WO2001029086A1 - Peptides presentant une activite antagoniste de la corticoliberine (crf) - Google Patents

Peptides presentant une activite antagoniste de la corticoliberine (crf) Download PDF

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Publication number
WO2001029086A1
WO2001029086A1 PCT/EP2000/010197 EP0010197W WO0129086A1 WO 2001029086 A1 WO2001029086 A1 WO 2001029086A1 EP 0010197 W EP0010197 W EP 0010197W WO 0129086 A1 WO0129086 A1 WO 0129086A1
Authority
WO
WIPO (PCT)
Prior art keywords
ala
peptides
crf
xaa
leu
Prior art date
Application number
PCT/EP2000/010197
Other languages
English (en)
Inventor
Dirk T. S. Rijkers
Jack A. J. Den Hartog
Rob M. J. Liskamp
Original Assignee
Solvay Pharmaceuticals B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP99203402A external-priority patent/EP1094072A1/fr
Application filed by Solvay Pharmaceuticals B.V. filed Critical Solvay Pharmaceuticals B.V.
Priority to AU11393/01A priority Critical patent/AU1139301A/en
Publication of WO2001029086A1 publication Critical patent/WO2001029086A1/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
    • C07K14/575—Hormones
    • C07K14/57509—Corticotropin releasing factor [CRF] (Urotensin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • A61P5/08—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH for decreasing, blocking or antagonising the activity of the anterior pituitary hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel peptides, to a method for the preparation of these novel peptides, to pharmaceutical compositions containing one or more of these peptides as an active component and to methods of using these in the treatment of a wide range of stress-related disorders.
  • the peptides have Corticotropin Releasing Factor (CRF) antagonist activity.
  • CRF a 41 amino acid peptide
  • POMC proopiomelanocortin
  • CRF antagonist compounds and compositions which can attenuate the physiological responses to stress-related phenomena, have potential therapeutic utility for the treatment of a wide range of stress-related disorders, such as depression, anxiety related diseases, post traumatic stress disorder, obsessive compulsive disorder, headache, eating and feeding disorders, anorexia nervosa, gastrointestinal diseases, irritable bowel syndrome, inflammatory diseases, immune suppression, HIV infections, Alzheimer's disease, hemorrhagic stress, drug and alcohol withdrawal symptoms, drug addiction and fertility problems
  • stress-related disorders such as depression, anxiety related diseases, post traumatic stress disorder, obsessive compulsive disorder, headache, eating and feeding disorders, anorexia nervosa, gastrointestinal diseases, irritable bowel syndrome, inflammatory diseases, immune suppression, HIV infections, Alzheimer's disease, hemorrhagic stress, drug and alcohol withdrawal symptoms, drug addiction and fertility problems
  • Astressin 16-41 the peptide derived from Astressin by N-terminal shortening to 25-am ⁇ no acids
  • Astressin 16-41 has a reduced CRF receptor affinity and an antagonistic activity which is at least a factor 1000 less in comparison with the known peptides of about 30 ammo acids, suggesting that further shortening will lead to a further reduction of activity
  • the invention relates to novel CRF antagonist peptides consisting of 12 to 16 ammo acids which bind to CRF receptors having the formula
  • Y is a hydrogen atom or an acyl group having up to 5 carbon atoms
  • A is absent or represents Gln-Leu-Ala-Gln, Leu-Ala-Gin, Ala-Ala-Gin
  • Xaa a is His or Ala
  • Xaa b is Lys or Ala
  • Xaa c is lie or Ala (SEQ ID NO 1-6), and salts thereof, with the proviso that Xaa c is not Ala when A is absent and that Xaa b is not Ala when A represents Leu-Ala-Gin
  • Y is acetyl and A is selected from the group having a length of 3 ammo acids
  • Suitable acids with which the peptides of the invention can form suitable acid addition salts are for example hydrochloric acid, sulphuric acid, phosphoric acid, nitric acid, and organic acids such as citric acid, fuma ⁇ c acid, maleic acid, tarta ⁇ c acid, acetic acid, benzoic acid, p-toluene sulphonic acid, methanesulphonic acid and naphthalene sulphonic acid
  • the peptides and their acid addition salts can be brought into pharmaceutical compositions suitable for administration by means of suitable processes using auxiliary substances such as liquid and solid carrier materials As mentioned the peptides described above and salts thereof have interesting CRF antagonistic activity The peptides are especially useful as CRF 1 antagonists In vitro receptor binding assay
  • the affinity of the compounds of the invention for the CRF receptor was determined by binding studies using membrane preparations of CC7-cells containing the human CRF receptor and [ 125 l]-ov ⁇ ne CRF as the ligand Separation of bound and free ligand is performed by filtration over glassfiber- filters, essentially as described by H Herdon et al, Soc Neurosci Abstracts
  • the antagonistic activity of the compounds of the invention was determined by functional studies using LVIP cells (Mol and Cell Neuroscience 2 331, 1991) containing the rat CRF receptor
  • the origin of these cells is the mouse L-cell-lme containing the cAMP responsive reporter gene construct coding for the enzyme ⁇ - galactosidase, which is subsequently transfected with the plasmid containing the genes coding for rat CRF Formation of cAMP is stimulated with rat CRF (10 8 M) for 3 hours
  • the increase of cAMP results in an increase in the production of ⁇ -galactosidase by stimulation of the reporter gene
  • O-nitrophenyl- ⁇ -D-Galactopyranoside is measured spectrophotomet ⁇ cally (405 nm) Antagonistic activity can be obtained after a 30 minutes pre-mcubation with putative antagonists and subsequent incubation with the (reference) agonist CRF for 3 hours and is expressed as pA 2 values
  • Peptides are synthesized on an Applied Biosystems 433A Peptide Synthesizer (Foster City, California, USA).
  • Analytical and preparative HPLC runs are performed on a Gilson HPLC workstation (Middleton, Wisconsin, USA), consisting of two Gilson 306 Pumps, a Gilson 811 C Dynamic Mixer, a Gilson 806 Manometric Module, a Gilson 119 UV ⁇ IS detector, a Gilson 402 Syringe Pump and a Gilson 233XL On-line Column Switching Autoinjector (Fraction collector).
  • Analytical HPLC runs are performed on Adsorbosphere XL C8, 5 ⁇ m particle size,
  • the samples are dissolved in water and approximately 1 ⁇ l of the solution, containing about 0.1 ⁇ g of the compound, is mixed with a microdroplet of glycerol on the FAB probe.
  • Compounds with a mass higher than m/z 3000 are measured by Electrospray lonization Mass Spectrometry (ESI-MS) on a Micromass Platform II (Micromass UK Ltd., Altrincham, United Kingdom) single quadrupole bench-top mass spectrometer operating in a positive ionization mode.
  • ESI-MS Electrospray lonization Mass Spectrometry
  • Micromass Platform II Micromass UK Ltd., Altrincham, United Kingdom
  • a cone voltage of 35 V is set, which produces predominantly (M + H) + ions with little evidence of fragmentation for this type of peptides.
  • Instrumental control, data acquisition and data processing are carried out using the MassLynx software package (version 2.3). The observed average m/z is compared with the calculated (M + H) + average mass.
  • the peptides are synthesized on TentaGel ® S RAM resin, 0.24 mmol/g (Rapp Polymere, Tubingen, Germany), functionalized with a 4-[(2',4'-d ⁇ methoxyphenyl)- am ⁇ no-methyl]phenoxyacetamido moiety (Rink amide linker) to obtain C- terminal amides (after TFA cleavage).
  • N Fmoc-protected amino acids and HOBt are purchased from Advanced ChemTech Europe (Machelen, Belgium).
  • the side chain protecting groups are chosen as: Arg(Pbf), Asn(Trt), Gln(Trt), Glu(OtBu), His(Trt) and Lys(Boc).
  • TIPS and EDT are supplied by Merck (Darmstadt, Germany). All other chemicals are at least analytical reagent grade.
  • the syntheses are controlled by Applied TM Biosytems SynthAssistTM version 2.0 formatted for Macintosh Coupling of N ⁇ -Fmoc ammo acids (1 mmol, 4 eq) are performed with HBTU/HOBt in the presence of DIPEA (2 mmol, 8 eq) in NMP for 45 mm Fmoc removal is carried out with 20% piperidme in NMP for 10 mm Any remaining ammo groups after incomplete coupling and the final ammo functionality, respectively, are acetylated by acetic anhydnde/DIPEA/HOBt in
  • Glu 1 and Lys 4 is mediated on resin with 3 eq. of BOP/HOBt in the presence of 9eq. of DIPEA in NMP for 16 hours. Both the allyl removal and the ring closure reaction are monitored by the Kaiser test for the presence of free amine functionalities. Generally, the allyl removal is complete after 16 hrs.
  • the peptides are cleaved from the resin and deprotected by treatment with TFA/H 2 O/TIS/EDT 85:8.5.2:4.5 v/v/v/v at room temperature for 3 hrs
  • the peptides are precipitated with diethyl ether/hexane 1 :1 v/v at -20 °C
  • the precipitates are decanted and subsequently washed with cold diethyl ether (three times) and finally lyophiiized from tert.BuOH/H 2 0 1 1 v/v or CH 3 CN/ H 2 0 1 -1 v/v
  • Crude lyophiiized peptides are desalted by size exclusion chromatography on Sephadex LH 20 (I. 135 cm, i.d.: 2 cm) (Pharmacia, Uppsala, Sweden) with
  • the peptides are eluted with a flow rate of 1.5 ml/mm using a linear gradient of buffer B (70% in 80 min) from 20% buffer A (buffer A: 0.1% TFA in H 2 O, buffer B: 0.1% TFA in CH 3 CN/H 2 0 8:2 v/v).
  • Peptide purity can be analyzed by analytical HPLC at a flow rate of 1 ml/mm using a lineair gradient of buffer B (100% in 30 min) from 100% buffer A (buffer A: 0.1% TFA in H 2 0; buffer B: 0.085% TFA in CH 3 CN/H 2 0 95:5 v/v) in case of the C8 column or a linear gradient of buffer B (70% in 40 min) from 80% buffer A (buffer A: 0.1% TFA in H 2 0; buffer B: 0.085% TFA in CH 3 CN/H 2 0 95:5 v/v) in case of the C8 column or a linear gradient of buffer B (70% in 40 min) from 80% buffer A (buffer A: 0.1% TFA in H 2 0; buffer B: 0.085% TFA in CH 3 CN/H 2 0 95:5 v/v) in case of the C8 column or a linear gradient of buffer B (70% in 40 min) from 80% buffer A (buffer
  • the peptide was deprotected and detached from the resin by treatment of TFA H 2 0 TIS/EDT 85:8.5:2:4.5 v/v/v/v. 400 mg of resin was suspended in 5 ml of the TFA solution and stirred for 3 hrs at room temperature. After this period the resin was filtered off and washed with 5 ml of TFA (twice 2.5 ml). The peptide was precipitated from the TFA solution by dilution in 100 ml diethyl ether/hexane 1 :1 v/v. The white precipitate was centhfuged and decanted. The peptide was washed by resuspending in diethyl ether and cent fugation (five times). Finally, the crude peptide was air-dried and dissolved in 50 ml tert.-BuOH/H 2 0 1 :1 v/v and lyophiiized. The yield of the crude peptide was 72%.
  • the crude peptide was purified by preparative HPLC. 50 mg of peptide was dissolved in 4 ml buffer A (0.1% TFA in H 2 O) and loaded onto the column
  • HOBT N-hydroxybenzotriazole
  • HBTU 2-(IH-benzotriazol-l-yl)-1 ,1 ,3,3-tetra- methyluronium tetrafluoroborate
  • BOP benzotriazole- 1-yl-oxy-tris-(dimethyl- amino)-phosphonium.

Abstract

L'invention concerne un certain nombre de nouveaux petits peptides composés de 12, 15 ou 16 amino acides. Ces peptides sont dérivés de l'Astressin, c'est-à-dire un peptide de 30 amino acides. De façon surprenante, on a découvert que ces petits peptides présentent également une bonne activité antagoniste du CRF.
PCT/EP2000/010197 1999-10-18 2000-10-17 Peptides presentant une activite antagoniste de la corticoliberine (crf) WO2001029086A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU11393/01A AU1139301A (en) 1999-10-18 2000-10-17 Peptides having corticotropin releasing factor (crf) antagonist activity

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP99203402A EP1094072A1 (fr) 1999-10-18 1999-10-18 Péptides ayant une activité antagoniste du facteur liberant la corticotropine (CRF)
EP99203402.5 1999-10-18
NL1013319 1999-10-18
NL1013319 1999-10-18

Publications (1)

Publication Number Publication Date
WO2001029086A1 true WO2001029086A1 (fr) 2001-04-26

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/010197 WO2001029086A1 (fr) 1999-10-18 2000-10-17 Peptides presentant une activite antagoniste de la corticoliberine (crf)

Country Status (2)

Country Link
AU (1) AU1139301A (fr)
WO (1) WO2001029086A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8623606B2 (en) 2006-08-22 2014-01-07 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Screening method for GPCR ligands

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108226318A (zh) * 2016-12-21 2018-06-29 鲁南制药集团股份有限公司 一种n-芴甲氧羰基-l-丙氨酸的分析检测方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5493006A (en) * 1993-06-16 1996-02-20 The Salk Institute For Biological Studies Cyclic CRF analogs
WO1996019499A2 (fr) * 1994-12-12 1996-06-27 The Salk Institute For Biological Studies Antagonistes cycliques ameliores du facteur de corticotropine
WO1998054221A1 (fr) * 1997-05-30 1998-12-03 The Salk Institute For Biological Studies Peptides cycliques antagonistes du facteur liberateur de corticotrophine (crf)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5493006A (en) * 1993-06-16 1996-02-20 The Salk Institute For Biological Studies Cyclic CRF analogs
WO1996019499A2 (fr) * 1994-12-12 1996-06-27 The Salk Institute For Biological Studies Antagonistes cycliques ameliores du facteur de corticotropine
WO1998054221A1 (fr) * 1997-05-30 1998-12-03 The Salk Institute For Biological Studies Peptides cycliques antagonistes du facteur liberateur de corticotrophine (crf)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8623606B2 (en) 2006-08-22 2014-01-07 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Screening method for GPCR ligands

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Publication number Publication date
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