WO1998018823A1 - Ice inhibiting peptides - Google Patents

Ice inhibiting peptides Download PDF

Info

Publication number
WO1998018823A1
WO1998018823A1 PCT/EP1996/004738 EP9604738W WO9818823A1 WO 1998018823 A1 WO1998018823 A1 WO 1998018823A1 EP 9604738 W EP9604738 W EP 9604738W WO 9818823 A1 WO9818823 A1 WO 9818823A1
Authority
WO
WIPO (PCT)
Prior art keywords
ice
peptide
seq
lane
amino acid
Prior art date
Application number
PCT/EP1996/004738
Other languages
French (fr)
Other versions
WO1998018823A8 (en
Inventor
Marta Muzio
Martino Introna
Alberto Mantovani
Original Assignee
Applied Research Systems Ars Holding N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to DK96937313T priority Critical patent/DK0938500T3/en
Priority to EEP199900178A priority patent/EE04782B1/en
Priority to BR9612755-4A priority patent/BR9612755A/en
Priority to KR10-1999-7003693A priority patent/KR100502211B1/en
Priority to AT96937313T priority patent/ATE277079T1/en
Priority to EP96937313A priority patent/EP0938500B1/en
Priority to JP51995298A priority patent/JP4018154B2/en
Priority to IL12966996A priority patent/IL129669A0/en
Priority to ES96937313T priority patent/ES2225893T3/en
Priority to EA199900432A priority patent/EA001866B1/en
Priority to PT96937313T priority patent/PT938500E/en
Priority to SI9630696T priority patent/SI0938500T1/en
Priority to CA002269638A priority patent/CA2269638C/en
Priority to UA99052981A priority patent/UA70291C2/en
Application filed by Applied Research Systems Ars Holding N.V. filed Critical Applied Research Systems Ars Holding N.V.
Priority to AU27023/99A priority patent/AU729946C/en
Priority to US09/297,369 priority patent/US6380162B1/en
Priority to DE69633463T priority patent/DE69633463T2/en
Priority to PCT/EP1996/004738 priority patent/WO1998018823A1/en
Priority to ARP970105024A priority patent/AR011269A1/en
Priority to ZA979712A priority patent/ZA979712B/en
Publication of WO1998018823A1 publication Critical patent/WO1998018823A1/en
Priority to NO19992079A priority patent/NO323517B1/en
Publication of WO1998018823A8 publication Critical patent/WO1998018823A8/en

Links

Classifications

    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • 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 a peptide capable of binding to ICE and/or to enzymes of the ICE family, which peptide consists essentially of the amino acid sequence of SEQ ID NO: l, in which Xaa is selected between Asp and Ala, optionally containing one or more amino acids at its N-terminal and/or C-te ⁇ ninal end.
  • ICE Interleukin-l ⁇ -Converting Enzyme
  • IL-l ⁇ mterleukin-l ⁇
  • pIL-l ⁇ 31 kDa precursor
  • EL-l ⁇ precursor protein is, therefore, cleaved by ICE in the mature and biologically active form.
  • ICE activity has been identified in monocytes and THP1 cells, which cleave pIL- l ⁇ at Asp 116 -Ala 117 as well Asp 27 -Gly 28 to yield products of 17.5 kDa and 28 kDa, respectively (3,4). Cleavage at each site is dependent on aspartic acid in the PI position (4,6,7).
  • cysteine proteases related to the Caenorhabditis elegans cell death protein ced-3 represent the effector components of the apoptotic machinery.
  • ICE was the first described homologue of CED-3 and it is known that overexpression of ICE or CED-3 in Rat-1 fibroblasts induced apoptosis (8). Further studies also suggest that proteases of the ICE family may play an important role in the apoptotic mechanism.
  • ICE seems to be a pIL-l ⁇ specific processing enzyme, because it does not cleave IL-l ⁇ or several other proteins containing many Asp-X bonds.
  • Interleukin EL- lcc and IL-l ⁇ are pleiotropic cytokines, which, although their sequences show scarce analogy, exert a variety of similar effects on different tissues and act on many human pathologies, in particular on the immunitary response of the organism and on inflammatory processes (9). Both proteins have a molecular weight of about 17.5 KDa and are previously synthesised as precursor molecules of larger size having a molecular weight of about 31 KDa.
  • IL-ls are potent inflammatory and pyrogenic cytokines that normally have beneficial effects but can also have extremely unhealthy effects for the organism.
  • IL-1 can, for example, participate in the pathogenesis of symptoms of the autoimmune pathologies like systemic lupus erithematosus and, in particular, they are involved as mediators to provoke damages to tissues as for example in rheumatoid arthritis.
  • Many of the biological effects of IL-1 are similar to those that can be observed during a septic event.
  • IL-ls have pleiotropic biological activities, many of which influence negatively the organism, the powerful effects of IL-1 should be under strict physiological control.
  • IL-1 synthesis is inhibited by anti-inflammatory cytokines, prostaglandins and glucocorticoids and the existence of multiple levels of inhibition of IL-1 points to the necessity of a strict control of this mediator.
  • IL-1RI There are two types of IL-1 receptors named EL-1RI and JL-IRII.
  • EL- IRE is a non- signalling DL-1 binding molecule which acts as a regulated decoy target for D -1
  • EL-l ⁇ , IL-l ⁇ , DL-lra recognise and bind to the same receptor on cell surface (EL-IR);
  • E_ -lra is a polypeptide which binds IL-1RI, and with less affinity IL-1RH, without any agonistic activity.
  • IL-lra production is induced in different cellular types, including mononuclear phagocytes, polymorphonuclear cells (PMN) and fibroblasts, by
  • IL-lra secreted DL-lra
  • icDL-lra intracellular DL-lra
  • sDL-lra and icIL-lra are generated from the same gene.
  • icEL-lra transcripts originate from an alternative starting site and from the splicing of a first alternative exon into an internal splice acceptor site located in the first exon of sIL- Ira.
  • the predicted proteins are thus identical except in their NH2 ends, where the first 21 amino acids of sIL-lra are substituted by four amino acids in icIL-lra. Expression of transcripts encoding sDv Ira and icE.- Ira is differently regulated.
  • IL-1 is involved in pathogenesis of many diseases it is evident the need of having available medicaments useful to limit the unhealthy effects of IL-1.
  • icIL-lra A new molecular form of icIL-lra has recently been identified and cloned (16 and PCT/EP95/04023). This molecule is generated by the in frame insertion of a new 63 bp exon between the first and the second exons of the icIL-lra specific form. This new transcript has been found to be expressed in fibroblasts, keratinocytes, activated monocytes and polymorphonuclear cells. Expression in COS cells revealed that this new molecule is mostly intracellular and has a molecular weight of approximately 25 kDa in SDS-PAGE. Such new molecule has been called icDL-lra type ⁇ (icIL-lra ⁇ ).
  • the main object of the present invention is to provide new peptides capable of binding to ICE, thus blocking the production of the active form of DL-1 ⁇ and/or, more generally, capable of binding to enzymes of the ICE family, thus blocking the activity of such enzymes.
  • the present invention relates to a peptide capable of binding to ICE and/or to enzymes of the ICE family, which peptide consists essentially of the amino acid sequence of SEQ ID NO: 1, in which Xaa is selected between Asp and Ala, as specifically reported in SEQ ID NO: 2 or 3.
  • the peptide also contains one or more amino acids at the N-terminal and/or the C-terminal end. Therefore, the peptide of the invention can be 19-40, preferably 19-25 amino acids long.
  • the peptide consists essentially of the amino sequence of SEQ D NO: 4 or 5.
  • a non-limiting list of cysteine proteases of the ICE family includes: CED-3 (17),
  • Another object of the present invention is to provide the peptide in substantially purified form in order to be suitable for use in pharmaceutical compositions as active ingredient in pathologies that require ICE inhibition and/or inhibition of enzymes of the ICE family.
  • Examples of pathologies in which the new antagonist according to the invention can be advantageously used for prophylactic, therapeutic or diagnostic uses are lethal bacterial and viral infections as well as autoimmune and inflammatory diseases. Specific examples include: rheumatoid arthritis, septic shock, acute myelomonocytic leukaemia, immunological reaction of transplantation against host, acquired immunodeficiency syndrome (ADDS), ulcerative colitis and multiple sclerosis. Further objects and advantages of the invention will be evident in the following description.
  • An embodiment of the invention is the administration of a pharmacological active amount of the peptide of the invention to subjects at risk of developing pathologies requiring ICE inhibition and/or inhibition of enzymes of ICE family or to subjects already showing such pathologies.
  • adrninistration compatible with the active principle can be used, but particularly preferred is the parenteral adrninistration because it permits to have, in short times, systemic effects. For this reason, it is preferable the administration of an intravenous bolus just before, during or after the surgical operation.
  • the dose of peptide to be a ⁇ inistered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient.
  • the dosage can be between 0.05 and 30 mg Kg body weight and the preferable dose is between 0.1 and 10 mg/Kg body weight.
  • the pharmaceutical composition for parenteral use can be prepared in injectable form comprising the active principle and a suitable vehicle.
  • Vehicles for the parenteral administration are well known in the art and comprise, for example, water, saline solution, Ringer solution and dextrose.
  • the vehicle can contain smaller amounts of excipients in order to maintain the solution stability and isotonicity.
  • the preparation of the cited solutions can be carried out according to the ordinary modalities and preferably the peptide content will be comprised between 1 mg/ml and 10 mg/ml.
  • Figure 1 shows a Northern analysis of ICE mRNA expression in COS transfected cells.
  • Total mRNA extracted by COS cells transfected with mock vector or with cDNA coding for human ICE was analysed by Northern analysis to evidence the expression of
  • lane 1 MOCK
  • lane 2 ICE
  • Figure 2 it shows a Western blotting analysis.
  • pEL-l ⁇ was incubated with a monocytes lysate prepared as described in literature (4); after 60 minutes of incubation at 37°C the mixture reaction was run on SDS-PAGE and the presence of precursor or mature EL-l ⁇ was evidenced by Western blotting.
  • lane 1 pEL-1
  • lane 2 pIL-1 + monocytes lysate.
  • Figure 3 it shows the amino acid sequences of the peptides under study.
  • Peptide A SEQ ID NO: 6
  • peptideC SEQ DD NO:4
  • Peptide X SEQ DD NO: 7
  • Peptide S SEQ DD NO: 5
  • Peptide S is identical to peptide C except for the presence of two Ala substituting the two
  • Peptide B (SEQ ID NO: 8) is a known ICE inhibitor (la).
  • Figure 4 it shows the inhibition of ICE activity by low concentrations of the peptides under study.
  • pDL-l ⁇ (5 ng) was incubated with ICE in the presence or absence of peptides (0.25 or 2.5 ⁇ M) and the presence of precursor or mature ⁇ .-l ⁇ was evidenced as described for Figure 2.
  • lane 1 pIL-1; lane 2: p ⁇ .-l + peptide A; lane 3: pIL-1 + peptide B; lane 4: pIL- 1 + peptide C; lane 5: pIL-l + ICE; lane 6: pDL-1 + ICE + peptide A (2.5 ⁇ M); lane 7: pIL-1 + ICE + peptide A (0.25 ⁇ M); lane 8: pIL-1 + ICE + peptide B (2.5 ⁇ M); lane 9: pIL- 1 + ICE + peptide B (0.25 ⁇ M); lane 10: pDL-1 + ICE + peptide C (2.5 ⁇ M); and lane 11: pK.-l + ICE + peptide C (0.25 ⁇ M).
  • Figure 5 It shows the inhibition of ICE activity by high concentrations of the peptides under study.
  • pIL-l ⁇ (5 ng) was incubated with ICE in the presence or absence of peptides (40 or 400 ⁇ M) and the presence of precursor or mature IL-l ⁇ was evidenced as described for Figure 2.
  • lane 1 pIL-1 + peptide A
  • lane 2 pEL-1 + peptide B
  • lane 3 pDL-1 + peptide C
  • lane 4 pE.- 1 + peptide X
  • lane 5 pDL-1
  • lane 6 pIL-l + ICE
  • lane 7 pIL-1 + ICE + peptide A (400 ⁇ M)
  • lane 8 pD.,-1 + ICE + peptide B (400 ⁇ M)
  • lane 9 pIL- 1 + ICE + peptide C (400 ⁇ M)
  • lane 10 pIL-1 + ICE + peptide X (400 ⁇ M)
  • lane 11 pIL-1 + ICE + peptide A (40 ⁇ M)
  • lane 12 pIL-1 + ICE + peptide B (40 ⁇ M)
  • lane 13 pIL-1 + ICE + peptide C (40 ⁇ M);
  • lane 14 pDL- 1
  • Figure 6 It shows the inhibition of ICE activity by peptides C and S.
  • pEL-l ⁇ (5 ng) was incubated with ICE in the presence or absence of peptides (100, 300 or 1,000 ⁇ M) and the presence of precursor or mature IL-l ⁇ was evidenced as described for Figure 2.
  • lane l pIL-l
  • lane 2 pIL-1 + peptide C
  • lane 3 pIL-1 + peptide S
  • lane 4 pIL-1 + peptide B
  • lane 5 pD ⁇ l + ICE
  • lane 6 pIL- 1 + ICE + peptide C (0.1 mM)
  • lane 7 pIL-1 + ICE + peptide C (0.3 mM)
  • lane 8 pIL-1 + ICE + peptide C (1 mM)
  • lane 9 pIL-1 + ICE + peptide S (0.1 mM)
  • lane 10 pIL-1 + ICE + peptide S (0.3 mM)
  • lane 11 pIL- 1 + ICE + peptide S ( 1 mM)
  • lane 12 pIL-1 + ICE + peptide B (1 mM).
  • Reagents The following commercially available reagents were used for culture and separation of cells:
  • ICE cDNA was obtained by RT-PCR, based on the published sequence (8a).
  • RT-PCR was performed as described (16) for 30 cycles at 95°C for 1 minute and
  • Oligonucleotides were obtained from Duotech (Milan, Italy). The sequences of oligos uses to selectively amplify ICE were as follows: 'TOR" ICE 1 : 5 '-AAAAGCCATGGCCGACAAGGTC- 3 ' (SEQ ID NO: 9)
  • COS cells cDNA coding for human ICE has been amplified by PCR The sequence was confirmed and the cDNA was subcloned into pSG5 expression vector.
  • COS cells were transfected with empty vector (mock) or vector containing ICE cDNA and after 48 hours, cells were analysed for the expression of ICE specific mRNA. As shown in figure
  • COS cells transfected with ICE cDNA, expressed high levels of ICE mRNA.
  • ICE activity Inhibition of ICE activity.
  • a source of ICE activity was prepared and its ability to cleave pDL-l ⁇ was tested by incubating the reaction at 37°C for 1 hour. The mixture was then run on SDS -PAGE and the presence of pILl- ⁇ or the mature form was evidenced by Western Blotting.
  • EL-1 converting enzyme requires aspartic acid for processing of the D ⁇ -l ⁇ precursor at two distinct sites and does not cleave 31 kDa IL-l ⁇ , J.
  • eiegans cell death gene ced-3 encodes a protein similar to mammahan interleukin- l ⁇ - converting enzyme, Cell, 75, 641-652, 1993; 18.Kumar et al., Induction of apoptosis by the mouse nedd2 gene, which encodes a protein similar to the product of the C. eiegans cell death gene ced-3 and the mammaUan interleukin- l ⁇ - converting enzyme, Genes Dev.
  • Nicholson et al Identification and inhibition of ICE/CED-3 protease necessary for mammahan apoptosis, Nature, 376, 37-43, 1995; 22.Tewari et al., Yama/CPP-32 ⁇ , a mammahan homologue of CED-3, is a CmA- inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase, Cell, 81, 801-809, 1995; 23.Faucheu et al., A novel human protease similar to the interleukin- l ⁇ -converting enzyme induces apoptosis in transfected cells, EMBO J., 14, 1914-1922, 1995; 24.Kamens et al., Identification and characterisation of ICH-2, a novel member of the interleukin- l ⁇ -converting enzyme family of cysteine proteases, J.
  • NAME APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Transplantation (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Hematology (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to a peptide capable of binding to ICE and/or to enzymes of the ICE family, which peptide consists essentially of the amino acid sequence of SEQ ID NO: 1 reported in the Sequence Listing, in which Xaa is selected between Asp and Ala, optionally containing one or more amino acids at its N-terminal and/or C-terminal end. It also relates to the use of the above peptide, for the preparation of pharmaceutical compositions active in pathologies requiring ICE inhibition and/or inhibition of enzymes of the ICE family.

Description

ICE INHIBITING PEPTIDES
FIELD OF THE INVENTION
The present invention relates to a peptide capable of binding to ICE and/or to enzymes of the ICE family, which peptide consists essentially of the amino acid sequence of SEQ ID NO: l, in which Xaa is selected between Asp and Ala, optionally containing one or more amino acids at its N-terminal and/or C-teπninal end.
It also relates to the use of the above peptide, for the preparation of pharmaceutical compositions active in pathologies requiring ICE inhibition and/or inhibition of enzymes of the ICE family. BACKGROUND OF THE INVENTION
ICE (Interleukin-lβ-Converting Enzyme) is a heterodimeric cysteine protease that has been recently purified and cloned (1). mterleukin-lβ (IL-lβ) is synthesised as an inactive 33 kDa or 31 kDa precursor (pIL-lβ); the fully active 17.5 kDa mature form of IL-lβ begins at Ala117 and seems to result from processing between Asp116 and Ala117 (2,3). EL-lβ precursor protein is, therefore, cleaved by ICE in the mature and biologically active form.
ICE activity has been identified in monocytes and THP1 cells, which cleave pIL- lβ at Asp116-Ala117 as well Asp27-Gly28 to yield products of 17.5 kDa and 28 kDa, respectively (3,4). Cleavage at each site is dependent on aspartic acid in the PI position (4,6,7).
It is becoming apparent that cysteine proteases related to the Caenorhabditis elegans cell death protein ced-3 represent the effector components of the apoptotic machinery. ICE was the first described homologue of CED-3 and it is known that overexpression of ICE or CED-3 in Rat-1 fibroblasts induced apoptosis (8). Further studies also suggest that proteases of the ICE family may play an important role in the apoptotic mechanism.
ICE seems to be a pIL-lβ specific processing enzyme, because it does not cleave IL-lα or several other proteins containing many Asp-X bonds.
Interleukin EL- lcc and IL-lβ are pleiotropic cytokines, which, although their sequences show scarce analogy, exert a variety of similar effects on different tissues and act on many human pathologies, in particular on the immunitary response of the organism and on inflammatory processes (9). Both proteins have a molecular weight of about 17.5 KDa and are previously synthesised as precursor molecules of larger size having a molecular weight of about 31 KDa. IL-ls are potent inflammatory and pyrogenic cytokines that normally have beneficial effects but can also have extremely unhealthy effects for the organism. They can, for example, participate in the pathogenesis of symptoms of the autoimmune pathologies like systemic lupus erithematosus and, in particular, they are involved as mediators to provoke damages to tissues as for example in rheumatoid arthritis. Many of the biological effects of IL-1 are similar to those that can be observed during a septic event. Recent studies demonstrated that the intravenous administration of IL-1 in doses from 1 to 10 ng/kg gives rise to fever, sleepiness, anorexia, generalised myalgia, arthralgia and cephalea.
Since IL-ls have pleiotropic biological activities, many of which influence negatively the organism, the powerful effects of IL-1 should be under strict physiological control.
IL-1 synthesis is inhibited by anti-inflammatory cytokines, prostaglandins and glucocorticoids and the existence of multiple levels of inhibition of IL-1 points to the necessity of a strict control of this mediator. There are two types of IL-1 receptors named EL-1RI and JL-IRII. EL- IRE is a non- signalling DL-1 binding molecule which acts as a regulated decoy target for D -1
(10-12).
An antagonist polypeptide for the receptor of IL-1 has been described up to now: the third known component until today of the family of the receptor-binding proteins is the antagonist for the DL-1 receptor (DL-lra) (13-15) . All three components
(EL-lα, IL-lβ, DL-lra) recognise and bind to the same receptor on cell surface (EL-IR);
EL- lot and D^-lβ binding to DL^IR transmit a signal, whilst DL-lra does not.
E_ -lra is a polypeptide which binds IL-1RI, and with less affinity IL-1RH, without any agonistic activity. IL-lra production is induced in different cellular types, including mononuclear phagocytes, polymorphonuclear cells (PMN) and fibroblasts, by
IgG, cytokines and bacterial products. Until now two molecular forms of IL-lra have been identified and cloned: 1) secreted DL-lra (sIL-lra) contains a classical leader sequence of 25 amino acids giving a mature protein of 152 amino acids; 2) intracellular DL-lra (icDL-lra) lacks a leader sequence thus allowing to predict that this protein remains intracellular. sDL-lra and icIL-lra are generated from the same gene. icEL-lra transcripts originate from an alternative starting site and from the splicing of a first alternative exon into an internal splice acceptor site located in the first exon of sIL- Ira. The predicted proteins are thus identical except in their NH2 ends, where the first 21 amino acids of sIL-lra are substituted by four amino acids in icIL-lra. Expression of transcripts encoding sDv Ira and icE.- Ira is differently regulated.
The biological significance of icIL-lra is still unclear.
Considering that IL-1 is involved in pathogenesis of many diseases it is evident the need of having available medicaments useful to limit the unhealthy effects of IL-1.
A new molecular form of icIL-lra has recently been identified and cloned (16 and PCT/EP95/04023). This molecule is generated by the in frame insertion of a new 63 bp exon between the first and the second exons of the icIL-lra specific form. This new transcript has been found to be expressed in fibroblasts, keratinocytes, activated monocytes and polymorphonuclear cells. Expression in COS cells revealed that this new molecule is mostly intracellular and has a molecular weight of approximately 25 kDa in SDS-PAGE. Such new molecule has been called icDL-lra type π (icIL-lraπ). Considering that icIL-lrall is an intracellular protein as well as ICE, the Applicant has also tested the ability of icIL-lrall to inhibit ICE activity. The results are reported in the Examples of this patent application and show that icD-^-lrall inhibits ICE activity. DESCRIPTION OF THE INVENTION The main object of the present invention is to provide new peptides capable of binding to ICE, thus blocking the production of the active form of DL-1 β and/or, more generally, capable of binding to enzymes of the ICE family, thus blocking the activity of such enzymes. So the present invention relates to a peptide capable of binding to ICE and/or to enzymes of the ICE family, which peptide consists essentially of the amino acid sequence of SEQ ID NO: 1, in which Xaa is selected between Asp and Ala, as specifically reported in SEQ ID NO: 2 or 3. Optionally, the peptide also contains one or more amino acids at the N-terminal and/or the C-terminal end. Therefore, the peptide of the invention can be 19-40, preferably 19-25 amino acids long.
In particular, according to one embodiment of the invention, the peptide consists essentially of the amino sequence of SEQ D NO: 4 or 5. A non-limiting list of cysteine proteases of the ICE family includes: CED-3 (17),
Nedd-2/ICH-l (18, 19), Yama/CPP-32/Apopain (20, 21, 22), Tx/ICH-2/ICE rel-D (23,
24, 25), ICE rel-iπ (25), Mch-2 (26), ICE-LAP3/Mch-3/CMH-l (27, 28, 29), ICE-
LAP-6 (30) and FLICE/MACH (31, 32).
Another object of the present invention is to provide the peptide in substantially purified form in order to be suitable for use in pharmaceutical compositions as active ingredient in pathologies that require ICE inhibition and/or inhibition of enzymes of the ICE family.
Examples of pathologies in which the new antagonist according to the invention can be advantageously used for prophylactic, therapeutic or diagnostic uses are lethal bacterial and viral infections as well as autoimmune and inflammatory diseases. Specific examples include: rheumatoid arthritis, septic shock, acute myelomonocytic leukaemia, immunological reaction of transplantation against host, acquired immunodeficiency syndrome (ADDS), ulcerative colitis and multiple sclerosis. Further objects and advantages of the invention will be evident in the following description.
An embodiment of the invention is the administration of a pharmacological active amount of the peptide of the invention to subjects at risk of developing pathologies requiring ICE inhibition and/or inhibition of enzymes of ICE family or to subjects already showing such pathologies.
Any route of adrninistration compatible with the active principle can be used, but particularly preferred is the parenteral adrninistration because it permits to have, in short times, systemic effects. For this reason, it is preferable the administration of an intravenous bolus just before, during or after the surgical operation. The dose of peptide to be aα inistered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient. The dosage can be between 0.05 and 30 mg Kg body weight and the preferable dose is between 0.1 and 10 mg/Kg body weight.
The pharmaceutical composition for parenteral use can be prepared in injectable form comprising the active principle and a suitable vehicle. Vehicles for the parenteral administration are well known in the art and comprise, for example, water, saline solution, Ringer solution and dextrose. The vehicle can contain smaller amounts of excipients in order to maintain the solution stability and isotonicity.
The preparation of the cited solutions can be carried out according to the ordinary modalities and preferably the peptide content will be comprised between 1 mg/ml and 10 mg/ml.
The present invention has been described with reference to the specific embodiments, but the content of the description comprises all modifications and substitutions which can be brought by a person skilled in the art without extending beyond the meaning and purpose of the claims. The invention will now be described by means of the following Examples, which should not be construed as in any way limiting the present invention. The Examples will refer to the Figures specified here below.
DESCRIPTION OF THE FIGURES
Figure 1: it shows a Northern analysis of ICE mRNA expression in COS transfected cells. Total mRNA extracted by COS cells transfected with mock vector or with cDNA coding for human ICE was analysed by Northern analysis to evidence the expression of
ICE specific mRNA. In particular, lane 1: MOCK; lane 2: ICE.
Figure 2: it shows a Western blotting analysis. pEL-lβ was incubated with a monocytes lysate prepared as described in literature (4); after 60 minutes of incubation at 37°C the mixture reaction was run on SDS-PAGE and the presence of precursor or mature EL-lβ was evidenced by Western blotting. In particular, lane 1: pEL-1; and lane 2: pIL-1 + monocytes lysate.
Figure 3: it shows the amino acid sequences of the peptides under study. Peptide A (SEQ ID NO: 6) and peptideC (SEQ DD NO:4) have been designed on the basis of icIL-lraπ sequence. Peptide X (SEQ DD NO: 7) is a randomly chosen peptide. Peptide S (SEQ DD NO: 5) is identical to peptide C except for the presence of two Ala substituting the two
Asp residues. Peptide B (SEQ ID NO: 8) is a known ICE inhibitor (la).
Figure 4: it shows the inhibition of ICE activity by low concentrations of the peptides under study. pDL-lβ (5 ng) was incubated with ICE in the presence or absence of peptides (0.25 or 2.5 μM) and the presence of precursor or mature π.-lβ was evidenced as described for Figure 2. In particular, lane 1: pIL-1; lane 2: pπ.-l + peptide A; lane 3: pIL-1 + peptide B; lane 4: pIL- 1 + peptide C; lane 5: pIL-l + ICE; lane 6: pDL-1 + ICE + peptide A (2.5 μM); lane 7: pIL-1 + ICE + peptide A (0.25 μM); lane 8: pIL-1 + ICE + peptide B (2.5 μM); lane 9: pIL- 1 + ICE + peptide B (0.25 μM); lane 10: pDL-1 + ICE + peptide C (2.5 μM); and lane 11: pK.-l + ICE + peptide C (0.25 μM).
Figure 5: It shows the inhibition of ICE activity by high concentrations of the peptides under study. pIL-lβ (5 ng) was incubated with ICE in the presence or absence of peptides (40 or 400 μM) and the presence of precursor or mature IL-lβ was evidenced as described for Figure 2. In particular, lane 1: pIL-1 + peptide A; lane 2: pEL-1 + peptide B; lane 3: pDL-1 + peptide C; lane 4: pE.- 1 + peptide X; lane 5: pDL-1; lane 6: pIL-l + ICE; lane 7: pIL-1 + ICE + peptide A (400 μM); lane 8: pD.,-1 + ICE + peptide B (400 μM); lane 9: pIL- 1 + ICE + peptide C (400 μM); lane 10: pIL-1 + ICE + peptide X (400 μM); lane 11: pIL-1 + ICE + peptide A (40 μM); lane 12: pIL-1 + ICE + peptide B (40 μM); lane 13: pIL-1 + ICE + peptide C (40 μM); and lane 14: pDL- 1 + ICE + peptide X (40 μM).
Figure 6: It shows the inhibition of ICE activity by peptides C and S. pEL-lβ (5 ng) was incubated with ICE in the presence or absence of peptides (100, 300 or 1,000 μM) and the presence of precursor or mature IL-lβ was evidenced as described for Figure 2. In particular, lane l: pIL-l; lane 2: pIL-1 + peptide C; lane 3: pIL-1 + peptide S; lane 4: pIL-1 + peptide B; lane 5: pD^l + ICE, lane 6: pIL- 1 + ICE + peptide C (0.1 mM); lane 7: pIL-1 + ICE + peptide C (0.3 mM); lane 8: pIL-1 + ICE + peptide C (1 mM); lane 9: pIL-1 + ICE + peptide S (0.1 mM); lane 10: pIL-1 + ICE + peptide S (0.3 mM); lane 11 : pIL- 1 + ICE + peptide S ( 1 mM); and lane 12: pIL-1 + ICE + peptide B (1 mM).
EXAMPLES
MATERIALS AND METHODS
Reagents The following commercially available reagents were used for culture and separation of cells:
Ficoll (Seramed , Berlin, Germany), Percoll (Pharmacia, Uppsala, Sweden), RPMI 1640
(Seramed, Berlin, Germany), FCS (HycloneLaboratories, Logan, UK), Glutamine
(Seramed, Berlin, Germany) and Hepes Merk, Darmastadt, Germany) Cells Mononuclear cells were obtained from the peripheral blood of human healthy donors by Ficoll gradient centrifugation. Purified monocytes were separated from mononuclear cells by Percoll gradient centrifugations at 2,000 rpm for 30 minutes at room temperature (10). COS-7 cells (purchased from ATCC, Rockville, MD, USA) and monocytes were cultivated in RPMI 1640 medium + 10% FCS - 2 mM glutamine + 20 mM Hepes. Human recombinant DL^lβ precursor was obtained from Cistron
Biotechnology, Pinebroom, NJ, USA. A polyclonal antibody reactive with both mature and precursors DL-lβ was generated in this laboratory.
PCR The ICE cDNA was obtained by RT-PCR, based on the published sequence (8a).
RT-PCR was performed as described (16) for 30 cycles at 95°C for 1 minute and
30 seconds, 55°C for 1 minute and 30 seconds and 72°C for 1 minute and 30 seconds.
Oligonucleotides were obtained from Duotech (Milan, Italy). The sequences of oligos uses to selectively amplify ICE were as follows: 'TOR" ICE 1 : 5 '-AAAAGCCATGGCCGACAAGGTC- 3 ' (SEQ ID NO: 9)
"REV" ICE 2: 5' -TCTCTTCACCCTGCCCACAGAC- 3' (SEQ ED NO: 10).
RESULTS
Expression of recombinant ICE enzyme in COS cells cDNA coding for human ICE has been amplified by PCR The sequence was confirmed and the cDNA was subcloned into pSG5 expression vector. COS cells were transfected with empty vector (mock) or vector containing ICE cDNA and after 48 hours, cells were analysed for the expression of ICE specific mRNA. As shown in figure
3, COS cells, transfected with ICE cDNA, expressed high levels of ICE mRNA.
A monocyte lysate, prepared as previously described (11), was also able to convert pIL-lβ into a mature form (Figure 2). Therefore, recombinant ICE or freshly isolated human monocytes were used as a source of ICE enzymatic activity for further experiments.
Inhibition of ICE activity. A source of ICE activity was prepared and its ability to cleave pDL-lβ was tested by incubating the reaction at 37°C for 1 hour. The mixture was then run on SDS -PAGE and the presence of pILl-β or the mature form was evidenced by Western Blotting.
Four different peptides were designed, synthesised and tested for their ability to inhibit ICE activity. These peptides are indicated in Figure 3 and were obtained by sohd phase synthesiser from Apphed Bio systems (Foster City, CA). Purity of these peptides was verified by high pressure liquid chromatography.
The tetrapeptide B (Bachem, Bubendorf, Switzerland) reported in Figure 3, which is a known ICE inhibitor (la), was used as positive control.
References
1. a) Thornberry et al, A novel cysteine protease is required for interleukin- lβ processing in monocytes, Nature, 356, 768-774, 1990; b) Ceretti et la., Molecular cloning of the interleukhi-lβ converting enzyme, Science, 256, 97-100, 1992;
2. Cameron et al., Amino acid sequence analysis of human IL-1. Evidence for biochemically distinct forms of DL-1, J. Exp. Med. , 162, 790-801, 1985;
3. Mosley et al., The interleukin- 1 receptor binds the human interleukin- lcc precursor but not the interleukin- lβ precursor, J. Bio Chem. , 262, 2941-2944, 1987; 4. Kostura et al., Identification of a monocyte specific pre-interleukin-lβ convertase activity, Proc. Natl Acad. Sci. USA , 86, 5227-5231, 1989;
5. Black et al., Activation of interleukin- lβ by a co-induced protease, FEBS Lett, 421, 386-390, 1989;
6. Howard et al., EL-1 converting enzyme requires aspartic acid for processing of the D^-lβ precursor at two distinct sites and does not cleave 31 kDa IL-lα , J.
Immunol., 147, 2964-2969, 1991;
7. Griffin et al., Ill Int. J. Mass. Spectrom. Ion. Phys., 11, 131-149, 1991;
8. Miura et al., Induction of apoptosis in fibroblasts by interleukin- lβ-converting enzyme, a mammalian homologue of the eiegans cell death gene ce -3, Cell, 75, 653- 660, 1993.
9. Dinarello, Interleukin- 1 and interleukin- 1 antagonism, Blood, 77, 1627-1652, 1991; lO.Colotta et al., Interleukin- 1 type II receptor: a decoy target for IL-1 that is regulated by IL-4, Science, 261, 472-475, 1993; 11. Sims et al., Interleukin- 1 signalling occurs exclusively via the type I receptor , Proc. Natl Acad. USA, 90, 6155-6159, 1993;
12.Colotta et al., Immunol. Today, 15, 562-566, 1994;
13.Hannum et al., Interleukin- 1 receptor antagonist activity of a human interleukin- 1 inhibitor, Nature, 343, 336-340, 1990; 14.Eisenberg et al, Primary structure and functional expression form complementary DNA of a human interleukin- 1 receptor antagonist, Nature, 343, 341-346, 1990; -il¬
ls. Carter et al., Purification, cloning, expression and biological characterisation of an interleukin- 1 receptor antagonist protein, Nature, 344, 633-638, 1990; lό.Muzio et al., Cloning and characterisation of a new isoform of mterleukin- 1 (IL-1) receptor antagonist (ILlra), J. Exp. Med. , 182623, 1995; 17. Yuan et al., The C. eiegans cell death gene ced-3 encodes a protein similar to mammahan interleukin- lβ- converting enzyme, Cell, 75, 641-652, 1993; 18.Kumar et al., Induction of apoptosis by the mouse nedd2 gene, which encodes a protein similar to the product of the C. eiegans cell death gene ced-3 and the mammaUan interleukin- lβ- converting enzyme, Genes Dev. 8, 1613-1626, 1994; 19.Wang et al, Ich-1, an 7ce/W'-3-related gene, encodes both positive and negative regulators of programmed cell death, Cell, 78, 739-750, 1994; 20.Fernandes-Alnemri et al, CPP-32, a novel human apoptotic protein with homology to Caenotiiaθditis eiegans cell death protein CDE-3 and mammahan interleukin- lβ- converting enzyme, J. Biol Chem., 269, 30761-30764, 1994; 21. Nicholson et al, Identification and inhibition of ICE/CED-3 protease necessary for mammahan apoptosis, Nature, 376, 37-43, 1995; 22.Tewari et al., Yama/CPP-32β, a mammahan homologue of CED-3, is a CmA- inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase, Cell, 81, 801-809, 1995; 23.Faucheu et al., A novel human protease similar to the interleukin- lβ-converting enzyme induces apoptosis in transfected cells, EMBO J., 14, 1914-1922, 1995; 24.Kamens et al., Identification and characterisation of ICH-2, a novel member of the interleukin- lβ-converting enzyme family of cysteine proteases, J. Biol. Chem., 270, 15250-15256, 1995; 25.Munday et al., Molecular cloning and proapoptotic activity of ICE rel-II and ICE rel- πi, members of the ICE/CED-3 family of cysteine proteases, J. Biol. Chem., 270, 15870-15876, 1995; 26.Fernandes-Alnemri et al., Mch-2, a new member of the apoptotic CED-3/ICE cysteine protease gene family, Cancer Res., 55, 2737-2742, 1995; 27.Duan et al., ICE-LAP3, a novel mammahan homologue of the Caenorfϊaδditis eiegans cell death protein CED-3, is activated during Fas- and TNF-induced apoptosis, J.
Biol. Chem., 2271, 35013-35035, 1996; 28.Fernandes-Alnemri et al., Mch-3, a novel human apoptotic cysteine protease highly related to CPP-32, Cancer Res., 55, 6045-6052, 1995;
29.Lippke etal., Identification and characterisation of CPP-32/Mch-2 homologue 1, a novel cysteine protease similar to CPP-32, J. Biol. Chem., Ill, 1825-1828, 1996; 30.Duan et al., ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B, J. Biol. Chem. , in press 1996 31.Muzio et al., FLICE a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95(Fas/Apo-l) Death-Inducing Signalling Complex, Cell, 85, 817-
827, 1996; 32.Boldin et al, Involvement of MACH, a novel MORT-1/FADD-interacting protease, in Fas/Aρo-1- and TNF receptor-induced cell death, Cell, 85, 803-815, 1996.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.
(B) STREET: 14, JOHN B. GORSIRAWEG
(C) CITY: CURACAO
(E) COUNTRY: THE NETHERLANDS ANTILLES
(F) POSTAL CODE (ZIP) : NONE
(G) TELEPHONE: 599-9-639300 (H) TELEFAX: 599-9-614129
(ii) TITLE OF INVENTION: ICE INHIBITING PEPTIDES (iii) NUMBER OF SEQUENCES: 10 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS :
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATIONS
(D) OTHER INFORMATION: /note= "Xaa is amino acid preferably selected between Asp and Ala. More preferably, it is Asp." (ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /no e= "Xaa is an amino acid preferably selected between Asp and Ala. More preferably, it is Asp." (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1:
Ala Xaa Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Glu Gly Glu Asp 1 5 10 15
Asn Ala Xaa
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Asp Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Glu Gly Glu Asp 1 5 10 15
Asn Ala Asp
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ala Ala Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Glu Gly Glu Asp 1 5 10 15
Asn Ala Ala (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Ala Leu Ala Asp Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Glu 1 5 10 15
Gly Glu Asp Asn Ala Asp Ser Lys 20
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Ala Leu Ala Ala Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Glu 1 5 10 15
Gly Glu Asp Asn Ala Ala Ser Lys 20
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Glu Gly Glu Asp Asn Ala Asp Ser Lys
1 5
(2) INFORMATION FOR SEQ ID NO : 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Phe Lys Asp Pro His Gly Leu Trp Lys Gly Leu Ser His 1 5 10
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI- SENSE: NO (ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATIO :1
(D) OTHER INFORMATION :/note= "Xaa is acetyl . "
(ix) FEATURE:
(A) NAME/KEY: Modified-site ( B ) LOCATION : 6
(D) OTHER INFORMATION :/note= "Xaa is -CHO."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Xaa Tyr Val Ala Asp Xaa 1 5
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide" (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
AAAAGCCATG GCCGACAAGG TC 22
(2) INFORMATION FOR SEQ ID NO : 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
TCTCTTCACC CTGCCCACAG AC 22

Claims

1. A peptide capable of binding to ICE and/or to enzymes of the ICE family, which peptide consists essentially of the amino acid sequence of SEQ ID NO: l, in which Xaa is selected between Asp and Ala, optionaUy containing one or more amino acids at the N-terminal and/or C-terminal end.
2. The peptide according to claim 1, consisting essentially of the amino acid sequence of SEQ ID NO: 2.
3. The peptide according to claim 1, consisting essentially of the amino acid sequence of SEQ ID NO: 3.
4. The peptide according to claim 1, consisting essentiaUy of the amino acid sequence of SEQ ID NO: 4.
5. The peptide according to claim 1, consisting essentiaUy of the amino acid sequence of SEQ ID NO: 5.
6. Use of the peptide according to claim 1, for medical use.
7. Use of the peptide according to claim 1, for the preparation of pharmaceutical compositions active in pathologies requiring ICE inhibition and/or inhibition of enzymes of the ICE family.
8. Use according to claim 7, characterised by the fact that the pathology is selected from the group of autoimmune diseases.
9. Use according to claim 7, characterised by the fact that the pathology is selected from the group consisting of lethal bacterial and viral infections.
10. Use according to claim 7, characterised by the fact that the pathology is selected from the group consisting of inflammatory diseases.
PCT/EP1996/004738 1996-10-31 1996-10-31 Ice inhibiting peptides WO1998018823A1 (en)

Priority Applications (21)

Application Number Priority Date Filing Date Title
US09/297,369 US6380162B1 (en) 1996-10-31 1996-10-31 Ice inhibiting peptides
BR9612755-4A BR9612755A (en) 1996-10-31 1996-10-31 Ice-inhibiting peptides
KR10-1999-7003693A KR100502211B1 (en) 1996-10-31 1996-10-31 ICE Inhibiting Peptides
AT96937313T ATE277079T1 (en) 1996-10-31 1996-10-31 ICE INHIBITING PEPTIDES
EP96937313A EP0938500B1 (en) 1996-10-31 1996-10-31 INTERLEUKIN-1beta-CONVERTING ENZYME (ICE) INHIBITING PEPTIDES
JP51995298A JP4018154B2 (en) 1996-10-31 1996-10-31 ICE inhibitory peptide
IL12966996A IL129669A0 (en) 1996-10-31 1996-10-31 Ice inhibiting peptides
ES96937313T ES2225893T3 (en) 1996-10-31 1996-10-31 PEPTIDES THAT INHIBIT THE CONVERTER ENZYME OF INTERLEUQUINA-1BETA (ICE).
EA199900432A EA001866B1 (en) 1996-10-31 1996-10-31 Peptide capable of binding to ice, and pharmaceutical compositions based thereon
PT96937313T PT938500E (en) 1996-10-31 1996-10-31 ICE INHIBITOR PEPTIDES
SI9630696T SI0938500T1 (en) 1996-10-31 1996-10-31 INTERLEUKIN-1beta-CONVERTING ENZYME (ICE) INHIBITING PEPTIDES
DK96937313T DK0938500T3 (en) 1996-10-31 1996-10-31 Peptides inhibiting interleukin-1 beta-converting enzyme (ICE)
UA99052981A UA70291C2 (en) 1996-10-31 1996-10-31 PEPTIDE CAPABLE OF BINDING TO INTERLEUKIN-1b-CONVEPEPTIDE CAPABLE OF BINDING TO INTERLEUKIN-1b-CONVERTING FERMENT (ICE) AND A PHARMACEUTICAL COMPOSITIRTING FERMENT (ICE) AND A PHARMACEUTICAL COMPOSITION CONTAINING IT ON CONTAINING IT
CA002269638A CA2269638C (en) 1996-10-31 1996-10-31 Ice inhibiting peptides
AU27023/99A AU729946C (en) 1996-10-31 Ice inhibiting peptides
EEP199900178A EE04782B1 (en) 1996-10-31 1996-10-31 Interleukin-1ß Converting Enzyme Peptide, Its Use and Pharmaceutical Composition
DE69633463T DE69633463T2 (en) 1996-10-31 1996-10-31 ICE INHIBITING PEPTIDES
PCT/EP1996/004738 WO1998018823A1 (en) 1996-10-31 1996-10-31 Ice inhibiting peptides
ARP970105024A AR011269A1 (en) 1996-10-31 1997-10-29 PEPTIDO ABLE TO BIND THE BETA CONVERTERING ENZYME OF INTERLEUKIN-1 (ICE), ITS USE TO PREPARE A MEDICINAL PRODUCT AND PHARMACEUTICAL COMPOSITION THAT INCLUDES IT.
ZA979712A ZA979712B (en) 1996-10-31 1997-10-29 ICE inhibiting peptides
NO19992079A NO323517B1 (en) 1996-10-31 1999-04-29 ICE-inhibiting peptides and pharmaceutical compositions comprising such, as well as the use of the peptides for drug preparation.

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP1996/004738 WO1998018823A1 (en) 1996-10-31 1996-10-31 Ice inhibiting peptides

Publications (2)

Publication Number Publication Date
WO1998018823A1 true WO1998018823A1 (en) 1998-05-07
WO1998018823A8 WO1998018823A8 (en) 1999-06-17

Family

ID=8166387

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1996/004738 WO1998018823A1 (en) 1996-10-31 1996-10-31 Ice inhibiting peptides

Country Status (20)

Country Link
US (1) US6380162B1 (en)
EP (1) EP0938500B1 (en)
JP (1) JP4018154B2 (en)
KR (1) KR100502211B1 (en)
AR (1) AR011269A1 (en)
AT (1) ATE277079T1 (en)
BR (1) BR9612755A (en)
CA (1) CA2269638C (en)
DE (1) DE69633463T2 (en)
DK (1) DK0938500T3 (en)
EA (1) EA001866B1 (en)
EE (1) EE04782B1 (en)
ES (1) ES2225893T3 (en)
IL (1) IL129669A0 (en)
NO (1) NO323517B1 (en)
PT (1) PT938500E (en)
SI (1) SI0938500T1 (en)
UA (1) UA70291C2 (en)
WO (1) WO1998018823A1 (en)
ZA (1) ZA979712B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2007016032A (en) * 2005-06-21 2008-03-10 Xoma Technology Ltd Il-1 beta binding antibodies and fragments thereof.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012022A1 (en) * 1994-10-13 1996-04-25 Applied Research Systems Ars Holding N.V. Intracellular isoform of the interleukin-1 receptor antagonist

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981713A (en) * 1994-10-13 1999-11-09 Applied Research Systems Ars Holding N.V. Antibodies to intereleukin-1 antagonists

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012022A1 (en) * 1994-10-13 1996-04-25 Applied Research Systems Ars Holding N.V. Intracellular isoform of the interleukin-1 receptor antagonist

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MUZIO M ET AL: "CLONING AND CHARACTERIZATION OF A NEW ISOFORM OF THE INTERLEUKIN 1 RECEPTOR ANTAGONIST", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 182, no. 2, 1 August 1995 (1995-08-01), pages 623 - 628, XP000564500 *
NICHOLSON D W ET AL: "IDENTIFICATION AND INHIBITION OF THE ICE/CED-3 PROTEASE NECESSARY FOR MAMMALIAN APOPTOSIS", NATURE, vol. 376, no. 6535, 6 July 1995 (1995-07-06), pages 37 - 43, XP000574812 *

Also Published As

Publication number Publication date
NO992079L (en) 1999-06-16
PT938500E (en) 2005-02-28
EP0938500B1 (en) 2004-09-22
SI0938500T1 (en) 2005-02-28
EA199900432A1 (en) 1999-10-28
KR100502211B1 (en) 2005-07-19
DE69633463D1 (en) 2004-10-28
EA001866B1 (en) 2001-10-22
IL129669A0 (en) 2000-02-29
JP4018154B2 (en) 2007-12-05
ZA979712B (en) 1999-02-05
CA2269638C (en) 2005-01-18
CA2269638A1 (en) 1998-05-07
NO323517B1 (en) 2007-06-04
EP0938500A1 (en) 1999-09-01
UA70291C2 (en) 2004-10-15
DE69633463T2 (en) 2006-02-23
KR20000052858A (en) 2000-08-25
AU2702399A (en) 1999-08-16
BR9612755A (en) 1999-10-19
AU729946B2 (en) 2001-02-15
EE04782B1 (en) 2007-02-15
AR011269A1 (en) 2000-08-16
WO1998018823A8 (en) 1999-06-17
US6380162B1 (en) 2002-04-30
ES2225893T3 (en) 2005-03-16
DK0938500T3 (en) 2005-01-17
EE9900178A (en) 1999-12-15
JP2001510452A (en) 2001-07-31
NO992079D0 (en) 1999-04-29
ATE277079T1 (en) 2004-10-15

Similar Documents

Publication Publication Date Title
Ghayur et al. Caspase-1 processes IFN-γ-inducing factor and regulates LPS-induced IFN-γ production
Van de Craen et al. Identification of a new caspase homologue: caspase-14
Stennicke et al. Caspases–controlling intracellular signals by protease zymogen activation
Zou et al. Molecular cloning of interleukin 1β from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site
Weining et al. A chicken homolog of mammalian interleukin‐1β: cDNA cloning and purification of active recombinant protein
Bank et al. Evidence for a crucial role of neutrophil-derived serine proteases in the inactivation of interleukin-6 at sites of inflammation
KR100682185B1 (en) Preparation of biologically active molecule
Miller et al. The interleukin‐1β converting enzyme family of cysteine proteases
EP0786002A1 (en) Intracellular isoform of the interleukin-1 receptor antagonist
CA2412730C (en) Method for preparing a physiologically active il-18 polypeptide
AU747005B2 (en) Modulators of the function of receptors of the TNF/NGF receptor family
AU729946C (en) Ice inhibiting peptides
EP0938500B1 (en) INTERLEUKIN-1beta-CONVERTING ENZYME (ICE) INHIBITING PEPTIDES
AU2001280442A1 (en) Method for preparing a physiologically active IL-18 polypeptide
US20020034812A1 (en) Caspase homologue
KR100496001B1 (en) Method of Making Polypeptides
MXPA99004046A (en) Ice inhibiting peptides
Johnson et al. Characterization of the avian Ich-1 cDNA and expression of Ich-1L mRNA in the hen ovary
Wardlow et al. Nucleotide sequence of equine caspase-1 cDNA
Young et al. Molecular characterization of interleukin-1Beta in the tammar wallaby (Macropus eugenii)
JP4010606B2 (en) Method for producing polypeptide
WO2003072724A2 (en) Human analogs of murine deubiquitinating protease genes
Velotti et al. SERINE ESTERASES IN NORMAL AND INTERLEUKIN-2 ACTIVATED RAT NK CELLS

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 96180489.0

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): BR CA CN EE IL JP KR LT LV MX NO SG SI UA US AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 27023/99

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2269638

Country of ref document: CA

Ref document number: 2269638

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1019997003693

Country of ref document: KR

ENP Entry into the national phase

Ref document number: 1998 519952

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: PA/a/1999/004046

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 1996937313

Country of ref document: EP

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 199900432

Country of ref document: EA

AK Designated states

Kind code of ref document: C1

Designated state(s): AU BR CA CN EE IL JP KR LT LV MX NO SG SI UA US

AL Designated countries for regional patents

Kind code of ref document: C1

Designated state(s): AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: PAT. BUL. 18/98 UNDER (81) ADD "AU"; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

WWE Wipo information: entry into national phase

Ref document number: 09297369

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1996937313

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1019997003693

Country of ref document: KR

WWG Wipo information: grant in national office

Ref document number: 27023/99

Country of ref document: AU

WWG Wipo information: grant in national office

Ref document number: 1996937313

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1019997003693

Country of ref document: KR