Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
  • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0212—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -N-C-N-C(=0)-, e.g. retro-inverso peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0802—Tripeptides with the first amino acid being neutral
  • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
  • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• • 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 refers to Gly-His-Lys (GHL) peptide derivatives having cytostimulant an cytoprotective activity, to their therapeutic use as well as to pharmaceutical compositions containing them.
• GBL Gly-His-Lys
• the stabilization strategy used until now is based on the modification of the N- or C-ends (or o equivalent groups of the amino acid side-chains): introduction, with ester- or amide- like bonds, o suitable residues on said ends, leaving the peptid backbone unchanged.
• Pierschbacher M. (WO 90/06767, La Jolla Cancer Res.Found.) proposes polypeptide polymers of Arg-Gly-Asp (RGD), conjugated with biodegradable polymeric matrices, such as jaluronic acid, chondroitin sulphate, heparan sulphate, etc..
• the said derivatives In comparison to the natural peptide, the said derivatives have an higher stability to hydrolysis by carboxypeptidases, i.e. the enzymes hydrolyzing the molecule starting from the carboxy-terminus (C- terminal) .
• the present invention provides a more favourable solution to the known problem in the stabilization of the GHL peptide.
• the derivatives of the invention provide an improvement to the applicative therapeutic fields (wound healing, ulcers and tissue damages of different etiology) and to cosmetic applications (increase of the subcutaneous fat; decrease of wrinkles and telangiectasia conditions; stimulation of hair growth) since, differently from the known derivatives disclosed by L.R. Pickart, they exhibit an higher and unexpected resistance to hydrolytic agents such as carboxypeptidases, aminopeptidases and esterases (the latter being able to hydrolyse the ester bond between GHL and an alcohol or amide residue, therefore exposing the resulting GHL to rapid subsequent degradation) .
• Gly is one of the fol lowing residues : - glycine ; sarcosine ; group of formula NH 2 -CH 2 NH- , [gem(Gly ) ] ;
• Lys is one of the following residues: - L-lysine;
• NH 2 R is hydrogen, straight or branched Cm - , . alkyl
• Gly, His and Lys cannot be contemporaneously the natural amino acids glycine, L-histidine and L-lysine.
• the invention also comprises the pharmaceutically acceptable salts and the copper complexes of the compound I.
• Preferred compounds of formula I are thos wherein:
• 1 - His or Lys is a residue of the corresponding aminoacid and R is H; 2 - His or Lys is a residue of the corresponding aminoacid and R is different from H;
• R are as defined in any one of the above point 1-4;
• 6 - Gly is a gem-diaminal residue as above define [gem(Gly) ] whereas one of the His and Lys residu is a residue m(His) or m(Lys) as above define whereas the other is a residue of the L or series and R is hydrogen;
• these may be used as therapeutic agents i pathological forms asking for a cytoprotective and/o cytostimulating activity.
• the compounds of formula I ar conveniently used for the preparation of a drug usefu in treating ulcers, scars, tissue damages of differen kind and more generally of drugs for the treatment o autoimmune disease.
• the compounds of the invention are formulate in suitable pharmaceutical compositions alone o in combination or with other useful activ principles.
• the dosages will be determined by the physicia and will anyhow depend on the pathology to be treated age, weight and conditions of the patient.
• Examples o pharmaceutical compositions are topical forms such a creams, ointments, gels, aspersory powders, medicate plasters, controlled release topical forms, loca injection (e.g. intraarticular) ; systemic forms, suc as injectable, or oral forms such as tablets, capsule or other conventionally known forms.
• the composition of the invention may be prepared by usual methods, suc as those disclosed in Remington's Pharmaceutica Sciences Handbook, Mack Pub. Co., NY, USA.
• Th disclosed solid-phase synthetic methods for th derivatives containing either D or L aminoacids ar those usually used in the peptide synthesis but the should not be intended to limit the different syntheti possibilities which can be used according to th available knowledges, such as, for instance, the synthesis in homogeneous phase, used herein for the derivatives esterified at the C-terminus.
• the analytical method used for the determination of the titer or the belonging to the steric series (L or D) of the single aminoacids contained in each tripeptide is based on the method of Noriyuki Nimura et al. in J. Chrom. 352 (1986), 169-177, suitably modified.
• the IR spectra were- recorded in D-O or KBr on Jasco Mod. Ft/IR 5000 apparatus.
• the FAB-MS (Fast Atom Bombardment Mass Spectroscopy) data were obtained with the VG-70-70 EQ- HF apparatus provided with a standard source, using Xe as gas, glycerol as matrix and temperature of 363°K. All the non retro-inverted samples not containing sarcosine showed a MH+ at 340 and those with sarcosine at 350, in full agreement with the proposed structure.
• Fmoc-D Lys(Boc)-R (R: p-benzyloxybenzyl alcohol resin), after treatment with PIP/DMF to remove the protect the group from ⁇ -amino group, added to the pentafluorophenyl activated ester Fmoc-His(Boc)-OPfp.
• Lys(Boc)-R treated with PIP/DMF, yielded His(Boc)-D Lys(Boc)-R which was conjugated with Fmoc-Gly-OPfp.
• the Gly-His-D Lys Cu (II) complex was prepared dissolving the peptide in water, adding an equimolar amount of monohydrate copper acetate and adjusting the pH with diluted sodium hydrate, under cooling. After centrifugation at low temperature, to make the solution clear, the product was lyophilized.
• Fmoc-Lys(Boc)-R (R: p-benzyloxybenzyl-alcohol resin), after treatment with PIP/DMF, was conjugated with Fmoc-DHis(Boc)-OH by means of dicyclo- hexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt).
• DCC dicyclo- hexylcarbodiimide
• HOBt hydroxybenzotriazole
• the reaction with Fmoc-Gly-OPfp and the subsequent deprotection with 90% TFA yielded the trifluoroacetate which, after addition of HC1 3:1 and lyophilization, yielded the tripeptide Gly-D His- Lys.3HC1.
• the corresponding acetate was obtained as disclosed in Example 1.
• the copper complex Gly-D His- Lys Cu (II) was prepared as disclosed in Example 1.
• Fmoc-D Lys(Boc)-R (R: p-bezyloxybenzyl-alcohol resin) was added, after treatment with PIP/DMF, to Fmoc-D His(Boc)-OH in the presence of DCC/HOBt and then to Fmoc-Gly-Opf.
• the recovery of the Gly-D His-D Lys.3HCl derivatives was carried out as in the previous examples.
• the Gly-D His-D Lys Cu(II) complex was obtained as disclosed in Example 1.
• the two derivatives were synthesized as in the previous examples, using the intermediates H-His(Boc)-D Lys(Boc)-R and H-D His(Boc)-Lys(Boc)-R in the conjugation with Fmoc-Sar-OPfp.
• the copper complexes were obtained as in Example 1. Chemico-physical characteristics: Aruninoacid content (three determinations) lit Sar: 1.01 ⁇ 0.03; His: 0.98 ⁇ 0.02; D Lys: 0.99+ 0.04 _10_: Sar: 0.97 ⁇ 0.02; D His: 1.01 ⁇ 0.03; Lys: 0.98+0.02
• Example 6 Gly-D His-D Lys-O-benzylester (j?)
• H-D Lys(Z)-0-Bzl and Boc-His(Tos) were conjugated in the presence of D.C.C..
• a suitable solvent hexane/ethyl acetate
• H-D Lys(Z)-0-Bzl and Boc-His(Tos) were conjugated in the presence of D.C.C..
• NaHC0_ a suitable solvent
• extraction and washing with water the organic phase was evaporated and the residue crystallized from hexane-ethyl acetate.
• the crystallized product was dissolved in 50% hydrofluoric acid in dichloromethane and the solvent was then removed.
• the residue dissolved in hexane/ethyl acetate and in the presence of D.C.C. was conjugated with Boc-Gly.
• the derivative D Lys (Z)-O-Oct, prepared a disclosed in Example 7, is reacted with Boc- His(Tos)-OH in the presence of D.C.C. and HOBT in th same conditions above reported; the obtained derivativ was condensed with Boc-Sar with D.C.C./HOBT and th crude product, dissolved in glacial acetic acid, wa hydrogenated with Pd/C and the final product purifie by chromatography.
• the complex Sar-D His-D Lys-O-benzylester Cu (II was obtained as disclosed in Example 1.
• the dipeptide obtained in form of amide, as above disclosed (1.386 mol) has been dissolved in 6 ml of CH,CN and 1.5 ml of H-,0 were then added; the bis-trifluoroacetoxy-iodobenzene reagent has been added thereto (820 mg; 2.087 mmol) and then 220 ⁇ l of pyridine.
• Example 6 was treated with 29 ml of trifluoroacetic acid and stirred at room temperature for 20 minutes, then evaporated, dissolved again in 10 ml of CH 3 CN and purified on preparative HPLC column:
• the surnatant was eluted through a Sephadex G- column, eluted with distilled water a lyophilized, to give 34.7 mg of blue powde consisting of the G-gH-mL-Cu.
• the product was obtained starting from t monophenyl ester of the 2-N-trifluoroacetylbuty malonic acid (described in Example 9) and H-D His(Bom) O-tBu in the presence of HOBT/DCC. After ac treatment, tBu-O-D His(Bom)-(R,S) mLys(TFA)-O-Ph obtained was transformed in HO- D His(Bom) -(R, mLys (TFA)-0- Ph which was reacted with glycinamide the presence of HOBT and DCC.
• Dunkin-Hartley guinea pigs were anesthetized and five square wounds (7x7 mm) were excised on the mid- back, completely removing the dermal surface, after shaving and cleaning the dorsal skin (F. Buffoni et al. - Pharmacol. Res. 2J5, suppl. 2, 332, 1992). The wounds, both in control and treated animals, were allowed to heal. Four, eight and eleven days after surgery, five animals/group were sacrificed and newly formed tissue was dissected and analyzed by histological and biochemical methods.
• biochemical parameters were determined: hydroxyproline production (index of collagen formation) (J.F. Woessner, Arch. Biochem. Biophys. 9_3, 440, 1961); protein content (O.H. Lowry et al., J. Biol. Chem. 193, 265, 1951); DNA content (C. Labarca et al., Anal. Biochem. 102, 344, 1980).
• Histology was performed after staining wit hematoxylin/eosin.
• mice/group 1) controls, receiving 20 ⁇ l of distilled water; 2) animals treated with 10 ⁇ g of the Cu (II) complexes described in Table 1 dissolved in 20 ⁇ l of distilled water; 3) animals treated with 10 ⁇ g of GHL-Cu(II) complex in 20 ⁇ l of distilled water.
• Administration route water solutions were applied onto the wound.
• the pieces of tissue removed from the wounds on the 4th day contain both the regenerated tissue and the scab (mainly cluster of dead cells) and thus the three analyzed parameters presented higher values than those at the 8th and the 11th day.
• the results obtained from the animals treated with the products hereinbefore described show higher chemotaxis and/or increased production of extracellular matrix in comparison to those obtained from animals untreated or treated with GHL-Cu(II).
• the experimental groups consisted of 6 animals/group.
• Administration route water solution applied onto the wound.
• Table 2 The results, shown in Table 2, indicate that the compounds described in the present application reduced the time of wound healing with respect to the native product. Therefore the histological analysis shows a complete restitutio ad integrum of the tissues and an angiogenetic effect similar to those obtained with physiological timing.
• the superoxide dismutase activity of the products hereinafter described was determined according to C. Beauchamp et al. (Anal. Biochem. 44_, 276, 1971). The method is based on the appearance of coloured species with a maximum of absorption at 560 nm by NBT (nitroblue tetrazolium) in the presence of superoxide ion. The reduction in the intensity of absorption at 560 nm in the presence of the product under investigation determines its SOD-like activity. This activity is expressed in units defined as the concentration of product, in nmoles/ml, able to induce 50% of the maximum inhibition. The results, reported in Table 3, show that the tested derivatives presented an activity equal to GHL-Cu(II) activity.
• the activity of the products under examination was determined in rabbit PRP (platelet rich plasma), according to N. Lad et al. (Br. J. Pharmacol. 6>9_, 3, 1980). TxB2 final titration was performed with the Thromboxane B2 125-1 Assay System (Amersham Life Science, U.K. ) .
• the stability of the derivatives described i Table 5 was tested in presence of mitochondrial leucineaminopeptidase (aminopeptidase M) , carboxypeptidase and human plasma from health volounteers.
• the residual quantity of each one of th tested products was determined at differen experimental times by HPLC using the above describe conditions.
• the results obtained after 5 and 35 min o exposure to the enzymatic action (phosphate buffer pH 7.4 at 37°C for both pure enzymes and undiluted plasma) are reported in Table 5.
• the data show a much highe resistance of the tested products to the enzyme, i comparison to native GHL.

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• 1992
  • 1992-08-04 IT ITMI921914A patent/IT1261646B/it active IP Right Grant
• 1993
  • 1993-07-28 EP EP93917672A patent/EP0656009A1/en not_active Withdrawn
  • 1993-07-28 AU AU47033/93A patent/AU4703393A/en not_active Abandoned
  • 1993-07-28 WO PCT/EP1993/002004 patent/WO1994003482A1/en not_active Application Discontinuation
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