US20060014693A1 - Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity - Google Patents
Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity Download PDFInfo
- Publication number
- US20060014693A1 US20060014693A1 US11/168,323 US16832305A US2006014693A1 US 20060014693 A1 US20060014693 A1 US 20060014693A1 US 16832305 A US16832305 A US 16832305A US 2006014693 A1 US2006014693 A1 US 2006014693A1
- Authority
- US
- United States
- Prior art keywords
- peptide
- growth factor
- bfgf
- pep1
- pdgf
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
Links
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 title claims abstract description 36
- 108010081589 Becaplermin Proteins 0.000 title claims abstract description 20
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 20
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 title claims abstract description 6
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 title claims abstract description 6
- 102000018233 Fibroblast Growth Factor Human genes 0.000 title claims abstract description 5
- 108050007372 Fibroblast Growth Factor Proteins 0.000 title claims abstract description 5
- 229940126864 fibroblast growth factor Drugs 0.000 title claims abstract description 5
- 230000002401 inhibitory effect Effects 0.000 title claims description 12
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 title claims 2
- 230000000694 effects Effects 0.000 title abstract description 26
- 238000000034 method Methods 0.000 claims description 13
- 230000012292 cell migration Effects 0.000 claims description 8
- 206010027476 Metastases Diseases 0.000 claims description 7
- 230000004663 cell proliferation Effects 0.000 claims description 7
- 230000009401 metastasis Effects 0.000 claims description 6
- 230000004614 tumor growth Effects 0.000 claims description 6
- 230000007170 pathology Effects 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 claims description 2
- 208000007536 Thrombosis Diseases 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 230000000144 pharmacologic effect Effects 0.000 claims 1
- 208000019553 vascular disease Diseases 0.000 claims 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 abstract description 34
- 238000001727 in vivo Methods 0.000 abstract description 10
- 238000000338 in vitro Methods 0.000 abstract description 9
- 230000033115 angiogenesis Effects 0.000 abstract description 8
- 241000283690 Bos taurus Species 0.000 abstract description 5
- 241000699670 Mus sp. Species 0.000 abstract description 5
- 210000002889 endothelial cell Anatomy 0.000 abstract description 4
- 210000000329 smooth muscle myocyte Anatomy 0.000 abstract description 4
- 108700038606 rat Smooth muscle Proteins 0.000 abstract description 3
- 108010088535 Pep-1 peptide Proteins 0.000 description 49
- 238000013508 migration Methods 0.000 description 18
- 230000005012 migration Effects 0.000 description 13
- 230000035755 proliferation Effects 0.000 description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 239000012894 fetal calf serum Substances 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 210000002469 basement membrane Anatomy 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 201000001320 Atherosclerosis Diseases 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000010232 migration assay Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000002269 spontaneous effect Effects 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 2
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 2
- 102100037362 Fibronectin Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 210000000709 aorta Anatomy 0.000 description 2
- 210000002403 aortic endothelial cell Anatomy 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 239000002975 chemoattractant Substances 0.000 description 2
- 230000035605 chemotaxis Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000001023 pro-angiogenic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 208000037803 restenosis Diseases 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 210000005167 vascular cell Anatomy 0.000 description 2
- 230000007998 vessel formation Effects 0.000 description 2
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108091008606 PDGF receptors Proteins 0.000 description 1
- 102000011653 Platelet-Derived Growth Factor Receptors Human genes 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 238000002399 angioplasty Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000001889 chemoattractive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 230000000437 effect on angiogenesis Effects 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000010595 endothelial cell migration Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000025366 tissue development Effects 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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 concerns the identification and the synthesis of a peptide, derived from the basic human fibroblast growth factor (bFGF), having the following primary structure:
- PEP1 Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu hereafter referred to as PEP1 (SEQ ID NO: 1).
- Said molecule showing analogy with a sequence of bFGF, namely inhibits in vitro as well as in vivo PDGF-BB and bFGF effects.
- RASMC primary rat smooth muscle cells
- BAEC primary bovine endothelial cells
- PEP1 might be used for the treatment of diseases with abnormal proliferation and migration of vascular cells such as restenosis after angioplasty, atherosclerosis, tumor growth and metastasis dissemination.
- PDGF-BB Platelet Derived Growth Factor
- bFGF basic Fibroblast Growth Factor
- PDGF-BB Platelet Derived Growth Factor
- bFGF basic Fibroblast Growth Factor
- Angiogenesis is a key process for tissue development, as well as tumor growth and dissemination, It is controlled by several factors modulating cell differentiation, proliferation and migration (Holash, J., 1999, Oncogene, 18, 5356-5362; Zetter, B. R. et al., 1998, Annu. Rev. Med., 49, 407-424).
- PDGF and bFGF are required for tumor cells growth in vitro, growth of solid tumors in vivo, as well as metastases dissemination (Shawver, L. K. et al., 1997, Clin. Cancer Res., 3, 1167-1177; Vignaud, J. M. et al., 1994, Cancer Res., 54, 5455-5463; Chandler, L. A. et al., 1999, Int. J. Cancer, 81, 451-458; Westphal, J. R. et al., 2000, Int. J. Cancer, 15,86 (6), 768-776).
- PDGF and bFGF are potential candidates for the treatment of proliferative diseases and angiogenesis-related disorders.
- PDGF-BB and BFGF play an unsuspected role in the modulation of their pro-angiogenic functions.
- the inhibitory role of bFGF on cell proliferation and migration in addition to its pro-angiogenic effect has been demonstrated (Facchiano, A. et al., 2000, J. Cell. Sci., 113, 2855-2863).
- PEP1 Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu
- bFGF derived from human bFGF turned out to be a strong inhibitor in vitro of bFGF, PDGF-BB and fetal calf serum (FCS) effects, such as cell proliferation and migration observed in primary rat smooth muscle cells (RASMC) and primary bovine endothelial cells (BAEC). Said activity has been observed at a dose as low as 10 nanograms/milliliter and PEP1 is not toxic at this dose in vitro.
- the heat-denatured and the scrambled version (with random aminoacid sequence) of PEP1 were used as control: both do not show any activity.
- PEP1 even show inhibitory activity in vivo; it is, indeed, able to inhibit angiogenesis in reconstituted basement membrane plugs, subcutaneously injected in CD1 mice.
- PEP1scr a scrambled version of the peptide
- FIG. 1 shows the results of dose-dependent experiments carried out on RASMC.
- RASMC proliferation induced by 10% FCS was evaluated after 48 hours, in the absence and in the presence of different PEP1 doses, ranging from 1 g/mi to 1 pg/ml;
- FIG. 2A shows PEP1 and PEPscr effect on RASMC proliferation induced by PDGF-BB (10 ng/ml);
- FIG. 2B shows PEP1 and PEP1scr effect on RASMC spontaneous proliferation in the presence of BSA
- FIG. 3A shows PEP1 and PEPscr effect on BAEC proliferation induced by PDGF-BB (10 ng/ml);
- FIG. 3B shows PEP1 and PEP1scr effect on BAEC spontaneous proliferation in the presence of BSA
- FIG. 4A shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by FCS (1%);
- FIG. 4B shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by PDGFD-BB (10 ng/ml);
- FIG. 4C shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by bFGF (10 ng/ml);
- FIG. 5A shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by EGF (10 ng/ml);
- FIG. 5B shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by aFGF (10 ng/ml);
- FIG. 5C shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by Fibronectin (10 ng/ml);
- FIG. 5D shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by VEGF (10 ng/ml);
- FIG. 6 shows PEP1 and PEP1scr effect on RASMC migration induced by PDGF-BB (10 ng/ml);
- FIG. 7 shows PEP1 and PEP1scr effect on angiogenesis induced by bFGF in reconstituted basement membrane plugs, subcutaneusly injected in CD1 mice.
- RASMC Primary rat aorta smooth muscle cells
- BAEC primary bovine aortic endothelial cells
- Chemoattractant factor were calf fetal serum (FCS) 10% or the following human recombinant factors: PDGF-BB, bFGF and vascular endothelial growth factor (VEGF).
- PEP1 PEPscr (scrambled control) diluted in water, were added to the growth factor solution at the reported final concentration.
- PEP1scr control does not show any activity when dispensed in both portion of the Boyden chamber.
- PEP1 effect on other chemoattractans was tested.
- PEP1 and PEP1scr do not affect Endothelial cell migration induced by aFGF or VEGF or EGF or Fibronectin ( FIGS. 5A, 5B , 5 C and 5 D), indicating that said molecule specifically affect bFGF and PDGF-BB.
- Proliferation assay was carried out on primary rat aorta SMC and on primary bovine aortic endothelial cells (BAEC).
- Cells were plated in six-well plates (1 ⁇ 10 5 cells/plate) and grown for 24 hours in Dulbecco Modified eagle's medium (DMEM) supplemented with 10% FBS, at 37° C. in 5% CO 2 . Then, the medium was replaced with DMEM medium containing 0.1% BSA for 24 hours. Subsequently, the medium was replaced with fresh medium containing either 0.1% BSA alone or 0.1% BSA with growth factors at 10 ng/ml final concentration or fetal calf serum (FCS) al 10%, in the absence or in the presence of PEP1 or control peptide. Each assay was carried out for mounting period of time up to a maximum time of three days and the cell were harvested and counted with hemacytometer.
- DMEM Dulbecco Modified eagle's medium
- FCS
- PEP1 was tested in dose-dependence experiments: RASMC proliferation induced by FCS 10%, was evaluated at 48 hours, in the presence and in the absence of different PEP1 doses, ranging from 1 ⁇ g/ml to 1 pg/ml ( FIG. 1 ).
- the heat-denatured PEP1 and the scrambled version of PEP1 were used as control.
- PEP1 showed a dose-dependent inhibitory activity, which reached 60% inhibitory effect at 10 ng/ml, while the control peptides did not show any activity. Consequently, the dose of 10 ng/ml was chosen for the following in vitro experiments.
- FIG. 2A shows the marked inhibition of proliferation induced by PDGF-BB.
- proliferation induced by PDGF-BB (10 ng/ml) was significantly inhibited in the presence of PEP1 at all time points.
- PEP1 block almost completely cell proliferation, while the control scrambled peptide (PEP1scr) is not effective at any time ( FIG. 2A ).
- PEP1 shows similar inhibitory effect on BAEC stimulated by bFGF (10 ng/ml) ( FIG. 3A ).
- Angiogenesis on reconstituted basement membrane plugs was carried out as previously reported (Muhlhauser, J., 1995, J. Circ. Res., 77,1077-1086). Briefly, reconstituted basement membrane plugs added with bFGF (150 ng/ml) alone or in the presence of PEP1 (10 micrograms/ml) were subcutaneusly injected in CD1 mice (female, 19 weeks age). bFGF induces the formation of capillary network within 7 days, therefore plugs were excised 7 days after injection and included in paraffin. Obtained slides were stained with trichrome-Masson staining procedure and analysed with an optical image analyzer and the number of vessels per mm 2 within plugs was quantified.
- FIG. 7 shows that PEP1 acts as strong inhibitor of blood vessel formation induced by bFGF (i.e. 46% inhibition vs bFGF alone). 10 animals were used as control (treated with bFGF alone) and 14 animals were treated with bFGF in the presence of PEP1. This experiment shows that PEP1 is able to markedly inhibit new-blood vessel formation induced by bFGF and indicates PEP1 as a good candidate for further in vivo studies.
- PEP1 showed a strong and specific inhibitory activity on mitogenic and chemoattractive properties of platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) in vitro.
- PDGF-BB platelet derived growth factor
- bFGF fibroblast growth factor
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Heart & Thoracic Surgery (AREA)
- Oncology (AREA)
- Cardiology (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A novel peptide, derived from the human fibroblast growth factor (bFGF), is identified. Said molecule is able to inhibit in vitro the effects of Platelet Derived Growth Factor (PDGF-BB) and basic Fibroblast Growth Factor (bFGF) on primary rat smooth muscle cells (RASMC) and primary bovine endothelial cells (BAEC). Said molecule is also able to inhibit in vivo angiogenesis CD1 mice.
Description
- This application is a division of co-pending application Ser. No. 10/077,746, filed on Feb. 20, 2002, the entire contents of which are hereby incorporated by reference.
- The present invention concerns the identification and the synthesis of a peptide, derived from the basic human fibroblast growth factor (bFGF), having the following primary structure:
- Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu hereafter referred to as PEP1 (SEQ ID NO: 1).
- Said molecule, showing analogy with a sequence of bFGF, namely inhibits in vitro as well as in vivo PDGF-BB and bFGF effects.
- More particularly, in vitro experimentation on primary rat smooth muscle cells (RASMC) and primary bovine endothelial cells (BAEC) indicated that said molecule is an efficient inhibitor of cell proliferation and migration at a dose that is not toxic for cells.
- Moreover, in vivo experimentation carried out on reconstituted basement membrane plugs, subcutaneously injected in CD1 mice demonstrated that said molecule strongly inhibits bFGF-induced angiogenesis.
- Reported results suggest that PEP1 might be used for the treatment of diseases with abnormal proliferation and migration of vascular cells such as restenosis after angioplasty, atherosclerosis, tumor growth and metastasis dissemination.
- Growth factors, such as Platelet Derived Growth Factor (PDGF-BB) and basic Fibroblast Growth Factor (bFGF) play a crucial role in the proliferation and differentiation of many cell types. In fact, increased levels and/or activity of these factors occur in several pathologies, including tumor growth and blood-vessel diseases like atherosclerosis.
- Platelet Derived Growth Factor (PDGF-BB) and basic Fibroblast Growth Factor (bFGF) are both essential for the phatogenesis of angiogenesis-related diseases since they directly modulate cell proliferation and migration within vascular wall—(Ross, R., et al. 1990, Science, 248, 1009-1012; Ross, R. 1993, Nature, 362, 801-809).
- Angiogenesis is a key process for tissue development, as well as tumor growth and dissemination, It is controlled by several factors modulating cell differentiation, proliferation and migration (Holash, J., 1999, Oncogene, 18, 5356-5362; Zetter, B. R. et al., 1998, Annu. Rev. Med., 49, 407-424).
- Several different molecules, such as antibodies neutralising PDGF and bFGF (Rutherford et al., Atherosclerosis, 1997, 45-51) and oligonucleotides inhibiting PDGF receptor expression (Sirois, M. G. et al., 1997, Circulation, 95, 669-676), were successfully used in vivo to inhibit diseases with abnormal proliferation and migration of vascular cells such as restenosis. Furthermore, specific inhibitors currently available are able to interfere with the receptor-binding or receptor dimerization or signaling (Heldin, C. H. et al., 1998, BBA, F79-F113).
- PDGF and bFGF are required for tumor cells growth in vitro, growth of solid tumors in vivo, as well as metastases dissemination (Shawver, L. K. et al., 1997, Clin. Cancer Res., 3, 1167-1177; Vignaud, J. M. et al., 1994, Cancer Res., 54, 5455-5463; Chandler, L. A. et al., 1999, Int. J. Cancer, 81, 451-458; Westphal, J. R. et al., 2000, Int. J. Cancer, 15,86 (6), 768-776).
- Inhibiting the activity and/or the signaling of PDGF and bFGF led to effective reduction of tumor growth and metastasis dissemination (Abramovich, R. et al., 1999, Br. J. Cancer, 79 (9-10), 1392-8; Bagheri-Yarmand, R. et al.,1998, Br. J. Cancer, 78 (1), 1118; Sola, F. et al, 1995, Invasion Metastasis, 15 (5-6), 222-231; Wang, Y. et al., 1997, Nature Med., 3, 887-893).
- Therefore, specific antagonists of PDGF and bFGF are potential candidates for the treatment of proliferative diseases and angiogenesis-related disorders.
- According to recent data collected by the same inventors, PDGF-BB and BFGF play an unsuspected role in the modulation of their pro-angiogenic functions. In particular, the inhibitory role of bFGF on cell proliferation and migration in addition to its pro-angiogenic effect, has been demonstrated (Facchiano, A. et al., 2000, J. Cell. Sci., 113, 2855-2863).
- Moreover, the factors regulating the protein-folding and the structure-biological function relationship has been investigated (Ragone, R. et al., 1987, Italian J. of Biochem., 36, 306-309; Facchiano, F. et al., 1988, CABIOS, 4, 2, 303-305; Ragone, R. et al., 1989, Protein Engineering, 2, 7, 497-504; Facchiano, A. M. et al., 1989, CABIOS, 5, 4, 299-303; Facchiano, A. M. et al., 1991, CABIOS, 7, 3, 395- 396; Facchiano, A. et al., 1993, J. Mol. Evol., 36 (5), 448-457; Benvenga, S. et al., 1993, EOS-J. of Immunol. and Immunopharm., 13 (1), 18-19; Facchiano, A., 1995, J. Mol. Evol., 40, 570-577; Facchiano, A., 1996, Trends in Genetics, 12(5), 168-169; Scarselli, M. et al., 1997, J. Peptide Sci., 3, 1-9; Benvenga, S. et al., 1999, Amyloid, 6 (4), 250-255; Facchiano, A. M., 1999, Protein Eng., 12 (10),893; Pozzetto, U. et al., 2000, Transplant Int., Suppl. n. 1, 13, S306-S310; Facchiano, A. M., 2000, Bioinformatics, 16 (3), 292-293).
- In the present invention, by investigating protein structure, regions of bFGF sequence potentially responsible of its biological activity have been identified. Among these regions, a peptide having the following primary structure:
- Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu (SEQ ID NO: 1; here referred to as PEP1), derived from human bFGF turned out to be a strong inhibitor in vitro of bFGF, PDGF-BB and fetal calf serum (FCS) effects, such as cell proliferation and migration observed in primary rat smooth muscle cells (RASMC) and primary bovine endothelial cells (BAEC). Said activity has been observed at a dose as low as 10 nanograms/milliliter and PEP1 is not toxic at this dose in vitro. The heat-denatured and the scrambled version (with random aminoacid sequence) of PEP1 were used as control: both do not show any activity.
- Moreover, PEP1 even show inhibitory activity in vivo; it is, indeed, able to inhibit angiogenesis in reconstituted basement membrane plugs, subcutaneously injected in CD1 mice.
- Accordingly with what previously detected, PEP1 synthesis was achieved by automatic synthetizer, using the standard technique named f-moc.
- After that, three different batches of PEP1 were tested and they gave similar results in the biological assays. Moreover, a scrambled version of the peptide (PEP1scr) was prepared and after used as negative control in all the experiments.
- Several in vitro and in vivo test were carried out on said molecule and they revealed the functional characteristics of said peptide.
- The results obtained are reported in the accompanying drawings:
-
FIG. 1 shows the results of dose-dependent experiments carried out on RASMC. RASMC proliferation induced by 10% FCS was evaluated after 48 hours, in the absence and in the presence of different PEP1 doses, ranging from 1 g/mi to 1 pg/ml; -
FIG. 2A shows PEP1 and PEPscr effect on RASMC proliferation induced by PDGF-BB (10 ng/ml); -
FIG. 2B shows PEP1 and PEP1scr effect on RASMC spontaneous proliferation in the presence of BSA; -
FIG. 3A shows PEP1 and PEPscr effect on BAEC proliferation induced by PDGF-BB (10 ng/ml); -
FIG. 3B shows PEP1 and PEP1scr effect on BAEC spontaneous proliferation in the presence of BSA; -
FIG. 4A shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by FCS (1%); -
FIG. 4B shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by PDGFD-BB (10 ng/ml); -
FIG. 4C shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by bFGF (10 ng/ml); -
FIG. 5A shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by EGF (10 ng/ml); -
FIG. 5B shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by aFGF (10 ng/ml); -
FIG. 5C shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by Fibronectin (10 ng/ml); -
FIG. 5D shows the effect in the presence or in the absence of PEP1 and PEPscr (10 ng/ml) on BAEC migration induced by VEGF (10 ng/ml); -
FIG. 6 shows PEP1 and PEP1scr effect on RASMC migration induced by PDGF-BB (10 ng/ml); -
FIG. 7 shows PEP1 and PEP1scr effect on angiogenesis induced by bFGF in reconstituted basement membrane plugs, subcutaneusly injected in CD1 mice. - This test was carried out on Primary rat aorta smooth muscle cells (RASMC) obtained from six-month old male Wistar rats following a well known technique (Sterpetti, A. V. et al., 1992, J. Vasc. Surg., 6, 16-20); primary bovine aortic endothelial cells (BAEC) were obtained according to previously described protocols (D'Arcangelo, D. et al., 2000, Circ. Res.,86,312-318).
- Cell migration is a key process for the development of new blood-vessels. Consequently, PEP1 effect on cell migration induced by several different chemoattractant factors has been evaluated mainly on endothelial cells (BAEC). Migration assays were carried out in modified Boyden chambers (Neuroprobe Inc.), following known standard techniques (Albini, A. et al., 1995, Int. J. Cancer,61,121-129; Facchiano, A. et al., 2000, J. Cell. Sci., 113, 2855-2863). Cells were dispensed in the upper portion of the Boyden chamber. Chemoattractant factor were calf fetal serum (FCS) 10% or the following human recombinant factors: PDGF-BB, bFGF and vascular endothelial growth factor (VEGF). PEP1 PEPscr (scrambled control) diluted in water, were added to the growth factor solution at the reported final concentration. Thus chemotaxis induced by bFGF (10 ng/ml), or PDGF-BB (10 ng/ml), or FCS (2%), in the absence or in the presence of 10 ng/ml PEP1 and PEP1scr, was evaluated.
- All the migration assays were carried out at 37° C. in 5% CO2, for a total time of 5 hours; then filters were removed, fixed with absolute ethanol and stained with toluidine blue. Cells migrated were counted at 400× magnification in 15 fields for each filter and the average number of cell/field was reported. All the experiments were performed at least 3 times in duplicate.
- The experiments show that, in every condition, PEP1 markedly inhibit, and in a rate more than 50%, BAEC migration, but PEP1scr do not have any effect (
FIG. 4A, 4B e 4C). When bFGF or PDGF-BB were-tested, PEP1 was either dispensed in the lower and in the upper portion of the Boyden chamber; a slightly better inhibitory activity was observed when it was dispensed in the lower portion of the Boyden chamber. - In contrast, PEP1scr control does not show any activity when dispensed in both portion of the Boyden chamber. To evaluate the specificity of said inhibitory effect, PEP1 effect on other chemoattractans was tested. PEP1 and PEP1scr do not affect Endothelial cell migration induced by aFGF or VEGF or EGF or Fibronectin (
FIGS. 5A, 5B , 5C and 5D), indicating that said molecule specifically affect bFGF and PDGF-BB. - Similar results were obtained in chemotaxis assays carried on RASMC induced by PDGF-BB and FCS. PEP1 inhibits RASMC migration (i.e. about 60%), while PEP1scr is inactive (
FIG. 6 ). - Proliferation assay was carried out on primary rat aorta SMC and on primary bovine aortic endothelial cells (BAEC). Cells were plated in six-well plates (1×105 cells/plate) and grown for 24 hours in Dulbecco Modified eagle's medium (DMEM) supplemented with 10% FBS, at 37° C. in 5% CO2. Then, the medium was replaced with DMEM medium containing 0.1% BSA for 24 hours. Subsequently, the medium was replaced with fresh medium containing either 0.1% BSA alone or 0.1% BSA with growth factors at 10 ng/ml final concentration or fetal calf serum (FCS)
al 10%, in the absence or in the presence of PEP1 or control peptide. Each assay was carried out for mounting period of time up to a maximum time of three days and the cell were harvested and counted with hemacytometer. - First of all, PEP1 was tested in dose-dependence experiments: RASMC proliferation induced by
FCS 10%, was evaluated at 48 hours, in the presence and in the absence of different PEP1 doses, ranging from 1 μg/ml to 1 pg/ml (FIG. 1 ). The heat-denatured PEP1 and the scrambled version of PEP1 were used as control. PEP1 showed a dose-dependent inhibitory activity, which reached 60% inhibitory effect at 10 ng/ml, while the control peptides did not show any activity. Consequently, the dose of 10 ng/ml was chosen for the following in vitro experiments. - The effect of PEP1 was tested on proliferation induced by PDGF-BB and bFGF (10 ng/ml each), in RASMC and BAEC.
FIG. 2A shows the marked inhibition of proliferation induced by PDGF-BB. In time course experiments, proliferation induced by PDGF-BB (10 ng/ml) was significantly inhibited in the presence of PEP1 at all time points. PEP1 block almost completely cell proliferation, while the control scrambled peptide (PEP1scr) is not effective at any time (FIG. 2A ). - Spontaneous proliferation (in the presence of bovine serum albumin, BSA) is not significantly affected by PEP1 nor by PEP1scr at any time, indicating that both molecules are not toxic per se at the tested doses on RASMC (
FIG. 2B ), nor on BAEC (FIG. 3B ) Moreover, PEP1 shows similar inhibitory effect on BAEC stimulated by bFGF (10 ng/ml) (FIG. 3A ). - Then the following in vivo experiment was carried out:
- Angiogenesis on reconstituted basement membrane plugs (named “Matrigel”, produced by Collaborative Biomedical Products, Beckton-Dickinson) was carried out as previously reported (Muhlhauser, J., 1995,J. Circ. Res.,77,1077-1086). Briefly, reconstituted basement membrane plugs added with bFGF (150 ng/ml) alone or in the presence of PEP1 (10 micrograms/ml) were subcutaneusly injected in CD1 mice (female, 19 weeks age). bFGF induces the formation of capillary network within 7 days, therefore plugs were excised 7 days after injection and included in paraffin. Obtained slides were stained with trichrome-Masson staining procedure and analysed with an optical image analizer and the number of vessels per mm2 within plugs was quantified.
-
FIG. 7 shows that PEP1 acts as strong inhibitor of blood vessel formation induced by bFGF (i.e. 46% inhibition vs bFGF alone). 10 animals were used as control (treated with bFGF alone) and 14 animals were treated with bFGF in the presence of PEP1. This experiment shows that PEP1 is able to markedly inhibit new-blood vessel formation induced by bFGF and indicates PEP1 as a good candidate for further in vivo studies. - In conclusion:
- 1) PEP1 showed a strong and specific inhibitory activity on mitogenic and chemoattractive properties of platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) in vitro.
- 2) Anti-angiogenic activity in vivo was demonstrated in assays carried out on reconstituted basement membrane plugs.
- These results indicate PEP1 as a good candidate for further investigation on animal models of tumor growth and metastasis as well as other vascular-based diseases.
Claims (7)
1. A method for inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) in a subject comprising administering to said subject an effective amount of a peptide, said peptide having the following primary structure:
Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu (Seq Id No: 1)
2. A method to affect cell proliferation in a subject comprising administering to said subject an effective amount of a peptide, said peptide having the following primary structure:
Asp-Pro-His-ILe-Lys-Leu-Gln-Leu-Gln-Ala-Glu (SEQ ID NO: 1).
3. The method according to claim 2 , where said method inhibits cell migration and tumor cell migration toward potential metastasis sites comprising administering an effective amount of said peptide.
4. The method according to claim 2 , wherein said method inhibits a primary tumor growth and metastasis comprising administering an effective amount of said peptide.
5. A method for the preparation of a pharmacological compound comprising adding a peptide of SEQ ID NO: 1 to an excipient.
6. A method for the treatment of vascular diseases in a subject comprising administering to said subject an effective amount of a peptide, said peptide having the following primary structure:
Asp-Pro-His-ILe-Lys-Leu-Gln-Leu-Gln-Ala-Glu (SEQ ID NO: 1).
7. The method according to claim 6 , wherein thrombotic events and related pathologies in a subject are treated by administering to said subject an effective amount of said peptide.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/168,323 US20060014693A1 (en) | 2001-02-21 | 2005-06-29 | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ITRM2001A000088 | 2001-02-21 | ||
IT2001RM000088A ITRM20010088A1 (en) | 2001-02-21 | 2001-02-21 | PEPTIDE ABLE TO INHIBIT THE ACTIVITY OF THE GROWTH FACTOR DERIVED FROM THE PLATES (PDGF-BB) AND OF THE GROWTH FACTOR DERIVED FROM THE FI |
US10/077,746 US7009036B2 (en) | 2001-02-21 | 2002-02-20 | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity |
US11/168,323 US20060014693A1 (en) | 2001-02-21 | 2005-06-29 | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/077,746 Division US7009036B2 (en) | 2001-02-21 | 2002-02-20 | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060014693A1 true US20060014693A1 (en) | 2006-01-19 |
Family
ID=11455243
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/077,746 Expired - Fee Related US7009036B2 (en) | 2001-02-21 | 2002-02-20 | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity |
US11/168,323 Abandoned US20060014693A1 (en) | 2001-02-21 | 2005-06-29 | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/077,746 Expired - Fee Related US7009036B2 (en) | 2001-02-21 | 2002-02-20 | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity |
Country Status (7)
Country | Link |
---|---|
US (2) | US7009036B2 (en) |
EP (1) | EP1414855A2 (en) |
JP (1) | JP2004529103A (en) |
AU (1) | AU2002241240B2 (en) |
CA (1) | CA2451089A1 (en) |
IT (1) | ITRM20010088A1 (en) |
WO (1) | WO2002066498A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9756360B2 (en) | 2011-07-19 | 2017-09-05 | Qualcomm Incorporated | Coefficient scanning in video coding |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070207154A1 (en) * | 2004-04-16 | 2007-09-06 | Martin Friedlander | Method of modulating vascularization |
KR20070111556A (en) * | 2005-10-24 | 2007-11-22 | (주)케어젠 | Peptides having the function of fibroblast growth factor and cosmetics using the same |
CN104693287A (en) * | 2015-03-31 | 2015-06-10 | 苏州普罗达生物科技有限公司 | Fibroblast growth factor inhibitory polypeptide and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5132408A (en) * | 1986-04-22 | 1992-07-21 | The Salk Institute For Biological Studies | Fibroblast growth factor antagonists |
US5252718A (en) * | 1986-04-22 | 1993-10-12 | The Salk Institute For Biological Studies | Fibroblast growth factor antagonists |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1018987B1 (en) * | 1997-04-04 | 2014-10-29 | Barnes-Jewish Hospital | Neocartilage and methods of use |
KR20010102556A (en) * | 1999-03-11 | 2001-11-15 | 추후제출 | Compositions and methods for treating cancer and hyperproliferative disorders |
AU5298900A (en) * | 1999-05-26 | 2000-12-12 | Regents Of The University Of California, The | Method of determining the three-dimensional shape of a macromolecule |
-
2001
- 2001-02-21 IT IT2001RM000088A patent/ITRM20010088A1/en unknown
-
2002
- 2002-02-19 JP JP2002566211A patent/JP2004529103A/en active Pending
- 2002-02-19 CA CA002451089A patent/CA2451089A1/en not_active Abandoned
- 2002-02-19 EP EP02707088A patent/EP1414855A2/en not_active Withdrawn
- 2002-02-19 WO PCT/IT2002/000098 patent/WO2002066498A2/en active Application Filing
- 2002-02-19 AU AU2002241240A patent/AU2002241240B2/en not_active Ceased
- 2002-02-20 US US10/077,746 patent/US7009036B2/en not_active Expired - Fee Related
-
2005
- 2005-06-29 US US11/168,323 patent/US20060014693A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5132408A (en) * | 1986-04-22 | 1992-07-21 | The Salk Institute For Biological Studies | Fibroblast growth factor antagonists |
US5252718A (en) * | 1986-04-22 | 1993-10-12 | The Salk Institute For Biological Studies | Fibroblast growth factor antagonists |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9756360B2 (en) | 2011-07-19 | 2017-09-05 | Qualcomm Incorporated | Coefficient scanning in video coding |
Also Published As
Publication number | Publication date |
---|---|
AU2002241240B2 (en) | 2008-05-29 |
ITRM20010088A0 (en) | 2001-02-21 |
WO2002066498A3 (en) | 2004-02-19 |
US20020119931A1 (en) | 2002-08-29 |
EP1414855A2 (en) | 2004-05-06 |
ITRM20010088A1 (en) | 2002-08-21 |
US7009036B2 (en) | 2006-03-07 |
CA2451089A1 (en) | 2002-08-29 |
JP2004529103A (en) | 2004-09-24 |
WO2002066498A2 (en) | 2002-08-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zachary et al. | Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding. | |
Arese et al. | Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals | |
Rong et al. | Met expression and sarcoma tumorigenicity | |
CN106456697B (en) | Peptide having fibrosis-inhibiting activity and composition containing the same | |
PL192537B1 (en) | Novel homoloques of fibroblast growth factor | |
US8911953B2 (en) | TDF-related compounds and analogs thereof, analogs and bioactive fragments | |
Lam et al. | Nuclear and nucleolar localization of parathyroid hormone‐related protein | |
JP2009502737A (en) | Antitumor drug containing R-spondin | |
CN103890003A (en) | Anti-fibrotic peptides and their use in methods for treating diseases and disorders characterized by fibrosis | |
Boyer et al. | Cyclic AMP distinguishes between two functions of acidic FGF in a rat bladder carcinoma cell line. | |
US20060014693A1 (en) | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (bFGF) activity | |
AU2002241240A1 (en) | Peptide inhibiting platelet derived growth factor (PDGF-BB) and fibroblast growth factor (BFGF) activity | |
Burton et al. | Growth factor expression during rat development: a comparison of TGF-beta 3, TGF-alpha, bFGF, PDGF and PDGF-R | |
CN117756909A (en) | Improved anti-aging compounds and their use in cancer treatment | |
Yamada et al. | Characterization of transforming growth factors produced by the insulin‐independent teratoma‐derived cell line 1246‐3A | |
US20240123033A1 (en) | THERAPEUTIC COMBINATIONS OF TDFRPs AND ADDITIONAL AGENTS AND METHODS OF USE FOR THE REVERSAL OF FIBROSIS | |
AU2012201060B2 (en) | TDF-related compounds and analogs thereof | |
US8367801B2 (en) | Proteinaceous compounds | |
Banks | The role of growth factors in tissue repair II: Epidermal growth factor | |
Brachet et al. | Nerve Growth Factor Synthesis and Biological Activity in Malignant Cells | |
KR20250028388A (en) | Peptide inhibitors and uses thereof | |
Moses et al. | Biological effects of transforming growth factors | |
JPH06189770A (en) | Expression promoter and its use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |