A pharmaceutical composition comprising emetine and mitoxantrone for the prevention or treatment of mammary gland cancer and a liposome containing this composition

Abstract

The description of the invention discloses a pharmaceutical composition comprising emetine at a concentration of not less than 0.01 μM and mitoxantrone at a concentration of not less than 0.003 μM for use in the treatment of mammary gland cancer and a liposome comprising at least three lipids including DPPC, DSPE-PEG(2000)amine and cholesterol containing the composition.

Description

[0001] A PHARMACEUTICAL COMPOSITION COMPRISING EMETINE AND MITOXANTRONE FOR THE PREVENTION OR TREATMENT OF MAMMARY GLAND CANCER AND A LIPOSOME CONTAINING THIS COMPOSITION

[0002] The object of the invention is a pharmaceutical composition for the prevention or treatment of cancer and diseases resulting from excessive cell proliferation, in particular mammary gland cancer, and a liposome containing the composition.

[0003] Cancer treatment can be local or systemic. Local treatment usually consists of removing the tumour or using radiotherapy. Systemic treatment affects other cells in the body in addition to the tumour cells. Such methods include chemotherapy, hormone therapy or targeted treatment, which involves the administration of intravenous or oral drugs that are tailored to the progression of the disease. Other methods of cancer treatment are also known from the state of the art.

[0004] Liposomes are commonly used as carriers in chemotherapy with cytostatic drugs, due to their ability to transport these drugs to their target sites in the body. They are spherical vesicles made up of one or more phospholipid bilayers surrounding a liquid interior. The lipid composition of liposomes is crucial for their stability and physicochemical properties. The choice of suitable lipids and their ratio affects the size, shape, stability and surface properties of liposomes. The size and surface properties of liposomes, on the other hand, are crucial for their bioavailability, stability and their selectivity of drug delivery to target tissues and cells (https: / / doi.Org / 10.1016 / j.addr.2012.09.037). Polyethylene glycol (PEG) is commonly used in the surface modification of liposomes to increase their stability in plasma, improve bioavailability and prolong circulation time (DOI: 10.1016 / j.jconrel.2013.07.026). In addition, the chain length of polyethylene glycol (PEG) can affect the site of drug accumulation in the human body and the targeting efficiency of a therapy (10.1016 / j.biomaterials.2011 .04.082). The most commonly used polyethylene glycol for modifying liposomes is a polymer terminated with a terminal methoxyl group (-O-CH3 ), the so-called MPEG. Other modifications such as a terminal amino group (-NH2) or a carboxyl group (-COOH) are usually used as intermediates for further modification with proteins or other cell homing molecules (https: / / doi.org / 10.1038 / s41598-021-86860-5) and their potential to generate surface charge in the changing tumour environment has so far been neglected. On the other hand, it is known that the surface charge of liposomes can influence where they accumulate in the body and, in particular, their accumulation in tumour tissues. Studies show that positively charged liposomes have better accumulation and penetration of the carrier in tumour tissues and better cellular uptake compared to inert and negatively charged nanoparticles

[0005] (https: / / doi.Org / 10.1016 / j.nantod.2016.04.008). The presence of a positive charge on the surface of liposomes is therefore crucial in ensuring efficient and specific transport into tumour tissues.

[0006] The essence of the pharmaceutical composition solution according to the invention is a composition comprising emetine at a concentration of not less than 0.003 pM and mitoxantrone at a concentration of not less than 0.01 pM for use in the treatment of mammary gland cancer.

[0007] Favourably, the concentration ratio of emetine and mitoxantrone is between 1 :3 and 1 :5.

[0008] The essence of the solution according to the invention for the modified liposome is that the liposome is composed of at least three lipids, including DPPC (1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine) in an amount of 48.6 to 70.7% by weight, cholesterol in an amount of 18.3 to 18,6% by weight, DSPE-PEG(2000)amine (ammonium salt of 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-N -[amino(polyethylene glycol)-2000]) in an amount of 10.6 to 10.7% by weight and contains emetine in a concentration of not less than 0.003 pM and mitoxantrone in a concentration of not less than 0.01 pM.

[0009] Favourably, the concentration ratio of emetine and mitoxantrone is between 1 :3 and 1 :5.

[0010] The main advantage of the solutions according to the inventions is that the composition used has a dual action, which leads to the arrest of growth or death of the cell by a synergistic combination of two mechanisms that would generally not be targeted by a single inhibitor, leading to the arrest of growth or death of the cancer cell. The synergism that occurs with emetine and mitoxantrone allows the use of much lower doses of the substances and limits the side effects of their use. The main advantage of modified liposomes as carriers for the compositions is that due to the positive charge generated by the presence of the terminal amino group, there is effective accumulation of the carrier with the drug in solid tumours, and thus anticancer therapy with these drugs is much more effective. This has been demonstrated in in vitro studies for emetine- and mitoxantrone-loaded nanoparticles. In the case of these studies, a 3-4 times higher treatment efficacy was achieved, resulting in tumour shrinkage at twice the dose compared to the free drugs.

[0011] The developed carriers allow emetine loading of up to above 95% for a drug to lipid ratio of 0.1 , which is twice the value reported in the literature. At higher initial drug concentrations, liposomes allow up to four times higher drug to lipid ratios than liposomes reported in the publications. The reason for such good loading is the specific lipid structure containing a terminal amino group in the polyethylene glycol molecule.

[0012] These carriers ensure that emetine is loaded inside the liposomes and not deposited on the surface, as indicated by the lack of significant changes in the Zeta potential of the liposomes after the drug loading process. Additional studies of emetine-loaded liposomes in the presence of human and rat albumin confirm the lack of sudden drug release under in vitro test conditions.

[0013] These carriers also allow for the binding of emetine in the presence of other drugs such as mitoxantrone without a drastic decrease in the %EE encapsulation value of emetine. The presence of a 5-fold excess of mitoxantrone (a drug from the anthracycline group) preserves the %EE of emetine at 95-99%, whereas literature values of liposomal carriers containing an anthracycline-emetine mixture at these concentrations indicate a decrease in %EE to values lower than 30%.

[0014] The carriers also provide a significant reduction in the toxicity of emetine encapsulated in liposomes compared to free emetine. They indicate much lower LC50 values for drugs encapsulated in carriers. The LC50 value calculated for emetine encapsulated in a liposome is 12-fold higher after 24 h of dosing than the value calculated for the free drug. This creates the possibility of significantly reducing the toxicity of this drug and thus enabling the use of this drug in anticancer therapies.

[0015] The solutions according to the inventions are illustrated by the following fabrication examples and drawings, where Fig. 1 .1 shows an example of the size distribution of the hydrodynamic radii of nanoCombo 6.2 liposomes after synthesis and purification (see fabrication description for details), measured by dynamic light scattering. Sample diluted 10 times; Fig. 1.2 - Example of size distribution of hydrodynamic radii of nanoCombo 6.3 liposomes after synthesis and purification, measured by dynamic light scattering. Fig. 1.3 - exemplary size distribution of hydrodynamic radii of nanoCombo 6.4 liposomes after synthesis and purification, measured by dynamic light scattering; Fig.1.4. - Example size distribution of hydrodynamic radii of nanoCombo 6.6 liposomes after synthesis and purification, measured by dynamic light scattering; Fig.1.5. - Example Zeta potential distribution of nanoCombo 6.2 liposomes after synthesis and purification, measured by dynamic light scattering; Fig.1..6. - Example Zeta potential distribution of nanoCombo 6.3 liposomes after synthesis and purification, measured by dynamic light scattering; Fig. 1.7. - Example Zeta potential distribution of nanoCombo 6.4 liposomes after synthesis and purification, measured by dynamic light scattering; Fig. 1.8. - Example Zeta potential distribution of nanoCombo 6.6 liposomes after synthesis and purification, measured by dynamic light scattering; Fig. 2.1 - Growth kinetics of CAL-148 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :5; Fig. 2.2

[0016] - Growth kinetics of CAL-148 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :3; Fig. 3.1 - Growth kinetics of CAL-51 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :5; Fig. 3.2 - Growth kinetics of CAL-51 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :3; Fig. 4.1 - Growth kinetics of Hs578T human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :5; Fig. 4.2

[0017] - Growth kinetics of Hs578T human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :3; Fig. 5.1 - Growth kinetics of MDAMB231 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :5; Fig. 5.2 - Growth kinetics of MDAMB231 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :3; Fig. 6.1 - Growth kinetics of MDAMB453 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :5; Fig. 6.2

[0018] - Growth kinetics of MDAMB453 human lung cancer tumours in mice treated with Emetine, Mitoxantrone and their combinations at a concentration ratio of 1 :3;

[0019] Example I Synthesis and physicochemical parameters of liposomes

[0020] A lipid solution in 99.8% ethanol with a total lipid concentration of 100mg / ml was prepared with the composition: DPPC / Cholesterol / DSPE- PEG(2000)NH2 in molar fractions of 0.65 / 0.32 / 0.026 and mass fractions of 0.70650 / 0.18607 / 0.1O743.The whole was heated for 15 minutes in a water bath at 60°C. Subsequently, a solution (NH )42 SO4 , at a concentration of 350mM in deionised water, was prepared in sterile urea. The solution was filtered using sterile syringe filters with a hydrophilic PTFE membrane with a pore diameter of 0.22 pm. The pH of the solution was titrated with 0.3M HCL solution until a pH of 5.3 was obtained.

[0021] Liposomes were prepared using a NanoAssemblr IGNITE microfluidic device™ with an NxGen cartridge. Flow parameters set: volume ratio of aqueous phase to organic phase 9:1 ; total flow rate 5ml / min, final volume of reaction mixture 9.05ml; initial loss 0.9ml; final loss 0.05ml. Six batches of liposomes were prepared. Liposomes were dialysed using MWCO 100kD pore diameter tubular dialysis membranes with a volume of 10ml, which were conditioned for 20 minutes each time in solutions of, respectively: 10% ethanol, deionised water and PBS buffer, pH 8.5.

[0022] A 5-fold PBS buffer concentrate pH 7.4 was prepared by dissolving 20 g NaCI, 3.6 g Na2 HPO4 (anhydrous), 0.775 g NaH2 PO X2H42 O (dihydrate), 0.5 g KCI in 0.5 L deionised water (milliQ). It was stirred on a magnetic stirrer for 30 min. The buffer was then diluted 5-fold and 0.1 M NaOH was added until a pH of 8.5 was obtained.

[0023] Liposomes were dialysed in a refrigerator (4°C) for 14 h, changing the external buffer after 2h and 6h.

[0024] Subsequently, starting solutions of emetine hydrochloride at 50mg / ml and mitoxantrone dihydrochloride in DMSO were prepared. Then, liposomes after dialysis in PBS at pH 8.5 were added with the emetine hydrochloride stock solution according to Table 1 and mixed. Mitoxantrone dihydrochloride stock solution was then added according to Table 1 and mixed. The bottle was tightly closed and heated in a water bath at 60°C for 15 minutes. After removal from the water bath, the liposomes were allowed to cool for 30 minutes. The vessel with liposomes was protected from light. The liposomes were then separated on a Sephadex G-25 packed chromatography column and the encapsulation rate (loading rate) of the drugs into the liposomes was determined by UV-Vis spectroscopy against a calibration curve using the principle of additivity of absorbance. Drug concentrations in liposomal fractions were determined by UV-Vis spectroscopy against calibration curves by dissolving 100uL of liposome sample in 850uL ethanol and 50uL 0.3M HCI (concentrations shown in Table 1). Hydrodynamic diameters and polydispersity indexes (PDI) and Zeta potentials of the nanoparticles were measured. All data are summarised in Table_1. Example distributions of hydrodynamic diameters with PDI coefficients and example distributions of Zeta potentials of the produced liposomes are summarised in Fig. 1.1 - Fig. 1.8.

[0025] Table 1 Physicochemical parameters of liposomes loaded with emetine hydrochloride (EM) and mitoxantrone dihydrochloride (MX) after synthesis.

[0026] Example II

[0027] Exploring synergies

[0028] The assay used emetine and mitoxantrone, which were dissolved in water and stored as 50mM stock solutions at -80°C. Cell lines were used in the assay as shown in Table 2.1 .

[0029] Table 2.1 List of cell lines detailing their origin

[0030] Cells were thawed from ampoules stored in liquid nitrogen and cultured in petri dishes in the appropriate culture medium according to Table 2.2, in a humidified 5% CO2 atmosphere at 37°C. Cells were passaged twice weekly using EDTA-Trypsin solution, pH 8. Counted cells with a viability of at least 90% as assessed with Trypan-Blue solution were used for anti-proliferation assays.

[0031] Table 2.2 List of cell lines and their corresponding culture medium

[0032] Twenty-four hours before the addition of the test compounds / combinations, cells were seeded into 384-well plates at 50pL / well and a density of 103cells / well, in the appropriate culture medium. After incubation overnight, cells were treated with different concentrations of compounds or combinations of compounds mixed at different molar ratios (described in the tables summarising the individual results, with a dilution factor of 3.16x. Solutions of the compounds were diluted in the test medium - RPMI-1640 and Opti-MEM in a 1 :1 ratio, and the medium was supplemented with 2 mM glutamine and 5% fetal bovine serum FBS. After 72h, colourimetric evaluation of cell growth was performed by SRB. Cells were fixed for 1 h with cold 25% (w / v) trichloroacetic acid (TCA) solution, washed five times with tap water, and stained with 0.4% (v / v) sulforhodamine B (SRB, solution in 1 % (v / v) acetic acid) for 30 min. Unbound dye was removed by washing the plates (4x) in 1 % (v / v) acetic acid, and the protein-bound dye was extracted with 10 mM unbuffered Tris base and the optical density (A = 540 pM) was determined in a BioTek Synergy H4 Hybrid computer microplate reader (BioTek Instruments USA). The entire washing / staining procedure was performed using a BioTek EL406 washing station (BioTek Instruments USA). The raw absorbance results were analysed in Microsoft Excel software using the formula: 100

[0033] Where:

[0034] %Zahlnh - inhibition of proliferation

[0035] Am - absorbance of wells without cells (medium) Ak - absorbance of control wells (cells treated with pure culture medium) Ap- absorbance of wells treated with compounds

[0036] The proliferation inhibition data were then used to calculate the IC50 - the concentration of the test compound that inhibits the proliferation of 50% of the tumour cell population, calculated using the GraphPadPrism 7.0 nonlinear model [Inhibitor] vs. response - Variable slope (four parameters). Each compound at the indicated concentrations was tested in triplicate in one experiment. Each experiment was repeated three times. The results obtained are presented in Tables 3.1 - 7.2 and Figs. 2.1 - 6.2.

[0037] Table 3.1 Inhibition of cell profiling of CAL148 lines for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :5 ratio in free and nanoparticle form including standard deviation

[0038] Table 3.2 Inhibition of cell profiling of CAL148 lines for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :3 ratio in free and nanoparticle form including standard deviation

[0039] Table 4.1 Inhibition of CAL51 cell profiling for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :5 ratio in free and nanoparticle form including standard deviation

[0040] Table 4.2 Inhibition of CAL51 line cell profiling for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :3 ratio in free and nanoparticle form including standard deviation

[0041] Table 5.1 Inhibition of cell profiling of line Hs578T for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :5 ratio in free and nanoparticle form including standard deviation

[0042] Table 5.2 Inhibition of cell profiling of line Hs578T for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :3 ratio in free and nanoparticle form including standard deviation

[0043] Table 6.1 Inhibition of cell profiling of MDAMB231 line for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :5 ratio in free and nanoparticle form including standard deviation

[0044] Table 6.2 Inhibition of cell profiling of MDAMB231 line for individual concentrations of Emetine, Mitoxantrone and their combinations in a 1 :3 ratio in free and nanoparticle form including standard deviation Table 7.1 Inhibition of cell profiling of MDAMB453 line for individual concentrations of Emetine, Mitoxantrone and their combinations in 1 :5 ratio in free and nanoparticle form including standard deviation

[0045] Table 7.2 Inhibition of cell profiling of MDAMB453 line for individual concentrations of Emetine, Mitoxantrone and their combinations in 1 :3 ratio in free and nanoparticle form including standard deviation

[0046] Mobile line: MDAMB453

Claims

Claims1 . Pharmaceutical composition consisting of emetine at a concentration of not less than 0.01 pM and mitoxantrone at a concentration of not less than 0.003 pM for the treatment of mammary gland cancer.

2. The composition according to claim. 1, characterised in that the concentration ratio of emetine and mitoxantrone is from 1 :3 to 1 :5.

3. A liposome characterised in that it is composed of at least three lipids, including DPPC (1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine) in an amount of 48.6 to 70.7% by weight, cholesterol in an amount of 18.3 to 18.6% by weight, DSPE-PEG(2000)amine (ammonium salt of 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-N [amino(polyethylene glycol)-2000]) in an amount of 10.6 to 10.7% by weight and contains emetine in a concentration of not less than 0.01 pM and mitoxantrone in a concentration of not less than 0.003 pM4. The liposome according to claim 3 characterized in that a concentration ratio of emetine and mitoxantrone of 1 :3 to 1 :5.

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