WO2022265510A1

WO2022265510A1 - Pharmaceutical composition for use in the treatment of metastatic epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma

Info

Publication number: WO2022265510A1
Application number: PCT/NL2022/050343
Authority: WO
Inventors: Gerard KRIELEN; Clemens LOWIK
Original assignee: Logick Energetics B.V.
Priority date: 2021-06-17
Filing date: 2022-06-17
Publication date: 2022-12-22

Abstract

The present invention relates to the use of pentosan polysulfate in combination with indomethacin and/or pioglitazone in the treatment of patients suffering from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, or patients at risk from metastases thereof, the present invention further relates to pentosan polysulfate in combination with indomethacin or pioglitazone, to a pharmaceutical composition and therapeutic combination thereof.

Description

PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT OF METASTATIC EPITHELIAL OVARIAN CANCER, METASTATIC FALLOPIAN TUBE CARCINOMA OR METASTATIC PRIMARY PERITONEAL CARCINOMA

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the use of pentosan poly sulfate in combination with indomethacin and/or pioglitazone in the treatment of patients suffering from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, or patients at risk from metastases thereof.

BACKGROUND OF THE INVENTION

Cancer starts when cells in the human body begin to grow out of control. Cells in nearly any part of the body can become aberrant cancer cells forming a tumor, and the aberrant cells can spread to other areas of the body such that further tumors may grow (metastases). There are more than 200 different types of cancer, classified based on the type of cancerous cell involved in the cancer and based on the organ(s) inflicted by the cancer. Cancer is one of the deadliest threats to human health. For many types of cancer, despite extensive R&D currently still no satisfactory solution is available with regard to treatment of the cancer or with regard to sufficient and satisfactory palliative care. Furthermore, different types of cancers vary widely in their severity and with regard to the survival rate of patients once diagnosed with the cancer. In 2015, 8.8 million people worldwide died from cancer. This is nearly 1 in 6 of all global deaths.

Ovarian cancer is defined as a tumor that forms in or on an ovary. Ovaries are female reproductive organs found on each side of the uterus in the female pelvis. The ovaries produce eggs (‘ova’) for reproduction. The eggs travel through the fallopian tubes into the uterus where the fertilized egg implants and develops into a fetus. The ovaries are also the main source of the female hormones estrogen and progesterone.

Ovarian cancer is the fifth most common cancer in women. It is usually diagnosed at an advanced stage and is the leading cause of death from gynaecologic cancers. The overall survival rate at five years is still very poor. Most patients are aged between 55 and 80 years.

Ovarian cancers are often referred to in relation to the extent of development of the cancer and to the localization of the cancer as well as to the responsiveness to certain anti-cancer drugs. Frequently, ovarian tumor cells spread to other sites within the body from the original tumor and form small localized clusters of cancer cells termed micrometastases. When they are left to continue to grow, they form larger metastases, which can depending on the treatment history are sensitive or resistant to common chemotherapy.

Ovarian cancer patients can, for example, in some cases be treated with platinum- based medicaments, whereas other ovarian cancer patients turn out not to respond to such therapy. Recurrent cancer refers to cancer that has come back after treatment. Platinum- sensitive recurrent ovarian cancer refers to the recurrence of active ovarian cancer in a patient at least six months after completing platinum-based therapy. Platinum-resistant recurrent ovarian cancer on the other hand refers to the recurrence of active ovarian cancer in a patient within six months after completing platinum-based therapy.

Chemotherapy for first-time ovarian cancer patients is most often a combination of two or more drugs, administered intravenously generally every three-weeks.

Administering combinations of drugs to an ovarian cancer patient rather than just one drug alone seems to be more effective in the initial treatment of ovarian cancer. The standard approach is the combination of a platinum compound, such as cisplatin or carboplatin, and either a taxane, such as paclitaxel or docetaxel or gemcitabine. Ovarian cancer often shrinks or even disappears upon the applied chemotherapy. However, remaining cancer cells may eventually begin to grow again (‘recurrence’) and may also have spread outside the area of the primary tumor and may have caused metastases.

In general, metastasis relates to a spread from an initial cancer site to a different site within a living body. This spread is caused by cancer cells which are able to circulate through the body towards other parts thereof. There they may form a secondary tumor, the metastatic tumor. The metastasis may have a small volume, but it is often widely spread through the body. Unfortunately, many ovarian cancer patients are only diagnosed at a relatively late stage, such that in many cases the disease has metastasized. For example, three-quarters of women who are newly diagnosed have late stage (III or IV) ovarian cancer. The cells of the metastasis often resemble the cells of the original tumor, and therefore reference is made to the initial tumor when characterizing the metastasis. They could therefore in principle be treated likewise, but as they are widespread, treatment may be cumbersome and to harmful to the body. Moreover, metastases can acquire new characteristics, for example due to previous treatment, making them unresponsive to anticancer drugs that are effective against the primary tumor and making them even more difficult to treat.

At this stage, the situation of the patients becomes very severe and the patients may suffer from more and more metastases and the consequences thereof. It is generally accepted that metastatic ovarian cancer is not curable and the aim of the present treatment are aimed at prolonging the overall survival, or at least the prolongation of the progression free survival of said patients.

Hence, a need remains for the provision of drugs that are able to prevent or treat metastatic epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma or at least further postpone progression and increase overall survival.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a pharmaceutical composition for use in the treatment of patients suffering from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, in particular metastatic forms thereof, wherein the composition comprises:

(a) pentosan polysulfate or a pharmaceutically acceptable salt thereof; and

(b) indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof; and

(c) one or more pharmaceutically acceptable excipients.

With the pharmaceutical composition of the present invention, it has now become possible to stop an ongoing metastasis, even if it has been pre-treated with for example a platinum-based compound, or to prevent metastasis in the first place if the patient is (at present) only suffering from a primary tumor.

A second aspect of the present invention relates to pentosan poly sulfate for use in the treatment of patients suffering from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, in particular metastatic forms thereof, the treatment comprising administering to said patients pentosan polysulfate or a pharmaceutically acceptable salt thereof and indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof.

A third aspect of the present invention relates to a pharmaceutical composition comprising:

(a) pentosan polysulfate or a pharmaceutically acceptable salt thereof; and

(c) one or more pharmaceutically acceptable excipients.

A fourth aspect of the present invention relates to a therapeutic combination comprising:

(a) a first unit dose comprising: pentosan polysulfate or a pharmaceutically acceptable salt thereof;

(b) a second unit dose comprising: indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof.

DEFINITIONS

The term “ chemotherapeutic compound ’ as used herein has its conventional meaning and refers to a compound useful in the treatment of cancer. The term ‘ pharmaceutical composition’ as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.

The term ‘ pharmaceutically acceptable ’ as used herein has its conventional meaning and refers to compounds, material, compositions and/or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

The term ‘ excipient’ as used herein has its conventional meaning and refers to a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing a granulate, solid or liquid oral dosage formulation. The term ‘sal? as used herein has its conventional meaning and includes the acid addition and base salts of the compound referred to.

The term ‘ treatment ’ as used herein has its conventional meaning and refers to curative, palliative and prophylactic treatment. The term 'unit dosage form ’ has its conventional meaning and refers to a dosage form which has the capacity of being administered to a subject, preferably a human, to be effective, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising the therapeutic agent. The term “ cancer ” as used herein has its conventional meaning and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors of micro metastases.

The term “ovarian cancer ” as used herein has its conventional meaning and refers to epithelial cancers that begin in the ovary, the fallopian tube, and the lining of the abdominal cavity, called the peritoneum. Hence, the term “ ovarian cancer ” refers to epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma.

The term “ metastasis’ ’ or “ metastatic’ ’ as used herein has its conventional meaning and refers to a cancer spread from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into the lymphatic and blood vessels, circulate through the bloodstream and grow in a distant focus (metastasize) in normal tissue elsewhere in the body. Metastasis can be local or distant.

The term “ progression free survival (PFS)” as used herein has its conventional meaning and refers to the time from treatment or randomization to first disease progression or death, PFS can be assessed by Response Evaluation Criteria in Solid Tumors (RECIST).

The term “ overall survival (OS) ” as used herein refers to the subject remaining alive for a defined period of time. DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a pharmaceutical composition for use in the treatment of patients suffering from ovarian cancer, i.e. epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, wherein the composition comprises:

(a) pentosan polysulfate or a pharmaceutically acceptable salt thereof; and

(c) one or more pharmaceutically acceptable excipients. In particular, the patients to be treated with the composition according to the present invention suffer from or are at risk of metastatic epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma. In this regard it is noted that the disease may also be already platinum-resistant. It has surprisingly been found that with the pharmaceutical composition of the present invention the formation of metastases originating from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma can be avoided or slowed down considerably. It has further been found that in case such metastases are present, the cells thereof can be trigged to differentiate into unharmful types of cells, such as brown/beige and likewise white fat cells. This way the (further) growth of metastases can be avoided or reduced, which would increase the progression free survival period of patients suffering from these types of cancer. In order to prevent metastasis to occur in the first place the pharmaceutical composition may be administered to patients suffering from primary epithelial ovarian cancer, primary fallopian tube carcinoma or primary peritoneal carcinoma. However, the effect of the pharmaceutical composition is the strongest in case of metastasized epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma.

The present inventors have surprisingly found that the combination of the present invention provides, compared to the expression of the sum of both compounds applied separately, an at least two-fold expression of certain genes (e.g., FABP4, CD36, Adipoq, PLIN1). Both ovarium cancer cell lines and breast-cancer cell lines show differentiation (change in cell type) of these cells into brown/beige and likewise white fat cells. As a consequence, ongoing metastasis is stopped. It has further been found that said cancer cells to dedifferentiate towards a stem cell which on its turn is differentiated into a brown/beige or white fat cell, under influence of the pharmaceutical composition according to the present invention.

As further elucidated in the experimental part of the present invention, the combination of the present invention stimulated synergistically the formation of adipocytes with brown/beige or white fat cells characteristics, that is an irreversible transfer of cancer cells with metastatic potential into harmless cells. It is noted that white adipocyte, beige adipocyte, and brown adipocyte clearly distinguish from one and another, such as being UCP1 negative or positive, size of lipid droplets, adipokine secretion, and so on. Hence formation of brown/beige and likewise white fat cells is not too difficult to establish. Such a transfer can be observed in the mesenchymal progenitor KS483 cell line, deposited atDSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, under accession number DSM ACC3286 on January 13, 2016, but other cell lines of comparable origin can be used as well, such as the 3T3-L1 cell line which may be obtained from Sigma- Aldrich, and also epithelial cancer cell lines with metastatic potential. Moreover, as is clear from the experimental part below, also ovarian cancer cell lines SKOV-3 (https://www.atcc.org/products/htb-77 ) and OVCAR-3 (https://www.atcc. org/en/search#q=OVCAR-3&sort=relevancy&numberOfResults=12 )show the same beneficial effect. Hence, patients with cancers that are known to metastasize or that already have one or more metastases, benefit from use of the pharmaceutical composition of the invention because metastasis can be prevented or ongoing metastasis can be stopped.

With the pharmaceutical composition according to the present invention, it will be possible to extend the progression free survival and even the overall survival of the patients treated by at least 3 months.

The pharmaceutical composition according to the present invention may be used to prevent metastasis of primary epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma. In these patients metastasis of the disease may not have occurred yet, but they may be at risk of such an event. It may also be the case that one expects that in fact micro-metastases are already present, but that they cannot (yet) been seen with imaging techniques. Moreover, patients wherein local or distant metastasis has been confirmed may benefit the most from the use of the pharmaceutical composition of the present invention. After all, for these patients only limited treatment options are available. The dose of the different constituents of the pharmaceutical composition range between 10 to 300 mg pentosan polysulfate and 10 to 200 mg indomethacin and/or 10 to 45 mg pioglitazone. In said formulation indomethacin or pioglitazone is preferably used. However, it may also be possible to combine the two. Said dosages are on a daily basis.

The pharmaceutical formulation according to the present invention may be formulated as an oral free dose, i.e. wherein each constituent is administered separately or as a fixed dose combination. In the latter case, the constituents of the pharmaceutical composition according to the present invention are administered in a single unit dose. Preferably, the pharmaceutical composition according to the present invention is an oral fixed dose combination.

In view of the ease of administration it is preferred that the pharmaceutical composition according to the present invention is formulated as a capsule or tablet. These tablets or capsules are preferably provided in a blister package, which may be placed in a secondary package together with the label.

The molar ratio of pentosan poly sulfate on the one hand and indomethacin or pioglitazone on the other hand generally ranges from 0.01:1 to 1 :0.1. This way sufficient synergistic effect of the different active ingredients is obtained. Optionally, said ratio ranges from 0.2:1 to 1:0.2, or from 0.33:1 to 1:0.33, or form 0.45:1 to 1:0.245, such as from 0.5:1 to 1:0.5, e.g. from 0.9:1 to 1:0.9.

The pharmaceutical composition according to the present invention comprises besides the active ingredients referred to also possible salts or solvates thereof. For a review on suitable salts, reference is made to “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim,

Germany, 2002).

A pharmaceutically acceptable salts of the active ingredients of the pharmaceutical composition of the present invention be readily prepared by mixing together solutions of said active ingredient and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised.

Oral administration may involve swallowing, so that the active ingredients of the pharmaceutical formulation of the present invention enter the gastrointestinal tract. Alternatively, buccal or sublingual administration may also be employed wherein said active ingredients enter the blood stream directly from the mouth.

Solid oral dosage forms which may be used within the context of the present invention include besides tablets and capsules amongst others caplets, lozenges, pills, mini-tablets, pellets, beads and granules packaged in sachets. Liquid oral dosage forms which may be used for the pharmaceutical preparation of the present invention include, but are not limited to drinks, solutions, beverages and emulsions.

The pharmaceutical composition for use in the present invention comprises besides the active ingredients referred to also an excipient, i.e. a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing granulate, solid or liquid oral dosage formulations.

Examples of categories of excipients include, but are not limited to, binders, disintegrants, lubricants, glidants, fillers and diluents. One of ordinary skill in the art may select one or more of the aforementioned excipients with respect to the particular desired properties of the granulate and/or solid oral dosage form by routine experimentation and without any undue burden. The amount of each excipient used may vary within ranges conventional in the art. The following references which are all hereby incorporated by reference disclose techniques and excipients used to formulate oral dosage forms. See “The Handbook of Pharmaceutical Excipients”, 4th edition, Rowe et al ., Eds., American Pharmaceuticals Association (2003); and “Remington: The Science and Practice of Pharmacy”, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2000).

A second aspect of the present invention relates to pentosan poly sulfate for use in the treatment of patients suffering from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, comprising administering to said patients pentosan polysulfate or a pharmaceutically acceptable salt thereof and indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof.

Pentosan polysulfate in combination with said other active ingredient is particularly effective when used in patients suffering from or at risk of metastatic epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma. In this regard it is noted that the combination of the present invention is also efficacious in platinum-resistant forms of said diseases.

Patients in need thereof may be administered 10 to 300 mg pentosan polysulfate or a pharmaceutically acceptable salt thereof; 10 to 200 mg indomethacin and/or 10 to 45 mg pioglitazone or pharmaceutically acceptable salts thereof. Said dose may be administered once daily, twice daily or three times per day

(a) pentosan polysulfate or a pharmaceutically acceptable salt thereof; and (b) indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof; and

(c) one or more pharmaceutically acceptable excipients.

Said pharmaceutical composition, per se may be formulated as has already been described above. It is particularly noted that it is preferred that the pharmaceutical composition according to the present invention is formulated as fixed dose combination in the form of a capsule or tablet. These tablets or capsules are preferably provided in a blister package, which may be placed in a secondary package together with the label.

A last aspect of the present invention relates to a therapeutic combination comprising

SUMMARY OF THE FIGURES

Figure 1 shows pictures of histological staining experiments, relating to the synergistic effect of the combination of Pentosan poly sulfate with Indomethacin. Figure 2 shows pictures of histological staining experiments, relating to the synergistic effect of the combination of Pentosan poly sulfate with Pioglitazone.

Figure 3 shows pictures of histological staining experiments, relating to the synergistic effect of the combination of Adequan with Indomethacin.

Figure 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.

Figures 5 shows a schematic structure of Thiazolidinedion and figure 6 of sulfonylurea.

Figures 7a, b, 8, 9a, b, 10, lla,b 12, 13a, b, and 14 show experimental results of the claimed composition and synergistic 15 effects thereof, figs. 15a-d show stimulation schemes, figs.l6a-c and 17a-d staining results, and fig. 18a,b synergistic effects.

Figures 19 and 20 show the experimental results of the combination of the present invention on ovarian cancer cells.

The present invention will be illustrated further by means of the following non- limiting examples EXAMPLES

EXAMPLE 1: COMBINATION OF PENTOSAN, INDOMETHACIN OR PIOGLITAZONE ON BREAST CANCER CELLS

Figure 1-3 show the effect of the combined use of a compound comprising a carbohydrate containing more than one sulphate (Adequan or Pentosan polysulfate), 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.

Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, C26H24N40) 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.

Figure 4 shows the effect of exposure of KS-483 cells to a compound comprising a carbohydrate and multiple sulphates (Adequan or Pentosan polysulfate), and Indomethacin or Pioglitazone. Similar experiments are performed on other cell lines, and on assays, showing similar results. 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 a-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. From experiments with up- and downregulated genes, and their activity, it is clear that a composition according to the present invention can differentiate mesenchymal (cancer) stem cells into beige/brown or white adipocytes. Activity measurements

KS-483 cells were used for activity measurements. From the RLU measured after exposure to single compounds it is clear that a composition according to the present invention lead to a significant increase in activity and transfer away from metastatic cells.

Experimental design cell culture

For the tests depicted in figures 7a-14 the following conditions were used:

Further experiments 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

Results

MTAECad cells were derived from the MMTV-Neu transgenic mouse, a genetic model of induced EMT. MTAECad cells were treated with the following compounds: Indomethacin (Indo), Pioglitazone (Pio) and Pentosan Polysulfate (PPS). Treatment and methods

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 MTAECad cells (treatment B). In our protocol MTAECad cells were stimulated with the following concentrations: PPS (5pg/ml), Indomethacin (50mM), and Pioglitazone (IOmM). Untreated cell lines were employed as negative controls. As read-outs of adipogenesis, several approaches were tested: 1. Oil red O staining by histochemistry

2. Nile red staining by immuno-fluorescence (IF)

3. RTqPCR (gene expression analysis)

Adiponectin (adipocyte marker)

- EpCam (marker for epithelial cells) Figures 15a-d show stimulation schemes; figures 15c and d according to the present invention.

Figure 16 shows Nile Red staining of the MTAECad treated cells. Induction of adipogenesis (indicated by the Nile 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: Control = untreated MTAECad cells.

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

Treatment C and D (fig. 15c-d)= PPS and indomethacin and PPS en Pioglitazone (the present combinations). 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 within MTAECad cell cultures there are cancer stem cells present that can be induced to terminally differentiate into (beige/brown) adipocytes. Interestingly, since cancer stem cells are the cells with high invasive and metastatic capacity, terminal differentiation into (beige/brown) adipocytes will stop metastasis.

Figure 16. Left Control (fig. 16a) untreated MTAECad cells that clearly show spindle shaped cells characteristics for epithelial cells that have undergone EMT. On the right (fig. 16b) MTAECad 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 MTAECad 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.

Fig. 17 shows Oil Red O staining of the MTAECad 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 although at a low level. Fig. 17a: control; fig. 17b indomethacin + pentosan polysulfate; fig. 17c pioglitazone; fig. 17d: pioglitazone + pentosan polysulfate.

EpCAM gene expression.

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

Steps performed when measuring gene expression using realtime PCR.

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.

Results

EpCam

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

MTAECad 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. 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.

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.

Cells treated as a positive control (treatment B) shown stimulated adiponectin gene expression. See figs.5 18a, b. Conclusion

Both Indomethacin and Pioglitazone in combination with Pentosan Polysulfate (PPS) are able to terminally differentiate cancer stem cells present in cultures of MTAECad cells breast cancer cells into adipocytes indicated by appearance in culture of islands of Nile Red positive lipid droplets containing adipocyte like cells that also express Adiponectin, measured by qPCR. Interestingly, apart from induction of islands of adipocytes the treated MTAECad cells showed a cuboidal epithelial phenotype this in contrast to the untreated control cells that have an elongated phenotype characteristic of EMT. This is either due to inhibition of EMT or induction of MET by the treatment. The cuboidal non-adipocyte cells that are visible are indicative of epithelial cells which was confirmed by the fact that we also found higher expression of the epithelial marker EpCAM compared to untreated control cells using qPCR.

Figures 7a, b, 8, 9a, b, 10, lla,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. 1 la,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. 1 lb 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.

EXAMPLE 2: COMBINATION OF PENTOSAN, INDOMETHACIN OR PIOGLITAZONE ON OVARIAN CANCER CELLS SKOV-3 ovarium carcinoma cells were seeded in petri dishes on day 0 in DMEM + Pen/Strep withlO % inactivated FCS, 24 hours after seeding cells were simulated with:

• Pentosan polysulfate: Compound A: 5 ug/ml

• Indomethacine: Compound B: 100 uM

• Pioglitazone: Compound C: 10 en 5 uM . · A + B and A + C

48 hours later the cells were stimulated again with the same compounds and combinations. 72 hours later the cells were fixed and stained with Hoechst 33342 to stain nuclei blue and with Nile red to stain lipid droplets red fluorescent. The following observations were made. In Figure 19 cultures of SKOV-3 with 5 microgram/ml Pentosan Polysulfate (PPS)

+ 10 micro-Molar Pioglitazone (Pio) around 70 islands were found containing Nile red fluorescent positive adipocytes. On the right hand site a clear island with red fluorescent adipocytes is visible (10 times magnification). On the left hand site at a magnification of 20 times adipocytes with blue Hoechst 33342 positive nuclei and Nile Red fluorescent lipid droplets are visible. SKOV-3 cells that did not form adipocytes have a cuboidal epithelial phenotype.

In Figure 20 cultures of SKOV-3 with 5 microgram/ml Pentosan Polysulfate (PPS) + 100 micro-Molar Indomethacin (Indo) around 10 islands were found containing Nile red fluorescent positive adipocytes. SKOV-3 cells that did not differentiate into adipocytes showed a cuboidal epithelial phenotype.

Conclusion: Similar to the MTAECad breast cancer cells, SKOV-3 ovarium cancer cells in culture in the presence of Pentosan poly sulfate + Pioglitazone or Pentosan poly sulfate + Indomethacin, both can induce islands of Nile red positive adipocytes most probably due to clonal expansion and differentiation of cancer stem cells. Both treated MTAECad breast cancer cells and SKOV3 ovarium cancer cells that do not differentiate into adipocytes cells will remain a cuboidal epithelial phenotype most probably by blocking EMT or induction of MET. Cancer stem cells are cells with a high invasive and metastatic capacity and the drug combinations mentioned above can induce already present cancer stem cells into innocent adipocytes and block EMT or induce MET inhibiting further cancer stem cell formation and thereby inhibit metastasis. EXAMPLE 3: COMBINATION OF PENTOSAN, INDOMETHACIN OR PIOGLITAZONE ON OVARIAN CANCER CELLS

The advantageous effect of the present combination of compounds in the treatment of ovarian cancer can be inferred from gene expression analysis performed in KS483 mouse mesenchymal progenitor cells treated with the compounds alone or in combination.

Notably, it was observed that the combination treatment was substantially more effective than any of the monotreatments alone in shifting the expression of genes involved in ovarian cancer progression and metastasis towards a less invasive and more differentiated phenotype.

For example, the expression of a tumour suppressor of a tumour suppressor gene LRRC4 that is known to interfere with E-cadherin-dependent cell invasion and metastasis in epithelial ovarian cancer (Zhao C, et al, Oncol. 2020 Feb 14; 10: 144. doi: 10.3389/fonc.2020.00144. PMID: 32117780; PMCID: PMC7033568), was increased 1-4 to 2-fold in response to the combination treatment as compared to the monotreatments.

Conversely, the expression levels of two oncogenes, CEMIP and Cadherin-17 (Cdhl7), known for their involvement in promoting of ovarian cancer development and progression, were consistently reduced following the treatments, which effect was substantially more pronounced for the combination treatments as compared to any of the treatments with a single compound alone.

CEMIP is a Wnt-related diagnostic marker which high expression correlates with a poor prognosis in patients, including patients suffering from ovarian cancer (Chen et ah, Biomed Pharmacother; 2022 Feb; 146: 112504. doi: 10.1016/j.biopha.2021.112504. Epub 2021 Dec 15.) and which is known to enhance ovarian cancer growth via the PI3K/AKT signaling pathway (Shen F, et ak, Biomed Pharmacother. 2019 Jun; 114: 108787. doi:

10.1016/j.biopha.2019.108787. Epub 2019 Mar 27 ). Cadherin-17 (Cdhl7) belongs to the 7D-cadherin superfamily and is involved in tumor invasion and metastasis. Importantly, its up-regulation and overexpression is associated with unfavourable prognosis and advanced stage epithelial ovarian cancer (Huang LP et al, Int J Gynecol Cancer; 2012 Sep;22(7): 1170-6. doi: 10.1097/IGC.0b013e318261d89c.). CEMIP expression levels decreased from 1.4-fold to 2.6-fold following the combination treatments, as compared to mono-treatments, while Cdhl7 expression levels decreased from to more than 2- to 3- fold.

The relative expression results for LRRC4, CEMIP, and Cdhl7 are summarised in the table below:

The results show that both, the specific downregulation of transcripts involved in ovarian cancer progression, like CEMIP and Cdhl7, as well as the specific upregulation of transcripts encoding for proteins that act as tumour suppressors in the context of ovarian cancer, like LRRC4, which are observed following a monotreatment with any of Pentosan (A), Indomethacin (B), or Pioglitazone (C) alone, are synergistically enhanced in combination.

5

Claims

1. Pharmaceutical composition for use in the treatment of patients suffering from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, wherein the composition comprises:

(a) pentosan polysulfate or a pharmaceutically acceptable salt thereof; and

(c) one or more pharmaceutically acceptable excipients.

2. Pharmaceutical composition for use according to claim 1, wherein the composition according to claim 1, wherein said patients suffer from or at risk of metastatic epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma.

3. Pharmaceutical composition for use according to any of the previous claims, wherein patients in need thereof are administered by means of said composition per day:

(a) 10 to 300 mg pentosan poly sulfate or a pharmaceutically acceptable salt thereof;

(b) 10 to 200 mg indomethacin and/or 10 to 45 mg pioglitazone or pharmaceutically acceptable salts thereof.

4. Pharmaceutical composition for use according to any of the previous claims, wherein the composition is formulated as an oral free dose or oral fixed dose combination, preferably as an oral fixed dose combination.

5. Pharmaceutical composition for use according to the previous claim, wherein the oral fixed dose combination is a solid oral dosage form, preferably a capsule or tablet.

6. Pharmaceutical composition for use according to any of the previous claims, wherein the molar ratio of pentosan poly sulfate to indomethacin or pioglitazone ranges from 0.01:1 to 1:0.1.

7. Pharmaceutical composition for use according to any of the previous claims, wherein the pharmaceutically acceptable excipient comprises a carrier, disintegrant or lubricant.

8. Pharmaceutical composition for use according to any of the previous claims, wherein the composition extends the progression free survival of the patient by at least 3 months.

9. Pharmaceutical composition for use according to any of the previous claims, wherein the treatment extends the progression free survival of said patient with at least 3 months.

10. Pentosan polysulfate for use in the treatment of patients suffering from epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma, comprising administering to said patients pentosan polysulfate or a pharmaceutically acceptable salt thereof and indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof.

11. Pentosan polysulfate for use according to the previous claim, wherein said patients suffer from or at risk of metastatic epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma.

12. Pentosan poly sulfate for use according to any of the previous claims, wherein a patient in need thereof is administered:

13. Pentosan polysulfate for use according to the previous claim, comprising administering to a patient in need thereof said dose once daily, twice daily or three times per day.

14. Pentosan poly sulfate for use according to any of the previous claims, wherein it is formulated as a pharmaceutical composition, more specifically as an oral free dose or oral fixed dose combination, preferably as an oral fixed dose combination.

15. Pharmaceutical composition comprising:

(a) pentosan polysulphate or a pharmaceutically acceptable salt thereof; and

(c) one or more pharmaceutically acceptable excipients.

16. Pharmaceutical preparation according to the previous claim, comprising:

17. Pharmaceutical composition according to any of the previous claims, wherein the composition is formulated as an oral free dose or oral fixed dose combination, preferably as an oral fixed dose combination.

18. Pharmaceutical composition according to the previous claim, wherein the oral fixed dose combination is a solid oral dosage form, preferably a capsule or tablet.

19. Pharmaceutical composition according to any of the previous claims, wherein the molar ratio of pentosan poly sulfate to indomethacin or pioglitazone ranges from 0.01:1 to 1:0.1.

20. Therapeutic combination comprising

21. Therapeutic combination according to the previous claim wherein the first unit dose comprises 10 to 300 mg pentosan polysulfate or a pharmaceutically acceptable salt thereof; and the second unit dose comprises 10 to 200 mg indomethacin and/or 10 to 45 mg pioglitazone or pharmaceutically acceptable salts thereof.

22. Therapeutic combination according to any of the previous claims, for use in the treatment of patients suffering from or at risk of metastatic epithelial ovarian cancer, metastatic fallopian tube carcinoma or metastatic primary peritoneal carcinoma.

23. Therapeutic combination according to any of the previous claims, wherein the unit dosages are formulated as solid oral dosage forms, preferably as capsules or tablets.

24. Method for treatment of patients suffering from ovarian cancer, in particular epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma wherein the treatment comprises administering to a patients in need thereof a therapeutically effective amount of:

(a) pentosan polysulfate or a pharmaceutically acceptable salt thereof; and

(b) indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof.

25. Method according to claim 24, wherein said patients suffer from or are at risk of metastatic ovarian cancer.

26. Method according to claim 24 or 25, wherein said patients are treated with a pharmaceutical composition or therapeutic combination according to any of the previous claims.

27. Use of (a) pentosan polysulfate or a pharmaceutically acceptable salt thereof and (b) indomethacin and/or pioglitazone or pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of ovarian cancer, in particular epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma.

28. Use according to claim 27, for the treatment of patients suffering from or at risk of metastatic ovarian cancer.