NL2013217B1 - New combination treatment for ovarian cancer. - Google Patents

Abstract

The invention relates to a combined preparation of xanthanodien and either or both of a taxane, preferably paclitaxel (Taxol®) and a platinum-containing anti-cancer drug, preferably cisplatin, for the simultaneous, separate or sequential use in therapy, preferably in the treatment of cancer and particularly ovarian cancer. The invention further comprises the use of such a combined preparation in the treatment of cancer, and a method of treating cancer with such a combined preparation.

Description

Title: New combination treatment for ovarian cancer

The invention relates to the field of cancer therapies, more particularly therapies for ovarian cancer and more specifically for combination therapies for use in the treatment of ovarian cancer.

Ovarian cancer, the silent killer, is the leading cause of death from gynecological malignancy. In 2012 it was diagnosed at an incidence of 1.3 cases per 10.000 inhabitants in the Western world, while in the USA 22,280 new cases were diagnosed. An estimated 15,500 women will die from the disease per year (the American Cancer Society). The cancer is mostly manifested at advanced stage (Stage III, IV), having spread beyond the ovaries to involve the peritoneal (abdominal) cavity. The serous papillary variants (>70%) of the ovarian epithelium form the largest subgroup (Scully, R.E. et al., 1998, in: Scully, R.E. et al. (eds.), Atlas of Tumor Pathology, 3rd Series, Washington DC, Armed Forces Institute of Pathology). With the current chemotherapy, with platinum derivates and/or taxol derivatives before or after surgery, most patients respond favorably. However relapses are common after this first line treatment. Relapses are most likely caused by a subset of cells that have the characteristics of cancer stem cells, self renewal properties and the ability to survive after completion of therapy (Clarke, M.F. et al., 2006, Cancer Res. 66:9339-9344; Guddati, A.K., 2012, Med. Oncol. 29:3400-3408). The general term “ovarian cancer” has been treated as a single disease entity with little stratification of histological or molecular subtypes, while a proportion of tumors may not arrive from ovarian tissue (Vaughan S. et al., 2011, Nat. Rev. Cancer 11:719-725). So far the prognosis for women with advanced disease remains poor and more efficacious approaches are badly needed. Taxol, paclitaxel, the first-fine chemotherapy agent, isolated from the bark of the western yew tree Taxus brevifolia, is an anti-microtubule agent stabilizing tubulin polymerization and causing cell arrest in the G2 and M phases of the cell cycle. Cisplatin is a chemotherapy drug that causes cross linking of DNA, which ultimately triggers apoptosis, which may be given in combination with taxol (Jekunen, A.P. et al., 1994, Br. J. Cancer 69:299-306).

Anew anti-cancer agent, eremophila-1(10)-ll(1230-dien-12,86-olide, (EPD or Xanthanodien) (Tanaka, N. et al, 1976, 24:1419-1421) of the eremophilanolide structure subtype has been isolated from Calomeria amaranthoides of the family Asteraceae (Compositae). This agent, a sesquiterpene lactone (SL), has been found to exhibit potent cytotoxic effects towards ovarian cancer cells in vitro and in vivo (van Haaften, C. et al., 2011, J. Exp. Chn. Cancer Res. 30:29-33; Duke, C.C. et al., 2011, Green Sust. Chem. 1:123-127, US 2013/0123352) and towards other cancers. SLs have been reported as being anti-cancer as well as anti-inflammatory agents and the majority of SLs are derived from the family Asteraceae. SLs are colorless and natural bitter compounds of the subfamily of terpenoids, with lipophilic character. This lipophilicity can facilitate penetration through the cell membrane, causing increased SL cytotoxicity in vitro. To date several SLs are studied in clinical trials (Ghantous A., et al., 2010, Drug Disc.

Today 15:668-678; Kreuger, M. et al., 2012, Anticancer Drugs 23:883-896).

Although all of the above-mentioned compounds have an effect on cancer cells, such as ovarian cancer cells, there is still need for new, more effective therapies.

The present inventor found a new combination therapy of xanthanodien (EPD) and taxol or cisplatin, or a combination of all three compounds for the treatment of cancers, more particularly ovarian cancers.

In the context of this specification, a "condition associated with hyperproliferative cellular division" refers to any clinical condition characterised by or otherwise involving an increased rate of cell division relative to a normal reference rate. Conditions associated with hyperproliferative cellular division include, but are not limited to: myeloproliferative syndromes such as Langerhans cell histiocytosis, mastocytosis, mixed myeloproliferative and myelodysplastic conditions, dermal proliferative conditions such as psoriasis, non-bullous congenital ichthyosiform erythroderma. Conditions associated with hyperproliferative cellular division also include cancer, whether benign or malignant, including haematopoietic malignant cancers. In particular, and in preferred embodiments, the term refers to ovarian cancer.

In the context of this specification, the terms "treatment" and "treating" refer to any and all uses which remedy a condition or disease or symptoms thereof, prevent the establishment of a condition or disease or symptoms thereof, or otherwise prevent or hinder or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever. The term “chemotherapy” means the treatment of disease by means of chemicals that selectively destroy cancerous tissue.

In the context of this specification, the term "therapeutically effective amount" includes within its meaning a non-toxic amount of each of the indicated compounds, alone or in combination, sufficient to provide the desired therapeutic effect. The exact amount will vary from subject to subject depending on the age of the subject, their general health, the severity of the disorder being treated and the mode of administration. It is therefore not possible to specify an exact "therapeutically effective amount", however one skilled in the art would be capable of determining a "therapeutically effective amount" by routine trial and experimentation.

In the context of this specification, the term "cytotoxic amount" is defined to mean an amount of one or more of the therapeutic compounds or combinations of compounds of the present invention that is toxic to the target cell once said compound or composition has associated with the cell. Generally, toxicity is indicated by statistically significant loss in cell viability.

The term "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer or other condition characterized by a hyperproliferation of cells.

In the context of this specification, "pharmaceutically acceptable salts" include, but are not limited to, those formed from: acetic, ascorbic, aspartic, benzoic, benzenesulfonic, citric, cinnamic, ethanesulfonic, fumaric, glutamic, glutaric, gluconic, hydrochloric, hydrobromic, lactic, maleic, malic, methanesulfonic, naphthoic, hydroxynaphthoic, naphthalenesulfonic, naphthalenedisulfonic, naphthaleneacrylic, oleic, oxalic, oxaloacetic, phosphoric, pyruvic, p-toluenesulfonic, tartaric, trifluoroacetic, triphenylacetic, tricarb ally lie, salicylic, sulfuric, sufamic, sulfanilic and succinic acid.

The terms "hyperproliferation" and "hyperproliferating" refer to the abnormal growth of a cell type, which can be cancerous or benign.

Generally, hyperproliferating cells exhibit a rate of cell division that is at least about ten percent greater than the rate of cell division exhibited by normal cells of that cell type.

The anti-cancer activity of Calomeria amaranthoides was previously reported by the present inventor in WO 2006/067603. The chemical constituents composition of aerial parts of C. amaranthoides have been examined once before by Zdero et al., 1991 Phytochemistry, Vol. 30,

No.8, pp 2643-2650. Further, the specific effects of eremophilanolide sesquiterpenes such as EPD against ovarian cancer was disclosed by the present inventor in US 2013/123352. EPD (synonym: xanthanodien) has been shown to completely kill ovarian cancer cells under in vitro conditions at concentrations below 10 pg/mL, such as 5 pg/mL. In addition, substantial killing of cancer cells is already observed at concentrations as low as of 1 pg/mL. Also a diasteroisomer of EPD, napthofuranone has been tested for anticancer effects (Cancer Chemother. Rep. 3(2), 1972).

One of the most advantageous characteristics of EPD is that it selectively kills cancer cells. This is an enormous advantage because standard chemotherapeutics for anti-cancer treatment, such as cisplatin and docetaxol both completely kill cancer cells and normal cells in parallel experiments conducted by the inventor.

Xanthanodien may be extracted from selected plants, for example Calomeria amaranthiodes, also known as: Humea elegans. Such extraction may be performed by steam destination as described in US 2013/123352. Alternatively, xanthanodien may be prepared from known starting materials according to literature procedures. See for example Zoretic et al., J. Org. Chem. (1982), 47, 1327, Tada et al. J. Chem. Soc. Perkin Trans. 1 (1993), 239 and W02006/067603.

Xanthanodien now is shown to act synergistically with other cytotoxic drugs, more particularly taxanes, hke paditaxel (Taxol®), platinum-containing anti-cancer drugs, hke cisplatin, carboplatin and oxaliplatin; and the combination of taxanes and platinum-containing anticancer drugs. In a preferred embodiment the taxane is pachtaxel and the platinum-containing anti-cancer drug is cisplatin. Accordingly, the present invention discloses three compositions: one composition of EPD and a taxane, preferably paclitaxel, the second composition of EPD and a platinum-containing anti-cancer drug, preferably cisplatin, and thirdly a combination of a taxane, a platinum-containing anti-cancer drug and EPD. Said compositions are useful in the treatment of cancer. The cancer may be selected from the group consisting of: gastrointestinal tumours, cancer of the liver and biliary tract, pancreatic cancer, prostate cancer, testicular cancer, blood cancer, lung cancer, skin cancer (for example melanoma), breast cancer, non-melanoma skin cancer (for example basal cell carcinoma and squamous cell carcinoma), ovarian cancer, uterine cancer, cervical cancer, cancer of the head and neck, bladder cancer, sarcomas and osteosarcomas,

Kaposi sarcoma, AIDS-related Kaposi sarcoma and renal carcinoma. In a preferred embodiment, the cancer is ovarian cancer.

Paclitaxel is known for its development of drug resistance; it is disrupting normal mitotic spindle formation and is arresting cell growth in the M-phase of the cell cycle (17). Earlier studies concluded that both dsplatin and paclitaxel arrest the cell cycle at Gi or G2+M. To measure the induction of apoptosis induced by cisplatin and paclitaxel in four cell lines, including OVCAR-3 and SK-OV-3, Gi arrest occurred more readily in OVCAR-3 and SK-OV-3 cells than in the other 2 cell lines (21). It has now been found that EPD in connection with a taxane and/or a platinum-containing anti-cancer compound has excellent properties. The effects of EPD combined with cisplatin or pachtaxel showed remarkable changes in the distribution of cells in the G2 +M-phase compared to the effects of cisplatin combined with pachtaxel.

The combined preparations of the present invention comprising EPD are useful as therapeutic agent in the treatment or prevention of conditions associated with hyperproliferative cellular division, such as cancer. The individual compounds making up the combined preparation of the present invention may together or separately suitably be administered to a subject (for example a human) in the form of pharmaceutical compositions. Pharmaceutical compositions include those suitable for enteral (including oral), parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intra-articular), inhalation (including oral or nasal inhalation optionally using metered dose pressurized aerosols, nebulizers or insufflators), rectal and topical (including dermal, transdermal, buccal, sublingual and intraocular) administration.

Taxanes (such as pachtaxel) and platinum-containing anti-cancer drugs (such as cisplatin) may suitably be administered in an intravenous infusion. If both a platinum-containing anti-cancer drug and a taxane are present in a combined preparation according to one of the embodiments of the present invention, preferably taxane administration precedes administration of the platinum-containing anti-cancer drug as investigated e.g. by Liebmann, J.E. et al., 1994, Oncol. Res. 6:25-31) or these compounds may be administered in an alternating scheme (Rowinsky, E.K. et al., 1991, J. Cbn. Oncol. 9:1692-1703). Alternatively, a platinum-containing anticancer drug may be administered intraperitonally following intravenous appbcation of a taxane (Markman, M. et al., 2001, J. Cbn. Oncol. 19:1001-1007).

The dosage of the individual compounds in the combined preparation in accordance with the present invention may be varied, although the amount of the active ingredients shall be such that a suitable dosage form is obtained. Hence, the selected dosage and the selected dosage form shall depend on the desired therapeutic effect, the route of administration and the duration of the treatment. Suitable dosage ranges for the combination are from the maximal tolerated dose for the single agent to lower doses, e.g. to one tenth of the maximal tolerated dose.

For the taxane compound and the platinum-containing anti-cancer compound the doses and administration routes that are normally used in cancer therapy may be used. A skilled practitioner will be able to determine suitable dosage schemes for treatment, depending on the general status of the subject to be treated, the severity of the disease and other factors that may influence efficacy of the treatment.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows the synergistic effects of combination treatment with EPD on ovarian cancer cell fines and normal fibroblasts. Relative viability is shown for each single compound. E= EPD, C=cisplatin, T=taxol. Strong synergism was observed between EPD and taxol for SK-OV-3 and JC, whereas for JC-pl the combination of EPD and cisplatin was found to be strong synergistic.

Figure 2 shows the cell cycle effects of EPD, taxol and cisplatin on the ovarian cancer cell lines, SK-OV-3 (Fig. 2A) and JC-pl (Fig. 2B), after 72 hrs, as measured by flow cytometry. Panel A: untreated cells; Panel B: EPD; Panel C: Taxol; Panel D: EPD+Taxol; Panel E: EPD+Cisplatin; Panel F: EPD+Taxol+Cisplatin. Figures are flow cytograms of cell populations stained with priopidium iodide.

Figure 3 shows the experiment wherein SK-OV-3 cells were untreated or treated with increasing concentrations of EPD (see Example below). A: 0 pg/mL EPD, G2M phase = 9.2%, B: 3 pg/mL EPD, G2M phase = 26.7%, C: 6 pg/mL EPD, G2M phase = 36.7%, D 9 pg/mL EPD, G2M phase = 54.9%. Histograms were generated with ModFit LT 4.0 using autolinearity, remotely controlled with WinList 7.1 (Verity Software House, Topsham, Maine).

EXAMPLE

Materials and methods

Reagents: EPD has been provided by the department of Pharmacy, Sydney University, NSW, Australia. In short: fresh leaves of a plant endemic to Australia, Calomeria amaranthoides, were steam distillated to get a high recovery of sesquiterpene-rich oil. The oil was fractionated by short-column vacuum chromatography to establish a 95% purity of EPD (10). Taxol (paclitaxel) and cisplatin were obtained from Sigma-Aldrich, USA.

Cell lines and Cell Cultures

Cell hnes (all of the serous subgroup) used in the assays were JC and JC- pi, earlier established (13) and OVCAR-3 and SK-OV-3 from the American Type Culture Collection (ATCC). Normal human skin fibroblasts, were provided by the department of Dermatology, Leiden University, The Netherlands.

The cell lines were grown in RPMI-1640, supplemented with 2mM L Glutamine (Gibco, Invitrogen, UK); 10% heat inactivated (H.I) fetal calf serum (FCS) (Sigma); and penicillin (50 units/mL) and streptomycin (50 pg/mL) (Invitrogen, UK). Normal skin fibroblasts were grown in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, UK), also supplemented with L-glutamine and 10% FCS. The cultures were maintained in a humidified atmosphere of 9% CO2 at 37°C.

The four human ovarian cancer cell lines were tested for their identity profile (ID), using a Cell ID ™ kit from Promega. SK-OV-3 and OVCAR-3 were compared to their known profile of the ATCC and JC and JC-pl were cross referenced.

In vitro cytotoxicity tests

In vitro cytotoxicity tests were performed using a non-fluorescent substrate, Presto Blue (Bio Source, Invitrogen, UK). Cells were seeded at approximately 16.000 cells/cm2 in 24-wells plates ( Costar, USA) in 1 mL medium/well. After 24 hrs, exponential growing cell cultures were treated in triplicate with the different, compounds EPD, taxol and cisplatin in 2 mL/ well fresh medium. Control (Bl) cells were untreated. The cells were incubated with the drugs for 72 hrs to ensure two doubling times of the cells. Cell viability was measured to determine the best doses for the assays. Different concentrations for each agent were used for each ovarian cancer cell line and for the normal fibroblasts. EPD was re-dissolved in dimethyl sulfoxide (DMSO), with final concentration of 0.02% DMSO. Combination of drugs used were: EPD + cisplatin, EPD + taxol; cisplatin + taxol and EPD + ciplatin + taxol. Final concentrations are given in Table I. After 72 hrs incubation, Presto Blue (10%) was added to the cultures. After 2-3 hrs of incubation the percent of cell viability was measured with a multi plate reader, Victor 3V (Perkin Elmer) by transferring 100 μΐ of the medium in a 96 well plate (Greiner Bio-one). Wave lengths used were 570 and 600 nm, with 570/8 and 610/10 filters. Viability was calculated relative to untreated cells.

Table 1 Drug concentrations used for each of the four ovarian cancer cell lines and normal fibroblasts

Statistical analysis

Statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, 111) to calculate means with error standard errors (SE).

Flow cytometry

The four ovarian cancer cell fines and normal fibroblasts were treated using the same conditions as described for the in vitro assays . Cells were seeded in 25 cm2 flasks and after 24 hrs drug treatment was initiated. After 72 hrs incubation with drugs, cells were prepared for flow cytometry. Seeding density was kept equal to the density used in the viability experiment (ca. 16.000/cm2), compensating for the difference in surface area. In short: cells were harvested using trypsin/EDTA, counted and transferred to FACS tubes (BD Falcon, Cat no 352052), and centrifuged (500g) for 5 min in 4° C. Supernatant was removed and fifty μΐ PBS was added to the cell pellet followed by 450 μΐ 100% cold methanol added drop-wise under constant swirling. Next, the cell suspensions were put for 20 min in the freezer. Then 500 μΐ cold PBS/Tw 0.05% was added and the cells were centrifuged (500g) for 5 min at 4° C. Supernatant was decanted and 1 mL PBA 1.0%/Tw 0.05% (PBA/Tw) was added to the pellets. After centrifuging, supernatant was decanted and 500 μΐ staining solution (PBA/Tw containing 0.1 % RNase (Sigma-Aldrich) and 100 μΜ propidium iodide (PI) (Sigma-Aldrich)) was added and the pellet, vortexed and incubated in a 37 0 C water bath for 30 min. Cells were kept at 4°C until flow cytometric analysis. In order to standardize the data, untreated cells of the different cell lines were used to calibrate the Gl position of untreated and treated cells.

For flow cytometric analysis a LSRII (BD Bio Sciences) was used with a 488 nm laser for excitation. Fluorescence was collected using a 610/20 nm band pass filter. Pulse-processing was used to collect 50.000 single cell events. During data storage, all events were included. Data was analyzed using WinList 7.1 (Verity Software House, Topsham, ME).

Results:

In vitro cytotoxicity tests

The percent relative cell viability of the four cell fines and normal fibroblasts, treated with EPD, cisplatin and taxol are shown in Figure 1. Both, the effects of single drugs, as well as the combination of the drugs were determined. All experiments were performed in triplicate for 72 hrs.

Treatment with EPD, taxol or cisplatin alone resulted in reduced viability in the cell fines, ranging between 41 to 93% of viable cells. Normal skin fibroblasts treated with taxol were affected mostly by taxol after 72 hrs. When combining EPD with cisplatin and or taxol, synergistic effects were found. In both SK-OV-3 and JC synergistic effects were found in the combination of EPD and taxol. Treatment of EPD and taxol in SK-OV-3 resulted in cell viability of 93% and 60% respectively. The combined effects were significantly lower than expected for SK-OV-3 with a 25% viability (p < 0.05). For JC the combined effect resulted in a viability of 26% (p < 0.05). This synergistic effect was not observed in combination with cisplatin.

In JC-pl a strong synergistic effect was detected for the combination EPD and cisplatin. Viability of the JC-pl cells for this combination treatment was 9%, where the additive effect of EPD and cisplatin was expected to be 20% (p < 0.05). Taxol with EPD did not have this synergistic effect. OVCAR-3, as well as the normal fibroblasts did not show any synergistic effects with the different combinations.

Cell ID of SK-OV-3 and OVCAR-3 matched the profiles of the ATCC, while JC and JC-pl are new and unique cell lines, not matching any of the known ID’s, Table 2.

Cell cycle analysis

The effects of EPD on the cell cycle in the four ovarian cancer cell fines were studied. Initially, a titration experiment with SKOV-3 was performed for 72 hrs, with a range of concentrations of EPD varying from 2-9 gg/mL. Untreated SK-OV-3 cells showed to be bi-modal in culture with a minor Gi population approximately at (relative) mean channel number (MCN) 501 and a major Gi population approximately at MCN 938 (Figure 2A, Panel A). Already at 3 pg/mL EPD treatment a clear accumulation of cells in the G2/M phase could be noted (Figure 2A, Panel B). This was dose-dependent and G2/M cell numbers increased at higher concentrations (Figure 3).

Furthermore, to assess also the effects of combinations of compounds on the cell fines, SK-OV-3 was chosen for the combination EPD and taxol and JC-pl was chosen for the combination EPD and cisplatin. Results are shown in Figure 2A and Figure 2B, respectively. The cell fines were treated with the same concentrations as given in Table 1. Also cells were counted before flow cytometry. After 72 hrs, 3.6 x 105 cells were counted with EPD treatment and 0.7 x 105 for the combination EPD+taxol, while EPD+cisplatin had 1.0 x 105 cells. Combination of EPD+taxol+cisplatin gave 0.5 x 105 cells. In cell fine SK-OV-3 a small peak (MCN = 1439) was noted in the S-phase (Figure 2A, Panel A), most likely caused by aggregates composed of a Gi cell of the minor population and a Gi cell of the major population. Arrest of cells occurred with treatment of EPD+taxol in the G2+M phase. Cell counts of cell line JC-pl with EPD after 72 hrs were: 0.7 x 105, for EPD+cisplatin: 0.3 x 105, for EPD+cisplatin+ taxol: 0.3 x 105. Arrest of cells in cell line JC-pl was noted after EPD treatment in the Gl. The combination EPD+ cisplatin showed arrest in G2M, as did the combination EPD+ cisplatin+taxol..

Table 2. Cell ID of the cell lines: JC and JC-pl

Sample THOI D21S11 D5S818 D13S317 D7S820 D16S539 CSF1PO AMEL vWA TPOX JC 6,9.3 28,31 11 8,14 10 11 11 X 17,19 8,11 JC-PI 6,9.3 28,31 11 8,14 10 11 11 X 17,19 8,11

Malignancy arising from the ovarian epithelium comprises the vast majority of cases of ovarian cancer (with the serous subtype as most common) besides sex cord and germ cell varieties. Relapses of ovarian cancer in patients with advanced epithelial ovarian cancer is still the main cause of ineffective chemotherapy and most likely due to remaining cancer stem cells, resistant to cisplatin and paclitaxel (14). In vitro studies to evaluate the cytotoxicity of taxanes and platinum agents have been widely conducted in the last decades and these treatments have demonstrated clinically equal efficacy (15,16).

In the present study it was focused on four ovarian cancer cell lines all of the serous subtype. First the three different compounds cisplatin, paclitaxel and EPD were tested for their IC50 values for each cell line; then combinations of cisplatin with EPD, paclitaxel with EPD or cisplatin, paclitaxel and EPD were studied.

The sesquiterpene lactone, EPD, has given new evidence to be of great interest as an anti-cancer agent. Not only has EPD proven from previous work to be cytotoxic (see US 2013/0123352)), the compound has now also been shown to have synergistic interactions with paclitaxel (cell lines SK-OV-3 and JC) and cisplatin (cell line JC-pl) (Figure 3). Paclitaxel is known for its development of drug resistance; it is disrupting normal mitotic spindle formation and is arresting cell growth in the M-phase of the cell cycle (17). Paclitaxel in combination with EPD however showed to improve cell death in the IC50 assays. The cell line JC-pl was established from the same patient as the cell hne JC, after the patient had become resistant to drug treatment (13). EPD might thus play an important role also in combination with cisplatin to resistant cancer cells (Figure 3). The cell line OVCAR.3, known for its isolation and characterization of stem-like or cancer-initiating cells and resistance to chemotherapy (18), did not show synergistic effects with any of the three compounds. It is known that many SLs are apoptosis inducers and although SL-induced apoptosis is not fully understood, it is believed that the a-methylene-6-lactone structure is essential for their apoptotic activity (19). Several experiments, using the Nicoletti method (20) were also performed with JC and JC-pl to measure apoptosis (data not shown). Earlier studies concluded that both cisplatin and paclitaxel arrest the cell cycle at Gi or G2+M. To measure the induction of apoptosis induced by cisplatin and paclitaxel in four cell lines, including OVCAR-3 and SK-OV-3, Gi arrest occurred more readily in OVCAR-3 and SK-OV-3 cells than in the other 2 cell lines (21). EPD has been studied for the first time for cell cycle analysis . Cells were plated in 252 flasks in the same concentration as in the 24 wells and were treated with the same doses.

In the experiment (SK-OV-3) with a range of concentrations of EPD, it was noted that with increase of EPD > 3gg/mL the G0/G1 diminished and the proportion of cells in the G2 +M- phase increased. The effects of EPD combined with cisplatin or paclitaxel showed remarkable changes in the distribution of cells in the G2 +M-phase compared to the effects of cisplatin combined with paclitaxel (Figure 3). Several of the SLs have demonstrated that their anti-cancer properties are also of interest for their selective targeting of cancer cells; in cancer clinical trials the SLs have targeted tumor and cancer stem cells, while sparing normal cells (22,23). In an in vivo experiment the effects of EPD as well as cisplatin on OVCAR-3 cells caused reduction of the abdomen size of mice. However, the mice treated with EPD could be kept for a much longer time than the mice treated with cisplatin (9). In the IC50 assays with normal skin fibroblasts, EPD at 3pg/mL did show some effects while in particular paclitaxel at 0.375 gg/mL had strong cytotoxic effects and less effects were shown with cisplatin at 2.5 gg/mL (Figure 3) . In the flow cytometry analyses of normal skin fibroblasts an increase of G2 + M in the cell cycle was noticed by treatment with 3 gg/mL EPD.

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Claims (10)

A combination preparation of xanthanodine and one or both of a taxane and a platinum-containing anti-cancer drug for the simultaneous, separate or sequential use in therapy. A combination preparation for use in therapy according to claim 1, wherein the preparation comprises xanthanodien and a taxane, preferably wherein said taxane is lease axel (Taxol®). The combination composition for use in therapy according to claim 1, wherein the composition comprises xanthanodine and a platinum-containing anti-cancer drug, preferably wherein said platinum-containing anti-cancer drug is cisplatin. Combination preparation for use in therapy according to any of the preceding claims, wherein the preparation comprises xanthanodien and a platinum-containing anti-cancer drug, preferably cisplatin, and a taxane, preferably paclitaxel (Taxol®). Combination preparation for use in therapy according to one of the preceding claims, wherein the therapy is the treatment of cancer, preferably treatment of ovarian cancer. The combination preparation for use in therapy according to claim 5, wherein the cancer shows resistance to chemotherapy and / or radiation. The combination preparation for use in therapy according to any of the preceding claims, wherein the platinum-containing anti-cancer drug is administered intravenously or intraperitoneally. A combination preparation for use in therapy according to any one of the preceding claims, wherein the taxane is administered intravenously. The combination preparation for use in therapy according to any of the preceding claims, wherein the xanthanodine is administered orally. A method for the treatment of cancer, preferably ovarian cancer, by the simultaneous, separate or sequential administration of xanthanodien and one or both of a taxane and a platinum-containing anti-cancer drug for the simultaneous, separate or sequential use in therapy.