ANTIMETASTATIC COMPOSITION COMPRISING AT LEAST ONE FLAVANOL-TYPE COMPOUND

Abstract

The invention relates to an anti-metastatic composition comprising at least one flavanol-type compound for the use thereof as a medicament, said at least one flavanol-type compound being present in the form of a complex formed by the creation of coordinate bonds with at least one basic amino acid and/or at least one derivative of a basic amino acid.

Claims

1-15. (canceled)

16. A method of preventing or treating cancer metastases in a subject, said method comprising, administering to said subject, a composition comprising at least one flavanol-type compound, wherein said at least one flavanol-type compound is (+)-catechin present in the form of a complex formed by the creation of coordinate bonds with at least one basic amino acid.

17. The method of claim 16, wherein said complex has a molar equivalence ratio between said (+)-catechin and said at least one basic amino acid of between 1:2 and 1:4.

18. The method of claim 16, wherein said at least one basic amino acid is lysine.

19. The method of claim 16, wherein said composition further comprises at least one acid.

20. The method of claim 19, wherein said acid is chosen from the group consisting of ascorbic acid, acetic acid, citric acid, hydrochloric acid, and mixtures thereof.

21. The method of claim 16, wherein said composition is characterized in that in a 0.01 molar solution at 25° C., it has a pH greater than or equal to 3, preferably of between 4 and 11, advantageously of between 4.5 and 7.5.

22. The method of claim 16, wherein said composition is in solid form.

23. The method of claim 22, wherein said composition is in water-soluble solid form.

24. The method of claim 23, wherein said composition is a powder or a table, a pessary or a suppository.

25. The method of claim 16, wherein said composition is administered orally.

26. The method of claim 16, wherein said composition further comprises one or more biocompatible excipients.

27. The method of claim 16, wherein said treatment is in combination with an anticancer treatment.

28. The method of claim 16, wherein said cancer is a liver, prostate, breast, uterine, testicular, bladder, kidney, lung, bronchial, bone, mouth, esophageal, stomach, pancreatic, colorectal or brain cancer.

29. The method of claim 16, wherein said cancer is a hepatocellular cancer.

30. The method of claim 16, wherein said cancer is a leukemia, a myeloma, a lymphoma or a melanoma.

31. The method of claim 16, wherein said composition is administered at a dosage of 250 mg to 750 mg/day.

32. The method of claim 16, wherein said composition treats circulating tumor cells.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0105] FIGS. 1A, 1B and 1C are graphs which reproduce respectively the percentage of SiHa-F3 tumor cells (F3) surviving (FIG. 1A), the percentage of cells having migrated (FIG. 1B) and the DCFDA fluorescence/protein content ratio (FIG. 10) for a treatment at variable concentrations of (+)-catechin (+C).

[0106] FIGS. 2A, 2B and 2C are graphs which reproduce respectively the percentage of SiHa-F3 tumor cells (F3) surviving (FIG. 2A), the percentage of cells having migrated (FIG. 2B) and the DCFDA fluorescence/protein content ratio (FIG. 20) for a treatment at variable concentrations of the (+)-catechin/lysine complex according to a 1:1 molar equivalence ratio.

[0107] FIGS. 3A and 3B are graphs which reproduce respectively the percentage of SiHa-F3 tumor cells (F3) surviving (FIG. 3A) and the percentage of cells having migrated (FIG. 3B) for a treatment of variable concentrations of the (+)-catechin/lysine complex according to a 1:2 molar equivalence ratio.

[0108] FIGS. 4A, 4B, 4C and 4D are graphs which reproduce the cell survival (measured by the crystal violet [CV] technique and expressed as % of non-treated SiHa-F3 supermetastatic cells) of tumor cells treated with epigallocatechin gallate (EGCG), (+)-catechin, (+)-catechin/lysineHCl 1:1 or (+)-catechin/lysineHCl (1:2) (concentrations of 100 nM, 1 μM, 10 μM, 100 μM, 500 μM or 1 mM).

[0109] FIGS. 5A, 5B, 5C and 5D are graphs which reproduce the cell survival (measured by the MTT technique and expressed as % of non-treated SiHa-F3) of tumor cells treated with epigallocatechin gallate (EGCG), (+)-catechin, (+)-catechin/lysineHCl 1:1 or (+)-catechin/lysineHCl (1:2) (concentrations of 100 nM, 1 μM, 10 μM, 100 μM, 500 μM or 1 mM).

[0110] FIGS. 6A, 6B, 6C and 6D are graphs which reproduce the DCFDA fluorescence/protein content ratio for a treatment of the SiHa-F3 tumor cells with epigallocatechin gallate (EGCG), (+)-catechin, (+)-catechin/lysineHCl 1:1 or (+)-catechin/lysineHCl (1:2) (concentrations of 100 nM, 1 μM, 10 μM, 100 μM, 500 μM or 1 mM).

[0111] FIGS. 7A, 7B, 7C and 7D are graphs which reproduce the cell migration (as % of non-treated SiHa-F3) of tumor cells treated with epigallocatechin gallate (EGCG), (+)-catechin, (+)-catechin/lysineHCl 1:1 or (+)-catechin/lysineHCl (1:2) (concentrations of 100 nM, 1 μM, 10 μM, 100 μM, 500 μM or 1 mM).

[0112] FIGS. 8A and 8B are photographs (A) and a graph (B) demonstrating the effect of (+)-catechin/lysineHCl 1:2 on melanoma tumor cells treated with rotenone (Rot 20 nM+C/L 1:2 10 μM) compared to treatment with rotenone all on its own (Rot 20 nM) on the formation of lung metastases in an experimental model. A group of animals was treated with DMSO alone and serves as a negative control.

[0113] FIGS. 9A and 9B are photographs (A) and a graph (B) showing the effect of (+)-catechin/lysineHCl 1:2 (Rot 20 nM+C/L 1:2 10 μM) and epigallocatechin-gallate/lysineHCl 1:2 (Rot 20 nM+EGCG/L 1:2 10 μM) on melanoma tumor cells during the formation of lung metastases in an experimental model. A group of animals was treated with rotenone alone (Rot 20 nM) and serves as a positive control. A group of animals was treated with DMSO alone and serves as a negative control.

[0114] FIGS. 10A and 10B are photographs (A) and a graph (B) showing the effect of catechin/lysineHCl 1:2 (Rot 10 nM+C/L 1:2 10 μM) or of epigallocatechin gallate (Rot 10 nM+EGCG 10 μM) on the formation of lung metastases in an experimental model.

EXAMPLES

[0115] Tests of cell migration and of survival of the treated cells were carried out with a model of super-invasive tumor cells obtained from a human cervical adenocarcinoma cell line, SiHa. The selection of the super-invasive SiHa-F3 cells (F3 on the graphs) was carried out after three consecutive invasions in vitro in permeable supports of Transwell® type coated with Matrigel® (Porporato P E et al., Cell Rep 2014, 8:754-766). Such a selection of particularly aggressive metastatic progenitor cells makes it possible to validate all the better the most active anti-metastatic products.

[0116] The actual migration of the tumor cells was measured in a Boyden chamber with 50 000 cells per well and 0.15% of fetal bovine serum (FBS) used as chemoattractant. The pore diameters of the porous membrane separating the two compartments of the Boyden chamber were fixed at 8 μm. After 16 hours of migration, the cells were first fixed with methanol for 3 min and then stained with crystal violet. The culture medium used was DMEM 4.5 g/l of glucose, GlutaMAX®, 1% of a solution of penicillin/streptomycin and 10% of fetal bovine serum.

[0117] The cell survival, in order to exclude any toxic effect of the products tested, was estimated either on the basis of the quantification of the cells stained with crystal violet, or by measuring the mitochondrial succinate dehydrogenase (SDH) activity on the reduction of the MTT tetrazolium salt (suspension of formazan obtained in DMSO and measurement of absorbance at 570 nm).

[0118] Moreover, an evaluation of the impact of the products tested on the level of reactive oxygen species (ROSs) observed was carried out according to the DCFDA technique after 60 minutes of pretreatment of the SiHa-F3 metastatic progenitor cells with the products tested.

[0119] In the figures, F3 signifies: control (non-treated cells); F3+C×M signifies: cells treated with (+)-catechin at a molar concentration of x; F3 C/L 1:1×M signifies: cells treated with the (+)-catechin/lysine complex according to a 1:1 molar equivalence ratio at a molar concentration of x; F3 C/L 1:2×M signifies: cells treated with the (+)-catechin/lysine complex according to a 1:2 molar equivalence ratio at a molar concentration of x.

[0120] In order to discuss the results obtained, the molar concentration of 1 μM was considered even though other values are reproduced on the graphs. This concentration is the most relevant in terms of the production of a medicament for which it is systematically sought to minimize the concentration of active compound. It remains no less the case that the other molar concentrations are just as suitable for the production of such a medicament, all the more so since the flavanol-type compounds have low toxicity and these concentrations are thus an integral part of the present invention.

Example 1: Treatment at Variable Concentrations of (+)-Catechin (+C)

[0121] As can be noted in FIG. 1A, a molar concentration of 1 μM of (+)-catechin (+C) does not affect the survival of the tumor cells, there by indicating that, at this concentration, the (+)-catechin is not toxic. FIG. 1B makes it possible to note that a molar concentration of 1 μM of (+)-catechin does not however make it possible to significantly decrease the number of tumor cells migrating from one compartment to the other of the Boyden cell. In the same sense, FIG. 10 shows that the (+)-catechin at this same concentration of 1 μM has no impact on the level of reactive oxygen species (ROSs) observed.

Example 2: Treatment at Variable Concentrations of the (+)-Catechin/Lysine Complex According to a 1:1 Molar Equivalence Ratio

[0122] As can be noted in FIG. 2A, a molar concentration of 1 μM of the (+)-catechin/lysine complex according to a 1:1 molar equivalence ratio (C/L 1:1) does not affect the survival of the tumor cells, thereby indicating that, at this concentration, the C/L 1:1 complex is not toxic. Moreover, FIG. 2B makes it possible to note that a molar concentration of 1 μM of C/L 1:1 complex makes it possible to significantly decrease by up to 30% the number of tumor cells migrating from one compartment to the other of the Boyden cell.

[0123] It was determined that this decrease in the percentage of migrating tumor cells is linked to a reduction of about and of at least 10% in the level of reactive oxygen species (ROSs) produced by the cells, this being under the effect of the C/L 1:1 complex.

Example 3: Treatment at Variable Concentrations of the (+)-Catechin/Lysine Complex According to a 1:2 Molar Equivalence Ratio

[0124] As can be noted in FIG. 3A, a molar concentration of 1 μM of the (+)-catechin/lysine complex according to a 1:2 molar equivalence ratio (C/L 1:2) does not affect the survival of the tumor cells, thereby indicating that, at this concentration, the C/L 1:2 complex is not toxic. Moreover, FIG. 3B makes it possible to note that a molar concentration of 1 μM of C/L 1:2 complex makes it possible to significantly decrease by up to 40% the number of tumor cells migrating from one compartment to the other of the Boyden cell.

[0125] It is clearly understood that the present invention is in no way limited to the embodiments described above and that many modifications can be introduced therein without departing from the context of the appended claims.

Example 4: Cytotoxicity of the (+)-Catechin/lysineHCl Derivatives Compared to Epigallocatechin Gallate on the Tumor Cells

[0126] The epigallocatechin gallate comes from Sigma Chemical Co, St Louis, the (+)-catechin was prepared pharmaceutically pure from Bloc Gambir and its complexes were obtained by combining therewith lysine hydrochloride in molar proportion.

[0127] The SiHa-F3 cells were initially generated as described in the article recently published in Cell Reports: Porporato P E et al., Cell Rep 2014, 8:754-766. The cells were maintained in culture and manipulated in DMEM, 4.5 g/I glucose, glutaMAX (Gibco), supplemented with 10% of fetal bovine serum.

[0128] Measurement of cell viability: 20 000 SiHa-F3 cells were seeded and treated with the indicated compounds overnight (16 h). The cells were then fixed for 3 min with methanol, stained with 0.23% crystal violet, and resuspended in DMSO in order to measure the absorbance at 595 nm (Victor X4 spectrophotometer, Perkin Elmer); or alternatively incubated for 3 h at 37° C. in a saturating solution of MTT in HBSS containing 10 mM of HEPES, and resuspended in DMSO in order to measure the absorbance at 630 nm (Victor X4 spectrophotometer, Perkin Elmer).

[0129] The SiHa-F3 cells were treated overnight (16 h) with epigallocatechin gallate (EGCG), (+)-catechin, (+)-catechin/lysineHCl 1:1 or (+)-catechin/lysineHCl (1:2) (concentrations of 100 nM, 1 μM, 10 μM, 100 μM, 500 μM or 1 mM) after which the cell survival was measured by staining with crystal violet (FIG. 4) or with MTT (FIG. 5).

[0130] As demonstrated in FIGS. 4 and 5, the catechin derivatives are less cytotoxic than epigallocatechin gallate.

Example 5: Antioxidant Activity of the Compounds

[0131] The epigallocatechin gallate, the (+)-catechin and its complexes combined with lysine hydrochloride and D,L-lysine and also the SiHa-F3 cells were obtained as described above.

[0132] Measurement of total cell ROSs: 20 000 SiHa-F3 cells were seeded before being treated for 1 h with the compounds indicated. The cells were then incubated for 30 min at 37° C. in a 1 μM solution of H2-DCFDA (Invitrogen) in HBSS containing 10 mM of HEPES, before measurement of the absorbance at 535 nm (Victor X4 spectrophotometer, Perkin Elmer).

[0133] The SiHa-F3 cells were treated for 2 h with epigallocatechin gallate (EGCG), (+)-catechin, (+)-catechin/lysineHCl 1:1 or (+)-catechin/lysineHCl (1:2) (concentrations of 100 nM, 1 μM, 10 μM, 100 μM, 500 μM or 1 mM), after which the cell production of ROSs was measured by fluorescence with DCFDA.

[0134] As can be noted in FIG. 6, all the catechin derivatives and the epigallocatechin gallate have a similar antioxidant activity on the tumor cells.

Example 6: The (+)-Catechin/lysineHCl Complexes Inhibit the Tumor Cell Migration

[0135] The epigallocatechin gallate, the (+)-catechin and its complexes combined with lysine hydrochloride and D,L-lysine and also the SiHa-F3 cells were obtained as described above.

[0136] Cell migration measurements: A 48-well reusable Boyden chamber (Neuroprobe) was used according to the manufacturer's instructions. Briefly, medium containing 0.15% of fetal bovine serum served as chemoattractant in the lower compartment, a polycarbonate membrane pierced with pores 8 μm in diameter separated the two compartments, and 50 000 SiHa-F3 cells in suspension in the presence of the indicated compounds were introduced into the upper compartment. The migration of the cells was quantified after an overnight period (16 h). The cells, having migrated, were fixed for 3 min in methanol, stained with 0.23% crystal violet for 30 min, and counted on photographs taken at a magnification of ×2.5 using an inverted-phase microscope (Axiovert and Mrc camera, Zeiss).

[0137] The SiHa-F3 cells were treated during a migration performed in a Boyden chamber with epigallocatechin gallate (EGCG), (+)-catechin, (+)-catechin/lysineHCl 1:1 or (+)-catechin/lysineHCl (1:2) (concentrations of 100 nM, 1 μM, 10 μM, 100 μM, 500 μM or 1 mM), after which the number of cells having migrated toward 0.15% of serum were counted. Results were expressed as % of the control.

[0138] As can be noted in FIG. 7, the epigallocatechin gallate and the (+)-catechin/lysineHCl complexes, but not the (+)-catechin, inhibit the migration of the cancer cells. Only the (+)-catechin/lysineHCl complex compounds inhibit the migration of the tumor cells independently of cell cytotoxicity that would have affected the count.

Example 7: H-Catechin/lysineHCl 1:2 Inhibits the Formation of Metastases

[0139] The B16F10 tumor cells come from the ATCC collection. The male 6- or 7-week-old C57BL/6 mice were acquired from the Janvier laboratories, and the protocols were applied with the agreement of the UCL ethics committee and while adhering to Belgian laws relating to animal care. Model of experimental metastases: the B16F10 cells were pretreated with DMSO (0.5%, rotenone carrier, negative control) or rotenone (respiratory chain complex I inhibitor and mitochondrial superoxide inducer as described in Porporato P E et al., Cell Rep 2014, 8:754-766, positive control), alone or in combination with the indicated compounds for 6 h. The cells were then detached, counted and resuspended in a proportion of 10×10.sup.6 cells/ml in HBSS without calcium or magnesium. 10.sup.6 cells (i.e. 100 μl of the suspension) were then injected into the caudal vein of C57BL/6 mice. 14 days after injection of the cells, the mice were sacrificed, the lungs were isolated and the metastases (spots positive for melanin) were counted under a dissection microscope.

[0140] FIG. 8 presents representative photographs (A) and a graph (B) quantifying the formation of lung metastases 14 days after injection of 10.sup.6 B16F10 murine melanoma cells into the caudal vein of syngenic mice. The cells were pretreated with 0.5% DMSO (DMSO, negative control) or with the respiratory chain complex I inhibitor and mitochondrial superoxide inducer rotenone at 20 nM alone (Rot 20 nM, positive control) or in combination with 10 μM (+)-catechin/lysineHCl 1:2 (Rot 20 nM+C/L 1:2 10 μM). n=7-9, one-way ANOVA with Bonferroni test, ** p<0.01.

[0141] It is noted that the (+)-catechin/lysineHCl 1:2 inhibits the formation of metastases in an intravenous injection experimental model. FIG. 9 shows representative photographs (A) and the graph (B) quantifying the formation of lung metastases 14 days after injection of 10.sup.6 B16F10 murine melanoma cells into the caudal vein of syngenic mice. The cells were pretreated with 0.5% DMSO (DMSO, negative control) or with the respiratory chain complex I inhibitor and mitochondrial superoxide inducer rotenone at 20 nM alone (Rot 20 nM, positive control) or in combination with 10 μM (+)-catechin/lysineHCl 1:2 (Rot 20 nM+C/L 1:2 10 μM) or with epigallocatechin gallate/lysineHCl 1:2 (Rot 20 nM+EGCG/L 1:2 10 μM). n=7-8, one-way ANOVA with Bonferroni test, ns p>0.05, ** p<0.01.

[0142] It was observed that, contrary to the epigallocatechin gallate/lysineHCl 1:2, the (+)-catechin/lysineHCl 1:2 inhibits the formation of metastases in an intravenous injection experimental model.

Example 8: H-Catechin/lysineHCl 1:2 but not Epigallocatechin Gallate Reduces the Formation of Lung Metastases in an Experimental Model

[0143] FIG. 10 shows representative photographs (A) and the graph (B) quantifying the formation of lung metastases 14 days after injection of 10.sup.6 B16F10 murine melanoma cells into the caudal vein of syngenic mice. The cells were pretreated with the respiratory chain complex I inhibitor and mitochondrial superoxide inducer rotenone at 10 nM in combination with epigallocatechin gallate (Rot 10 nM+EGCG 10 μM) or with 10 μM (+)-catechin/lysineHCl 1:2 (Rot 10 nM+C/L 1:2 10 μM). n=5-9, Student's t test, ns p>0.05, * p<0.05.

[0144] It was observed that the (+)-catechin/lysineHCl 1:2 inhibits the formation of metastases more effectively than the epigallocatechin gallate 1:2 in an intravenous injection experimental model.

[0145] Statistics:

In all of the experiments and examples 1 to 8, one-way ANOVA was applied followed by the appropriate multiple comparison tests (Dunnett for the in vitro tests, Bonferroni for the in vivo tests), and the Student's t test for the comparison of the two conditions presented in FIG. 10. A p-value <0.05 was considered to be statistically significant (*); ** indicates p<0.01; *** p<0.005. Ns (not significant) indicates that the p-value was >0.05. The columns of the in vitro graphs represent the mean of each group, the points of the in vivo graphs each represent one mouse and the horizontal bar represents the mean of each group. N indicates the number of times the experiment was reproduced independently, and n indicates the total number of replicates.

Example 9: Anti-Metastatic Effect in a Model of Spontaneous Metastases

[0146] In order to study the metastatic process as a whole, a model of spontaneous metastases from a primary tumor is used (Porporato P E et al. Cell Rep 2014; 8:754-766). For example, in a model, 1 000 000 moderately metastatic murine melanoma cells (B16F10) or strongly metastatic murine melanoma cells (B16M4b, described in Porporato P E et al., Cell Rep 2014, 8:754-766), optionally carrying a luminescent or fluorescent reporter, are injected subcutaneously or intradermally into the flank of syngenic C57BL/6 mice. The anti-metastatic and/or antitumor activity of a compound of catechin type is evaluated by administering this compound to the animals, for example intraperitoneally or intravenously (injection) or orally (gavage, incorporation into food or into drinking water) and by comparing it to its carrier, according to a defined dosage regimen scheme (administration frequency and dose).

[0147] The growth of the primary tumor is carefully monitored by means of repeated measurements (for example every 2-3 days) of the tumor diameter by means of a caliper and/or of luminescence or fluorescence imaging in vivo if the expression of a reporter by the tumor cells enables this. When the mean tumor diameter reaches preferentially 10 mm (typically between days 10 and 14 for the abovementioned tumor lines), the primary tumor is excised with the double objective of keeping the mouse alive and of stimulating the development of the metastases (Porporato P E et al., Cell Rep 2014, 8:754-766; Gabri M R et al., Clin Cancer Res 2006; 12:7092-7098). The period of time between the surgeries on the first mouse and the last mouse to develop a primary tumor of required size is as short as possible (typically 5 days for the abovementioned tumor lines). The excision of a possible regrowth of the primary tumor is carried out as often as necessary, when this regrowth reaches the size of about 10 mm in diameter. In order to evaluate the metastatic dissemination, the mice are sacrificed in the various groups when one of the following events occurs: 1°) a first animal presents a state of suffering exceeding the ethical limits, 2°) a first animal dies without prior symptoms and exhibits numerous and/or bulky metastatic lesions, 3°) the monitoring of the luminescence or of the fluorescence in vivo indicates a considerable metastatic dissemination, if the cells express an appropriate reporter, 4°) a period of typically 30 to 40 days post-injection for the B16M4b mice or of typically 60 to 90 days for the B16F10 mice. In these melanoma models, the metastases essentially occur in the lungs and the lymph nodes (inguinal, axillary, mediastinal) and less frequently in the liver, on the peritoneum, and possibly the brain. For the quantification of the number of lung metastases, insufflation of the lungs by intratracheal injection of a saline solution is performed. The lung metastases (spots positive for the black melanin pigment) are counted using a dissection microscope 1. Alternatively, the ex vivo luminescence or fluorescence imaging of the organs removed and/or a histological analysis are carried out. In the case of a considerable variability in the size of the primary tumor, the results are standardized for each animal by means of the area under the curve of growth of the primary tumor until resection thereof.

[0148] It is observed that the catechin-type derivatives according to the invention have an anti-metastatic effect.