Glucocorticoid receptor-targeted formulations for the treatment of colorectal cancer and preparation thereof

Abstract

The present disclosure provides an anti-cancer lipid-based composition that kills very aggressive colorectal cancer cells. This composition is a concoction of an anti- cancer agent, 5-Fluorouracil and a glucocorticoid receptor (GR)-targeting cationic lipid delivery system, D1X. The uptake studies of these formulations have showed that formulations with dexamethasone enter into nucleus, bind GRE region and upregulate CYP3a5 gene. The intensity of upregulation is better than liposome without dexamethasone. Same results found in in vivo studies, the D1X5-FU shows better tumor regression and survivability than FOLFOX and FOLFIRI. The biodistribution studies showed that D1X5-FU targets only tumor tissues, not other organs. The pilot studies of tumor regression by other liposomal formulations- D1XLeucovorin, D1XOxaliplatin has showed better tumor regression than FOLFOX and FOLFIRI.

Description

GLUCOCORTICOID RECEPTOR-TARGETED FORMULATIONS FOR THE TREATMENT OF COLORECTAL CANCER AND PREPARATION THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to a synergistic anti-cancer composition for drug delivery for colorectal cancer treatment and a process for the preparation thereof. More particularly, the present invention relates to the enhanced non-viral delivery of drug molecules to cancer cells utilizing expressed glucocorticoid receptors.BACKGROUND OF THE INVENTION

[0002] Colorectal cancer is one of the major causes of cancer-related deaths across the globe. At the moment, surgical resection is the major curative treatment for nonmetastasized CRC, but it is very difficult to treat more advanced cases. There is no defined therapy for complete cure of aggressive and metastatic colorectal cancer. 5-Fluorouracil (5-FU) is a widely used drug for colon cancer treatment and has a response rate of around 20% when it is used alone to treat patients (Violette et. al., International Journal of cancer(2002)vol. 98, pg. no. 498-504, Kurokawa et. al.,. Journal of gastroenterology (2012), Mohelnikova-Duchonova et. al., World J Gastroenterol (2074), vol. 20(30), pg. no. 10316-10330).

[0003] 5-FU exerts its cytotoxic effect by inhibiting thymidylate synthase (TS), which is recognized as the rate- limiting enzyme in de novo pyrimidine biosynthesis (Nakamura et. al., Gastric Cancer (2014), vol. 17, pg.no. 188-195). TS is inhibited by 5-fluoro-20-deoxyuridinemonophosphate (FdUMP) formed from 5-FU in the presence of the folate co-factor 5,10-methylenetetrahydrofolate (CH2FH4), leading to inhibition of DNA synthesis (Kurokawa et. al., Journal of gastroenterology (2012), Mohelnikova-Duchonova et. al., World J Gastroenterol (2014), vol. 20(30), pg. no. 10316-10330).

[0004] Drug resistance is the major drawback of chemotherapy. It is caused by repeated use of drugs. There are different mechanisms of drug resistance including amplification or mutation of drug target, mutation of transporter proteins,overexpression of multidrug resistance proteins, drug efflux, etc. ( Violette et. al., International Journal o / cancer (2002)vol. 98, pg. no. 498-504, Lee et.al., Mol. Cells (2014), vol. 37(7), pg. no. 540-546).

[0005] Nowadays, 5-FU and folinic acid i.e. leucovorin are used in combination with other anticancer drugs such as oxaliplatin (a DNA cross-linking agent) or irinotecan (a topoisomerase I inhibitor) (the combination being termed as FOLFOX or FOLFIRI respectively) which shows synergistic or additive anticancer effect and improves the response rate by about 40-50% and overall survival (Kurokawa et. al., Journal of gastroenterology (2012), Guo et. al., ACS Nano, (2020) vol. 14, pg.no. 5075-5089, Miller et. al., J Gastrointest Surg (2017) 21(11), 1831-1840). Here folinic acid is not an anticancer drug but it sensitizes cells and enhances cytotoxicity of 5-FU, but it does not affect cytotoxicity of oxaliplatin (Guo et. al., ACS Nano, (2020) vol. 14, pg.no. 5075-5089), or irinotecan. In spite ofbetter survival, patients are affected by side-effects such as myelotoxicity, neurotoxicity or gastrointestinal toxicity due to long course of treatment and high doses (Guo et. al., ACS Nano, (2020) vol. 14, pg.no. 5075-5089, Miller et. al., J Gastrointest Surg (2017) 21(11), 1831-1840). There is a high need to reduce the side effects of this combination therapy using new strategies.

[0006] Glucocorticoid receptor (GR) is a nuclear hormone receptor residing ubiquitously in almost all cells including most cancer cells because of its vital role in gluconeogenesis. This receptor in a ligand-bound state acts as a transcription factor in nucleus through binding to glucocorticoid response elements (GRE) in the promoter sequences of various GR responsive genes and regulates their transcription.

[0007] Dexamethasone (dex), a synthetic glucocorticoid (GR hormone-like molecule) exhibits antiproliferative effect on tissues of various origins (Corroyer, S. E. et al. 1997, Endocrinology, 138, 3677-3685; Ramalingam, A. et al. 1997, Mol. Endocrinology, 11, 577-586; Rider, L. G. et al. 1996, J. Immunol., 157, 2374-2380; Goya, L. et al. 1993, Mol. Endocrinology, 7, 1121-1132; Wattenberg, L. W., and Estensen, R. D.. 1996, Cancer Res., 56, 5132-5135; Greenberg A. K. et al. 2002, Am. J. Respir. Cell Mol. Biol., 27, 320-328) as well as regulates and controlsmetabolism, development, inflammation, cell growth, proliferation and differentiation (Yamamoto K. R. 1985, Annu. Rev. Genet. 19, 209-252; Cole, T. J. et al. 1995, Genes Dev., 9, 1608-1621; Rogatsky, I. et al. 1997, Mol. Cell Biol., 17, 3181-3193). GR-mediated glucocorticoid- signaling potentiates a possible hypoxia related pathway leading to inflammation. As an anti-inflammatory agent, dexamethasone inhibits hypoxia inducible factor (HIF-1), which has direct role in mediating angiogenesis through up-regulation of VEGF (Leonard, M. W. et al. 2005, J. Immunol., 174, 2250-2257). Hence, dexamethasone (dex) is a very important and inexpensive drug-like substitute used in various pathological conditions. As a gene carrier agent, dexamethasone- spermine conjugate was used to deliver genes to airway epithelial with concurrent reduction of inflammation (Gruneich J. A. 2004, Gene Ther,l l, 668-674).

[0008] Proliferation of cancer cells increases their glucose consumption and produces lactate by glycolysis in aerobic condition (Warburg effect), which promotes drug efflux. GCs induce gluconeogenesis by using pyruvate and lactate as substrate. Consequently, if GCs are used in cancer treatment, the drug resistance may be reversed. Cancer is an inflammatory disease and glucocorticoids reduce inflammation; considering this mechanistic relationship of glucocorticoids with cancer cells, glucocorticoid based liposomal formulations are being prepared and explored.

[0009] A cationic lipid formulation (namely, DX) was prepared based on the idea that dexamethasone possesses close structural resemblance with cholesterol (a commonly used co-lipid in many cationic lipid formulations used for non-viral based gene delivery). Hence, it can be accommodated in cationic lipid formulation along-side cholesterol. It has also been shown that the lipid content of this formulation DX, upon treatment to cells lead to selective localization within cancer cells’ nuclei. But importantly, no nuclear localization of lipid content was observed in non-cancer cells. This observation of nuclear delivery of lipid content selectively to cancer cells led to reformulation of DX by associating a lipophilic anti-cancer drug in the lipid phase.

[0010] In that invention, any drug especially lipophilic anti-cancer drug which can induce cell death may be incorporated in the DX formulation as a means to induce killing of cancer cells via, for example, apoptosis or sensitizing aggressive and relapsing cancer phenotypes to drug treatment. Here, different classes of anticancer pharmacological agents may be incorporated with a variable ratio with respect to other lipid-phase components. The non-limiting examples are as follows: a) drugs that act on DNA topoisomerases; b) DNA-alkylating agents; c) drugs acting on transcription machinery, d) drugs reactivating apoptotic proteins such as p53, e) drugs down-regulating metastasis inducing gene, f) drugs up-regulating tumor suppressor gene / miRNAg) drugs regulating developmental pathways that plays role in cancer progression, such as Notch, Wnt-P-catenin pathways etc.

[0011] In this respect, the applicants own patent application 2849DEL2012 discloses the utilization of glucocorticoid receptor (GR)-targeted cationic liposomal delivery systemas described in 1936 / DEL / 2006; PCT / IN2007 / 000367, Molecular Therapy (2009), 17, 623-631) discloses targeting and delivering genetic and drug cargo to Cancer stem cells (CSC) or aggressive cancer cells. The above said invention is cationic lipid-based and it can carry dual cargo, drugs and genes, simultaneously. The formulation has dualsaturated chain cationic lipid (DO) to deliver estrogen receptor targeted anticancer drugs ESC8 and nutlin, and NRP-lShRNA plasmid. It targets CSC in breast and pancreatic cancer respectively via a non- developmental pathway i.e., through the cytoplasmic protein glucocorticoid receptor (GR)(Ahmad et al., Molecular Pharmaceutics(2016),vol. 13, pg no. 1081-1088, US Patent No. 9,861,653 B2; EP Patent No. EP2895149B 1). Later, another molecule was synthesized where onesaturated aliphatic chain of DO cationic lipid was replaced with an unsaturated aliphatic carbon chain (DI). When DI lipid replaced DO lipid in DX formulations, the formulation was termed D1X. This D1X was co-formulated with ESC8 drug which exhibited high fusogenicity, lessergenotoxicity in mice with better antitumor efficacy than earlier lipoplex, clearly indicating the favourable role of unsaturated lipid chain in developing efficient, GR-targeted, anti-tumor delivery system. (Rachamalla HK et al., Nanotoxicology (2019), vol.13(9) pg. no. 1161-1175). The D1X formulation wasfurther co-formulated with multiple drugs such as bortezomibwhich isused to treat various malignancies of blood. This formulation showed reduction of tumour burden when used against humanoid models of B-lymphoma / Waldenstrom macroglobulinemia (WM), a known blood cancer(Madamsetty et. PA., Leukemia & lymphoma. (2020), vol. 61(10), pg. no. 2399-2408).

[0012] The potent lipoplex formulation of DlX-ESC8-anti-miR Hsp90 was also used to treat colon tumor generated in mice orthotopically (Jinka et. Al., Biomedical materials (2021 ), vol. 16(2), pg. no. 024105).

[0013] In one of the closest prior art US9827196, it is noticeable that along with cationic liposomal formulation of an anticancer drug, paclitaxel, inventors have mentioned administering human subjects at least one further active agent, such as chemotherapeutic drug e.g., 5 -fluorouracil (5-FU), anti-hypersensitivity agent e.g., dexamethasone and / or heat, and / or radiation, and / or cryotherapy. These combinations of treatments are done either simultaneously, separately or sequentially. It is clearly evident that the extra active agents are administered along with other physical conditions and liposomal-paclitaxel but not as liposomally coencapsulated formulation combination of all active agents. Inventors neither explicitly mentioned that possibility nor showed any data about how that possibility could lead to pharmaceutically beneficial effect.

[0014] In contrast to the existing art (Indian Application No. 1936 / DEL / 2006, PCT / IN2007 / 000367, EP-2061514-A2 and Mukherjee, A. et al. 2009, Mol.Ther., 17, 623-631 and US9827196), the current invention is a liposomal formulation consisting of cationic lipid with one of its aliphatic chains carrying unsaturation(Dl), anticancer drug such as 5-fluorouracil (5-FU) and dexamethasone to target glucocorticoid receptor (GR). The formulation D1X5-FU leads to effective targeting and killing of tumor cells with increased survivability of tumor-bearing mice than combination drugs FOLFOX or FOLFIRI. The formulation shows increased pro-apoptotic proteins and reduced pro-inflammatory protein NF-kB when compared to free drug, 5-FU, or due to combination therapy FOLFOX and FOLFIRI. It is noticeable in the present invention that no additional genetic drug is needed for effective treatment of colorectal cancer, especially withrespect to a reference literature(Jinka et. al., Biomedical materials (2021), vol. 16(2), pg. no. 024105). In the reported literature it was shown that a hydrophobic, anticancer molecule (ESC8) encapsulated in a dexamethasone-associated cationic liposomal formulation, which is electrostatically complexed with anticancer genetic drug, anti-miR Hsp90 plasmid [i.e., DlX-ESC8-anti-miR Hsp90] induced effective anti-colorectal tumor effect.

[0015] The present disclosure can also be projected as platform for many other kinds and classes of drugs. This would typically to repurpose their use and avoid drug resistance that these drugs might face in multiple cancers including colon cancer especially in advanced stages. As an example, niclosamide, an antihelminthic drug finds its use against colon cancer as repressor of metastatic gene (such as S100A4) (WO2012143377A1) or promoter of tumor suppressor microRNAs such as miR200 family members or repressor of Notch proteins (Suliman et al. International Journal of Molecular Medicine (2016), vol. 38(3), pg. no. 776-784). However, the advanced stages of colon cancer where drug resistance is paramount, the use of niclosamide is not clear. Niclosamide is used mostly alone or in liposomal or polymeric formulations but are not used along with drug sensitizing formulations such as the present dexamethasone-associated formulation. In this regard, critical use of niclosamide class of drugs in dexamethasone- associated formulation is envisioned to enhance its applicability in aggressive and drug resistant colon cancer phenotypes.OBJECTIVES OF THE INVENTION

[0016] The objective of the current invention is to induce higher cytotoxicity in colorectal cancer cells than that in normal cells.

[0017] Another objective is to target nucleus, by binding to GRE region and therefore to sensitize cells leading to enhance drug toxicity.

[0018] Thus, the main objective of the present invention is to provide a synergistic anti-cancer composition for enhanced non-viral delivery of drug molecules to cancer cells utilizing expressed glucocorticoid receptors.

[0019] Another objective of the present invention is to provide a synergistic anti-cancer composition for drug delivery for colorectal cancer treatment.

[0020] Another objective of the present invention is to provide a process for the preparation of synergistic anti-cancer composition for drug delivery for colorectal cancer treatment.

[0021] Yet another objective is to evaluate these compositions against well- known colorectal cancer combination drug therapies FOLFOX and FOLFIRI.SUMMARY OF THE INVENTION

[0022] Accordingly, the present invention provides a synergistic liposomal anti-cancer composition for simultaneous non- viral delivery of an anti-cancer drug to glucocorticoid receptor expressing cancer cells comprising: i. a cationic lipid [N,N-Bis(2-hydroxyethyl)-N-octadecyloctadec-9-en-l- aminiumchloride] ; ii. cholesterol; iii. dexamethasone; iv. 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) DSPE-PEG and v. an anti-cancer drug.

[0023] In an embodiment of the present invention the mole ratio of cationic lipid: cholesterol: dexamethasone: anti-cancer drug: DSPE-PEG is in the range of 1 : 1 : 0.5 : 0.25 : 0.02 to 1 : 1 : 0.5 : 0.5 : 0.02.

[0024] In a preferred embodiment of the present invention the anti-cancer drug is a lipophilic drug selected from ESC8, paclitaxel, docetaxel or niclosamide. In another embodiment of the present invention, anti-cancer drug is a lipophilic drug selected from ESC8, or niclosamide.

[0025] In a preferred embodiment of the present invention the anti-cancer drug is a hydrophilic drug selected from 5-Fluorouracil, leucovorin, capecitabin or oxaliplatin. In another embodiment of the present invention, anti-cancer drug is ahydrophilic drug selected from 5-Fluorouracil, leucovorin, oxaliplatin, or combinations thereof.

[0026] In an embodiment of the present invention the composition inhibits growth of aggressive cancer cells selected from colorectal cancer, breast cancer, pancreatic cancer, melanoma, and myeloma.

[0027] In an embodiment of the present invention the composition is for intra-venous, intra-muscular or intra-peritoneal administration.

[0028] The present invention provides a process for the preparation of synergistic composition as claimed in claim 1, wherein the said process comprising the steps of: a) dissolving a cationic lipid [N,N-Bis(2-hydroxyethyl)-N-octadecyloctadec- 9-en-l-aminiumchloride], cholesterol, dexamethasone and DSPE-PEG in a solvent followed by removing solvent using nitrogen gas to obtain a lipid film; b) keeping the lipid film as obtained in step (a) under vacuum for a period ranging between 3-4 hours at a room temperature of 25-35°C to obtain dried lipid film; c) hydrating the dried lipid film as obtained in step (b) by keeping dried film in 5% glucose solution for a period ranging between 10-12 hrs followed by vortexing for a period ranging between 1-2 min and encapsulating an anti-cancer drug to obtain the synergistic composition, wherein the mole ratio of cationic lipid: cholesterol: dexamethasone: the anti-cancer drug: DSPE-PEG in the composition is in the range of 1 : 1 : 0.5 : 0.25 : 0.02 to 1 : 1 : 0.5 : 0.5 : 0.02.

[0029] In an embodiment of the present invention the solvent for dissolving a cationic lipid[N,N-Bis(2-hydroxyethyl)-N-octadecyloctadec-9-en-l- aminiumchloride], cholesterol, dexamethasone and DSPE-PEG is chloroform.

[0030] These and other features, aspects, and advantages of the present subject matter will become better understood with reference to the following description. This summary is provided to introduce a selection of concepts in asimplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS:

[0031] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0032] Figure 1. shows the effect of liposomal formulations on CT 26. WT cell line, in accordance with an embodiment of the present disclosure.

[0033] Figure 2. shows the effect of liposomal formulations on CT 26.WT cell line, in accordance with an embodiment of the present disclosure.

[0034] Figure 3. shows the effect of liposomal formulations on HCT-15 cell line, in accordance with an embodiment of the present disclosure.

[0035] Figure 4. shows the effect of liposomal formulations on CT 26.WT cell line, in accordance with an embodiment of the present disclosure.

[0036] Figure 5. shows the effect of liposomal formulations on HCT-15 cell line, in accordance with an embodiment of the present disclosure.

[0037] Figure 6. shows the effect of liposomal formulations on HEK-293T cell line, in accordance with an embodiment of the present disclosure.

[0038] Figure 7. Shows the effect of niclosamide liposomal formulation on CT26.WT and HEK293, in accordance with an embodiment of the present disclosure.

[0039] Figure 8. shows the effect of D1X5-FU formulation on CYP3A5 upregulation, in accordance with an embodiment of the present disclosure.

[0040] Figure 9. illustrates the quantitative uptake analysis of liposomal formulations by flow cytometry, in accordance with an embodiment of the present disclosure.

[0041] Figure 10. illustrates the qualitative uptake analysis of liposomal formulations by confocal, in accordance with an embodiment of the present disclosure.

[0042] Figure 11. illustrates the qualitative uptake analysis of liposomal formulations by confocal, in accordance with an embodiment of the present disclosure.

[0043] Figure 12. illustrates the Ki 67 staining of proliferating cells, in accordance with an embodiment of the present disclosure.

[0044] Figure 13. shows biodistribution study of D1X5-FU and D15-FU formulations, in accordance with an embodiment of the present disclosure.

[0045] Figure 14. illustrates tumor regression studies of orthotopic colon-tumor- bearing mice, in accordance with an embodiment of the present disclosure.

[0046] Figure 15. illustrates tumor regression studies of orthotopic colon-tumor- bearing mice, in accordance with an embodiment of the present disclosure.

[0047] Figure 16. shows the western blot analysis of tumor lysate, in accordance with an embodiment of the present disclosure.

[0048] Figure 17. illustrates body weight monitoring of female Balb / C mice, in accordance with an embodiment of the present disclosure.

[0049] Figure 18. illustrates body weight monitoring of male Balb / C mice, in accordance with an embodiment of the present disclosure.

[0050] Figure 19. illustrates feed intake study of female Balb / C mice, in accordance with an embodiment of the present disclosure.

[0051] Figure 20. illustrates feed intake study of male Balb / C mice, in accordance with an embodiment of the present disclosure.

[0052] Figure 21. illustrates organ weight monitoring of female Balb / C mice, in accordance with an embodiment of the present disclosure.

[0053] Figure 22. illustrates organ weight monitoring of male Balb / C mice, in accordance with an embodiment of the present disclosure.

[0054] Figure 23. illustrates blood hematological parameters monitoring of female Balb / C mice, in accordance with an embodiment of the present disclosure.

[0055] Figure 24. illustrates blood hematological parameters monitoring of male Balb / C mice, in accordance with an embodiment of the present disclosure.

[0056] Figure 25. shows total platelets count in blood of male and female Balb / C mice, in accordance with an embodiment of the present disclosure.

[0057] List of AbbreviationsDETAILED DESCRIPTION OF THE INVENTION

[0058] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions

[0059] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0060] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0061] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. Throughout this specification, unless the context requires otherwise the word “comprise”, and variations, such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0062] The term “including” is used to mean “including but not limited to”, “including” and “including but not limited to” are used interchangeably.

[0063] The term “cationic lipid” refers to the lipids that are amphiphilic molecules, which consist of a hydrophilic and a hydrophobic region connected by a linker structure. In an aspect of the present disclosure, there is provided a synergistic liposomal anti-cancer composition for simultaneous non-viral delivery of an anti-cancer drug to glucocorticoid receptor expressing cancer cells comprising: a cationic lipid, wherein the cationic lipid is N,N-Bis(2-hydroxyethyl)- N-octadecyloctadec-9-en-l-aminiumchloride.

[0064] In the structural formulae given herein and throughout the present disclosure, the following terms have been indicated meaning, unless specifically stated otherwise.

[0065] In the present invention, a new potent synergistic anti-cancer composition has been developed by encapsulating anti-cancer drug within a cationic liposome. This provides not only distinct improvement in the anti-cancer efficiency of the composition over the individual use of the anti-cancer drug butalso showed exceptionally efficient killing of highly aggressive colorectal cancer cells ubiquitously expressing glucocorticoid receptors (GR). The formulation simultaneously facilitates drug delivery to GR-expressing cells as well as helps increasing cell number receiving drug.

[0066] A glucocorticoid pharmacological agent, dexamethasone is incorporated into cationic liposome. Dexamethasone, a glucocorticoid receptor (GR) targeting ligand is used in the invention to induce drug sensitivity in drug resistant cancer cells through an unique process of GR-mediated nuclear localization of liposome. Dexamethasone, one of the most potent synthetic glucocorticoids, at mole ratios 0.1-2 with respect to cationic lipid, has been shown to facilitate the non-viral delivery of a variety of genetic constructs capable of performing their function (including apoptotic cell death) in human cancer cells.

[0067] In contrast to the existing art, the present invention emphasizes coencapsulation of active agents, such as dexamethasone (a glucocorticoid receptor targeting ligand acting as drug sensitizer) and 5-FU (sole anticancer agent) within the same cationic liposomal formulation. These liposomal formulation combination [namely, D1X5-FU] has exhibited a highly beneficial pharmaceutical effect which was not shown in prior art. The D1X5-FU showed highly efficacious anti-colorectal tumor effect that is much better than the anti-colorectal tumor effect of not only free 5-FU (the first line of treatment for earliest stage colon cancer) but also of 5-FU- associated three drug combination regimens prevalent for treating aggressive and advanced stages of colorectal tumor. These are FOLFOX (folinic acid, 5-FU and oxaliplatin) and FOLFIRI (folinic acid, 5-FU and irinotecan). Clearly, simultaneous delivery of GR-ligand (as a drug- sensitizer) and the first-generation drug, 5-FU together in a cationic liposome has repurposed the use of a first-generation drug, potentially obliterating the use of three drug combination regimen that too at l / 6th5-FU dosage used in colorectal tumor treatment.

[0068] In contrast to the existing art, the present invention uses a hydrophilic drug, 5-FU along with dexamethasone-associated cationic liposomal formulation. This simple combination eliminated the additional use of nucleic acidbased anticancer drug for treating colorectal tumor.

[0069] In fact, what drugs are to be associated along with this dexamethasone-associated formulation to get enhanced anticancer effect in comparison to the respective free drug is an inventive contribution of the present invention. Dexamethasone-associated liposomes upon targeting GR localize to nucleus. Most of the drugs as mentioned in the present invention are those chosen drug which while in association with this dexamethasone-associated liposomal formulation not only showed nuclear localization but also showed the anticancer synergism. Well known colon cancer drugs, capecitabin and irinotecan when liposomally encapsulated with this dexamethasone-associated cationic liposomal formulation, did show nuclear localization (Figure 9) but did not show any better pharmaceutical effect, rather performed worse than when it is associated with dexamethasone-free cationic liposomal formulation.

[0070] The use of specific amount of DSPE-PEG along with DI makes the formulation much clearer, which helps in injecting the drug cargo (molecule with or without gene). Moreover, use of DSPE-PEG with DI along with drug led to much less toxicity as compared to formulation that carries DODEAC and DSPE- PEG [Nano toxicology (2019), vol.13(9) pg. no. 1161-1175].

[0071] In addition to hydrophilic drugs, hydrophobic drugs like WP1066 (non-approved drug-like molecule) and niclosamide (an FDA approved antihelminthic drug) were respectively co-formulate with this dexamethasone- associated formulation. Both these drugs are known STAT3 inhibitors and also target and inhibit multiple cancer implicated pathways. These are used against various cancer models but are yet to be approved for treating cancer. It has been found that dexamethasone-associated WP1066 liposomal formulation had no selectivity of killing compared to WP1066 liposomal formulation (without Dex) as well as pristine drug WP1066 (data not shown). However, a very clear distinction of selectivity of cell killing by dexamethasone-associated niclosamide formulation (DIX-Niclo) has been found in cancer cells with respect to non-cancer cells (Figure 7). Similar extent of selectivity between cancer and non-cancer cells could not be seen following the treatment of niclosamide liposomal solution (without dexamethasone) (Dl-Niclo) or pristine niclosamide (Niclo).

[0072] Treating highly aggressive, drug resistant and relapsing cancer, a hallmark trait of cancer stem cells, is a challenge with any existing treatment regimen. The composition of the present invention targets and kills colorectal cancer cells via GR pathway. The inclusion of anticancer drug such as 5-FU in the glucocorticoid receptor (GR)- targeted liposomal formulation D1X acts against aggressive and drug resistant cells. This was however, not observed when these components are not simultaneously associated in the formulation or by the individual treatment of known anticancer drugs. The present invention exhibits how a first-generation anticancer drug, 5-FU (or its kinds), which is meant for treating the 1ststage of colorectal cancer can be repurposed to potentially use against aggressive, advanced stage of cancer while exhibiting better antitumor effect compared to even multi-drug combination regimen. Moreover, the pharmaceutical effect of this formulation shows that the use of any nucleic acid-based drug can be supplemental but not essential for better anticancer effect.

[0073] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.EXAMPLES

[0074] The following examples are given by way of illustration therefore should not be construed to limit the scope of the present invention.MATERIALS AND METHODSCell lines

[0075] CT26.WT and HCT-15 cells were maintained in RPMI 1640 containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 1% sodium pyruvate, 2.5% HEPES, and 2mM L-glutamine. HEK-293T cell line was maintained in DMEM. CT26.WT cell line was obtained from Department of Biochemistry, Indian institute of science, Bangalore.Example 1Preparation of D1X5-FU liposomes:

[0076] DI [N,N-Bis(2-hydroxyethyl)-N-octadecyloctadec-9-en-l- aminiumchloride], the cationic lipid) (645 pg), cholesterol (the co-lipid) (386 pg), dexamethasone (a GR-targeting synthetic ligand) (196 pg), DSPE-PEG 2000 (55.8 pg) were premixed in a molar ratio of 1:1:0.5:0.02 in chloroform. The organic solvent was removed under a thin flow of dry air / N2 to get a thin layer of lipid film. The lipid film was further dried under a high vacuum for 4hrs at 27°C. The obtained dried film was hydrated using 1ml autoclaved milliQ premixed with 32.51 pg of 5- Fluorouracil and kept overnight at room temperature. After thin film hydration, the formulation was vortexed for 5 min to form vesicles. These vesicles were multilamellar vesicles. To encapsulate water soluble 5-flurouracil, 5 cycles of freeze-thaw were done at temperature 80°C and 37°C followed by 3 cycles of extrusion (Avanti polar). Following this, the liposomes were filtered using centrifugal filter of 10,000 MWCO. The free unentrapped drug was filtered down whereas the liposome with encapsulated drug remained as residue. The resulting liposome formulation was named D1X5-FU, which contained cationic lipid,cholesterol, dexamethasone, 5 -Fluorouracil and DSPE-PEG as 1 mM : 1 mM : 0.5 mM : 0.25 mM :0.02mM. Similarly, a liposomal formulation without dexamethasone was prepared and named as D15-FU [DI: Cholesterol: 5- Fluorouracil: DSPE-PEG, 1 : 1 : 0.02 molar ratio]. The same procedure was followed to make respective formulations for other hydrophilic drugs such as for Capecitabin [DIXCapecitabine, DICapecitabine], for oxaliplatin [DIXOxaliplatin, DI Oxaliplatin], for leucovorin [DIXLeucovorin and DI Leucovorin]. Briefly, all the molecules except the drugs were premixed in chloroform, dried while maintaining DI: cholesterol: Dex: DSPE-PEG 2000 molar ratio as 1:1:0.5:0.02 or DI: cholesterol: DSPE-PEG 2000 as 1:1:0.02. This was followed by hydration with premixed, respective aqueous solutions of hydrophilic drugs, freeze-thaw cycles, extrusions and filtration to remove unentrapped drugs. The effort was to maintain the ratio of components as 1:1:0.5:0.25:0.02 in DI: cholesterol: Dex: drug: DSPE- PEG 2000 or as 1:1:0.25:0.02 in Dl:cholesterol:drug; DSPE-PEG 2000, where respective drugs were capecitabine, oxaliplatin, leucovorin. The anticancer activity of these liposomal formulations was compared with free 5-FU, FOLFOX (5- Fluorouracil- 32.51 pg + Leucovorin- 118.4 pg + Oxaliplatin-21.1 pg) and FOLFIRI (5-Fluorouracil- 32.51 pg + Leucovorin- 118.4 pg + Irinotecan- 70.3 pg).Preparation of DIX-Niclo liposomes:

[0077] DI, the cationic lipid (645 pg), cholesterol (the co-lipid) (386 pg), dexamethasone (a GR-targeting synthetic ligand) (196 pg) were dissolved in chloroform, Niclosamide (164 pg) was dissolved in ethanol and were premixed in a molar ratio of 1:1:0.5:0.5. The organic solvent was removed under a thin flow of dry air / N2 to get a thin layer of lipid film. The lipid film was further dried under a high vacuum for 4h at 27°C. Then the lipid mixture was dissolved in 50 pL ethanol. The mixture of lipid in ethanol was rapidly injected in 950 pL of autoclaved, milliQ water and then the solution was sonicated in 85Hz bath sonicator for 1-2 min depending on the haziness, if any in the solution.Example 3: in-vitro cell Culture study

[0078] Cancer cells (CT26.WT and HCT 15) were seeded plates in RPMI 1640 media containing 10% Fetal Bovine serum (FBS) at a concentration of 5000 cells / well of 96-well cell culture. Following a minimum incubation time of 16h at 37°C, the cells were treated in triplicate continuously for 48 hours. Cells were treated with following treatment groups:1. D1X5-FU, D15-FU, 5-FU, FOLFOX and FOLFIRI2. DIXOxp, DIOxp, Oxp and FOLFOX3. DIXLeuco, DlLeuco, Leuco, FOLFOX and FOLFIRI4. DIXCape, DiCape, Cape and XELOX.5. DIX-Niclo, Dl-Niclo, NicloMTT assay was carried out to check the cytotoxicity of various formulations.Example 4

[0079] Figure 1 demonstrates that the delivery of oxaliplatin through D1X formulation (DIXOxp) had better anticancer effect towards CT- 16 colorectal cancer cells compared to DIOxp or pristine treatment of Oxp. DIXOxp is exhibiting modestly better anticancer effect compared to multidrug regimen (FOLFOX), which carries folinic acid, 5-FU and oxaliplatin.

[0080] In contrast, DIXCape (carrying capecitabin or Cape) did not show any better anticancer effect than free drug Cape or multidrug regimen (XELOX), which contains capecitabin and oxaliplatin (Figure 2).

[0081] Figure 3 demonstrates that the delivery of leucovorin (Leuco) in D1X formulation (DIXLeuco) induced better anticancer effect than DlLeuco or free drug, Leuco. In fact, in higher concentration of Leuco, DIXLeuco had modestly better anticancer effect compared to multidrug regimens, FOLFOX [folinic acid (leuco), 5-FU and oxaliplatin] and FOLFIRI [folinic acid, 5-FU and irinotecan].

[0082] Figure 4 and 5 demonstrates that the delivery of 5-Fluorouracil through D1X formulation (D1X5-FU) had maximum anticancer effect towards colorectal cancer cells (CT-26 and HCT-15). It was notable that the treatment with naked drug in respective concentration had the least anticancer effect under samecondition. It was observed that, when single drug 5-FU encapsulate in D1X liposome showed better anticancer effect than 5-FU in combination with other two drugs Oxaliplatin and leucovorin (FOLFOX) or Irinotecan and leucovorin (FOLFIRI). It was observed that D1X-FU shows better anticancer activity than the combination of three different drugs in FOLFOX and FOLFIRI formulations. This indicated that the DX-formulation should contain the naked drug constitutively in it to show maximum anti-cancer effect to the colorectal cancer cells.

[0083] In contrast, D1X5-FU had least killing effect in non-cancer cell HEK-293 compared to D15-FU, free drug 5-FU and the multidrug regimens (FOLFOX) and (FOLFIRI) at a given concentration of 5-FU (Figure 6).

[0084] Using hydrophobic drug, niclosamide similar results were obtained. DIX-Niclo had the most killing effect in colon cancer cell but had least killing effect in non-cancer cells (HEK293T). The non-dexamethasone formulation, Dl- Niclo exhibited similar colon cancer cell killing but has relatively more toxic effect on non-cancer cell (HEK293T) compared to the effect of DIX-Niclo. The pristine drug niclosamide (Niclo) has distinctly no difference in toxicity to both cancer and non-cancer cells in all treated concentrations, indicating no selectivity in its action (Figure 7).Cellular uptake study:

[0085] A previous report showed that liposomes with dexamethasone have high affinity towards cancer cell line. At the time of inflammation, glucocorticoids enter into cell and bind to their cytoplasmic receptors. After binding of ligands, receptors dimerize, and the exposed nuclear localization sequence allows the receptors with ligand cargo to enter into nucleus through nuclear pore. The homodimer of ligand-bound GR binds the GRE (glucocorticoid responsive element) promoter region and upregulated or downregulated certain GRE genes of CYP 3a5 and CYP 3a4 family. To check this RT-PCR studies were done, in which it was observed that the free dex was upregulating CYP 3a5 and dex in formulation D1X was showing more upregulation of CYP 3a5 in cancer cells. D1X5-FU showed significantly more upregulation of CYP 3a5 than D1X (Figure 8). After bindingwith receptor, it was able to translocate to nucleus and upregulate the expression of GRE genes especially CYP3a5 gene. It was well known fact that CYP3a5 was involved in drug metabolism and hence it may be predicted that the upregulation of this gene was increasing the active form of 5-FU in the cell by metabolizing the prodrug. This might be the probable reason behind the increase in cytotoxicity with D1X5-FU, though same quantity of 5-FU was provided in each formulation.

[0086] Then Rhodamine PE was formulated into all the liposome formulations and treated to RKO, HCT-15 and CT26.WT cell lines. The uptake activity was studied by Fluorescent cytometer and confocal microscopy. 2.5, 5 and 10 pM of D1X5-FU liposomes were treated to RKO cell line and quantitative uptake were studied by FACS. It was observed that uptake was increasing as increase in liposomal concentration (Figure 9). HCT-15 cell line were treated with 2.5 pM of D1X5-FU liposome, and more uptake was observed in cells treated with D1X5-FU than D15-FU.

[0087] Eiposome is cationic in nature and cell membrane is generally negatively charged. Therefore, electrostatic interaction allows liposome to enter inside the cells.

[0088] So, nuclear localization of the formulations were checked by confocal microscopy and it was observed that D1X5-FU was present in both cytoplasm and nucleus, whereas D15-FU remains in cytoplasm (Figure 10 and Figure 11). This was due to affinity of GR towards GRE. As per color intensity of microscopic images it could be said that, the uptake of D1X5-FU was more in cytoplasm as well as in nucleus than D15-FU.Example 5Ki 67 staining of proliferating cells.

[0089] Cancer is an unwanted proliferation of cells. So to check the effect of the liposomal formulation on proliferation of CT 26.WT cells, cells were treated with 2.5 pM of D1X5-FU, D15-FU, 5-FU, FOEFOX and FOLFIRI for 12 hours. Cells were treated with Ki67 primary antibody and then with PE conjugated secondary antibody. Red fluorescence shows proliferation of cells. It was observedthat, Ki 67 expression was significantly decreased in cells in DlX5-FU-treated group compared to cells of other treated and untreated group s(Figure 12).Example 6In-vivo tumor model study

[0090] In Vivo study: a. The NIH strain Balb / C mice were purchased from CSIR Center for molecular biology, Hyderabad. All experiments were done by following Institutional Animal Ethical Committee guidelines with protocol number IICT-IEAC-68-2019. 5 x 105CT-26 cells were orthotopically injected into cecal wall of cecum. All experiments were done after 20 days of surgery.

[0091] Biodistribution study: b. NIR DiR dye was encapsulated in D1X5-FU and D15-FU formulations and treated to tumor bearing mice. The images of live mice and organs were taken by IVIS machine. It was observed that DiR dye-containing liposomal formulation was mostly present in tumor and liver but DiR-containing D15-FU was present in all organs. This data proves that Dex incorporated liposomes are successfully targeting tumor (Figure 13).Tumor regression studies and immunohistochemistry studies:

[0092] After 20 days of surgery mice were segregated into different group for tumor regression and survivability studies and treatments were started (n=5). 5 injections were given in alternative days. Mice grouped for regression studies were sacrificed on the next day of last injection and cecum weight of all the were taken. Treatment groups were: a) i) 5% glucose (as untreated group) ii) DIXOxp iii) DIOxp iv) Naked Oxaliplatin v) FOLFOX (Pilot study, n=2). In all treatment formulations Oxaliplatin was injected at 2.2 mg / kg of mouse body weight. b) i) 5% glucose (as untreated group) ii) DIXLeuco iii) DlLeuco iv) Naked Leucovorin v) FOLFOX vi) FOLFIRI (Pilot study, n=l). In all treatment formulations leucovorin was injected at 10 mg / kg of mouse body weight. Here, itwas observed that DIXOxp and DIXLeuco shows better tumor regression than other treatment groups (Figure 14). c) i) 5% glucose (as untreated group) ii) D1X5-FU iii) D15-FU iv) Naked 5-FU v) FOLFOX vi) FOLFIRI. In all treatment formulations 5-FU was injected at 10 mg / kg of mouse body weight. Here, D1X5-FU shows better tumor regression than free 5-FU and even better than combination drugs FOLFOX and FOLFIRI (Figure15).Western blots studies:

[0093] To elucidate the possible mechanism behind the anti-cancer effect of D1X5-FU in in vivo settings, western blot analysis of tumor lysates obtained from mice treated with D1X5-FU, DI 5-FU, 5-FU, FOLFOX, FOLFIRI were performed. High BAX / BCL2 ratio with distinct upregulation of cleaved caspase-3 was observed in tumor lysate obtained from treated mice. The D1X5-FU treatment upregulated p-P53 Significant downregulation of NF-kB and vimentin was also observed in tumor lysate obtained from D1X5-FU and D15FU treated mice. The D1X5-FU treatment downregulated STAT 3 expression.

[0094] Taken together, these data indicate that D1X5-FU liposome induces intrinsic pathway of apoptosis and diminishes NF-kB levels in tumor mass which resulted in enhanced drug sensitivity and efficient tumor growth inhibition (Figure16).Example 7:In vivo toxicity studies:

[0095] With regard to potent biomedical applications of the liposomal formulations and to further establish the usage of the cationic lipid and GR based liposomes in the commercial market and phase clinical trials it is mandatory to perform some toxicity related studies.

[0096] It was observed that the mice in all groups were observed to be healthy with no mortality and any evident changes in gross pathological characteristics till 90 days of observation period. No unusual changes in the behavior or in locomotor activity and no evident toxic effects were observed during 90-day study period.

[0097] Body weight of animals were observed during study period and there was no such difference observed in both female (Figure 17) and male (Figure 18). The amount of feed consumed by the groups under treatment was almost like the control group and followed a similar pattern up to 30 days but after that it was increased slightly in D1X5FU female (Figure 19) and both D1X5-FU and FOLFOX male treatment groups (Figure 20).

[0098] The organ indices for liver, kidney, heart, brain, stomach, lungs, cecum and spleen to determine the possible effect of D1X5-FU and FOLFOX at the injected doses was also calculated. The results indicate no significant variation in the corresponding organ indices treated with 10 mg kg-1b.w. dose as compared with vehicle control group suggesting the nontoxic nature of D1X5-FU (Figure 21 and 22).

[0099] After completion of the study tenure, blood was collected from mice through retro orbital sinus for estimation of various hematological and biochemical parameters. Various blood hematological parameters are RBC: red blood cells, WBC: white blood cells, MCHC: mean corpuscular hemoglobin concentration, MCH: mean corpuscular hemoglobin, MCV: mean corpuscular volume, HCT: hematocrit, HGB: hemoglobin, LYMPHO: lymphocytes, MONO: monocytes, EOS: eosinophils, NEU: neutrophils (Figure 23, 24) and PLT: platelets (Figure 25). All the parameters in the D1X5-FU and FOLFOX treated groups displayed nonsignificant statistical alteration compared with control values, which are in the reference of range according to the literature (Wolford et al. J Toxicol Environ Health, 1986, 18(2), 161-188).The descriptions of the figures are as follows:Figure 1. Effect of liposomal formulations on CT 26. WT cell line:[000100] CT26.WT cells were treated continuously with different concentrations of DIXOxp, DIOxp, Oxp as a free drug and FOLFOX for 48 hours. Percentage viability values are shown as a mean of 3 results obtained from individual cytotoxicity experiments.Figure 2. Effect of liposomal formulations on CT 26. WT cell line:[000101] CT 26.WT cells were treated continuously with different concentration of DIXCape, DiCape, Cape as a free drug and XELOX for 48 hours. Percentage viability values are shown as a mean of 3 results obtained from individual cytotoxicity experiments.Figure 3. Effect of liposomal formulations on HCT-15 cell line:[000102] HCT-15 cells were treated continuously with different concentration of DIXLeuco, DlLeuco, Leuco as a free drug, FOLFOX and FOLFIRI for 48 hours. Percentage viability values are shown as a mean of 3 results obtained from individual cytotoxicity experiments.Figure 4. Effect of liposomal formulations on CT 26. WT cell line:[000103] CT26.WT cells were treated continuously with different concentration of D1X5-FU, D15-FU,5-FU as a free drug, FOLFOX and FOLFIRI for 48 hours. IC 50 values are shown as a mean of 3 results obtained from individual cytotoxicity experiments.Figure 5. Effect of liposomal formulations on HCT-15 cell line:[000104] HCT-15 cells were treated continuously with different concentration of D1X5-FU, D15-FU,5-FU as a free drug, FOLFOX and FOLFIRI for 48 hours. IC 50 values are shown as a mean of 3 results obtained from individual cytotoxicity experiments.Figure 6. Effect of liposomal formulations on HEK-293T cell line:[000105] HEK-293T cells were treated continuously with different concentration of D1X5-FU, D15-FU,5-FU as a free drug, FOLFOX and FOLFIRI for 48 hours. IC 50 values are shown as a mean of 3 results obtained from individual cytotoxicity experiments.Figure 7. Effect of niclosamide liposomal formulations on CT26.WT and HEK293T cell lines.[000106] Both CT26.WT and HEK293T cells were treated respectively with different niclosamide-associated liposomal formulations Dl-Niclo and DIX-Niclo or pristine drug niclosamide (Niclo) at different effective Niclo concentrations. * denotes p<0.05; ** denotes p<0.01[000107] Figure 8. Effect of D1X5-FU formulation on CYP3A5 upregulation:(a) RT-PCR analysis of CYP3A5 and 18Smessenger RNAs in HCT-15 cells in untreated control (I), free dex (II), D1X (III), D1X5-FU (IV). Here cells were treated with 2.5 pM concentration of above formulations for 12 hours.Figure 9. Quantitative uptake analysis of liposomal formulations by flow cytometry:[000108] RKO cells were treated with 2.5 pM, 5 pM and 10 pM of Rhodamine-PE-conjugated D1X5-FU liposomal formulation for 6 hours. Uptake study was done by flow cytometry (BD sciences instrument).Figure 10. Qualitative uptake analysis of liposomal formulations by confocal: [000109] Confocal microscopic images of cellular uptake of Rhodamine-PE- conjugated DIXCape, DIXOxp, D1X5-FU, DIXLeuco uptake in HCT-15 cell line after 6h of treatment.Figure 11. Qualitative uptake analysis of liposomal formulations by confocal: [000110] Confocal microscopic images of cellular uptake of Rhodamine-PE- conjugated D1X5-FU and D15-FU uptake in HCT-15 cell line after 6h of treatment.Figure 12. Ki 67 staining of proliferating cells.[000111] CT 26. WT cells were treated with 2.5 pM of D1X5-FU, D15-FU, 5-FU, FOLFOX and FOLFIRI for 12 hours. Cells were treated with Ki67 primary antibody and then PE conjugated secondary antibody. Red fluorescence shows proliferation of cells.Figure 13. Biodistribution study of D1X5-FU and D15-FU formulations:[000112] In vivo and ex vivo biodistribution of NIR dye labeled- D1X-5FU and D15-FU liposomes following 24 h of injection, (a) Ex vivo biodistribution study of D1X-5FU and D15-FU liposomes in different organs (I, colon; II, liver; III, kidneys; IV, spleen; V, lungs; and VI, heart) of tumor-bearing mice. The image clearly shows an intense localization of D1X-5FU vesicles in the colon tumor area as compared to DI 5-FU treated colon tumor and a (b) Histogram showing the ratio of radiant efficiency (ROI) of D1X-5FU and D15-FU vesicles in different organswith respect to total ROI in the same mice either bearing a tumor or are healthy mice.Figure 14. Tumor regression studies of orthotopic colon-tumor-bearing mice. [000113] Tumor bearing Balb / C female mice were treated with DIXOxp, DIXLeuco, oxaliplatin, leucorvorin as free drugs FOLFOX and FOLFIRI. Representative image showing colon tumor sizes in different treatment groups. Colons were harvested on 10th day after beginning treatment.Figure 15. Tumor regression studies of orthotopic colon-tumor-bearing mice. [000114] Tumor bearing Balb / C female mice were treated with D1X5-FU, D15-FU,5-FU as a free drug, FOLFOX and FOLFIRI. b) Representative image showing colon tumor size in different treatment groups. Colons were harvested on 10th day after beginning treatment wherein a=UT, b= D1X5-FU, c= D15-FU, d= 5-FU, e= FOLFOX, f= FOLFIRI, g= healthy mice cecum, (b) Histogram representing cecum weights normalized to corresponding body weights of mice as well as to normal cecum with that of the body weight of healthy mice.Figure 16. Western blot analysis of tumor lysate:[000115] Western blot analysis of tumor either kept untreated or treated with D1X5-FU, D15-FU,5-FU as a free drug, FOLFOX and FOLFIRI.Figure 17. Body weight monitoring of female Balb / C mice:[000116] Healthy female Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice body weight were monitored up to 90 days and compared with control (untreated) group.Figure 18. Body weight monitoring of male Balb / C mice:[000117] Healthy male Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice body weight were monitored up to 90 days and compared with control (untreated) group.Figure 19. Feed intake study of female Balb / C mice:[000118] Healthy female Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice feed intake were monitored up to 90 days and compared with control (untreated) group.Figure 20. Feed intake study of male Balb / C mice:[000119] Healthy male Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice feed intake were monitored up to 90 days and compared with control (untreated) group.Figure 21. Organ weight monitoring of female Balb / C mice:[000120] Healthy female Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice were sacrificed after 90 days and different organs weight were checked and compared with control (untreated) group.Figure 22. Organ weight monitoring of male Balb / C mice:[000121] Healthy male Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice were sacrificed after 90 days and different organs weight were checked and compared with control (untreated) group.Figure 23. Blood hematological parameters monitoring of female Balb / C mice: [000122] Healthy female Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice were sacrificed after 90 days and different blood hematological parameters were checked.Figure 24. Blood hematological parameters monitoring of male Balb / C mice: [000123] Healthy male Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice were sacrificed after 90 days and different blood hematological parameters were checked.Figure 25. Total platelet count in blood of male and female Balb / C mice:[000124] Healthy female Balb / C mice were treated with D1X5-FU (5- FU:10mg / kg body weight) and FOLFOX. The mice were sacrificed after 90 days and total platelets count in blood were checked.ADVANTAGES OF THE INVENTION• The process of the present invention can be exploited for preparing cationic lipid based drug transfer reagents containing glucocorticoid receptor binding dexamethasone in the formulation.• The composition disclosed herein can be used to deliver a pharmacologically active drug molecule selectively into cancer cells for therapeutic use.• The formulation can be used in gene therapy protocols to deliver a therapeutically useful protein to a cell or for delivering nucleic acids encoding therapeutically useful protein molecules.• The dexamethasone-associated lipid-based formulation can be formulated with ionic / non-ionic, lipophilic and hydrophilic therapeutic agents including many anticancer agents but not limited to ESC8, taxol™, irinotecan, nutlin, 5- Fluorouracil, oxaliplatin, leucovorin, niclosamide etc. therapeutic agent(s).

Claims

We claim:

1. A synergistic liposomal anti-cancer composition for simultaneous non-viral delivery of an anti-cancer drug to glucocorticoid receptor expressing cancer cells comprising: i. a cationic lipid [N,N-Bis(2-hydroxyethyl)-N-octadecyloctadec-9-en-l- aminiumchloride] ii. cholesterol; iii. dexamethasone; iv. 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG); and v. an anti-cancer drug.

2. The composition as claimed in claim 1, wherein cationic lipid: cholesterol: dexamethasone: anti-cancer drug: DSPE-PEG are in a mole ratio range of 1 : 1 : 0.5 : 0.25 : 0.02 to 1 : 1 : 0.5 : 0.5 : 0.02.

3. The composition as claimed in claim 1, wherein the anti-cancer drug is a lipophilic drug selected from ESC8, paclitaxel, docetaxel or niclosamide.

4. The composition as claimed in claim 1, wherein the anti-cancer drug is a hydrophilic drug selected from 5-Fluorouracil, leucovorin, capecitabin or oxaliplatin.

5. The composition as claimed in claim 1, wherein the composition inhibits growth of aggressive cancer cells selected from colorectal cancer, breast cancer, pancreatic cancer, melanoma or myeloma.

6. The composition as claimed in claim 1, wherein the composition is for intravenous, intra-muscular or intra-peritoneal administration.

7. A process for the preparation of synergistic composition as claimed in claim 1, wherein the process comprising the steps of:a) dissolving a cationic lipid [N,N-Bis(2-hydroxyethyl)-N-octadecyloctadec-9- en-l-aminiumchloride], cholesterol, and DSPE-PEG in a solvent followed by removing solvent using nitrogen gas to obtain a lipid film; b) keeping the lipid film as obtained in step (a) under vacuum for a period ranging between 3-4 hours at a room temperature of 25-35°Cto obtain dried lipid film and c) hydrating the dried lipid film as obtained in step (b) by keeping dried film in 5% glucose solution for a period ranging between 10-12 hrs followed by vortexing for a period ranging between 1-2 min and encapsulating an anti-cancer drug, wherein the mole ratio of cationic lipid: cholesterol: dexamethasone: the anti-cancer drug: DSPE-PEG is in a range of 1 : 1 : 0.5 : 0.25 : 0.02 to 1 : 1 : 0.5 : 0.5 : 0.02.

8. The process as claimed in claim 7, wherein the anti-cancer drug is a lipophilic drug selected from ESC8, paclitaxel, docetaxel and niclosamide.

9. The process as claimed in claim 7, wherein the anti-cancer drug is a hydrophilic drug selected from 5 -Fluorouracil, leucovorin, capecitabin and oxaliplatin.

10. The process as claimed in claim 7, wherein the solvent is chloroform.

Images