WO2005085286A2

WO2005085286A2 - Peptides with antiangiogenic and antitumor activities

Info

Publication number: WO2005085286A2
Application number: PCT/EP2005/001945
Authority: WO
Inventors: Francesco Chillemi
Original assignee: Francesco Chillemi
Priority date: 2004-02-27
Filing date: 2005-02-24
Publication date: 2005-09-15
Also published as: WO2005085286A3; ITMI20040364A1

Abstract

The peptide with the following sequence: Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys(Acm)-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala-Phe-Leu-Ser-Ser-Arg-Leu-Gln-Asp-Leu-Tyr-Ser-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala, its analogues and derivatives, are useful for the preparation of medicaments with antiangiogenic and antitumor activities

Description

PEPTIDES WITH ANTIANGIOGENIC AND ANTITUMOR ACTIVITIES

The present invention relates to peptides with antiangiogenic and antitumor activities having a sequence corresponding to fragments of human endostatin. BACKGROUND OF THE INVENTION Angiogenesis is the process of outgrowth of new capillaries from preexisting blood vessels. This phenomenon occurs in various physiological and pathological conditions and is particularly involved in tumor growth and in formation and maintenance of metastasis. Angiogenesis is a complex multistep process that includes proliferation, migration and differentiation of endothelial cells, with parallel degradation events of extra-cellular matrix, formation of tubules and "sprouting" of new capillaries. Endostatin is a C-terminal fragment of XVIII collagen with molecular weight of 20 kDa, that specifically inhibits endothelial cells proliferation in vitro and angiogenesis and tumor growth in vivo. In particular, systemic administration of recombinant endostatin causes regression of tumors in mice. However, administration - and consequently production - of large amounts of endostatin are necessary to observe these effects. Availability of molecules endowed with biological activity comparable to endostatin, but having smaller dimensions and higher stability, may be extremely useful. Peptides with a sequence corresponding to murine endostatin described by Folkman in WO 97/15666, have been disclosed in WO 99/29855, WO 99/48924 and WO 00/63249. In particular, WO 99/29855 (Beth Israel Deaconess Medical Center) discloses mutants and peptides of murine endostatin (deletion of 9 amino acids 176-184 in the C-terminal region) and characterized by the sequence SYIVLCIE(168-175) in C-terminal region. WO 99/48924 (Children's Medical Center, Ben-Sasson) discloses peptides having from about 10 to about 28 amino acids deriving from the sequence (angiogenic homology region), corresponding to 36-70 region of human endostatin. Hybrid peptides containing 10-11 amino acids corresponding to endostatin AHR sequence and other 10-11 amino acids corresponding to the sequence of other proteins (TSP-1, TSP-4; TSP = thrombospondin) are therein described in detail. WO 00/63249, in the Applicant's name, discloses the fragments corresponding to sequences 1-39, 40-89, 90-134, 135-184 of murine endostatin. Some of said fragments are more active than the whole endostatin molecule. WO 02/68457 describe the fragments 6-49, 50-92, 93-133 and 134-178 of human endostatin with a homology of about 86% as regards that murine. Recently, Chillemi et al. have shown that fragments 6-49 and 134-178 possess antiangiogenic activity unlike the central fragments 50-92 and 93-133 that are devoid of antiangiogenic activity (J. Med. Chem. 46, 4165-4172, 2003). Morbidelli et al. (Clinical Cancer Research, 9, 5358-5369, 2003) studying the activity of five fragments of murine endostatin (1-39, 40-89, 90- 134, 135-184 and 135-184 with a disulfϊde bond between the cysteines 135 and 165) have found that the endostatin molecule also contains a pro- angiogenic domain (peptide 90-134) with an activity comparable to and even higher than that of VEGF . DISCLOSURE OF THE INVENTION It has now been found that the peptide corresponding to sequence 16-67 of human endostatin shows not only antiangiogenic activity markedly higher than endostatin itself and the above cited known peptides, but also possesses antitumor al activity. Therefore the invention relates to said peptide and analogues and derivatives thereof, pharmaceutical compositions containing them and the use thereof for the preparation of medicaments with antiangiogenic and antitumor activities. DETAILED DISCLOSURE OF THE INVENTION The peptide sequence of the invention is the following: Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe- Gln-Cys(Acm)-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe- Arg-Ala-Phe-Leu-Ser-Ser-Arg-Leu-Gln-Asp-Leu-Tyr-Ser-Ile-Val-Arg-Arg- Ala-Asp-Arg-Ala. The invention also comprises the derivatives of said peptide obtained for example by substitution of natural amino acids with the corresponding amino acids of the D series and/or by derivatization of hydroxy, thio or basic functional groups of serine, threonine, cysteine, tyrosine, arginine residues and/or by functionalization of the terminal NH₂ (for example, by acylation with acetyl groups) and/or by retro-inversion of one or more peptide bonds, according to known techniques which allow to stabilize peptides against hydrolytic enzymes, therefore improving the pharmacokinetic characteristics. The present invention also includes derivatives or analogues of peptide

16-67 obtained by conservative substitutions of amino acids, typically one to ten substitutions, preferably one to five and more preferably one to three conservative substitutions. A conservative substitution is for example the substitution of a basic amino acid with another basic amino acid, of an acid amino acid with another acid amino acid or of a neutral amino acid with another neutral amino acid. The present invention also includes peptides that possess one to five additional amino acids at the N- or C-terminal ends, preferably selected from those present at the corresponding positions of endostatin sequence (for example the amino acids at the position 11-15 and 68-72). The peptides object of the present invention can be prepared with methods and reactions conventionally used in peptide synthesis. The protection of amino groups in the amino acids can be carried out by use of 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), trityl (Trt) moieties and others commonly used in the peptide chemistry. The carboxyl group can be protected by means of tert-butyl ester, benzyl ester, p-methoxybenzyl ester and others conventionally used for said purposes. The protective groups can be removed according to processes known in literature, such as by treatment with trifluoroacetic acid, anhydrous hydrofluoric acid, piperidine and the like. The amino acids can be condensed by using active esters such as pentafluorophenyl ester (OPfp), 3-hydroxy-4-oxo-3,4-dihydro-l,2,3- benzotriazine ester (ODhbt), or carboxy-activators such as benzotriazol-1-yl- oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBop), 2-(lH- benzotriazol-l-yl-l,l,3,3-tetramethyl)uronium tetrafluorob orate (TBTU) and the other activators conventionally used for this type of reactions. The purification of the polypeptides described in the present invention can also be carried out according to known techniques of protein chemistry, such reverse phase HPLC, gel filtration, ion exchange chromatography and preparative electrophoresis. More particularly, the peptides can be prepared using the solid phase peptide synthesis and the automatic synthesizer Biolynx plus, mod 4170 by Novabiochem (Nottingham, Great Britain) (A. Dryland and R.C. Sheppard, J. Chem. Soc, Perkin 1, 125, 1986). The protection of the α-amino groups in the amino acids can be carried out by use of 9-fluorenylmethoxycarbonyl (Fmoc). The functional groups of amino acids side chains are protected using the following protective groups: tert-butyl for aspartic acid, serine, threonine and tyrosine; 2,2,4,6,7- pentamethyl-dihydro-benzofuran-5-sulfonyl for arginine; acetamidomethyl for cysteine. The synthesis is gradually carried out starting from the C-terminal Fmoc-amino acid, attached by an ester bond to a resin consisting of polyethylene oxide grafted to a polystyrene matrix and functionalized by a 4-hydroxymethyl-phenoxyacetic acid residue (E. Bayer, Angew. Chem., 103, 117, 1991). Fmoc is removed by using a solution of piperidine in dimethylformamide (DMF). Pentafluorophenyl esters of Fmoc-amino acids are generally used for condensation reactions. In the case of arginine the carboxylic group was activated by PyBop in the presence of diisopropylethylamine, with three hour reaction times. To maximize the reaction yields, a five equivalent excess of Fmoc-amino acid is used. The times of deprotection and condensation reactions are automatically determined by the synthesizer; the technician will select the acylation times only in the case of activation with PyBop. The peptide is cleaved from the solid carrier, at the same time removing all the protective groups, by acidolysis with a mixture having the following composition: 80% TFA, 5% H₂O, 2.5% ethanedithiol, 2.5% phenol and 5% thioanisole. The resulting crude polypeptides are purified by reverse phase semipreparative HPLC, using a column "Jupiter" (250 x 10 mm) C, 10 μ (Phenomenex, USA) and an Aktabasic apparatus 100 mod. 18-1405 (Amersham Pharmacia Biotech, Freiburg). Solvent A: 90% of 0.1% trifluoroacetic acid and 10% of acetonitrile; solvent B: 90% of acetonitrile and 10% of 0.1% trifluoroacetic acid. Gradient: from solvent A to solvent B in 65 minutes. Flow rate: 5 ml/minute. Detection at λ = 226 nm. 20-25 mg of product are loaded for each run. The main fractions are collected and freeze-dried. The purified polypeptides are characterized by specific rotation, amino acid analysis and electrospray mass spectrometry. EXAMPLE 1 Asn-Ser(tBu)-Pro-Leu-Ser(tBu)-Gly-Gly-Met-Arg(Pbf)-Gly-Ile- Arg(Pbf)-Gly-Ala-Asp(OtBu)-Phe-Gln-Cys(Acm)-Phe-Gln-Gln-Ala-Arg(Pbf)- Ala-Val-Gly-Leu-Ala-Gly-Thr(tBu)-Phe-Arg(Pbf)-Ala-Phe-Leu-Ser(tBu)- Ser(tBu)-Arg(Pbf)-Leu-Gln-Asp(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ile-Val- Arg(Pbf)-Arg(Pbf)-Ala-Asp(OtBu)-Arg(Pbf)-Ala- resin. 500 mg (0.1 mmol) of Fmoc-Ala-resin were suspended in 25 ml of DMF and after 2 hours they were loaded into the reaction column. The Fmoc-amino acid-resin was then subjected to the following treatments: a) washings with DMF; b) removal of Fmoc by treatment with a 20% piperidine solution in DMF; c) washings with DMF; d) condensation with the suitable Fmoc-amino acid active ester (5 equivalents) in the presence of N-hydroxy-benzotriazole (5 equivalents) as catalyst, with the addition of an anionic dye (Novachrome, Calbiochem-Novabiochem AG, Laufelfingen, Switzerland) for automatically monitoring the reaction time. Only in the case of Fmoc-Arg(Pbf), the carboxyl was activated by using PyBop (1 equivalent), 1-hydroxybenzotriazole (1 equivalent) and N-diisopropylethylamine (2 equivalent), without addition of dye (reaction time 4 hours). All coupling reactions were repeated to maximize the reaction yields; e) washings with DMF. This cycle of operations was repeated with the suitable Fmoc-amino acid to finally obtain the protected resin-dipentacontapeptide. The product was then placed in a sintered glass funnel and washed in succession with DMF, tert-amyl alcohol, acetic acid, tert-amyl alcohol, methylene chloride and dry diethyl ether. 1276 mg of protected resin- dipentacontapeptide were obtained. EXAMPLE 2 Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe- Gln-Cys(Acm)-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe- Arg-Ala-Phe-Leu-Ser-Ser-Arg-Leu-Gln-Asp-Leu-Tyr-Ser-Val-Ile-Arg-Arg- Ala- Asp- Arg- Ala. 1276 mg of protect resin-dipentacontapeptide were suspended in 200 ml of a mixture having the following composition: 80% TFA, 5% H₂O, 2.5% ethanedithiol, 2.5% phenol and 5% thioanisole; the mixture was reacted for 2 hours with occasional stirring. After filtration under vacuum, the resin was washed with TFA (2 x 50 ml). The filtrate was slowly added with dry ethyl ether to precipitate the polypeptide. The product was filtered, repeatedly washed with dry diethyl ether and finally dried under vacuum over KOH. The crude compound was purified by semipreparative HPLC as described above. Main fraction was pooled and lyophilized to obtain 98 mg of pure dipentacontapeptide. [α]²⁰ _D = - 62.5 (c = 0.2, H₂O) Mass spectrum: molecular peak = 5760 Amino acid analysis: Asp = 3.89 (4); Thr = 0.93 (1); Ser = 4.85 (5); Glu = 4.05 (4); Pro = 0.91(1); Gly = 6.19 (6); Ala = 6.94 (7); Cys = 0.89 (1); Val = 2.03 (2); Met - 0.91 (1); He = 2.11(2); Leu - 5.07 (5); Tyr = 1.06 (1); Phe = 4.12 (4); Arg = 7.88 (8). The peptide of Example 2 exhibit potent antiangiogenic activities, inhibiting cell migration, cell proliferation and chemotaxis using human umbilical vein endothelial cells; peptide 16-67 is also active in the matrigel plug assays in vitro and vivo. When tested in vivo on tumor growth, peptide 16-67 demonstrated to be more effective than full-length endostatin. For therapeutic applications, the peptides of the invention or non-toxic salts or derivatives will be formulated in pharmaceutical compositions in mixture with a suitable diluent or carrier. These compounds may be administered by parenteral route: subcutaneously, intramuscularly or intravenously. The dosage of the active ingredient may vary from 0.1 to 1 mg/kg/die.

Claims

1. Peptide with the following sequence: Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe- Gln-Cys(Acm)-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe- Arg-Ala-Phe-Leu-Ser-Ser-Arg-Leu-Gln-Asp-Leu-Tyr-Ser-Ile-Val-Arg-Arg- Ala-Asp-Arg-Ala, its analogues and derivatives.

2. Use of the peptide of claim 1 for the preparation of medicaments with antiangiogenic and antitumor activities.

3. Pharmaceutical compositions containing the peptide of claim 1 in mixture with a suitable carrier.