Novel triglycerides obtained from Macrocybe titans extract, their clinical uses and production methods

Abstract

The present invention relates to novel triglycerides of formula 2R-TG (C18:2, 9z, 12z; C16:0; C18:1, 9z) extracted from an extract of the fungus Macrocybe titans. Its pharmaceutical use and various clinical applications in the treatment of diseases controlled by the actin cytoskeleton are also contemplated. Its use as an antitumor agent for cancer diseases is primarily contemplated. Finally, the present invention relates to a method for obtaining a Macrocybe titans extract containing the triglycerides of the present invention.

Description

Technical Field

[0001] Description Field of the Invention The present invention belongs to the biomedical sector. More particularly, the present invention encompasses extracts of fungal origin and their clinical uses. In particular, the present invention refers to extracts of the Macrocybes titans fungus containing triglycerides that act as modifiers of the biological response leading to important clinical uses. Similarly, a method for obtaining said extracts is provided.

Background Art

[0002] Background of the Invention Cancer is a serious disease with a high incidence rate. In 2012, 14.1 million new cancer diagnoses were made worldwide, and 8.2 million people died due to this group of diseases. It is estimated that by 2030, there will be approximately 23.6 million new cases per year (Cancer Research UK. Worldwide cancer statistics. https: / / www cancer researchukorg / health-professional / cancer-statistics / worldwide-cancer#heading-Zero 2018). Apart from the heavy human toll, this disease also has a strong impact on the health economies of developing countries. For example, the cost associated with cancer treatment in the United States in 2017 was $147.3 billion (NIH. Cancer statistics. https: / / www cancer gov / about-cancer / understanding / statistics 2018). There are several fronts in the fight against cancer. First, prevention has been clearly identified as the most effective form of therapeutic intervention, but prevention requires strong political and social constraints to change harmful habits and avoid the main causes of cancer (Vineis P, Wild CP. Global cancer patterns: causes and prevention. Lancet 2014; 383:549-57). However, the development of new drugs that can contain previously detected diseases is also important (Magalhaes LG, Ferreira LLG, Andricopulo AD. Recent Advances and Perspectives in Cancer Drug Design. An Acad Bras Cienc 2018; 90:1233-50).

[0003] Traditionally, one way to obtain compounds with therapeutic properties was the screening of natural products. As a result, up to 60% of the drugs currently used to fight cancer or infectious diseases have a natural origin (Rates SM. Plants as source of drugs. Toxicon 2001; 39:603-13; Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461-77). Furthermore, these natural products can become leading compounds, from which families of similar compounds can be created using synthetic chemistry techniques and rational engineering, and thus new functions can be obtained that were not present in the original compounds (Hamburger M, Hostettmann K. Bioactivity in plants: The link between phytochemistry and medicine. Phytochemistry 1991; 30:3864-74). In traditional Chinese and Japanese medicines, the therapeutic uses of fungi are well-known, and these organisms serve as a source of propagation for pharmacologically active compounds, providing substances with the ability to antibacterial, antiviral, antitumor, antiallergic, immunomodulatory, anti-inflammatory, etc. (Lindequist U, Niedermeyer TH, Julich WD. The pharmacological potential of mushrooms. Evid Based Complement Alternat Med 2005; 2:285-99; Strader CR, Pearce CJ, Oberlies NH. Fingolimod (FTY720): a recently approved multiple sclerosis drug based on a fungal secondary metabolite. J Nat Prod 2011; 74:900-7).

[0004] Through a joint study with the National Institute of Biodiversity (INBio) in Costa Rica, the inventors of the present invention studied the antitumor capacity of various extracts of natural fungi obtained from the tropical forests of this country in order to search for new natural products with therapeutic uses. As a result, they identified a novel compound from the Macrocybe titans fungus that has the ability to halt tumor growth in vivo. The genus Macrocybe classifies seven species distributed in the tropical and subtropical regions of the world that produce large fruit bodies reaching up to 1 meter in diameter and 18 kg in weight (Pegler DN, Lodge Dj, Nakasone KK. The pantropical genus Macrocybe gen. nov. Mycologia 1998; 90:494-504). The Macrocybe titans species is the only species of this genus found in the American continent (Ramirez NA, Niveiro N, Michlig A, Popoff OF. First record of Macrocybe titans (Tricholomataceae, Basidiomycota) in Argentina. Check List 2017; 13:153-8). They are edible species (Razaq A, Nawaz R, Khalid AN. An Asian edible mushroom, Macrocybe gigantea: its distribution and ITS-rDNA based phylogeny. Mycosphere 2016; 7:525-30), but their use as therapeutic fungi has not been described in the literature.It has only been suggested that some macrocybe titans polysaccharides can inhibit melanoma cell migration (Milhorini SDS, Smiderle FR, Biscaia SMP, Rosado FR, Trindade ES, Iacomini M. Fucogalactan from the giant mushroom Macrocybe titans inhibits melanoma cells migration. Carbohydr Polym 2018; 190:50-6) and that the mycelium of Macrocybe gigantea contains antibacterial compounds (Gaur T, Rao PB. Analysis of Antibacterial Activity and Bioactive Compounds of the Giant Mushroom, Macrocybe gigantea (Agaricomycetes), from India. Int J Med Mushrooms 2017; 19:1083-92).

Brief Description of the Drawings

[0005]

Figure 1

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Mode for Carrying Out the Invention

[0006] Detailed Description of the Invention The inventors of the present invention have identified a novel compound from the macrocybe titans fungal extract that has the ability to arrest tumor growth in vivo. The main aspect of the present invention provides the compound which is a triglyceride of formula 2R-TG(C18:2, 9z, 12z;C16:0;C18:1, 9z) (hereinafter TG10) represented by the following scheme.

[0007]

Chemical formula

[0008] This compound acts as a biological response modifier (BRM) because it specifically alters the actin cytoskeleton. Therefore, it has direct applications in all such phenomena controlled by this component of the cytoskeleton, and in particular, the following non-exclusive pathways: i) changes in cell morphology, ii) cell motility (including metastasis), iii) muscle contraction, iv) modification of cell-cell junctions, v) control of endocytosis and phagocytosis, vi) embryogenesis, vii) control of gene expression, viii) control of the immune system, ix) defense against pathogens (bacteria, fungi, viruses, parasites, etc.), and x) control of apoptosis. Due to its properties, in a second aspect of the present invention, the TG10 triglyceride of the present invention is provided for use as a medicament. TG10 triglyceride has a beneficial effect on all such diseases associated with the actin cytoskeleton. Accordingly, another aspect of the present invention provides the use of triglyceride in the treatment of actin cytoskeleton control diseases such as, but not limited to, amyloidosis, retinitis pigmentosa, infectious diseases, kidney diseases, and congenital deafness syndromes.

[0009] The triglyceride of the present invention has been found to be effective in reducing the size of human tumors. Accordingly, another main aspect of the present invention provides the use of TG10 triglyceride, alone or in combination with other therapies (radiotherapy, chemotherapy, immunotherapy, etc.), in the treatment of cancer diseases in both animal and human patients. Furthermore, the inventors of the present invention have shown that since triglyceride preferentially binds to tumor cells, in the context of diagnostic imaging devices (PET, fluorescence, etc.), it can be used together with a tracer (fluorescent, radioactive, etc.) to locate tumors in living organisms, both in animal patients and human patients. Said affinity of triglyceride for tumor cells also makes it possible to direct drugs or nanoparticles towards tumor cells as a therapeutic treatment.

[0010] In another main aspect of the present invention, a pharmaceutical composition comprising the triglyceride of the present invention is provided. As described above, the inventors of the present invention obtained triglyceride from the extract of Macrocybe titans. Accordingly, another main aspect of the present invention is a method for obtaining an extract of Macrocybe titans, comprising the following steps: a. Subjecting a sample of Macrocybe titans fungus to extraction with 95% ethanol, b. Treating the extract obtained in a) with ultrasound for 30 to 60 minutes, preferably 30 minutes, c. Maintaining the extract treated in b) at a temperature of -20°C to -80°C, preferably -20°C, for 24 to 48 hours, preferably 24 hours, d. Obtaining the precipitate phase of the extract thus treated, and After performing alkaloid separation from the precipitate phase of the extract obtained in e. d), column chromatography was carried out using a polymer resin with an 8:2 IP:CH2L2 mobile phase at pH 12 to yield a fraction of the extract containing the triglyceride of formula 2R-TG(C18:2, 9z, 12z;C16:0;C18:1, 9z). Provided is a method comprising the above.

[0011] Once the triglyceride of the present invention has been identified and its formula determined, it is also possible to obtain it by utilizing its chemical synthesis instead of extracting it from its original source, which is advantageous from the viewpoints of economy, reproducibility, and pharmacological safety.

Example

[0012] Example 1. Repeated screening by toxicity analysis and separation of new fractions by chromatography Extracts (aqueous, ethyl, acidic) were obtained from nine macrofungal species collected by INBio in the Costa Rican rainforest. All of them were subjected to cytotoxicity tests with two human cell lines: A549 (lung adenocarcinoma) and NL20 (immortalized, non-tumor cell line, obtained from lung epithelium). Cells were seeded in 96-well plates at a concentration of 2,000 (A549) or 10,000 (NL20) cells per well. Immediately after the cells adhered to the bottom, serial concentrations of each extract were added and incubated at 37 °C for 5 days in an atmosphere containing humidity (85% relative humidity) and 5% CO2. Eight replicates (wells) were examined for each concentration. At the end of the incubation process, 20 μl of Cell Titer (Promega) was added to each well and left for 4 hours to form the colored formazan salt. Colorimetric quantification was performed using a plate reader (POLARstar Omega) at a wavelength of 490 nm according to a previously published protocol (Coderch C, Diaz de CM, Zapico JM, Pelaez R, Larrayoz IM, Ramos A, et al. In silico identification and in vivo characterization of small molecule therapeutic hypothermia mimetics. Bioorg Med Chem 2017;25:6597-604). The selection criteria for the extracts consisted of choosing those that were toxic to tumor cells even at low concentrations while taking into account the integrity of non-tumor cells, or at least providing a wide therapeutic concentration range between both cell lines. The extracts selected in the first round were fractionated with different column and chromatograph protocols. Each new peak was separated and subjected to toxicity analysis again. This process was repeated as many times as necessary to finally obtain a fraction pure enough that most of its compounds could be identified by analytical chemistry methods.

[0013] Results: Among all the extracts analyzed in the first round, the extract corresponding to the M. titans species had the widest therapeutic concentration range as all A549 cells were destroyed even at 0.1 mg / ml, while NL20 cells survived even at a concentration of 0.42 mg / ml (Figure 1). Among the various extracts of this species, the extract obtained by extraction with 95% ethanol, treated with ultrasonic waves for 30 minutes, and kept at -20 °C for 24 hours, especially the precipitate phase after this treatment, was the most effective. Due to the lipid nature of this extract, it was decided to perform column chromatography with a polymer resin (HP20-SS) using various mobile phases after alkaloid separation. All the obtained fractions were subjected to toxicity tests again. The fraction that maintained the best therapeutic concentration range corresponded to the fraction obtained with a mobile phase of pH 12 and 8:2 IPA:CH2Cl2.

[0014] Example 2. Chemical characterization of the molecule responsible for the antitumor activity. The chemical structure of the antitumor product was elucidated using the fractions obtained in the previous experiment. For this purpose, 1 H, 13 nuclear magnetic resonance (NMR) analysis by 1H, 13C and DEPT-135, mass spectrometry (Q-TOF), and two-dimensional HMQC and COSY experiments were performed.

[0015] Results: 1 The 1H-NMR study suggested that the product contained many alkyl protons, a series of α-protons from alcohols, and some olefinic protons. The distribution of these protons suggested that the natural extract contained triglycerides (TG) mixed with some of its component fatty acids. The triplet at 2.77 ppm was found to be the two protons of the methylene located between two double bonds, meaning a double unsaturation of one fatty acid (Figure 2). 13The C and DEPT-135 experiments enabled the assignment of the product carbon (Figure 3). The two-dimensional experiments confirmed the assignment of the carbon and proton of TG. On the other hand, accurate mass determination (MS-QTOF) enabled the identification of the fatty acids constituting TG. All of this led to the suggestion that TG contains three different fatty acids (C18:1, 9z), palmitic acid (C16:0), and a double-unsaturated fatty acid with 18 or 20 carbons. However, since the sample was not pure and it was difficult to accurately assign the order and size of the fatty acids, a series of similar TGs were synthesized and it was decided to experimentally determine the antitumor activity of each one. For some TGs with optical activity, two enantiomers were synthesized (Table 1). The toxicity test showed that only TG10 [2R-TG(C18:2, 9z, 12z;C16:0;C18:1, 9z)] (Figure 4) among all the synthesized TGs exhibited antitumor activity against A549, suggesting that it is not toxic to normal cells (NL20). The other TGs had no recognizable activity on cells. It should be noted that the S enantiomer (TG9) of the active TG (TG10) also had no effect on the cells, suggesting a high degree of specificity regarding this activity.

[0016]

Table 1

[0017] Example 3. In vitro test: Modification of the actin cytoskeleton. Many antitumor substances exert their effects by modifying the cytoskeleton of tumor cells and acting on either microtubules or actin filaments (Zhang S, Menche D, Zahler S, Vollmar AM, Liebl J, Forster F. In vitro anti-cancer effects of the actin-binding natural compound rhizopodin. Pharmazie 2015;70:610-5.). To examine whether compound TG10 has a similar mechanism of action to other antitumor drugs, tumor cells (A549) and non-tumor cells (NL20) were treated with 0.03 mg / ml of TG10 for 24 hours. At the end of this time, the cells were fixed with 10% formaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and exposed to 1:200 Bodipy-phallacidin and 1:1000 DAPI (Molecular Probes) for 1 hour. Images were obtained with a confocal laser microscope (TCS SP5, Leica).

[0018] Results: As expected, both A549 and NL20 cells had typical stress fibers (composed of actin filaments) in their cytoplasm before treatment. After TG10 treatment, A549 cells completely lost their polymerized actin fibers, which migrated to the cell ends where small filopodia were formed. In contrast, NL20 (non-tumor) cells maintained their filamentous actin-like structures even after TG10 treatment (Figure 4). These results confirmed that TG10 acts on the actin cytoskeleton of tumor cells.

[0019] Example 4. In vivo assay: Human lung cancer xenograft in immunosuppressed mice. The usual way to demonstrate that a novel anti-tumor drug works in vivo is to perform a xenograft experiment in which human tumor cells are injected into immunodeficient mice (Zitvogel L, Pitt JM, Daillere R, Smyth MJ, Kroemer G. Mouse models in oncoimmunology. Nat Rev Cancer 2016;16:759-73). In this case, 20 male mice of the NOD scid gamma strain (NSG, Stock No. 005557, The Jackson Laboratory) were used. All of them were subcutaneously injected with 10 million A549 cells and left for 2 weeks until the tumors became measurable. Two groups of 10 mice each were randomized and labeled as the control group (diamonds) and the TG10 treatment group (squares). From this point, intratumoral injections of 100 μl of PBS (control group) or 0.1 mg / ml of TG10 in PBS (treatment group) were performed three times a week. Before each injection, the tumor volume was measured with calipers. The mice were euthanized when the tumors reached a size incompatible with animal welfare.

[0020] Results: TG10 injection was started on day 15 after the initial injection of tumor cells. From this point, the size of the tumors injected with PBS continued to increase gradually until they reached the maximum size allowed on day 48 (for ethical and animal welfare reasons). On the other hand, the tumors injected with TG10 continued to grow at a slower rate than the growth rate of the tumors in the control group (Figure 5). At the end of the experiment, the tumors in the control group had an average volume of 2900 mm 3 while the treated tumors had an average volume of 1600 mm 3 . Two-way analysis of variance (ANOVA) was performed and it was found that there was a significant difference due to treatment between the volumes of the treated tumors and the control tumors (<0.05). Another aspect of the present invention may be as follows. 〔1〕Triglyceride of formula 2R-TG(C18:2, 9z, 12z;C16:0;C18:1, 9z). 〔2〕The triglyceride according to 〔1〕above for use as a medicament. 〔3〕The triglyceride according to 〔2〕above for use in the treatment of diseases controlled by the actin cytoskeleton. 〔4〕The triglyceride according to 〔3〕above, wherein the disease controlled by the actin cytoskeleton is selected from amyloidosis, retinitis pigmentosa, infectious diseases, kidney diseases and congenital deafness syndrome. 〔5〕The triglyceride according to 〔2〕above for use in the treatment of cancer diseases. 〔6〕The triglyceride according to 〔1〕above for use in tumor diagnosis using image processing technology. 〔7〕The triglyceride according to 〔6〕above for use in combination with a tracer. 〔8〕A pharmaceutical composition comprising the triglyceride according to 〔1〕above. 〔9〕A Macrocybe titans extract containing the triglyceride according to 〔1〕above. 〔10〕A method for obtaining the extract of Macrocybe titans according to 〔9〕above, comprising the following steps: a. Subjecting a sample of Macrocybe titans fungus to extraction with 95% ethanol. b. Treating the extract obtained in a) with ultrasonic waves for 30 to 60 minutes. c. Maintaining the extract treated in b) at a temperature of -20°C to -80°C for 24 to 48 hours. d. Obtaining the precipitate phase of the extract thus treated, and e. After performing alkaloid separation from the precipitate phase of the extract obtained in d), using a mobile phase of 8:2 IP:CH at pH 12 and performing column chromatography with a polymer resin to obtain a fraction of the extract containing the triglyceride according to 〔1〕above. 2 L 2 The method as described above. ​

Claims

1. Triglyceride of formula 2R-TG(C18:2, 9z, 12z; C16:0; C18:1, 9z).

2. A pharmaceutical composition for use in the treatment of cancer diseases, comprising the triglyceride according to Claim 1.

3. A pharmaceutical composition for use in tumor diagnosis using image processing technology, comprising the triglyceride according to Claim 1.

4. The pharmaceutical composition according to Claim 3, for use in combination with a tracer.

5. A method for obtaining a Macrocybe titans extract comprising the triglyceride according to Claim 1, comprising the following steps: a. Subjecting a sample of Macrocybe titans fungus to extraction with 95% ethanol, b. Treating the extract obtained in a) with ultrasound for 30 to 60 minutes, c. Maintaining the extract treated in b) at a temperature of -20°C to -80°C for 24 to 48 hours, d. Obtaining the precipitate phase of the thus-treated extract, and e. After performing alkaloid separation from the precipitate phase of the extract obtained in d), using an 8:2 IPA:CH 2 Cl 2 Performing column chromatography with a polymer resin using a mobile phase at pH 12 to provide a fraction of the extract containing the triglyceride according to Claim 1 comprising the said method.

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