SYNERGISTIC FORMULATION OF MICROALGAL EXTRACTS FOR TREATING BREAST CANCER
Technical description of this Malaysian patent
FIELD OF INVENTION
The present invention relates to composition/formulations of Microalgal Extracts for treating breast cancer. More specifically, the present invention relates to a novel synergistic formulation/composition for the treatment against breast cancer cells, comprising of anti estrogen Tamoxifen drug and Microalgal Crude Extracts (MCEs) combined in predefined ratios and ethanol and water as green/eco-friendly solvents to provide strong cytotoxicity against MCF-7 and 4T1 cancer cells, with reduced cytotoxicity against normal Vero cells.
BACKGROUND OF THE INVENTION
Breast cancer is a heterogeneous disease, characterized by the proliferation and abnormal differentiation of malignant immature cells which often carry aberrations and deregulate genes (El-Kassas & El-Sheekh, 2014). It is a global health problem with an estimated age standardized incidence rate of 37.4 per 100,000 and age-standardized mortality rate of 13.2 per 100,000 women (International Agency, 2002). Interest in personalized medicine has moved research into understanding the genetic pathways of drug metabolism and the role of biomarkers for therapeutic decisions on individual patient. Although the current breast cancer therapies are improving rapidly, the major challenge is still in overcoming the drug resistance and treatment-related toxicities (Tan et al., 2008). The therapeutic management of breast cancer include the use of tamoxifen, TMX (an anti- estrogen drug in hormonal therapy) and chemotherapy drugs such as doxorubicin (a cytotoxic antibiotic) and paclitaxel (an anti-microtubule agent), both of which have been widely used for estrogen-dependent breast cancer and metastatic breast cancer (Cuzick et al., 2011; Padmavathi et al., 2006). However, the use of these drugs are often associated with some undesirable long-term side effects such as myelosuppression that affect the immune system, serious irreversible cardiotoxicity, and endometrial cancer with thromboembolic events 2 (Hosseini et al., 2017; Lazzeroni & DeCensi, 2013; Padmavathi et al., 2006). TMX or trans- 1-[p-b-(dimethyl-amino) ethoxy- phenyl]1,2-diphenyl-1-butene is a substituted trans-isomer of triphenylethylene. It belongs to the class of compounds with anti-estrogen activity, used in chemotherapy and chemo-prevention of breast cancer (Jordan, 2006; Morrow & Jordan, 1995). It is classified as a selective estrogen receptor modulator because it acts as an estrogen agonist in some tissues and as an antagonist in others (Jones & Buzdar, 2004). Due to its lipophilic nature, TMX strongly incorporates into biomembranes and interacts with lipids and proteins. TMX has been shown to interact very strongly with mitochondria isolated from the rat liver and the mammary cancer cells. These interactions can influence the mitochondrial bioenergetics (Marek et al., 2011). Like many small molecule inhibitors, TMX is not a highly selective drug. It has been reported to display anti-tumor activity against estrogen-receptor-negative breast cancers (ER-ve), and other unrelated cancers (Sun et al., 2012). TMX, a selective estrogen receptor modulator, is one of the most common anti-cancer drug in breast cancer treatment. It is a frequently-prescribed medication, widely used in all stages of breast cancer, for chemoprevention of breast cancer and the treatment of early and advanced stage estrogen receptor–positive breast cancer (Olopade et al., 2008). It acts as an anti-estrogen on breast cancer tissue, but as an estrogen, upon other tissue such as the uterus (Akim et al., 2013). Apart from its use as an endocrine therapy to combat breast cancer, TMX is also prescribed as an obviation treatment among high risk communities. Given as adjuvant, TMX significantly reduces the recurrence of contralateral breast cancer and improves the 10- year survival of breast cancer patients (Alkner et al., 2009). TMX causes rapid changes in membrane permeability which can lead to cell death and reduced cell viability. TMX also rapidly inhibits estrogen-dependent protein kinase C in MCF-7 cells and induces rapid mitochondrial death in estrogen receptor-positive MCF-7 cells (Hassan et al., 2018). Despite its wide-spread pharmacological uses, TMX suffers from low solubility and low selectivity (Kim et al., 2010). Further, the side effects of TMX include liver cancer, blood clotting, retinopathy and corneal opacities which is dose-dependent (Akim et al., 2013).TMX therapy has been attributed to the intrinsic or acquired resistance of the tumor to the effects 3 of estrogen receptor blockade. The inter-individual genetic variability plays a critical role not only in determining the toxicity from therapy but also in determining the benefits (Tan et al., 2008). TMX undergoes extensive metabolism via the CYP pathway to several primary and secondary metabolites. Some of these metabolites exhibit more potent anti-estrogenic effects than the TMX itself on breast cancer cells. One of the key enzymes is CYP2D6 that metabolizes TMX to a more active metabolite, endoxifen (Tan et al., 2008). Several variants in the CYP2D6 gene also result in the poor metabolizer phenotype, which is associated with negative side-effects (Olopad et al., 2008). The side effects include liver cancer, blood clotting, retinopathy and corneal opacities which is dose dependent (Akim et al., 2013). Thus, the long-term usage of TMX puts patients at increased risk of having thromboembolic events and uterine malignancies (Kim et al., 2010). The long-term use of TMX can cause endometrial and hepatic cancer with severe side effects, mainly because of TMX’s cytostatic (G0/G1 arrest) and cytotoxic (induction of apoptosis) action on cells. This dual effect suggests that TMX targets the checkpoint between cell cycle progression and apoptosis. TMX has been used experimentally to induce aneuploidy and hepatic cancer in animals, and to stimulate the formation of DNA adducts in rat hepatic cells (Petinari et al., 2004). Breast cancer has been found attenuated by an appreciable amount of natural substances including phytochemicals and dietary substances which affect cell proliferation, cell differentiation, angiogenesis, apoptosis, and a few other cellular transduction pathways. More effective and safer natural agents could improve the efficiency of breast cancer treatment (Zingue et al., 2018). The discovery and development of new chemopreventive drugs against breast cancer need to consider the safety and efficacy aspects to improve the breast cancer therapeutics management and reduce the high cost and pain of patients (Steward & Brown, 2013). It is therefore becoming imperative to search for new alternatives, strategies and formulations in the development of breast cancer prevention agents (Senthilraja & Kathiresan, 2015). There is a growing need for the development and or discovery of highly potential bioactive compounds from natural sources due to the resistance to chemical drugs (Alghazeera et al., 2016). Natural compounds with their high structural diversity, represent an invaluable source 4 for the development of powerful therapeutics. These exhibit high effectiveness by addressing the targets that exert central functions in most organisms (Hakkim et al., 2016). The anticancer activity of plant compounds for example may have high bioavailability and affinity to the target, with little loss of entropy when they bind to a protein. With conformational flexibility in aqueous and lipophilic environments, plant compounds may act as good alternative anti-cancer agents (McCullagh, 2008). There has been growing interest in marine bioprospecting, where potent natural compounds such as terpenes, steroids, alkaloids, and polyketides, have been discovered from marine organisms. Currently there are seven drugs of marine origin on the market, four of which are anticancer drugs, and close to 26 marine natural products are in clinical trials, of which 23 are anti-cancer compounds (Jaspars et al., 2016). Approximately 60% of the drugs used in hematology and oncology have their origin in natural sources, and one third of the most sold in the market are either natural compounds or derivatives thereof (Dyshlovoy & Honecker, 2015). Microalgae are eukaryotic unicellular plants that contribute up to 40% of global primary productivity (Martínez Andrade et al., 2018). They are excellent sources of pigments, lipids, carotenoids, omega-3 fatty acids, polysaccharides, vitamins and other fine chemicals, and there is an increasing demand for their use as nutraceuticals and food supplements (Abdullah et al., 2017; 2016; 2015). Some microalgae species such as Chlorella ellipsoidea, Chlorella sorokiniana, Cocconeis scutellum, Dunaliella tertiolecta, Chaetoceros calcitrans, Amphidinium carterae, Navicula incerta and Phaeodactylum tricornutum have been reported as having anti-cancer activity (Martínez Andrade et al., 2018). Nannochloropsis oculata extracted by 80% methanol (NOM) and hexane fraction (NOMH) exhibit anti-inflammatory activities against lipopolysaccharide-stimulated RAW 264.7 macrophages cells with IC50 value less than 6.25 μg/mL. Moreover, 90% n-hexane column elution of NOMH (NOMH90) shows marked cytotoxic effect on the HL-60 cells with IC50 value of 23.58 ± 0.09 μg/mL (Sanjeewa et al., 2016). Six microalgal methanolic crude extracts (Isochrysis galbana, Chaetoceros calcitrans, Scenedesmus quadricauda, Chlorella vulgaris, N. oculata and Tetraselmis tetrathele) are active in inhibiting the lipid peroxidation of linoleic acid. I. galbana and C. calcitrans show the highest antioxidant activity (>90%) indicating the presence of active 5 compounds for protection from lipid peroxidation (Natrah et al., 2007). N. oculata hexane and chloroform fractions exhibit the strongest anti-inflammatory activity (Lauritano et al., 2016). Chlorella ellipsoidea and C. vulgaris show cytotoxicity against HCT116 with the IC50 values of 40.73 ± 3.71 and 40.31 ± 4.43 μg/mL, respectively (Cha et al., 2008). The aqueous crude extract of C. vulgaris shows the highest antioxidant activity for inhibition scavenging (68.5%), highest phenolic content (3.45 mg/ mL) and antimicrobial activities (Dantas et al., 2015). The major challenge with cytotoxic compound is to retain its cytotoxic activity against cancer cells, without giving a threat to normal cells and if its cellular toxicity mechanism is via necrosis or apoptosis (Reyna-Martinez et al., 2018). Many chemotherapeutic agents are selectively toxic to tumor cells as they increase oxidant stress and enhance these already stressed cells beyond their limit (Moungjaroen et al., 2006). The systemic complications could be minimized by decreasing the peripheral distribution and by increasing the localization in the tumor by means of incorporating TMX into Nanostructured Lipid Carrier (NLC) (How et al., 2013). The use of a lower TMX dose and a better delivery system could enhance its efficiency in breast cancer treatment (Akim et al., 2013). The combined therapy of drugs with a carrier or natural products such as Microalgal Crude Extracts (MCEs) is attractive as it may improve the penetration of drugs into tumor cells, enhances their ability of tumor targeting, and reduces their side effects (Gul-e-Saba & Abdullah, 2015; Abdullah et al., 2014). Antitumor activity of microalgal compounds has been attributed to their lipophilicity to cross the lipophilic membranes and interact with proteins involved in apoptosis. Several microalgal compounds induce DNA-dependent DNA polymerases inhibition, cyclins expression alteration, or major transduction pathways interference (Baudelet et al., 2013) and these compounds may trigger immune response stimulation, as well as cytotoxicity against several cancer cell-lines (Lin et al., 2017; Shanab et al., 2012). Accordingly, there remains a need to develop a novel synergistic formulation/composition for anti-cancer treatment against breast cancer cells, comprising sufficient amounts of antiestrogen drug, namely Tamoxifen (TMX) and crude extracts of Nannochloropsis oculata, Tetraselmis suecica and Chlorella sp. combined together in predefined ratios, providing 6 strong cytotoxicity against MCF-7 and 4T1 cancer cells, with reduced cytotoxicity against normal Vero cells.
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