Use of parasites and extracellular vesicles obtained from parasites in cancer treatment

Abstract

The present invention relates to the use of parasites and extracellular vesicles obtained from parasites for cancer treatment. The aim of the present invention is to use parasites and extracellular vesicles obtained from parasites in cancer treatment and to load active substances onto the exosomes by using the drug loading capacity of the exosomes and thus to carry the specific drug directly to the target cancer cells without causing any side effects on healthy cells and thereby to increase the bioavailability of the drug to achieve the desired effect in the tumor-specific target area. Within the scope of the present invention, in particular, the infantile leishmania parasite is used as a source of extracellular vesicles.

Description

Technical Field

[0001] This invention relates to the use of parasites and extracellular vesicles obtained from parasites for cancer treatment purposes. Background Technology

[0002] Cancer is one of the most dangerous and common diseases today. It is estimated that there were 14.1 million new cancer cases worldwide in 2012 [1]. Despite the many different chemical and biological agents that have been tried in cancer treatment, the treatments developed are inadequate due to their low efficacy and high side effects. For example, chemotherapy is the most commonly used treatment for many cancers, such as breast cancer, but it causes serious damage to many healthy tissues in the body, such as bone marrow, hair and digestive system [2]. The inadequacy of treatments has forced scientists to try new approaches in the field of cancer. In these trials, exogenous agents are expected to yield effective results.

[0003] As used within the scope of this application, the term "hypertrophic disease" refers to a malignant tumor or a physiological condition characterized by uncontrolled cell growth, such as cancer. In this context, the terms "hypertrophic disease" and "cancer" are used interchangeably. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma sarcoma, and leukemia.

[0004] • The cancer referred to in this article refers to a type of cancer composed of epithelial cells.

[0005] • The lymphomas used in this article describe a type of cancer that develops from lymphocytes.

[0006] • The term "blastoma" as used in this article describes a type of cancer that develops from progenitor cells (also known as embryonic cells).

[0007] • The sarcoma used in this article describes a type of cancer that arises from transformed cells derived from mesenchyme.

[0008] • The leukemia used in this article describes a type of cancer that originates in the bone marrow and results in a large number of abnormal white blood cells.

[0009] More specific examples of cancer types include breast cancer, prostate cancer, colorectal cancer, skin cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, gastrointestinal cancer, pancreatic cancer, glioblastoma, vulvar cancer, cervical cancer, endometrial cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, bladder cancer, kidney cancer, salivary gland cancer, thyroid cancer, and various head and neck cancers.

[0010] Surgery and / or chemotherapy are frequently used to treat cancer. While surgery can produce complete and effective results in some cases, chemotherapy is often administered after surgery to prevent the presence of cancer cells that may survive for a long time and lead to cancer formation. In most cases, chemotherapy causes numerous side effects by destroying both healthy and cancerous cells. Cells with high proliferation rates in the body are most affected by chemotherapy; these include hair cells, blood cells produced in the bone marrow, and cells of the digestive system.

[0011] The following are some of the side effects that are frequently observed after chemotherapy:

[0012] • Fatigue: While this is primarily caused by anemia resulting from the fact that blood cells are affected, the cause may also be psychological.

[0013] • Nausea and vomiting: While this may be due to drug sensitivity, it can also have psychological causes.

[0014] Hair loss: Hair loss, especially due to its rapid growth and adverse effects of chemotherapy, is one of the most important causes of depression in patients.

[0015] • Decreased blood cell count: Bone marrow affected by chemotherapy results in a significant reduction in blood cells. Due to this reduction, insufficient oxygen is delivered to tissues, leading to numerous adverse effects such as a weakened immune system and difficulty in blood clotting.

[0016] • Oral ulcers: Chemotherapy drugs can sometimes cause inflammatory sores in the mouth. During treatment, patients should avoid extremely hot or cold drinks and pay special attention to their oral hygiene.

[0017] • Diarrhea or constipation: Diarrhea or constipation can be observed due to the cellular response of the digestive system to different chemotherapeutic agents. This condition (whose effects can be primarily alleviated through diet) can, in some cases, lead to severe diarrhea requiring intravenous fluid administration.

[0018] • Skin and nail changes: Chemotherapy drugs have side effects such as darkening of the skin, peeling, redness, or dryness. Brittle nails or darkening of the nails may also be observed. Special attention should be paid to peeling skin, as it can cause open wounds in immunocompromised patients.

[0019] • Sleep problems: Although they are usually caused by psychological reasons, the body cannot rest, especially during chemotherapy treatment. This not only reduces the effectiveness of chemotherapy, but also further disrupts the patient's mental health.

[0020] To increase the success rate of treatment, chemotherapy needs to be combined with surgery and other methods, and the richness and unpredictability of its side effects vary from patient to patient, which has led scientists to seek new treatment methods.

[0021] Nauts et al. (1953) had already demonstrated that microorganisms could induce antitumor responses. They worked on the idea of ​​using weakened or genetically modified non-pathogenic organisms as antitumor agents [3]. They discovered Toxoplasma gondii ( Toxoplasma gondii ) and Acanthamoeba karyotes ( Acanthamoeba castellanii They exhibited antitumor activity. In light of these developments, a new therapeutic field using microorganisms emerged in oncology, and research and development have been conducted under the name of "biological therapy," a term first used in 1931.

[0022] Baird et al. (2013) treated B16F10 mouse melanoma with attenuated Toxoplasma gondii parasite and demonstrated that a strong CD8+ T cell-induced antitumor response occurred in mice given Toxoplasma gondii [9].

[0023] Bose et al. (2016) have concluded in their study that heat-degraded Leishmania donovani ( Leishmania donovani Apoptosis of HepG2 liver cancer cells was induced by the p53-dependent mitochondrial pathway mediated by reactive oxygen species (ROS)

[10] .

[0024] Yang et al. (2017) tested the exogenous organism isolated from mice infected with Plasmodium in a Lewis lung cancer model and observed that it inhibited tumor angiogenesis

[11] .

[0025] Exogens have already gained prominence in initial studies for their availability in targeted therapy by delivering load contents (which are specific to their source cells) to improve current treatments and reduce side effects.

[0026] Extracellular vesicles are small sacs involved in intercellular transport of substances and are separated from the cytoplasm by at least one lipid bilayer. Exosomes (a type of extracellular vesicle) are vesicles released by many organisms, such as higher eukaryotes and plants, and contain lipid bilayer membranes of varying sizes. The importance of these vesicles lies in their ability to transmit information to other cells to influence cellular function. Signaling via exosomes is achieved by means of many different classes of biomolecules, including proteins, lipids, nucleic acids, and sugars

[12] . Because exosomes carry the surface proteins of the cells that produce them, they target such cell types in which they are delivered in vivo systems. These properties make exosomes suitable for carrying nucleic acids for drug delivery, bioactive substances, and gene therapy. Another unique aspect of exosomes is their specificity to cells and the current physiological state of those cells, in which they generate the signals and loads they carry. Exosomes from different organisms, exosomes from different cell types of the same organism, and exosomes from the same cell under different conditions exhibit different properties.

[0027] Each cell produces foreign bodies for its own purposes. Cancer cells and their foreign bodies have been found to create favorable environments for themselves in the body

[13] , escape the immune system[14, 15], and use foreign bodies for metastasis

[16] . Stem cell foreign bodies

[17] and especially foreign bodies from dendritic cells, which are immune system cells[18, 19], have many activities in the body’s response to cancer. Another source of foreign bodies that produce successful results is plants. Foreign bodies obtained from lemons have been successfully used for therapeutic purposes in a model of chronic myeloid leukemia

[20] .

[0028] All eukaryotic cells produce their own foreign bodies, including eukaryotic parasites. Leishmaniasis is a general term for a group of vector-borne diseases transmitted by Leishmania protozoa (…). Leishmania protozoan Leishmaniasis is transmitted to humans through the bites of female sandflies infected with parasites. According to the World Health Organization, leishmaniasis is prevalent in more than 60 countries worldwide, particularly in Southern Europe, the Middle East, and North Africa, including Turkey and surrounding regions.

[0029] European Patent Document No. EP1687025 (an application known in the art) discloses a vaccine composition comprising interleukin-18 and a saponin adjuvant. The combination therapies disclosed within the scope of the invention have been identified as being effective for the treatment or prevention of infectious diseases, cancer, autoimmune diseases, and related conditions.

[0030] Chinese patent document CN102988417 (an application known in the art) discloses the use of worm parasite biological agents in the prevention and control of diseases. These parasite preparations affect regulatory T cell function. Diseases are treated by altering regulatory T cell activity through the administration of parasite preparations. Examples of diseases treated in this way include Th1 or Th2-related cancers. Summary of the Invention

[0031] The object of this invention is the use of parasites and extracellular vesicles obtained from parasites in the treatment of cancer. Parasitic exosomes directly induce responses in cancer cells without causing side effects on healthy cells. Furthermore, by utilizing the drug-carrying capacity of the exosomes, active substances can be loaded onto the exosomes, thereby delivering specific drugs to target cells and thus improving drug bioavailability, achieving the desired effect in tumor-specific target areas. Within the scope of this invention, in particular *Leishmania infantis* (… Leishmania infantum Parasites are used as a source of extracellular vesicles (exogenous bodies). Detailed Implementation

[0032] The accompanying drawings illustrate the use of "parasites and extracellular vesicles obtained from parasites in the treatment of cancer," developed to achieve the objectives of this invention, wherein:

[0033] Figure 1 This is a diagram illustrating the apoptosis effect of U87 cells incubated with two different doses of Leishmania infantis parasite.

[0034] Figure 2 This is a view of an optical microscope photograph showing the parasite infection in U87 cells after 8 hours of incubation with the parasite in a culture medium.

[0035] Figure 3 This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of U87 cells.

[0036] Figure 4 This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of A172 cells.

[0037] Figure 5 This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of SHSY5Y cells.

[0038] Figure 6This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of SH4 cells.

[0039] Figure 7 This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of 22RV cells.

[0040] Figure 8 This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of HDF cells.

[0041] Figure 9 This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of HaCaT cells.

[0042] Figure 10 This is a diagram illustrating the effect of eight different doses of exogenous organisms obtained from Leishmania infantis parasites administered for 24, 48, and 72 hours on the cell viability of astrocytes.

[0043] Figure 11 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from the infantile Leishmania protozoan parasite for 48 hours on Bax gene expression in SHSY5Y neuroblastoma cells.

[0044] Figure 12 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from the infantile Leishmania protozoan parasite for 48 hours on Bcl-2 gene expression in SHSY5Y neuroblastoma cells.

[0045] Figure 13 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from Leishmania infantis parasite for 48 hours on Caspase-3 gene expression in SHSY5Y neuroblastoma cells.

[0046] Figure 14 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from Leishmania infantis parasite for 48 hours on p53 gene expression in SHSY5Y neuroblastoma cells.

[0047] Figure 15 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from Leishmania infantis parasite administered 3 hours prior on p21 gene expression in SHSY5Y neuroblastoma cells.

[0048] Figure 16This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from the infantile Leishmania parasite 3 hours after administration on the expression of the p21 gene in astrocytes.

[0049] Figure 17 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from the infantile Leishmania protozoan parasite 3 hours after administration on p53 gene expression in SHSY5Y neuroblastoma cells.

[0050] Figure 18 This is a diagram illustrating the effect of a single dose administered 3 hours after the exogenous organism obtained from the infantile Leishmania parasite on the expression of the p53 gene in astrocytes.

[0051] Figure 19 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from the infantile Leishmania protozoan parasite 3 hours after administration on the expression of the Caspase-3 gene in SHSY5Y neuroblastoma cells.

[0052] Figure 20 This is a diagram illustrating the effect of a single dose of an exogenous organism obtained from the infantile Leishmania protozoan parasite 3 hours after administration on the expression of the Caspase-3 gene in astrocytes.

[0053] This invention relates to the use of parasites and extracellular vesicles derived from parasites in the treatment of vesicular diseases. The term "vesicular disease" refers to a malignant tumor or a physiological condition characterized by uncontrolled cell growth, and specifically to cancer. Within the scope of this invention, the terms "vesicular disease" and "cancer" are used interchangeably. A vesicular disease can be at least one type of cancer selected from the group consisting of: breast cancer, prostate cancer, colorectal cancer, skin cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, gastrointestinal cancer, pancreatic cancer, sarcoma, blastoma, lymphoma, glioblastoma, neuroblastoma, vulvar cancer, cervical cancer, endometrial cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, leukemia, bladder cancer, kidney cancer, salivary gland cancer, thyroid cancer, and various head and neck cancers. Within the scope of this invention, cancer treatment is performed by killing the following cells: U87 cancer cells and A172 cancer cells, which are glioblastoma cell lines; SHSY-5Y cancer cells, which are neuroblastoma cell lines; SH4 cancer cells, which are melanoma cell lines; and 22RV cancer cells, which are prostate cell lines.

[0054] This invention relates to the use of Leishmania species parasites and extracellular vesicles obtained from said parasites in the treatment of cancer, as they selectively kill cancer cells. In developing this invention, it has been observed that said parasites and extracellular vesicles obtained from said parasites are highly lethal to cancer cells but have almost no side effects on healthy cells.

[0055] Within the scope of this invention, it becomes possible to load active substances onto exogenous bodies and deliver target cell-specific drugs by utilizing the drug-carrying capacity of exogenous bodies, thereby improving drug bioavailability and achieving the desired effect in the target region.

[0056] A distinguishing feature of this invention is the use of exogenous bodies isolated from Leishmania species in the treatment of cancer. In the research mentioned under the heading "Background Art," the direct use of parasites is a serious obstacle to their use in treatment because they can cause disease. For this invention, the risk of disease that may occur with the direct use of parasites is prevented by using exogenous bodies isolated from parasites.

[0057] In this invention, various parasites, including *Leishmania infantis* and foreign bodies obtained from said parasites, are used in cancer treatment. Within the scope of this invention, the parasites used herein include the following species: *Acanthamoeba* sp. (*Acanthamoeba karyotes* sp.) Acanthamoeba castellanii ), Echinococcus spp., E. histolytica spp., Ancylostoma brazilense, A. caninum, A. ceylanicum, Uncinaria stenocephala, Angiostrongylus cantonensis, Ascaris, Giardia, Leishmania spp. (Leishmania africana) L. Arabica ), L. archibaldi , L. aristedesi Leishmania brasiliensis ( L. braziliensis ), Chagas Leishmania ( L. chagasi ), Colombian Leishmania ( L. colombiensis ), L. Deanei Leishmania donovani ( L. donovani ), L. enrietii , L. equatorensis , L. forattinii Gahamilishmania ( L. Garnhami ), Gerbil Leishmania ( L. gerbil ), Guyana Leishmania ( L. guyanensis ), L. herreri Leishmania hesperidin ( L. hertigi), Leishmania infantis ( L. infantum ), L. killicki , L. lainsoni Large Leishmania ( L. major ), Leishmania melanogaster ( L. Mexicana ), L. naiffi Panama Leishmania ( L. panamensis ), Peruvian Leishmania ( L. peruviana ), L. pifanoi , L. shawi , L. tarentolae Tropical Leishmania ( L. tropica ), Dulan Leishmania ( L. turanica ), Venezuelan Leishmania ( L. venezuelensis )), Plasmodium genus ( Plasmadium spp. (Plasmodium falciparum) P. falciparum ), Plasmodium vivax ( P. vivax ), Plasmodium ovale ( P. ovale The invention relates to the use of extracellular vesicles obtained from one or more of these species, individually or in combination, wherein combinations of these species allow exogenous bodies obtained from different parasites to interact in cancer treatment.

[0058] Within the scope of this invention, *Leishmania infantis* is selected as the parasite used to characterize extracellular vesicles obtained from said parasite. Its application in cancer treatment can be as a culture medium for the parasite.

[0059] Within the scope of this invention, parasites and extracellular vesicles obtained from parasites, said parasite antigens (heat-inactivated or the direct antigen itself), genetic material (mRNA, small RNA, mitochondrial DNA, DNA fragments), and gene-transferred and transfected parasites (plasmid transfer, lentivirus, electroporation with SV40 antigen) are used for CRISPR (a method involving nucleotide editing) cancer therapy. These are then encapsulated into nanocarrier systems for cancer therapy.

[0060] Within the scope of this invention, parasites and extracellular vesicles obtained from parasites are separated by using at least one of the following separation methods: separation with an aqueous two-phase system (ATPS), fractional centrifugation, ultracentrifugation, sucrose gradient ultracentrifugation, polymerization precipitation, ultrafiltration, separation by chromatographic methods (affinity chromatography (antibody and peptide affinity chromatography), size separation chromatography (size exclusion chromatography)), separation with microbeads, and precipitation based on ionic charge (charge-based precipitation) and salting out.

[0061] A preferred two-phase liquid separation method within the scope of this invention includes the following steps:

[0062] - Collect the culture medium of the parasites, from which extracellular vesicles will be isolated.

[0063] Centrifuge at 2,000 g to 10,000 g for 5–20 minutes to remove unwanted substances such as cell residue and parasites from the culture medium.

[0064] - After centrifugation, particles with a size of 220 nm and larger are removed by filtration.

[0065] - The vesicle-protein mixture obtained by centrifugation was transferred to a two-phase liquid system containing a PEG phase and a DEX phase for separation.

[0066] - By utilizing the chemotaxis of PEG relative to proteins and DEX relative to phospholipid membrane structures, non-vesicle proteins, cellular lipids, and other impurities are removed from vesicles.

[0067] - Obtain the isolated vesicles.

[0068] Formulations developed within the scope of this invention and containing parasites and extracellular vesicles derived from parasites comprise nanocarrier systems selected from the group consisting of: vesicles, emulsion systems, biological and chemical nanoparticles (polymerized nanoparticles, solid lipid nanoparticles), inorganic nanoparticles (metal nanoparticles), lipid vesicle systems (liposomes, lipid vesicles, and liposomes), dendritic polymers, polymer-drug conjugates, micelles, and carbon nanotubes. The formulation comprises at least one active compound and binary or ternary combinations thereof as additional active substances, said active compound being selected from the group consisting of active compounds exhibiting antiparasitic, antibacterial, antiviral, antitumor, and / or cytotoxic and / or antimetastatic activities.This formulation comprises at least one pharmaceutical agent and a binary or ternary combination thereof and / or encapsulation as an active compound exhibiting antibacterial activity, said pharmaceutical agent being selected from the group comprising: amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, gerdemycin, terbufos, rifaximin, cephalosporin, ertapenem, doripenem, imipenem, meropenem, cephadroxyle, cefazolin, cefotaxime, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, ceftoranone, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefbutidine, cefbufen, cefazolin, cefepime, cefurox ... Archimonium, Travancin, Dabafancin, Otavacin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Dierythromycin, Erythromycin, Roxithromycin, Acetylosin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Pocizolide, Redazolamide, Terizolamide, Amoxicillin, Ampicillin, Aloxicillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Meropenem, Methicillin, Nafcillin, Oxyxacillin, Penicillin G, Penicillin V, Piperacillin, Temoxicillin, Ticarcillin, Clavulanate, Sulbactam, Triazobactam, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemimifloxacin, Levofloxacin, Lomifloxacin, Moxifloxacin, Nalidixic Acid (acid), norfloxacin, ofloxacin, trovafloxacin, gapfloxacin, sparfloxacin, timafloxacin, sulfamidosulfonamide, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfonamide, sulfasalazine, sulfisoxazole, trimethoprim, demethylchlorocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, isoniazid, pyrazinamide, rifampin, rifabutin, rifapentine, streptomycin, arsenamin, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, acanthromycin, quinupristin, dalfopristin, thiamphenicol, tegafur, tinidazole, trimethoprim.This formulation comprises at least one pharmaceutical agent and a binary or ternary combination thereof and / or encapsulation as an active compound exhibiting antiviral activity, said pharmaceutical agent being selected from the group consisting of: abacavir, acyclovir, adefovir, amantadine, ampranavir, ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, kombivir, dolutegravir, darunavir, delavudine, doxorinosine, doxorubicin, eduksudine, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoliver, famciclovir, fomivirex, furazonavir, phosphonic acid, phosphonet, ganciclovir, ibasitabin, imunovir, idoxuridine, imiquimod, indinavir, inosine, type I interferon, type II interferon Interferon type III, interferon, lamivudine, lopinavir, lopinavir, maravirok, morpholine guanidine, meticaxane, nelfinavir, nevirapine, nitrozolomide, novir, oseltamivir, pegylated interferon alpha-2a, pencilovir, pelamivir, plekonaril, podofilotoxin, protease inhibitors, nucleoside analogs, ralgetavir, ribavirin, amantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, telapirvir, tenofovir, telanavir, trifluridine, trisivir, triamantadine, trovada, valacyclovir, valacyclovir, vidarabine, tarivirine, zalcitabine, zanamivir, zidovudine. This formulation comprises at least one agent and a binary or ternary combination thereof and / or encapsulation as an active compound exhibiting antiparasitic activity, said agent being selected from the group consisting of: nitrozole, melarisinol, efornithine, metronidazole, tinidazole, mitefonine, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbazine, ivermectin, niclosamide, praziquantel, albendazole, rifampin, amphotericin B, fumarazol, furazolidone, nifurozone, nitrozole, ornidazole. Zolpidem, Paromomycin Sulfate, Pentamidine, Pirimethamine, Tinidazole, Albendazole, Mebendazole, Thiabendazole, Fenbendazole, Trichlorobendazole, Flubendazole, Abacrotin, Ethiazide, Ivermectin, Sulamine, Pyrantel Pyrantel, Levamisole, Nitrosalinomycin, Nitrozole, Hydroxychlorozadamine, Monetatel, Dequiteril, Amphotericin B, Urea Antimonyamine, Sodium Antimony Gluconate, Meglumine Antimony, Paromomycin, Mitefocin, Fluconazole, Pentamidine.This formulation comprises at least one agent, or a binary or ternary combination thereof and / or encapsulation thereof, in combination with an extracellular vesicle and / or nanocarrier system, as an active compound exhibiting antitumor activity. The agent is selected from the group consisting of: cyclophosphamide, ifosfamide, temozolomide, capecitabine, 5-fluorouracil, methotrexate, gemcitabine, pemetrexed, mitomycin, bleomycin, epirubicin, doxorubicin, etoposide, paclitaxel, irinotecan, docetaxel, vincristine, carboplatin, cisplatin, oxaliplatin, bevacizumab, cetuximab, gefitinib, imatinib, trastuzumab, denosumab, rituximab, sunitinib, zoledronic acid, abiraterone, anastrozole, bicalutamide, exemestane, goserelin, medroxyprogesterone acetate, octreotide, tamoxifen. Xifen, Bendamustine, Carmustine, Chlorobutazone, Lomustine, Melphalan, Procarbazine, Strepzocin, Fludarabine, Raltitrexed, Actinomycin D, Dermatomycin, Doxorubicin, Mitoxantrone, Eribulin, Topotecan, Vincristine, Vinorelbine, Afatinib, Aflibercept, Crizotinib, Dabrafenib, Interferon, Ipilimumab, Lapatinib, Nivolumab, Perimumab, Pembrolizumab, Pertozhuumab, Sorafenib, Trastuzumab (emtansine), Temsorilimus, Vemurafenib, Ibandronic Acid, Pamidronate, Besarotin, Busereline, Ciprofloxacin, Degarelix, Leucovorin, Fluvestrantraniliprole, Lanreitide, Lenalidomide, Letrozole, Leuproreline, Medroxyprogesterone acetate, Mesna, Thalidomide, Vincristine. In addition, the formulation contains at least one active substance. The active substance includes the substances defined and listed above.

[0069] The administration method of the above-described pharmaceutical composition for treatment includes at least one method selected from the group consisting of: parenteral, intravenous, intradermal, subcutaneous, intraperitoneal, local, intrathecal, intranasal, intravenous, ocular, vaginal, urethral, ​​transdermal, sublingual, subarachnoid, rectal, periodontal, perineal, epidural, perarticular, oral, intratympanic, intratumoral, intrapulmonary, intrasynovial, intramuscular, intraovarian, intradural, intracavernosal, intracoronary, intracerebral, epidural, skin, buccal, and dental.

[0070] Within the scope of this invention, extracellular vesicles are used as adjuvants in cancer treatment. Pharmaceutical compositions disclosed within the scope of this invention can be formed by incorporating extracellular vesicles obtained from parasites into at least one or a combination of two or more of aluminum hydroxide, aluminum phosphate, tocopherol, an emulsion system containing 3D-MPL, cholesterol, and CG oligonucleotides.

[0071] For the purposes of this invention, the generation of parasites and parasitic extracellular vesicles that can be used for cancer treatment includes the following steps.

[0072] 1. Parasite culture

[0073] The proflagellates of Leishmania infantis (MHOM / MA / 67 / ITMA-P263) were incubated in RPMI medium (containing heat-inactivated 10% fetal bovine serum, 2 mM L-glutamine, 20 mM HEPES, 100 U / ml penicillin, and 100 µg / ml streptomycin) at 27°C. They reached the logarithmic developmental stage (10^6). 6 Parasites present in doses of 1 ml or less are infectious.

[0074] 2. Cultured parasites were treated with glioblastoma carcinoma cells, and the amount of apoptosis was measured using annexin V assay.

[0075] The prepared parasites were added to glioblastoma U87 cells in culture medium. The parasites, applied at two different doses (50 and 100 times the amount applied to U87 cells), were incubated for 8 hours, after which the parasites were removed from the U87 cells. The next day, mortality was measured by flow cytometry after the addition of annexin V solution.

[0076] 3. Collecting parasite culture medium

[0077] Within the scope of this invention, parasitic extracellular vesicles are separated by a selection of methods including separation via a two-phase liquid system, fractionation centrifugation, ultrafiltration, chromatography, polymer-based separation, and microbead separation. Among these, separation via a two-phase liquid system achieves the purest separation of extracellular vesicles, and therefore this separation method is preferred within the scope of this application.

[0078] The separation method using a two-phase liquid system for separating extracellular vesicles of parasites, as used within the scope of this invention, includes the following steps:

[0079] • Collect the culture medium from the parasites, and then isolate the extracellular vesicles from it.

[0080] • Centrifuge at 2,000 g to 10,000 g for 5–20 minutes to remove unwanted substances such as cell residue and parasites from the culture medium.

[0081] • After centrifugation, particles with a size of 220 nm and larger are removed by filtration.

[0082] • The vesicle-protein mixture obtained by centrifugation is transferred to a two-phase liquid system containing a PEG phase and a DEX phase for separation.

[0083] • By utilizing the chemotaxis of PEG relative to proteins and DEX relative to phospholipid membrane structures, non-vesicle proteins, cellular lipids, and other impurities are removed from vesicles.

[0084] • Obtain isolated vesicles.

[0085] 4. MTS

[0086] Cell viability was measured on days 1, 2, and 3 after seeding cells at 5000 cells / well in Dulbecco modified Eagle's medium (DMEM) (containing 10% fetal bovine serum (Invitrogen) and 1% PSA (Biological Industries, Beit Haemek, Israel)) in 96-well plates (Corning Glasswork, Corning, NY). Cell viability was measured using the 3-(4,5-dimethylthiazolyl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS) method (CellTiter96 Aqueous One Solution; Promega, Southampton, UK). 10 μl of MTS solution was added to cells in 100 μl of growth medium, and they were incubated in the dark for 2 hours. After the incubation process, cell viability was observed by measuring absorbance at a wavelength of 490 nm using an ELISA plate reader (Biotek, Winooski, VT).

[0087] 5. Quantitative real-time polymerase chain reaction (RT-PCR)

[0088] Cells were seeded at 150,000 cells / well in Dulbecco modified Eagle's medium (DMEM) (containing 10% fetal bovine serum (Invitrogen) and 1% PSA (Biological Industries, Beit Haemek, Israel)) into 96-well plates (Corning Glasswork, Corning, NY). The next day, the cells were incubated with different doses (10–40 μg / ml) of parasitic exogenous organisms. At 3 and 48 hours post-incubation, the expression of p21, p53, caspase 3, Bcl-2, and Bax genes was observed in the cells. In summary, RNA was isolated from the cells using Trizol®, total RNA concentration was measured by NanoDrop spectrophotometry, and cDNA analysis was then performed using a cDNA kit (Roche). RT-PCR was performed as follows: 39 cycles were performed using the SYBR Green Kit (Thermofisher Science) with incubation at 95°C for 15 s, at 58°C for 1 min, and at 72°C for 30 s. GAPDH was used as the housekeeping gene.

[0089] One of the differences between this invention and the prior art is the application of parasites and exogenous bodies isolated from parasites, and a significant difference in the source species from which they are isolated and the application of exogenous bodies from parasites. These exogenous bodies are merely a portion of the chemicals released extracellularly by cells. Within the scope of this invention, the activity of parasites and their exogenous bodies against cancer cell lines and healthy cell lines has been demonstrated. Parasites were applied at 50-fold and 100-fold increases to glioblastoma U-87 cells, and the apoptosis rate, i.e., mortality rate, of the cancer cells was evaluated. At the end of 8 hours of incubation, a mortality rate of up to 30% was observed at both doses. Figure 1 Meanwhile, approximately 68% of cells that underwent apoptosis (i.e., death) were infected with the infantile Leishmania protozoan parasite. Figure 2 After applying not only parasites but also exogenous bodies secreted by these parasites and isolated from the culture medium at specific concentrations (0.78-50 µg / ml) to certain cancer cells and healthy cells, cell viability was examined for 3 days. Decreased cell viability and apoptosis were observed in U87 glioblastoma cells (over 40%), SHSY5Y neuroblastoma cells (over 90%), A172 glioblastoma cells (over 45%), SH4 melanoma cells (over 90%), and 22RV prostate cells (over 90%), while no effect was observed on the viability of healthy cell lines HaCaT, HDF, and astrocytes. Figure 3-10 ).

[0090] SHSY-5Y neuroblastoma cells were treated with a 10 µg / ml dose of exogenous material, and after incubation for 48 hours, the expression of p53, caspase 3, Bcl-2, and Bax genes was measured by quantitative real-time polymerase chain reaction (RT-PCR). The results showed a significant increase in the expression of p53, caspase 3, and Bax genes, and a decrease in the expression of Bcl-2 gene in SHSY-5Y neuroblastoma cells. Figure 11-14 A 40 µg / ml dose of exogenous material was applied to SHSY-5Y neuroblastoma and astrocyte (NHA) cells, and changes in p21, p53, and caspase 3 gene expression were evaluated after 3 hours of incubation. A significant increase in p21 and caspase 3 gene expression was observed in SHSY-5Y neuroblastoma cells, while concluding that p21, p53, and caspase 3 gene expression was decreased in astrocyte (NHA) cells. Figure 15-20 Based on the experimental results, this invention is applicable to the development of formulations that have no cytotoxic effect on healthy cells while causing the death (apoptosis) of a large number of cancer cells.

[0091] The advantages of this invention can be described as follows:

[0092] • This invention exhibits almost complete activity against different types of cancer within 24 hours and has no side effects on healthy cells.

[0093] • Because parasites, especially Leishmania species, are extremely open to genetic modification, their extracellular vesicles can be modified through gene intervention as needed to improve their activity against cancer and the yield obtained from parasites.

[0094] • By utilizing the drug-carrying capacity of exogenous bodies, active substances can be loaded onto the exogenous body, thereby enhancing the bioavailability of the drug by delivering specific drugs to target cells and achieving the desired effect in tumor-specific target areas.

[0095] • The extracellular vesicles used in this invention are products that can be mass-produced at an affordable price.

[0096] References

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Claims

1. Use of extracellular vesicles obtained from a parasite in the preparation of a medicament for treating aplastic diseases, wherein the parasite is Leishmania infantis ( L. infantum The term "hyperplastic disease" refers to a malignant tumor or a physiological condition described by uncontrolled cell growth, wherein the hyperplastic disease is at least one cancer selected from the group consisting of: prostate cancer, glioblastoma, neuroblastoma, and melanoma.

2. The use according to claim 1, wherein the extracellular vesicles obtained from the parasite are separated using at least one of the following separation methods: separation using an aqueous two-phase system, fractional centrifugation, ultracentrifugation, sucrose gradient ultracentrifugation, polymerization precipitation, ultrafiltration, separation by chromatographic methods, separation by microbeads, and precipitation and salting out based on ionic charge.

3. The use according to claim 1, wherein the extracellular vesicles obtained from the parasite are separated by a separation method via a two-phase liquid system, the method comprising the following steps: - Collect the culture medium of the parasites and isolate the extracellular vesicles from it. Centrifuge at 2,000 g to 10,000 g for 5–20 minutes to remove unwanted substances from the culture medium, including cell residues and parasites. - After centrifugation, particles with a size of 220 nm and larger are removed by filtration. - The vesicle-protein mixture obtained by centrifugation was transferred to a two-phase liquid system containing a PEG phase and a DEX phase for separation. - By utilizing the chemotaxis of PEG relative to proteins and DEX relative to phospholipid membrane structures, non-vesicle proteins, cellular lipids, and other impurities are removed from vesicles. - Obtain the isolated vesicles.

4. Use of the extracellular vesicles as defined in claim 1 in the preparation of an adjuvant for cancer treatment, wherein the cancer is selected from the group consisting of: prostate cancer, glioblastoma, neuroblastoma, and melanoma.

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