This application is a national stage of PCT/FR99/03208, filed Dec. 22, 1999, which claims priority to French application 98/16264 filed Dec. 22, 1998.
The present invention relates to peptides which have strong inhibitory activity for human topoisomerase II alpha, and which find an application in therapeutics, in particular as antitumor agents.
DNA topoisomerase II is an enzyme which is essential to the life of eukaryotic cells. It changes the topology of the DNA by transient double-stranded cleavage of a DNA double helix, through which it passes another DNA helix. In addition to the biological advantage of this enzyme, there is a pharmacological advantage, since it is the preferred target of many antitumor agents (Corbett and Osheroff, Chem. Res. Toxicol., 6, 585, 1993).
One of the major problems of current anticancer chemotherapy consists of the lack of specificity of the antitumor agents targeting topoisomerase II. In order to resolve this problem, we have developed a novel class of peptide inhibitors of topoisomerase II, which interact selectively with this enzyme and block its catalytic activity.
Frxc3xa9re Gallois et al. have described, in Eur. J. Biochem., 249, 142, 1997, a topoisomerase II fragment, corresponding to sequence 1013 to 1041 of human topoisomerase II alpha which has inhibitory activity for topoisomerase II.
The present invention is directed toward providing peptides which have greater inhibitory activity.
A subject of the present inventionis thus a peptide having the following amino acid sequence:
The expression xe2x80x9cpharmaceutically acceptable protected derivativexe2x80x9d is intended to mean, in particular, derivatives comprising pharmaceutically acceptable N-protective groups.
Pharmaceutically acceptable N-protective groups are, in particular, the groups which protect against N-terminal attack by exopeptidase enzymes. As examples of such groups, mention may be made of acyl groups, such as t-butyloxycarbonyl (Boc), tert-amyloxycarbonyl (+Aoc), benzyloxycarbonyl, benzoyl, acetyl, formyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl or cyclo pentylcarbonyl groups.
The peptides according to the invention can be prepared conventionally by peptide synthesis in liquid or solid phase, by successive coupling of the various amino acid residues which have to be incorporated (from the N-terminal end toward the C-terminal end in liquid phase, or from the C-terminal end toward the N-terminal end in solid phase), and the N-terminal ends and reactive side chains of which have been blocked beforehand.
As examples of groups which block the N-terminal ends, mention may be made of: Boc, Bpoc, Fmoc.
A subject of the present invention is also a pharmaceutical composition comprising, as an active ingredient, a peptide according to the invention.
Such compositions can be used as an antiviral, antibacterial, antiparasitic or anticancer agent, such as for example in the treatment of tumors or of parasitic diseases, leishmaniasis or Chagas disease, or in the treatment of respiratory tract diseases, such as otitis or pneumonias.
Prior vectorization of the peptide may prove to be advantageous since it may promote contact between the peptide and the target cells. The methods of vectorization are known to those skilled in the art. One of these methods consists in encapsulating the peptide in liposomes, such as cationic liposomes. It is known that these liposomes ensure additional protection of the peptide against degradation in vivo. Thus, the slow release of the active ingredient in the targeted areas is therefore very clearly promoted. The first step of the process of transfecting the targeted cells with the peptide, or of the process of peptide release, involves the formation of a complex between the lipid wall of the liposome and the peptide.
After fusion of the respective membranes of the liposome and of the cell, the peptide is released into the cytoplasm.
The preparation of such liposomes is described in detail in xe2x80x9cLiposome technologyxe2x80x9d, Gregoriadis (CFC Press, NY 1984), in xe2x80x9cLiposomesxe2x80x9d, Ostro (Macel Dekker, 1987) or in the publication by Lichtenberg et al., which appeared in xe2x80x9cMethods Biochem. Anal. 33: 337-462, 1988xe2x80x9d. In the liposomes, the peptide is either dispersed, contained or otherwise trapped, in corpuscles consisting of concentric aqueous layers adhering to lipid layers. The peptide is either contained in the aqueous phase or contained in the lipid phase, and optionally in each of the two phases, depending on its solubility. The lipid phase can, in particular, comprise phospholipids such as lecithin or sphingomyelin, steroids such as cholesterol, surfactants such as decetyl phosphate, stearylamine or phosphatidic acid, and/or other hydrophobic materials. The diameter of the liposomes preferably varies between 15 nm and 5 xcexcm.
It is also possible to envision vectorization by incorporating the peptide into biodegradable microspheres.
The preparation of the compositions of the invention is carried out conventionally and depends on the method of administration envisioned.
According to a preferred embodiment of the invention, the peptides optionally incorporated into a vector are administered transdermally, transmucosally or intratumorally. The compositions of the invention are preferably in the form of injectable solutions or suspensions.
As a pharmaceutically acceptable vehicle, use will be made essentially of buffers, a phosphate buffered saline solution (PBS) ,or any other solution provided that it has a physiologically acceptable pH.
The active ingredient can also be incorporated into a matrix or a vehicle in the form of hydrated gel, for example a gel based on a propylene oxide/ethylene oxide copolymer, which is liquid below room temperature and gelatinous at a temperature equal to or higher than room temperature. Before administration, the gel can optionally be fluidized.
In order to enable delayed release of the active ingredient, it is possible, as a variant, to formulate the peptide in a sustained-release composition. Such compositions have been described in the literature. To this end, the peptide can in particular be combined with polylactic acid polymers.
All these compositions can be applied locally by direct injection, or released from implants, or diffused locally using a suitable pump.
A subject of the present invention is also a method for treating a patient with a tumor, which comprises the administration, to this patient, of an effective amount of a peptide according to the invention.
The amount of peptide to be administered depends on the cancerous pathological condition of the individual to be treated, on the seriousness (presence or absence of metastases) of the disease, on the chemotherapy protocol selected, on the choice of a first or second line treatment and on the duration of the treatment, and also on the general condition of the patient. The daily doses can be between 0.1 mg/kg and 10 mg/kg per day or per week, depending on the number of treatment cycles for each therapy.