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WO1990006944A1 - 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
WO1990006944A1
WO1990006944A1 PCT/EP1989/001472 EP8901472W WO9006944A1 WO 1990006944 A1 WO1990006944 A1 WO 1990006944A1 EP 8901472 W EP8901472 W EP 8901472W WO 9006944 A1 WO9006944 A1 WO 9006944A1
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WIPO (PCT)
Prior art keywords
group
pro
peptide
ala
tyr
Prior art date
Application number
PCT/EP1989/001472
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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 WO1990006944A1 publication Critical patent/WO1990006944A1/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

  • New TNF peptides Description 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.
  • Y for a group -Z, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NH-CHQ-CO-U-Z or
  • G represents a hydrogen atom or an amino protecting group
  • Z represents an OH or NH 2 group or a carboxy protecting group
  • V is a peptide chain of 1-10 naturally occurring ⁇ -amino acids and 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) mean, 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.
  • 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 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. cleaved.
  • the conditions under which the various types of resin can be split off are known from the literature. Acid and palladium-catalyzed cleavage reactions are used most frequently, especially those
  • Deprotection of the peptide can be useful if certain derivatization reactions or a cyclization are to be carried out.
  • 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 for
  • neoplastic diseases and autoimmune diseases Treatment of neoplastic diseases and autoimmune diseases as well as for the control and prophylaxis of infections, inflammation 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 ⁇ 103 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 of MEM Earle 1x (Boehringer,
  • non-essential 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 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 lx (Boehringer, Mannheim), 50 ml FCS heat-inactivated at 56 ° C for 30 min, 5 ml L-glutamine (200 mM), 5 ml 100x nonessential amino acids, 3 ml IM Hepes buffer pH 7.2 and 500 ⁇ ⁇ gentamycin (50 mg / ml).
  • the MCF-7 culture medium contained 500 ml MEM Dulbecco lx (Boehringer, Mannheim), 100 ml heat-inactivated (30 min, 56 ° C) FCS, 5 ml L-glutamine (200 mM) and 5 ml lOOx nonessential amino acids.
  • 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, and 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 I-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 ⁇ 106 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.
  • 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 Gamma Counter 1272 (LKB Wallac). 5. After the correction of the measured values to the non-specific binding, the 50% Kompetitionswert was based on the total binding, defined and the sample concentration to 50% at the submitted 1 25 j-rhu TNF concentration Competition of 125 I-rhu-TNFBindung leads, determined as the competitive activity of the sample examined.
  • proteogenic amino acids are abbreviated in the examples with the well-known three-letter code.
  • Aad ⁇ -aminoadipic acid
  • Ac acetic acid
  • Ade 10-aminodecanoic acid
  • Ahp 7-aminoheptanoic acid
  • Ahx 6-aminohexanoic acid
  • Ano 9-aminononanoic acid
  • Aoc 8-aminooctanoic acid
  • Bai ß-alanine
  • Hey homocysteine
  • Hly homolysin
  • Orn ornithine
  • Dap 2,3-diaminopropionic acid.
  • the peptide resin obtained according to Ia was dried in vacuo 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 indole formyl group, preferably ethanedithiol (0.5 ml / g resin), condensation was carried out 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
  • 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
  • the peptide resin obtained according to Ib was dried in vacuo and then, depending on the amino acid composition, subjected to one of the following cleavage procedures (Wade, Tregear, Howard Florey Fmoc-Workshop Manual, Melbourne 1985).
  • the purity of the end products obtained was determined using analytical HPLC (stationary phase: 100 ⁇ 2, 1 mm VYDAC C-18, 5 ⁇ , 300 ⁇ ; mobile phase CH 3 CN / H 2 O gradient, buffered with 0.1% TFA, 40 ° C). Amino acid analysis and fast atom bombardment mass spectroscopy were used for characterization.
  • the N-terminus was acetylated (steps 2-4 and 8-9 according to Alb).
  • the peptide resin obtained was dried in vacuo; the yield was 0.7 g.
  • the crude peptide (268 mg) obtained after the TFA cleavage according to AIII was purified by gel filtration (SEPHADEX® G - 10) and medium pressure chromatography (cf. AIV; 40-60%; 0.25% min -1 ). There were 147 mg
  • H-Ser-Ala-Glu-Ile-Asn-Leu-Pro-Lys-Tyr-Leu-Asp-Phe-Ala-Glu-OH 11.
  • Boc-Pro-OH Boc-Hcy (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
  • the resin thus obtained was subjected to RF cleavage according to AH.
  • the lyophilized crude product was in 1 l of a 1 molar aqueous
  • the crude product (321 mg) obtained after the TFA cleavage according to AIII was dissolved in 500 ml of degassed DMF. After adding 0.2 ml of triethylamine, 0.2 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 (128 mg) was deprotected with HF according to A II and by medium pressure chromatography (cf. AIV; 25 - 45% A; 0.25% min -1 )
  • the N-terminus was acetylated (steps 2 to 4 and 8 to 9 were carried out according to Alb).
  • the crude peptide obtained after TFA cleavage according to AII I was dissolved in 500 ml degassed DMF. After addition of 210 mg of NaHCO 3 , 0.2 ml of diphenylphosphoryl azide was added at -25 ° C., the mixture was stirred at -25 ° C. for 2 h 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 (127 mg) was deprotected with HF according to A II and purified by medium pressure chromatography (cf. AIV; 55-75% A; 0.25% min -1 ). 78 mg of pure product were obtained.

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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)
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  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
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  • Genetics & Genomics (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

L'invention concerne de nouveaux peptides ayant la formule X-A-Y, dans laquelle A, X et Y ont la notation donnée dans la description, et leur procédé de production. Ces nouveaux peptides sont utiles pour traiter des maladies.
PCT/EP1989/001472 1988-12-12 1989-12-02 Nouveaux peptides derives du facteur de necrose de tumeurs (tnf) WO1990006944A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3841754A DE3841754A1 (de) 1988-12-12 1988-12-12 Neue tnf-peptide
DEP3841754.5 1988-12-12

Publications (1)

Publication Number Publication Date
WO1990006944A1 true WO1990006944A1 (fr) 1990-06-28

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ID=6368953

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Application Number Title Priority Date Filing Date
PCT/EP1989/001472 WO1990006944A1 (fr) 1988-12-12 1989-12-02 Nouveaux peptides derives du facteur de necrose de tumeurs (tnf)

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EP (1) EP0447429A1 (fr)
JP (1) JPH04502308A (fr)
CA (1) CA2005057A1 (fr)
DE (1) DE3841754A1 (fr)
WO (1) WO1990006944A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0521918A1 (fr) * 1990-03-19 1993-01-13 Peptide Technology Ltd Peptides a action anti-tumorale

Citations (2)

* 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
EP0250000A2 (fr) * 1986-06-20 1987-12-23 BASF Aktiengesellschaft Polypeptides, leur préparation et utilisation

Patent Citations (2)

* 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
EP0250000A2 (fr) * 1986-06-20 1987-12-23 BASF Aktiengesellschaft Polypeptides, leur préparation et utilisation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0521918A1 (fr) * 1990-03-19 1993-01-13 Peptide Technology Ltd Peptides a action anti-tumorale
EP0521918A4 (en) * 1990-03-19 1993-05-05 Peptide Technology Ltd Anti-tumour peptides

Also Published As

Publication number Publication date
CA2005057A1 (fr) 1990-06-12
JPH04502308A (ja) 1992-04-23
DE3841754A1 (de) 1990-06-13
EP0447429A1 (fr) 1991-09-25

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