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WO1990006941A1 - Nouveaux peptides derives du facteur de necrose de tumeurs (tnf) - Google Patents

Nouveaux peptides derives du facteur de necrose de tumeurs (tnf) Download PDF

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Publication number
WO1990006941A1
WO1990006941A1 PCT/EP1989/001468 EP8901468W WO9006941A1 WO 1990006941 A1 WO1990006941 A1 WO 1990006941A1 EP 8901468 W EP8901468 W EP 8901468W WO 9006941 A1 WO9006941 A1 WO 9006941A1
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WO
WIPO (PCT)
Prior art keywords
group
peptide
asn
leu
tnf
Prior art date
Application number
PCT/EP1989/001468
Other languages
German (de)
English (en)
Inventor
Hans-Joachim Boehm
Lothar Daum
Andreas Haupt
Bernhard Schmied
Nigel Walker
Johann-Christian Zechel
Original Assignee
Basf Aktiengesellschaft
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 Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1990006941A1 publication Critical patent/WO1990006941A1/fr

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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-Asn-B-Y I, wherein
  • A is Asp or Asn
  • B means Gin or Ser, ⁇ for a group G-NH-CHM-CO-, G-NH-CHM-CO-W-, G-R-NH-CHM-CO- or
  • Y represents 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 Y
  • G represents a hydrogen atom or an amino protecting group
  • Z represents an OH or NH 2 group or a carboxyl protecting group
  • M and Q are hydrogen atoms or one of the groups
  • 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.
  • 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. So you can build the peptides 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. Both in the sequential structure and in the fragment coupling, 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.
  • the peptide is typically built up sequentially on the polymeric support using the Boc or Fmoc protective group technique, the growing peptide chain at the C-terminus being covalently linked to the insoluble resin particles (see Figs. 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'-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, Tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of the solvents mentioned.
  • 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.
  • the new peptides show good cytotoxic properties. Another part of the peptides has a high affinity for the cellular TNF receptor without, however, having any cytotoxic activity. They therefore represent TNF antagonists. In competition with natural TNF, they bind to the cellular TNF receptor and thus suppress the TNF effect.
  • the new peptides prove to be valuable medicinal products that are used to treat neoplastic and autoimmune diseases, as well as to fight and prevent infections, inflammations and
  • 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.
  • Cytotoxicity of the peptide was determined by incubating the cell line in the presence of the peptide. In a second experiment, you incubate the
  • the agonistic evaluation of the new peptides is based on their cytotoxic effects on TNF-sensitive cells (e.g. L929, MCF-7,
  • the L929 and MCF-7 test was performed as follows: 1. 100 ⁇ l of culture medium with 3 to 5 ⁇ 10 3 freshly trypsinized, exponentially growing L929 cells (mouse) or MCF-7 cells (human) were placed in the wells of a
  • the L929 culture medium contained 500 ml MEM Earle 1 ⁇ (Boehringer, Mannheim), 50 ml heat-inactivated (30 min, 56 ° C.) fetal calf serum (FCS), 50 ml L-glutamine (200 mM), 5 ml 100 ⁇
  • non-essential amino acids 3 ml 1M 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 violet solution had the following composition:
  • 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 for 30 min at 56 ° C heat-activated FCS, 5 ml L-glutamine (200 mM), 5 ml 100 ⁇ nonessential amino acids, 3 ml IM Hepes Buffer pH 7.2 and 500 ⁇ l gentamycin (50 mg / ml).
  • the MCF-7 culture medium contained 500 ml MEM Dulbecco 1x (Boehringer, Mannheim), 100 ml heat-inactivated (30 min, 56 ° C) FCS, 5 ml L-glutamine (200 mM) and 5 ml 100x nonessential amino acids.
  • 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 .
  • 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 violet solution had the one given in II.3
  • the crystal violet 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) 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 (eg U937) compete.
  • the medium contained 500 ml PBS (Boehringer, Mannheim), 10 ml heat-inactivated (30 min, 56 ° C) FCS and 100 mg sodium azide.
  • rhu-TNF lactoperoxidase method according to Bolton
  • NBS nonspecific binding
  • the 125 iodine-labeled rhu-TNF (1 ng 125 J-rhu-TNF in 100 ⁇ l medium) with a 200-fold excess of non-radioactively labeled rhu-TNF (200 ng rhu-TNF mixed in 100 ul medium).
  • 100 ⁇ l of medium with 2 ⁇ 10 6 U937 cells (human) were pipetted into the reaction vessels and mixed.
  • the reaction vessels (test volume 300 ⁇ l) were incubated at 0 ° C. for 90 min. After 45 minutes, the reaction batches were mixed again. 4.
  • the cells were centrifuged for 5 min at 1800 rpm and 4 ° C., washed 3 times with medium, transferred quantitatively into counting tubes and the cell-bound radioactivity was determined in a Clini Gamrna Counter 1272 (LKB Wallac). 5. After correcting the measured values for the non-specific binding, based on the total binding, the 50% competition value was defined and the sample concentration was 50% competition at the 1 25 J-rhu-TNF concentration
  • proteogenic amino acids are in the examples with the known proteogenic amino acids
  • Abs 4-aminobutyric acid
  • Ac acetic acid
  • Ahp 7-aminoheptanoic acid
  • Ahx 6-aminohexanoic acid
  • Bal ß-alanine
  • Hey homocysteine
  • Hly homolysin
  • Orn ornithine
  • 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. 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
  • the 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 was evaporated to dryness. The residue was crystallized from DMF / Et 2 O or MeOH / Et 2 O.
  • Boc-Asn-OH Boc-Cys (pMB) -OH
  • 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
  • Anion exchanger (BIORAD ® 3 ⁇ 4A, chloride form) added. After 30 minutes, the ion exchange resin was filtered off, the filtrate was concentrated to 100 ml on a rotary evaporator and then lyophilized. All solvents used were previously saturated with nitrogen to prevent possible oxidation of the free cysteine residues.
  • the crude product was purified by gel chromatography (SEPHADEX G-15) and medium pressure chromatography (cf. AIV; 10-30% A; 0.25% min -1 ). 71 mg of pure product were obtained.
  • Example 56 1.16 g Boc-Lys (Cl-Z) -MBHA resin (substitution ⁇ 0.43 mmol / g), corresponding to a batch size of 0.5 mmol, were in accordance with Ala with 2 mmol
  • the crude product (317 mg) obtained after the HF cleavage according to All was dissolved in 500 ml of degassed DMF, 0.2 ml of triethylamine and (at -25 ° C.) 0.20 ml of diphenylphosphoryl azide were added. The mixture was stirred at 25 ° C. for 2 hours, kept at -20 ° C. for 2 days, at 4 ° C. for 2 days and at room temperature for 2 days and then evaporated to dryness.
  • the crude peptide was purified by gel chromatography (SEPHADEX ® LH 20) and medium pressure chromatography (cf. AIV; 10-25% A; 0.25% min -1 ). There were 101 mg
  • the crude product (1.22 g) obtained after the TFA cleavage according to AIII was purified according to AIV (414 mg). 200 mg was dissolved in 250 ml degassed DMF. After the addition of 0.24 ml of NEt 3 , 0.24 ml of diphenylphosphoryl azide was added at -25 ° C. and the mixture was stirred at -25 ° C. for 2 h. The mixture was then stored at -20 ° C. for 2 days, at 4 ° C. for 2 days and at room temperature for 2 days. The mixture was then evaporated to dryness and the crude peptide was purified by gel chromatography (SEPHADEX ® LH 20). The isolated monomer (143 mg) was deprotected with HF according to All and purified by medium pressure chromatography (cf. AIV; 5-25% A; 0.25% min -1 ). 89 mg of pure product were obtained.
  • Boc-Leu-Merrifield resin substituted by Boc-Arg (Tos) -OH according to Ala with 2 mmol each of Boc-Gln-OH
  • Boc-Asn-OH implemented. After the synthesis was complete, the peptide resin was deprotected at the N-terminal (steps 1-3 according to Ala) and then dried in vacuo. The yield was 0.85 g.

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  • 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)
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  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Pain & Pain Management (AREA)
  • Transplantation (AREA)
  • Rheumatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouveaux peptides ayant la formule X-A-Asn-B-Y, dans laquelle A, B, X et Y ont la signification donnée dans la description, et leur procédé de production. Ces nouveaux peptides sont utiles pour traiter des maladies.
PCT/EP1989/001468 1988-12-12 1989-12-02 Nouveaux peptides derives du facteur de necrose de tumeurs (tnf) WO1990006941A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3841764A DE3841764A1 (de) 1988-12-12 1988-12-12 Neue tnf-peptide
DEP3841764.2 1988-12-12

Publications (1)

Publication Number Publication Date
WO1990006941A1 true WO1990006941A1 (fr) 1990-06-28

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PCT/EP1989/001468 WO1990006941A1 (fr) 1988-12-12 1989-12-02 Nouveaux peptides derives du facteur de necrose de tumeurs (tnf)

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EP (1) EP0447472A1 (fr)
JP (1) JPH04502155A (fr)
CA (1) CA2005281A1 (fr)
DE (1) DE3841764A1 (fr)
WO (1) WO1990006941A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4340111A1 (de) * 1993-11-22 1995-05-24 Schering Ag Tumor-Nekrose-Faktor-alpha inaktivierende Peptide
EP1880737A1 (fr) * 1994-08-19 2008-01-23 La Region Wallonne Composés, composition pharmaceutique et dispositif de diagnostic les comprenant et leur utilisation
WO2015044900A1 (fr) * 2013-09-27 2015-04-02 Aurigene Discovery Technologies Limited Composés immunomodulateurs thérapeutiques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986002381A1 (fr) * 1984-10-15 1986-04-24 Cetus Corporation Facteur de necrose de tumeurs humaines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986002381A1 (fr) * 1984-10-15 1986-04-24 Cetus Corporation Facteur de necrose de tumeurs humaines

Also Published As

Publication number Publication date
JPH04502155A (ja) 1992-04-16
EP0447472A1 (fr) 1991-09-25
DE3841764A1 (de) 1990-06-13
CA2005281A1 (fr) 1990-06-12

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