  • Inhibitors of TGF ⁇ activity are identified by contacting a TGF ⁇ or a TGF ⁇ receptor with a candidate compound.
  • a control assay with the corresponding TGF ⁇ or TGF ⁇ receptor — in the absence of the candidate compound - is run in parallel.
  • a decrease in activity in the presence of the candidate compound compared to the level in the absence of the compound indicates that the compound is a TGF ⁇ inhibitor.
  • Screening systems to be used for identification of candidate compounds comprise in vitro studies using TGF ⁇ as a cytokine which induces chemotaxis of leukocytes.
  • Compounds to be tested for TGF ⁇ blocking activity will be added together with TGF ⁇ in the chemotactic assays.
  • the screen can also be done in vivo by injection of S. pneumoniae type 3 (10 7 colony forming units) into the cisterna magna of normal wild type mice treated with ceftriaxone (100 mg/kg intraperitoneal ⁇ ).
  • Candidate compounds will be injected intravenously or into the cisterna magna of infected mice.
  • Efficiency of the candidate compounds will be evaluated by following the clinical course of bacterial meningitis of infected mice and by analyzing their cerebrospinal fluid (number of leukocytes and bacteria; albumin concentration) and by counting the number of intracerebral hemorrhages.
  • TGF ⁇ exerts potent chemotactic activity on PMN and monocytes in vitro and, upon injection into joints, induces a synovial inflammatory reaction (J. B. Allen et al., J. Exp. Med. 171:231 , 1990).
  • PMN chemotaxis the reports on the chemotactic property of TGF ⁇ can be reproduced (Fig. 6). This contrasts, however, with the observation in vivo when using mice lacking TGF ⁇ receptor mediated signaling in PMN and macrophages. These mice were generated by crossing floxed TGF ⁇ RII mice with lys-CRE mice (Fig. 1).
  • TGF ⁇ RII-/- mice Mice with impaired expression of TGF ⁇ RII are termed phag-TGF ⁇ RII-/- and are shown to be defective in the response of activated macrophages and PMN to TGF ⁇ (Fig. 2 and 6). TGF ⁇ fails to exert chemotactic responses in vitro on PMN derived from TGF ⁇ RII-/- mice (Fig. 6).
  • the basis of the invention is the observation that in TGF ⁇ RII-/- mice infected with S. pneumoniae and treated with antibiotics, recruitment of PMN and macrophages into the CNS is much more efficient than in antibiotic treated control mice with intact TGF ⁇ signaling (Fig. 3).
  • PMN from phag-TGF ⁇ RII-/- mice are not hindered by TGF ⁇ from being attracted in vitro or in vivo into the CNS in S. pneumoniae mediated meningitis (Fig. 3).
  • Higher numbers of phagocytes in the CNS are associated with improved clearance of bacteria in the CNS (Fig. 3).
  • secondary CNS damage including vasculitis and cerebral hemorrhages are prevented (Fig.
  • the principle of the present invention also applies to infected skin ulcers, e g. seen in patients with diabetes mellitus.
  • infected skin ulcers the expression of TGF ⁇ hinders efficient PMN migration to the site of infection and bacterial killing.
  • TGF ⁇ By blocking the action of TGF ⁇ locally in the skin the host response to infections in leg ulcers can be improved.
  • TGF ⁇ is a major risk factor for the impaired host response to infections and therefore blocking its action is helpful to treat skin ulcers and CNS infections.
  • TGF ⁇ Rllflox/flox mice were obtained from Stefan Karlsson and Per Leveen, Lund University, Sweden (P. Leveen et al., Blood 100(2): 560-8, 2002), and LysMcre were provided by lrmgard F ⁇ rster, University of D ⁇ sseldorf, Germany and are available at The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609 USA.
  • the Cre-bearing deleter mice express cre-recombinase under control of the murine lysozyme genes in macrophages and PMN (B. E. Clausen et al., Transgenic Research 8: 265-277, 1999).
  • the cre-recombinase binds cooperatively to 34 bp recombination sequences termed locus of crossing over in phage P1 (loxP) which have been introduced in exon 4 of the TGF ⁇ receptor gene.
  • These mice are termed TGF ⁇ Rllflox/flox (P. Leveen et al., loc.cit.).
  • the TGF ⁇ Rllflox/flox mice were crossed with LysMcre to yield a homozygous TGF ⁇ Rllflox/flox background and heterozygous background for LysMcre. Cre non-expressing littermates were used as controls.
  • the genotype of the off-springs was determined by PCR using the primer pair P3/P4 (P3: 5 1 -TATGGACTGGCTGCTTTTGTATTC-3 1 (SEQ ID NO:1) and P4: 5 1 -TGGGGATAGAGGTAGAAAGACATA-3 • (SEQ ID NO:2)) to distinguish floxed alleles (575 bp) from wild type (422 bp) (P. Leveen et al., loc.cit.).
  • the primer combinations Cre8/ Mlys1/Mlys2 (Cre8: ⁇ '-CCCAGAAATGCCAGATTACG-S' (SEQ ID NO:3); Mlysi: 5'-CTTGGGCTGCCAGAATTTCTC-3' (SEQ ID NO:4); Mlys2: 5'- TTACAGTCGGCCAGGCTGAC-3' (SEQ ID NO:5)) result in LysMcre amplicons of 700 bp and 1700 bp and in wild type amplicons of 350 bp.
  • the mice were bred under specific pathogen-free (SPF) conditions with screening of ubiquituous pathogens during the breeding of the animals.
  • SPPF pathogen-free
  • M-MuLV reverse transcriptase M-MuLV reverse transcriptase
  • TGF ⁇ RII exon boundary 3/4 (Mm01348770_m1)
  • TGF ⁇ RII exon boundary 6/7 (Mm00436978_m1) were used.
  • Detection of TGF ⁇ RII mRNA was performed by using the forward primer P1: 5'-ACATTACTCTGGAGACGGTTTG-S' (SEQ ID NO:6) and the reverse primer P2: ⁇ '-GGTAGTGTTCAGCGAGCCATCTT-S' (SEQ ID NO:7). All real-time PCR reactions were performed and analysed on an ABI Prism 7700 Sequence Detection SystemTM (Perkin Elmer Applied Biosystems). The reactions for the target and the endogenous control (18s rRNA, Applied Biosystems, Nr. 4310893) were performed in separate tubes, and the comparative CT method was used for standardization.
  • Meningitis was induced by transcutaneous injection of 15 ⁇ l of a bacterial suspension containing 107 colony forming units (cfu) per ml of Streptococcus pneumoniae type 3 into the cisterna magna under short-term anesthesia with halothane. Mice were clinically examined, weighed, put into cages, and allowed to wake up. The clinical score comprises the following criteria: a) presence of tremor, piloerection, and seizures; b) spontaneous motor activity; c) vigilance; d) body proprioception; e) a beam balancing test; and f) a postural reflex test. In healthy animals, the score was 0, infected animals that died within the observation period received 16 points.
  • mice 24 hours after infection, mice were evaluated clinically and treated with the antibiotic ceftriaxone (100 mg/kg intraperitoneally). 48 hours after infection, mice were again clinically evaluated, re-weighed, and the body temperature was measured via a rectal probe. Then, mice were anaesthetized with 100 mg/kg ketamine and 5 mg/kg xylazine. Subsequently, a catheter was inserted into the cisterna magna to measure intracranial pressure (ICP) and to de-termine CSF leukocyte counts. Subsequently, blood samples were taken by transcardial puncture.
  • ICP intracranial pressure
  • mice were perfused transcardially with 15 ml of ice-cold phosphate-buffered saline (PBS) containing 10 U/ml heparin. The brains were removed and rapidly frozen.
  • PBS ice-cold phosphate-buffered saline
  • Cerebella were dissected and homogenized in sterile saline. Cerebellar homogenates were diluted serially in sterile saline, plated on blood agar plates, and cultured for 24 h at 37°C with 5% CO 2 .
  • BBB blood-brain barrier
  • mice brain homogenates were examined for infiltration by albumin, an abundant serum protein that is normally excluded from the brain by the intact BBB, using ELISA.
  • lysis buffer 10 mM Hepes, pH 7.9, 10 mM KCI, 1.5 mM MgCI 2 , and a mixture of protease inhibitors including phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin A; 0.5 ⁇ g protein per well
  • mice brains were cut in a frontal plane into 10 ⁇ m thick sections. Beginning from the anterior parts of the lateral ventricles, 9 serial sections were photographed with a digital camera in 0.3 mm intervals throughout the ventricle system. The areas of the lateral ventricles and the third ventricle were measured (Image tool, UTHSCSA, Texas, USA) and the volume of the ventricle size was estimated (ventricle area / 9 pictures x 0.3 mm). Haemorrhagic spots were counted and the bleeding area was measured.
  • mice brains Histology Cryosections of mice brains were HE and hemacolor stained. In addition, selected brains were formaline fixed, paraffin embedded, and stained with HE and hemacolor.
  • peritoneal exudate cells were recovered 20 h (PMN) or 2 days (macrophages) after injection of 1 ml 3% Brewer thioglycollate medium (Sigma, Buchs, Switzerland).
  • the peritoneal cavity was flushed with 10 ml of HBSS (without Ca, Mg) / 1% BSA, 15 mM EDTA, the cells were collected by centrifugation at 1200 rpm and resuspended to 1x10 6 cells/ml X-Vivo 15 medium (BioWhittaker, Cambrex, Belgium) / 2 mM glutamine.
  • FACS-sorting Thioglycollate-elicited peritoneal exudate cells were resuspended in FACS buffer (2% FCS, 5 mM EDTA, in PBS) and incubated with anti- mouse CD16/32 (Fc-block from BD Pharmingen) for 5 min, then stained with anti-Gr-1 FITC (BD Pharmingen) and anti-mouse CD11b PE (BD Pharmingen) for 20 min. The Gr-1high/CD11b+ cells were sorted by an FACStar Plus (Becton Dickinson).
  • the plates were incubated at 37°C and 5% CO 2 for 1 h. At the end of the incubation, the remaining cells on the upper membrane surface were carefully removed with a cotton swab.
  • Migratory cells attached on the lower part of the filter were stained with DAPI (Molecular Probe, Netherlands) at 1 ⁇ M dilution for 20 min at 37°C and thereafter fixed in 4% paraformaldehyde in PBS.
  • Migrated neutrophils were counted with a square graticule (Leica Microsystems Wetzlar GmbH, Germany) in five visual fields per filter (three horizontal and two vertical fields crossing the middle of the filter) at 20Ox magnitude.
  • TNF ⁇ synthesis by macrophages Thioglycollate-elicited PEC were seeded at a density of 5x10 5 cells/well in a 24-well tissue culture plate in DMEM / 10% FCS / 2 mM glutamine. After 24 h, the medium was changed to X-Vivo 15 / 2 rtiM glutamine and the macrophages were incubated over night at 37°C, 5% CO 2 . The cells were prestimulated with 20 ng/ml TGF ⁇ i (R&D Systems) for 2 h, and then 0.01 ng LPS/ml (055:B5; Sigma, Buchs, Switzerland) was added for another 6 h. TNF ⁇ content in culture supernatants was measured by a TNF ⁇ ELISA Kit (BioSource Europe, Nivelles, Belgium).

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Abstract

The invention relates to the use of a transforming growth factor beta (TGFβ) blocker in the manufacture of medicaments for treating infectious CNS diseases and infections of ulcers of the skin. Moreover, the invention relates to a method of screening for a compound effective in the treatment of infectious CNS diseases and/or infections of ulcers of the skin. The method comprises contacting a candidate compound with a TGFβ or a TGFβ receptor and choosing candidate compounds which selectively reduce activity of TGFβ or the TGFβ receptor.

Description

Blockers of transforming growth factor beta and its receptors for the treatment of infectious diseases
Field of the invention
This invention relates to the treatment of infectious diseases of the central nervous system (CNS) and the skin with particular focus on bacterial meningitis and infected leg using blockers of transforming growth factor beta (TGFβ)
Background of the invention
Transforming growth factor beta (TGFβ)
TGFβ is a multifunctional cytokine involved in the regulation of proliferation, differentiation, migration and other functions of many different cell types Three isoforms are present in mammals, i.e. TGFp1, TGFβ2 and TGFβ3, which, to a great extent, show overlapping functions. TGFβ is secreted in a latent form, which needs an activation step mediated by proteases or thrombospondins before it can bind to its specific type I and type Il serin/threonin kinase receptors TGFβ mediates the association of TGFβ receptor type I (TGFβRI) and TGFβRII, which triggers TGFβRII to phosphorylate and activate TGFβRI. Thereby, downstream signaling involving receptor-activated SMAD transcription factors (SMAD2 and SMAD3) become phosphorylated, associate with co-SMADs (SMAD4), and the formed complex moves into the nucleus and associates with transcriptional co- activators or co-repressors Inhibitory SMADs, including SMAD7, induced by TGFβ family ligands and other cytokines function in a negative feedback loop to reduce TGFβ induced signaling
Target mutation of either TGFβ,, TGFβRII or downstream signaling molecules (SMAD3) lead to a severe impairment of immune homeostasis with inflammatory lymphocyte infiltration in multiple organs and auto-antibody production Overexpression of TGFβ has been observed in human malignancy and often correlates with disease progression Mice which lack TGFβ signaling in T cells show a highly impaired anti-tumor response. TGFβ plays a pivotal role in the propagation of wound healing and in the development of excessive fibrosis, as seen in patients with scleroderma, in glomerular scarring in glomerulonephritis, or in the fibrosis of the lung as seen in idiopathic pulmonary fibrosis or secondary conditions due to side-effects of drugs such as methotrexate or exposure to inhaled antigens. TGFβ inhibitors are considered for treating such diseases. Indeed, experimental strategies aimed at interfering with the activity of TGFβ have proved therapeutically successful in cancer models and in fibrosis of the lung and kidney. The specific use of TGFβ inhibitors in the treatment of infections in the CNS or in infections of skin ulcers has not been investigated yet.
Infectious diseases of the CNS
This invention focuses on the treatment of infections of the CNS including bacterial meningitis, a disease occurring worldwide (approximately 1.2 million cases a year) with a high mortality rate and incidence of neurologic sequelae. Even in industrial countries, and despite early and intensive treatment with antibacterial antibiotics and intensive care, death occurs in 25% of adults. The major causes of bacterial meningitis in Western Europe and the United States are Streptococcus pneumoniae and Neisseria meningitis. The introduction of Haemophilus influenzae conjugated vaccines provoked a declining incidence of invasive H. influenzae infections. Central in the host response to bacteria are polymorphonuclear leukocytes (PMN), which, in the case of bacterial meningitis, are attracted into the subarachnoid space by chemokines produced by meningeal cells, ependymal cells, and choroids plexus epithelium. The process of migration of PMN is initiated by adhesion of the cells to selectins and other adhesion molecules on endothelial cells of the CNS vasculature.
Besides the elimination of bacteria, PMN and macrophages are also essential in the resolution of fungal infections and protozoan diseases of the CNS. Examples of frequently seen fungal CNS diseases are infections with aspergillus and Candida; and Toxoplasma gondii serves as an example of protozoan CNS infections. These non-bacterial infections of the CNS may typically be present in a subacute fashion with focal neurologic signs such as seizures or altered mental status due to meningoencephalitis. Since the clearance of protozoan or fungal infectious agents depends on the activation of macrophages by T-lymphocytes, infections of the CNS with these pathogens are most frequently seen in T-cell deficiency such as HIV-infections.
Treatment of CNS infections with bacteria, fungus or parasites focuses on the elimination of the infective agent on the one hand, and on the restriction of bystander injury on neurons exerted by the inflammatory response on the other hand. Based on experimental studies, it has been suggested to include anti-inflammatory strategies, namely the administration of corticosteroids, of inhibitors of apoptosis, of radical scavengers, glutamate antagonists, inhibitors of NFkB, TNFα and IL-1 converting enzymes, endothelin and MMP, and to use antibodies to CD18, which inhibit leukocyte function and downregulate the host response to infections (see e g in R Nau and W Bruck, Trends in Neurosci 25 38-45, 2002)
Skin Ulcers
Skin ulcers are common and cause a substantial burden to the patient The major causes of skin ulcers comprise arteriosclerosis, postthrombotic venous insufficiency, diabetes mellitus, and chronic renal insufficiency In a given patient a combination of these risk factors is seen Given the chronicity of the aforementioned diseases also the skin ulcers are very often chronic, and their complete healing difficult to achieve The major complication of skin ulcers is their infection Despite intensive antibiotic treatment and adequate debridement, amputation of the affected foot is often required Local immunosuppression which is due to poor invasion of PMN into the skin ulcers and defective bacterial clearance of PMN in patients with diabetes provide the basis for their impaired elimination of bacteria and the risk of chronicity of the infection, which counteracts the healing process
While the host response to infection is very efficient in the normal skin, this is not the case in skin ulcers in vascular diseases and metabolic disorders In these circumstances local immunosuppression is seen in the skin, the low capacity of PMN migration and function resembling the one seen in CNS infections such as infective meningitis and meningoencephalomyelitis
Similarities in CNS infections and skin ulcers
Elimination of pathogens require leukocytes to migrate to the site of infection and eliminate the pathogen This process is hindered in the CNS because the blood brain barrier (BBB) prevents the easy travel from the blood to the CNS parenchyma In the skin of patients with arteriosclerosis, venous insufficiency, diabetes mellitus and renal insufficiency, migration of leukocytes into chronic ulcerations are hindered because of angiopathy and scarring of the lesions
Summary of the invention
The present invention relates to a method of treating infectious CNS diseases and infections of skin ulcers comprising administering a TGFβ blocker, and the use of such blockers in said treatment and in the manufacture of medicaments for treating infectious CNS diseases and infections of ulcers of the skin.
The invention further relates to a method of screening for a compound effective in the treatment of infectious CNS diseases and/or infections of ulcers of the skin comprising contacting a candidate compound with a TGFβ or a TGFβ receptor, and choosing candidate compounds which selectively reduce activity of TGFβ or the TGFβ receptor . The invention further relates to compounds selected by these methods of screening.
Brief description of the Figures
Fig. 1: Generation of mice with phagocyte-specific deletion of TGFβ receptor II.
A) Breeding scheme: TGFβRllflox/flox mice were crossed with LysMcre mice in order to obtain phagocyte-specific TGFβRII deficiency. B) Detection of deletion by RT-PCR: Deletion of exon 4 in total RNA of FACS-sorted
Gr-1high, CD11 b+ phag-TGFβRII-/- neutrophils shown by RT-PCR analysis. The primers SEQ ID NO:6 and SEQ ID NO:7 were used to verify the deletion of exon 4.
Fig. 2: Deletion of TGFβRII in phagocytes determined by Taqman real-time PCR. A) The presence of exon 3;4 and 6;7 was measured in total RNA of phag-TGFβRII-/- and control mice. Expression of exon 3;4 and 6;7 was normalized to 18S RNA, and the ratio of exon 3;4 to 6;7 in TGFβRllflox/flox mice was set as 100%.
B) Low level of expression of runx3, a TGFβ-induced gene in PMN of phag-TGFβRII-/- mice. Runx3 was normalized to 18S RNA. C) TGFβ inhibits the LPS induced TNFα secretion in macrophages of TGFβRllflox/flox mice, but not in macrophages of phag-TGFβRII-/- mice. Macrophages were pre-stimulated for 2 h with TGFβ followed by a 6 h incubation period with 0.01 ng LPS/ml. TNFα content was determined by ELISA.
Fig. 3: Deletion of TGFβRII in phagocytes results in increased numbers of leukocytes in the CNS and more efficient bacterial killing. Lane a: uninfected TGFβRllflox/flox mice Lane b: infected TGFβRllflox/flox mice Lane c: infected phag-TGFβRII-/- mice In phag-TGFBRII-/- mice infected with S pneumoniae and treated with ceftriaxone, recruitment of leukocytes into the cerebrospinal fluid (CSF) is much more efficient than in ceftriaxone-treated TGFβRllflox/flox (A, y-axis CSF leukocyte count, n cells per μl) The higher CSF leukocyte numbers are associated with reduced cerebellar bacterial titers, indicating an improved clearance of bacteria in phag-TGFβRII-/- mice (B, y-axis cerebellar bacterial titers, log cfu per organ)
*p<0 05, compared to ceftriaxone-treated TGFβRllflox/flox #p<0 05, compared to uninfected TGFβRllflox/flox controls Data are expressed as means±SDV
Fig 4 Deletion of TGFβRII in phagocytes prevents intracranial hemorrhages in bactenal meningitis
Representative images of brain cryosections obtained from an uninfected TGFβRllflox/flox (a), an infected TGFβRllflox/flox (b), and an infected phag-TGFβRII-/- mouse (c) In phag- TGFβRII-/- mice, meningitis-induced intracerebral hemorrhages are only barely detectable in comparison to TGFβRllflox/flox mice Both infected mouse strains showed an enlargement of the lateral ventricles (hydrocephalus), compared to uninfected TGFβRllflox/flox controls
Fig 5 Deletion of TGFβRII in phagocytes lowers secondary intracranial complications in bacterial meningitis
Lane a uninfected TGFβRllflox/flox mice
Lane b infected TGFβRllflox/flox mice
Lane c infected phag-TGFβRII-/- mice
Meningitis-associated intracranial complications including a rise in intracranial pressure (A, y-axis intracranial pressure, mm Hg) and vasogenic brain edema (B, brain albumin content, ng per μg protein), as assessed by albumin content in the CNS, are less pronounced in infected ceftriaxone-treated phag-TGFβRII-/- mice than in the corresponding TGFβRllflox/flox
*p<0 05, compared to ceftriaxone-treated TGFβRllflox/flox #p<0 05, compared to uninfected TGFβRllflox/flox controls Data are expressed as means±SDV
Fig 6 While TG Fβ is a chemoattractant for neutrophils, pretreatment of the cells with the cytokine reduces their chemotaxis
Migration of thioglycollate-elicited PMN of TGFβRllflox/flox mice (closed bars), and of phag-TGFβRII-/- mice (grey bars) in response to TGFβ Human rTGFβi was added at various concentrations to the lower chamber of Transwell plates; PMN were added in the upper chamber and incubated for 1 h. Migrated cells were counted in five high power fields, and average values are depicted, n = number of migrated cells.
Detailed description of the invention
The present invention relates to a method of treating infectious CNS diseases and infections of ulcers comprising administering a TGFβ blocker, and the use of such blockers in said treatment and in the manufacture of medicaments for treating infectious CNS diseases and infections of ulcers of the skin. TGFβ blockers diminish or prevent TGFβ receptor signaling in PMN and macrophages. By this approach, PMN and monocytes-macrophages are recruited more efficiently from the blood to the CNS or the skin, and become more potent phagocytic cells. These effects result in improved elimination of bacteria, fungus and parasites causing meningitis or meningoencephalitis or encephalitis, and decrease injury of the CNS. Furthermore, the effect supports clearance of pathogens in infected skin ulcers, e.g. in diabetes.
Infectious CNS diseases considered are, for example, bacterial meningitis and CNS infections with fungus or protozoan pathogens such as infections with aspergillus, Candida and Toxoplasma gondii. Infections of ulcers considered are, for example, such infections in patients suffering of arteriosclerosis, postthrombotic venous insufficiency, diabetes mellitus, and chronic renal insufficiency.
TGFβ blockers are compounds which inhibit the production of TGFβ or the activation of TGFβ from its latent form, inhibit the production or the activation of TGFβ receptors, partly or entirely inactivate active TGFβ, inhibit binding of TGFβ to its receptors, or inhibit the TGFβ mediated receptor downstream signaling pathway.
TGFβ production can be inhibited by anti-sense oligodeoxynucleotides or siRNAs to the three TGFβ isoforms. Prevention of the formation of active TGFβ can be achieved by targeting thrombospondin (TSP1 and/or TSP2) with anti-thrombospondin antibodies or with inhibitors which interfere with the synthesis of thrombospondins. Thrombospondins are matricellular glycoproteins, which activate latent TGFβ. The active form of TGFβ can also be inhibited by polypeptides and fragments thereof, which are related to the human TGFβ latent binding protein family, and thereby bind to the active TGFβ protein. Examples of such compounds considered as TGFβ blockers according to the invention are described in WO 00/12551 (Eh Lilly). A further example of a compound considered as TGFβ blocker according to the invention is a binding composition which specifically and/or selectively binds TGFβi, TGFβ2 and/or TGFβ3, and which neutralizes TGFβi , i.e. a compound comprising antigen binding sites of anti-TGFβ Fabs as described in WO 2005/010049 (Eh LiIIy)
Targeting of the TGFβ pathway can be achieved by the administration of neutralizing antibodies to TGFβ or TGFβ receptors or by the therapeutic use of proteins or synthetic compounds, which bind TGFβ, and thereby prevent its binding to TGFβ receptors The following antibodies have been described and are considered as TGFβ blockers according to the invention Metelimumat, a monoclonal antibody to TGFβi , which inhibits the function of TGFβi (from Cambridge Antibody Technology); GC-1008, a humanized monoclonal antibodies to TGFβi, TGFβ2 and TGFβ3 (from Cambridge Antibody Technology), Lerdelimumat, a monoclonal antibody to TGFβi (from Cambridge Antibody Technology, Cordeiro M F , Curr Opin MoI Ther 5 199-203, 2003) Antibodies to TGFβ have been used successfully in animal studies to lower scarring in experimental glomerulonephritis (W.A Border et al , Nature 360 361-364, 1992) Alternatively, peptides comprising parts of TGFβ can be used as TGFβ blockers according to the invention, for example the peptides comprising amino acids 49-58 of TGFβ2, amino acids 41-65 of
TGFβi , or amino acids 41-65 of TGFβ3, since they are capable of blocking TGFβ effects (US 6'500'920, Univ of St Louis) Further examples of TGFβ blockers according to the invention are compounds which by binding to TGFβ interfere with TGFβ receptor activation, such as proteins of the family of small leuciπe-πch proteoglycans, which have a high affinity for TGFβ One small leucine-rich proteoglycan is decorin which is composed of repeated motifs with leucine residues flanked by cysteine-rich regions. Decorin transgene expression in vivo inhibits TGFβ induced pulmonary fibrosis Small leucine-rich proteoglycans which can be used to bind and neutralize TGFβ also include other proteins such as biglycan, fibromoduhn and lumican Compositions of these proteins are provided by Alcon Lab lnc , and are examples of TGFβ blockers according to the invention
On the level of the membrane receptor, TGFβ binding can be inhibited with soluble TGFβ receptors An example of such TGFβ blockers according to the invention are the soluble TGFβ type III receptor fusion proteins comprising all or a portion of the Fc tail of human IgG covalently linked to all or an active portion of a splice variant of the extracellular domain of human TGFβ or activin type Il or type Il B-receptor (WO 2005/028517, Gen Hospital Corp.) Interference with TGFβ mediated signaling is achieved with (1) the overexpression of SMAD7 by virus-mediated gene transfer, which results in the inhibition of the TGFβ receptor mediated phosphorylation of SMAD transcription factors required for the transcription of TGFβ regulated genes, and (2) small organic compounds, which block TGFβ receptor kinases, and thereby activate of SMAD proteins. These small organic compounds, which are considered TGFβ blockers according to the invention, comprise novel pyrazole compounds and related dihydropyrrolo pyrazoles, and are highly potent inhibitors of the TGFβ receptor kinase by binding to the ATP-binding pocket of the kinase. Such compounds with a high degree of selectivity for the TGFβ receptor, and their in vivo application are described by S -B Peng et al , Biochemistry 44 2293-2304, 2005. Another example of a TGFβ blocker according to the invention, a further TGFβ receptor kinase blocker, is Ly 2157299 from EIi Lilly & Company, which modulates TGFβ Rl function by inhibiting SMAD2 phosphorylation. Other compounds considered as TGFβ blockers according to the invention, which interfere with TGFβ receptor signaling, include
(1) quinazoline derivatives (WO 2004/081009, Millennium Pharmaceuticals lnc );
(2) 4-(pyridιn-4-ylamino)-quinoline derivatives (WO 2004/112710, Millennium Pharmaceuticals Inc.), (3) Thiazole derivatives (WO 02/62793, Glaxo Group Ltd.); (4) SB-4314542, a small molecular inhibitor of the TGFβ receptor type I which blocks phosphorylation and nuclear translocation of SMAD proteins (J Rich, Duke University Medical Center), (5) HTS466284, a small molecule inhibitor of the TGFβ receptor type I which inhibits downstream signaling events (E T Akporiaye and M Rausch, Department of Microbiology and Immunology, University of Arizona), (6) N-acetyl-seryl-aspartyl-lysyl- proline (Ac-SDKP) which inhibits effects of TGFβ by interfering with nuclear translocation of SMAD proteins (described by K. Kanasaki et al., J. Am. Soc. Nephrol 14: 863-872, 2003)
Gene therapy can make use of vectors harboring cDNA, which encode for proteins encoding for TGFβ or TGFβ receptor genes or for genes involved in the activation or inactivation of TGFβ or of TGFβ receptors or which encode genes mediating TGFβ receptor signaling An example of the latter are vectors which lead to overexpression of inhibitory SMAD proteins such as SMAD7, and are likewise considered TGFβ blockers according to the invention TGFβ blockers according to the invention are also widely known TGFβ inhibitors, for example, as disclosed in Patent Applications WO 92/00330, WO 99/48904, WO 99/50296, WO 00/12551, WO 02/26935, WO 02/62793, WO 2004/010929, WO 2004/056352, WO 2004/081009, WO 2004/112710, WO 2005/010049, WO 2005/019422, WO 2005/023248, WO 2005/028517, WO 2005/039570, US 6'500'920, US 6'806'358, US 6'841 '542 and JP 08333249, which are incorporated herewith by reference However, the invention is not restricted to the TGFβ inhibitors disclosed therein, but extends to all TGFβ inhibitors
Preferred TGFβ blockers according to the invention are
A TGFβ binding proteins, TGFβ peptides and antibodies to TGFβ and TGFβ receptors GC-1008, a humanized monoclonal antibody to TGFβi, TGFβ2 and TGFβ3 (Cambridge Antibody Technology), Lerdehmumat, a monoclonal antibody to TGFβi (Cambridge Antibody Technology), SB-4314542, a small molecular inhibitor of the TGFβ receptor type I which blocks phosphorylation and nuclear translocation of SMAD proteins (J Rich, Duke University Medical Center, Hjelmeland M D et al , MoI Cancer Ther 3 737-745, 2004, Boyer A N and M Korc, J Biol Chem 280 21858-21866, 2005), a binding composition (1021 , 2471, and 3821), which specifically and/or selectively binds TGFβi , TGFβ2 and/or TGFβ3, and which neutralizes TGFβi , comprising antigen binding sites of anti-TGFβ Fabs designed 1021 , 2471 and 3821 (WO 2005/010049, Eh Lilly), peptides comprising amino acids 49-58 of TGFβ2, amino acids 41-65 of TGFβi , or amino acids 41-65 of TGFβ3, capable of blocking TGFβ effects (US 6'500'920, Univ St Louis), and small leucine-πch proteoglycans, which can be used to bind and neutralize TGFβ, including decorin, biglycan, fibromodulin and lumican (Alcon Lab lnc )
B Soluble TGFβ receptors
Soluble TGFβ type III receptor fusion proteins comprising all or a portion of the Fc tail of human IgG covalently linked to all or an active portion of a splice variant of the extracellular domain of human TGFβ or activin type Il or type Il B-receptor (WO 2005/028517, Gen Hospital Corp )
C Blockers of the TGFβ receptor kinase
Pyrazole compounds and related dehydropyrrolo-pyrazoles (S -B Peng et al , Biochemistry 44 2293-2304, 2005, EIi Lilly & Company), the TGFβ receptor kinase blocker Ly 2157299 (from EIi Lilly & Company); quinazoline derivatives (WO 2004/081009, Millennium Pharmaceuticals Inc.);
4-(pyridin-4-ylamino) quinoline derivatives (WO 2004/112710, Millennium
Pharmaceuticals Inc.); thiazole derivatives (WO 02/62793, Glaxo Group Ltd.);
SB-4314542, a small molecular inhibitor of the TGFβ receptor type I which blocks phosphorylation and nuclear translocation of SMAD proteins (J. Rich, Duke University
Medical Center);
HTS466284, a small molecule inhibitor of the TGFβ receptor type I which inhibits downstream signaling events (ET. Akporiaye and M. Rausch, Department of Microbiology and Immunology, University of Arizona); and
N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which inhibits effects of TGFβ by interfering with nuclear translocation of SMAD proteins (K. Kanasaki et al., J. Am. Soc.
Nephrol. 14: 863-872, 2003).
D: Other compounds which inhibit TGFβ receptor mediated signaling
A cytoxazon derivate which inhibits the TGFβ signal transduction pathway (WO
2005/039570, RIKEN KK).
Nitrogen-containing heterocyclic compounds and other aromatic organic compounds (WO 2004/056352, Scios Inc.)
E: Other compounds
Substituted p-benzoyloxy- or p-phenyloxycarbonyl-phenylamidines und -guanines, inhibitors for release, activation and synthesis of TGFβ (JP 08333249, ONO Pharma Co.).
F: TGFβ and TGFβ receptor gene targeting
Small interfering ribonucleic acid (siRNA) molecules which reduce expression of the TGFβ type Il receptor (WO 2005/019422, Univ. of Illinois Found.);
TGFβi and TGFβ2 antisense constructs (from Antisense Pharma GmbH); and RNA ligand TGFβi selected from a group of 137 sequences identified by the SELEX
Method (WO 99/489004, Nexstar Pharma Inc.).
G: Overexpression of TGFβ inhibitory proteins and of proteins which interfere with the intracellular TGFβ signaling cascade SMAD6 and SMAD7 nucleic acids and polypeptides (WO 99/50296, EIi Lilly); and vectors comprising nucleic acids which encode the hLTBP-3 polypeptide (WO 00/12551 , EIi Lilly & Company).
Particularly preferred TGFβ blockers according to the invention are: - antibodies to TGFβ and TGFβ receptors;
- the TGFβ receptor kinase blocker Ly 2157299 (EIi Lilly);
- Pyrazole compounds and related dehydropyrrolo-pyrazoles (S. -B. Peng et al., Biochemistry 44: 2293-2304, 2005, EIi Lilly);
- quinazoline derivatives (WO 2004/081009, Millennium Pharmaceuticals Inc.); - 4-(pyridin-4-ylamino) quinoline derivatives (WO 2004/112710, Millennium Pharm. Inc.);
- decorin (Alcon Lab Inc.);
- thiazole derivatives (WO 02/62793, Glaxo Group Ltd.);
- a cytoxazon derivate which inhibits the TGFβ signal transduction pathway (WO 2005/039570, RIKEN KK); and - nitrogen-containing heterocyclic compounds and other aromatic organic compounds (WO 2004/056352, Scios Inc.).
Most preferred TGFβ blockers according to the invention are:
- antibodies to TGFβ and TGFβ receptors; - the TGFβ receptor kinase blocker Ly 2157299 (EIi Lilly); and
- decorin (Alcon Lab Inc.).
One aspect of the invention relates to a method of treating infectious diseases of the CNS or infected skin ulcers comprising administering TGFβ blockers as defined hereinbefore in a quantity effective against the CNS or skin disease to a mammal in need thereof, for example to a human requiring such treatment. The treatment may be for prophylactic or therapeutic purposes. For the administration, the TGFβ blocker is preferably in the form of a pharmaceutical preparation comprising the TGFβ blocker in chemically pure form and optionally a pharmaceutically acceptable carrier and optionally adjuvants. The TGFβ blocker is used in an amount effective against the infectious CNS disease or the infected skin ulcer. The dosage of the active ingredient depends upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, the mode of administration, and whether the administration is for prophylactic or therapeutic purposes. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 1 mg to approximately 500 mg, preferably from approximately 10 mg to approximately 100 mg, of a TGFβ inhibitor or TGFβ receptor inhibitor
Pharmaceutical compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as subcutaneous, intravenous, intramuscular or injections into the cerebrospinal fluid (CSF) compartment are especially preferred. The pharmaceutical compositions comprise from approximately 1% to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient
For parenteral administration including injections into the CSF preference is given to the use of solutions of the TGFβ blockers, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example, can be made up shortly before use The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known perse, for example by means of conventional dissolving and lyophilizing processes.
For oral pharmaceutical preparations suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, and also binders, such as starches, cellulose derivatives and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, flow conditioners and lubricants, for example stearic acid or salts thereof and/or polyethylene glycol Tablet cores can be provided with suitable, optionally enteric, coatings Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The capsules may contain the active ingredient in the form of granules, or dissolved or suspended in suitable liquid excipients, such as in oils
Transdermal application is also considered, for example using a transdermal patch, which allows administration over an extended period of time, e g. from one to twenty days. Another aspect of the invention relates to the use of TGFβ blockers as described hereinbefore in the treatment of infectious CNS diseases and infected skin ulcers and in the manufacture of medicaments for treating these diseases. Such medicaments are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing.
The TGFβ blocker can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of fixed combinations of a TGFβ blocker and one or more other therapeutic agents known in the treatment of infectious CNS diseases and infected skin ulcers, the administration being staggered or given independently of one another, or being in the form of a fixed combination.
Possible combination partners considered are anti-infective compounds.
The invention further relates to a method of screening for a compound effective in the treatment of infectious CNS diseases and infected skin ulcers comprising contacting a candidate compound with a TGFβ or a TGFβ receptor and choosing candidate compounds which selectively reduce the activity of TGFβ. The invention further relates to compounds selected by these methods of screening.
Inhibitors of TGFβ activity are identified by contacting a TGFβ or a TGFβ receptor with a candidate compound. A control assay with the corresponding TGFβ or TGFβ receptor — in the absence of the candidate compound - is run in parallel. A decrease in activity in the presence of the candidate compound compared to the level in the absence of the compound indicates that the compound is a TGFβ inhibitor.
Screening systems to be used for identification of candidate compounds comprise in vitro studies using TGFβ as a cytokine which induces chemotaxis of leukocytes. Compounds to be tested for TGFβ blocking activity will be added together with TGFβ in the chemotactic assays. The screen can also be done in vivo by injection of S. pneumoniae type 3 (107 colony forming units) into the cisterna magna of normal wild type mice treated with ceftriaxone (100 mg/kg intraperitoneal^). Candidate compounds will be injected intravenously or into the cisterna magna of infected mice. Efficiency of the candidate compounds will be evaluated by following the clinical course of bacterial meningitis of infected mice and by analyzing their cerebrospinal fluid (number of leukocytes and bacteria; albumin concentration) and by counting the number of intracerebral hemorrhages.
Concepts and Evidence behind the Invention Elimination of pathogens require leukocytes to migrate to the site of infection and eliminate the pathogen. This process is hindered in the CNS because the blood brain barrier (BBB) prevents the easy travel from the blood to the CNS parenchyma. In the skin of patients with arteriosclerosis, venous insufficiency, diabetes mellitus and renal insufficiency, migration of leukocytes into chronic ulcerations are hindered because of angiopathy and scarring of the lesions. The data provided hereinbelow strongly support the concept that TGFβ suppresses the migration of leukocytes much rather than, as commonly believed, it provokes leukocyte chemotaxis.
In the migration and chemotaxis of phagocytes, TGFβ exerts potent chemotactic activity on PMN and monocytes in vitro and, upon injection into joints, induces a synovial inflammatory reaction (J. B. Allen et al., J. Exp. Med. 171:231 , 1990). When testing TGFβ on PMN chemotaxis in vitro, the reports on the chemotactic property of TGFβ can be reproduced (Fig. 6). This contrasts, however, with the observation in vivo when using mice lacking TGFβ receptor mediated signaling in PMN and macrophages. These mice were generated by crossing floxed TGFβRII mice with lys-CRE mice (Fig. 1). Mice with impaired expression of TGFβRII are termed phag-TGFβRII-/- and are shown to be defective in the response of activated macrophages and PMN to TGFβ (Fig. 2 and 6). TGFβ fails to exert chemotactic responses in vitro on PMN derived from TGFβRII-/- mice (Fig. 6).
The basis of the invention is the observation that in TGFβRII-/- mice infected with S. pneumoniae and treated with antibiotics, recruitment of PMN and macrophages into the CNS is much more efficient than in antibiotic treated control mice with intact TGFβ signaling (Fig. 3). PMN from phag-TGFβRII-/- mice are not hindered by TGFβ from being attracted in vitro or in vivo into the CNS in S. pneumoniae mediated meningitis (Fig. 3). Higher numbers of phagocytes in the CNS are associated with improved clearance of bacteria in the CNS (Fig. 3). As a consequence, secondary CNS damage including vasculitis and cerebral hemorrhages are prevented (Fig. 4), and the increase in intracerebral pressure and albumin concentration is much lower in phag-TGFβRII-/- mice compared to control mice (Fig. 5). Clinical scores which correlate with the number of intracerebral hemorrhages and the extent of elevation of the intracerebral pressure is significantly better in phag-TGFβRII-/- mice than in control mice (Fig. 4 and 5).
The experimental results show that by blocking TGFβ receptor signaling in phagocytes, clearance of bacteria is highly improved in mice with experimental bacterial meningitis, and thereby secondary brain damage is dramatically reduced. The basic mechanism behind these findings is that in contrast to common belief TGFβ produced in vivo does not improve, but rather suppresses the migration of phagocytes induced by chemokines or bacterial cell wall products.
In the light of the new finding of PMN to be less responsive to the effects of chemotactic factors in vivo, the principle of the present invention also applies to infected skin ulcers, e g. seen in patients with diabetes mellitus. In infected skin ulcers, the expression of TGFβ hinders efficient PMN migration to the site of infection and bacterial killing. By blocking the action of TGFβ locally in the skin the host response to infections in leg ulcers can be improved.
These findings show that TGFβ is a major risk factor for the impaired host response to infections and therefore blocking its action is helpful to treat skin ulcers and CNS infections.
Examples
Generation of phag-TGFβRII-/- mice TGFβRllflox/flox mice were obtained from Stefan Karlsson and Per Leveen, Lund University, Sweden (P. Leveen et al., Blood 100(2): 560-8, 2002), and LysMcre were provided by lrmgard Fδrster, University of Dϋsseldorf, Germany and are available at The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609 USA. The Cre-bearing deleter mice express cre-recombinase under control of the murine lysozyme genes in macrophages and PMN (B. E. Clausen et al., Transgenic Research 8: 265-277, 1999). The cre-recombinase binds cooperatively to 34 bp recombination sequences termed locus of crossing over in phage P1 (loxP) which have been introduced in exon 4 of the TGFβ receptor gene. These mice are termed TGFβRllflox/flox (P. Leveen et al., loc.cit.). The TGFβRllflox/flox mice were crossed with LysMcre to yield a homozygous TGFβRllflox/flox background and heterozygous background for LysMcre. Cre non-expressing littermates were used as controls. The genotype of the off-springs was determined by PCR using the primer pair P3/P4 (P3: 51-TATGGACTGGCTGCTTTTGTATTC-31 (SEQ ID NO:1) and P4: 51-TGGGGATAGAGGTAGAAAGACATA-3• (SEQ ID NO:2)) to distinguish floxed alleles (575 bp) from wild type (422 bp) (P. Leveen et al., loc.cit.). The primer combinations Cre8/ Mlys1/Mlys2 (Cre8: δ'-CCCAGAAATGCCAGATTACG-S' (SEQ ID NO:3); Mlysi: 5'-CTTGGGCTGCCAGAATTTCTC-3' (SEQ ID NO:4); Mlys2: 5'- TTACAGTCGGCCAGGCTGAC-3' (SEQ ID NO:5)) result in LysMcre amplicons of 700 bp and 1700 bp and in wild type amplicons of 350 bp. The mice were bred under specific pathogen-free (SPF) conditions with screening of ubiquituous pathogens during the breeding of the animals.
RNA Analysis
RNA was isolated from PMN by TRIzol (Invitrogen, Life Technologies) and reverse transcribed by M-MuLV reverse transcriptase (Roche, Rotkreuz, Switzerland). For Taqman real-time PCR, the Applied Biosystems assays-on-demand for runx3
(Mm00490666_m1), TGFβRII exon boundary 3/4 (Mm01348770_m1) and TGFβRII exon boundary 6/7 (Mm00436978_m1) were used. Detection of TGFβRII mRNA was performed by using the forward primer P1: 5'-ACATTACTCTGGAGACGGTTTG-S' (SEQ ID NO:6) and the reverse primer P2: δ'-GGTAGTGTTCAGCGAGCCATCTT-S' (SEQ ID NO:7). All real-time PCR reactions were performed and analysed on an ABI Prism 7700 Sequence Detection System™ (Perkin Elmer Applied Biosystems). The reactions for the target and the endogenous control (18s rRNA, Applied Biosystems, Nr. 4310893) were performed in separate tubes, and the comparative CT method was used for standardization.
Mouse meningitis model
Meningitis was induced by transcutaneous injection of 15 μl of a bacterial suspension containing 107 colony forming units (cfu) per ml of Streptococcus pneumoniae type 3 into the cisterna magna under short-term anesthesia with halothane. Mice were clinically examined, weighed, put into cages, and allowed to wake up. The clinical score comprises the following criteria: a) presence of tremor, piloerection, and seizures; b) spontaneous motor activity; c) vigilance; d) body proprioception; e) a beam balancing test; and f) a postural reflex test. In healthy animals, the score was 0, infected animals that died within the observation period received 16 points. 24 hours after infection, mice were evaluated clinically and treated with the antibiotic ceftriaxone (100 mg/kg intraperitoneally). 48 hours after infection, mice were again clinically evaluated, re-weighed, and the body temperature was measured via a rectal probe. Then, mice were anaesthetized with 100 mg/kg ketamine and 5 mg/kg xylazine. Subsequently, a catheter was inserted into the cisterna magna to measure intracranial pressure (ICP) and to de-termine CSF leukocyte counts. Subsequently, blood samples were taken by transcardial puncture. After deep anesthesia with ketamine, mice were perfused transcardially with 15 ml of ice-cold phosphate-buffered saline (PBS) containing 10 U/ml heparin. The brains were removed and rapidly frozen.
Cerebellar and blood bacterial titers
Cerebella were dissected and homogenized in sterile saline. Cerebellar homogenates were diluted serially in sterile saline, plated on blood agar plates, and cultured for 24 h at 37°C with 5% CO2.
Determination of the blood-brain barrier (BBB) integrity
To assess BBB integrity, mouse brain homogenates were examined for infiltration by albumin, an abundant serum protein that is normally excluded from the brain by the intact BBB, using ELISA. Maxisorb plates (Nunc, Wiesbaden, Germany) were coated and incubated for 60 min at room temperature (RT) with a mouse albumin specific rabbit polyclonal antibody (Acris, Bad Nauheim, Germany), diluted in coating buffer (0.05 M sodium carbonate, pH=9.6) to a concentration of 0.5 μg/ml. Plates were washed with washing buffer (50 mM Tris, 0.14 M NaCI, 0.05% Tween 20, pH=8.0) and blocked using blocking buffer (50 mM Tris, 0.14 M NaCI, 1% BSA, pH=8.0). Mouse brain protein extracts diluted in lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCI, 1.5 mM MgCI2, and a mixture of protease inhibitors including phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin A; 0.5 μg protein per well) were transferred to assigned wells, and plates were incubated for 60 min at RT. Bound albumin was detected using a goat polyclonal peroxidase- conjugated anti-mouse albumin anti-body, diluted in sample conjugate buffer (50 mM Tris, 0.14 M NaCI, 1% BSA, 0.05% Tween 20, pH=8.0) to a concentration of 0.1 μg/ml. Plates were incubated for 60 min at RT. Enzyme substrate reagent (R&D Systems, Wiesbaden, Germany) was added to the wells and incubated for 10 min at RT. The colorimetric reaction was stopped by adding 2 M sulfuric acid and absorbance was read at 450 nm.
Analysis of cerebral bleeding and hydrocephalus
Mice brains were cut in a frontal plane into 10 μm thick sections. Beginning from the anterior parts of the lateral ventricles, 9 serial sections were photographed with a digital camera in 0.3 mm intervals throughout the ventricle system. The areas of the lateral ventricles and the third ventricle were measured (Image tool, UTHSCSA, Texas, USA) and the volume of the ventricle size was estimated (ventricle area / 9 pictures x 0.3 mm). Haemorrhagic spots were counted and the bleeding area was measured.
Histology Cryosections of mice brains were HE and hemacolor stained. In addition, selected brains were formaline fixed, paraffin embedded, and stained with HE and hemacolor.
Analysis of chemotaxis of PMN
Thioglycollate-elicited peritoneal exudate cells (PEC) were recovered 20 h (PMN) or 2 days (macrophages) after injection of 1 ml 3% Brewer thioglycollate medium (Sigma, Buchs, Switzerland). The peritoneal cavity was flushed with 10 ml of HBSS (without Ca, Mg) / 1% BSA, 15 mM EDTA, the cells were collected by centrifugation at 1200 rpm and resuspended to 1x106 cells/ml X-Vivo 15 medium (BioWhittaker, Cambrex, Belgium) / 2 mM glutamine. FACS-sorting: Thioglycollate-elicited peritoneal exudate cells were resuspended in FACS buffer (2% FCS, 5 mM EDTA, in PBS) and incubated with anti- mouse CD16/32 (Fc-block from BD Pharmingen) for 5 min, then stained with anti-Gr-1 FITC (BD Pharmingen) and anti-mouse CD11b PE (BD Pharmingen) for 20 min. The Gr-1high/CD11b+ cells were sorted by an FACStar Plus (Becton Dickinson). To assess chemotaxis of PMN, the chemoattractants fmlp (Sigma, Buchs, Switzerland), and human rTGFβi (R&D Systems, Minneapolis, MN) were diluted in X- Vivo 15 medium / 2 mM glutamine / G-CSF (10 ng/ml) at the indicated concentrations and transferred into the lower chamber of Transwell plates (Corning-Costar). Thioglycollate-elicited peritoneal exudate cells were recovered after 20 h and seeded into the upper chamber of Transwell plates (3 mm pore size, 105 cells/insert in 100 ml X-Vivo 15 medium / 2 mM glutamine/G- CSF (10 ng/ml)). The plates were incubated at 37°C and 5% CO2 for 1 h. At the end of the incubation, the remaining cells on the upper membrane surface were carefully removed with a cotton swab. Migratory cells attached on the lower part of the filter were stained with DAPI (Molecular Probe, Netherlands) at 1 μM dilution for 20 min at 37°C and thereafter fixed in 4% paraformaldehyde in PBS. Migrated neutrophils were counted with a square graticule (Leica Microsystems Wetzlar GmbH, Germany) in five visual fields per filter (three horizontal and two vertical fields crossing the middle of the filter) at 20Ox magnitude.
Detection of cytokines TNFα synthesis by macrophages: Thioglycollate-elicited PEC were seeded at a density of 5x105 cells/well in a 24-well tissue culture plate in DMEM / 10% FCS / 2 mM glutamine. After 24 h, the medium was changed to X-Vivo 15 / 2 rtiM glutamine and the macrophages were incubated over night at 37°C, 5% CO2. The cells were prestimulated with 20 ng/ml TGFβi (R&D Systems) for 2 h, and then 0.01 ng LPS/ml (055:B5; Sigma, Buchs, Switzerland) was added for another 6 h. TNFα content in culture supernatants was measured by a TNFα ELISA Kit (BioSource Europe, Nivelles, Belgium).

Claims

Claims
1. Use of a transforming growth factor beta (TGFβ) blocker in the manufacture of medicaments for treating infectious CNS diseases and infections of ulcers of the skin.
2. Use according to claim 1 wherein the TGFβ blocker diminishes or prevents TGFβ receptor signaling in PMN and macrophages.
3. Use according to claim 1 wherein the infectious CNS disease is bacterial meningitis and CNS infections with aspergillus, Candida and Toxoplasma gondii.
4. Use according to claim 1 wherein the infections of ulcers are such infections in patients suffering of arteriosclerosis, postthrombotic venous insufficiency, diabetes mellitus, and chronic renal insufficiency.
5. Use according to claim 1 wherein the TGFβ blocker is a compound which inhibits the production of TGFβ or the activation of TGFβ from its latent form, inhibits the production or the activation of TGFβ receptors, partly or entirely inactivates active TGFβ, inhibits binding of TGFβ to its receptors, or inhibits the TGFβ mediated receptor downstream signaling pathway.
6. Use according to claim 1 wherein the TGFβ blocker is selected from the group consisting of TGFβ binding proteins, TGFβ peptides and antibodies to TGFβ; soluble TGFβ receptors; blockers of the TGFβ receptor kinase; a cytoxazon derivate which inhibits the TGFβ signal transduction pathway; compounds targeting TGFβ and TGFβ receptor gene; and compounds causing overexpression of TGFβ inhibitory proteins and of proteins which interfere with the intracellular TGFβ signaling cascade.
7. Use according to claim 1 wherein the TGFβ blocker is selected from the group consisting of
GC-1008, a humanized monoclonal antibodies to TGFβi , TGFβ2 and TGFβ3; Lerdelimumat, a monoclonal antibody to TGFβi ;
SB-4314542, a small molecular inhibitor of the TGFβ receptor type I which blocks phosphorylation and nuclear translocation of SMAD proteins; a binding composition (1021 , 2471 , and 3821), which specifically and/or selectively binds
TGFβi, TGFβ2 and/or TGFβ3, and which neutralizes TGFβi , comprising antigen binding sites of anti-TGFβ Fabs designed 1021 , 2471 and 3821 ; peptides comprising amino acids 49-58 of TGFβ2, amino acids 41-65 of TGFβi, or amino acids 41-65 of TGFβ3, capable of blocking TGFβ effects; small leucine-rich proteoglycans, which can be used to bind and neutralize TGFβ, including decorin, biglycan, fibromodulin and lumican; soluble TGFβ type III receptor fusion proteins comprising all or a portion of the Fc tail of human IgG covalently linked to all or an active portion of a splice variant of the extracellular domain of human TGFβ or activin type Il or type Il B-receptor; pyrazole compounds and related dehydropyrrolo-pyrazoles blocking the TGFβ receptor kinase; the TGFβ receptor kinase blocker Ly 2157299; quinazoline derivatives blocking the TGFβ receptor kinase; 4-(pyridin-4-ylamino) quinoline derivatives blocking the TGFβ receptor kinase; thiazole derivatives blocking the TGFβ receptor kinase;
SB-4314542, a small molecular inhibitor of the TGFβ receptor type I which blocks phosphorylation and nuclear translocation of SMAD proteins;
HTS466284, a small molecule inhibitor of the TGFβ receptor type I which inhibits downstream signaling events;
N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which inhibits effects of TGFβ by interfering with nuclear translocation of SMAD; a cytoxazon derivate which inhibits the TGFβ signal transduction pathway; nitrogen-containing heterocyclic compounds and other aromatic organic compounds which inhibit the TGFβ signal transduction pathway; substituted p-benzoyloxy- or p-phenyloxycarbonyl-phenylamidines und -guanines, which are inhibitors for release, activation and synthesis of TGFβ; small interfering ribonucleic acid (siRNA) molecules which reduce expression of the TGFβ type II; TGFβi and TGFβ2 antisense constructs; and
RNA ligand TGFβi identified by the SELEX Method.
SMAD6 and SMAD7 nucleic acids and polypeptides; and vectors comprising nucleic acids which encode the hLTBP-3 polypeptide.
8. Use according to claim 1 wherein the TGFβ blocker is selected from the group consisting of antibodies to TGFβ and TGFβ receptors; the TGFβ receptor kinase blocker Ly 2157299; pyrazole compounds and related dehydropyrrolo-pyrazoles blocking the TGFβ receptor kinase; quinazoline derivatives blocking the TGFβ receptor kinase; 4-(pyridin-4-ylamino) quinoline derivatives blocking the TGFβ receptor kinase; decorin; thiazole derivatives blocking the TGFβ receptor kinase; a cytoxazon derivate which inhibits the TGFβ signal transduction pathway; and nitrogen-containing heterocyclic compounds and other aromatic organic compounds which inhibit the TGFβ signal transduction pathway.
9. Use according to claim 1 wherein the TGFβ blocker is selected from the group consisting of antibodies to TGFβ and TGFβ receptors; the TGFβ receptor kinase blocker Ly 2157299; and decorin.
10. A method of treating infectious CNS diseases and infections of skin ulcers comprising administering a TGFβ blocker in an effective amount to a patient in need thereof.
11. A method of screening for a compound effective in the treatment of infectious CNS diseases and/or infections of ulcers of the skin comprising contacting a candidate compound with a TGFβ or a TGFβ receptor, and choosing candidate compounds which selectively reduce activity of TGFβ or the TGFβ receptor.
12. A compound selected by the method of claim 11.
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