EP0447432A1 - Peptides tnf - Google Patents

Peptides tnf

Info

Publication number
EP0447432A1
EP0447432A1 EP90900120A EP90900120A EP0447432A1 EP 0447432 A1 EP0447432 A1 EP 0447432A1 EP 90900120 A EP90900120 A EP 90900120A EP 90900120 A EP90900120 A EP 90900120A EP 0447432 A1 EP0447432 A1 EP 0447432A1
Authority
EP
European Patent Office
Prior art keywords
ala
leu
val
pro
arg
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP90900120A
Other languages
German (de)
English (en)
Inventor
Hans-Joachim Boehm
Lothar Daum
Andreas Haupt
Bernhard Schmied
Nigel Walker
Johann-Christian Zechel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
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 BASF SE filed Critical BASF SE
Publication of EP0447432A1 publication Critical patent/EP0447432A1/fr
Withdrawn legal-status Critical Current

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
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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 new peptides derived from tumor necrosis factor (TNF), their production and their use as medicaments.
  • TNF tumor necrosis factor
  • TNF TNF-related protein kinase
  • mice In addition to its cytotoxic properties, TNF is one of the main participants in inflammatory reactions (Pharmac. Res. 5, 129, 1988). The involvement of TNF in septic shock (Science 229, 869, 1985) and graft versus host disease (J. Exp. Med. 166, 1280, 1987) was shown in the animal model.
  • the invention relates to peptides of the formula I
  • A is Glu, Pro or Gin
  • B represents Gly, Glu, Asn or Asp
  • E means Gin or Ser
  • GR-NH-CHM-CO-W- and Y for a group -Z, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NH-CHQ-CO-UZ or -V- NH-CHQ-CO-UZ, where in X and YG represents a hydrogen atom or an amino protecting group,
  • Z represents an OH or NH 2 group or a carboxyl protecting group or G and Z together also represent a covalent bond or the group
  • V is one of the peptide chains
  • M and Q are hydrogen atoms or one of the groups
  • M and Q together form a - (CH 2 ) c -SS- (CH 2 ) d , - (CH 2 ) e -CO-NH- (CH 2 ) f - or - (CH 2 ) e -NH-CO- ( CH 2 ) g -NH-CO- (CH 2 ) f bridge (with c and d meaning a number from 1 to 4, e and f a number from 1 to 6 and g a number from 1 to 12) , and their salts with physiologically acceptable acids.
  • the peptides of formula I are made up of L-amino acids, but they can contain 1 to 2 D-amino acids.
  • the side chains of the trifunctional amino acids can carry protective groups or be unprotected. In particular, the following can be mentioned as physiologically acceptable acids:
  • Methanesulfonic acid acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid,
  • Glucuronic acid oxalic acid, ascorbic acid, acetylglycine.
  • the new compounds can be prepared by methods known in peptide chemistry.
  • the peptides can be built up sequentially from amino acids or by fragment linking of suitable small peptides. In the sequential construction, the peptide chain is gradually extended by one amino acid each, starting at the C terminus. When coupling fragments, fragments
  • the fragments in turn being able to be obtained by sequential construction from amino acids or in turn by fragment coupling.
  • the cyclic peptides are obtained by a cyclization reaction carried out in high dilution.
  • the building blocks must be linked by forming an amide bond. Enzymatic and chemical methods are suitable for this.
  • the coupling reagents can be used alone or in combination with additives such as N, N'-dimethyl-4-aminopyridine (DMAP), N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine .
  • DMAP N, N'-dimethyl-4-aminopyridine
  • HOBt N-hydroxybenzotriazole
  • HOOBt N-hydroxybenzotriazine
  • HSu N-hydroxysuccinimide
  • 2-hydroxypyridine 2-hydroxypyridine
  • protective groups can be dispensed with, chemical synthesis requires reversible protection of the reactive functional groups of the two reactants which are not involved in the formation of the amide bond.
  • three protecting group techniques known from the literature are preferred: the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (Boc) and the 9-fluorenylmethyloxycarbonyl (Fmoc) protective group technique.
  • the protective group of the ⁇ -amino function of the chain-extending building block is designated in each case.
  • the side chain protecting groups of the trifunctional amino acids are chosen so that they are not necessarily split off together with the ⁇ -amino protecting group.
  • Fmoc protective group technology are set up, the reactants being brought to reaction in solution, and processes in which, similar to the Merrifield technique mentioned, a reactant is reacted to an insoluble polymeric carrier (hereinafter also called resin).
  • resin an insoluble polymeric carrier
  • the peptide is typically built up sequentially on the polymeric support using the Boc or Fmoc protective group technique, the growing peptide chain being covalent at the C-terminus with the insoluble resin particles (see Fig. 1 and 2). This procedure allows reagents and by-products to be removed by filtration, making recrystallization of intermediate products unnecessary.
  • the protected amino acids can be bound to any suitable polymers which are only insoluble in the solvents used and must have a stable physical form which enables easy filtration.
  • the polymer must contain a functional group to which the first protected amino acid can be bound by a covalent bond.
  • a wide variety of polymers are suitable for this purpose, e.g.
  • All solvents which prove inert under the reaction conditions are suitable for peptide synthesis in solution, in particular water, N, N'-D ⁇ methylformamid (DMF), dimethylsulfox ⁇ d (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, Tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of the solvents mentioned.
  • DMF N, N'-D ⁇ methylformamid
  • DMSO dimethylsulfox ⁇ d
  • DCM dichloromethane
  • THF Tetrahydrofuran
  • NMP N-methyl-2-pyrrolidone
  • the peptide synthesis on the polymeric support can be carried out in all inert organic solvents in which the amino acid derivatives used are soluble; however, solvents which additionally have resin-swelling properties, such as DMF, DCM, NMP, acetonitrile and DMSO, and mixtures of these solvents are preferred.
  • Rejection reactions can be used in transplants. Simple experiments can be used to determine the mode of action of the individual peptides.
  • Simple experiments can be used to determine the mode of action of the individual peptides.
  • the agonistic evaluation of the new peptides is based on their cytotoxic effects on TNF-sensitive cells (e.g. L929, MCF-7, A204, U937).
  • TNF-sensitive cells e.g. L929, MCF-7, A204, U937.
  • L929 and MCF-7 test was carried out as follows
  • MCF-7 cells human were placed in the wells of a
  • the plate was incubated overnight at 37 ° C in the incubator.
  • the air saturated with water vapor in the incubator contained 5 vol% CO 2 .
  • the L929 culture medium contained 500 ml of MEM Earle 1x (Boehringer,
  • Calf serum 50 ml L-glutamine (200 mM), 5 ml 100 ⁇ nonessential amino acids, 3 ml IM Hepes buffer pH 7.2 and 50 ml gentamycin (50 mg / ml).
  • the MCF-7 culture medium contained 500 ml of MEM Dulbecco 1x
  • the percentage of surviving cells in the cultures treated with peptide dilution was determined by means of crystal violet staining.
  • the liquids were removed from the wells by knocking off the test plate. 50 ⁇ l of crystal violet solutions were pipetted into each well.
  • the crystal vietiette solution had the following composition:
  • the crystal vietiette solution remained in the wells for 20 min and was then also knocked off.
  • the plates were then washed 5 times each by immersion in water to remove the non-cell-bound dye.
  • the cell-bound dye was extracted from the cells by adding 100 ul reagent solution (50% ethanol, 0.1% glacial acetic acid, 49.9% water) to each well. 4. By shaking the plates for 5 min each was obtained
  • the antagonistic evaluation of the peptides is based on their
  • TNF-sensitive cells e.g. L929, MCF-7, A204, U937.
  • the L929 culture medium contained 500 ml MEM Earle 1 ⁇ (Boehringer, Mannheim), 50 ml FCS heat-inactivated for 30 min at 56 ° C., 5 ml L-glutamine (200 mM), 5 ml 100 ⁇ non-essential amino acids, 3 ml 1M Hepes Buffer pH 7.2 and 500 ⁇ l gentamycin (50 mg / ml).
  • the MCF-7 culture medium contained 500 ml of MEM Dulbecco 1x
  • the culture plate was then incubated for 48 hours at 37 ° C. in an atmosphere of water vapor-saturated air with 5 vol.% CO 2 . 3.
  • the percentage of surviving cells in the solution-treated cultures was determined by crystal violet staining.
  • the liquids were removed from the wells by knocking off the test plate. 50 ⁇ l of crystal violet solutions were pipetted into each well.
  • the crystal vietiette solution remained in the wells for 20 min and was then also knocked off. The plates were then washed 5 times each by immersion in water to remove the non-cell-bound dye.
  • cell-bound dye was added by adding 100 ul
  • Reagent solution (50% ethanol, 0.1% glacial acetic acid, 49.9% water) was extracted from the cells into each well.
  • rhu-TNF control defined the 50% competition value and the sample concentration, which leads to 50% competition of the rhu-TNF cytotoxicity at the presented rhu-TNF concentration, was determined as the antagonistic activity of the examined sample.
  • Indicator cells e.g. U937 compete.
  • the competition receptor binding test was carried out as follows:
  • proteogenic amino acids are in the examples with the known proteogenic amino acids
  • Aoc 8-amino octanoic acid
  • Ape 5-aminopentanoic acid
  • Hey homocysteine
  • Orn ornithine
  • Dap 2,3-diaminopropionic acid.
  • the peptide resin obtained according to la was dried in a vacuum and transferred into a reaction vessel of a Teflon HF apparatus (from PENINSULA). After adding a scavenger, preferably anisole (1 ml / g resin), and in the case of tryptophan-containing peptides of a thiol to remove the indolene formyl group, preferably ethanedithiol (0.5 ml / g resin), the mixture was condensed with cooling with liquid N 2 hydrogen fluoride ( 10 ml / g resin). The mixture was allowed to warm to 0 ° C and stirred at this temperature for 45 min.
  • a scavenger preferably anisole (1 ml / g resin)
  • tryptophan-containing peptides of a thiol to remove the indolene formyl group, preferably ethanedithiol (0.5 ml / g resin)
  • the hydrogen fluoride was then stripped off in vacuo and the residue was washed with ethyl acetate in order to remove remaining scavengers.
  • the peptide was extracted with 30% acetic acid, filtered and the filtrate lyophilized.
  • peptide resin (Pam or Merrifield resin) was suspended in DMF (15 ml / g resin) and, after addition with hydrazine hydrate (20 equivalents), stirred for 2 days at room temperature. For working up, the resin was filtered off and the filtrate
  • Boc-Glu (OBZl) -OH Boc-Pro-OH implemented.
  • Boc-Glu (OChx) -OH Boc-Cys (pMB) -OH implemented. After the synthesis was complete, the N-terminus was acetylated (steps 1-6 and 14-16 were carried out according to Ala).
  • the peptide resin obtained was dried in vacuo; the yield was 1.3 g.
  • Acetic acid was added and the pH was then adjusted to 8.4 with aqueous ammonia. 0.01 N was slowly added under an argon atmosphere

Landscapes

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

Abstract

Peptides correspondant à la formule: X-A-B-E-Leu-Y, où A, B et X et Y ont les significations indiquées dans la description; et méthode de production correspondante. Ces peptides servent à combattre certaines maladies.
EP90900120A 1988-12-12 1989-12-02 Peptides tnf Withdrawn EP0447432A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3841767A DE3841767A1 (de) 1988-12-12 1988-12-12 Neue tnf-peptide
DE3841767 1988-12-12

Publications (1)

Publication Number Publication Date
EP0447432A1 true EP0447432A1 (fr) 1991-09-25

Family

ID=6368962

Family Applications (1)

Application Number Title Priority Date Filing Date
EP90900120A Withdrawn EP0447432A1 (fr) 1988-12-12 1989-12-02 Peptides tnf

Country Status (5)

Country Link
EP (1) EP0447432A1 (fr)
JP (1) JPH04502306A (fr)
CA (1) CA2005052A1 (fr)
DE (1) DE3841767A1 (fr)
WO (1) WO1990006939A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993001211A1 (fr) * 1991-07-05 1993-01-21 Peptide Technology Limited Peptide neutralisant la toxicite du facteur de necrose tumorale alpha (fnt) et/ou la toxicite des lipopolysaccharides (lps)
DE4341471A1 (de) * 1993-12-02 1995-06-08 Schering Ag Tumor-Nekrose-Faktor-alpha-inaktivierende CDR-Peptide
FR2825154B1 (fr) * 2001-05-22 2004-01-30 Univ Compiegne Tech Composes capables de moduler l'activite et de stimuler la production d'un anticorps catalytique

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3211263A1 (de) * 1981-03-31 1983-01-27 Otsuka Pharmaceutical Co. Ltd., Tokyo Human-interferon verwandte peptide, antigene und antikoerper, sowie verfahren zu deren herstellung
EP0190127A1 (fr) * 1984-08-10 1986-08-13 MERCK PATENT GmbH Agents polypeptides immunotherapeutiques
EP0205038A1 (fr) * 1985-05-29 1986-12-17 Suntory Limited Polypeptide, son procédé de préparation, micro-organisme et utilisation pharmaceutique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9006939A1 *

Also Published As

Publication number Publication date
CA2005052A1 (fr) 1990-06-12
JPH04502306A (ja) 1992-04-23
DE3841767A1 (de) 1990-06-13
WO1990006939A1 (fr) 1990-06-28

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