FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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, 3 ^rd Series, Washington DC, Armed Forces Institute of Pathology). With the current chemotherapy, with platinum derivates and/or paclitaxel 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®, or paclitaxel, the first-line 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 M phase 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 paclitaxel (Jekunen, A.P. et al., 1994, Br. J. Cancer 69:299-306).

A new anti-cancer agent, eremophila- l(10)- l l(13)-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. Clin. 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 hpophihc character. This lipophihcity 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.

SUMMARY OF THE INVENTION

The present inventor found a new combination therapy of xanthanodien (EPD) and paclitaxel or cisplatin, or a combination of all three compounds for the treatment of cancers, more particularly ovarian cancers. In a first aspect, the present invention provides a combined preparation of xanthanodien and either or both of a taxane and a platinum- containing anti-cancer drug for the simultaneous, separate or sequential use in therapy.

In a preferred embodiment of this combined preparation for use in therapy according to the present invention, the preparation comprises xanthanodien and a taxane, preferably said taxane is paclitaxel (Taxol®).

In another preferred embodiment of this combined preparation for use in therapy according to the present invention, the preparation comprises xanthanodien and a platinum-containing anti-cancer drug, preferably said a platinum -containing anti-cancer drug is cisplatin.

In yet another preferred embodiment of this combined preparation, the preparation comprises xanthanodien, a platinum-containing anti-cancer drug, preferably cisplatin, and a taxane, preferably paclitaxel (Taxol®).

In still another preferred embodiment of this combined preparation for use in therapy according to the present invention, the therapy is cancer treatment, preferably treatment of ovarian cancer. Preferably, the cancer exhibits resistance to chemotherapy and/or radiation.

In still another preferred embodiment of the combined preparation according to the invention, the platinum-containing anti-cancer drug is administered intravenously or intraperitoneally.

In still another preferred embodiment of the combined preparation according to the invention, the taxane is administered intravenously.

In yet another preferred embodiment of the combined preparation according to the invention, the xanthanodien is administered orally.

In another aspect, the present invention provides a method for treating cancer, preferably ovarian cancer, by simultaneous, separate or sequential administration of xanthanodien and either or both of a taxane and a platinum-containing anti-cancer drug. In one embodiment, said method comprises the step of administering to a subject suffering from cancer, preferably ovarian cancer, a therapeutically effective amount of xanthanodien, preferably via the oral route of administration, and further comprising the step of simultaneously, separately or sequentially

administering to said subject a therapeutically effective amount of a taxane, preferably paclitaxel, preferably via the intravenous route of

administration, and/or a therapeutically effective amount of a platinum - containing anti-cancer drug, preferably cisplatin, preferably via the intravenous or intraperitoneal route of administration. DESCRIPTION OF THE DRAWINGS

Figure 1 shows the synergistic effects of combination treatment with EPD on ovarian cancer cell lines and normal fibroblasts. Relative viability is shown for each single compound. E= EPD, C=cisplatin,

T=paclitaxel. Strong synergism was observed between EPD and Paclitaxel for SK-OV-3 and JC, whereas for JC-pl the combination of EPD and cisplatin was found to be strongly synergistic. Concentrations are given in Table 1.

Figure 2 shows the synergistic drug combinations in JC and JC-pl. A: Comparison of expected and observed relative viability of JC after combination treatment with EPD and paclitaxel. B: Lower doses of EPD and cisplatin were found to be antagonistic for JC-pl, whereas the higher doses showed synergistic effects.

Figure 3 shows the synergistic drug combinations induce apoptosis. A: EPD and paclitaxel in single treatments induced apoptosis. Combination treatment showed an even higher caspase 3 activity. B: Cisplatin treatment alone did not induce apoptosis. Combination treatment of EPD and cisplatin showed a strong induction of caspase 3 activity. Inhibitor: Ac-DEVD-CHO; RFU: Relative Fluorescence Units.

Figure 4 shows the cell cycle effects of EPD, paclitaxel and cisplatin on the ovarian cancer cell lines SK-OV-3 (Fig. 4A) and JC-pl (Fig. 4B), after 72 hrs, as measured by flow cytometry. Panel A: untreated cells; Panel B: EPD; Panel C: paclitaxel; Panel D: EPD + paclitaxel; Panel E: EPD + cisplatin; Panel F: EPD + paclitaxel + cisplatin. Figures are flow

cytograms of cell populations stained with priopidium iodide.

Figure 5 shows the experiment wherein SK-OV-3 cells were untreated or treated with increasing concentrations of EPD (see Example below). A: 0 μg/mL EPD, G2/M phase = 9.2%; B: 2 μg/mL EPD; C: 3 μg/mL EPD, G2/M phase = 26.7%; D: 6 μg/mL EPD, G2/M phase = 36.7%; E: 9 μg/mL EPD, G2/M phase = 54.9%. Histograms were generated with ModFit LT 4.0 using autolinearity, remotely controlled with WinList 7.1 (Verity Software House, Topsham, Maine).

Figure 6 shows the sell cycle effects of EPD, paclitaxel and cisplatin on normal fibroblasts. A: untreated cells; B: EPD; C: pachtaxel; D: cisplatin; E: EPD + pachtaxel; F: EPD + cisplatin; G: paclitaxel + cisplatin; H: EPD + paclitaxel + cisplatin.

DETAILED DESCRIPTION OF THE INVENTION

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 amaranthoides, 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 WO2006/067603.

Xanthanodien now is shown to act synergistically with other cytotoxic drugs, more particularly taxanes, like paclitaxel (Taxol®), platinum -containing anti-cancer drugs, like cisplatin, carboplatin and oxaliplatin; and the combination of taxanes and platinum -containing anticancer drugs. In a preferred embodiment the taxane is paclitaxel 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 hver 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 (Wang X, et al., Cancer Cell Internat 2013; 13: 77-85). 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 OVCAE-3 and SK-OV-3 cells than in the other 2 ceU lines (Gibb RK, et al., Gynecol Oncol 1997; 65: 13-22). 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 paclitaxel showed remarkable changes in the distribution of cells in the G2 M -phase compared to the effects of cisplatin combined with paclitaxel.

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 paclitaxel) 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. Clin. Oncol. 9: 1692-1703). Alternatively, a platinum -containing anticancer drug may be administered intraperitonally following intravenous application of a taxane (Markman, M. et al., 2001, J. Clin. Oncol. 19: 1001- 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.

Suitable effective dosages for EPD in the use of killing cancer cells (cytotoxic amount) is in the range of 0.01-100 μg/ml. Therapeutically effective dosages of EPD for use in treating cancer are about 0.000 lmg to about lOOOmg per kg body weight of the treated subject per 24 hours, preferably 0.01-100 mg/kg body weight, most preferably 0.1-10 mg/kg body weight, both in single drug preparations as well as in synergistic

combinations according to this invention.

For the taxane compound paclitaxel and the platinum -containing anti-cancer compound cisplatin 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.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate aspects and preferred embodiments thereof, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

The invention will now be illustrated by the following example, which is provided by way of illustration and not of limitation and it will be understood that many variations in the methods described and the amounts indicated can be made without departing from the spirit of the invention and the scope of the appended claims.

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 (Duke CC, et al., Green and Sustainable Chem 2011; 1: 123-127). Paclitaxel (Taxol®) and cisplatin were obtained from Sigma-Aldrich, USA. Cell lines and Cell Cultures

Cell lines (all of the serous subgroup) used in the assays were JC and JC- pi (Van Haaften-Day C, et al., Cancer Res 1983; 43: 3725-3731) 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, LUMC, The Netherlands.

The cell lines were grown in RPMI-1640, supplemented with 2mM L Glutamine (Gibco, Invitrogen, UK), 10% heat inactivated fetal calf serum (FCS) (Sigma), penicillin (50 units/mL) and streptomycin (50 μg/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 an incubator with humidified atmosphere at 37°C with 9% CO2. 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/cm ² 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, paclitaxel 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. Combinations of drugs used were: EPD + cisplatin, EPD + paclitaxel; cisplatin + paclitaxel and EPD + cisplatin + paclitaxel. 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 610 nm, with 570/8 and 610/10 filters. Viability was calculated relative to untreated cells. Synergistic drug interactions were evaluated using the simplified version of the Bliss independence model (Wong M, et al., Mol Cancer Ther. 2012; 11: 1026-35).

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

Cone. SK-OV-3 JC JC-pl OVCAR-3 Fibroblasts μg/mL

EPD 3.0 3.0 5.0 1.25 3.0 cisplatin 2.5 2.0 2.75 0.75 2.5 paclitaxel 0.02 0.375 0.225 0.004 0.375 Statistical analysis

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

Apoptosis

Cells were seeded as described for the in vitro cytotoxicity tests. 24 hrs after seeding the following drug concentrations were added for the cell line JC: 3.15 pg/mL EPD and 0.52 pg/mL paclitaxel, for the cell line JC-pl: 6.5 pg/mL EPD and 3.39 pg/mL cisplatin. Apoptosis was measured after 24 hrs of drug treatment using the Caspase 3 Assay kit (Sigma-Aldrich, Cat.nr CASP3F) according to the manufacturer's specifications.

Flow cytometry

The four ovarian cancer cell lines and normal fibroblasts were treated using the same conditions as described for the in vitro cytotoxicity assays. Cells were seeded in 25 cm ² 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/cm ² ), 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 (500 xg) for 5 min in 4° C. Supernatant was removed and 50 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 (500 xg) 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 pellets were 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 Gi 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

To verify cell line identity, short tandem repeat (STR) profiling was performed on the ovarian cancer cell lines. 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, both from the same patient, JC-pl derived from a pleural effusion after cisplatin treatment. JC and JC-pl showed identical STR profiles, not matching any of the known cell lines from the ATCC, Table 2. Table 2. STR profiles of the cell lines: JC and JC-pl

Marker

Sample 1 2 3 4 5 6 7 8 9 10

JC 6,9.3 28,31 11 8, 14 10 11 11 X 17, 19 8, 11

JC-P1 6,9.3 28,31 11 8, 14 10 11 11 X 17, 19 8, 11

STR Marker identification: 1: THOI; 2: D21S11; 3: D5S818; 4: D13S317; 5:

D7S820; 6: D16S539; 7: CSF1PO; 8: AMEL; 9: vWA; 10: TPOX. Based on dose response curves the concentrations of EPD, cisplatin and paclitaxel were selected for the four cell lines and the normal fibroblasts as indicated in Table 1, above.

The relative cell viability of the four cell lines and normal fibroblasts, treated with EPD, cisplatin and paclitaxel are shown in Figure 1. Treatment with EPD, cisplatin or paclitaxel alone resulted in reduced viability in the cell lines, ranging between 41 to 93% of viable cells. Normal skin fibroblasts were affected mostly by paclitaxel after 72 hrs. The combination treatments of EPD with cisplatin and paclitaxel showed increased activity in the different cell lines. To evaluate whether these combination treatments were synergistic drug interactions, a simplified version of the Bliss independence model was applied (Wong M, et al., Mol Cancer Ther. 2012; 11: 1026-35). When two drugs exert their effects independent to one another, the resulting relative viability after drug treatment is expected to equal the product of the relative viability after treatment with the individual drugs. When the observed viability is higher than the expected based on the individual drug effects, the compounds inhibit each others effects; antagonism. However, when observed viability after combination treatment is lower than the expected viabihty, then this is an indication of a synergistic drug interaction.

When combining EPD with cisplatin and or paclitaxel, synergistic effects were found. In both SK-OV-3 and JC synergistic effects were found in the combination of EPD and paclitaxel. Treatment of EPD and paclitaxel 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% viabihty (p < 0.05). The combination of EPD and paclitaxel in JC cells was also found to be synergistic with a resulting viabihty of 26% (p < 0.05). No synergistic effect was observed when combining EPD and cisplatin in JC. In JC-pl a strong synergistic effect was detected for the combination EPD and cisplatin. Viabihty 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). Paclitaxel with EPD did not have a synergistic effect. OVCAE-3, as well as the normal fibroblasts did not show any synergistic effects with the different combinations. Due to the striking differences in synergistic interactions observed between EPD and paclitaxel in JC and between EPD and cisplatin in JC-pl, the synergistic interactions were further

investigated.

Synergistic interactions between EPD, paclitaxel and cisplatin in JC and JC-pl

To further investigate the differences in response to the

combinations of drugs in JC and JC-pl, a range of drug combinations was tested for both cell lines. Drug concentrations were selected based on dose- response curves of the individual drugs. Similar to the previous viability tests, using single drug combinations, the expected viability of combination treatment was calculated. Comparison of the observed viability with the expected viability is shown in Figure 2. For all conditions tested, EPD and paclitaxel showed synergistic effects in JC. For JC-pl the lower

concentrations showed an antagonistic drug interaction between EPD with cisplatin. At the highest four doses synergistic interactions were observed, indicating a critical point in the synergistic combinations between these two drugs.

Synergistic drug combinations induce apoptosis

To identify the mechanism of growth inhibition of the synergistic interaction between EPD in combination with cisplatin and paclitaxel, apoptosis measurements were performed. Apoptosis was measured using a fLuorimetric caspase 3 activity assay. EPD and paclitaxel alone induced apoptosis, cisplatin did not. The combination treatment of JC and JC-pl with EPD and the respective synergistic drug showed an increase in caspase 3 activity (Figure 3). Addition of Ac-DEVD-CHO, a caspase 3 inhibitory small molecule completely abolished the signal in all conditions. This confirmed that the synergistic drug combinations induce caspase 3 mediated apoptosis. Flow cytometry

Having established evidence for the synergistic interaction between EPD, cisplatin and paclitaxel in ovarian cancer cell lines, we further sought to elucidate the effects of these compounds. First, the effect of EPD on the cell cycle was studied in SK-OV-3. Untreated SK-OV-3 cells showed to be bi- modal in culture with a minor Gl population approximately at (relative) mean channel number (MCN) 501 and a major Gl population approximately at MCN 938 (Figure 4A). Already at 3 pg/mL EPD treatment, a clear accumulation of cells in the G2/M phase could be noted (Figure 4B). This was dose dependent and G2/M cell numbers increased at higher

concentrations (Figure 5).

To assess the effects of combinations of compounds on the cell lines therefore we performed cell cycle analysis on all four cell lines as well as normal fibroblasts. Conditions chosen for this experiment were identical to those of the initial cytotoxicity tests. Results are shown in Figure 4. After 72 hrs, 6.25 x 10 ⁵ cells were counted for SK-OV-3 with EPD treatment and 2.84 x 10 ⁵ for the combination EPD + paclitaxel, while EPD + cisplatin had 2.89 x 10 ⁵ cells. Combination of EPD + paclitaxel + cisplatin gave 2.40 x 10 ⁵ cells. Cell counts of cell line JC-pl with EPD after 72 hrs were: 3.52 x 10 ⁵ , for EPD + cisplatin: 3.40 x 10 ⁵ and for EPD + cisplatin + paclitaxel: 3.29 x 10 ⁵ . For all cell lines the percent of viable cells and the number of viable cells per mL was plotted against the viability results from Figure 1. Both the percent of viable cells and the number of viable cells per mL showed a strong correlation for JC, JC-pl and SK-OV-3 (Figure 1).

In cell line SK-OV-3 a small peak (MCN = 1439) was noted in the

S-phase (Figure 4A), most likely caused by aggregates composed of Gl cells of the minor population and Gl cells of the major population. Treatment of EPD + paclitaxel resulted in arrest of cells in the G2/M phase. Arrest of cells in cell line JC-pl was observed after EPD treatment in the Gl. The combination EPD + cisplatin showed arrest in G2/M, as did the combination EPD + cisplatin + paclitaxel.

The effects of EPD on the cell cycle distribution of normal fibroblasts was much less than the effects of paclitaxel and/or cisplatin, but at 3 pg/mL EPD treatment increase in G2/M occurred (Figure 6).