WO2005085286A2 - Peptides with antiangiogenic and antitumor activities - Google Patents
Peptides with antiangiogenic and antitumor activities Download PDFInfo
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- WO2005085286A2 WO2005085286A2 PCT/EP2005/001945 EP2005001945W WO2005085286A2 WO 2005085286 A2 WO2005085286 A2 WO 2005085286A2 EP 2005001945 W EP2005001945 W EP 2005001945W WO 2005085286 A2 WO2005085286 A2 WO 2005085286A2
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 32
- 230000001772 anti-angiogenic effect Effects 0.000 title claims abstract description 10
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 7
- 102000004196 processed proteins & peptides Human genes 0.000 title description 17
- 239000003814 drug Substances 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 24
- 235000001014 amino acid Nutrition 0.000 description 21
- 150000001413 amino acids Chemical class 0.000 description 21
- 102400001047 Endostatin Human genes 0.000 description 14
- 108010079505 Endostatins Proteins 0.000 description 14
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 9
- -1 hydroxy, thio Chemical group 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 8
- 239000012634 fragment Substances 0.000 description 7
- MSXVEPNJUHWQHW-UHFFFAOYSA-N 2-methylbutan-2-ol Chemical compound CCC(C)(C)O MSXVEPNJUHWQHW-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 241001529936 Murinae Species 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 4
- 101500026378 Homo sapiens Endostatin Proteins 0.000 description 4
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- 210000004899 c-terminal region Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 3
- 230000033115 angiogenesis Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
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- 230000035484 reaction time Effects 0.000 description 3
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000010933 acylation Effects 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011894 semi-preparative HPLC Methods 0.000 description 2
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 1
- VUCNQOPCYRJCGQ-UHFFFAOYSA-N 2-[4-(hydroxymethyl)phenoxy]acetic acid Chemical group OCC1=CC=C(OCC(O)=O)C=C1 VUCNQOPCYRJCGQ-UHFFFAOYSA-N 0.000 description 1
- SONUFGRSSMFHFN-IMJSIDKUSA-N Asn-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O SONUFGRSSMFHFN-IMJSIDKUSA-N 0.000 description 1
- 108010001463 Collagen Type XVIII Proteins 0.000 description 1
- 102000047200 Collagen Type XVIII Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101100202932 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) tsp-4 gene Proteins 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 101100208249 Rattus norvegicus Thbs4 gene Proteins 0.000 description 1
- 239000012317 TBTU Substances 0.000 description 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000034196 cell chemotaxis Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001023 pro-angiogenic effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 125000005500 uronium group Chemical group 0.000 description 1
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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
Definitions
- the present invention relates to peptides with antiangiogenic and antitumor activities having a sequence corresponding to fragments of human endostatin.
- 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.
- systemic administration of recombinant endostatin causes regression of tumors in mice.
- 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.
- 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 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.
- 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. studying the activity of five fragments of murine endostatin (1-39, 40-89, 90- 134, 135-184 and 135-184 with a disulf ⁇ de 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 .
- 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 NH 2 (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
- 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.
- 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
- 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 ⁇ -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).
- DMF dimethylformamide
- Pentafluorophenyl esters of Fmoc-amino acids are generally used for condensation reactions.
- the carboxylic group was activated by PyBop in the presence of diisopropylethylamine, with three hour reaction times.
- 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% H 2 O, 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 ⁇ (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 ⁇ 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.
- Fmoc-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.
- an anionic dye Novachrome, Calbiochem-Novabiochem AG, Laufelfingen, Switzerland
- 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.
- 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.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ITMI20040364 ITMI20040364A1 (en) | 2004-02-27 | 2004-02-27 | PEPTIDES WITH ANTIANGIOGENIC AND ANTI-TUMOR ACTIVITY |
ITMI2004A000364 | 2004-02-27 |
Publications (2)
Publication Number | Publication Date |
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WO2005085286A2 true WO2005085286A2 (en) | 2005-09-15 |
WO2005085286A3 WO2005085286A3 (en) | 2006-03-02 |
Family
ID=34917541
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2005/001945 WO2005085286A2 (en) | 2004-02-27 | 2005-02-24 | Peptides with antiangiogenic and antitumor activities |
Country Status (2)
Country | Link |
---|---|
IT (1) | ITMI20040364A1 (en) |
WO (1) | WO2005085286A2 (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999048924A1 (en) * | 1998-03-24 | 1999-09-30 | The Children's Medical Center Corporation | Endostatin derived peptides with anti-angiogenic and anti-cancer activity |
WO2000063249A1 (en) * | 1999-04-15 | 2000-10-26 | Universita' Degli Studi Di Milano | Polypeptides derived from endostatin exhibiting antiangiogenic activity |
WO2000067771A1 (en) * | 1999-05-06 | 2000-11-16 | The Burnham Institute | Antiangiogenic endostatin peptides, endostatin variants and methods of use |
WO2002068457A2 (en) * | 2001-02-27 | 2002-09-06 | Universita' Degli Studi Di Milano | Antiangiogenic peptides derived from endostatin |
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2004
- 2004-02-27 IT ITMI20040364 patent/ITMI20040364A1/en unknown
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2005
- 2005-02-24 WO PCT/EP2005/001945 patent/WO2005085286A2/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999048924A1 (en) * | 1998-03-24 | 1999-09-30 | The Children's Medical Center Corporation | Endostatin derived peptides with anti-angiogenic and anti-cancer activity |
WO2000063249A1 (en) * | 1999-04-15 | 2000-10-26 | Universita' Degli Studi Di Milano | Polypeptides derived from endostatin exhibiting antiangiogenic activity |
WO2000067771A1 (en) * | 1999-05-06 | 2000-11-16 | The Burnham Institute | Antiangiogenic endostatin peptides, endostatin variants and methods of use |
WO2002068457A2 (en) * | 2001-02-27 | 2002-09-06 | Universita' Degli Studi Di Milano | Antiangiogenic peptides derived from endostatin |
Non-Patent Citations (1)
Title |
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CHILLEMI FRANCESCO ET AL: "Studies on the structure-activity relationship of endostatin: Synthesis of human endostatin peptides exhibiting potent antiangiogenic activities." JOURNAL OF MEDICINAL CHEMISTRY, vol. 46, no. 19, 11 September 2003 (2003-09-11), pages 4165-4172, XP002350488 ISSN: 0022-2623 cited in the application * |
Also Published As
Publication number | Publication date |
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WO2005085286A3 (en) | 2006-03-02 |
ITMI20040364A1 (en) | 2004-05-27 |
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