PHARMACEUTICAL COMPOSITION FOR USE IN PREVENTING OR STOPPING METASTASES

Abstract

The invention relates to a composition, and a medicament for preventing or stopping of metastases, in particular metastases originating from epithelia cancer, such as skin cancer, lung cancer, stomach cancer, brain cancer, breast cancer, colo-rectal cancer, prostate cancer, pancreas cancer, head/neck cancer, and esophagus cancer. It has been found that the present composition irreversibly converts cancer cells with metastatic potential into harmless cells with no metastatic potential.

Claims

1. A pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis, comprising (i) a first compound comprising at least one carbohydrate and containing more than one sulphate, in combination with (ii) a second compound selected from the group of thiazolidinediones, NSAIDs, sulphonylureas, and indoles.

2. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis according to claim 1, comprising at least one of pentosan sulphate, and adequan.

3. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis according to claim 1 2, comprising at least one of indomethacin and pioglitazone.

4. The pharmaceutical composition a treatment selected from in the prevention of metastasis and stopping of ongoing metastasis according to claim 1, wherein the first compound and second compound are provided in a molar ratio of 0.01:1 to 1:0.01.

5. The pharmaceutical composition a treatment selected from in the prevention of metastasis and stopping of ongoing metastasis according to claim 1, wherein the composition further comprises (iii) at least one pharmaceutically acceptable carrier.

6. The pharmaceutical composition a treatment selected from in the prevention of metastasis and stopping of ongoing metastasis according to claim 1, wherein the active pharmaceutical ingredients are in one dosage form.

7. The pharmaceutical composition a treatment selected from in the prevention of metastasis and stopping of ongoing metastasis according to claim 1, comprising at least one of pentosan sulphate and adequan, and at least one of indomethacin and pioglitazone.

8. The pharmaceutical composition a treatment selected from in the prevention of metastasis and stopping of ongoing metastasis according to claim 1, wherein the pharmaceutical composition comprises separate dosage forms for individual pharmaceutical active ingredients, and wherein the composition is in the form of at least one of a tablet, a capsule, a repository, and an injectable.

9. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis according to claim 8, wherein the composition is in the form of at least one of a tablet and a capsule suitable for oral administration.

10. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis according to claim 1, formulated in a form, which, depending on its use, allows targeting of at least one of specific cells, tissues, and organs.

11. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis according to claim 1, wherein the cancer originates from epithelial tissue.

12. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis according to claim 11, wherein the cancer is selected from skin cancer, lung cancer, stomach cancer, brain cancer, breast cancer, kidney cancer, liver cancer, colo-rectal cancer, bladder cancer, gall bladder cancer, prostate cancer, pancreas cancer, head/neck cancer, melanomas, and esophagus cancer.

13. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis according to claim 1 for use as a medicament by administering said medicament in an effective amount to cancer cells with metastatic potential for a sufficient period.

14. The pharmaceutical composition for a treatment selected from the prevention of metastasis and stopping of ongoing metastasis of claim 13, wherein the administration is to a pet or mammal comprising said tissue, wherein the mammal preferably is a human with a cancer that has metastatic potential.

15. A medicament comprising a pharmaceutical composition comprising (i) a first compound comprising at least one carbohydrate and containing more than one sulphate, in combination with (ii) a second compound selected from the group of thiazolidinediones, NSAIDs, sulphonylureas, and indoles in a treatment selected from for prevention of metastasis and for stopping of ongoing metastasis.

16. The pharmaceutical composition according to claim 1, wherein the first compound comprising at least one carbohydrate and containing more than one sulphate is selected from multiple Sulphur containing agents with a molecular weight of <10 kDa.

17. The pharmaceutical composition according to claim 6, wherein the dosage form comprises 1-10 mg active ingredients/kg body weight.

Description

SUMMARY OF THE FIGURES

[0080] FIG. 1 shows pictures of histological staining experiments, relating to the synergistic effect of the combination of Pentosan sulphate with Indomethacin.

[0081] FIG. 2 shows pictures of histological staining experiments, relating to the synergistic effect of the combination of Pentosan sulphate with Pioglitazone.

[0082] FIG. 3 shows pictures of histological staining experiments, relating to the synergistic effect of the combination of Adequan with Indomethacin.

[0083] FIG. 4 shows in vitro measurement in relative light units (RLU) of PPAR transcriptional activity in KS-483 cells transfected with a dual PPAR reporter system and exposed to different compounds.

[0084] FIGS. 5 shows a schematic structure of Thiazolidinedion and FIG. 6 of sulfonylurea.

[0085] FIGS. 7a,b, 8, 9a,b, 10, 11a,b 12, 13a,b, and 14 show experimental results of the claimed composition and synergistic effects thereof, FIGS. 15a-d show stimulation schemes, FIGS. 16a-c and 17a-d staining results, and FIG. 18a,b synergistic effects.

DETAILED DESCRIPTION OF THE FIGURES

[0086] The invention will be elucidated with the following non-limiting examples and experiments.

[0087] FIG. 1-3 show the effect of the combined use of a compound comprising a carbohydrate containing more than one sulphate (Adequan or Pentosan sulphate), and Indomethacin or Pioglitazone. The concentrations of active compounds used to stimulate the cells are given in the pictures. The active compounds were added to the medium, and thereafter the cells were suspended in the medium. After 3 days, the cells were stained with Oil red O or Nile Red staining.

[0088] Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, C26H24N4O) is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections. The pictures show central nuclei, increasing lipid formation and droplets by the combined use of the composition of the invention, and therefore is indicative of conversion of cancer cells with metastatic potential into harmless cells. Nile red (also known as Nile blue oxazone) is a lipophilic stain accumulating in lipid globules inside the cell. Nile Red fluoresces strongly when partitioned into lipids, but practically not at all in an aqueous solution.

[0089] FIG. 4 shows the effect of exposure of KS-483 cells to a compound comprising a carbohydrate and a sulphate (Adequan or Pentosan sulphate), and Indomethacin or Pioglitazone. Similar results are obtained with other cell-lines.

[0090] Similar experiments are performed on other cell lines, on organoids, and on assays, showing similar results.

[0091] Examples and Experiments

[0092] The genes in KS-483 cells were assessed for up- or downregulation after stimulation with the compounds. Briefly, KS-483 cells were cultured under standard culturing conditions in α-MEM medium supplemented with glutamax, penicillin/streptomycin and 10% heat-inactivated Fetal Calf Serum (FCS). Similar experiments were conducted with other cell lines, such as the 3T3-L1 cell line.

[0093] From experiments with up- and downregulated genes, and their activity, it is clear that a composition, comprising (i) a compound comprising at least one carbohydrate and containing more than one sulphate, in combination with (ii) indomethacin and/or pioglitazone blocked metastasis development.

[0094] Activity Measurements

[0095] KS-483 cells were used for activity measurements. From the RLU measured after exposure to single compounds it is clear that a composition, comprising (i) a compound comprising at least one carbohydrate and containing more than one sulphate and indomethacin and/or pioglitazone lead to a significant increase in activity and transfer away from metastatic cells.

[0096] The invention has been described by reference to certain embodiments discussed above. It will be recognized that these embodiments are susceptible to various modifications and alternative forms well known to those of skill in the art without departing from the scope of the invention. Accordingly, although specific embodiments have been described, these are examples only and are not limiting upon the scope of the invention, which is defined in the accompanying claims.

[0097] Experimental Design Cell Culture

[0098] For FIGS. 7a-14 the following conditions were used:

TABLE-US-00001 Protocol 3T3-L1 Protocol 3T3-L1 Protocol KS-483 cell line αMEM cell line DMEM cell line αMEM 10% heat 10% heat 10% heat inactivated FCS inactivated FCS inactivated FCS Compound A - Compound A - Compound A - 5.0 μg/ml 0.5 μg/ml 5.0 μg/ml Compound B - Compound B - Compound B - 50 μM 50 μM 5.0 μM Compound C - Compound C - Compound C - 1.0 μM 1.0 μM 1.0 μM Compound A + B Compound A + B Compound A + B Compound A + C Compound A + C Compound A + C In the of presence of: 0.25 μM Dexameth- asone & 1 ug/ml Insulin

[0099] Further Experiments

[0100] Background

[0101] The overall rationale of the in vitro experiments conducted to test the efficacy of the combined present compounds in the treatment of cancer was mainly based on their capacity to induce terminal adipogenesis and transition into beige/brown fat cells, following experimental conditions of a previous publication with different compounds but similar endpoints (Ishay-Ronen et al., Cancer Cell 2019) https://doi.org/10.1016/j.ccell.2018.12.00

[0102] Results.

[0103] MTΔECad cells were derived from the MMTV-Neu transgenic mouse, a genetic model of irreversible EMT. MTΔECad cells were treated with the following compounds: Indomethacin (Indo), Pioglitazone (Pio) and Pentosan Polysulphate (PPS)

[0104] Treatments & Methods

[0105] According to the protocol established by the Ishay-Ronen et al. study, inventors employed a combination of insulin, dexamethasone and Rosiglitazone for 3T3-L1 cells (treatment A), and of Rosiglitazone and BMP2 for MTΔECad cells (treatment B).

[0106] For the stimulation of MTΔECad cells the following concentrations were used: PPS (5 μg/ml), Indomethacin (50 μM), en Pioglitazone (10 μM).

[0107] Untreated cell lines were employed as negative controls. As read-outs of adipogenesis, several approaches were tested: [0108] 1. Oil red O staining by histochemistry. [0109] 2. Nile Red staining by immuno-fluorescence (IF) [0110] 3. RTqPCR (gene expression analysis) [0111] Adiponectin (adipocyte marker) [0112] EpCam (marker for epithelial cells) FIGS. 15a-d show stimulation schemes; FIGS. 15a-b according to prior art, and FIGS. 15c-15d according to the present invention.

[0113] The FIG. 16 shows Nile Red staining of the MTΔECad treated cells. Induction of adipogenesis (indicated by the red staining), epithelialization (i.e. re-acquisition of epithelial features, i.e. MET; indicated by morphological changes from spindle-shaped to cuboid cells) and induction of adipogenesis is shown in both conditions:

[0114] Control=untreated MTΔECad cells.

[0115] Treatment B (FIG. 15ba)=Rosiglitazone and BMP2 (according to Ishay-Ronen et al., Cancer Cell 2019)

[0116] Treatment C and D (FIG. 15c-d)=PPS and indomethacin and PPS en Pioglitazone (the present combinations).

[0117] Although the images below are only indicative of the processes triggered by the different treatments and do not allow their quantitative evaluation, they point to the fact that the two processes, that is adipogenesis and transition into fat cells can occur independently of each other. Interestingly, this is thought to prevent and even ‘cure’ metastasis formation.

[0118] FIG. 16. Left Control (FIG. 16a) untreated MTΔECad cells that clearly show spindle shaped cells characteristics for epithelial cells that have undergone EMT. On the right (FIG. 16b) MTΔECad cells of which many are differentiated into adipocytes as a positive control (red stained) by treatment B (FIG. 16b) and by treatment D (FIG. 16c). Important to note is that the MTΔECad cells that did not differentiate into adipocyte by treatment B or D have a more cuboidal phenotype characteristic of epithelial cells in contrast to the spindle shape seen in the untreated control cells. Arrows indicate abundantly present fat cells.

[0119] FIG. 17 shows Oil Red O staining of the MTΔECad treated cells. Under these culture conditions, adipocyte formation has been found after stimulation with the combination of Indomethacin/PPS and Pioglitazone/PPS stimulated cells. Pioglitazone alone can stimulate adipocyte formation at a low level. FIG. 17a: control; FIG. 17b indomethacine+pentosan; FIG. 17c pioglitazone; FIG. 17d: pioglitazone+pentosan.

[0120] EpCAM Gene Expression.

[0121] Inventors studied the effect of treatment of MTΔECad cells with the different compounds and combinations on EpCAM expression as a marker for epithelial cells and Adiponectin expression as a marker for adipocytes.

[0122] qPCR

[0123] Steps performed when measuring gene expression using real-time PCR.

[0124] RNA was first isolated and characterized for quantity and integrity. During a two-step reaction, cDNA was first synthesized and then used as a PCR template. The housekeeping gene GAPDH was used for normalizing the results. A normalization factor has been used for calculating each individual sample. Dividing the fluorescent data by its normalization factor produced the normalized data, which was followed by statistical analysis.

[0125] Results

[0126] EpCam

[0127] Indomethacin and Pioglitazone clearly stimulated EpCAM expression. PPS on its own had no significant effect. However when PPS was combined with Indomethacin or Pioglitazone there was a synergistic effect. 3T3-L1 pre-adipogenic cells unstimulated or stimulated towards adipocytes (treatment A) were used as a control because they are mesenchymal cells and therefore do not express EpCAM, data not shown. Cells treated as a positive control (treatment B) show stimulated EpCAM gene expression.

[0128] MTΔECad treated cells show synergetic upregulation of EpCam gene expression after stimulation with the combination Indomethacin/PPS or Pioglitazone/PPS, This indicates that MTΔECad treated cells express more epithelial characteristics, since EpCam is only expressed on epithelial cells and not on mesenchymal cells.

[0129] Adiponectin

[0130] MTΔECad treated cells shown synergetic upregulation of adiponectin gene expression after stimulation with the combination Indomethacin/PPS or Pioglitazone/PPS, indicating that the treated cells express adipocyte characteristics, since adiponectin expression is restricted to adipocytes.

[0131] 3T3-L1 pre-adipogenic cells were stimulated towards adipocytes (treatment A) and used as a control, Adiponectin expression was up-regulated after stimulation, data not shown.

[0132] Indomethacin and Pioglitazone obviously stimulated adiponectin gene expression and a clearly synergetic effect is shown after stimulation with PPS in combination with Indomethacin or Pioglitazone. PPS on its own had no effect on adiponectin gene expression.

[0133] Cells treated as a positive control (treatment B) shown stimulated adiponectin gene expression. See FIGS. 18a,b. Conclusion

[0134] Both Indomethacin and Pioglitazone in combination with Pentosan Polysulfate (PPS) are able to terminally differentiate MTΔECad cells breast cancer cells into adipocytes but at the same time they can induce MET (mesenchymal-to-epithelial transition). This was shown by appearance in culture of Nile Red positive lipid droplets containing adipocyte like cells that also express Adiponectin, measured by qPCR. However, at the same time cuboidal non-adipocyte cells are visible indicative of epithelial cells which was confirmed by the fact that we also found expression of the epithelial marker EpCAM using qPCR.

[0135] FIGS. 7a,b, 8, 9a,b, 10, 11a,b, 12, 13a,b and 14 show experimental results of the claimed composition and synergistic effects thereof. FIGS. 7a,b show the effect of compounds on Perilipin-1 gene expression in 3T3-L1 cells; in FIG. 7a the effect of the compounds alone, and in FIG. 7b the effect of compounds stimulated in presence of Insulin/Dexamethasone. FIG. 8 shows the effect of compounds on Perilipin-1 gene expression in KS-483 cells. FIGS. 9a,b show the effect of compounds on FABP4 gene expression in 3T3-L1 cells; in FIG. 9a the effect of the compounds alone, and in FIG. 9b the effect of compounds stimulated in presence of Insulin/Dexamethasone. FIG. 10 shows the effect of compounds on FABP4 gene expression in KS-483 cells. FIGS. 11a,b show the effect of compounds on CD-36 gene expression in 3T3-L1 cells; in FIG. 11a the effect of the compounds alone, and in FIG. 11b the effect of compounds stimulated in presence of Insulin/Dexamethasone. FIG. 12 shows the effect of compounds on CD-36 gene expression in KS-483 cells. FIGS. 13a,b show the effect of compounds on Adiponectin gene expression in 3T3-L1 cells; in FIG. 13a the effect of the compounds alone, and in FIG. 13b the effect of compounds stimulated in presence of Insulin/Dexamethasone. FIG. 14 shows the effect of compounds on Adiponectin gene expression in KS-483 cells.