MXPA06009310A - The use of o-atp for the treatment of diseases involving angiogenesis - Google Patents

Abstract

The use of o-ATP as a pharmacological agent useful for the treatment of diseases in whose onset or progression angiogenesis is involved, such as ocular diseases, atherosclerotic processes or tumors.

Description

THE USE OF OXIDIZED TRIFOSPHATE ADENOSIN FOR THE TREATMENT OF DISEASES INVOLVING ANGIOGENES1S The present invention relates in general to substances that act in angiogenesis. More precisely, the invention relates to the use of o-ATP for the treatment of pathologies requiring inhibition of angiogenesis.

BACKGROUND OF THE INVENTION Angiogenesis The proliferation of endothelial cells is responsible for the process of formation of new blood vessels, known as angiogenesis. The newly formed vessels provide nutrients and oxygen to the tissue cells where angiogenesis occurs. The angiogenetic process is useful, for example, for wound healing, because the regenerating tissues need an adequate blood supply. On the contrary, angiogenesis is harmful in the case of tumor diseases, because the blood supply facilitates the proliferation of tumor cells. In addition, neoangiogenesis is harmful when it develops in the atherosclerotic plaques; in fact, in these structures the generation of new vessels due to the production of VEGF (vascular endothelial growth factor) through endothelial cells and other cells, such as monocytes / macrophages, supports the preservation of the same plaques. Therefore, the inhibition of endothelial cell proliferation, or anti-angiogenic activity, is of notable interest in anti-tumor and antiatherosclerotic therapies.

Biological activity of o-ATP The oxidized form of ATP, known as o-ATP, is characterized by the presence of two aldehyde groups at the 2 'and 3' positions of the ribofuranosyl ring. It can be prepared by treating ATP with a periodic acid salt, as described in P.N. Lowe et al., "Preparation and chemical properties of periodate-oxidized adenosine triphosphate and some related compounds", Biochemical Society Transactions, vol. 7: 1131-1133, 1979. o-ATP is commonly used as an affinity tag for nucleotide enzyme sites (Easterbrook-Smith, B., Wallace, JC &Keech, DB (1976) Eur. J. Biochem. 62, 125-130), thanks to its ability to react with non-protonated lysine residues that are present in the nucleotide sites, forming Schiff bases or dihydromorpholine derivatives (Colman, RF (1990) in The Enzymes - Sigman, DS, and Boyer, PD , eds-Vol 19, pp. 283-323, Academic Press, San Diego). It has also been used to study platelet activation and inhibit ATP-induced stimulation of chicken muscle (Pearce, PH, Wright, JM Egan, CM &Scrutton, MC (1978) Eur. J. Biochem. 88, 543-554 Thomas, SA, Zawisa, MJ, Lin, X. & Hume, R.l. (1991) Br. J. Pharmacol. 103, 1963-1969). In addition, in macrophage cell lines, o-ATP proved able to block ATP-induced permeabilization of the plasma membrane, reduce the level of exogenous ATP hydrolysis through membrane ecto-ATPases, and inhibit cell-induced dilation by ATP, vacuolization and lysis (Murgia et al.The Journal of Biological Chemistry, (1993) by The American Society for Biochemistry and Molecular Biology, Inc., Vol. 268, No. 11, pp 8199). It has been suggested that o-ATP has an antagonistic activity in the P2z / P2X7 purinergic receptor, due to the fact that the release of IL-1β (interleukin 1β) (which depends on LPS = lipopolysaccharide) from microglial cells expressing P22 / P2X7 is selectively inhibited by o-ATP (Ferrari D. et al., J. Exp. Med., (1997) Vol. 185, N. 3, Page 579-582). WO 02/11737, in the name of the applicant, describes the antiinflammatory and analgesic effect of o-ATP, using the unilateral inflammation of rat paw caused by injection of Freund's complete adjuvant (CFA) as the experimental model.

DETAILED DESCRIPTION OF THE INVENTION In vitro tests on human umbilical vein endothelial cells (HUVEC) have shown that o-ATP induces a significant reduction in its proliferative capacity, even in the presence of a mitogen. The effect of o-ATP was greater than that induced by vasostatin, a known antiangiogenic compound. Therefore, it is an object of the present invention to use o-ATP for the inhibition of angiogenesis. In particular, the invention provides a medicament containing o-ATP as the active principle, useful for the treatment of pathologies, whose initiation or advancement involves angiogenesis. The angiogenesis-mediated diseases that can benefit from treatment with o-ATP according to the invention, include ocular diseases induced by neovascularization, such as diabetic retinopathy, macular degeneration, proliferative vitreoretinopathy, glaucoma, atherosclerotic processes and tumors, such as carcinomas, lymphomas, leukemia, sarcomas, melanomas, gliomas, neuroblastomas, and other solid tumors. For therapeutic use, o-ATP can be formulated with pharmaceutically acceptable carriers and excipients, and can be administered through the oral, topical or parenteral route. The pharmaceutical forms suitable for the different administration routes comprise tablets, pills, capsules, granules, powders, suppositories, syrups, solutions, suspensions, creams, ointments, gels, pastes, lotions, emulsions, sprays. Pharmaceutical compositions can be prepared as described in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., NY, USA, XVII Ed. The amount of active substance per unit dose ranges from 0.01 to 100 mg per Kg of body weight, which will be administered once a day or more according to the type and severity of the pathology. In general, the daily dose will vary from 1 to 300 mg, preferably from 10 to 100 mg. In another embodiment, the invention relates to combinations of o-ATP and other biologically active substances for the treatment of pathologies mediated by angiogenesis. According to a preferred embodiment, o-ATP is used in combination with antitumor substances such as alkaloids, antibiotics, cytotoxic or cytostatic compounds, antimetabolites, antihormonal agents, alkylating agents, peptides, biological response modulators, cytokines. Alternatively, o-ATP is used in combination with anti-atherosclerotic substances, preferably with lipid-lowering drugs or statins. The different active substances can be administered either simultaneously or separately. The choice of the specific combination of active substances, their dosage and method of administration depend on the specific disease, its resistance to pharmacological treatments, patient tolerance and other variables that will be determined on a case-by-case basis.

EXAMPLE 1 Proliferation test Human endothelial cells (HUVEC) were isolated from the umbilical vein, counted and plated at a constant number in 96-well plates. Cells were cultured as described in (Jaffe, EA (1984) Biology of Endothelial Cells, Martinus Nighoff Publisher, Boston, USA, pp 1-260), with or without VEGF (control) (50 ng / ml), in the presence of o-ATP (100 μM), and o-ATP + VEGF. After 24 hours of culture with or without stimulus, the cells were washed and counted with an optical microscope using a Burker camera. The results are reported in figure 1 and represent the mean ± SD of 10 experiments.

EXAMPLE 2 Permeability test Transwell cameras were used for cell cultures (0.4 μm polycarbonate filters, Costar). In summary, confluent endothelial cells, in monolayer, were exposed to VEGF, o-ATP, ATP (300 μM), ATP + o-ATP, o-ATP + VEGF (in the concentrations previously indicated) for one hour and washed completely . 125 I-labeled albumin (NEN, Boston, MA) was added to the upper compartment; cold albumin (1.5 mg / ml) was added to the culture medium to minimize transcytosis. One hour after the addition of albumin labeled with 125 I to each well, samples were taken from the lower compartment. The radioactivity of the samples was measured with a gamma counter (Packard, Steriing, VA). The results, reported in Figure 2, represent the mean ± SD of 10 independent experiments and are expressed as a percentage of migrated endothelial cells.

Claims (6)

NOVELTY OF THE INVENTION CLAIMS

1. - Use of o-ATP for the preparation of a drug with anti-angiogenic activity.

2. The use as claimed in claim 1, for the treatment of diseases which in their onset or progress involve angiogenesis.

3. The use as claimed in claims 1-2, for the treatment of ocular diseases, atherosclerotic processes or tumors.

4. The use as claimed in claim 3, for the treatment of carcinomas, lymphomas, leukemias, sarcomas, melanomas, gliomas, neuroblastomas. 5.- Therapeutic preparation containing o-ATP in combination with an antitumor substance selected from cytotoxic or cytostatic compounds, antimetabolites, alkaloids, antibiotics, alkylating agents, peptides, biological response modulators and cytokines, for simultaneous, separate or consecutive use in the treatment of tumors. 6.- Therapeutic preparation containing o-ATP in combination with an antiatherosclerotic substance selected from lipid and statin reducing drugs, for simultaneous, separate or consecutive use in the treatment of atherosclerotic processes.