Angiogenesis, the outgrowth of new capillaries from pre-existing vessels, is essential for physiological and pathological conditions including tumor growth and metastasis (1-3). Angiogenesis is a complex multistep process that includes proliferation, migration and differentiation of endothelial cells, degradation of extracellular matrix, microtubule formation and sprouting of new capillary branches (3,4)
Endostatin is a 22 kDa C-terminal fragment of collagen XVIII that specifically inhibits endothelial proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo (5). Systemic administration of non refolded precipitated protein expressed in E. coli caused growth regression of experimental tumors in mice (5,6). We reported that human full-length endostatin produced by E. coli expression system is able to block fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) induced proliferation and migration of microvascular endothelial cells (7).
The production of large amount of endostatin can be difficult. Thus the availability of smaller molecule with sequence homology with endostatin endowed with biological activity can be useful.
The present invention relates to polypeptides comprising ten to sixty amino acid residues with a sequence corresponding or homologous to that or endostatin, having angiogenesis-inhibiting activity, useful for the treatment of angiogenesis-dependent tumours.
The present invention also relates to a process for the preparation of said polypeptides.
The invention further relates to pharmaceutical formulations containing one or more of said polypeptides.
Endostatin is a protein having antiangiogenic activity, isolated by J. Folkman and M. O'Reilly (EP 0 857 210).
Examples of the polypeptides prepared according to the invention are the following.    (I) nonatriacontapeptide: His-Thr-His-Gln-Asp-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Thr-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys(tBu)-Phe-Gln-Gln-Ala-Arg-Ala (SEQ ID NO: 1)    (II) pentacontapeptide: Val-Gly-Leu-Ser-Gly-Thr-Phe-Arg-Ala-Phe-Leu-Ser-Ser-Arg-Leu-Gln-Asp-Leu-Tyr-Ser-Ile-Val-Arg-Arg-Ala-Asp-Arg-Gly-Ser-Val-Pro-Ile-Val-Asn-Leu-Lys-Asp-Glu-Val-Leu-Ser-Pro-Ser-Trp-Asp-Ser-Leu-Phe-Ser-Gly (SEQ ID NO: 2)    (III) pentatetracontapeptide: Ser-Gln-Gly-Gln-Val-Gln-Pro-Gly-Ala-Arg-Ile-Phe-Ser-Phe-Asp-Gly-Arg-Asp-Val-Leu-Arg-His-Pro-Ala-Trp-Pro-Gln-Lys-Ser-Val-Trp-His-Gly-Ser-Asp-Pro-Ser-Gly-Arg-Arg-Leu-Met-Glu-Ser-Tyr (SEQ ID NO: 3)    (IV) pentacontapeptide: Cys-Glu-Thr-Trp-Arg-Thr-Glu-Thr-Thr-Gly-Ala-Thr-Gly-Gln-Ala-Ser-Ser-Leu-Leu-Ser-Gly-Arg-Leu-Leu-Glu-Gln-Lys-Ala-Ala-Ser-Cys-His-Asn-Ser-Tyr-Ile-Val-Leu-Cys(tBu)-Ile-Glu-Asn-Ser-Phe-Met-Thr-Ser-Phe-Ser-Lys (SEQ ID NO: 4).
These polypeptides have remarkable antiangiogenic activity in the in vitro inhibition test on endothelial cells proliferation and migration (16). More particularly, nonatriacontapeptide I turned out to be equipotential to endostatin.
The process for the preparation of the polypeptides of the invention is based on the following general methods and reactions used in peptide synthesis.
Amino groups of the amino acids can be protected by use of the 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z), trityl (Trt) groups and other groups commonly used in peptide chemistry.
The carboxylic group can be protected by means of the tert-butyl ester, benzyl ester, p-methoxybenzyl ester and others conventionally used for said purposes.
These protective groups, as it will be illustrated in is detail in the examples, can be removed according to processes known in literature, such as by treatment with trifluoroacetic acid, anhydrous hydrofluoric acid, piperidine and the like, according to circumstances.
The amino acids can be condensed by using active esters such as pentafluorophenyl ester (OPfp), 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzothiazine ester (ODhbT), or carboxy-activators such as benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBop), 2-(1H-benzotriazol-1-yl-1,1,3,3-tetramethyl)-uronium tetrafluoroborate (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 as reverse phase HPLC, gel filtration, ion exchange chromatography and preparative electro-phoresis.
For example, the process of the present invention can be carried out as follows, using the solid phase peptide synthesis and the automatic synthesizer Biolynx plus, mod. 4170 by Novabiochem (Nottingham, Great Britain) (17).
The protection of the α-amino groups in the amino acids is carried out by use of 9-fluorenylmethoxycarbonyl (Fmoc). The functional groups of the amino acids side chains are protected using the following protective groups: tert-butyl for aspartic acid, glutamic acid, serine, threonine and tyrosine; tert-butoxycarbonyl for lysine and trypthophan; trityl for histidine; 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl for arginine; tert-butyl for cysteine of polypeptide I and for the third cysteine of polypeptide IV; trityl for the two other cysteines of polypeptide IV.
The synthesis is gradually carried out starting from the C-terminal Fmoc-amino acid, attached to a resin by an ester bond, consisting of polyethyleneoxide grafted to a polystyrene matrix and functionalized by a 4-hydroxymethyl-phenoxyacetic acid residue (18). Fmoc is removed by using a solution of piperidine in dimethylformamide (DMF). Pentafluorophenyl esters of Fmoc-amino acids are generally used for the condensation reactions. In the case of serine and threonine, the use of ODhbt esters was preferred, whereas in the case of arginine and histidine 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 resin, at the same time removing all the protective groups, by acidolysis with trifluoroacetic acid in the presence of 5% anisole and 1% ethanedithiol.
The resulting crude polypeptides are purified by reverse phase semipreparative HPLC, using a column (250×10 mm) filled with Source TM 15 RPC (Pharmacia Biotech AB, Uppsala, Sweden). Polypeptides are eluted with a linear gradient from 0% to 60% of acetonitrile in 0.1% aqueous TFA, at a 5 ml/min flow rate with detection at 226 nm; 10-15 mg of product are loaded for each run.
The main fractions are collected and freeze-dried.
The purified polypeptides are characterized by amino acid analysis and electrospray mass spectrometry with a Finnigan Mat apparatus mod. LCQ.
The present invention also relates to the pharmaceutical compositions comprising the polypeptides of the invention or a non toxic salt thereof, in mixture with a suitable diluent or carrier.
The following examples further illustrate the invention without limiting it.