RGD-analog non-peptidic molecules having anti-adhesive,...

Details RGD-analog non-peptidic molecules having anti-adhesive,... RGD-analog non-peptidic molecules having anti-adhesive,...

2002-02-20

2003-09-30

Kumar, Shailendra (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carboxylic acids and salts thereof

C562S560000, C562S440000, C548S540000, C514S423000, C514S563000, C514S564000, C514S565000

Reexamination Certificate

active

06627769

ABSTRACT:

The present invention concerns the identification of new synthetic molecules able to mimic peptides with biological activity, i.e. peptide-like molecules or peptidomimetic molecules. More particularly, the present invention relates to the identification of new molecules with no intramolecular peptidic bonds and able to mimic the RGD motif. In fact, it is known that the RGD motif is a potent inhibitor, in vivo and in vitro, of cellular adhesion.
In vitro experimentation on rat smooth muscle cells (RASMC) demonstrated that these new peptidomimetic molecules are efficient inhibitors of RASMC proliferation and migration (modification of cell migration and proliferation occurs in many vascular diseases such as atherosclerosis and restenosis which have a high social impact) and could be used as inhibitors of tumor cells migration towards potential metastatic sites.
The lack of proper peptide bounds makes said molecules less sensible to in vivo proteolytic degradation. It is well known that proteolytic degradation is a catabolic mechanism which usually reduce or block in vivo biological effects of peptidic molecules having in vitro activity.
Therefore, the peptidomimetic molecules, object of the invention, are potentially more stable and consequently show higher in vivo activity than the corresponding template peptide used for their design.
Vascular diseases represent one of the major cause of morbidity and mortality in western countries. Ischemic diseases are mainly due to atherosclerosis, thrombotic events, and other phenomena such as restenosis after angioplasty. In addition, vascular changes lead to neovascularization during solid tumours growth. For this reason, molecules active in controlling the growth of vascular cells and vessel walls are largely investigated for the potential benefits in a variety of socially relevant diseases.
It has been demonstrated that angiogenesis depends on the adhesion of vascular cells to the extracellular matrix and that the protein so called &agr;
v
&bgr;
3
integrin is required for this process (Brooks et al. Science, 1994, 264, 569-571).
Consequently, peptides containing the ARG-GLY-ASP motif “arginine-glycine-aspartic acid” (RGD motif), which are integrin-inhibitors, may exert anti-angiogegenic properties (Friedlander. et al., Science 1995, 270, 1500-1502; Woodard et al., J. Cell Sci. 1998, 1930-1935) and anti-neoplastic activity (Hammes. et al., Nat.Medicine, 1996, 2, 529-533;.Carron. et al. Cancer Res. 1998, 1930-1935) and anti-metastatic activity (Fujii. et al., Oncology Res. 1996, 8, 333-342).
Moreover, it has been shown that RGD-containing proteins and RGD-containing short peptides modulate blood coagulation, cell growth, cell migration and cell adhesion to extracellular matrix (Rouslhti et al., Annu Rev Cell Dev Biol. 1996, 12, 697-715; Ojima et al., Bioorganic & Medecin Chemistry 1997, 3, 337-360; Wang et al., Curr.Med.Chem., 2000, 7, 74-80).
Besides that, RGD-analogs have been shown to induce apoptosis under different experimental conditions (Modlich et al., Lab.Invest. 1996, 74, 771-780; Yeh et al., Blood 1998, 92, 3268-3276; Buckley et al., Nature 1999, 397, 534-539).
Considering these data, RGD-analogs peptides can efficiently induce apoptosis of tumour cells and of actively proliferating vascular cells.
The use of small biologically active peptides is currently under strong investigation, due to the relative cost of synthesis and to the restricted in vivo side effects of these small peptides in comparison with the parent whole proteins. Unfortunately, small peptides are usually unstable molecules; they are degradable by proteases and they can be recognised as non-self by the immune system and thus eliminated rapidly loosing their biological effects. Therefore, according to the present invention, once the active site of specific proteins was identified, the following step consisted in the identification of molecules mimicking said active site, but having different chemical characteristics (i.e. peptides bounds or antigenic activity) in order to reduce their catabolism and consequently increase their activity in vitro as well as in vivo.
Nowadays large investigation is on RGD-containing peptides, because, as already mentioned, they show strong activity in vitro as well in vivo as inhibitors of integrin-mediated diseases.
Non-peptidic analogs of RGD-peptides are designed to obtain molecules which are more stable in vivo, lacking of proteolitic-sites and undergoing a slower catabolic degradation in vivo (Wang et al., Curr.Med.Chem., 2000, 7, 74-80; Horton et al., Exp.Nephrol., 1999, 7, 178-184). Said peptides-like molecules are named peptidomimetics since they mimic the original compound, but the absence of peptidic bonds, avoid their degradation by proteases and consequently they are more stable in vivo.
The use of this novel approach (peptidomimetics) represent a new and spreading field of research, supported by new chemical compounds available on the market. The use of specific software able to detect similar shapes or three-dimensional structures and similar electronic distributions in different molecules allows to identify the building blocks most suitable to be used as alternative to the amino acids for the construction of peptidomimetic molecules.
Object of the present invention is a novel family of synthetic non-peptidic molecules, able to mimic the RGD motif and to inhibit cellular adhesion, migration and proliferation: said family of molecules is at least equally or more active in vitro in comparison to the corresponding template, and potentially more stable in vivo.
According to the present invention, in the first step a peptidic molecule has been chosen as template to design peptidomimetic ore peptide-like molecules in the following steps. For this purpose, RGD-containing peptides (“arginine-glycine-aspartic acid”), involved in several biological functions, as modulation of adhesion between cells and between cells and extracellular matrix, have been chosen. Therefore, the three-dimensional structure of said polypeptides was obtained.
Consequently, synthetic molecules belonging to the same family were designed, containing a motif similar to the RGD of the template, having potential biological activity but lacking of peptide bonds in order to be less sensible to proteolitic degradation in vivo.
After said design step, the more interesting molecule of the family, i.e. the easiest to be produced and the one showing highest similarity to the template, was synthesised and tested in vitro.
Said RGD-analog molecule with no intramolecular peptidic bonds, was tested in smooth muscle rat cells, and showed the capability to strongly inhibit cellular adhesion to extra-cellular matrix and to inhibit PDGF-induced (platelet derived grow factor) cellular migration and proliferation.
These data indicate that this new molecule might be used as inhibitor of abnormal migration and proliferation of vascular cells that occur in several vascular diseases such as atherosclerosis and restenosis, as well as of migration of neoplastic cells toward the potential metastatic sites.
Designing RGD-peptidomimetics
According to the previous observations, RGD-containing peptides were chosen as templates to design peptidomimetic molecules.
This choice was made considering carefully the reports on the biological activity of RGD-containing peptides available in literature and using useful and specific databases for the identification of known structural conformations (PDB databases).
Once the three-dimensional conformation of the RGD motif in the template peptide to be used in the simulation was identified, the designing process of similar molecules was performed and molecules belonging to the same family and showing the same features were obtained.
The molecules belonging to the same family are showed as follows:
Mimic-polypeptide 1: (H
2
N—RmRmD—NH
2
)
Mimic-polypeptide 2: (H
2
N—RphacRmD—NH
2
)
Mimic-polypeptide 3 (H
2
N—RvRmD—NH
2
)
Mimic-polypeptide 4 (H
2
N—RpRmD—NH
2
):
Mimic-polypeptide 5 (H
2
N—RIRmD—NH
2
)
Mimic-polypeptide 6 (H
2
N—RfRmD—NH
2
)

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