WO2021171083A1

WO2021171083A1 - Combination of niclosamide and doxorubicin

Info

Publication number: WO2021171083A1
Application number: PCT/IB2020/061921
Authority: WO
Inventors: Dhirendra S. Katti; Garima LOHIYA
Original assignee: Indian Institute Of Technology Kanpur
Priority date: 2020-02-26
Filing date: 2020-12-15
Publication date: 2021-09-02
Also published as: IN202011008213A

Abstract

The present invention presents a therapeutic combination Niclosamide with Doxorubicin for improved treatment of breast cancer. Niclosamide, an FDA approved anti-helminthic drug is repurposed as a Wnt signaling inhibitor for cancer therapy along with doxorubicin which is a first line treatment for breast cancer. The therapeutic combination of Niclosamide and doxorubicin caused synergistically enhanced death of breast cancer cells irrespective of their clinical subtype (viz. HR positive, HER2 positive and triple negative). Since single agent therapy proves to be inefficient in treating cancer and combination therapy is the need of the hour in clinics, such a combination therapy holds great potential to be an effective treatment option for breast cancer.

Description

COMBINATION OF NICLOSAMIDE AND DOXORUBICIN

Field of the Invention

The present invention relates to a synergistic combination of Niclosamide and Doxorubicin for an improved treatment of cancer.

Background of the Invention

According to World Health Organization (WHO) estimations, breast cancer accounts for maximum cancer related deaths amongst women both globally as well as in India. Further, the number of incidences is increasing at a very fast speed which is the major driver of global breast cancer therapeutics market. “According to the report from the study titled “Breast cancer therapeutics market: global market analysis, insights and forecasts”, the global Breast Cancer Therapeutics Market, which stood at US$ 17,868.8 Million in 2018, will reach US$ 38,573.5 Million by the end of 2026. If these figures prove true, the global Breast Cancer Therapeutics Market will rise at a CAGR of 10.2% between 2018 and 2026.” Growing awareness amongst the female population about the disease and availability of novel treatment options are another major driver of the breast cancer market. In India also, many non-profit and charitable organizations have come forward to bring awareness in the Indian population about the disease and its treatment options. Further, many healthcare market players are investing and putting their efforts to bring effective cure for breast cancer into the market which also drives the breast cancer therapeutics market. Overall, breast cancer therapeutic market is very large both globally as well as in India.

Breast cancer is an adenocarcinoma which originates from the breast tissue. Clinically, there are three sub-groups of breast cancer viz., hormone receptor (HR) positive, Human Epidermal Growth factor receptor (HER2) positive and triple negative breast cancer (TNBC). HR stands for hormone receptor. HR+ means that tumor cells have receptors for the hormone estrogen or progesterone, which can promote the growth of HR+ tumors.

HER2 stands for human epidermal growth factor receptor. HER2+ means that tumor cells make high levels of a protein called HER2/neu, which has been shown to be associated with certain aggressive types of breast cancer and often treated with target-based therapy.

Triple-negative breast cancer (TNBC) is cancer that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein. These results mean the growth of the cancer is not fueled by the hormones estrogen and progesterone, or by the HER2 protein. So, triple-negative breast cancer does not respond to hormonal therapy medicines or medicines that target HER2 protein receptors.

Conventional treatment options for breast cancer include surgery, radiation therapy and chemotherapy. Along with these options, specific hormone therapy and targeted therapy is available for HR positive and HER2 positive cases respectively. However, cancer cells develop resistance against these therapies leading to failure of the treatment and tumor recurrence. Further, molecular heterogeneity, characteristics of breast cancer confounds chemotherapeutic approaches as conventional anticancer agents tend to be inefficient in treating the complex tumour mass containing cells with different molecular signatures. Dysregulated cell signaling pathways which are commonly found in cancer further complicates the situation as it has been found that dysregulated signaling pathways imparts enhanced proliferation, migration, invasion and chemoresistance to cancer cells and are implicated in poor clinical outcomes. Wnt signalling is one such dysregulated pathway in all subtypes of breast cancer, wherein, upregulated Wnt signaling has been shown to be associated with tumorigenesis, metastasis and chemoresistance of cancer cells leading to a poor clinical outcome. JP2015502958A discloses a method comprising combination therapy for treating cancer comprising Wnt pathway inhibitors in combination with MAPK pathway inhibitors, wherein, the Wnt pathway inhibitor is an antibody which specifically binds at least one frizzled (FZD) protein or portion thereof.

CN106714822A discloses a method of treating cancer comprising administering to a subject a therapeutically effective amount of a Wnt pathway inhibitor and a therapeutically effective amount of a mitotic inhibitor, wherein, the Wnt pathway inhibitor and the mitotic inhibitor are administered using a staggered dosing schedule and the Wnt pathway inhibitor is administered first; and wherein, the Wnt pathway inhibitor is: (a) an antibody that specifically binds at least one human Frizzled (FZD) protein, or (b) a soluble receptor comprising the Fri domain of a human FZD protein.

WO2016094457A1 discloses a method for treating basal-like breast cancer in a subject comprising determining an expression level of FOXC1 in a population of tumor cells obtained from the subject; and administering one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, to the subject to treat the cancer if the population of tumor cells expresses FOXC1. The proteasome inhibitor and Wnt inhibitors were bortezomib and iCRT3 respectively, wherein, the basal-like breast cancer includes triple negative breast cancer, ER positive breast cancer, HER2 positive breast cancer, claudin-low cancer.

US20180244783A1 discloses method of treating cancer comprising administering to a subject a therapeutically effective amount of a Wnt pathway inhibitor and a therapeutically effective amount of an immunotherapeutic agent, wherein, the Wnt pathway inhibitor is an antibody.

Given the high attrition rates, substantial costs and slow pace of new drug discovery and development, repurposing of old drugs to treat both common and rare diseases is increasingly becoming an attractive proposition because it involves the use of de -risked compounds, with potentially lower overall development costs and shorter development timelines Such drug repurposing promises faster access of drugs to patients while reducing costs in the long and difficult process of drug development. An example of such drugs is niclosamide an FDA approved anti-helminthic drug which is repurposed as a Wnt signaling inhibitor for cancer therapy.

W02004006906A2 discloses a method for treating a patient having a cancer or other neoplasm by administering to the patient a niclosamide, or a structural or functional analog thereof, and optionally, one or more antiproliferative agents in an amount effective to inhibit the growth of the neoplasm.

US20140294957A1 discloses a method for treating and/or preventing cancer metastasis, comprising administering to a subject in need thereof a therapeutically effective amount of niclosamide or a niclosamide derivate.

JP2015528437A discloses a novel therapeutic use of niclosamide for the treatment of cancer in a combination with an alkylating agent. Furthermore, niclosamide or one of its derivatives can be used for the treatment of solid tumors characterized by low expression of NFKBIA.

Yin L in Oncotarget (2016) Jul 5; 7(27):42126-42138; reported that niclosamide produces significant radiosensitizing effects by inhibiting Wnt/p-catenin signaling in Triple-negative breast cancer (TNBC) cells.

Liu J in Tumor Biol. (2016) Jul; 37(7):9825-35; reported niclosamide treatment in combination with or without cisplatin significantly inhibited Akt, ERK, and Src signaling pathways. In vivo study showed that niclosamide or combination with cisplatin could repress the growth of xenografts originated from either 231-CS or 231-CR cells, with prominent suppression of Ki67 expression. These findings suggested that niclosamide might serve as a novel therapeutic strategy, either alone or in combination with cisplatin, for triple-negative breast cancer treatment, especially those resistant to cisplatin.

Numerous treatment options for breast cancer proves to be inefficient as cancer cells develop resistance towards chemotherapeutic agents, cause tumor relapse and finally leads to very poor overall survival. The multiple signaling pathways such as mTOR, Wnt, STAT3, NF-KB are dysregulated in cancer. This highlights the need to develop a well rationalized combination therapy that makes use of an agent which targets dysregulated signaling pathways along with a conventional anti-cancer agent which may prove to be efficient in improving efficacy and clinical outcome of the treatment. Niclosamide is known to target all these dysregulated pathways.

Niclosamide is a multi-purpose drug found to be effective against multiple cell signaling pathways which are aberrantly expressed in cancer and are responsible for enhanced cancer cell proliferation, survival, metastasis and reduced apoptosis such as STAT-3, m-TOR, NF-KB pathway etc. finally leading to poor treatment outcome. Therefore, in breast cancer proposed combination can be tested for its efficacy in down regulating these pathways also along with Wnt signaling.

The present invention focuses on combination of Niclosamide with Doxorubicin against breast cancer cells. Such combination has not been reported till now.

Doxorubicin is a chemotherapeutic drug also known as Anthracy cline. It slows or stops the growth of cancer cells by blocking an enzyme called topoisomerase 2, which is required by cancer cells to divide and grow.

The inventors of the present invention found that combination of Niclosamide with Doxorubicin shows synergistic effect and prove to be an efficient treatment option for multiple cancer types. The present invention establishes that Niclosamide can potentiate the action of Doxorubicin against breast cancer cell lines if provided in a combination.

Object of the Invention:

It is an object of the present invention is to provide a therapeutic combination effective to potentiate the activity of Doxorubicin against breast cancer cell lines.

It is further an object of the invention is to provide a therapeutic combination comprising Niclosamide and Doxorubicin in their therapeutically active amount, against all the breast cancer subtypes.

It is further an object of the present invention is to provide Niclosamide (Nic) and doxorubicin (Dox) combination for different therapeutic regimens.

It is yet another object of the present invention is to provide a kit comprising therapeutic form of Niclosamide and therapeutic form of Doxorubicin and instructions for the use of both drugs in combination.

Summary of the Invention

The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key /critical elements of the invention or to delineate the scope of the invention.

It is an aspect of the present invention to provide an effective therapeutic approach against all the subtypes of breast cancer, namely HER2, HR+ and TNBC.

It is another aspect of the present invention to provide a combination comprising therapeutically active amounts of Doxorubicin and Niclosamide. Brief description of figures:

Figure 1 illustrates in vitro cytotoxicity of a) Niclosamide (Nic) and b) Doxorubicin (Dox) against MDA MB-231 breast cancer cells at different doses for 24h.

Figure 2 illustrates in vitro cytotoxicity of a) Doxorubicin (Dox) and b) Niclosamide (Nic) against MDA-MB-231 breast cancer cells at different doses for 48h.

Figure 3 illustrates in vitro cytotoxicity of combination of Doxorubicin and Nicloasmide against MDA-MB-231 breast cancer cells at different doses when given sequentially (24h of Nicloasmide treatment followed by 24h of Doxorubicin treatment). A) Illustrates cell viability contour plot and B) illustrates combination index contour plot for the combination of Doxorubicin and Nicloasmide against MDA-MB-231 breast cancer cells.

Figure 4 illustrates in vitro cytotoxicity of combination of Doxorubicin and Nicloasmide against MDA-MB-231 breast cancer cells at different doses for 48h when given simultaneously. A) Cell viability contour plot. B) Combination index contour plot.

Figure 5 illustrates in vitro cytotoxicity of a) Nicloasmide and b) Doxorubicin against SKBR3 breast cancer cells at different doses for 24h.

Figure 6 illustrates in vitro cytotoxicity of a) Nicloasmide and b) Doxorubicin against SKBR3 breast cancer cells at different doses for 48h.

Figure 7 illustrates in vitro cytotoxicity of combination of Doxorubicin and Nicloasmide against SKBR3 breast cancer cells at different doses when given sequentially (24h of Nicloasmide treatment followed by 24h of Doxorubicin treatment). A) Cell viability contour plot. B) Combination index contour plot. Figure 8 illustrates in vitro cytotoxicity of combination of Doxorubicin and Nicloasmide against SKBR3 breast cancer cells at different doses for 48h when given simultaneously A) Cell viability contour plot. B) Combination index contour plot.

Figure 9 illustrates in vitro cytotoxicity of a) Doxorubicin (Dox) and b) Niclosamide (Nic) against MCF7 breast cancer cells at different doses for 24h.

Figure 10 illustrates in vitro cytotoxicity of a) Doxorubicin (Dox) and b) Niclosamide (Nic) against MCF7 breast cancer cells at different doses for 48h.

Figure 11 illustrates in vitro cytotoxicity of combination of Doxorubicin and Nicloasmide against MCF7 breast cancer cells at different doses when given sequentially (24h of Nicloasmide treatment followed by 24h of Doxorubicin treatment). A) Cell viability contour plot. B) Combination index contour plot.

Figure 12 illustrates in vitro cytotoxicity of combination of Doxorubicin and Nicloasmide against MCF7 breast cancer cells at different doses for 48h when given simultaneously. A) Cell viability contour plot. B) Combination index contour plot.

Figure 13 illustrates gene expression analysis of Wnt/p-catcnin signaling markers LEF & CCND1 after treatment with indicated doses of Nicloasmide in MDA- MB-231 cells, wherein, normalization has been done with GAPDH expression.

Figure 14 illustrates gene expression analysis of Wnt/p-catenin signaling markers LEF & CCND1 after combination treatment of Nicloasmide and Doxorubicin in MDA-MB-231 cells for 48h, wherein, normalization was done with GAPDH expression. Figure 15 illustrates a) Cell cycle analysis b) reactive oxygen species (ROS) analysis of MDA-MB-231 cells after single and combination treatment of Doxorubicin and Nicloasmide at indicated doses.

Figure 16 illustrates apoptosis analysis of MDA-MB-231 cells after single and combination treatment of Doxorubicin and Nicloasmide at indicated doses for 24h. a) Sequential combination in which each agent was incubated for 24h and b) Simultaneous combination in which individual agents as well as combination was given for 48h.

Figure 17 illustrates gene expression analysis of Wnt/p-catenin signaling markers TCF, cMYC & CCND1 after treatment with indicated doses of Nic in SKBR3 cells, wherein, normalization was done with GAPDH expression.

Figure 18 illustrates gene expression analysis of Wnt/p-catenin signaling markers TCF, cMYC & CCND1 after combination treatment of Nic and Dox in SKBR3 cells for 48h, wherein, normalization was done with GAPDH expression.

Figure 19 a) illustrates cell cycle analysis of SKBR3 cells after single and combination treatment of Dox and Nic at indicated doses.

Figure 19 b) illustrates reactive oxygen species (ROS) analysis of SKBR3 cells after single and combination treatment of Dox and Nic at indicated doses.

Figure 20 illustrates apoptosis analysis of SKBR3 cells after single and combination treatment of Dox and Nic at indicated doses for a) Sequential combination in which each agent was incubated for 24h and b) Simultaneous combination in which individual agents as well as combination was given for 48h. Figure 21 illustrates gene expression analysis of Wnt/p-catenin signaling markers after treatment with indicated doses of Nic for 24h in MCF7 cells, wherein, normalization was done with GAPDH expression.

Figure 22 a) illustrates cell cycle analysis of MCF7 cells after treatment of Dox and Nic at indicated doses.

Figure 22 b) illustrates reactive oxygen species (ROS) analysis of MCF7 cells after treatment of Dox and Nic at indicated doses.

Figure 23 illustrates apoptosis analysis of MCF7 cells after single and sequential treatment of Dox and Nic at indicated doses.

All the analysis illustrated in above figures is the results of mean values from three experimental repeats.

Detailed Description of the Invention:

The present invention is directed to a synergistically active combination for use in cancer treatment, preferably breast cancer comprising Niclosamide, a non- conventional drug which has been repurposed in cancer treatment and Doxorubicin, a chemotherapeutic agent.

The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of various embodiments of the present disclosure as defined by the claims and their equivalents. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the various embodiments described herein can be made without departing from the scope and spirit of the present disclosure. In addition, descriptions of well-known functions and constructions may be omitted for clarity and conciseness. The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the present disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of various embodiments of the present disclosure is provided for illustration purpose only and not for the purpose of limiting the present disclosure as defined by the appended claims and their equivalents.

It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component surface" includes reference to one or more of such surfaces.

The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising" used herein specify the presence of stated features, integers, steps, operations, members, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, members, components, and/or groups thereof. Also, expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

Described herein, is a combination comprising Niclosamide and its combination effective against all the breast cancer subtypes.

Doxorubicin is the most commonly used anticancer drug in combinations for the treatment of several solid tumors, for example breast cancer. Doxorubicin inhibits topoisomerase activity, affect regulation of gene expression, and produce free radical damage to DNA.

The present invention discloses the effectivity of Niclosamide in combination with Doxorubicin against breast cancer subtypes.

The inventors of the present invention have proposed that Niclosamide, repurposed as Wnt inhibitor potentiates the anticancer activity of Doxorubicin against breast cancer cell lines, when provided in a combination.

Thus it is an aspect of the present invention to provide a combination comprising Niclosamide with Doxorubicin effective against breast cancer cells.

Surprisingly, it has been found by the inventors that the combination of Niclosamide and Doxorubicin is synergistically effective against all the subtypes of breast cancer cell, viz. Hormone receptor (HR) positive, Human Epidermal Growth factor receptor (HER2) positive and triple negative breast cancer (TNBC). In an embodiment of the present invention, there is provided a combination, against different breast cancer subtypes.

The present invention provides an effective combination comprising Niclosamide and Doxorubicin against the breast cancer subtypes, wherein, the breast cancer subtypes are Hormone receptor (HR) positive, Human Epidermal Growth factor receptor (HER2) positive and triple negative breast cancer (TNBC).

The efficiency of the combination of Niclosamide with Doxorubicin against different breast cancer subtypes have been analyzed at many combinatorial ratios at various therapeutically effective concentrations of Niclosamide and Doxorubicin. Surprisingly, various combinatorial ratios having therapeutically effective concentrations of Niclosamide and Doxorubicin are found to cause synergistically enhanced cell deaths for all three subtypes of breast cancer cells, when compared to treatment with either of the agents alone.

In an embodiment of the present invention, there is provided a combination comprising Niclosamide and Doxorubicin against all the subtypes of breast cancer cells.

The present invention provides a combination comprising Niclosamide and Doxorubicin, wherein, the combination is effective against Hormone receptor (HR) positive subtype, Human Epidermal Growth factor receptor (HER2) positive subtype and triple negative breast cancer (TNBC) subtypes. The hydrophilic salt anthracycline glycoside antibiotic Doxorubicin hydrochloride (DOX-HC1), exhibits low oral bioavailability due to active efflux from intestinal P-glycoprotein receptors so, is administered via an intravenous (IV) injection through a central line or a peripheral venous line.

While Niclosamide is administered orally when used as an anti-helminthic, it may also be formulated with surfactants such as polysorbate 80 to enable intra-venous administration. This is based on an extrapolation of other hydrophobic drugs that have been formulated using the above method. Other studies on Niclosamide have reported its administration either orally or intraperitonially or by intravenous route.

The present invention provides a combination comprising therapeutically effective concentrations of Doxorubicin and Niclosamide, wherein, Doxorubicin is administered by intravenous route and wherein, Niclosamide is administered orally or intraperitonially. The breast cancer cell lines (HR+, HER2+ and TNBC subtypes) are treated with multiple concentrations of Nic and Doxorubicin in both of the therapeutic regimens, i.e., simultaneous and sequential therapeutic regimens.

In the first approach, the drugs are given sequentially i.e. cells are first treated with various concentrations of Niclosamide for 24h and then Doxorubicin is added and cells are treated for another 24 h.

In the second regimen, both the drugs are given simultaneously i.e. cells are treated with multiple concentrations of Niclosamide and Doxorubicin at the same time for 48h.

The present inventors found that the combination of Nic and Doxorubicin causes synergistically enhanced death of all subtypes of breast cancer cells with combination index (Cl) of <0.5 at both the treatment regimens and at multiple concentrations.

The present invention provides a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin, wherein, the combination is effective in sequential therapeutic regimen, wherein, the sequential therapeutic regimen comprises administration of Niclosamide for 24 hours followed by administration of Doxorubicin for 24 hours. The present invention provides a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin, wherein, the combination is effective in simultaneous therapeutic regimen, wherein, the simultaneous therapeutic regimen comprises administration of Niclosamide and Doxorubicin at the same time for 48h. The term “therapeutic effective concentration” as used in present embodiment can be defined as concentration of drugs to achieve a desired therapeutic effect with lower doses or lower toxicity drugs, to show a synergistic effect.

In an embodiment of the present invention, there are provided a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin, causing synergistically enhanced cell death of all the breast cancer subtypes in both of the therapeutic regimens.

The present invention in an aspect provides a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin, against all the subtypes of breast cancer cells; wherein, the combination is administered in either of the therapeutic regimens (i.e. sequential and simultaneous incubation of agents) at multiple combinatorial concentrations.

Such versatile combination which is effective against all the breast cancer subtype at multiple concentrations and schedules has not been reported yet.

It is thus an aspect of the invention to provide dosing of Niclosamide followed by dosing of Doxorubicin as an effective therapeutic combination against all the subtypes of breast cancer.

It is yet another aspect of the invention to provide simultaneous dosing of Niclosamide and Doxorubicin as an effective therapeutic combination against all the subtypes of breast cancer.

In further embodiment of the invention, the mechanism of the synergism is unveiled. The inhibitory effect of Niclosamide on Wnt signaling pathway is determined in both the cases, for Niclosamide alone and for combination with Doxorubicin by treating MDA-MB-231 cell lines, SKBR3 cell lines and MCF7 cell lines in both sequential and simultaneous therapeutic regimens.

MDA-MB-231 cell lines represents as a model of triple-negative breast cancer (TNBC); SKBR3 cell lines as a model of HER2 positive breast cancer; and MCF7 cell lines as a model of hormone receptor positive breast cancer.

The downregulation of major transcription factors and target genes of Wnt signaling including CCDN1 (cyclin Dl) by Niclosamide have been observed for all the three breast cancer subtypes.

Since cyclin Dl regulates transition of cells from G0/G1 to S phase of the cell cycle, therefore cell cycle is further analyzed after single and combination therapy of both the drugs.

The cell cycle analysis of both Niclosamide alone and the combination of Niclosamide with Doxorubicin is found to show G0/G1 arrests in MDA-MB-231 cells, SKBR3 cell lines and MCF7 cell lines when compared to Doxorubicin alone.

In an embodiment of the invention there is provided a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin causing cancer cells arrest at G0/G1 phase of cell cycle for all the breast cancer subtypes.

The present invention provides a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin, wherein, the combination is effective to causes cancer cells arrest at G0/G1 phase of cell cycle in Triple Negative Breast Cancer (TNBC), Hormone Receptor Positive (HR+) breast cancer and Human Epidermal Growth Factor Receptor 2 positive (HER2+) breast cancer subtypes.

It is known that cells are more prone to apoptosis in G1 checkpoint as compared to G2 checkpoint of DNA repair if they further encounter any DNA damage conditions such as reactive oxygen species (ROS).

Anthracyclines such as Doxorubicin are known to generate ROS, therefore, after cell cycle analysis ROS generation analysis are determined for single treatment of Niclosamide alone and Doxorubicin alone and for combination comprising Niclosamide and Doxorubicin in both of the therapeutic regimens.

The present inventors found that both Niclosamide and Doxorubicin alone and the combination of Niclosamide with Doxorubicin produce significantly enhanced amount of ROS in MDA-MB-231 cell lines, SKBR3 cell lines and MCF7 cell lines which make the G0/G1 arrested cells more prone to apoptosis.

In an embodiment of the present invention there is provided a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin for enhancing the production of ROS all the breast cancer subtypes.

The present invention provides a combination comprising therapeutically effective concentrations of Niclosamide and Doxorubicin; wherein, the combination enhances the ROS production in Triple Negative Breast Cancer (TNBC), Hormone Receptor Positive (HR+) breast cancer and Human Epidermal Growth Factor Receptor 2 positive (HER2+) breast cancer subtypes.

To further establish the cytotoxic effect of the combination, cell apoptosis are analyzed in MDA-MB-231 cell lines, SKBR3 cell lines, and MCF7 cell lines after single vs combination (sequential as well as simultaneous) therapy in various concentration and combinatorial ratios. In an embodiment of the invention there are provided synergistically active combinations comprising effective concentrations of Niclosamide and Doxorubicin enhancing the cell apoptosis in triple-negative breast cancer (TNBC) cell lines; SKBR3 cell lines; and MCF7 cell lines.

Table 1 presents the various combinations comprising effective concentrations of Niclosamide and Doxorubicin tested for cell cytotoxicity analysis.

Table 1: Various effective concentrations of Niclosamide and Doxorubicin tested for cell cytotoxicity analysis

The percentages of cells in late stage apoptosis are found statistically higher in case of combinations comprising Niclosamide and Doxorubicin in all the cell lines for both sequential and simultaneous therapeutic regimen, when compared to the effect of single agents (Niclosmide alone or Doxorubicin alone). In an embodiment of the present invention, there is provided the effective concentrations of synergistically active combination comprising Niclosamide and Doxorubicin against MDA-MB-231 cell lines which is against triple-negative breast cancer (TNBC) in sequential and simultaneous therapeutic regimens.

The present invention provides a combination comprising 700-1400nM concentration of Niclosamide and 50-1000nM concentration of Doxorubicin is found synergistically active against MDA-MB-231 cell lines in both sequential and simultaneous therapeutic regimens.

The present invention provides a synergistically active combination comprising Niclosamide and Doxorubicin against MDA-MB-231 cell lines for simultaneous therapeutic regimens, wherein, the combination comprises 700-1400nM concentration of Niclosamide and 50-1000nM concentration of Doxorubicin.

The present invention provides a synergistically active combination comprising Niclosamide and Doxorubicin against MDA-MB-231 cell lines for sequential therapeutic regimens, wherein, the combination comprises 700-1400nM concentration of Niclosamide and 50-1000nM concentration of Doxorubicin.

The present invention thus provides a synergistically active combination comprising Niclosamide and Doxorubicin effective against breast cancer (TNBC) cells; wherein, the combination comprising 700-1400nM of Niclosamide and 50- lOOOnM of Doxorubicin is effective for both simultaneous and sequential therapeutic regimens.

In an embodiment of the present invention, there is provided the effective concentrations of synergistically active combination comprising Niclosamide and Doxorubicin against SKBR3 (HER2 positive breast cancer) cell lines in sequential and simultaneous therapeutic regimens.

The present invention provides a synergistically active combination comprising Niclosamide and Doxorubicin against SKBR3 cell lines for simultaneous therapeutic regimens, wherein, the combination comprises 300-600nM concentration of Niclosamide and 100-400nM concentration of Doxorubicin.

The present invention provides a synergistically active combination comprising Niclosamide and Doxorubicin against SKBR3 cell lines for sequential therapeutic regimens, wherein, the combination comprises 300-600nM concentration of Niclosamide and 25-200nM concentration of Doxorubicin.

The present invention thus provides a synergistically active combination comprising Niclosamide and Doxorubicin effective against HER2 positive breast cancer cells; wherein, the combination comprising 300-600nM of Niclosamide and 100-400nM of Doxorubicin is effective for simultaneous therapeutic regimens and wherein, the combination comprising 300-600nM of Niclosamide and 100-400nM of Doxorubicin is effective for sequential therapeutic regimen.

In an embodiment of the present invention, there is provided the effective concentrations of synergistically active combination comprising Niclosamide and Doxorubicin against MCF7 (hormone receptor positive breast cancer) cell lines in sequential and simultaneous therapeutic regimens.

The present invention provides a synergistically active combination comprising Niclosamide and Doxorubicin against MCF7 cell lines for simultaneous therapeutic regimens, wherein, the combination comprises 50-100nM concentration of Niclosamide and 50-500nM concentration of Doxorubicin.

The present invention provides a synergistically active combination comprising Niclosamide and Doxorubicin against MCF7 cell lines for sequential therapeutic regimens, wherein, the combination comprises 800-1200nM concentration of Niclosamide and 400-1200nM concentration of Doxorubicin.

The present invention thus provides a synergistically active combination comprising Niclosamide and Doxorubicin effective against hormone receptor positive breast cancer cells; wherein, the combination comprising 50-100nM of Niclosamide and 50-500nM of Doxorubicin is effective for simultaneous therapeutic regimens and wherein, the combination comprising 800-1200nM of Niclosamide and 400- 1200nM of Doxorubicin is effective for sequential therapeutic regimen.

In addition to the cell apoptosis, the over expressing of Wnt signaling imparts chemoresistance to cancer cells, further downregulation of the same makes cancer cells more chemo-sensitive, thereby Niclosamide is found to enhance the cytotoxicity of Doxorubicin in case of combination therapy.

In an embodiment of the present invention, there is provided a Wnt signaling inhibitor which enhances the cytotoxicity of Doxorubicin against triple-negative breast cancer (TNBC); HER2 positive breast cancer; and hormone receptor (HR+) positive breast cancer subtypes, wherein, the Wnt signaling inhibitor is provided in combination with Doxorubicin; wherein, the Wnt signaling inhibitor is Niclosamide; and wherein, the combination is effective in both sequential and simultaneous treatment. Thus, it presents an aspect of the invention is to provide a mechanism or technique to enhance the cytotoxicity of Doxorubicin against all the subtypes of breast cancer, wherein, Doxorubicin is provided in combination with Niclosamide; wherein, the combination is cytotoxic than either of the individual agent and; wherein, the combination is applicable in both sequential and simultaneous treatment.

The invention also encompasses a kit comprising therapeutically effective therapeutic forms of Niclosamide and Doxorubicin and instructions for the use of both drugs in combination against all the subtypes of breast cancer; wherein, Niclosamide is administered either sequentially or simultaneously with Doxorubicin.

The cytotoxic effect of the combination of Niclosamide (Nic) and Doxorubicin (Dox) against various cancer cell lines and at various dose regimens will now be described with reference to the accompanying experiment and illustrated graphs provided to assist in a comprehensive understanding of the invention. It includes specific details of the process to assist in that understanding. It is assumed that a person skilled in the art can make use of the above description in its widest scope. The preferred embodiment and example is regarded merely as descriptive but in no way limiting disclosures.

Examples

Experiment 1: Cytotoxicity assessement of Niclosamide (Nic) and Doxorubicin (Dox) on Breast Cancer Cells

To assess the cytotoxicity of individual agents, triple negative breast cancer cell line, MDA-MB-231; HER2 positive breast cancer cell line, SKBR3; and hormone receptor positive breast cancer cell line MCF7 were treated with different concentrations of niclosamide and doxorubicin for 24h and 48h. Briefly, 5,000 cells/well were seeded in 96 well cell culture plate and post 24h of seeding, cells were treated with different concentrations of Dox and Nic for 24h and 48h. Dox stock was made in saline and Nic stock was made in DMF. Appropriate vehicle controls were kept in case of Nic and maximum vehicle cone used was 0.1%. Post treatment, cells were washed twice with IX PBS and cell viability was assessed using resazurin assay in which cells were incubated with resazurin (Sigma Aldrich) in complete media at a cone of 0.02mg/ml for 5h and then fluorescence is measured at excitation of 540nm and emission of 600nm. Afterwards, IC50 values were calculated through non-linear regression analysis using Graph pad Prism software. Figure 1 and 2 illustrates in vitro cytotoxicities of Niclosamide and Doxorubicin against MDA-MB-231 breast cancer cells at different doses for 24 hours and 48 hours respectively. Figure 5 and Figure 6 illustrates in vitro cytotoxicities of Niclosamide and Doxorubicin against SKBR3 breast cancer cells at different doses for 24 hours and 48 hours respectively. Figure 9 and 10 illustrates in vitro cytotoxicities of Niclosamide and Doxorubicin against MCF7 breast cancer cells at different doses for 24 hours and 48 hours respectively.

Experiment 2: Cytotoxicity assessement of combination of Niclosamide (Nic) and Doxorubicin (Dox) on breast cancer cells

To assesss the cytotoxicity of combination of Nic and Dox on breast cancer cell lines(MDA-MB-231, SKBR3 and MCF7), similar protocol was followed as discussed above. Briefly, 5,000cells/well in 96well cell culture plate were treated with differenent concentrations of Nic and Dox in sequential (Nic for 24h followed by Dox for another 24h) and simultanoeus treatment (Nic and Dox together for 48h) regimens and cytotoxicity of the combination was evaluated through resazurin assay.

The combination index (Cl) of various combinatorial concentrations are calculated by calculating the effective concentrations. of individual drugs (EC_F) which is capable of causing the same percentage of cell death(lOO-F) as caused by combination of Nic and Dox using following formula-

where,

EC_F = effective cone of single drug needed to cause same percentage of cell death as caused by combination of drugs

F= percentage of viable cells left after treatment with combination (sequential or simultaneous) of Nic and Dox

100-F = percentage of dead cells after treatment with combination (sequential or simultaneous) of Nic and Dox H= hill slope of non-linear regression of single drug

EC5_O= effective cone of single drug required to cause 50% cell death, also known as IC50

After calculating the ECF for both Nic and Dox (ECFM_C) and (ECFD_OX) for each combinatorial cell death, Cl for that pair of combination of Nic and Dox are calculated using following formula

where,

CI(F) = combination index of combination of Nic and Dox which caused (100- F) percentage of cell death

Nic (Fcombo) = concentration of Nic required to cause (100-F) % of cell death when used in combination

EC (FNic) = concentration of Nic required to cause (100-F) % of cell death when used individually Dox (Fcombo) = concentration of Dox required to cause (100-F) % of cell death when used in combination

EC (FDox) = concentration of Dox required to cause (100-F) % of cell death when used individually

We found that combination of Nic and Dox caused synergistically enhanced death of all subtypes of breast cancer cells i.e. combination index <1 at many concentrations in both sequential(Niclosamide followed by Doxorubicin) (Figure 3, 7, 11) as well as simultaneous (Figure 4, 8, 12) treatment regimens.

Experiment 3: Elucidating mechanism of synergism between Niclosamide and Dox

Since, Niclosamide is a Wnt signaling inhibitor, the Wnt signaling is assessed in various breast cancer cell lines, MDA-MB-231 cells after treatment with 900nM and 1200nM of Niclosamide both individually and in combination with 400nM Doxorubicin; SKBR3 cells after treatment with 600nM of Niclosamide both individually and in combination with lOOnM Doxorubicin, and MCF7 cells after treatment with 900nM of Niclosamide both individually and in combination with 400nM Doxorubicin. The downregulation of Wnt signaling caused by Niclosamide has been observed in all the breast cancer cell lines for both the therapeutic regimens.

Experiment 3.1: Gene expression analysis of Wnt signaling markers

Gene expression analysis of various Wnt signaling markers was performed in all breast cancer cell lines after treatment with selected concentrations of Nic either alone or in combination with Dox through real time qPCR. The total RNA was extracted using TRI reagent® (Sigma Aldrich, USA) according to manufacturer’s protocol. Briefly, treated cells were trypsinized and lysed with 700 pL TRIZOL followed by addition of 140 pL of chloroform and centrifugation at 13000 rpm at 4°C for 15min. The aqueous phase obtained after centrifugation was pipetted out into fresh 1.5mL micro-centrifuge tubes and total RNA was pelleted down by adding equal volume of isopropanol and centrifugation at 13000 rpm for 15min at 4°C. The RNA pellet obtained was washed two times with 75% chilled ethanol and allowed to dry at 37°C to remove excess ethanol. Dried pellet was resuspended in RNase free water and quantified for total RNA using a Nanodrop (Thermo Scientific, USA). cDNA was synthesized from 500ng of total RNA using a cDNA reverse transcription kit (Applied Biosystems, USA) according to manufacturer’s protocol. Afterwards, quantitative PCR was performed in Step One Plus qPCR machine (Applied Biosystems, USA) using SYBR Green Master Mix and the relative expression of the target gene was calculated using AACt method by normalizing the Ct of the target gene to the average Ct of the housekeeping gene GAPDH. The fold change expression of the target gene was calculated as 2 ^{( DDa)} with respect to untreated controls.

The results as illustrated in curve plots provided in Figure 13 and 14 shows that Nic causes downregulation of Wnt signaling in MDA-MB-231 cells for both the conditions, Niclosamide alone and when in combination with Doxorubicin.

Similarly, the results as illustrated in curve plots provided in Figure 17 and 18 shows that Nic cause downregulation of Wnt signaling in SKBR3 cells and Figure 21 shows that Nic cause downregulation of Wnt signaling in MCF7 cell lines.

Experiment 3.2: Cell cycle analysis

Cell cycle analysis has been studied in all breast cancer cells after single and combination (sequential and combination) treatment of Nic and Dox using Muse® Cell cycle kit (Merck, Millipore, USA) according to manufacturer’s protocol. Briefly, treated cells were trypsinized, transferred into 1.5mL micro-centrifuge tubes and pelleted down. Cells pellets were once washed with IX PBS and resuspended in 50 pL of IX PBS. Resuspended cells were added dropwise into a tube containing lmL of ice-cold 70% ethanol and left for at least 3h for fixation at -20°C. Afterwards, ethanol-fixed cells were centrifuged and washed twice in 1XPBS. Finally, cells were suspended in 200 pL of Muse® cell cycle reagent and incubated for 30min at room temperature and thereafter analyzed in Guava® Muse® Cell Analyzer (Merck, Millipore, USA).

The result of the analysis has been provided in curve plot of Figure 15 (a), which shows the efficacy of both Niclosamide and combination of Niclosamide and Doxorubicin in causing G0/G1 cell cycle arrest in MDA-MB-231 cells when compared to Doxorubicin alone. Similarly, Figure 19 (a) and 22 (a) shows the efficacy of both Niclosamide and combination of Niclosamide and Doxorubicin in causing G0/G1 cell cycle arrest in SKBR3 cells and MCF7 cells respectively when compared to Doxorubicin alone.

Experiment 3.3: Reactive oxygen species (ROS) analysis

Nic and Dox treated cells have been analyzed for ROS using DCFDA (Thermo Scientific, USA). Briefly, 5,000 cells/well were seeded in 96well black cell culture plates and post 24h of seeding, cells were washed once with HBSS. Afterwards, cells were incubated with 50 mM of DCFDA in HBSS for 45min in CO2 cell culture incubator maintained at 37 °C. Cells were washed again with HBSS after incubation and treated with indicated doses of Nic and Dox for 4h and 8h. Fluorescence is then measured using multiplate reader (Synergy, Hybrid H4) without removing treatment with excitation at 485nm and emission at 535nm.

The result of the analysis have been provided in curve plots of Figure 15 (b), 19 (b) and 22 (b) which confirm the enhancement in production of ROS in MDA- MB-231 cells, SKBR3 cells and MCF7 cells respectively and which makes the G0/C1 cell cycle arrest more prone to apoptosis.

Experiment 3.4 Apoptosis analysis

Apoptosis analysis has been performed in cells treated with indicated doses of Nic and Dox using Muse® Annexin V and Dead Cell kit (Merck, Millipore, USA) according to manufacturer’s protocol. Briefly, treated cells were washed with IX PBS, trypsinized and transferred into 1.5mL micro-centrifuge tubes. 100 pL of cell suspension was mixed with 100 pL of Muse® Annexin V and Dead Cell reagent and incubated for 20min at room temperature. Afterwards, cells are analyzed in Guava® Muse® Cell Analyzer (Merck, Millipore, USA).

The result as illustrated in curve plots of Figure 16, Figure 20 and Figure 23 which show high percentage of cells in late stage apoptosis in case of combination (both sequential and simultaneous) of Niclosamide and Doxorubicin when compared to single agentsin MDA-MB-231 cells, SKBR3 cells and MCF7 cells respectively. Advantage of Invention:

The present invention provides a therapeutic combination of Doxorubicin and Niclosamide as Wnt signaling inhibitor that is synergistically effective against all the subtypes of breast cancer cell lines. The therapeutic combination of the present invention can be administered by patient of breast cancer cell line in either mode, sequential or simultaneous in any therapeutic regimen. Since Wnt signaling is upregulated in many other cancer types, the present invention can be used for treating all such cancers. Further, along with Wnt signaling, Niclosamide downregulates various other pathways known to be dysregulated in cancer cells such as mTOR, STAT3, NF-KB. Therefore, apart from breast cancer, the combination therapy based on Niclosamide and Doxorubicin can be used for many other cancer types as well.

Claims

CLAIMS:

1. A combination comprising therapeutically effective concentrations of Doxorubicin and Niclosamide.

2. The combination as claimed in claim 1, wherein, the combination is effective in the treatment of breast cancer.

3. The combination as claimed in claim 2, wherein, the breast cancer is selected from HER2 positive breast cancer, hormone receptor positive (HR+) breast cancer, and triple negative breast cancer (TNBC).

4. A therapeutic combination comprising Niclosamide and Doxorubicin; wherein, the combination enhances the ROS production; wherein, the combination is effective in any therapeutic forms and in any therapeutic regimens, i.e., sequential and simultaneous.

5. The combination as claimed in claim 1, wherein, Niclosamide and Doxorubicin is administered sequentially, Niclosamide followed by Doxorubucin.

6. The combination as claimed in claim 1, wherein, Niclosamide and Doxorubicin is administered simultaneously.

7. A combination as claimed in claim 1, wherein, the combination effective against Triple Negative breast cancer cells comprises 700-1400 nM concentration of Niclosamide and 50-1000 nM concentration of Doxorubicin, and wherein, the combination is effective in both therapeutic regimens, i.e., sequential and simultaneous.

8. A combination as claimed in claim 1 effective against (HER2) positive breast cancer cells wherein, the combination comprises 300-600nM concentration of Niclosamide and 100-400nM concentration of Doxorubicin for simultaneous therapeutic regimen; and wherein, the combination comprises 300-600 nM of Niclosamide and 25-200 nM of

Doxorubicin for sequential therapeutic regimen.

9. A combination as claimed in claim 1 effective against (HR+) breast cancer cells wherein, the combination comprises 50-100nM of Niclosamide and 50-500nM of Doxorubicin for simultaneous therapeutic regimens; and wherein, the combination comprises 800-1200 nM of Niclosamide and 400-1200 nM of Doxorubicin for sequential therapeutic regimens.

10. The combination as claimed in preceding claims, wherein, Doxorubicin is administered by intravenous route and wherein, Niclosamide is administered orally, intraperitonially or by intravenous route.

11. The combination as claimed in preceding claims, wherein, in sequential therapeutic regimens Niclosamide is provided prior to Doxorubicin.

12. A kit comprising (i) a combination comprising Doxorubicin and Niclosamide and (ii) manual for administration of the combination, wherein, the combinations comprise effective concentrations of Niclosamide and Doxorubicin against triple negative breast cancer, (HR) positive breast cancer and (HER2) positive breast cancer subtypes.

13. A method for treating breast cancer by administering a combination comprising therapeutically active amounts of Doxorubicin and Niclosamide.

14. A method for enhancing the cytotoxicity of Doxorubicin against breast cancer cell lines, the method comprising administering Niclosamide in combination with Doxorubicin.