U.S. patent application number 11/168323 was filed with the patent office on 2006-01-19 for peptide inhibiting platelet derived growth factor (pdgf-bb) and fibroblast growth factor (bfgf) activity.
This patent application is currently assigned to PROVINCIA ITALIANA DELLA CONGREGAZIONE DEI FIGLI. Invention is credited to Angelo Facchiano, Antonio Facchiano, Francesco Facchiano.
Application Number | 20060014693 11/168323 |
Document ID | / |
Family ID | 11455243 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060014693 |
Kind Code |
A1 |
Facchiano; Antonio ; et
al. |
January 19, 2006 |
Peptide inhibiting platelet derived growth factor (PDGF-BB) and
fibroblast growth factor (bFGF) activity
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.
Inventors: |
Facchiano; Antonio; (Roma,
IT) ; Facchiano; Francesco; (Roma, IT) ;
Facchiano; Angelo; (Napoli, IT) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET
2ND FLOOR
ARLINGTON
VA
22202
US
|
Assignee: |
PROVINCIA ITALIANA DELLA
CONGREGAZIONE DEI FIGLI
ROMA
IT
DELL' IMMACOLATA CONCEZIONE-ISTITUTO DERMOOPATICO DELL'
IMMACOLATA
|
Family ID: |
11455243 |
Appl. No.: |
11/168323 |
Filed: |
June 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10077746 |
Feb 20, 2002 |
|
|
|
11168323 |
Jun 29, 2005 |
|
|
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Current U.S.
Class: |
514/8.2 ;
514/9.1 |
Current CPC
Class: |
C07K 14/503 20130101;
A61P 9/00 20180101; A61P 35/04 20180101; A61P 7/02 20180101; A61K
38/00 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/015 |
International
Class: |
A61K 38/10 20060101
A61K038/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 2001 |
IT |
RM2001A000088 |
Claims
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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] 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.
[0002] 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:
[0003] Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu hereafter
referred to as PEP1 (SEQ ID NO: 1).
[0004] Said molecule, showing analogy with a sequence of bFGF,
namely inhibits in vitro as well as in vivo PDGF-BB and bFGF
effects.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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).
[0010] 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).
[0011] 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).
[0012] 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).
[0013] 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).
[0014] Therefore, specific antagonists of PDGF and bFGF are
potential candidates for the treatment of proliferative diseases
and angiogenesis-related disorders.
[0015] 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).
[0016] 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).
[0017] 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:
[0018] 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.
[0019] 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.
[0020] Accordingly with what previously detected, PEP1 synthesis
was achieved by automatic synthetizer, using the standard technique
named f-moc.
[0021] 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.
[0022] Several in vitro and in vivo test were carried out on said
molecule and they revealed the functional characteristics of said
peptide.
[0023] The results obtained are reported in the accompanying
drawings:
[0024] 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;
[0025] FIG. 2A shows PEP1 and PEPscr effect on RASMC proliferation
induced by PDGF-BB (10 ng/ml);
[0026] FIG. 2B shows PEP1 and PEP1scr effect on RASMC spontaneous
proliferation in the presence of BSA;
[0027] FIG. 3A shows PEP1 and PEPscr effect on BAEC proliferation
induced by PDGF-BB (10 ng/ml);
[0028] FIG. 3B shows PEP1 and PEP1scr effect on BAEC spontaneous
proliferation in the presence of BSA;
[0029] 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%);
[0030] 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);
[0031] 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);
[0032] 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);
[0033] 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);
[0034] 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);
[0035] 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);
[0036] FIG. 6 shows PEP1 and PEP1scr effect on RASMC migration
induced by PDGF-BB (10 ng/ml);
[0037] FIG. 7 shows PEP1 and PEP1scr effect on angiogenesis induced
by bFGF in reconstituted basement membrane plugs, subcutaneusly
injected in CD1 mice.
IN VITRO PEP1 ACTIVITY ASSAY
[0038] 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).
Migration Assay
[0039] 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.
[0040] All the migration assays were carried out at 37.degree. C.
in 5% CO.sub.2, 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.times. 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.
[0041] 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.
[0042] 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.
[0043] 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
[0044] 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.times.10.sup.5 cells/plate) and grown
for 24 hours in Dulbecco Modified eagle's medium (DMEM)
supplemented with 10% FBS, at 37.degree. C. in 5% CO.sub.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.
[0045] 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 .mu.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.
[0046] 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).
[0047] 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).
[0048] Then the following in vivo experiment was carried out:
Angiogenesis on Reconstituted Basement Membrane Plugs
[0049] 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 mm.sup.2 within plugs was quantified.
[0050] 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.
[0051] In conclusion:
[0052] 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.
[0053] 2) Anti-angiogenic activity in vivo was demonstrated in
assays carried out on reconstituted basement membrane plugs.
[0054] 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.
Sequence CWU 1
1
1 1 11 PRT Homo sapiens 1 Asp Pro His Ile Lys Leu Gln Leu Gln Ala
Glu 1 5 10
* * * * *