Composition for treating paclitaxel-resistant breast cancer and method for preparation thereof

Abstract

The compound IIIM-152 and its derivatives have been reported as potent stem cell modulators by down-regulating beta-catenin signalling. TNBCs are highly aggressive cancers and chemotherapy remains one of the primary treatment options. However, CSCs remains non-responsive to the Paclitaxel treatment leads to disease recurrence and metastasis and is one of the major clinical challenges. The Combinatorial treatment of IIIM eliminates the Paclitaxel Resistant Cancer Stem Cells, whereas Paclitaxel only treatment is unable to target CSCs subpopulation. Similarly, a combination of Paclitaxel-IIIM152, showed significant reduction in tumor burden and decrease in number of CSCs in excised tumours. The combination of IIIM-152 and Paclitaxel proves to be clinically efficacious in treating the Paclitaxel non-responsive highly aggressive residual cancer stem cells to achieve long lasting treatment. The invention also provides a method for preparing the composition comprising Paclitaxel and IIIM-152.

Description

[0001] COMPOSITION FOR TREATING PACLITAXEL-RESISTANT BREAST CANCER AND METHOD FOR PREPARATION THEREOF

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a composition for treating paclitaxel-resistant breast cancers. The composition comprises Paclitaxel and IIIM-152 in the effective range of the ratio of 1:5 to 1:10 along with suitable additives. The present invention also relates to the method for preparing the composition comprising Paclitaxel and IIIM-152.

[0004] BACKGROUND AND PRIOR ART OF THE INVENTION:

[0005] Triple-Negative Breast Cancer

[0006] Triple-negative breast cancer, a special subtype of breast cancer with distinct clinical features, has a poorer prognosis and is characterized by larger tumor volume, a higher degree of proliferation, early risk of relapse, prevalence of visceral and metastatic progression, and aggressive behaviour. Gene expression analysis has shown that nearly 70% of all TNBC tumors are basal-like and thus initially TNBCs were often used as surrogates for basal-like breast cancer due to the absence of ER, PR, and Her2 but a substantial number of basal-like tumors express ER / PR or HER2 and at least one other basal molecular marker, mainly cytokeratin 5 and 6 (CK5 / 6), CK14, CK17, caveolin 1 / 2, and EGF receptor (EGFR). TNBCs are usually ductal or medullary or invasive or metaplastic and grade III tumors with elevated mitotic rates and p53 mutations. It is more common in younger women and is correlated with BRCA-1 mutation in general. TNBC cells spread from the local site to the bones, lungs, liver, and brain where the survival rate declines from 90 to 20%. TNBC is itself biologically heterogeneous and is further sub-classified into seven subclasses based primarily on comprehensive gene expression profile and prediction analysis of microarray-50 (PAM50) classifier- Basal-like-l(21%), Immunomodulatory (23%), Mesenchymal (20%), Basal-like-2 (8%), Mesenchymal stem-like (10%), Luminal androgen receptor (1%), and Unstable (17%). Consequently, TNBCs constitute a distinct histopathological category and therefore pose a major challenge for the diagnosis and treatment of aggressive breast cancers. Therefore, the main aim of Triple-Negative Breast Cancer investigators is to define the prognostic factors or targets that can be useful for the treatment of this particular tumor subtype. While advanced therapies exist, the prognosis for this advanced stage is poor. Chemotherapy is the sole treatment option for TNBC management owing to the absence of precise targets and acquired resistance to available radio and chemo agents is huge clinical challenge. Current Treatment options in TNBC and the need for new agents:

[0007] The chemotherapeutic s currently available, such as paclitaxel, cisplatin, cyclophosphamide, doxorubicin, fluorouracil, etc, target conventional mechanisms of TNBC. Besides these, irinotecan, trabectedin, and ABI-007 were also seen effective in TNBCs. In addition, preclinical and clinical trials involving inhibition of poly-ADP ribose polymerase (PARP) enzymes with PARP inhibitors such as Iniparib and Olaparib, have shown some potential in early trials, which was further augmented when provided in combination with gemcitabine and carboplatin. The monoclonal antibody cetuximab is also used to combat TNBC. Erlotinib and lapatinib have also been used against TNBC. For TNBC cases, the combination of erlotinib, tyrosine-kinase inhibitors (TKI), with docetaxel or carboplatin culminated in a 40% pathologic complete response (pCR). TNBCs are commonly considered to be more aggressive than that other subtypes and therefore undergo increased glycolysis, metformin demonstrated impressive results by induction of AMP-activated protein kinase. Bevacizumab, a mAb against VEGF-A, alone and in combination with paclitaxel enhanced response rate (RR) in metastatic TNBC to 48%. Other angiogenic inhibitors Avastin and Docetaxel (A VADO) lowered the risk by 47%. Therapeutic targets that regulate the cell cycle and DNA damage response in TNBC such as checkpoint kinase 1 / 2 (CHK1 / 2) has received significant attention in TP53 mutated TNBC tumors. In women with BRCA mutation, cisplatin had a higher degree of pathological complete response (pCR). A bevacizumab neoadjuvant combination with cisplatin exhibited 15% of pCR. In combination with auxiliary cytotoxic agents, epirubicin or 5-FU, cisplatin showed a response rate of 40%. However, in metastatic TNBC cases, chemoresistance and lack of responsiveness to chemotherapies are responsible for 90% of drug failures.

[0008] Claim of this invention is based on Current Clinical Challenges and Shortcoming of available treatment options:

[0009] Although conventional therapies are successful to a certain extent, they often have certain shortcomings. Chemotherapy does not guarantee complete tumor elimination, thus increasing metastasis risks. Many cancer cells evolve with various mechanisms to acquire resistance to these agents. Major clinical challenge with these therapies is the nonresponding stem- like cells called cancer stem cells (CSCs), a fraction of cells established to be responsible for tumor metastasis drug resistance, and recurrence. Overview of Cancer Stem cell in Breast cancer and need to discover new agents to eliminate these cells.

[0010] Current invention is based on Targeting Cancer Stem Cells (CSCs) in TNBCs to achieve effective and long lasting therapeutic outcome to eliminate drug resistant cancer stem cells. Recent studies suggest that cancer stem cells play an important role in tumorigenesis and tumor biology of TNBC. CD44+ / CD24- and ALDH1+ breast cancer stem cells are enriched in TNBC and may contribute to the propensity of TNBC for chemotherapy resistance and tumor metastasis. There is new evidence to support the evaluation of cancer stem cells in TNBC for diagnostic purposes. Targeting cancer stem cells may also be a promising, novel strategy for the treatment of TNBC. Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are characterized as a subpopulation of self-renewing cancer cells that possess a strong tumorigenic capacity and can undergo multilineage differentiation to produce all forms of malignant cells. CSCs accelerate tumor development and are known to be a source of tumor relapse and disease progression, possibly by their therapy resistance and metastatic ability. Despite all the advances and the utilization of modem treatments, cancer cannot be fully treated in its late stages. While chemotherapeutic agents currently used can reduce tumor mass, recurrence is normal. Therefore, CSCs are being investigated widely in multiple cancers.

[0011] Pathological significance of cancer stem cells in breast cancer disease Tumor aggressiveness / metastasis

[0012] There is growing evidence that CSCs are involved in metastatic breast cancer development. This is particularly important considering that the majority of deaths from cancer are attributed to secondary lesions that have spread from the primary tumor. Immunohistochemistry of breast cancer cells collected from the bone marrow utilizing the CD44highCD24- / low phenotype indicates that metastatic tumors had a significantly higher proportion of CSCs relative to the primary site. CSCs isolated by ALDH activity has been shown to mediate metastasis in both in vitro and xenograft studies of inflammatory breast cancer (IBC) models. In addition, the presence of ALDH+ cells in tumors in IBC patients was associated with both early metastasis onset and reduced overall survival. CSCs were also suggested to modify the structure of the tissue by inducing epithelial remodeling. This disturbance of normal tissue structure may be another mechanism by which CSCs may lead to metastasis. Therapy resistance and tumor recurrence

[0013] A possible mechanism for understanding the recurrence of breast cancer is the resistance of CSCs to chemo therapy / radiotherapy. The CSCs population is enriched during neoadjuvant chemotherapy, indicating that CSCs are more resistant than the bulk of the tumor. Treatment with chemotherapeutic medications (paclitaxel or 5-fluorouracil) with both SUM159 and SUM149 cells contributed to enrichment in the percentage of stem-like cells. The association between EMT and CSCs is also applicable to therapeutic resistance, as cells undergoing EMT are more chemotherapeutic resistant. Cells derived from tumors with Her2-antigen dysfunction undergoing EMT had upregulated protein pump expression (BCRP and PGP) associated with drug resistance. These cells were thus protected from mitoxantrone and etoposide pharmacological treatments. The mesenchymal tumor cells also had elevated levels of DNA repair enzyme and were resilient to ionizing radiation.

[0014] Induction of CSC differentiation: Differentiation Therapy

[0015] Differentiation therapy is another strategy for targeting specifically CSCs. CSCs are usually locked into an undifferentiated state, their capacity for self-renewal and their differentiation potential render them extremely tumorigenic. Therefore, eliminating this blockade and pushing them back into differentiated constantly dividing cells could eradicate them in conjunction with chemotherapy and thus minimize the risk of tumor relapse. The first breakthrough was seen when all-trans retinoic acid (ATRA) was used to treat acute promyelocytic leukemia. ATRA induced the differentiation and rapid division of leukemic promyelocytes into granulocytes. This strategy's success has led to the assumption that differentiation therapy may be used for addressing other cancers. In the case of CSCs, this strategy will cause their exit from the CSC state into the more differentiated or epithelial state (126). Several studies have documented that CSCs lack markers of differentiation. For example, breast, colon, and prostate CSCs lack epithelial differentiation markers cytokeratin, whereas glioblastoma CSCs lack glial fibrillary acidic protein.

[0016] CSC differentiation can be an effective clinical strategy, since the bulk of the tumor has less potential for proliferation and is more responsive to chemotherapy or radiation therapy. Potential way-out that induce quiescent CSCs to transform into more mature tumor cells involve stimulation of various signalling pathways, such as morphogen-driven signalling cascades, modifying gene expression profiles with microRNAs, and epigenetic differentiation therapy. Piccirillo et al., for example, used BMP signalling to cause differentiation of CSCs in models of human brain cancer. Moreover, administration of BMP4 to glioblastoma cultures in vitro or to human brain cancer in vivo has resulted in differentiation of glioblastomas and substantially decreased the number of CD133 + cells.

[0017] Methyl 2-(2-(3-methylbenzoyl)-5-( / n-tolyl)-7H-imidazol-l-yl)acetate (IIIM-152), an amino acid-based imidazole:

[0018] A small molecule able to selectively target and eliminate Paclitaxel Resistant Cancer Stem Cells (CSCs) in TNBCs

[0019] The anticancer efficacy of imidazoles and imidazole-based derivatives has been evaluated in various cancers. For instance, imidazole-based molecules inhibited tumor growth in a panel of the National Cancer Institute's 60 human cancer cell lines. Remarkable cytotoxic activities were shown against various cell lines by a novel series of imidazoles. The trisubstituted- imidazoles decreased the expression of cyclin DI and increased the cleavage of PARP. Furthermore, imidazoles have been reported to act against tumor resistance and relapse.

[0020] A recent study showed the effectiveness of imidazoles in anti-cancer therapy and abrogation of tumor resistance by modulation of microtubule dynamics. Another study indicated that imidazole inhibited colon cancer cell growth through activation of apoptosis and cell cycle arrest. These findings provide a strong rationale for evaluating the chemoprevention property of imidazoles in clinical trials.

[0021] Increasing data support the CSC hypothesis, which suggests that a small proportion of CSCs drive and maintain a variety of cancers. The theory of CSCs has far-reaching clinical significance for cancer therapies and prevention. Recent findings suggest that CSCs have the potential to induce tumor resistance and relap se / recurrence. Recent findings suggest that CSCs have the potential to elicit tumor resistance and relapse / recurrence. The lack of effectiveness of existing chemotherapies in advance and metastatic cancer demands different strategies to particularly address CSC population. Therefore, therapies aimed towards both differentiated cancer cells and CSCs will offer advantages in the treatment of such diseases. Researchers also observed that certain molecules, such as salinomycin, curcumin, are effective chemoprevention compounds against CSCs. We have therefore used both in vitro and in vivo methods to assess how imidazoles work against breast CSCs, relying on the chemopreventive nature of imidazoles and the consequences of the CSC hypothesis.

[0022] Various techniques have been established for in vitro breast CSC isolation and characterization. Mammosphere culture was first used by Dontu et al. to isolate and enhance mammalian stem / progenitor cells, based on the propensity of stem / progenitor cells to thrive in serum-free suspension, whereas differentiated cells do not survive in the same environment. By using this method, we have demonstrated that Methyl 2-(2-(3- methylbenzoyl)-5-(m-tolyl)-lH-imidazol-l-yl)acetate (IIIM-152) significantly inhibited the mammosphere formation of both MDA-MB-231 and SUM159 Triple-negative Breast cancer cell lines. Another strategy is to use cell makers such as CD44+CD24- / lowlin- and ALDH positive to differentiate mammary stem / progenitor cells from differentiated cancer cells. It has been documented that as fewer as 500 ALDH-positive cells could develop breast tumors in less than 40 days, while 50,000 ALDH-negative cells did not generate a breast tumor. ALDH-positive cells and CD44+CD24- / lowlin- have been reported as narrow overlaps with the greatest tumorigenic potential, developing tumors from as minimal as 20 cells. In comparison, ALDH-positive cells without the CD44+CD24- / lowlin- marker have been able to develop tumors from 1,500 cells, whereas 50,000 CD44+CD24- / lowlin- negative cells have not. Therefore, we used the Aldefluor Assay to assess the potential of IIIM-152 to target stem / progenitor cells of TNBCs. We have demonstrated that IIIM-152 could suppress cancerinitiating ALDH-positive cells in vitro by 63% to 90% in MDA-MB-231 and SUM159 cells respectively. Of special interest, IIIM-152 concentrations inhibiting stem / progenitor cells in both the mammosphere assay and the CD44+ / CD24- Aldefluor assays had only marginal effects on the bulk population of breast cancer cell lines, which suggests selective targeting of stem / progenitor cells by IIIM-152.

[0023] The injection of mouse breast cancer cells into the mammalian fat pad of immunodeficient BALB / c mice offers an effective and resilient in vivo model for breast cancer studies. By using this BALB / c tumor model, we have shown that IIIM-152 was able to take down breast CSCs in vivo. Intravenous injection of IIIM-152 for 2 weeks decreased tumor volume in primary BALB / c mice and the population of ALDH-positive cell population of the tumor by 50%. IIIM-152 has many advantages as a chemoprevention agent, such as excellent bioavailability and minimal toxicity. The IIIM-152 pharmacokinetics was conducted by intravenous (IV) and oral (PO) routes at a 50 mg / kg dose. Following intravenous administration of IIIM-152 at a 50 mg / kg dosage, the mean plasma clearance (Cl) was found to be high (0.59L / h / kg) with an elimination half-life (tl / 2) of 5.7h. The volume of distribution at a steady-state was reported to be 8.7 L / kg and the Mean plasma concentration (AUClast) was found to be 27492 ng. h / mL. Following a single oral gavage injection of IIIM- 152 to male BALB / C mice, the median time to reach the maximum plasma concentration was discovered to be 11.4h with an initial plasma concentration (Co) of 7256 ng / mL. The volume of distribution at a steady-state was reported at 2.1 1 / kg and the Plasma exposure (AUClast) was found to be 32310 ng.h / mL. Similarly, no mortality and signs of toxicity such as loss of body weight, food consumption, relative organ weight, or gross pathology of vital organs during treatment with different doses as compared with control in the toxicity study. Despite, the promising anti-CSC activity, IIIM-152 was also found to be safe up to the dose of 200 mg / kg in 14 days of repeated-dose studies in mice.

[0024] Imidazole compounds were shown to interfere with the Wnt / p-Catenin self-renewal pathway in Triple-Negative Breast Cancer. Jeong et al. previously reported that imidazole increased the inhibitory phosphorylation and subsequent degradation of P-catenin in melanoma. In consistent with this study, we revealed that IIIM-152 was able to down-regulate the Wnt / p- catenin self-renewal pathway in TNBCs, and IIIM-152-induced P-catenin phosphorylation and (Ser33 / Ser37 / Thr41) and proteasome degradation was likely through GSK3P activation.

[0025] In conclusion, we have shown that IIIM-152 was able to target breast CSCs as evaluated by the mammosphere formation assay, Aldefluor assay, CD44+24- assay, and tumor growth upon 4T1 implantation in BALB / C mice. Furthermore, our research reported the downregulation of the Wnt / p-catenin self-renewal pathway by IIIM-152 as one of the plausible mechanisms for its efficacy. All these findings endorse the use of IIIM-152 for chemoprevention against breast cancer. These findings provide a convincing rationale for preclinical and clinical assessment of IIIM-152 for breast cancer therapies.

[0026] OBJECTIVE OF THE INVENTION:

[0027] An objective of the present invention is to provide a combitorial treatment of Paclitaxel and IIIM152 used in the effective ratio in the rage of 1:5 to 1: 10, for eliminating Paclitaxel nonresponsive (resistant) Triple Negative Breast Cancer Stem Cells (CSCs).

[0028] Another objective of the present invention is to provide a method for preparation of composition comprising Paclitaxel and IIIM152.

[0029] Further an objective of the present invention is to provide a method for treating Paclitaxel resistant Cancer Stem Cells in TNBCs by combination of Paclitaxel and IIIM152.

[0030] Yet another objective of the present invention is to provide use of combination of Paclitaxel and IIIM152 for treatment of Paclitaxel resistant Cancer Stem Cells in TNBCs. SUMMARY OF THE INVENTION:

[0031] Accordingly, the present invention provides a novel combination of Paclitaxel and IIIM152 when used at the ratio in the range of the range of 1-5 : 1-10, was found effective against Paclitaxel resistant Cancer Stem Cells in TNBCs.

[0032] The combination was found safe in mice as mice were healthy and no mortality was observed.

[0033] Further, the present invention also provides a method for preparation of composition comprising Paclitaxel and IIIM152.

[0034] IIIM152 showed encouraging PKPD profile.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS:

[0036] Fig 1 shows the general structure of representative of compounds referred to in Table 1 and Table 2.

[0037] DETAILED DESCRIPTION OF THE INVENTION:

[0038] The present invention relates to a composition for treating paclitaxel-resistant breast cancers. These derivatives show to be useful against Paclitaxel-resistant triple-negative breast cancer and in combination showed efficacy against drug-resistant CSCs.

[0039] In an embodiment of the invention, it provides a composition for treating paclitaxel-resistant triple negative breast cancer consisting of Paclitaxel and IIIM-152 in the ratio in the range of 1-5: 1-10.

[0040] In another embodiment of the invention, it provides a method for preparing the composition comprising: a) Providing Paclitaxel b) Providing IIIM152 c) Dissolving DMSO (1 part) + Tween-80 (1 Part) and 0.9% Normal Saline (8 parts) making final composition as 1:1:8 to get a buffer solution. d) Dissolving paclitaxel and IIIM152 in the buffer solution to obtain the composition.

[0041] Yet another embodiment of the invention provides a method of treatment of paclitaxel- resistant triple negative breast cancer by using combination of of Paclitaxel and IIIM-152 in the ratio in the range of 1-5: 1-10. Further an embodiment of the invention provides use of combination of Paclitaxel and IIIM- 152 in the ratio in the range of 1-5: 1-10 for treating paclitaxel-resistant triple negative breast cancer.

[0042] EXAMPLES

[0043] The following examples are provided to illustrate the present invention and should not be construed to limit the scope of the present invention

[0044] Example 1:

[0045] General procedure for the preparation of compounds (IA1-30):

[0046] Scheme 1. A reaction vessel was charged with 2-oxoaldehydes (1 mmol), amino acid alkyl ester hydrochloride (1.2 mol %), selenium dioxide (1.2 mmol), and pyridine (1.5 mmol) in 3 mL of ACN solvent. The reaction mixture was stirred at room temperature for 2-4 h. Upon completion of the reaction, confirmed by thin-layer chromatography, the crude mixture was filtered and purified by column chromatography using silica gel (100-200 #) with ethyl acetate and hexane as an eluent to afford the desired compounds (IA1-30) in 60-89 % yields.

[0047] General procedure for the preparation of compounds (2A1-4):

[0048] Scheme 2. A reaction vessel was charged with 2-oxoaldehydes (1 mmol), amino acid alkyl ester hydrochloride (1.2 mol %), selenium dioxide (1.2 mmol), and pyridine (1.5 mmol) in 3 mL of ACN solvent. The reaction mixture was stirred at room temperature for 2-4 h. After 4 hours, add NaOH (1.1 mmol) and stir the reaction again for 4-6 h at room temperature. Upon completion of the reaction, confirmed by thin-layer chromatography, the crude mixture was filtered and purified by column chromatography using silica gel (100-200 #) with ethyl acetate and hexane as an eluent to afford the desired compounds (2A1-4) in 56-84 % yields.

[0049] General procedure for the preparation of compounds (2A1-4):

[0050] Scheme 3. A reaction vessel was charged with 2-oxoaldehydes (Cyclic, Acyclic, and, Heterocyclic 1 mmol), amino acid alkyl ester hydrochloride (Substituted Thioesters, Amides 1.2 mol %), selenium dioxide (1.2 mmol) and pyridine (1.5 mmol) in 3 mL of ACN solvent. Upon completion of the reaction, confirmed by thin-layer chromatography, the crude mixture was filtered and purified by column chromatography using silica gel (100-200 #) with ethyl acetate and hexane as an eluent to afford the desired compounds (2A5-17) in 49-78 % yields.

[0051] Table 1. Compound of formula 1A of general formula 1

[0052] Table 2. Compound of formula 2A of general formula 1 In vitro cell line activity:

[0053] Cell culture, growth conditions, and treatments.

[0054] Human breast cancer cell lines MDA-MB-231, SUM159, and 4T1 mouse mammary carcinoma cell lines were purchased from American Type Culture Collection(ATCC). Both MDA-MB-231 and SUM 159 cell lines are negative for estrogen receptor (ER), and progesterone receptor (PR) and does not have Her2 overexpression. MDA-MB-231 and 4T1 were maintained in RPMI 1640(Invitrogen) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). SUM159 was maintained in Ham’s F12 medium (Invitrogen) supplemented with 5% fetal bovine serum, 1% antibiotic-antimycotic (Gibco), 5pg / mE insulin (Gibco), Ipg / mE hydrocortisone (Sigma- Aldrich). PTEN and P53 knockdown MCF10A cell lines were established by transduction with shRNA lentiviral particles purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were grown in a CO2 incubator (Thermocon Electron Corporation, USA) at 37oC in an atmosphere of 95% air and 5% CO2 with 98% humidity. Different molecules were dissolved in DMSO and were delivered to cell cultures in a complete medium.

[0055] Propidium Iodide and paclitaxel were purchased from Invitrogen. LiCl was purchased from Fisher Scientific and MG 132 from TOCRIS Bioscience.

[0056] Antibodies against oct4, sox2, cyclin DI, P-catenin, phospho-b-catenin ser33 / ser37 / Thr41, phospho-GSK3P ser9, E-cadherin, Vimentin, Zebl, ATG5, and p62 were purchased from cell signalling technology. Antibodies against snail and LC3 II were purchased from Sigma Aldrich.

[0057] Results:

[0058] (1) IIIM-152 inhibits proliferation and induces cell cycle inhibition of triple-negative breast cancer cells in an apoptosis-independent manner.

[0059] (IA) The antiproliferative effect of IIIM-152 was measured by SRB assay on triple-negative breast cancer cell lines MDA-MB-231 and SUM159. MDA-MB-231 and SUM159 cells were treated with different concentrations of IIIM-152 for a time period of 24-96 hours. IIIM-152 proved to be a non-toxic compound and exhibited higher IC50 values of more than lOOpM even when cells were treated for longer time periods spanning up to 96 hours.

[0060] (IB) To determine whether the decrease in cell proliferation was due to cell-cycle inhibition, MDA-MB-231 and SUM159 cells were treated with increasing concentrations of IIIM-152 for 72 hours and subjected to cell cycle analysis by flow cytometry. IIIM-152 inhibited proliferation and induced Gl-phase cell cycle arrest in triple-negative breast cancer cells (1C)IIIM-152 was found to arrest the cells in the Go / Gl phase of the cell cycle, which was also confirmed by a drastic decrease of cyclin DI expression using western blot analysis.

[0061] Table 1A IC50 values of IIIM-152 against TNBC cell lines Table IB Cell Cycle Inhibitory potential of IIIM-152 in TNBC cell lines

[0062] Table -1C Protein expression of Cell Cycle regulators in TNBC cell lines treated with IIIM-152

[0063] (2) HIM- 152 effectively inhibits the growth of breast cancer stem / progenitor cells in vitro and attenuates paclitaxel-induced augmentation of the Stem cell population in TNBCs. It has been shown that in breast carcinomas, a cell population with high ALDH activity, high CD44+ / 24- expression, and ability to form mammospheres is capable of enriching tumorigenic stem / progenitor cells. This sub-population of cells has been attributed to be responsible for breast cancer progression, tumor relapse and chemotherapeutic resistance. IIIM-152 was shown to reduce the number of ALDH+ expressing cells, CD44+ / 24- expression besides decreasing the number and size of mammospheres formed in

[0064] MDA-MB-231 and SUM159 cells in both time and dose-dependent manner.

[0065] (2A)To evaluate the suppressive effects of IIIM-152 on mammosphere formation, we exposed primary MDA-MB-231 and SUM159 mammospheres to varying concentrations of IIIM-152 and then cultured them in an additional passage (secondary mammospheres) in the absence of the compound. IIIM-152 remarkably declined the number and size of primary spheres. A significant decrease was also observed in the number and size of secondary mammospheres in the second generation and no cells continued to the third generation. (2B) A drastic reduction in the expression of pluripotency markers 0CT4 and SOX2 was also observed. These findings, therefore, demonstrate that IIIM-152 can suppress the breast cancer stem / progenitor cell population in-vitro. An important observation is that IIIM-152 inhibits breast cancer stem / progenitor cells at the concentration (10-80pmol / l) without affecting the bulk population of cancer cells, thus implying that IIIM-152 preferentially targets cancer stem cells compared to bulk cancer cells.

[0066] (2C) Similarly SUM159 tumorspheres were treated with IIIM-152, paclitaxel, and a combination of both agents to evaluate their effect on the formation of secondary mammospheres. Strikingly, tumorosphere efficiency was decreased by IIIM-152 and increased by paclitaxel. Similarly, combination of IIIM-152 and paclitaxel resulted in a marked reduction of tumor sphere formation.

[0067] (2D)In contrast, treatment with cytotoxic agent paclitaxel increased the percentage of Aldefluor+ cells, consistent with CSCs being resistant to conventional chemotherapeutic agents. Importantly IIIM-152 attenuated paclitaxel-induced augmentation of ALDH+CSCs.

[0068] Table-2A

[0069] Inhibitory effect of IIIM-152 on Mammo sphere formation

[0070] Inhibitory effect of IIIM-152 on Secondary Mammosphere formation Table-2B

[0071] Effect of IIIM 152 on proteins regulating pluripotency of TNBC

[0072] Table-2C IIIM- 152 eliminates paclitaxel resistant mammospheres at combination of Paclitaxel (lOnM) and IIIM152 (40pM) Table -2D IIIM-152 effectively effects growth of breast cancer stem progenitor cells

[0073] (3) IIIM-152 suppresses Epithelial-mesenchymal transition (EMT) in triple negative breast cancer cells.

[0074] To investigate the effect of IIIM-152 on the process of EMT, MDA-MB-231 and SUM159 cells were treated with different concentrations of IIIM-152 for 72 hours and analyzed for the expression of EMT components, including snail, zebl, vimentin, and E-cadherin by western blotting. The expression of snail, zebl, and vimentin was decreased whereas the expression of E-cadherin was increased significantly. We observed a 4-fold decrease in a snail, a 3-fold decrease in zebl, a 2-fold decrease in vimentin, and a 2-fold increase in E-cadherin. Table -3A IIIM-152 suppresses Epithelial-mesenchymal transition (EMT) in triple negative breast cancer cells

[0075] (4) IIIM-152 reduces breast cancer stem / progenitor cells in vivo.

[0076] (4A) To determine whether IIIM-152 could reduce breast CSCs in vivo, we used 4T1 Balb / c mouse mammary carcinoma model. 4T1 cells were injected in mammary fat pad and after two weeks animals were injected on alternate days with 50mg / kg of IIIM-152. Treatment with compound continued for 2 weeks, we found 50% inhibition in tumor volume compared to 0.9% NaCl solution control group. Moreover, IIIM-152 had no apparent toxicity as animals remained healthy throughout the experiment and no body weight loss was observed.

[0077] (4B)We analyzed the ALDH levels in isolated tumor cells from the animals by Aldefluor assay. IIIM-152 significantly reduced the ALDH-positive population by more than 50% compared to control mice.

[0078] Table -4A HIM 152 significantly decreased the tumor volume in BALb / c mice Table -4B in vivo treatment of IIIM-152 significantly reduced the Cancer Stem Cell population in excised Mouse tumors.

[0079] (5) Pharmacokinetics of IIIM-152

[0080] Since novel compounds have to be explored for their pharmacokinetic behaviour, which is one of the essential components of the drug discovery and development process. Herein, we have administered IIIM-152 in mice via oral and i.v routes followed by a collection of blood through retro-orbital plexus and the plasma concentration was determined by LC-MS / MS system (Model name). Plasma concentrations at different time points along with the pharmacokinetic parameters are given in Table. The pharmacokinetic analysis of IIIM 152 was based on plasma concentration versus time profile analysis using non-compartmental methods. The primary pharmacokinetic parameters used to assess dose proportionalities were the area under the curve (AUCO-t) and maximum plasma concentration (Cmax). Wherein, IIIM-152 was well absorbed following oral administration with a T max and C max of 30 min and 13836 ng / ml respectively after an oral dose of 40 mg / kg. Further, AUCO-infinity, after oral administration was 104085 ng / h / ml, and the AUCO-t, was 93309 ng h / ml. On the other hand, IIIM 152 showed shorter half (15 min) when administered intravenously with a high clearance rate i.e 15.46 L / h / kg. Following intravenous administration at a dose of 20 mg / kg, AUCO-t and AUC0-co obtained were 550.6 and 576.5 ng / ml, respectively. IIIM-017 also displayed a higher volume of distribution of 8.7 L / kg and a medium clearance of 0.59 L / h / kg. The oral bioavailability of IIIM-152 w.r.t i.v administration in male BALB / c mice was found to be approximately 90%. Thus, IIIM-152 exhibited a promising pharmacokinetic profile and excellent oral bioavailability. Pharmacokinetics study

[0081] Downregulation of Wnt / p-catenin pathway by HIM- 152 in triple negative breast cancer cells.

[0082] The Wnt / b-catenin pathway is mainly responsible for maintaining stem cell self-renewal and is mostly dysregulated in triple-negative breast cancers. Since Cyclin DI is a direct downstream target of P-catenin and is required for progression through the G1 phase of the cell cycle. As we observed that IIIM-152 downregulated cyclin DI and arrested cells in the G1 phase of the cell cycle in MDA-MB-231 and SUM159 cells. This prompted us to check whether downstream targets of wnt / p-catenin are downregulated by IIIM-152.

[0083] (6A) As shown IIIM-152 significantly decreased the expression levels of P-catenin and cyclin DI besides decreasing the phosphorylation of GSK3P at ser9 increasing the activity of GSK3P, thereby destabilizing P-catenin.

[0084] (6B) Proteasome inhibitor MG132 was also used to confirm the increased expression levels of phosphorylated P-catenin at Ser33 / Ser37 / Thr41 by GSK3p. GSK3b phosphorylation of P- catenin at ser33 / ser37 / Thr41 renders it to ubiquitin-mediated proteasome degradation.

[0085] (6C) To further confirm the P-catenin downregulation by IIIM-152 a GSK3b inhibitor, LiCl(10umol / l) was used to reverse the IIIM-152-induced P-catenin phosphorylation. LiCl inactivates GSK3b by phosphorylation at ser9 which further stabilizes and reduces the phosphorylation of P-catenin at ser33 / ser37 / thr41. Taken together these results suggest that the downregulation of wnt / p-catenin might contribute to the inhibitory effects of IIIM-152 on breast cancer stem cells. This warrants further studies to establish the conclusive role of this downregulation in the inhibition of breast CSCs by IIIM-152.

[0086] Table 6A IIIM-152 significantly decreased the expression levels of p-catenin and cyclin

[0087] DI besides increasing the activity of GSK3 by decreasing GSK3p-ser9 phosphorylation , thereby destabilizing p-catenin in TNBC Cells lines. Fig 6B Effect of IIIM-152 treatment on the level of p-p-Catenin 33 / 37 / 41) under the treatments of MG132 and LiCl

[0088] Fig 6C- IIIM-152 neutralizes the effect of activation of Catenin by LiCl.

[0089] ADVANTAGES OF THE PRESENT INVENTION

[0090] • The advantage of our current finding is to address the major clinical challenge in treating TNBCs for which the only available treatment options are radio and chemotherapy, and Paclitaxel remain remains among the first- line therapeutic options. However, recurrence of tumor occurs due to resistance shown by CSCs, so we claim that newly identified small molecule IIIM-152, is able to eliminate paclitaxel-resistant CSCs responsible for drug resistance and therapeutic failure.

[0091] • The combinatorial treatment of Paclitaxel and IIIM-152 was able to reduce the tumor burden in mice and overcome the drug resistance by targeting CSCs. • In in vitro studies we found the optimum dose of combination for effective elimination of Paclitaxel and IIIM152 treated cells was lOnM and 40pM, respectively. Similarly, in mice experiment we used effective combination of Paclitaxel and IIIM152, to target drug resistant stem like cells.

[0092] • IIIM 152 is a Non-toxic molecule with a promising Pharmacokinetic Profile.

Claims

WE CLAIM:

1. A composition for treating paclitaxel-resistant triple negative breast cancer comprising of Paclitaxel and IIIM-152 in the ratio in the range of 1-5: 1-10.

2. A method for preparing the composition as claimed in claim 1 comprising: e) Providing Paclitaxel f) Providing IIIM152 g) Dissolving DMSO (1 part) + Tween-80 (1 Part) and 0.9% Normal Saline (8 parts) making final composition as 1:1:8 to get a buffer solution. h) Dissolving paclitaxel and IIIM152 in the buffer solution to obtain the composition.

3. A method of treatment of paclitaxel-resistant triple negative breast cancer, the method comprising; administering the composition of Paclitaxel and IIIM-152 as claimed in claim 1.

4. Use of composition of Paclitaxel and IIIM-152 as claimed in claim 1, in treating paclitaxel-resistant triple negative breast cancer.

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