PHARMACEUTICAL COMPOSITION FOR CANCER TREATMENT, PROCESS FOR ITS PREPARATION, AND ITS USE

Abstract

The use of the C-terminal region of one or more phytocystatin(s) for the curative or prophylactic treatment of tumors and/or inhibition of metastases in mammals or other vertebrates. In one embodiment, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable carrier and a polypeptide containing only the C-terminal region of one or more phytocystatin(s). In another embodiment, the composition of the invention comprises a gene construct for the in vivo expression of said polypeptide.

Claims

1. (canceled)

2. (canceled)

3. A pharmaceutical composition for cancer treatment of mammalian and/or other vertebrate comprising: a pharmaceutically acceptable carrier; and one or more polypeptides comprising one or more sequences having 70% or greater similarity to SEQ ID No: 6, or one or more sequences having 70% identity or greater with SEQ ID No: 6; or one or more gene constructs useful for the expression in mammals and/or other vertebrates of one or more polypeptides as defined above.

4. The composition according to claim 3, wherein said polypeptide comprises an arrangement of two or more fused, repeated or sequenced polypeptide sequences of different legumain-selective ligand carboxy-terminal domains of mammalian and/or other vertebrate.

5. The composition according to claim 3, wherein said gene construct comprises an arrangement of two or more nucleotide sequences encoding said polypeptides fused in phase, repeated or in sequence of the same or different nucleotide sequences having equal or greater similarity to 70% with SEQ ID No: 5, or with identity equal to or greater identity to 70% with SEQ ID No: 5, for administration and expression of the polypeptide(s) directly into the mammalian/vertebrate organism.

6. The composition according to claim 3, wherein it is in the oral, sublingual, injectable, transdermal, topical, inhalable or suppository dosage form.

7. The composition according to claim 3, wherein it comprises a gene construct or expression vector comprising: one or more nucleotide sequences having 70% or greater similarity to SEQ ID No: 5, or one or more nucleotide sequences having 70% identity or greater with SEQ ID No: 5; a functional promoter in mammals and/or other vertebrates linked to the coding nucleotide sequence described above; and a nucleotide sequence selected from: a transcription terminator nucleotide sequence; a selection marker; a secretion signal sequence; a sequence facilitating export; a nucleotide sequence encoding another protease inhibitory polypeptide sequence; combinations thereof, or still a plasmid comprising such sequences, wherein any of the nucleotide sequences defined above is heterologous.

8. The composition according to claim 3, wherein said polypeptide is combined with one or more candidate molecules with legumain linker(s).

9. (canceled)

10. A method for the cancer treatment of mammals and/or other vertebrates comprising the use of a pharmaceutical composition comprising: one or more polypeptides comprising one or more sequences having equal or greater than 70% of similarity to SEQ ID No: 6, or one or more sequences having an equal or greater than 70% of identity with SEQ ID No: 6; or one or more gene constructs useful for the expression of one or more of said polypeptides, wherein the mammals and/or other vertebrates are treated with a pharmaceutically effective amount of the pharmaceutical composition.

11. A process for the preparation of a pharmaceutical composition for cancer treatment of mammalian and/or other vertebrate cancer comprising a mixing step for mixing: one or more polypeptides comprising one or more sequences having equal or greater than 70% similarity to SEQ ID No: 6, or one or more sequences having an identity equal or greater than 70% identity with SEQ ID No: 6; or one or more gene constructs useful for the expression in mammals and/or other vertebrates of one or more of said polypeptides; and a pharmaceutically acceptable carrier.

12. The composition according to claim 4, wherein it is in the oral, sublingual, injectable, transdermal, topical, inhalable or suppository dosage form.

13. The composition according to claim 5, wherein it is in the oral, sublingual, injectable, transdermal, topical, inhalable or suppository dosage form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] FIG. 1 shows schematic representations of embodiments of the invention: in A) there is shown an artificial/unnatural polypeptide sequence of SEQ ID No: 6, comprising a carboxy-terminal region derived from rice phytocystatin; in B) there is shown a sequence of another single recombinant polypeptide resulting respectively from the combination of two carboxy-terminal regions of papaya cystatin, followed by a carboxy-terminal region of a corn cystatin and a carboxy-terminal region of a rice cystatin.

[0097] FIG. 2 shows a schematic representation of gene construct embodiments of the present invention, indicated: in A) 21, a plasmid pCAMBIA, which enables expression in both plants and bacteria for the expression of a polypeptide encoded by SEQ ID No. 6; 22, an origin of replication; 23, a promoter operably linked to the encoding sequence; 24, the coding sequence SEQ ID No: 5; and 25, a reporter gene (GFP), the transcription terminator region; in B) there is shown the schematic representation of the plasmid pRSFDuet-1, which provides for the co-expression of two encoding sequences of interest. This vector contains two multiple cloning sites (MCS), each preceded by a promoter T7lac and a ribosome binding site (rbs).

[0098] FIG. 3 shows a graph indicative of the number of cells U343 after 5 days of treatment with one embodiment of the invention comprising a recombinant rice phytocystatin (O. sativa) containing only the carboxy-terminal portion. On the axis y, the total number of cells is represented and, on the axis x the results of each tested condition are represented, respectively: control cell U343 (without administration of the composition of the invention); and cells U343 that received treatments of 1, 10 and 50 micromolar of said polypeptide.

[0099] FIG. 4 shows a graph indicative of the number of cells A172 after 5 days of treatment with the composition indicated in the description of FIG. 3. On the axis y the total number of cells is represented and, on the axis x are represented the results of each condition tested, respectively: control cells A172 (without administration of the composition of the invention); and cells A172 that received treatments of 1, 10 and 50 micromolar of said polypeptide.

[0100] FIG. 5 shows a graph indicative of the number of cells C6 after 5 days of treatment with the composition indicated in the description of FIG. 3. On the axis y the total number of cells is represented and, on the axis x are represented the results of each condition tested, respectively: control cells C6 (without administration of the composition of the invention); and cells C6 that received treatments of 1, 10 and 50 micromolar treatments of said polypeptide.

[0101] FIG. 6 shows a graph indicative of the number of cells U138 after 5 days of treatment with the composition indicated in the description of FIG. 3. On the axis y the total number of cells is represented and, on the axis x are represented the results of each condition tested, respectively: control cells U138 (without administration of the composition of the invention); and cells U138 that received treatments of 1, 10 and 50 micromolar of said polypeptide.

[0102] FIG. 7 shows an illustrative graph of cumulative cell proliferation over time for glioma cells U343 subjected to treatment with the composition indicated in the description of FIG. 3. On the axis y cumulative cell duplication is represented and, on the axis x represents the time in days. They are shown: in A, the proliferation/control curve, that is, without the administration of the active; in B, C and D the curves relative to the administration of said polypeptide, respectively at concentrations 1, 10 and 50 micromolar.

[0103] FIG. 8 shows an illustrative graph of cumulative cell proliferation over time for glioma cells A172 subjected to treatment with the composition indicated in the description of FIG. 3. On the axis y cumulative cell duplication is represented and, on the axis x the time in days is represented. They are shown: in A, the proliferation/control curve, that is, without the administration of the active; in B, C and D the curves relative to the administration of said polypeptide, respectively at concentrations 1, 10 and 50 micromolar.

[0104] FIG. 9 shows an illustrative graph of cumulative cell proliferation over time for glioma cells C6 subjected to treatment with the composition indicated in the description of FIG. 3. On the axis y, cumulative cell duplication is represented, and, on the axis x is represented the time in days. They are shown: in A, the proliferation/control curve, that is, without the administration of the active; in B, C and D the curves relative to the administration of said polypeptide, respectively at concentrations 1, 10 and 50 micromolar.

[0105] FIG. 10 shows an illustrative graph of cumulative cell proliferation over time for glioma cells U138 subjected to treatment with the composition indicated in the description of FIG. 3. On the axis y, cumulative cell duplication is represented, and, on the axis x is represented the time in days. They are shown: in A, the proliferation/control curve, that is, without the administration of the active; in B, C and D the curves relative to the administration of said polypeptide, respectively at concentrations 1, 10 and 50 micromolar.

[0106] FIG. 11 shows a schematic representation of the gene sequence data of rice phytocystatin with carboxy-extended terminal. The representation of the complete nucleotide sequence of the locus in rice chromosome 1, indicated: the white regions correspond to the introns and the shaded regions correspond to the exons.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0107] Legumains are found primarily located in mammalian lysosomes, but have also been found in association with the extracellular matrix. Endogenously, legumains can be inhibited by cystatin; however, animal cystatins additionally inhibit some cathepsin class proteases, thus lacking a specific activity. The present invention provides a composition for the legumains selective binding and/or inhibition of mammalian and/or other vertebrate.

[0108] The present invention has as an inventive concept common to its various objects the use, for preparing a medicament for cancer treatment of mammalian or other vertebrate, of a polypeptide comprising one or more sequences having similarity equal to or greater than 70% with SEQ ID No: 6, or one or more sequences with identity equal to or greater than 70% with SEQ ID No: 6.

[0109] Said polypeptide resembles phytocystatin(s), but comprises only selective ligand carboxy-terminal domain of legumain, a type of protease, modulating its activity and/or exclusive inhibitor of legumain(s), unlike known animal cystatins, which also inhibit other targets. In the present invention, it is surprisingly shown that said polypeptide is useful for cancer treatment of mammalian and/or other vertebrate by acting as suppressor of tumor and/or metastasis formation.

[0110] Cancer cannot be considered a single disease in its etiology, there being more than 100 different types named by the organ or cell type in which the disease begins. Characterized by a disordered growth of cells, cancer cells can invade other tissues, spreading through the body through metastases. According to data collected and released by the National Institutes of Health (NIH), WHO (World Health Organization) and INCA (Instituto Nacional do Cancer), in 2012 there were 14.1 million new cases of cancer and 8.2 million deaths in the world. Prospects for 2030 include 21.4 million new cases and 13.2 million cancer deaths globally. Given these high numbers, cancer is a major public health problemdeeply affecting at least 1 million people diagnosed per year.

[0111] Cancer treatments are advancing, saving lives and extending the survival rate of people with various types of cancer. Although most causes of cancer are known to be multi-factorial and range among subjects, some molecular pathways, which are already known, are similar in some types of cancer. One of these pathways relates to the altered and increased activity of cysteine proteases of the legumains type. Independently, this increased activity has already been detected in different types of cancer including breast, colon, lung, prostate, ovary, and kidneys, among others. In most of these studies, tumors presenting a greater activity of legumains are associated with a high level of metastases and a worse prognosis of survival.

[0112] Thus, recent works have been attempting to develop drugs that specifically aim legumains. In general, there is a greater focus for synthetic prodrug research and some studies focus on the use of animal cystatin as legumain inhibitors. However, animal cystatin, although currently under intense study, provides low specificity for the inhibition of animal legumains. In this way, the research and development of other molecular entities continues to be an important demand for the development of new strategies for the cancer treatment.

[0113] The wide diversity of cancer types in which an increase of legumain-like proteases in association with aggressiveness and metastasis formation is found justifies efforts and investments in the development of effective inhibitors of low or no side effects and with therapeutic potential. In this context, the present invention meets, among others, the objectives of the Federal Government's incentive (Decree No. 6,041, February 2007) for the generation and national production of new biomolecules and/or recombinant proteins of therapeutic interest.

[0114] The present invention provides solutions to the problems of the prior art by providing a candidate useful for cancer treatment of mammalian or vertebrate through a polypeptide of plant origin.

[0115] For purposes of the present invention the following definitions are used:

[0116] Recombinant or modified polypeptide: In the context of the present patent application, non-natural polypeptide, or recombinant polypeptide, or modified polypeptide is to be understood as a non-naturally occurring polypeptide.

[0117] Target organism: In the context of the present patent application, the term target organism means those organisms which receive the gene construct of the invention, so that the gene expression occurs in the target organism. Said target organism includes, but is not limited to, bacteria, yeasts and other fungi, plants or other plant cells, animal, mammals and/or other vertebrates cells.

[0118] Pharmaceutical composition: In the context of the present patent application, pharmaceutical composition is to be understood as any and all compositions containing an active ingredient, for prophylactic, palliative and/or curative purposes, acting in a manner to maintain and/or recovering homeostasis, and may be administered topically, parenterally, enterally, intrathecally, orally, intravenously, intranasally, intravitreally and intramuscularly, intracerebrally, intracerebroventricularly and intraocularly in its administration and/or formulation.

[0119] Pharmaceutically acceptable formulation or carrier: In the context of the present patent application, a pharmaceutically acceptable formulation or carrier is understood to mean a formulation containing pharmaceutically acceptable excipients and carriers well known to those skilled in the art, as it is the development of convenient doses and treatments for use in particular compositions which may be described in a variety of treatment regimens, including oral, topical, parenteral, intravenous, intranasal, intravitreal and intramuscular, intracerebral, intracerebroventricular and intraocular and its administration and/or formulation.

[0120] Cellular disorder: In the context of the present patent application, cellular disorders is to be understood as any cellular disorder which alters the normal physiological functions of a cell or tissue, especially mammals or other vertebrates. In the context of the present patent application the term encompasses any biochemical or molecular change resulting in or is associated with altered expression and/or activity of legumain(s) in cells or tissues, including neoplasms.

[0121] The present invention contributes first, to scientific knowledge about the activity of plant cysteine protease inhibitors for cancer treatment of mammalian or other vertebrate. Throughout the evolution of organisms, several genes and proteins remained with related functions between the two realms. However, although it is known that legumains are involved in the process of developing tumors and metastases and that cystatins have the ability to inhibit the activity of these proteases, to the best of the inventors' knowledge there was at present no available report of the approach to use and effectively to test only the C-terminal region of plant phytocystatins for cancer treatment in mammalians. Perhaps the fact that plant inhibitors have progressively acquired the specific inhibitory property only for legumains explains this gap. The present invention provides an innovative approach by extending these types of analyzes across the realms of organisms, providing for the identification of biomolecules with therapeutic potentials. In addition, plant systems have several advantages over others in the production of new drugs because they require much less effort and investments in preclinical testing than conventional systems.

[0122] Among other objects, the present invention provides a pharmaceutical composition for cancer treatment of mammalian and/or other vertebrate, the composition comprising: [0123] a pharmaceutically acceptable carrier; and [0124] one or more polypeptides comprising one or more polypeptide sequences having a similarity equal to or greater than 70% with SEQ ID No: 6, or one or more sequences having identity equal to or greater than 70% with SEQ ID No: 6.

[0125] The following examples illustrate some of the embodiments of the invention without, however, limiting the scope thereof.

Example 1a Composition for Cancer Treatment in Mammals and/or Other VertebratesComprising Recombinant Polypeptide

[0126] Carboxy-extended phytocystatins gene regions of previously known plants were selected for evaluation of the functionality of the resulting recombinant polypeptides. The carboxy-extended phytocystatins genes of the plants were obtained from: arabidopsis (Arabidopsis thaliana), papaya (Carica papaya), soybean (Glycine max), rice (Oryza sativa), corn (Zea mays) and eucalyptus (Eucaliptus grandis). The choice of the genes from these species was based on previously presented phylogenies (Christoff and Margis 2014; Christoff 2015), in order to contemplate the inclusion of the species that represent a good sampling of the diversity of phytocystatins in plants. The encoding sequences of the C-terminal regions of phytocystatins from different plants were inserted into the target vectors using the restriction enzyme system of each vector and subsequent ligation with DNA ligase.

[0127] In the composition of the invention, the polypeptide is selected from among different variants, among which has higher inhibitory activity (Ki) against mammalian or human legumain. The element common to the different embodiments and composition of the invention is a polypeptide comprising a specific region of a carboxy-extended phytocystatin, independently of the parent plant or being a synthetic polypeptide. In this embodiment, the composition of the invention comprises a recombinant phytocystatin extract from rice (O. sativa) containing only the carboxy terminal portion, as indicated in SEQ ID No: 6 and FIG. 1, part A).

Example 2Gene Construct

[0128] The gene construct of the invention is useful for the expression of one or more polypeptides useful for cancer treatment of mammalian or other vertebrate. Said gene construct comprises: [0129] one or more nucleotide sequences with similarity equal to or greater than 70% with SEQ ID No: 5, or one or more nucleotide sequences with identity equal to or greater than 70% with SEQ ID No: 5; [0130] a functional promoter in the target organism, linked to the encoding nucleotide sequence described above; and [0131] a nucleotide sequence selected from: a transcription terminator nucleotide sequence; a selection marker; a secretion signal sequence; a sequence facilitating export; a nucleotide sequence encoding another protease inhibitory polypeptide sequence; combinations thereof, or yet a plasmid comprising such sequences,
wherein any of the nucleotide sequences defined above being heterologous.

[0132] In this embodiment, schematically illustrated in FIG. 2A, the gene construct of the invention comprises as a polypeptide encoding sequence SEQ ID No.5, as shown in Example 1.

[0133] It should be noted that each expression system provides advantages and disadvantages, with the choice of the most efficient system for the expression of the biomolecules being in each case a matter of weighing the amount of protein recovered and/or the degree of purity thereof. Similarly, the gene construct of the invention may be directed to expression of the recombinant polypeptide of the invention in bacteria, yeast, plants, for subsequent administration, or still directly to mammals or other vertebrates.

Example 3Composition Comprising Recombinant Polypeptide in Arrangement of Fused Sequences

[0134] In this embodiment, the polypeptide comprises a fused sequence arrangement with four carboxy-terminal domains of phytocystatins, two of which are repeat papaya followed by one of corn and another of rice, as schematically illustrated in FIG. 1, part B). Said sequence arrangement is also selective ligand of legumain(s), being useful in the modulation and/or inhibition of its activity. The preparation of this composition is made according to the process of the invention, which comprises a mixing step of: [0135] one or more polypeptides comprising one or more sequences having similarity equal to or greater than 70% with SEQ ID No: 6, or one or more sequences having identity equal to or greater than 70% with SEQ ID No: 6; or [0136] one or more gene constructs useful for the expression in mammals and/or other vertebrates of one or more of said polypeptides; and [0137] a pharmaceutically acceptable carrier.

Example 4Tests on Mammalian Cancer Lines

[0138] The composition of the invention has surprisingly been shown to provide curative or prophylactic treatment of a variety of cell disorders, notably cancer. The composition of this embodiment of the invention comprises: human cell culture medium as vehicle; and a phytocystatin recombinant polypeptide of rice (O. sativa) containing only the carboxy terminal portion (FIG. 1, part A)), having been administered to mammalian cells. Experiments with the administration of this embodiment of the invention composition in glioma cells have shown surprising and very promising results, as reported below.

[0139] In an experiment carried out with 4 different glioma lines, being 3 human lines and one murine line (C6) were plating in 24-well single-plate plates and treated the following day with the composition of the invention containing said polypeptide at concentrations of 1 uM, 10 uM and 50 uM. The treatment last 5 days with the presence of the composition of the invention and on the fifth day the medium was changed by one medium without the composition of the invention, the evaluation being done over time. Those skilled in the art will appreciate that the vehicle used in this embodiment of the invention can be replaced by other, according to the cell and/or organism to be treated and is well known in the art.

[0140] The results, shown in FIGS. 3-6, allow to conclude that: (i) under the conditions and cells tested, the 1 uM and 10 uM doses do not seem to have much long-term effect on these 4 lines tested; (ii) the dose of 50 uM gives evidence of an antitumor effect on glioma cells in the short term, although for some lines the effect is not maintained in the long term and the proliferative ability of the treated population appears to be restored; (iii) the 50 uM dose provided the complete elimination of one of the most resistant glioma lines, which are non-responsive to most of the available chemotherapeutic treatments.

[0141] The graphs of FIGS. 7-10 illustrate the cell proliferation profiles of the lines treated with the composition of the invention, demonstrating promising effects. FIGS. 3-7 show the reduction of cell proliferation in cells U138. FIGS. 5 and 9 show the reduction of cell proliferation in cells C6. FIGS. 6 and 10 show the very substantial reduction of cell proliferation in cells U343, which are cells of glioma lines more chemotherapy resistant available in the art.

[0142] Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments encompassed within the scope of the appended claims.