Patent Description:
Cancer is an abnormal growth of cells and there are several types of cancer, including breast cancer, skin cancer, lung cancer, colon cancer, prostate cancer, brain cancer and lymphoma. Brain tumors are abnormal growth of cells in the brain. High grade gliomas (HGG) or malignant glioma is a deadly form of human cancer. 'Cancer stem cells' (CSC) within solid tumors that compel tumor formation and growth are reported to confer resistance to conventional chemotherapy and radiotherapy. The prognosis for patients with high-grade gliomas ( GBM-glioblastomas) is generally poor (in patients with IDH mutations which is a feature in younger patients, the survival is poor). Despite combined modality treatment including surgical resection and radiation, a minority of patients live beyond <NUM> years. However, various chemotherapeutic agents used as combination therapy targeting multiple pathways are becoming a fast growing area of research.

Selective estrogen receptor modulators (SERM) used as anti-tumoral agents include raloxifene, tamoxifen or toremifine. Tamoxifen ((Z)-<NUM>[p[(<NUM>,<NUM>-diphenyl-<NUM>-butenyl)phenoxyl]-N,N-dimethyl amine citrate) is a potent estrogen receptor (ER) antagonist that has been extensively used to treat ER-positive breast cancer. Tamoxifen is a selective estrogen receptor modulator (SERM, indicated to treat women diagnosed with hormone-receptor-positive, early-stage breast cancer after surgery to reduce the risk of recurrence. Tamoxifen also is used to reduce breast cancer risk in women who haven't been diagnosed but are at higher-than-average risk for the disease. However, tamoxifen is not indicated in hormone-receptor-negative breast cancer.

Histone deacetylase inhibitors (HDACi) like valproic acid, sodium butyrate, vorinostat, romidepsinor and trichostatin A (TSA) are known to be effective anticancer agents through epigenetic modulation. Valproic acid (Valproate; VPA) or valpromide (VPM) or sodium valproate or valproate semisodium forms are medications primarily used to treat epilepsy and prevention of seizures.

Prostate apoptosis response-<NUM> (Par-<NUM>) is a naturally occurring tumor suppressor protein that is capable of inducing apoptosis. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, extracellular or secretory Par-<NUM> induces apoptosis by binding to membrane GRP78. Though secretory Par-<NUM> induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored.

Certain prior arts disclose certain combination of drugs used for treating cancer. For instance, <CIT> discloses a method for treating hormone resistant breast cancer comprising administration of HDAC inhibitors and hormone targeted drugs. However, US'<NUM> fails to disclose the use of such combination for treating malignant gliomas by targeting Par-<NUM>-GRP78 pathway.

<CIT> discloses method of treating estrogen receptor positive breast cancer comprising administration of combination comprising HDAC inhibitor in combination with hormonal therapy wherein the HDAC inhibitor is not valproic acid. However, US'<NUM> fails to disclose the combination of tamoxifen and valproic acids for treating malignant gliomas.

Publication <NPL>, is drawn to inferences of phase II clinical trials and concludes that the combination of histone deacetylase inhibitor vorinostat and estrogen receptor (ER) antagonist tamoxifen is well tolerated and exhibits encouraging activity in reversing hormone resistance. The publication aims to provide a solution to hormone therapy-resistant breast cancer by suggesting use of HDAC inhibitors (exemplified as vorinostat) could re-sensitise tamoxifen-resistant cells to hormone therapy.

Publication <CIT> discloses valproic acid (VPA) and its salts in combination with hormonal therapy, including antiestrogens, aromatase inhibitors, and selective estrogen receptor modulators, for treatment of cancer, such as human ERa -positive breast cancer cells. However nowhere mentions the activity of the disclosed combination against glioma or malignant gliomas and only provides a combination for activity against the treatment and prevention of breast cancer.

Publication <CIT> discloses valproic acid (VPA), carbamazepine, and other HDAC inhibitors in combination with hormonal therapy, including antiestrogens and aromatase inhibitors, on human ERα-positive breast cancer cells, however nowhere mentions the activity of the disclosed combination against glioma or malignant gliomas and only provides a combination for activity against the treatment and prevention of breast cancer.

The publication of <NPL>, studies paradoxical role of HDACs as tumor suppressors, the treatment of tumor cells with HDAC inhibitors (HDACi) that induces a range of effects including apoptosis, cell cycle arrest, differentiation and senescence, modulation of immune response, and altered angiogenesis. The composition comprising histone deacetylase inhibitor (HDACi) and an estrogen receptor (ER) antagonist is not disclose there, nor the specific dose range of HDACi is disclosed in detailed. Noemi Arrighetti et al. discloses glioma treatment with valproate however is completely silent of combination of valproate and tamoxifene for the treatment of glioma. There is also no synergy between those two compounds disclosed in the treatment of glioma.

The above cited prior arts disclose combinations of HDAC inhibitor with hormone targeted drugs in breast cancer. There remains a need for effective novel combination of HDAC inhibitor and hormone targeted drugs having anticancer activity against drug resistant malignant gliomas. Therefore, there is a need to develop new strategies for controlling the cell growth and killing of cancerous cells in high grade gliomas or malignant gliomas.

An object of the invention is to provide a novel, synergistic composition of HDAC inhibitor and estrogen receptor (ER) antagonist having anticancer effects by targeting Par4/GRP78 pathway.

The present invention is based on a phamaceutical synergistic composition comprising histone deacetylase inhibitors (HDACi) selected from valproic acid, sodium valproate, valproate semisodium, valpromide, in combination with estrogen receptor (ER) antagonist which is tamoxifen for use in a method of treating gliomas.

The scope of the present invention is defined in the claims; any other disclosure in the present description not protected by the appended claims, regardless of whether it is indicated as being disclosed, preferred or exemplified, is provided for information purposes, only.

Further disclosed histone deacetylase inhibitors (HDACi) are from the group comprising valproic acid, sodium butyrate, vorinostat, romidepsinor and trichostatin A (TSA). The disclosed estrogen receptor (ER) antagonist are from the group comprising tamoxifen, raloxifene, fulvestrant, and torimefene.

The compositions of the invention are for use in a method of treating gliomas, in particular high grade gliomas or malignant gliomas.

The present invention is based on synergistic composition comprising histone deacetylase inhibitors (HDACi) selected from valproic acid, sodium valproate, valproate semisodium, valpromide in combination with a hormone-targeted drug i.e. estrogen receptor (ER) antagonist which is tamoxifen for use in the treatment of cancer selected from gliomas.

The invention relates to a composition comprising histone deacetylase inhibitor (HDACi) and an estrogen receptor (ER) antagonist for use in a method of treatment of cancer, wherein the cancer is selected from glioma, malignant glioma, high grade glioma, and wherein histone deacetylase inhibitor (HDACi) is selected from valproic acid, sodium valproate, valproate semisodium, valpromide, and wherein estrogen receptor (ER) antagonist is tamoxifen.

In the preferred pharmaceutical composition for use the histone deacetylase inhibitor (HDACi) is the valproic acid in the range of <NUM> to <NUM>, more preferably at <NUM> to <NUM> and most preferably at <NUM> to <NUM>.

In the preferred pharmaceutical composition for use the tamoxifen is in the range of <NUM>µg/ml to <NUM>µg/ml, more preferably at <NUM>µg/ml to <NUM>µg/ml.

Preferably the pharmaceutical composition for use shows the effect in RTK/Ras/PI3K signaling pathway, pRB signaling pathway, PI3K/AKT/mTOR pathway, p53 pathway, Par-<NUM>/GRP78, preferably in Par-<NUM>/ GRP78 pathway.

The pharmaceutical composition for use preferably , further comprises excipients selected from the group comprising preservatives, buffering agents, salts, carriers, diluents.

The present invention is capable of being administered to a subject. A "Subject" herein is any mammal, preferably the subject is a human. The subject includes within its scope patients or any diseased mammal or human.

The present invention is based on combined synergistic combination effect of histone deacetylase inhibitor (HDACi) wherein histone deacetylase inhibitor (HDACi) is selected from valproic acid, sodium valproate, valproate semisodium, valpromide with estrogen receptor (ER) antagonist which is tamoxifen. Without being limited to the theory, the novel composition for use of the present invention induces its anticancer effects through intervention of signal transduction pathway in gliomas. It hence envisages that this composition for use will also be effective in other pathways, preferably in Par-<NUM>/ GRP78 pathway.

The synergistic composition for use may also affect other signal transduction pathways such as RTK/Ras/PI3K signaling pathway, pRB signaling pathway, PI3K/AKT/mTOR pathway, p53 pathway, Par-<NUM>/GRP78 pathway individually or in combination thereof as promising targets for killing of cancer cells.

The histone deacetylase inhibitors (HDACi) may be selected from the group comprising valproic acid, sodium valproate, valproate semisodium, valpromide, sodium butyrate, vorinostat, romidepsinor and trichostatin A (TSA), preferably valproic acid.

The concentration of valproic acid/valpromide may be used in the range of <NUM> to SmM, more preferably at <NUM> to <NUM> and most preferably at <NUM>-<NUM>.

The estrogen receptor (ER) antagonist may be selected from the group comprising tamoxifen, raloxifene, fulvestrant, and torimefene, preferably tamoxifen.

The concentration of tamoxifen may be used in the range of <NUM>µg/ml to <NUM>µg/ml, more preferably at <NUM>µg/ml to <NUM>µg/ml.

Surprisingly, it has been found that the composition of tamoxifen and valproic acid is synergistic and induce apoptosis in glioma cells in a dose-dependent manner that has been demonstrated in vitro on various brain tumor cell lines such as LN-<NUM>, T98G [T98-G], LN-<NUM>, M059K, M059J, U-<NUM>, U-<NUM>, or a composition thereof.

Additional ingredients may be included with the composition for use of the present invention having anticancer effect on gliomas. Such ingredients may be any actives that may be used for treatment of cancer.

In reference embodiment, the present invention discloses a composition comprising the combination of histone deacetylase inhibitor (HDACi) and estrogen receptor (ER) antagonist for use in a method of treatment of cancer, along with pharmaceutically acceptable excipients selected from the group comprising preservatives, buffering agents, salts, carriers, diluents. Such additional ingredients include other active agents, preservatives, buffering agents, salts, carriers, diluents, or other pharmaceutically acceptable ingredients. As mentioned VPM also works though it is not a HDAC inhibitor. The finding suggests that the anti-cancer effect is independent of its activity as HDAC inhibitor.

There is disclosed a composition comprising the combination, for treating cancer, drug-resistant tumors, cancer stem cells, malignant gliomas or high-grade gliomas by administering a pharmaceutically effective amount to a subject.

The synergistic combination comprising histone deacetylase inhibitor (HDACi), preferably valproic acid and estrogen receptor (ER) antagonist, preferably tamoxifen may induce the expression of intrinsic and secretory pro-apoptotic protein Par-<NUM> which further interacts with the cell surface receptor GRP78 (glucose-regulated protein <NUM>) to induce cancer cell apoptosis in a specific manner.

The described combination is used for treatment of cancer, drug-resistant tumors, cancer stem cells, malignant gliomas or high-grade gliomas.

There is described a composition comprising the combination of histone deacetylase inhibitors (HDACi) and an estrogen receptor (ER) antagonist, for treating cancer, drug-resistant tumors, cancer stem cells, malignant gliomas or high-grade gliomas by administering a pharmaceutically effective amount to a subject or for its effect in RTK/Ras/PI3K signaling pathway, pRB signaling pathway, PI3K/AKT/mTOR pathway, p53 pathway, Par-<NUM>/GRP78, preferably in Par-<NUM>/ GRP78 pathway.

The histone deacetylase inhibitors (HDACi) is selected from the group comprising valproic acid, sodium valproate, valproate semisodium, valpromide, sodium butyrate, vorinostat, romidepsinor and trichostatin A (TSA), preferably valproic acid and is present in the range of <NUM> to SmM, more preferably at <NUM> to <NUM> and most preferably at <NUM> to <NUM>.

The estrogen receptor (ER) antagonist is selected from the group comprising tamoxifen, raloxifene, fulvestrant, and torimefene, preferably tamoxifen and is in the range of <NUM>µg/ml to <NUM>µg/ml, more preferably at <NUM>µg/ml to <NUM>µg/ml and most preferably at <NUM>µg/ml to <NUM>µg/ml.

There is also described a method of intervening in RTK/Ras/PI3K signaling pathway, pRB signaling pathway, PI3K/AKT/mTOR pathway, p53 pathway, Par-<NUM>/GRP78, preferably in Par-<NUM>/ GRP78 pathway by administration of the disclosed combination.

The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

MTT assay was employed for assessing the combined action of tamoxifen (TAM) and VPA. LN-<NUM> cells and LN-<NUM> were seeded in <NUM> well plate (<NUM> cells/100ul/well) in complete medium and cultured for <NUM> hr in two culture models-monolayer and 3D culture. Cells were treated with tamoxifen (TAM) at <NUM> concentrations- 5µg/ml, 10µg/ml and 15µg/ml either individually or in composition with Valproic Acid (VPA) (<NUM>) for <NUM> hr. MTT was added and after <NUM> hr the crystals were solubilized using DMSO. Absorbance was recorded at <NUM> (test filter) and <NUM> (reference filter). The % cell viability in treated cells was calculated considering the readings in untreated cells as <NUM>%. The results are presented at <FIG>. From <FIG> it is evident that the effect of TAM was dose dependent in monolayer, TAM was toxic at 15µg/ml in LN-<NUM> cells. More importantly it can be seen that VPA enhanced the effect of TAM (<NUM>µg/ml) significantly in LN-<NUM> cells cultured as monolayer culture and that MCS of LN-<NUM> cells were resistant to TAM treatment. Furthermore, VPA enhanced the effect of TAM at 10µg/ml in ML and at <NUM>µg/ml in MCS of LN-<NUM> cells. MCS of LN <NUM> cells were resistant to TAM treatment and VPA enhanced the effect significantly in cells treated with TAM at <NUM>µg/ml.

From <FIG> it is evident that the composition of the present invention is synergistic.

LN-<NUM> cells were seeded in <NUM> well plate (<NUM> million cells/well) in complete medium and grown as 2D (monolayer) or 3D (MCS) cultures. Cells were treated with tamoxifen (TAM) -<NUM>µg/ml either individually or in combination with Valproic Acid (VPA) (<NUM>). Monolayer were treated for <NUM> hr and MCS were treated for <NUM> hr. Cells were fixed and stained with propidium Iodide and analysed by flowcytometry for cell cycle analysis. Data was acquired for <NUM>,<NUM> cells on linear scale (FL2A) and the cell population in pre-G0/G1 phase representing the apoptotic population was measured. The results are presented at <FIG>. From <FIG>. , it can be seen that VPA enhanced the apopototic effect of TAM (10µg/ml) significantly in monolayer culture of LN-<NUM> cells treated for <NUM> hr. VPA enhanced the apopototic effect of TAM (10µg/ml) significantly in MCS culture of LN-<NUM> cells treated for <NUM> hr. In <FIG>, the synergistic effect of the combination being the effect of apoptosis. Statistically significant difference is observed between apoptosis induced by TAM alone Vs. TAM + VPA is clearly seen.

LN-<NUM> cells (ML) were seeded in <NUM> well plate (<NUM> cells/100ul/well) in complete medium and cultured for <NUM> hr. Cells were treated with tamoxifen (TAM) at <NUM>µg/ml either individually or in combination with Valproic Acid (VPA) (<NUM>) or VPM (<NUM>) for <NUM> hr. MTT was added and after <NUM> hr the crystals were solubilized using DMSO. Absorbance was recorded at <NUM> (test filter) and <NUM> (reference filter). The % cell viability with treatment was calculated with readings in untreated cells considered as <NUM> %. The results are presented at <FIG>. It can be seen from <FIG> that there was no significant difference in the effect induced by VPA and VPM in combination with TAM (10µg/ml and 15µg/ml) on cell viability in LN-<NUM> cells treated for <NUM> hr.

LN-<NUM> cells were seeded in <NUM> well plate (<NUM> million cells/well) and treated with TAM (10µg/ml) and VPA (<NUM>) either individually or in combination for <NUM> hr.

Cell lysates were assessed for expression of intracellular prostate apoptosis response (Par)-<NUM> and secretory Par-<NUM> was measured in supernatants. Western blotting was done using <NUM>% gels and the proteins were identified using specific antibodies followed by probing with luminol-based Enhanced Chemiluminescent (ECL) western blotting substrate. The results are presented at <FIG>. From <FIG>, it can be seen that both cellular and secretory Par-<NUM> was enhanced with TAM and a combination of TAM and VPA further enhanced the level indicating a synergistic effect.

Claim 1:
A pharmaceutical composition comprising histone deacetylase inhibitor (HDACi) and an estrogen receptor (ER) antagonist for use in a method of treatment of cancer,
wherein the cancer is selected from glioma, malignant glioma, high grade glioma, and
wherein histone deacetylase inhibitor (HDACi) is selected from valproic acid, sodium valproate, valproate semisodium, valpromide, and
wherein estrogen receptor (ER) antagonist is tamoxifen.