EP1187624A1 - Antagonistes cyclises d'interleukine-6 a squelette contrainte par conformation - Google Patents

Antagonistes cyclises d'interleukine-6 a squelette contrainte par conformation

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Publication number
EP1187624A1
EP1187624A1 EP00929763A EP00929763A EP1187624A1 EP 1187624 A1 EP1187624 A1 EP 1187624A1 EP 00929763 A EP00929763 A EP 00929763A EP 00929763 A EP00929763 A EP 00929763A EP 1187624 A1 EP1187624 A1 EP 1187624A1
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European Patent Office
Prior art keywords
arg
lys
phe
leu
amide
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English (en)
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Vered Hornik
Eran Hadas
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Peptor Ltd
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Peptor Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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]
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to conformationaUy constrained bac bone-cyclized IL-6 antagonists, and to pharmaceutical compositions containing same.
  • Interleukin-6 is a member of the helical cytokine family. IL-6 is produced by almost all cell types in response to a variety of different stimuli including bacterial (LPS) and viral infections, cancer, and other cytokines (e.g. IL-1). IL-6 is a pleiotropic factor, it participates in numerous processes and is thus associated with numerous disorders (for a review see Hirano T . , Intern. Rev. Immunol. 16:249, 1998).
  • Bioactivity of IL-6 requires interaction of the cytokine, IL-6, its receptor (IL-6R) and a transmembrane signal transducer known as glycoprotein 130 (gpl30), and formation of a hexameric complex containing two units of each protein.
  • the outcome of the complex formation is dimerization of gpl30, which by itself is sufficient for obtaining IL-6 like bioactivity (Fourcin, et al., J. Biol. Chem. 271: 11756, 1996.).
  • Several other cytokines also use gpl30 for signal transduction. These include: interleukin-11 (IL-11), ciliary neurotrophic factor (CNTF) , leukocyte inhibitory factor (LIF) , oncostatin M (OSM) .
  • IL-11 interleukin-11
  • CNTF ciliary neurotrophic factor
  • LIF leukocyte inhibitory factor
  • OSM oncostatin M
  • IL-6 levels increases early during bacterial and viral infections. IL-6 induces production of acute phase proteins which are thought to participate in the defense of the host organism against tissue damage and infection. The acute phase response is considered to be the systemic inflammatory reaction to infection and injury.
  • IL-6 also amplifies the immune system through its multiple growth and differentiation activities such as induction of B cell differentiation, replication of bone marrow progenitor cells, and augmentation of T lymphocytes, including enhancement of cytotoxic T lymphocytes.
  • IL-6 levels are increased during stress. IL-6 in rabbits is directly responsible for elevation of body temperature. High IL-6 levels in burn patients correlates with mortality. Elevated IL-6 levels are associated with traumatic events and allograft rejection.
  • a massive loss of bone mass is reported in women at there postmenopausal stage. Increased bone resorption and increased osteoclast activity in postmenopausal osteoporosis have been * linked with IL-6.
  • the production of IL-6 is elevated in bone marrow cells at this stage correlating the fact that estrogen down regulates IL-6 gene expression.
  • Estrogen loss induced by ovariectomy in mice enhances osteoclast development and this change can be prevented by antibodies to IL-6.
  • Several experiments including an IL-6 knockout mice model, treatment with anti IL-6 antibodies or with IL-6 antisense demonstrate that elevated levels of IL-6 plays a critical role in the formation of osteoclastic cells. As such, dysregulation of IL-6 activity in bone cells leads to the development of pathological disease .
  • IL-6 in immune disorders Elevated levels of IL-6 in cardiac myxoma and cervical carcinoma are associated with autoimmunity indications such as: production of anti-nuclear factor, rheumatoid factors, elevated immune complexes, arthritis and nephritis.
  • Castelman's disease patients suffer form fever, anemia, hyper- ⁇ -globulinemia, and an increase in acute phase proteins.
  • rheumatoid arthritis such as plasma cell infiltration into synovial tissues, autoantibody production, and polyclonal hyper- ⁇ -globulinemia can be explained by increased IL-6 production observed in synovial tissue.
  • Higher than normal levels of IL-6 have been detected in sera of patients with active SLE. Increased plasma levels of IL-6 were observed in psoriasis patients.
  • IL-6 is a growth factor of the AIDS associated Kaposi's sarcoma (Murakamin-Mori, et al. Int. Immunol. 8: 595, 1996).
  • the soluble form of the IL-6 receptor (sIL-6Ra) is a potent growth factor for AIDS-associated Kaposi's sarcoma (KS) cells.
  • the soluble form of gpl30 is antagonistic for sIL-6Ra-induced AIDS-KS cell growth.
  • high IL-6 levels are associated with weight loss in AIDS.
  • IL-6 An autocrine role for IL-6 has been reported in several types of cancer, among which are renal cell carcinoma, Hodgkin and non-Hodgkin' s lymphoma, chronic lymphocytic leukemia, and acute myeloid leukemias.
  • Plasmacytoma and myeloma cells require IL-6 for growth.
  • Treatment of primary plasma cell leukemia with anti-IL-6 antibodies improves the patient's clinical status throughout the treatment.
  • IL-6 deficient mice are completely resistant to plasmacytoma induction.
  • Multiple myeloma is a malignant proliferation of plasma cells derived from a -single clone. It is manifested in a number of organ dysfunctions and symptoms of bone pain or fractures, hypercalcemia, renal failure, susceptibility to infection, anemia and bleeding. The disease typically follows a chronic course for 2 to 5 years before progressing into an acute terminal phase.
  • IL-6 has also a regulatory role in activation of Matrix Metalloproteinases (MMPs) .
  • MMPs are enzymes that are capable degrading the basement membrane components. As such MMPs are refereed as key enzymes in Extra Cellular Matrix remodeling, tumor invasion and metastasis.
  • IL-6 inhibitors have clinical value. As indicated above there are a number of clinical situations where IL-6 inhibitors could be of therapeutic use. Most of the attempts to produced inhibitors to IL-6 reported in the literature in the past, used proteins. In general, proteins are not very suitable as drugs, due to their immunogenic potential, high cost, and the necessity for parenteral administration. The various attempts to use proteins to inhibit IL-6 are described below.
  • Monoclonal antibodies and antibody fragments Monoclonal antibodies and antibody fragments :
  • mAbs monoclonal antibodies
  • the drawbacks in the use of antibodies against IL-6 are that the mAb traps the IL-6 in an immune complex in the circulation (May et al., J. Immunol. 151, 3225, 1993), thereby increasing its half-life 200-fold (Lu et al., Blood 86, 3123,1995).
  • the immune complexes are thus serve as long term, slow release deposits of IL-6.
  • the presence of high levels of circulating immune complexes could result in their precipitation in the basal lamina in the kidneys or in the joints, which could lead to kidney failure or arthritis.
  • Attempts to block IL-6 with monoclonal antibodies have been reported for the following diseases: AIDS associated syndromes and lymphoma (Emilie, et al .
  • Minibodies which utilize the hypervariable loop structure of antibodies, capable of inhibiting IL-6 bioactivity were recently reported (Martin et al., The EMBO J. 13, 5303, 1994). Binding of IL-6 to a minibody molecule should create a complex that is small enough to be secreted from the kidney, thereby decreasing the risk of creating slow release IL-6 deposits. Minibody-IL-6 complexes may not be recognized as immune complexes, thereby decreasing the chances for kidneys and arthritic problems. The minibodies could still be immunogenic and it is unlikely that they will be orally available. So far, minibodies with sufficiently high affinity for IL-6 have not been obtained.
  • IL-6 mutants were selected for desired activity using phage display systems.
  • Super active mutants were reported (Toniatti, et al . , The EMBO J. 15: 2726, 1996) as well as mutants which retain the capacity for binding of the IL-6R but lose the ability for interaction with the gpl30 and thus could serve as functional antagonists of IL-6 bioactivity (Savino et al., The EMBO J. 13,5863, 1994; Sporeno, et al., Blood 87: 4510, 1996) .
  • the danger in clinical use of such mutants is the formation of antibodies that would recognize both the mutated and the native molecules. Such antibodies could block the bioactivity of IL-6 long after the treatment is terminated thereby exposing the patients to danger associated with lack of IL-6.
  • CTNF and IL-6 antagonists which are heterodimer proteins comprising a soluble specificity determining cytokine receptor component and the extracellular domain of a ⁇ receptor component.
  • the inventors claim an IL-6 antagonist, capable of binding IL-6 to form a nonfunctional complex, comprising: soluble IL-6R and the extracellular domain of gpl30.
  • IL-6 inhibitors entail the elimination of IL-6 dependent tumors, such as multiple myeloma. This goal can be achieved by the use of IL-6-toxin conjugate (Jean and Murphy, Prot. Eng. 4, 989, 1991). Malignant cells that have receptor for IL-6 would bind the toxin via the IL-6 portion of the conjugate and would be eliminated by toxin activity. Toxicity to all non-malignant cells that also express the IL-6 receptor is a dangerous possibility. Since IL-6 is required for development of normal humoral and cellular immune response, it is possible to speculate that treated patients would immunocompromised.
  • Grube and Cochran identified a regulatory domain of the IL-6 receptor (J. Biol. Chem. 269: 20791, 1994) .
  • the region is from the extramembranal domain of the IL-6R and it is involved in the regulation of IL-6 signal transmission.
  • a synthetic peptide, corresponding to residues 249-264 of the IL-6R inhibits IL-6-dependent cell mitogenesis and IL-6-stimulated acute phase response without affecting ligand binding.
  • IL-6 inhibitory activity it is an object of the present invention to provide novel peptide analogs, which are characterized in that they incorporate building units with bridging groups attached to the alpha nitrogens of alpha amino acids, having IL-6 inhibitory activity.
  • these compounds are backbone cyclized IL-6 antagonists comprising a peptide sequence of five to twenty amino acids that incorporates at least one building unit, said building unit containing one nitrogen atom of the peptide backbone connected to a bridging group comprising an amide, thioether, thioester or disulfide, wherein the at least one building unit is connected via said bridging group to form a cyclic structure with a moiety selected from the group consisting of a second building unit, the side chain of an amino acid residue of the sequence or the N-terminal amino acid residue.
  • the peptide sequence incorporates six to twelve amino acids, having IL-6 inhibitory activity.
  • Bioactivity of IL-6 requires each of the molecules in the tripartite complex, i.e. IL-6, IL-6R and gpl30 signal transducer, to interact with the two other partners.
  • the objectives of the present invention will be achieved by peptides that inhibit any one of interactions in the complex as follows : a) Peptides derived from IL-6 that interfere with IL-6-IL-6R interaction or with IL-6/gpl30 interaction. b) Peptides derived from IL-6R that interfere with IL-6-IL-6R interaction or with IL-6R/gpl30 interaction. c) Peptides derived from gpl30 that interfere with IL-6/gpl30 or with IL-6R/gpl30 interactions.
  • the segment of the IL-6R spanning residues 247-271 is currently a most preferred embodiment for development of conformationaUy constrained backbone cyclized peptide analogs to be used as an inhibitor of IL-6 activity.
  • more preferred backbone cyclized analogs are decapeptide and nonapeptide antagonists of IL-6 with improved activity and metabolic stability. Additional more preferred analogs may advantageously include at least one D-isomer of amino acids in their sequence.
  • the backbone cyclized analogs of the present invention are derived from, or mimic the sequence of the IL-6R molecule, preferably related to residues 247-271 of the IL-6R amino acids sequence. Additional preferred analogs are derived from the sequence of the IL-6 molecule.
  • n and n are 1 to 5;
  • X designates a terminal carboxy acid, amide or alcohol group
  • R 249 is Trp, (L) or (D)Lys, (L) or (D) Tyr or (D)Phe;
  • R 250 is Arg
  • R 251 is (L) or (D)Leu or Lys
  • R 252 is (L) or (D)Arg
  • R 254 is Ala
  • R 255 is (D)- or (L)- Leu or is Lys
  • R 256 is absent or is (L) or (D) Arg;
  • R 257 is (L) or (D) Tyr;
  • R 258 is Ala
  • Y is amide, thioether, thioester or disulfide.
  • n designates a terminal carboxy acid, amide or alcohol group
  • R 249 is Trp , ( D ) Lys or ( D ) Phe ;
  • R 250 is Arg ;
  • R 252 is ( D ) Arg ;
  • R 253 is ( D ) - or ( D - Phe ;
  • R 255 is (D)- or (L) - Leu; R 256 is absent or is Arg; R 257 is (D) Tyr; R 258 is Ala; and Y 2 is amide, thioether, thioester or disulfide.
  • the currently most preferred backbone cyclized IL-6 antagonists of the invention which are derived from the IL-6 receptor molecule are as follows:
  • Trp-Arg-Lys- (D) Arg-Phe-AlaC3-Leu-Arg- (D) Tyr-AlaN3-NH 2 designated herein as PTR-5045
  • PTR 5045 was also found to be active in-vivo in prevention of IL-6 induced pathology, and to be metabolically functional bio-stable.
  • additional preferred analogs are derived from the sequence of the IL-6 molecule.
  • the region of the IL-6 molecule spanning loop AB and helix D is currently a most preferred embodiment for development of conformationaUy constrained backbone cyclized peptide analogs to be used as an inhibitor of IL-6 activity.
  • additional more, preferred backbone cyclized analogs are hexapeptides antagonists of IL-6 with improved activity and metabolic stability. Additional more preferred analogs may advantageously include at least one D-isomer of amino acids in their sequence.
  • a preferred embodiment of the present invention relating to peptides derived from IL-6, has the following formula:
  • n and n are 1 to 5;
  • X designates a terminal carboxy acid, amide or alcohol group
  • R 1 is (D)Bip, Gin, Lys, Lys(ZCL) or Dab;
  • R 2 is (D)Lys, Gly, Ala or Trp
  • R 3 is Orn, 4PyrAla, (L) or (D)Dab, (D)Arg , Lys or Dpr;
  • R 4 is Lys, Lys(ZCL), Arg, Arg(Mtr) or (D)Glu;
  • R 5 is Asn, Trp or (D)Ala;
  • R 6 is Arg, (p-N02)Phe, (L) - or (D)- Trp, Gin, Abu or Glu;
  • Another preferred embodiment of the present invention relating to peptides derived from IL-6, has the following formula:
  • X designates a terminal carboxy acid, amide or alcohol group
  • R 3 is Dpr, 4PyrAla or (L)- or (D)- Arg; R 4 is HomArg, Orn or Lys ; R 5 is (D)Gln or (L)- or (D)- Trp; R 6 is (D- or (D)- Gin or (p-N02)Phe; and Y 2 is amide, thioether, thioester or disulfide.
  • the currently most preferred backbone cyclized IL-6 antagonists of the invention which are derived from the IL-6 molecule are as follows:
  • LysC3-Lys-4PyrAla-Orn-TrpN2- (p-N02) Phe-NH 2 denoted 70003-40; LysC4-Lys- (D)Arg-Lys-TrpN3-Gln-NH 2 denoted 70003-33; (D) PheC2- (D) Bip-Arg-Lys- (D) TrpN2-Gln-NH 2 denoted 70003-81.
  • the backbone cyclized peptide analogs of the present invention incorporates two such N ⁇ - ⁇ -functionalized amino acid derivatives which may be linked to one another to form N-backbone to N-backbone cyclic peptide analogs.
  • Additional preferred analogs of the invention can be constructed with two or more cyclizations, including N-backbone to N-backbone, as well as backbone to side-chain or any other peptide cyclization.
  • Backbone cyclized analogs of the present invention may be used as pharmaceutical compositions and in methods for the treatment of disorders including: cancers (including multiple myeloma/plasmacytoma) , autoimmune diseases (including rheumatoid arthritis, multiple sclerosis, SLE and diabetes), infectious diseases (bacterial and viral infection, septic shock), inflammatory diseases (including pancreatitis), immune deficiency diseases (including AIDS), hematologic diseases (e.g., leukemia, lymphoma), allergic diseases, organ transplantation reactions, Castelman's disease, Lennart's T-cell lymphoma, Non-Hodgkin's lymphoma, Cardiac myxoma, mesangial proliferative glomerulonephritis, polyclonal B-cell activation conditions, abnormal acute phase protein production conditions .
  • cancers including multiple myeloma/plasmacytoma
  • autoimmune diseases including rheumatoid arthritis, multiple sclerosis
  • compositions comprising pharmacologically active backbone cyclized IL-6 antagonist and a pharmaceutically acceptable carrier or diluent represent another embodiment of the invention, as do the methods for the treatment of a mammal in need thereof with a pharmaceutical composition comprising an effective amount of an IL-6 antagonist according to the invention.
  • compositions of the invention are useful for therapy of cancers (including multiple myeloma/plasmacytoma) , autoimmune diseases (including rheumatoid arthritis, multiple sclerosis, SLE and diabetes) , infectious diseases (bacterial and viral infection, septic shock), inflammatory diseases (including pancreatitis), immune deficiency diseases (including AIDS) , hematologic diseases (e.g., plasma cell dyscrasias, leukemia, lymphoma), allergic diseases, organ transplantation reactions, Castelman's disease, Lennart's T-cell lymphoma, Non-Hodgkin's lymphoma, Cardiac myxoma, mesangial proliferative glomerulonephritis, polyclonal B-cell activation conditions, abnormal acute phase protein production conditions, and osteoporosis using such compositions.
  • cancers including multiple myeloma/plasmacytoma
  • autoimmune diseases including rheumatoid arthritis,
  • compositions according to the present invention advantageously comprise at least one backbone cyclized peptide analog which includes at least one D-isomer of amino acids in its sequence.
  • These pharmaceutical compositions may be administered by any suitable route of administration, including topically or systemically .
  • Preferred modes of administration include but are not limited to parenteral routes such as intravenous and intramuscular injections, as well as via nasal or oral ingestion.
  • Figure 1 is a schematic drawing depicting known active IL-6 inhibitory and non-active peptides derived from the IL-6R.
  • Figure 2 describes the effect of backbone cyclic peptide analogs on B16.F10.9 melanoma cells growth.
  • Figure 3 describes in-vivo effects of IL-6 antagonists on IL-6 mediated acute-phase responses: a) IL-6 serum levels, b) fibrinogen plasma levels and c) changes in body weight, in normal and IL-6 knockout mice.
  • Figure 4 is a chart describing the biological activity and selectivity of backbone cyclized peptide antagonists of IL-6, in TF1 cells maintained with IL-6 or GM-CSF.
  • stable compound or “stable structure” is meant herein a compound that -L-s sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • substituted means that any one or more hydrogen atoms on the designated moiety is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound.
  • any variable for example R, X, Z, etc.
  • its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • peptide indicates a sequence of amino acids linked by peptide bonds.
  • the IL-6 peptide antagonists of this invention comprise a sequence of amino acids of 4 to 24 amino acid residues, preferably 6 to 16 residues, each residue being characterized by having an amino and a carboxy terminus.
  • a "building unit” indicates an N ⁇ derivatized ⁇ amino acid of the general Formula No. 5: -N-CH ( R' ) -CO-
  • X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene;
  • R' is an amino acid side chain, optionally bound with a specific protecting group;
  • G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, and alkyl halides; which is incorporated into the peptide sequence and subsequently selectively cyclized via the functional group G with one of the side chains of the amino acids in said peptide sequence or with another ⁇ -functionalized amino acid derivative .
  • the building units are abbreviated by the three letter code of the corresponding modified amino acid followed by the type of reactive group (N for amine, C for carboxyl) , and an indication of the number of spacing methylene groups.
  • N for amine
  • C for carboxyl
  • PheN3 designates a modified phenylalanine group with an amino reactive group and a three carbon methylene spacer .
  • backbone cyclic peptide or “backbone cyclized peptide” denotes an analog of a linear peptide which contains at least one building unit that has been liked to form a bridge via the alpha nitrogen of the peptide backbone to another building unit, or to another amino acid in the sequence.
  • PTR denotes a reference number assigned to a backbone cyclic peptide analog that is synthesized, purified and fully characterized (e.g., by HPLC, MS, capillary electrophoresis, by amino acid analysis for peptide content and amino acid ratio determination) .
  • the superscript numbers following the amino acids refer to their position numbers in the native IL-6 receptor or in the IL-6 molecule.
  • AcOH refers to acetic acid
  • Ada refers to adamantanacetyl
  • Adac refers to adamantanecarbonyl
  • Alloc refers to allyloxycarbonyl
  • AIDS refers to acquired immune deficiency syndrome
  • Boc refers to the t-butyloxycarbonyl radical
  • BOP refers to benzotriazol-1-yloxy-tris- (dimethylamino) phosphonium hexafluorophosphate
  • BSA refers to bovine serum albumin
  • Cbz refers to the carbobenzyloxy radical
  • CNTF refers to ciliary neurotrophic factor
  • DCC refers to dicyclohexylcarbodiimide
  • DCM refers to Dichloromethane
  • Dde refers to
  • DIEA diisopropyl-ethyl amine
  • DMF dimethyl formamide
  • DPPA diphenylphosphoryl azide
  • Dtc 5, 5-dimethylthiazolidine-4-carboxylic acid
  • EDT refers to ethanedithiol
  • ESI-MS electrospray ionization mass spectrometry
  • Fmoc refers to the fluorenylmethoxycarbonyl radical
  • HBTU 1-hydroxybenztriazolyltetramethyl-uronium hexafluorophosphate
  • HF hydrofluoric acid
  • HOBT refers to
  • Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate PyBrOP refers to Bromo-tris- pyrrolidino-phosphonium hexafluorophosphate
  • RA Rheumatoid arthritis
  • RP Rheumatoid arthritis
  • SLE SLE
  • SLE system lupus ethrythematosus
  • TBTU refers to
  • tBu 2- (lH-benzotriazole-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate
  • tBu refers to the tertiary butyl radical
  • TFA refers to trifluoroacetic acid
  • TIS refers to tri-isopropyl-silane .
  • amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent and convergent synthetic approaches to the peptide sequence are useful in this invention.
  • Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by "D" before the residue abbreviation.
  • Abu refers to 2-aminobutyric acid
  • Aib refers to
  • IL-6 plays a pivotal role in mediating immune responses, acute-phase reactions and hematopoiesis .
  • loss of IL-6 regulation, or its overexpression may be involved in a number of pathological conditions.
  • elevated IL-6 levels are detected in bacterial, parasite and viral infections, including HIV, as well as in chronic autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and multiple sclerosis.
  • IL-6 is implicated in the pathology of various neoplasms, such as multiple myeloma, leukemia, Kaposi's sarcoma, renal cell carcinoma and cardiac myxoma.
  • IL-6 has been recognized as the major cytokine required for growth of multiple myeloma tumors and is also possibly involved in the n tumor-associated toxicity in multiple myeloma patients.
  • backbone cyclized IL-6 peptide antagonists of the present invention is expected to be beneficial in treating a variety of IL-6 associated diseases, many of which are currently treated with immunomodulators or immunosuppresants .
  • Such IL-6 associated diseases include: a) Multiple myeloma/plasmacytoma. b) Autoimmune diseased (including, but not limiting to, rheumatoid arthritis, multiple sclerosis, SLE and diabetes) . Infection diseases (bacterial and viral infection, septic shock) .
  • Immune deficiency diseases including AIDS.
  • Hematologic, diseases e.g., plasma cell dyscrasias, leukemia, lymphoma.
  • the search comprises mainly rational design and combinatorial libraries screening using multiple parallel synthesis (MPS) approaches.
  • the suggested IL-6 inhibitor is non-cytotoxic as compared with the currently utilized cytotoxic drugs.
  • the effect of the IL-6 antagonist would be specific to multiple myeloma cells and a small subset of IL-6 dependent cells, where the other cytotoxic drugs are non-selective and kill all types of dividing cells in the body.
  • the suggested IL-6 antagonist is small and thus non-immunogenic by nature, as compared to the potentially immunogenic antibodies, antibody fragments and minibodies.
  • the suggested IL-6 antagonist could be modified to be orally bioavailable . It is unlikely that proteins (antibodies, fragments or minibodies) would be orally available.
  • PTRs peptide analogs
  • X designates a terminal carboxy acid, amide or alcohol group
  • R 249 is Trp, (L) or (D)Lys, (L) or (D) Tyr or (D)Phe;
  • R 250 is Arg
  • R 251 is (L) or (D)Leu or Lys
  • R 252 is (L) or (D)Arg
  • R 253 is (D)- or (L) - Phe;
  • R ,256 is absent or is (L) or (D) Arg;
  • R ,2"57' is (L) or (D) Tyr;
  • R 258 is Ala
  • Y 2 is amide, thioether, thioester or disulfide.
  • n and n are 1 to 5;
  • X designates a terminal carboxy acid, amide or alcohol group
  • R 249 is Trp, (D)Lys or (D)Phe;
  • R 250 is Arg
  • R 251 is Lys or (D)Leu;
  • R 252 is (D)Arg
  • R 253 is (D)- or (D- Phe;
  • R 254 is Ala
  • R 255 is (D)- or (L) - Leu;
  • R 256 is absent or is Arg
  • R is (D) Tyr
  • R" JO is Ala
  • Y 2 is amide, thioether, thioester or disulfide.
  • a most preferred compound according to this embodiment is denoted PTR 5045 wherein the residues are as follows:
  • PTR 5041 Another preferred compound according to this embodiment is denoted PTR 5041 wherein the residues are as follows:
  • R ,2 ⁇ 49 is (D)Lys
  • R 250 is Arg
  • R 251 is (D) Leu
  • R 252 is (D)Arg
  • R 254 is Ala
  • R 255 is Leu
  • R is Arg
  • R is (D) Tyr
  • R 256 is Ala
  • Y is amide
  • PTR 5043 Another preferred compound according to this embodiment is denoted PTR 5043 wherein the residues are as follows:
  • R 249 is (D) Phe
  • R 250 is Arg
  • R 251 is (D)Leu
  • R 252 is (D)Arg
  • R 253 is (D)Phe
  • R 254 is Ala; R 255 is Leu; R 256 is absent; R 257 is (D)Tyr; R 258 is Ala; and Y 2 is amide.
  • Another preferred embodiment of the present invention relating to peptides derived from IL-6, has the following formula:
  • n and n are 1 to 5;
  • X designates a terminal carboxy acid, amide or alcohol group
  • R 1 is (D)Bip, Gin, Lys, Lys(ZCL) or Dab;
  • R 2 is (D)Lys, Gly, Ala or Trp
  • R 3 is Orn, 4PyrAla, (L) or (D)Dab, (D)Arg , Lys or Dpr;
  • R 4 is Lys, Lys(ZCL), Arg, Arg(Mtr) or (D)Glu;
  • R 5 is Asn, Trp or (D)Ala
  • R 6 is Arg, (p-N02)Phe, (L) - or (D)- Trp, Gin, Abu or Glu;
  • Y 2 is amide, thioether, thioester or disulfide.
  • Another preferred embodiment of the present invention relating to peptides derived from IL-6, has the following formula:
  • n and n are 1 to 5;
  • X designates a terminal carboxy acid, amide or alcohol group
  • R 1 is (D) Phe or Lys; R i s ( D ) Cit , Lys or ( D ) Bip ;
  • R 3 is Dpr, 4PyrAla or (L) - or (D)- Arg;
  • R 4 is HomArg, Orn or Lys
  • R 5 is (D)Gln or (L) - or (D)- Trp;
  • R 6 is (L)-,or (D)- Gin or (p-N02)Phe;
  • Y 2 is amide, thioether, thioester or disulfide
  • X is -NH 2 or -OH and the bridg ng group extends between the two building units.
  • N- ⁇ -functionalized amino acid derivatives which may be linked to one another to form N-backbone to N-backbone cyclic peptide analogs .
  • backbone cyclization The most striking advantages of backbone cyclization are:
  • the bridge can be designed in such a way as to minimize interaction with the active region of the peptide and its cognate receptor. This decreases the chances of the cyclization arm interfering with recognition and function, and also creates a site suitable for attachment of tags such as radioactive tracers, cytotoxic drugs, light capturing substances, or any other desired label.
  • bond type e.g., amide, disulfide, thioether, thioester, etc.
  • Peptide analogs can be constructed with two or more cyclizations, including N-backbone to N-backbone, as well as backbone to side-chain or any other peptide cyclization.
  • the second cyclization can be formed by incorporating at least one additional building unit into a peptide sequence and linking it to another building unit, to the amino acid side chain or to any of the peptide terminals.
  • the second cyclization can be a side-chain to side-chain (including di-sulfide bond), or a side-chain to terminal cyclization.
  • backbone cyclized IL-6 antagonists of the present invention which were identified by screening of individual backbone cyclized peptide analogs that were synthesized and assayed for inhibition of IL-6 activity, are 10-100 folds more active than the linear IL-6 inhibitory peptides previously described.
  • the backbone cyclic peptides of this invention are novel analogs which mimic the IL-6R inhibitory domain of residues 249-258.
  • the amino acid sequence of the backbone cyclic analogs is based on what was identified as the most active inhibitory fragment of the IL-6R (Grube and Cochran, ibid) .
  • Additional analogs mimic a non-continuos region of the IL-6 molecule, comprising the .contact residue of IL-6 to its receptor. These analogs have an additional advantage of having molecular weights of around 1000 dalton.
  • the present innovative backbone cyclic analogs preferably include 5 to 20 amino acids with special amino acid modifications. Specifically, at least one amino acid in each analog is a D-isomer of the amino acid.
  • novel backbone cyclic peptide analogs are their metabolic stability as tested in vi tro against degradation of the most aggressive enzyme mixture in the body (e.g., renal homogenate) .
  • PTR 5045 is stable under these conditions for up to 24 hours.
  • the previously described peptide analogs derived from the IL-6R, and fragments of the native protein, are significantly less stable metabolically.
  • Cyclization performed with a solution containing 3 equivalents PyBOP and 6 equivalents DIEA in 7 ml NMP. For 0.5-2 hours with shaking. Cyclization is monitored by ninhydrine test and repeated if necessary.
  • An individual purification method for each backbone cyclic peptide is developed on analytical HPLC to give the maximum isolation of the cyclic peptide from other crude components.
  • the analytical method is usually performed using a C-18 Vydac column 250X4.6 mm as the stationary phase and water/ACN containing 0.1% TFA mixture gradient.
  • the preparative method is designed by implying the analytical separation method on the 2" C-18 Vydac preparative method.
  • the peak containing the cyclic peptide is collected using a semi-automated fraction collector.
  • the collected fractions are injected to the analytical HPLC for purity check.
  • the pure fractions are combined and lyophilized.
  • the combined pure lyophilized material is analyzed for purity by HPLC, MS and capillary electrophoresis and by amino acid analysis for peptide content and amino acid ratio determination.
  • the MPS procedure is used as the routine peptide development procedure. Individual peptides, or groups of a few peptides, are synthesized, in 96-wells microtiter plates equipped with filters that allow passage of solvent but not of solid phase matrix. A simple and efficient valve apparatus that enables simultaneous closing and opening of all the valves (produced by Millipore) is used.
  • the system utilizes an approach in which each well is equipped with a solvent permeable membrane at the bottom that does not allow passage of particles above a certain size. The process allows one to place resin in the wells, perform reaction in solvent, and remove the solvent from all the wells simultaneously by applying vacuum.
  • These special plates, which are available in the standard 96 well format allow the parallel synthesis of 96 peptides simultaneously.
  • the synthesis scale of the procedure is in the range of 1-5 ⁇ mole per well.
  • the peptides are routinely dissolved in 1 ml of water to yield a theoretical crude concentration of 1-5 mM (depending on synthesis scale) .
  • Monitoring of chemical quality of the resulting peptides is performed by ESI-MS analysis. Analysis of several plates prepared on different occasions by different operators indicated a general success rate of about 80% as judged by the presence of the desired peptide mass in the crude preparation. Further analysis of a peptides from MPS is carried out by LC-MS .
  • Amount of amino acid per coupling per well 26 ⁇ mol Amino acid in NMP concentration: 650 mM Amino acid volume used: 40 ⁇ l PyBroP amount: 26 ⁇ mol
  • this step is performed after completing the assembly, by addition of 180 ⁇ l solution of 1.5 g Pd(PPh 3 ) 4 in 20 ml CH 2 C1 2 containing 5% AcOH + 2.5% NMM. Cyclization- this step is performed by addition of 100 ⁇ l solution of PyBoP in NMP + DIEA.
  • the resin is transferred into a deep well microtiter plate, to each well 300 ⁇ l of TFA solution containing 2.5% TIS, 2.5% H 2 0, 2.5% EDT are added. Removal of the TFA is performede by lyophilization. After cleavage the peptides are purified by SepPak.
  • IL-6 dependent cell lines such as the murine T1165 and B9, or the human TFl and XGl.
  • inhibition of IL-6 can be monitored by following the IL-6 induced differentiation of A375, B16.F10.9 and Ml cells which results in continued growth of the cells. Measuring IgG secretion by CESS cell line can also be used for monitoring IL-6 inhibitory activity.
  • Five types of in vitro bioassays for IL-6 inhibition using different murine and human cell lines were used in different stages of the search, for screening of inhibitory peptides.
  • Bioassay using B9 cells B9 (murine myeloma) cells require IL-6 for growth. Inhibition of IL-6 results in cell death. The assay procedure was performed as described in Halimi et al. (ibid). Bioassay using T1165 cells
  • T1165 (murine myeloma cells) require IL-6 for growth and die if IL-6 is omitted or inhibited. The validity of the assay was demonstrated by inhibition of IL-6 bioactivity using rabbit IL-6 antiserum.
  • the F10.9 sub-line of B16 (murine melanoma) expresses gpl30 but not IL-6R.
  • the B16.F10.9 cell assay was routinely used as a confirmation assay for the T1165 or TFl assay which were the primary screening assays (at different stages of the search) . Evaluation of the results in this assay is possible using two methods.
  • One method uses vital dyes for monitoring cell growth.
  • the second method comprises visual observation of the cell morphology and establishing a cut off point based on the following observations: Cells which are not treated with IL-6 create a monolayer that covers almost all the surface area. Treatment with IL-6 causes growth arrest and a morphological change: the cells become very narrow and elongated as compared to the more spread out shape of the non-treated cells.
  • Treatment of the cells with IL-6 and an inhibitory peptide apparently completely restores cell growth but does not completely restores the cell morphology. Treatment of cells with IL-6 and a non-inhibitory peptide results in cells that appear to be very similar to the IL-6 treated cells. Based on such observations, the results of the assay are reported as the last concentration of the peptide that cause almost complete inhibition of the effect of IL-6. Bioassay using A375 cells
  • the A375 are human melanoma cells.
  • IL-6 induces differentiation of A375 cells which is associated with growth arrest similar to the phenomenon observed with the B16F10.9 murine melanoma cells.
  • inhibition of IL-6 results in continued growth of the cells which is easily quantifiable. In such case toxicity of peptides would register as a false negative rather than false positive. This conditions is preferable during screening process.
  • the molecules involved in the bio-response i.e. IL-6, IL-6R and gpl30 are all of human origin thus ensuring authenticity of the tested peptide bioactivity.
  • A375 cells Another advantage of the A375 cells is their ability to be induced by other cytokines that share the gpl30 signal transducer, i.e. leukemia inhibitory factor (LIF) and oncostatine M (OSM) .
  • LIF leukemia inhibitory factor
  • OSM oncostatine M
  • LIF alone does not affect A375 cells, unless the respective receptor (LIF-R) is also added.
  • LIF-R LIF-R
  • OSM is highly effective alone.
  • the availability of this assay system allows us to test, the specificity of our IL-6 inhibitory peptides vs . other cytokines of the family, in an array of assays all using a single cell line. Assay performance is described in Savino et al. (ibid).
  • the assay was validated using rabbit IL-6 antiserum which was used to inhibit the bioactivity of IL-6. Testing of crude peptide preparations, as well as high concentrations of purified peptides resulted in marked toxicity to the cells. Further results indicated that the toxicity is dose dependent and decreases with increasing dilution of the crude peptide preparation, or at low concentration of the purified peptides. It is therefore anticipated that the assay can be used for screening of analogs with nM activity.
  • TFl cells human erythroleukemia cells require IL-6 for growth and thus inhibition of IL-6 bioactivity results in cell death, similar to T1165 cells.
  • the origin of all relevant molecules is human .
  • Assay procedure is based on the procedure described in Fourcin et al. (ibid) .
  • the amount of viable cells at the end of the assay is determined by staining the cells with WST reagent as well as by measuring thymidine corporation ( 3 H-T) .
  • Binding assays are intended to measure the direct effect of the peptide on formation of the IL-6 active hexamer. Unlike the bioassays, the use of this assay could clearly demonstrate the mode of activity of the tested peptides.
  • a simple format for such assay would be as follows: IL-6, IL-6R and soluble gpl30 would be mixed in solution together with the test peptide. Capture of the putative hexamer would be achieved by an anti-gpl30 antibody and detection of the bound complex would be achieved by antibody to either IL-6 or IL-6R. A separate assay performed in order to test the interference of the peptide in the IL-6/IL-6R interaction.
  • Assays of similar format are used for testing the inhibition specificity of the peptide to the IL-6 bioactive complex by replacing the IL-6-IL-6R complex with commercially available cytokines and receptors of the other cytokines known to utilize the gpl30 signal transducer.
  • the prime clinical target for the IL-6 antagonist is multiple myeloma.
  • In-vivo model systems are performed in mice inoculated with murine IL-6 dependent myeloma cell lines. Nude mice grafted with human multiple myeloma cells are also used.
  • Other in-vivo assays which are used for testing the inhibitory effects of the backbone cyclized IL-6 antagonists are: IL-6 mediated acute-phase response, IL-6 mediated adjuvant arthritis, and pancreatitis induced by taurocholic acid as described in the following examples.
  • Fmoc-Lys (Boc) -OH, Fmoc-Arg (pmc) -OH, Fmoc-Trp (Boc) -OH were carried out with 4 eq (1.76 mmol) of the amino acid +PyBrop (4 equivalents, 1.76 mmol)+ DIEA (8 equivalents, 3.52 mmol) in NMP (10 ml) for 1.5 hour at room temperature. Reaction completion was monitored by the qualitative ninhydrin test (Kaiser test) . After each coupling, the peptide-resin was washed with NMP (5 times with 15 ml NMP, 2 minutes each) .
  • the coupling of Fmoc- (D) Tyr (t-Bu) -OH to AlaN3 building unit was carried out by use of 4eq of amino acid + a mixture of TPTU and ToPPyU(4 eq, 1.76 mmol) in 10 ml NMP + 8 eq DIEA, double coupling: first coupling 2h, second coupling overnight.
  • the coupling of Fmoc-Phe-OH to AlaC3 building unit was carried out by the same manner. Coupling completion was monitored by HPLC .
  • the Allyl/Aloc protecting groups were removed by reaction with Pd(PPh 3 ) 4 and acetic acid 5%, morpholine 2.5% in CH 2 C1 2 , under argon, for 2 hours at room temperature.
  • Example 2 the -effect of backbone cyclic peptide analogs on B16.F10.9 melanoma cells growth.
  • Peptides were added to B16.F10.9 melanoma cells in the presence of 200 ng/ml IL-6 and 125 ng/ml sIL-6R. Incubation for three days. (Peptide concentration was calculated for average molecular weight of 1500 Da.
  • PTR 5049 Trp-Arg-Lys- (D) Arg-Phe-AlaC3-Leu-Arg-Tyr-AlaN3-NH 2
  • PTR 4041 Lys-GlyC2-Leu-Ile-Gln-Leu-Phe-GlyN3-Lys-Lys-NH 2
  • the results described in figure 2 show that PTR 5045 and PTR 5041 fully block IL-6 activity at concentration of about 250 nM while PTR 5049 and PTR 4041 are not active.
  • IL-6 Systemic and localized inflammation elicit a general reaction in the organism, known as the acute phase response, which includes fever, loss of body weight, hypoglycemia, and changes in the serum levels of several plasma proteins produced by the liver.
  • IL-6 is an important mediator of the acute-phase reaction (see Heinrich et.al., Biochemistry J. 1 265:621, 1990 for a comprehensive review) , together with IL-1 and TNF- ⁇ • It has been shown previously that sterile tissue damage caused by injection of turpentine induces an acute-phase reaction, and that IL-6 is an essential mediator of this response (Rokita et.al., Cytokine 5:454, 1993). To confirm this role of IL-6 in this phenomenon we used IL-6-deficient mice generated by gene targeting (knock out). Method and Results
  • IL-6 knock-out mice -/- mice
  • C57/Black mice mice were used.
  • Sterile tissue damage was induced by subcutaneous injection of turpentine, 0.1 ml, into both hind limbs.
  • IL-6 was determined in the serum by ELISA (QuantikineM, R&D) .
  • Fibrinogen was determined in citrated plasma, by the calcium method.
  • IL-6 levels rose 7-fold following turpentine injection in normal, but not in IL-6 deficient mice ( Figure 3a) .
  • Fibrinogen levels rose 3- to 4-fold following turpentine injection in normal, but not in IL-6 deficient mice ( Figure 3b) .
  • mice received 1 or 10 mg/kg of each peptide analog
  • PTR 5045 reduces by about half the effect mediated by IL-6 on body weight during acute inflammation. The difference between PTR 5045 and the control peptide is significant also if taking only the 1 mg/kg dose.
  • Example 4 IL-6 mediated adjuvant arthritis.
  • the objective was to establish an in-vivo model of rheumatoid arthritis (RA) , for testing the activity of Interleukin-6 (IL-6) inhibitors.
  • RA rheumatoid arthritis
  • IL-6 Interleukin-6
  • the adjuvant arthritis model was chosen because it has clinical and pathological similarities to RA in humans, and based on the putative role that IL-6 plays in this inflammatory condition.
  • Rheumatoid arthritis is a chronic, multi-system autoimmune disease, mainly characterized by a persistent inflammatory synovitis, usually involving peripheral joints in a symmetric distribution.
  • IL-6 has been implicated in RA, since increased levels were found in serum and synovial fluid of RA patients, and these levels were correlated with clinical parameters of inflammation (Miltenburg et al., British J. Rheumatology
  • Adjuvant induced arthritis in rats is used as a model for human RA because adjuvant arthritic animals develop the inflammatory and immunologic features which are observed in RA patients. Furthermore, IL-6 levels are elevated in the serum of arthritic rats, and the levels correlate with inflammation, and with the progress of the syndrome.
  • the animals were injected intradermally at the base of the tail.
  • the arthritis developed two weeks after immunization, and involved the small joints of the extremities.
  • the rats were then subjected to a clinical scoring of the inflamed joints, and to a histo-pathological evaluation.
  • the rate of success in inducing arthritis is greater than 85%.
  • Dexamethasone treatment completely abolished the clinical signs of arthritis, and can therefore be used as a positive control in this model. Delivery of the dexamethason treatment was successfully achieved by employing osmotic mini-pumps, which could also be used for the continuous administration of peptides .
  • the adjuvant arthritis model can serve as a disease model to determine the pharmacological activity of anti-IL-6 treatments.
  • the testing is based on scoring of clinical parameters at the onset of the disease (day 12) .
  • Example 5 Pancreatitis Induced by Taurocholic Acid.
  • the objective is to establish an in-vivo model of acute pancreatitis for testing the activity of IL-6 inhibitors.
  • the taurocholic acid induced pancreatitis model has clinical and pathological similarities to severe necrotizing acute pancreatitis .
  • pancreatitis In its severe form acute pancreatitis has clear systemic manifestations such as: circulatory failure, metabolic acidosis, ascites, hyperglycemia, hyperlipidemia, and ultimately a multisystem organ failure.
  • IL-6 has been implicated in acute pancreatitis, since elevated serum levels were more predictive of disease severity or lethality as compared with C-reactive protein in patients with acute pancreatitis (Leser et al., Gastroenterology 101:782:5, 1991).
  • Taurocholic acid induced pancreatitis in rats is used as a model for human pancreatitis because pancreatitis animals develop the biochemical and pathological features which are observed in pancreatitis patients.
  • IL-6 levels were elevated in the serum of pancreatitis rats.
  • pancreatitis was induced by the infiltration of 0.5 ml of 10% sodium taurocholate, into several sites of the pancreatic parenchyma with a 30G needle. A progressive detergent effects took place, which resulted in a diffuse pancreatic necrosis, and a high mortality rate.
  • the measured parameters were serum levels of the pancreatic enzymes amylase and lipase, serum IL-6, and mortality.
  • a histopathological evaluation of the pancreas was performed.
  • a mortality rate of 60-80% was found in the pancreatitis animals, with no mortality in the sham control group.
  • IL-6 is elevated in animal models of pancreatitis.
  • the taurocholate induced pancreatitis model can serve as a disease model to determine the pharmacological activity of anti-IL-6 treatments.
  • the testing is be based on measuring levels of pancreatic enzymes in the serum, mortality, and histopathological scoring,
  • Example 6 Synthesis and in vitro activity of further preferred backbone cyclized IL-6 antagonist PTRs.
  • PTR-5005 appears to be active on TFl cells.
  • Table 4 Summary of synthesis and bioactivity of certain preferred PTRs .
  • the average activity of the peptides found following the above procedure is estimated to be over 100 ⁇ M as estimated from the results of the MPS experiments.
  • Table 6 Summary of activity of certain preferred analogs derived from the IL-6R.
  • BTyr is a mixture of DTyr and LTyr, and thus these wells contain 2 peptides each.
  • Example 7 Design, synthesis, screening and identification of additional preferred backbone cyclized analogs derived from the IL-6 molecule.
  • NBU represents a building unit with amine reactive group
  • CBU represents a building unit with carboxyl reactive group.
  • the libraries were screened for binding of soluble IL-6.
  • the information obtained was used for synthesis of backbone cyclic peptides in MPS format. Analyses for biological activity were performed on the TFl human cell line. Eighteen backbone cyclic peptides from the first MPS plate exhibit more then 45% inhibition at 1:10 dilution. Since the endpoint of the bioassay used is cell death, there is a need to check the specificity of the killing action, and to examine whether the cell death is due to inhibition of the IL-6 signaling pathway. The peptide analogs were therefore analyzed for their ability to induce non-specific cell death when the same cells were maintained with GM-CSF (instead of IL-6) as growth factor.

Abstract

La présente invention concerne de nouveaux peptides qui sont des antagonistes cyclisés d'interleukine-6 à squelette contrainte par conformation. L'invention concerne également des procédés de synthèse d'antagonistes d'IL-6. L'invention concerne en outre des compositions pharmaceutiques comprenant des antagonistes d'IL-6, et des procédés d'utilisation desdites compositions.
EP00929763A 1999-06-01 2000-05-28 Antagonistes cyclises d'interleukine-6 a squelette contrainte par conformation Withdrawn EP1187624A1 (fr)

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