+

WO1997048728A1 - Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6 - Google Patents

Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6 Download PDF

Info

Publication number
WO1997048728A1
WO1997048728A1 PCT/NL1997/000345 NL9700345W WO9748728A1 WO 1997048728 A1 WO1997048728 A1 WO 1997048728A1 NL 9700345 W NL9700345 W NL 9700345W WO 9748728 A1 WO9748728 A1 WO 9748728A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
peptides
amino acids
receptor
amino acid
Prior art date
Application number
PCT/NL1997/000345
Other languages
English (en)
Inventor
Leonardus Andrianus Maria Govardus VAN LEENGOED
Kasper Hubertus Nicolaas Hoebe
Original Assignee
Koster, Henk, Wilhelmus
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
Application filed by Koster, Henk, Wilhelmus filed Critical Koster, Henk, Wilhelmus
Priority to AU31930/97A priority Critical patent/AU3193097A/en
Priority to EP97927475A priority patent/EP0928293A1/fr
Priority to JP10502722A priority patent/JP2000516574A/ja
Publication of WO1997048728A1 publication Critical patent/WO1997048728A1/fr

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
    • C07K14/705—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]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of cytokines.
  • Cytokines are substances that are produced by cells of the immune system and are involved in regulation of humoral and cellular immune reactions and inflammatory 5 responses. Many cytokines are known, and all exert influence on various reactions in the body in a complicated fashion. To illustrate their interdependency and the intricate web of relationships that exist between cytokines, one often speaks about the "cytokine network”.
  • Interleukine 6 IL-6 is a cytokine which has many effects upon mammalian cells.
  • a specific cell surface receptor that consists of a specific ⁇ -subunit of with a molecular weight of approximately 80 kD and a common ⁇ -subunit of 5 approximately 130 kD, also named gpl30.
  • the gpl30 ⁇ chain is also involved in signal transduction of interleukin-11 (IL-11) , leukemia inhibitory factor (LIF) , oncostatin M (OM) , ciliairy neurotrophic factor (CNTF) , and cardiotrophin-1 (CT-1) (P.B. Sehgal, Ling Wang, Ravi 0 Rayanade et al . , pp 1-14; volume 762, Annals of the New York Academy of Sciences; 1995) .
  • IL-11 interleukin-11
  • LIF leukemia inhibitory factor
  • OM oncostatin M
  • CNTF ciliairy neurotrophic factor
  • CT-1 cardiotrophin-1
  • IL-6 is an extremely pleiotropic cytokine, and its activities include: induction of Ig production by B cells, stimulation of B and T cell growth, differentia- 5 tion of T cells and macrophages, induction of acute phase protein production by hepatocytes, multilineage hematopoesis, osteoclast formation, maturation of megakaryocytes, and platelet production.
  • IL-6 also effects the central nervous system: IL-6 is an endogenous 0 pyrogen and can induce ACTH production by the pituitary, finally resulting in increased glucocorticoid levels in the circulation. IL-6 exerts its activity by triggering a transmembrane receptor that is present on all target cells.
  • IL-6 signaling cascade Specific steps in the IL-6 signaling cascade are the binding to the low affinity ⁇ -chain (CD126) .
  • the complex of IL-6 and ⁇ -chain binds with the high affinity signal transducing ⁇ -chain (GP130, CD130) .
  • GP130, CD130 high affinity signal transducing ⁇ -chain
  • IL-6 levels are increased in various diseases, and it is postulated that these increased levels play a causative role in the pathogenesis of these diseases.
  • Examples of diseases where increased levels of IL-6 are found are multiple myeleoma, AIDS lymphoma, polyclonal B cell activation as observed in AIDS, rheumatoid arthritis, cardiac myxoma and Castleman's disease, mesangial proliterative glomerulonephritis, psoriasis, cancer- associated cachexia, postmenopausal osteoporosis, sepsis, multiple system organ failure, alcohol cirrhosis, and diseases of the central nervous system like Alzheimer, among others.
  • Evidence for the causative role of IL-6 in the pathogenesis of some of the above mentioned diseases has come from from phase I/II clinical trials with IL-6 neutralizing monoclonal antibodies.
  • Treatment with anti- IL-6 monoclonal antibodies reversed fever, acute phase proteins, night sweats, bone destruction, and cachexia.
  • Treatment of a patient with Castleman disease with anti- IL-6 monoclonal antibodies reduced acute phase protein levels, fever, anemia, thrombocytosis, and hypergamma- globulinemia. Improvement of patients was also observed in patients with rheumatoid arthritis. Apparently, reduction of IL-6 activity in these patients resulted in improvement of the clinical signs of their disease.
  • monoclonal antibodies directed to IL-6.
  • monoclonal antibodies are usually not of human origin and repeated administration of non-human monoclonal antibodie generally leads to immune responses against the constant part of the antibodies, since this is foreign to the body of the patient.
  • This immunereaction to the monoclonal antibodies used in the treatment is, first of all, counterproductive to the therapeutic treatment itself.
  • the monoclonal antibodies used will be rendered ineffective by the reaction with the antibodies produced by the immune system.
  • repeated administration of non-human monoclonal antibodies may elicit such severe immune reactions that they will be detrimental to the patient.
  • Inhibitors or antagonists based upon mutagenesis of IL-6 have also been proposed, such as IL-6.Q160E /T163P (Brakenhoff, J., de Hon,F., Fontaine, V., et al; J.Biol.Chem. ; 269:86-93 (1994)) , and IL-6.Q159E/T162P (Ehlers, M., de Hon, D. , Klaasse Bos, H, et al . , J. Biol. Chem. ; 270:8158-8163 (1995)) . It has been shown with these mutant proteins that receptor binding of IL-6 and signal transduction of IL-6 can be separated in vitro.
  • mutant proteins are also foreign to the body of the patient to be treated and will also elicit an unwanted and unfavourable immune response that generally is detrimental to the treatment.
  • mutant proteins may only be partly effective, in that, although they may effectively block or inhibit specific IL-6 activities, at the same time they may exert other effects on the cytokine network with additional, still remaining, reactive sites present on these proteins. Therapeutic treatment with such reagents would then elicit other, yet unpredictable, side effects.
  • a great disatvantage of earlier reported mutant IL-6 and IL-6 receptor antagonists is that these molecules, instead of inhibiting IL-6 in vivo , act as carrier and increase the half-life time and result in an increase of IL-6 activity in vivo.
  • these mutant IL-6 and IL-6 receptor antagonists have a low affinity to their target molecules and will likely act as an immunogen.
  • antibodies raised to IL-6 stabilize IL-6 and result in an increased IL-6 production. Accumulation of circulating IL-6 as stable IL-6-anti-IL-6 complexes as a result of treatment with these antibodies to IL-6, will occur as no renal filtration can be expected. Repeated use of nonhumanized IL-6 antibodies to human patients will most likely induce antibody production to these antibodies, and result in formation of immune-complexes (Heremans,H. , Dillen,C, et al J. Immunol. 22,2395-2401) .
  • the present invention provides a solution to the above illustrated problems without hampering the possibility of therapeutic treatment of IL-6 related disease.
  • the above methods to inhibit IL-6 activity by antibodies or mutants differ greatly from the invention as described here: peptides that antagonize or agonize IL-6 at the binding site to the receptor in three ways: at the IL-6 part, at the ⁇ -receptor part, and at the gpl30-receptor part.
  • These antagonists and agonists and combinations of these antagonists and/or agonists as multimeric peptides or as single peptides with defined pharmacokinetic characteristics gives a powerful tool to manage IL-6 bioactivity.
  • immune responses to the treatment do not occur. Further, the occurence of unpredictable side effects is greatly minimized.
  • the invention provides synthetic peptides that interact with the receptor site of IL-6 or with IL-6 receptors ( ⁇ and ⁇ ) present at target cells.
  • the invention further provides synthetic peptides that, when combined, interact with the receptor site of IL-6 as well as with IL-6 receptors ( ⁇ and ⁇ ) present at target cells.
  • a mixture of these peptides is particulary valuable as the pharmacological properties of the peptides can be adjusted to obtain a maximal desired effect.
  • half-life time can be prolonged by inserting unnatural amino-acids into the synthetic peptides.
  • the antagonizing or agonizing activity of the peptides is increased by producing di- or multi-meric peptides directed to one or more receptor sites.
  • Such di- or multimeric peptides can for instance be made by linking the peptides via one or more amino-acids such as lysine (Tarn, PNAS 1988, 85: 5409-5413) .
  • the distribution of the peptides into target organs can be optimized by adjusting the hydrophilic or lipophylic nature of peptides or by binding of these peptides onto peptides that interact with specific organ markers.
  • the peptides provided can be bound onto the solid phase of membranes or filters that are connected into an extra- corporal blood circulation circuit of the patient . A more efficient clearance of IL-6 and/or soluble IL-6 receptors can in that way be achieved.
  • Such synthetic peptides can be derived from (A) IL- 6, or derived from (B) the receptor ⁇ -chain of IL-6 (IL- 6R ⁇ , CD126) , or from (C) the receptor ⁇ -chain of IL-6 (IL-6R ⁇ , GP130, CD130) and exhibit antagonistic and agonistic activity against the various components and steps of the IL-6 signaling cascade.
  • the peptides were found by testing sets of overlapping amino acid sequences from the published human IL6 (Hirano, T., Yasukawa, K. , Harada, H. , et al . ; Nature 324, 73-76 (1986) ; Yasukawa, R., Hirano, T.
  • Murakami, M., Saito, M. ,- Cell 63:1149-1157 (1990)) were tested in an assay for antagonistic or agonistic IL-6 activity.
  • the peptides provided by the invention all exhibit antagonistic or agonistic IL-6 activity against the IL-6 signaling cascade as measured in an IL-6 assay.
  • the peptides of the present invention are too small to generate immune responses. Further, they are too short to contain additional reactive sites, so that the antagonistic and, in addition, the agonistic peptides can advantageously be used to treat patients to counteract and adjust elevated IL-6 levels.
  • amino acids in all antagonistic or agonistic peptides described below are identified by the one letter code, in which the N- terminal (head) amino acid is listed first (on the left) and the C-terminal (tail) amino acid is listed last (on the right) .
  • the antagonistic peptides derived from IL-6 preferably comprise at least 5 consecutive amino acids selected from the following 3 regions that were identified as RYILDGISALRK, STKVLIQFLQKKAKNL, and I- LRSFKEFLQSSLRALRQM.
  • the antagonistic peptides derived from the receptor ⁇ -chain of IL-6 preferably comprise at least 5 consecutive amino acids selected from the following 3 regions that were identified as QLSCFRKSPLSNWC,
  • the agonistic peptides derived from the receptor ⁇ -chain of IL-6 preferably comprise at least 5 consecutive amino acids selected from the following region that was identified as EWGPRSTPSLTTKAVLLVRKFQNSPAED.
  • the antagonistic peptides derived from the receptor ⁇ -chain of IL-6 preferably comprise at least 5 consecutive amino acids selected from the following 4 regions that were identified as PEKPKNLSCIVNEGKKMRCE- WDGGR, NFTLKSEWATHKFADCKAKRDTPTS, WVEAENALGKVTSDH, and PVYKVKPNPPHNLSVIN.
  • Relatively short peptides (as short as a string of 5 amino acids) that are selected from any of the above peptides, or peptides of no more than 30 amino acids long which show antagonistic or agonistic activity as measured in an IL-6 assay and have at least one string of at least 5 amino acids in common with the peptides from groups A, B or C, are also peptides of the present invention.
  • the peptides according to the invention can vary in length.
  • the peptides comprising a string of at least 5 amino acids which are in common with the peptides from groups A, B, and C can be modified by replacing one or a few amino acids in said string by other amino acids.
  • Such amino acids can be selected from any of the naturally occuring amino acids, but also amino acids that normally do not occur in nature can be used as replacement amino acid.
  • the choice of the replacing amino acid can for example be guided by comparing IL-6 or IL-6 receptor sequences from other species than humans or by selecting amino acids that lead not to extreme functional or conformational changes of the selected peptide, but also other selection methods can be used.
  • the present invention relates in a first aspect to a peptide containing at least 5 amino acids and at most 30 amino acids that exhibits antagonistic activity directed against IL-6 and/or against the ⁇ -chain of the IL-6 receptor and/or against the ⁇ -chain of the IL-6 receptor.
  • the present invention relates in another aspect to a peptide containing at least 5 amino acids and at most 30 amino acids that can exhibit antagonistic or agonistic IL-6 activity, depending on the concentration in which it is used.
  • An example of such peptides are peptides selected with as basis with the amino acid sequence EWGPRSTPSLTTKAVLLVRKFQNSPAED as found in the ⁇ - chain of the IL-6 receptor.
  • Agonistic activity was observed in the in vitro bioassay in a concentration range from 7.5 to 120 ⁇ g/ml peptide.
  • these peptides had an antagonistic effect upon the biological activity of IL-6 in the bioassay.
  • the agonistic peptides can be used in vivo in concentrations that are relatively equivalent but not necessarily the same as when used in vitro.
  • the invention provides combinations of peptides, either provided as a simple mixture of several, possibly modified, peptides selected from groups A, B or C, or, provided as, possibly modified, peptides selected from groups A, B, or C that are linked, with direct chemical bonds or using spacer molecules, head to tail, or head to head, or tail to tail, or via side chains of the amino acids present in the selected peptides.
  • Such combinations of peptides are for example using the peptides SLTTKAV and ILRSFKEFLQSS, or WVEAENALGKVTSDH and RYILD, or KAVLLVRK and KAVLLVRK, but many other combinations of two or more peptides can be selected from the peptides listed in groups A, B or C.
  • Such combinations of peptides can advantageously be used to counteract the events occuring in the IL-6 signaling cascade, such as disrupting the binding of IL-6 to the ⁇ -chain by simultanous competing at both the IL-6 and the ⁇ -chain binding site, or simultanous competing at the binding sites of the IL-6/ ⁇ -chain complex and the ⁇ -chain.
  • the peptides of the invention can suitably be used in a medicinal or pharmaceutical preparation for therapeutic or prophylactic purposes. Further, they can be used in protocols to remove circulating IL-6 from the blood of diseased patients via dialysis methods in which the peptides are bound to a solid phase. Passing blood or blood filtrates along the thus bound peptides will result in clearance of IL-6 that will bind to the peptide at the solid phase. Also the peptides according to the invention may be added to blood or blood filtrates and (ir ⁇ reversibly bind to IL-6 or IL-6 receptor molecules and thus render these inactive before they re-enter the body. Also, the peptides can be used in diagnostic tests, i.e.
  • IL-6 agonistic peptides can completely or partially replace IL-6 that is added to cell-cultures, for example IL-6 is used to grow or culture IL-6 dependant cells, like B-cel.l hybridomas to which IL-6 as growth factor is often added, bot also cell-cultures in general will benefit from the addition of agonistic IL-6 peptides.
  • the IL-6 agonistic peptides administered to humans or animals can be used to enhance the immune response of an host exposed to a specific immunogenic substance.
  • the IL-6 agonistic peptides can be administered to humans or animals to increase the responsiveness of the immune system of the host.
  • a specific use is in pharmaceutical preparations for topical or intramammary application. When these agonistic peptides are combined with IL-6 antagonists as described, excess of IL-6 can be inhibited without loss of basal IL-6 signal transduction.
  • Antibodies specifically directed against the peptides, and their corresponding anti-idiotypic antibo ⁇ dies, are part of the invention. Such antibodies can for example be administered to patients treated earlier with the peptides, to counteract the effect of the peptides on the patient. Such antibodies can be used in the above described dialysis protocols and diagnostic tests. Synthesis of the peptides may be acomplished according to the available methods in the art. The synthesis of the exemplified peptides was done according to Valerio et al . (Int. J. Peptide Res., 42:1-9 (1993) and/or Valerio et al . (Int. J. Peptide Res., 44:148-165 (1994) ) . Methods for large scale production of syntethic peptides and the purification thereof are well known in the art. The invention is illustrated in the following experimental part . Experimental part
  • the peptides of the examples which were intended for identifying active centers in the IL-6 and IL-6 receptor molecules were synthesized using a method according to Valerio et al . (Int. J. Peptide Res., 42:1-9 (1993) and/or Valerio et al . (Int. J. Peptide Res., 44:148-165 (1994)) .
  • Multimeric peptides (four branched) were synthetized by the solid-phase method and using of a dispersed system with branching oligolysines as a scaffolding for incorporation of the synthetized antagonistic peptides (Tarn, J.P.; Proc. Natl. Acad. Sci . USA, 85:5409-5413 (1988) .
  • a set of overlapping peptides, each twelve amino acids long (each consecutive peptide shifts one amino acid, so consecutive peptides have 11 amino acids in common) derived from human IL-6 sequence (Hirano, T., Yasukawa, K. , Harada, H., et al . ; Nature 324, 73-76 (1986) ; Yasukawa, R., Hirano, T., Watanabe Y., et al . ; EMBO J. 6:2939-2945 (1987) , were incubated with cells (B9) at 37°c. After one hour, recombinant human IL-6 (CLB, Amsterdam, The Netherlands) was added at 3 different concentrations (2.5 U/ml, 5 U/ml and 10 U/ml) .
  • a set of overlapping peptides, each twelve amino acids long (each consecutive peptide shifts one amino acid, so consecutive peptides have 11 amino acids in common) derived from human IL6Ra (Yamasaki, K. , Taga, T., Hirata, Y., et al . ; Science 241:825-828 (1988)) or gpl30 (Hibi, M. , Murakami, M., Saito, M.
  • B9 mouse hybridoma cells were collected during their logarithmic growth phase in IL-6 free media and suspended at a concentration of 1*10 5 cells/ml in DMEM+HT medium containing 5% FCS. Fifty ⁇ l of each IL-6 dilution was combined with each of the synthetized peptides representing IL-6 sequences and incubated for 1 hour at 37'C. This mixture was added in duplicate to 50 ⁇ l of the B9 cell suspension in flat-bottommed 96-well tissue culture plates (Greiner) and incubated at 37 °c and 5% CO2 for 72 h.
  • IL-6 activity was assessed by using 3- (4,5- dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT, Sigma) . After addition of 25 ⁇ l of MTT (5mg/ml dissolved in PBS) to each well and further incubation at 37 °c for 4 h, 100 ⁇ l of lysis buffer (20% w/v SDS in 50 % dimethyl formamide) was added. Thereafter, incubation was continued over night at 37'c and the next morning absor-schule was read at 578 nm.
  • MTT 3- (4,5- dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide
  • Toxi.oity testing of peptiHP>B Three separate test were performed to determine whether the synthetized peptides excert toxic effect in vitro upon erythrocytes (A) , or polymorphonuclear cells (B) , or hepatocytes (C) .
  • SRBC Sheep red blood cells
  • SRBC Sheep red blood cells
  • a 1% (v/v) suspension of erythrocytes was prepared in veronal-buffered saline that contained gelatin (GVS: 0.032% gelatin in 3.9 mM barbitone sodium, 1 mM MgS0 4 , 0.38 mM CaCl 2 , and 145.6 mM NaCl) .
  • GRS veronal-buffered saline that contained gelatin
  • GRS 0.032% gelatin in 3.9 mM barbitone sodium, 1 mM MgS0 4 , 0.38 mM CaCl 2 , and 145.6 mM NaCl
  • Twofold dilutions of the synthetized peptides 50 ⁇ l were made in U-shaped microtiter plates (Greiner Labortechnik) and
  • Porcine polymorphonuclear cells were isolated from pig blood (Cruijsen, T.L.M., Van Leengoed, L.A.M.G. et al.; Infect. Immun. 60:4867-4871 (1992)) . Twofold dilutions of the synthetized peptides (50 ⁇ l) were made in flat-bottomed microtiter plates (Greiner Labortechnik) and 50 ⁇ l of the PMN suspension (2*10 6 cells/ml) were added to each well. Plates were sealed, gently mixed and incubated for 6 hours at 37° and 5% CO2 • Thereafter, plates were examined for cytotoxicity by nigrosine dye exclusion. Non of the synthetized peptides was toxic for PMN.
  • Porcine hepatocytes were isolated from liver of pigs based on Seglens 1 method (Seglen, P.O.; Methods Cell Biol 13:29-83 (1976)) and adapted according to Monshouwer M. , et al . (Toxicol . Applied Pharmacol . in press) . Hepatocytes were suspended in Williams' medium E to a concentration of 10 6 cells/ml. From this suspension 1.5 ml was put into each well of 12-well tissue culture plates (Costar) and incubated for 12 h at 37 * C. Adherent hepatocytes were examined for their viability and nonadherent hepatocytes were discarded.
  • Each synthetized peptide was mixed with Williams' medium E (at dilutions of 1:50 and 1:100) and added to wells with adherent hepatocytes. After another 24 h incubation at 37"C viability was assessed by using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT, Sigma) . After addition of 1.5 of MTT (1- mg/ml dissolved in Williams' medium E) to each well and further incubation at 37'c for 30 min, 1 ml of lysis buffer (0.8 M HCL in isopropanol) was added. Thereafter, plates were mixed for 10 min and absorbance was read at 560 nm. None of the synthetized peptides proved to affect the viability of the hepatocytes.
  • Porcine hepatocytes were isolated from liver of pigs based on Seglens ' method (Seglen, P.O.; Methods Cell Biol 13:29-83 (1976)) and adapted according to Monshouwer M. , et al . (Toxicol. Applied Pharmacol.in press) . Hepatocytes were suspended in Williams' medium E to a concentration of 10 6 cells/ml. From this suspension 1 ml was put into each well of 12-well tissue culture plates (Costar) and incubated for 12 h at 37'C. Adherent hepatocytes were examined for their viability and nonadherent hepatocytes were discarded.
  • Each synthetized peptide was mixed (at dilutions of 1:50 and 1:100) with Williams' medium E containing IL-6 (1000 U/ml) and added to wells with adherent hepatocytes. Also a negative (containing no IL-6 and without synthetized peptides in the medium) and positive control (containing 1000 U/ml IL-6 in the medium) were prepared and tested. After an incubation period of 24 hours, the medium was removed and for each well CYP450 dependent enzyme activity of intact monolayers of hepatocytes was determined. CYP450 enzym assay.
  • CYP450 dependent enzym activity using testosterone (250 ⁇ M) as substrate, was determined as previously described by Van 't Klooster et al . (Bioch. Pharmacol .46,-1781-1790 (1993)) . Briefly, testosteron was mixed with Williams' medium E without fetal calf serum and added to the wells with hepatocytes. After 30 min incubation at 37 'C and 5%C ⁇ 2/ hydroxylated testosteron metabolites in the medium were quantified by HPLC.
  • the mobile phase consisted of buffer A (12% methanol, 75% milli Q water) and buffer B (64% methanol, 6% acetonitril, 30% milli Q water) . With these buffers an elution gradient was generated; 10-58% B from 0-45 minutes; 58-59% from 45-50 minutes; 59-10% from 50-53 minutes, with a flow rate of 0,8 ml/min. Metabolites were detected spectrofotometrically at 254 nm.
  • Inhibition of IL-6 dependant downregulation of cytochrome P450 was determined by comparing the relative concentration of hydroxylated testosteron metabolites in medium from adherent hepatocytes incubated with synthetized peptides and IL-6, and the relative concentration of hydroxylated testosteron metabolites in medium from positive and negative control hepatocyte monolayers .
  • results Peptides derived from hIL-6, hgpl30 (the ⁇ -chain of the IL-6 receptor) and hIL6Ra (the ⁇ -chain of the IL-6 receptor) were analysed for antagonistic IL-6 activity.
  • hIL-6 peptides 3 regions were identified which inhibited IL-6 activity in an IL-6 assay (fig. 2) .
  • Peptide 31, 119-123 and 167-174 represent the identified regions (RYILDGISALRK, resp. STKVLIQFLQKKAKNL, resp. ILRSFKEFLQSSLRALRQM) .
  • hIL6Ra For hIL6Ra, also 3 regions were identified which inhibited IL-6 activity in an IL-6 asay (fig. 3) .
  • Peptide 6-9, 24-33 and 80-89 represent the identified regions (QLSCFRKSPLSNWC, resp. PRSTPSLTTKAVLLVRKFQNS, resp. MCVASSVGSKFSKTQTFQGC) .
  • the identified peptides with anti-IL-6 activity were not lytic for erythrocytes and not toxic for polymorpho- nuclear cells and not toxic for primary hepatocyte culture cells.
  • Peptide 21-37 represent the region EWGPRSTPSLTTKAVLLVRKFQNSPAED of the IL-6Ra sequence
  • Agonistic activity was observed in a concentration range from 7.5 to 120 ⁇ g/ml peptide. These peptides induced prolife rative growth of the IL-6 dependant cell line B9, and when combined with IL-6 enhanced proliferation of the B9 cell line was examined, and thus the biological activity of IL-6 was enhanced. At a concentration of j_ ⁇ l20 ⁇ g/ml these agonistic peptides had an antagonistic effect upon the biological activity of IL-6. The identified peptides with agonistic IL-6 activity were not lytic for erythrocytes and not toxic for polymorphonuclear cells and not toxic for primary hepatocyte culture cells.
  • Synthetized peptides from the regions PVYKVKPNPP ⁇ HNLSVIN, WVEAENALGKVTSDH, and MCVASSVGSKFSKTQTFQGC inhibit IL-6 regulated downregulation of cytochrome P-450 of hepatocytes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention porte sur des peptides dérivés de l'IL-6 et du récepteur de l'IL-6 et présentant une activité agoniste ou antagoniste vis-à-vis de l'IL-6. Lesdits peptides ont une longueur d'au moins cinq acides aminés sélectionnés parmi les séquences: RYILDGISALRK, STKVLIQFLQKKAKNL, ILRSFKEFLQSSLRALRQMQLSCFRKSPLSNVVC, PRSTPSLTTKAVLLVRKFQNS, MCVASSVGSKFSKTQTFQGC, PEKPKNLSCIVNEGKKMRCEWDGGR, NFTLKSEWATHKFADCKAKRDTPTS, WVEAENALGKVTSDH, EWGPRSTPSLTTKAVLLVRKFQNSPAED or PVYKVKPNPPHNLSVIN. Les peptides sélectionnés et leurs combinaisons peuvent servir dans le traitement, la prévention, la détection ou le diagnostic de maladies liées à l'IL-6 ou à épurer le sang ou les produits sanguins des molécules d'IL-6 ou de leurs récepteurs.
PCT/NL1997/000345 1996-06-20 1997-06-19 Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6 WO1997048728A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU31930/97A AU3193097A (en) 1996-06-20 1997-06-19 Il-6 and il-6-receptor derived peptides having il-6 antagonistic or agonistic activity
EP97927475A EP0928293A1 (fr) 1996-06-20 1997-06-19 Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6
JP10502722A JP2000516574A (ja) 1996-06-20 1997-06-19 Ｉｌ―６アンタゴニストまたはアゴニスト活性を有するｉｌ―６およびｉｌ―６レセプター誘導ペプチド

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP96201720 1996-06-20
EP96201720.8 1996-06-20

Publications (1)

Publication Number Publication Date
WO1997048728A1 true WO1997048728A1 (fr) 1997-12-24

Family

ID=8224097

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL1997/000345 WO1997048728A1 (fr) 1996-06-20 1997-06-19 Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6

Country Status (4)

Country Link
EP (1) EP0928293A1 (fr)
JP (1) JP2000516574A (fr)
AU (1) AU3193097A (fr)
WO (1) WO1997048728A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998036061A3 (fr) * 1997-02-13 1998-11-12 Univ Manchester Reduction de la fibrose et/ou de la cicatrisation par inhibition de l'activite induite par le recepteur de l'interleukine 6
EP0972780A1 (fr) * 1998-05-18 2000-01-19 Applied Research Systems ARS Holding N.V. Peptides antagonistes de l'Il-6
WO2001016166A3 (fr) * 1999-08-27 2001-06-21 Us Health Polypeptides comprenant des domaines de recepteur d'interleukine-6 se liant a un ligand, acides nucleiques correspondants, anticorps, compositions, et leurs procedes d'utilisation
EP1218404A1 (fr) * 1999-09-15 2002-07-03 Medvet Science Pty. Ltd. Motif de liaison d'un recepteur
FR2833011A1 (fr) * 2001-12-04 2003-06-06 Univ Claude Bernard Lyon Nouvelle proteine a activite inhibitrice de l'il-6
EP1334731A1 (fr) * 2000-10-25 2003-08-13 Chugai Seiyaku Kabushiki Kaisha Agents preventifs ou therapeutiques contre le psoriasis renfermant l'antagoniste de l'il-6 comme substance active
US6841533B1 (en) 1995-12-07 2005-01-11 Peptor Limited Conformationally constrained backbone cyclized interleukin-6 antagonists
WO2007060647A3 (fr) * 2005-11-25 2007-07-19 Trinity College Dublin Methode de detection de sepsie
WO2008113597A1 (fr) * 2007-03-22 2008-09-25 Helmholtz Zentrum Muenchen Deutsches Forschungszentrum Fuer Gesundheit Und Umwelt Gmbh Peptides utilisés pour traiter le myélome multiple
US8295392B2 (en) 2005-12-14 2012-10-23 Nec Corporation Digital communication system, indoor unit, and outdoor unit
WO2014174517A1 (fr) * 2013-04-25 2014-10-30 Carmel-Haifa University Economic Corp. Peptides anti-inflammatoires synthétiques et leur utilisation

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02188600A (ja) * 1989-01-17 1990-07-24 Chugai Pharmaceut Co Ltd Ｂｓｆ↓２アンタゴニスト
EP0426857A1 (fr) * 1989-02-08 1991-05-15 Kuraray Co., Ltd. Peptide et adsorbant le comprenant immobilise sur un support
US5210075A (en) * 1990-02-16 1993-05-11 Tanabe Seiyaku Co., Ltd. Interleukin 6 antagonist peptides
WO1995004075A1 (fr) * 1993-07-28 1995-02-09 Medvet Science Pty. Ltd. Antagonistes de facteurs de croissance hematopoietique
JPH07324097A (ja) * 1994-05-30 1995-12-12 Daicel Chem Ind Ltd インターロイキン６拮抗剤、及びペプチド類または医薬として許容されるその塩類
JPH07324098A (ja) * 1994-05-30 1995-12-12 Daicel Chem Ind Ltd インターロイキン６吸着材、及びペプチド類または生理学的に許容されるその塩類

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02188600A (ja) * 1989-01-17 1990-07-24 Chugai Pharmaceut Co Ltd Ｂｓｆ↓２アンタゴニスト
EP0426857A1 (fr) * 1989-02-08 1991-05-15 Kuraray Co., Ltd. Peptide et adsorbant le comprenant immobilise sur un support
US5210075A (en) * 1990-02-16 1993-05-11 Tanabe Seiyaku Co., Ltd. Interleukin 6 antagonist peptides
WO1995004075A1 (fr) * 1993-07-28 1995-02-09 Medvet Science Pty. Ltd. Antagonistes de facteurs de croissance hematopoietique
JPH07324097A (ja) * 1994-05-30 1995-12-12 Daicel Chem Ind Ltd インターロイキン６拮抗剤、及びペプチド類または医薬として許容されるその塩類
JPH07324098A (ja) * 1994-05-30 1995-12-12 Daicel Chem Ind Ltd インターロイキン６吸着材、及びペプチド類または生理学的に許容されるその塩類

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
C. MORTON ET AL.: "Solution structure of synthetic peptides corresponding to the C-terminal helix of interleukin-6.", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 219, no. 1-2, 15 January 1994 (1994-01-15), BERLIN, GERMANY, pages 94 - 107, XP000645297 *
DATABASE WPI Week 9035, Derwent World Patents Index; AN 90-266224, XP002027136 *
DATABASE WPI Week 9607, Derwent World Patents Index; AN 96-065476, XP002027135 *
DATABASE WPI Week 9607, Derwent World Patents Index; AN 96-065477, XP002027134 *
M. KALAI ET AL.: "Participation of two Ser-Ser-Phe-Tyr repeats in interleukin-6 (IL-6) binding sites of the human IL-6 receptor.", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 238, no. 3, June 1996 (1996-06-01), BERLIN, GERMANY, pages 714 - 723, XP000645294 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6841533B1 (en) 1995-12-07 2005-01-11 Peptor Limited Conformationally constrained backbone cyclized interleukin-6 antagonists
WO1998036061A3 (fr) * 1997-02-13 1998-11-12 Univ Manchester Reduction de la fibrose et/ou de la cicatrisation par inhibition de l'activite induite par le recepteur de l'interleukine 6
US6599875B1 (en) 1998-05-18 2003-07-29 Applied Research Systems Ars Holding N.V. IL-6 antagonist peptides
EP0972780A1 (fr) * 1998-05-18 2000-01-19 Applied Research Systems ARS Holding N.V. Peptides antagonistes de l'Il-6
WO1999060013A3 (fr) * 1998-05-18 2000-03-09 Applied Research Systems Peptides antagonistes de il-6
US6838433B2 (en) * 1998-05-18 2005-01-04 Applied Research Systems Ars Holding N.V. IL-6 antagonist peptides
WO2001016166A3 (fr) * 1999-08-27 2001-06-21 Us Health Polypeptides comprenant des domaines de recepteur d'interleukine-6 se liant a un ligand, acides nucleiques correspondants, anticorps, compositions, et leurs procedes d'utilisation
US6664374B1 (en) 1999-08-27 2003-12-16 The United States Of America As Represented By The Department Of Health & Human Services Polypeptides comprising IL-6 ligand-binding receptor domains
EP1218404A4 (fr) * 1999-09-15 2004-04-07 Medvet Science Pty Ltd Motif de liaison d'un recepteur
EP1218404A1 (fr) * 1999-09-15 2002-07-03 Medvet Science Pty. Ltd. Motif de liaison d'un recepteur
EP1334731A1 (fr) * 2000-10-25 2003-08-13 Chugai Seiyaku Kabushiki Kaisha Agents preventifs ou therapeutiques contre le psoriasis renfermant l'antagoniste de l'il-6 comme substance active
EP1334731A4 (fr) * 2000-10-25 2004-07-28 Chugai Pharmaceutical Co Ltd Agents preventifs ou therapeutiques contre le psoriasis renfermant l'antagoniste de l'il-6 comme substance active
US8562990B2 (en) 2000-10-25 2013-10-22 Chugai Seiyaku Kabushiki Kaisha Method of treating psoriatic arthritis with an IL-6 receptor antibody
US8597644B2 (en) 2000-10-25 2013-12-03 Chugai Seiyaku Kabushiki Kaisha Method for treating psoriasis by administering an antibody to interleukin-6 receptor
US7320792B2 (en) 2000-10-25 2008-01-22 Chugai Seiyaku Kabushiki Kaisha Preventives or remedies for psoriasis containing as the active ingredient IL-6 antagonist
FR2833011A1 (fr) * 2001-12-04 2003-06-06 Univ Claude Bernard Lyon Nouvelle proteine a activite inhibitrice de l'il-6
WO2007060647A3 (fr) * 2005-11-25 2007-07-19 Trinity College Dublin Methode de detection de sepsie
US8295392B2 (en) 2005-12-14 2012-10-23 Nec Corporation Digital communication system, indoor unit, and outdoor unit
WO2008113597A1 (fr) * 2007-03-22 2008-09-25 Helmholtz Zentrum Muenchen Deutsches Forschungszentrum Fuer Gesundheit Und Umwelt Gmbh Peptides utilisés pour traiter le myélome multiple
WO2014174517A1 (fr) * 2013-04-25 2014-10-30 Carmel-Haifa University Economic Corp. Peptides anti-inflammatoires synthétiques et leur utilisation
US9850278B2 (en) 2013-04-25 2017-12-26 Carmel-Haifa University Economic Corp. Synthetic anti-inflammatory peptides and use thereof

Also Published As

Publication number Publication date
EP0928293A1 (fr) 1999-07-14
AU3193097A (en) 1998-01-07
JP2000516574A (ja) 2000-12-12

Similar Documents

Publication Publication Date Title
Weiergräber et al. Soluble human interleukin‐6 receptor: expression in insect cells, purification and characterization
JP2783361B2 (ja) ほ乳類サイトカイン、ｉｌ−１１
Gearing et al. The IL-6 Signal Transducer, gp130: An Pncostatin M Receptor and Affinity Converter for the LIF Receptor
CA2056997C (fr) Cytokine humaine, interleukine-9
Tanabe et al. Identification of a new mouse beta-chemokine, thymus-derived chemotactic agent 4, with activity on T lymphocytes and mesangial cells.
US6355476B1 (en) Nucleic acid encoding MIP-1α Lymphokine
US5574138A (en) Epithelium-derived T-cell factor
IE883523L (en) Interleukin-1 Receptors
JPH09504030A (ja) Ｃｎｔｆ族アンタゴニスト
JP2007267750A (ja) 可溶性ｉｌ−ｔｉｆ／ｉｌ−２２レセプターまたはｉｌ−ｔｉｆ／ｉｌ−２２に結合する結合タンパク質をコードする単離された核酸分子、およびその使用
Bossu et al. Transfected type II interleukin-1 receptor impairs responsiveness of human keratinocytes to interleukin-1
EP0928293A1 (fr) Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6
JPH10511266A (ja) ヒト・インターロイキン−１１受容体
Baynes et al. Serum form of the erythropoietin receptor identified by a sequence-specific peptide antibody [see comments]
US5726036A (en) Granulocyte-macrophage colony-stimulating factor receptor and derivatives thereof
JP2000500644A (ja) Ｉｌ−６活性を阻害する合成ペプチド
US5180678A (en) Uses of substantially pure interleukin-9 receptor
JPH08510907A (ja) リンパ球化学誘引因子およびその用途
JP2863265B2 (ja) インターロイキン１インヒビター
AU601168B2 (en) Novel cell growth regulatory factor
WO1991014767A1 (fr) Procede de croissance et proliferation ameliorees de mastocytes
Schwabe et al. Differential expression and ligand-induced modulation of the human interleukin-6 receptor on interleukin-6-responsive cells.
Robb et al. T-cell growth factor: purification, interaction with a cellular receptor, and in vitro synthesis
EP0544719A1 (fr) Production de cytokine
JP3622208B2 (ja) 骨粗鬆症治療方法及び治療薬

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL

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: 1997927475

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1997927475

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1997927475

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载