WO2010106135A1

WO2010106135A1 - Combined use for the treatment of ovarian carcinoma

Info

Publication number: WO2010106135A1
Application number: PCT/EP2010/053537
Authority: WO
Inventors: Claudio Pisano; Franco Zunino
Original assignee: Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.
Priority date: 2009-03-20
Filing date: 2010-03-18
Publication date: 2010-09-23

Abstract

A pharmaceutical composition containing as active ingredient a therapeutically effective amount of a derivative of the retinoids having general Formula (I) wherein : R represents H or adamantyl; R' represents OR"'; R" represents, COOH, COO R^IV CONHOH, CONH, CONH R^IV; R"' represents H or alkyl,; R^VI represents H or alkyl; its optically active forms such as enantiomers, diastereoisomers and its racemate forms, as well as pharmaceutically acceptable salts, and the proteasome inhibitor bortezomib, for the treatment of ovarian carcinoma, drug-resistant forms included. The compounds according to the present invention can be administrated in a co-ordinated or combined manner. The combination is also used for the preparation of a medicament for the treatment of ovarian carcinoma.

Description

Combined use for the treatment of ovarian carcinoma

FIELD OF THE INVENTION

The present invention relates to the pharmaceutical field and in particular to the combined use of a derivative of the retinoids with a proteasome inhibitor (bortezomib) for the inhibition of tumor growth and migration, in particular for the treatment of ovarian tumor and/or carcinoma. BACKGROUND OF THE INVENTION

A fundamental stage in the biology of the tumour cell is the acquisition of metastasising capability. The tumour cells that metastasise are able to loose adherence to the surrounding structures, invade blood and lymphatic vessels and colonise other tissues at a distance where they can continue to reproduce themselves.

In the medical field, for treating cancer are widely used combinations of different anticancer agents. In fact most of the therapeutical protocols provide for the combined use of different antineoplastic agents; this procedure allows to enhance the treatment efficacy because the individual feedback to the agents can change according to the agent adopted.

The standard treatment for most advanced cancers is multidrug therapy and several methods have been developed in order to try to establish an efficient combination of drugs for the treatment of the different tumour pathologies [Zinner et al., 2009; 8(3); p.521-532].

The use of association of antitumoral agents is known in the art : in particular Pisano et al. [Annals of Oncology, 2007; 18(9); p. 1500-1505] discloses the combined use of ST1926 and cisplatin in the treatment of ovarian cancer, while Aghajanian et al. [J. Clin. Oncology, 2005; 23(25); p. 5943-5949] describes the use of bortezomib and carboplatin for the treatment of similar pathologies. Vitamin A and its biologically active derivatives, retinal and retinoic acid, play an important role in vision, are necessary in the reproductive system, act as morphogenic agents during embryonic growth and regulate the growth and differentiation of a vast range of cell types at the basis of the growth of an organism [M.Sporn, A. Roberts, D.Goodman, The Retinoids, Raven Press, New York 1994] . The biological action of retinoic acid and its derivatives is mediated by the interaction with nuclear receptors belonging to two families: the first named RAR (retinoic acid receptor) and the second named RXR (retinoid X receptor) [P.Chambon, FASEB J., 1996, 10, 940-54] . Retinoids, whether natural or synthetic vitamin A analogues, exercise a great influence over cellular proliferation, differentiation and apoptosis: these properties are amply exploited in the control of tumoral and dermatological pathologies, and pathologies linked to an altered angiogenesis.

Retinoids useful for treating cancer or having antiangiogenic activity are already known [S. Dallavalle et ai, Expert Opinion on Therapeutic Patents, 2005; 15(11); 1625-1635].

It is well known in the art that some synthetic retinoids can induce apoptosis in cancer cells [Lotan R., J. Biol . Regul . Homeost. Agents, 2003; 17, 12-28; Garattini E., Gianni M, Terao M ., Curr. Pharm. Des., 2004; 10 :433- 448.] .

Those derivatives of the retinoids, in particular the ones containing the adamantyl moiety, represent a series of potentially useful agents characterized by proapoptotic activity in a large variety of tumor cells and antitumor activity with minimal acceptable side effects. Recently, it has been showed that a novel adamantyl retinoid, (2E)-3-[3'-

(l-adamantyl)-4'-hydroxy-[l,l'-biphenyl]-4-yl]-2-propenoic acid, hereinafter shortly ST1926, is a potent inducer of apoptosis in ovarian carcinoma cells (WO 03/0118089).

The molecular mechanism involved in ST1926 action suggested the involvement of genotoxic stress as a relevant aspect of the mechanism of the proapoptotic activity.

It is known that a typical feature of cellular response to ST1926 is the activation of p53 and the modulation of genes involved in DNA damage response, however, both p53-dependent and p53-independent pathways appear to be implicated in apoptosis induction by ST1926. ST1926 showed to induce high level of apoptosis in ovarian carcinoma cells at sub-micromolar concentrations [Parrella E., Gianni M ., Fratelli M . et al., MoI . Pharmacol ., 2006; 70 :909-924] .

Although the detailed mechanism of action of ST1926 and related molecules remains to be defined, several lines of evidence support that the genotoxic stress is a critical event mediating drug-induced apoptosis [Zuco V., Zanchi C, Cassinelli G. et al ., Cell Death Diff., 2004; 11 : 280-289; Zuco V., Zanchi C, Lanzi C. et al., Neoplasia 2005; 7 : 667-677; Ortiz M . A., Bayon Y., Lopez-Hernandez FJ., Piedrafita FJ., Drug . Resistance Updates, 2002; 5 : 162- 175] . Another compound, which is a biphenyl-4-yl-acrylohydroxamic acid derivative, E-3-(4'-hydroxy-biphenyl-4-yl)-N-hydroxy- acrylamide, hereinafter shortly ST2782, has shown a significant antiproliferative activity on different tumor cell lines (WO2007/000383).

The chemical compound [(lR)-3-methyl-l-({(2S)-3-phenyl-2-[(pyrazin-2- ylcarbonyl) amino]propanoyl}amino)butyl]boronic acid, shortly named bortezomib (originally codenamed PS-341; marketed as Velcade by Millennium Pharmaceuticals) is the first therapeutic proteasome inhibitor to be tested in humans.

Bortezomib is a dipeptide boronic acid and chymotryptic site-selective inhibitor of the 2OS proteasome. It is approved in the U.S. for treating relapsed multiple myeloma and mantle cell lymphoma. In multiple myeloma, complete clinical responses have been obtained in patients with otherwise refractory or rapidly advancing disease. Among the mechanisms of action recently attributed to proteasome inhibitors, there are the induction of cell cycle arrest, of aggresome formation and endoplasmic reticulum stress, and the terminal unfolded protein response [Orlowski and Kuhn 2008, Hideshima

2005, Nawrocki 2005, Obeng 2006, Neubert 2008].

The boron atom in bortezomib binds the catalytic site of the 2OS proteasome with high affinity and specificity. In normal cells, the proteasome regulates protein expression and function by degradation of ubiquitinylated proteins, and also cleanses the cell of abnormal or misfolded proteins.

While multiple mechanisms are likely to be involved, proteasome inhibition may prevent degradation of pro-apoptotic factors, permitting activation of programmed cell death in neoplastic cells dependent upon suppression of pro- apoptotic pathways.

The antitumoral activity of bortezomib is known in the art. In WO/2008/091620 is described a combination therapy for treating cancer and other neoplams including romidepsin and a proteasome inhibitor like bortezomib.

WO/2008/057456 is related to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of a histone deacetylase (HDAC) inhibitor such as suberoylanilide hydroxamic acid

(SAHA), or a pharmaceutically acceptable salt or hydrate thereof, and a second amount of one or more anti-cancer agents, including bortezomib.

According to this method the effect of the HDAC inhibitor and the anti-cancer agent may be additive or synergistic.

DESCRIPTION OF THE INVENTION

Our intention was to explore the effect of the combination of a derivative of the retinoids (ST2782) with the proteasome inhibitor bortezomib in three ovarian carcinoma cell lines, including the parental IGROV-I cell line and two variants exhibiting resistance to Pt compounds. The IGROV-1/OHP and IGROV-1/Ptl cells, selected for resistance to oxaliplatin and cisplatin respectively, carried p53 gene with mutations in hotspot codons implicating loss of p53 protein function.

Several studies has been carried out to discover a method to identify the best "cocktail" of drugs to be used in the multidrug therapies, but no good results have been obtained at the moment. For this reason the selection of the drugs for the multidrug is made on the basis of their antitumoral activity and in some cases the association with another anticancer agent does not cause a synergistic effect in the treatment of the tumor pathology, especially in the treatment of resistant forms of tumour, which is already an important problem to be solved. It has unexpectedly been found that the combined use of this retinoid compound, such as the compounds of formula (I) as detailed below, and the proteasome inhibitor bortezomib improve efficacy in terms of tumor growth without substantial increase of toxicity.

It is therefore the main object of the present invention a pharmaceutical composition containing as active ingredient a therapeutically effective amount of a derivative of the retinoids having general Formula (I) wherein :

(D

R represents H or adamantyl; R' represents OR"';

R" represents, COOH, COO R^IV CONHOH, CONH, CONH R^IV; R"' represents H or alkyl,; R^VI represents H or alkyl; its optically active forms such as enantiomers, diastereoisomers and its racemate forms, as well as pharmaceutically acceptable salts, and the proteasome inhibitor bortezomib, for the treatment of ovarian carcinoma, drug-resistant forms included.

A further object of the present invention is the use of a derivative of the retinoids having general Formula (I) and the proteasome inhibitor bortezomib, or a pharmaceutically acceptable salts thereof, for the treatment of ovarian carcinoma. The compounds according to the present invention can be administrated in a co-ordinated or combined manner.

What is meant by combined use of the aforesaid compounds is, indifferently, either the co-administration, i.e. the substantially concomitant or sequential supplementation of a Formula (I) compound and of bortezomib, or the administration of a composition comprising the aforesaid active ingredients in combination and in a mixture optionally further comprising one or more excipients or diluents pharmaceutically acceptable.

A further object of the invention described herein is the use of a pharmaceutical composition as described above for the preparation of a medicine for the treatment of ovarian cancer.

The active substances described in the present invention, or a pharmaceutically acceptable salts thereof, may also be used in form of a hydrate or include other solvents used for crystallization, also known as solvates.

What is meant by pharmaceutically acceptable salt of compound described in the present invention is any salt of the latter with an acid that does not give rise to toxic or side effects.

Non-limiting examples of such salts are: chloride, bromide, orotate, aspartate, acid aspartate, acid citrate, magnesium citrate, phosphate, acid phosphate, fumarate and acid fumarate, magnesium fumarate, lactate, maleate and acid maleate, oxalate, acid oxalate, pamoate, acid pamoate, sulphate, acid sulphate, glucose phosphate, tartrate and acid tartrate, glycerophosphate, mucate, magnesium tartrate, 2-amino-ethanesulphonate, magnesium 2-amino-ethanesulphonate, methanesulphonate, choline tartrate, trichloroacetate, and trifluoroacetate. A list of FDA-approved pharmaceutically acceptable salts is given in the publication Int. J. of Pharm. 33 (1986), 201-217.

The pharmaceutical composition according to the present invention may contain suitable pharmaceutical acceptable carriers, biologically compatible vehicles suitable for administration to an animal (for example, physiological saline) and eventually comprising auxiliaries (like excipients, stabilizers or diluents) which facilitate the processing of the active compounds into preparations which can be used pharmaceutical.

The pharmaceutical composition according to the present invention may be formulated in any acceptable way to meet the needs of the mode of administration. The use of biomaterials and other polymers for drug delivery, as well the different techniques and models to validate a specific mode of administration, are disclosed in literature.

Any accepted mode of administration can be used and determined by those skilled in the art. For example, administration may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, oral, or buccal routes.

"Therapeutically effective amount" is an amount effective to achieve the medically desirable result in the treated subject. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice or rats.

The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The dosage administered will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. The total dose required for each treatment may be administered by multiple doses or in a single dose.

Administration will be carried out according to the decision of the medical doctor, according to type of the tumor to be treated, patient's conditions and any other considerations pertaining the exercise of medical science. The dose for the retinoid may be from 0.01 mg/kg to 100 mg/kg, preferably 0.05 mg/kg to 50 mg/kg and more preferably from 30 mg/kg to 40 mg/kg.

The dose for bortezomib may be administered to a human according to standard protocols. All the compounds described in the present invention are disclosed in

WO 03/011808, WO 07/000383 and WO96/013266 both for their preparation and pharmacological profile. Specific reference is made to these documents for disclosure of the compounds described in the present invention. Preferred compounds of formula (I) are comprised in the following group:

E-3-(4'-hydroxy-biphenyl-4-yl)-N-hydroxy-acrylamide (ST2782); (2E)-3-[3'-(l-adamantyl)-4'-hydroxy-[l,l'-biphenyl]-4-yl]-2-propenoic acid (ST1926); and E-4-(3-(l-adamantyl)-4-methoxyphenyl)cinnamic acid (ST1898). Further features of the present invention will be evident with reference to the following examples and attached figures wherein : Figure 1 : Cell cycle distribution of IGROV-I (A), IGROV- 1/OHP (B) and

IGROV- 1/Ptl (C) cells following 24h exposure to ST2782 or bortezomib or to the combined treatment. Cells were harvested and than fixed, stained with propidium iodide and analyzed by flow cytometry. One experiment representative of three is reported.

Figure 2 : Cell cycle distribution of IGROV-I (A), IGROV- 1/OHP (B) and

IGROV- 1/Ptl (C) cells following 48h exposure to ST2782 or bortezomib or to the combined treatment. Cells were harvested and than fixed, stained with propidium iodide and analyzed by flow cytometry. One experiment representative of three is reported.

Figure 3 : Analysis of apoptosis induction after 72h exposure to ST2782 or bortezomib or to the combined treatment. Apoptosis was measured by TUNEL assay and representative dot plots showing viable cells (FL< 10²) and apoptotic cells (FL > 10²) are presented for IGROV-I (A), IGROV- 1/OHP (B) and IGROV- 1/Ptl (C) cells, respectively. The percentage of TUNEL-positive cells are indicated. One experiment representative of three is reported. (D) Levels of apoptosis expressed as a percentage of the total cell population. Each data point represents the mean (± SD) of three independent experiments.

Figure 4 : Western blot analysis in IGROV-I (A), IGROV- 1/OHP (B) and IGROV- 1/Ptl (C) cells following 24h exposure to ST2782, or bortezomib or to the combined treatment. Western blot analysis of selected proteins was performed using total protein extracts and control loading is shown by β- tubulin. Experiment was repeated three times and a representative image is reported . Figure 5 : Western blot analysis in IGROV-I, IGROV-1/OHP and IGROV-

1/Ptl cells following 48h exposure to ST2782, or bortezomib or to the combined treatment. Western blot analysis of selected proteins was performed using total protein extracts and control loading is shown by β-tubulin. Experiment was repeated three times and a representative image is reported.

The following examples further illustrates the invention. EXAMPLES

Method a) Cell lines and cell sensitivity to drugs

The human ovarian carcinoma cell lines IGROV-I, IGROV-1/OHP and IGROV- 1/Ptl were used in this study. The resistant variants, IGROV-1/OHP and IGROV-1/Ptl, were selected by continue exposure of IGROV-I cells to increasing oxaliplatin and cisplatin concentrations respectively, and they carried a p53 gene with mutations in hot spot codon implicating loss of p53 protein function. The cell lines were cultured in RPMI-1640 medium (BioWhitaker, Verviers, Belgium) supplemented with 10% fetal bovine serum (Invitrogen Italia, San Giuliano Milanese, Italy). The cell sensitivity to drugs was measured by using a growth-inhibition assay based on cell counting . Exponentially growing cells were seeded in duplicate into six-well plates and, 24 h later, exposed to drugs for 72 h. At the end of treatment, cells were harvested and counted with a cell counter (Coulter Electronics, Luton, UK). IC₅₀ is defined as the drug concentration producing 50% inhibition of cell growth as compared with control. At least 3 independent experiments were performed for each drug or type of treatment. The effect of the combination was evaluated according to the method of Kern et al. [Kern 1988]. An R index of 1 (additive effect) or lower indicated the absence of synergism. Synergysm was defined as any value of R > 1 [Romanelli 1998] . ST2782 and bortezomib (Velcade, Millennium Pharmaceuticals, Inc., Boston, MA, USA) were dissolved in dimethylsulfoxide and diluted in water. b) Apoptosis and cell cycle analysis

Exponentially growing cells were seeded in 25 cm² flasks and, 24 h later, they were exposed to different concentration of bortezomib or ST2782 or to the combination of both drugs for 24, 48 or 72 h. At the end of treatment floating and adherent cells were harvested for detection of apoptotic cells or cell cycle analysis. For cell cycle analysis, cells were fixed and stained with a propidium iodide (PΙ)-containing solution (30 mg/ml PI, 66 U/ml RNase A in PBS). The cell cycle perturbations were measured by using a flow cytometer (Becton Dickinson, Mountain View, CA). Samples were analyzed for DNA content, and cell cycle distributions were calculated using Modfit software (Becton Dickinson). Apoptosis was evaluated by TUNEL (Terminal deoxynucleotidyl tranferase dUTP Nick End Labeling) assay (Roche, Mannheim, Germany). After harvesting, cells were fixed in paraformaldehyde, permeabilized in a solution of 0.1% Triton X-100 in 0.1% sodium citrate, and then incubated in the TUNEL reaction for 1 h. After washing, samples were analyzed by flow cytometry using Cell Quest software (Becton Dickinson). c) Western blot analysis Western blot analysis was carried out according to the following brief description (for any further details see Perego et al. MoI Pharm. 1999; 55:528- 534) : samples were fractionated by SDS-PAGE and blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried non fat milk and then incubated overnight at 4°C with antibodies to p21^WAF1 (NeoMarkers, Fremont, CA), cleaved Caspase 3 (Aspl75, Cell Signaling Technology, Danvers, MA), p53 (DO-7 clone, DAKO, Glostrup, Denmark), Bax (SIGMA Chemicals Co., Saint Louis, MO) Bcl-2 (DAKO), PARP (Clontech, Palo Alto, CA). An anti-tubulin antibody (SIGMA Chemicals Co.) was used as control for loading. Antibody binding to blots was detected by chemiluminescence (Amersham Pharmacia Biotech., Cologno Monzese, Italy). Three independent experiments were performed. Example 1

Growth-inhibition assays and drug combination studies Sensitivity of ovarian carcinoma cells to ST2782 and to the proteasome inhibitor bortezomib was assessed by growth-inhibition assay following exposure to the drugs for 72 h. ST2782 exhibited a comparable antiproliferative effect in the tested cell lines, with IC₅₀ values in the micromolar range (Table 1).

The proteasome inhibitor was substantially more potent, presenting a marked anti-proliferative effect in all the tumor cell lines tested, the IC₅O values being in the nanomolar range (Tablel).

Using a simultaneous combination treatment with different concentrations of ST2782 and a subtoxic concentration of the proteasome inhibitor bortezomib (< 30%; 0.005 μM for IGROV-I and IGROV-1/Ptl cells and 0.0025 μM for IGROV- 1/OHP cells, respectively), we observed a synergistic interaction that was more marked in platinum- resistant sublines compared to parental cells, as indicated by the Kern Index values (R Kern Index, obtained with 10 μM ST2782, of 1.75 ± 0.35 in IGROV-I, 3 ± 0.9 in IGROV- 1/OHP and 2.6 ± 0.5 in IGROV- 1/Ptl, respectively). Table 1. Sensitivity of IGROV-I, IGROV-1/OHP and IGROV-1/Pt cells to ST2782 and BORTEZOMIB³

a Cell sensitivity was assessed by growth-inhibition assay.

Cells were exposed to drugs for 72h and counted at the end of treatment. Values are the mean (± SD) of at least 3 independent experiments. b IC₅O represents the drug concentration producing 50% decrease of cell growth.

Example 2

Cell cycle perturbation analysis

The analysis of cell cycle perturbations was performed on cells exposed to each single agent, ST2782 (10 and 3 μM) or bortezomib (0.005 μM), or to their combination for 24 and 48 h. In IGROV-I cells bortezomib caused a stable Gl accumulation both 24 and 48 h after drug exposure (Fig. IA and 2A). On the contrary, the higher concentration (10 μM) of ST2782 determined a decrease in the percentage of Gl phase cells and an increase in the S phase, together with a certain increase in the sub-Gl DNA content, whereas the lower concentration (3 μM) only delayed progression of IGROV-I cells through G2/M (Fig. IA and 2A). Simultaneous exposure to the two drugs appeared to produce a marked increase of the subdiploid population both at 24 and 48 h (Fig. IA and 2A). The modifications of cell cycle distribution observed in IGROV- 1/OHP cells were more marked regarding the effect of ST2782 alone (i.e., Gl phase emptying and enrichment of S phase population), whereas bortezomib did not produce the early Gl accumulation found in sensitive parental cells (Fig. IB and 2B). Thus, IGROV-1/OHP cells exposed to the drug combination could advance towards S and G2/M phase (Fig. IB and 2B). Besides, a more marked increase in the sub-Gl DNA content was found in IGROV-1/OHP cells 48 h after the exposure to ST2782 (± bortezomib), thus suggesting a massive induction of apoptotic cell death (Fig. IB and 2B). A similar response to ST2782 and to the combination was observed in

IGROV- 1/Ptl cells, which exhibited a marked increase of sub-Gl cells, in spite of a different cell cycle distribution in untreated cells (Fig. 1C and 2C). Example 3 Analysis of apoptotic cell death To determine whether the drug interaction resulted in an enhancement of apoptotic cell death, as suggested by cell cycle perturbations analysis, we performed cytofluorimetric analysis of fragmented DNA of apoptotic cells by TUNEL assay. Apoptosis was determined at 72 h after drug exposure, when the observed increase of the subdiploid population was more marked (Fig 1 and 2). In IGROV-I cells, the combined treatment of 0.005 μM bortezomib with ST2782 (10 or 3 μM) produced only a marginal increase of apoptosis (8 %; Fig. 3A and 3D) as respect to the treatment with the single agents, whereas in the platinum resistant cells the increase in the percentage of apoptotic cells following the combined drug exposure was remarkably higher and also dependent on the ST2782 concentration (28 % with 3 μM ST2782 and 36.4 % with 10 μM ST2782 in IGROV-1/OHP; 16 % with 3 μM ST2782 and 53,5 % with 10 μM ST2782 in IGROV- 1/Ptl, respectively; Fig. 3B, 3C and 3D). Example 4 Western blot analysis

To examine the possible contribution of specific pathways to the effects observed in cell cycle and apoptosis analysis, we investigated the modulation of factors implicated in apoptosis through Western blot experiments. The effect of 24 h ST2782 exposure on p53 expression was an evident down- regulation in the three cell lines (Fig. 4A, 4B and 4C). After 24 h drug treatment, bortezomib alone triggered an increase in expression of p21^WAF1 only in IGROV-I parental cells, whereas ST2782 alone or in combination with the proteasome inhibitor determined a p21^WAF1 induction in all tested cell lines (Fig. 4A, 4B and 4C). Thus, in IGROV-I cells the level of induction of p21^WAF1 following exposure to the combination resulted wider than in resistant cells, presumably as a result of up-regulation both by bortezomib and ST2782. The increased expression of the multidomain proapoptotic protein Bax was particularly evident in IGROV-1/OHP cells 48 h after exposure to 10 μM ST2782 (Fig. 5), and more slightly in IGROV- 1/Ptl cells 24 h after exposure to 10 μM ST2782, whereas no induction of Bax was observed in IGROV-I cells (Fig. 4C, 4A and 5). Since the regulation of mitochondria is considered a primary target for damage by bortezomib and also by ST2782, we evaluated the expression of Bcl-2, but no modulation of the protein expression was noticed under our experimental conditions (Fig 4A, 4B and 4C). We also investigated typical hallmarks of apoptosis, such as cleavage of caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). We observed the appearance of a cleaved form of caspase 3 in the three cell lines. In the parental cell line, the phenomenon was more weakened as compared with the resistant cells, where the cleavage was particularly clear following 24 h exposure to 10 μM ST2782 in the presence or absence of bortezomib (Fig. 4A, 4B and 4C). Also the kinetics of PARP cleavage resulted different in the three cell lines, because it was already evident in IGROV-1/OHP cells 24 h after exposure to drug combination (both with 10 and 3 μM ST2782), whereas in IGROV-1/Ptl cells the cleaved protein appeared later (48 h) with minor intensity and only with 10 μM ST2782 (Fig . 4b and 5). In Pt drug-sensitive IGROV-I cells, no cleavage of PARP was detected, neither 24 or 48 h after drug exposure (Fig . 4A and 5). Discussion

In the present invention, we show that a synergistic interaction between ST2782 and bortezomib can be obtained in Pt drug-resistant cells carrying a mutant p53 gene. In such cell lines, the combination of the two drugs led to a strong increase in apoptosis as compared to single agents, and was accompanied by caspase 3 and PARP cleavage, as well as by induction of the pro-apoptotic protein Bax (Fig. 3, 4 and 5).

In the p53-defective ovarian carcinoma models (IGROV- 1/Ptl and IGROV- 1/OHP), a p53-independent induction of p21^WAF1 by ST2782 was observed similarly to what exhibited by the parental p53 wt cell line (IGROV-I; Fig . 4). Even if we do not want to be bound by any theory, Gl accumulation induced by the proteasome inhibitor in cells carrying a wt p53 (Fig. 1 and 2) appears to play a role in protecting them from ST2782-induced apoptosis, whereas the drug combination resulted particularly active in p53-mutant cells (Fig. 3). Thus, the apoptotic effect and synergistic interaction between ST2782 and bortezomib resulted moderate in IGROV-I cells, even though the antiproliferative and apoptotic effect of ST2782 alone was more evident in such a cell line (Table 1; Fig. 3D). The Pt drug-resistant cells appeared to have lost the p53-dependent induction of p21^WAF1 by bortezomib, differently from what reported for IGROV-I parental cells (Fig. 4). Therefore, the synergistic interaction observed in the studied cell systems could be explained by the type of cell response (in particular by drug-induced cell cycle perturbations). In this context, we also found that ST2782 produced dose-dependent down-regulation of p53 in all the three cell lines (Fig. 4). Such an effect appears dependent on the stimulation of 26S proteasome activity. Thus, a stimulatory effect of ST2782 on proteasome activity may underlie the synergism between ST2782 and bortezomib, because the apoptotic effect of

ST2782 could be favoured by proteasome inhibition.

An additional explanation of the synergistic interaction may evoke the effect of ST2782 on HDAC6, a predominantly cytosolic HDAC which acts as a deacetylase for HSP90. Hyperacetylation of this molecular chaperone, by reducing its function may lead to impairment of the correct proteolytic activity of proteasome, further inhibited by bortezomib.

In summary, the examples reported above show that bortezomib potentiates the apoptotic effect of ST2782 by suppressing the stimulatory effect of the same compound on proteasome activity. This phenomenon could be the basis for the synergistic interaction between the two drugs in all the three cell lines.

In conclusion, these results provide evidence of a relationship between p53 mutational status and response to a combined treatment of ST2782 and bortezomib in ovarian carcinoma cell lines, and supporting clinical applications of the combination in mutant p53 ovarian tumors.

Claims

CLAIMS l. A pharmaceutical composition containing as active ingredients both a compound of Formula (I):

(D wherein

R represents H or adamantyl; R' represents OR"';

R" represents, COOH, COO R^IV CONHOH, CONH, CONH R^IV; R"' represents H or alkyl,; R^VI represents H or alkyl; and the proteasome inhibitor bortezomib.

2. Use of a compound of Formula (I):

(I) wherein

R represents H or adamantyl;

R' represents OR"'; R" represents, COOH, COO R^IV CONHOH, CONH, CONH R^IV;

R"' represents H or alkyl;

R^VI represents H or alkyl; its optically active forms suche as enantiomers, diastereoisomers and its racemate forms, as well as pharmaceutically acceptable salts, and the proteasome inhibitor bortezomib, or a pharmaceutically acceptable salts thereof, for the treatment of ovarian carcinoma, drug-resistant forms included.

3. The use according to claim 2, in which the Formula (I) compound is (2E)-3-[3'-(l-adamantyl)-4'-hydroxy-[l,l'-biphenyl]-4-yl]-2-propenoic acid.

4. The use according to claim 2, in which the Formula (I) compound is E-3- (4'-hydroxy-biphenyl-4-yl)-N-hydroxy-acrylamide.

5. The use according to claim 2, in which the Formula (I) compound is E-4-(3-(l-adamantyl)-4-methoxyphenyl)cinnamic acid.

6. The use according to claim 2, in which the administration of the Formula

(I) compound and the bortezomib is sequential.

7. The use according to claim 2, in which the administration of the Formula (I) compound and the bortezomib is simultaneous.

8. The use according to claim 2, in which the compound of Formula I and brotezomib are packaged in a single pharmaceutical form or in separate containers for sequential administration.

9. The use according to claim 8, in which the Formula (I) compound can be administrated orally, parenterally, intravenously and/or transdermally.

10. The use according to claim 8, in which the bortezomib can be administrated orally, parenterally, intravenously and/or transdermally.

11. The use according to claim 2, in which the medicament is in solid or liquid form, suitable for oral or parenteral administration in the form of tablet, sachet, capsule or vial.