WO2022019583A1 - Therapeutic agent for exosome-mediated disease - Google Patents

Abstract

Disclosed is a therapeutic agent for an exosome-mediated disease, for example a cancer such as metastatic cancer, e.g., a solid cancer.

Description

Therapeutic agents for exosome-mediated diseases

The present disclosure relates to a therapeutic agent for an exosome-mediated disease, and more particularly, to an exosome-mediated disease containing botulinum toxin as an active ingredient, for example, a therapeutic agent for cancer.

Exosomes are a type of extracellular vesicles, and have a size of approximately 100 nm in diameter. They are surrounded by a lipid bilayer and are secreted by most cells. Exosomes are generated as an intraluminal membrane vesicle (ILV) within the endocytic system and are secreted during the fusion of the cell membrane and the multivesicular body (MVB). It has been reported that exosomes contribute to maintaining tissue homeostasis by regulating cell-cell communication. Exosomes released from cancer cells are involved in cancer progression. Proliferative cancer cells (1) expand cancer tissue using angiogenesis, (2) acquire the ability to migrate and invade, and (3) acquire the ability to evade attack from immune cells, and ultimately metastatic lesion indicates the formation of Research results to date show that exosomes are involved in each of these processes. International Publication No. WO2010/056337 discloses a method for detecting and diagnosing various cancers by quantifying exosomes isolated from a biological sample such as blood from a subject.

Botulinum toxin (Botulinum neurotoxin, BoNT) is a polypeptide product of the anaerobic bacterium Clostridium botulinum, and is a toxic substance that specifically acts on nerve cells. Botulinum toxin is a toxic substance that intrinsically causes lethality, but it is associated with a number of conditions including: It is used to treat urologic disease, migraine, and the like.

International Publication No. WO2006/025976 discloses a method for treating atypical tissues such as hyperplastic tissues, cysts and neoplasms by topical administration of botulinum toxin to or near cancer. In addition, International Publication No. WO2010/062955 discloses that a therapeutically effective amount of botulinum toxin is administered in combination with an anticancer drug or anticancer therapy to a non-neoplastic area near a neoplasm, but a therapeutically effective amount of botulinum toxin is A non-penetrate method for inhibiting the growth or metastasis of a neoplasm is disclosed.

It was not known about the suppression of exosome secretion of botulinum toxin or the therapeutic effect of exosome-mediated diseases.

A first object of the present disclosure is to provide a composition for treating an exosome-mediated disease, comprising a botulinum toxin as an active ingredient.

A second object of the present disclosure is to provide a method for treating an exosome-mediated disease by administering a therapeutically effective amount of a botulinum toxin to a subject in need thereof.

A third object of the present disclosure is to provide a botulinum toxin for use as a therapeutic agent for exosome-mediated diseases.

A fourth object of the present disclosure relates to the use of botulinum toxin as a therapeutic agent for exosome-mediated diseases.

A first aspect of the present disclosure relates to a composition for treating an exosome-mediated disease, comprising a botulinum toxin as an active ingredient.

A second aspect of the present disclosure relates to a method of treating an exosome-mediated disease by administering to a subject in need thereof a therapeutically effective amount of a botulinum toxin.

A third aspect of the present disclosure relates to a botulinum toxin for use as a therapeutic agent for exosome-mediated diseases.

A fourth aspect of the present disclosure relates to the use of botulinum toxin as a therapeutic agent for exosome-mediated diseases.

As used herein, the term 'botulinum toxin' refers to a botulinum toxin protein molecule produced by bacteria or recombinantly, including all serotypes and variants or fusion proteins thereof.

The Clostridium botulinum strain produces immunologically distinct neurotoxins (types A-G). The molecular masses of type A-G neurotoxin (NTX) are about 150 kDa (7S). In culture media and foods with acidic conditions, NTX combines with non-toxic components to form large complexes called progenitor toxins (PTX). PTX with molecular weights of 900 kDa (19S), 500 kDa (16S) and 300 kDa (12S) are found. 12S toxin consists of NTX and a nontoxic nonhemagglutinin (named NTNH) having no hemagglutinin activity, and 19S and 16S toxin consists of NTX, NTNH and hemagglutinin. Type A strains produce three types of toxins (19S, 16S and 12S). Type B, C and D strains produce 16S and 12S toxins. Under alkaline conditions, PTX dissociates into NTX and non-toxic components, 19S and 16S toxins dissociate into NTX and NTNH and non-toxic components (complex) of hemagglutinin, and 12S toxins dissociate into NTX and NTNH. In the present specification, the botulinum toxin may be of any of the above-described forms, for example, may have a molecular weight of 19S, 16S, 12S, or 7S.

Botulinum toxin includes botulinum toxin derivatives. The botulinum toxin derivative may be a natural botulinum toxin having activity or a recombinant prototype botulinum toxin, or a derivative comprising one or more chemical or functional modifications on a partial chain. For example, the botulinum toxin derivative may be a modified toxin having one or more amino acids deleted, modified or substituted. The botulinum toxin may be a recombinant peptide, a fusion protein, or a hybrid neurotoxin prepared, for example, from subunits or domains of different toxin serotypes. The botulinum toxin may also be part of a whole molecule having toxin activity, and may be used in combination or as part of a conjugated molecule, eg, a fusion protein.

In one aspect, the botulinum toxin is a botulinum toxin that does not comprise a chemical or functional modification, e.g., a natural botulinum toxin or a recombinant proto-botulinum toxin or a portion thereof, e.g., a complexed botulinum toxin (e.g., 19S toxin) or a non- complexed botulinum toxin (eg, 7S toxin).

'Administering' or 'administering' refers to a step of providing a pharmaceutical composition or an active ingredient to a subject. The pharmaceutical composition may be administered via a variety of suitable routes.

“Pharmaceutical composition” means a formulation in which the active ingredient may be botulinum toxin.

The term 'formulation' means that in addition to the active ingredient, at least one additional ingredient is present in the pharmaceutical composition, such as albumin (human serum albumin or recombinant human albumin) and/or sodium chloride. A pharmaceutical composition is a formulation suitable for administration to a subject, such as a human patient. The pharmaceutical composition may be in lyophilized form, for example a solution formed after reconstitution of the lyophilized pharmaceutical composition with saline or water, or in the form of a solution that does not require reconstitution. Pharmaceutical compositions may be liquid or solid. The pharmaceutical composition may be free of animal-derived proteins and/or albumin.

'therapeutically effective amount' means the level, amount or concentration of a pharmaceutical composition comprising an agent, e.g., botulinum toxin or botulinum toxin, required to treat a disease, disorder or condition without causing significant adverse or adverse side effects do.

'Treat', 'treating' or 'treatment' refers to, for example, healing of wounded or damaged tissue, or altering, altering, enhancing, ameliorating, ameliorating and/or modifying an existing or recognized disease, disorder or condition. Means alleviation or reduction (including partial reduction, significant reduction, near complete reduction and complete reduction), resolution or prevention (temporary or permanent) of a disease, disorder or condition, by beautification, so as to achieve a desired therapeutic outcome do. As used herein, 'treatment' is a concept including prevention. By 'prevention' is meant delaying the onset of a disease, disorder or condition. Prevention may be considered complete if the onset of the disease, disorder or condition is delayed for a predetermined period.

In one aspect 'treatment' refers to the treatment of a disease, disorder, or medical condition in a patient, such as a mammal (especially a human), comprising one or more of the following:

(a) preventing the occurrence of said disease, disorder, or medical condition, i.e., preventing recurrence of said disease or medical condition, or prophylactic treatment of a patient pre-disposed to said disease or medical condition;

(b) ameliorating said disease, disorder, or medical condition, i.e., eliminating or regressing said disease, disorder, or medical condition in a patient, including antagonizing the effect of another therapeutic agent;

(c) suppressing the disease, disorder, or medical condition, ie, slowing or arresting the development of the disease, disorder, or medical condition in a patient; or

(d) alleviating the symptoms of said disease, disorder, or medical condition in the patient;

The present disclosure responds to the discovery that botulinum toxin or a composition comprising the same may be useful as a therapeutic agent for exosome-mediated diseases, such as cancer, for example, metastatic cancer, for example, solid cancer by inhibiting exosome secretion. it is based

In one embodiment, the botulinum toxin can be used without limitation of serotype, type A (BoNT/A), B (BoNT/B), C (BoNT/C), D (BoNT/D), E (BoNT/E) ), F(BoNT/F), G(BoNT/G), H(BoNT/H), X(BoNT/X), Enterococcus sp. botulinum toxin J (eBoNT/J) and mosaic botulinum toxin and/or variants thereof It may be selected from the group consisting of. Examples of mosaictoxins include BoNT/DC, BoNT/CD and BoNT/FA. For example, type A can be used.

In one aspect, the botulinum toxin is selected from various BoNT/A subtypes, eg, A1, A2, A3, A4, A5, A6, A7, A9, A10; BoNT/B subtypes such as B1, B2, B3, B4, B5, B6, B7, B8, Bnp and Bbv; BoNT/C subtypes such as C and CD; BoNT/D subtypes such as D and DC; BoNT/E subtypes such as E1, E2, E3, E4, E5, E6, E7, E8, E9; BoNT/F subtypes such as F1, F2, F3, F4, F5, F6, F7; and from the BoNT/G subtype, eg, subtype G. BoNT subtypes include chimeric BoNTs, such as BoNT/DC, BoNT/CD, BoNT/FA, and the like.

According to one aspect of the present disclosure, a botulinum toxin or a composition comprising the same may be for treating an exosome-mediated disease.

In an embodiment, the exosome-mediated disease may be cancer, a viral infection, a neurological disease or a rheumatic disease, and in particular the exosome-mediated disease may be cancer.

In an embodiment, the cancer may be metastatic cancer.

In an embodiment, the cancer may be a solid tumor. Non-limiting examples of solid cancer include breast cancer, lung cancer, head or neck cancer, colorectal cancer, esophageal cancer, laryngeal cancer, stomach cancer, liver cancer, pancreatic cancer, bone cancer, skin cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, perianal cancer, Colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer , chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain tumor, such as glioma (eg, astrocytoma) , glioblastoma, oligodendroglioma, ependymoma, germ cell tumor, meningioma, brainstem glioma, pituitary adenoma, schwannoma, congenital tumor, craniopharyngoma, metastatic brain tumor. For example, solid cancer may be one or more selected from the group consisting of melanoma, breast cancer, lung cancer, pancreatic cancer, brain tumor and prostate cancer, but is not limited thereto.

In one embodiment, the botulinum toxin can be used without limitation of serotype, and type A, B, C, D, E, F, G, H, or a combination thereof, for example type A, can be used.

In one aspect, the composition comprising botulinum toxin may further comprise one or more pharmaceutically acceptable excipients or additives.

Pharmaceutically acceptable excipients or additives include stabilizers, ionic compounds, surfactants, buffers, lyophilization protectants, or combinations thereof, such as amino acids (eg methionine), salts (eg NaCl). ), buffers, nonionic surfactants (e.g. polysorbate, e.g. polysorbate 20), sugars (e.g. disaccharides such as sucrose), sugar alcohols (e.g. sorbitol), or their It can be a combination.

The composition may not contain albumin or animal-derived components or polysaccharides.

Said compositions include, for example, those disclosed in WO2009-008595 A1 or WO2012-134240 A2, the contents of which are incorporated herein by reference in their entirety.

The compositions include, without limitation, commercially available pharmaceutical compositions containing botulinum toxin, such as, for example, BOTOX® (botulinum toxin type A neurotoxin complex with human serum albumin and sodium chloride) (Allergan, Inc.) (Allergan, Inc: Irvine, CA, USA), DYSPORT® (a powder reconstituted with 0.9% sodium chloride prior to use, Clostridium botulinum type A toxin hemagglutinin with human serum albumin and lactose in the formulation complex) (Ipsen Limited (Berkshire, UK)), MYOBLOC™ (injection solution containing botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride (approximately pH 56)) (Solstice Neurosciences) , Inc. (Solstice Neurosciences, Inc: South San Francisco, CA, USA)), Medytoxin® (a lyophilized powder formulation containing Clostridium botulinum toxin type A, human serum albumin and sodium chloride) (Medytox, Korea), Inno Tox® (Liquid formulation containing Clostridium botulinum toxin type A, L-methionine, polysorbate 20, sodium chloride and water for injection) (Medytox, Korea), Coretox® (Clostridium botulinum toxin type A (150kD) ), polysorbate 20, lyophilized powder formulation containing sucrose and sodium chloride) (Medytox, Korea). In an embodiment, the composition may be Medytoxin®, Innotox®, Coretox®, or one or more combinations thereof, but is not limited thereto.

In one embodiment, the composition may be formulated in any form, such as a solid or liquid formulation, for example, a lyophilized powder, a liquid, or a pre-filled syringe formulation.

In one embodiment, the composition may be a formulation for topical administration. For example, topical administration may be intratumoral administration or peritumoral administration, and typically may be accomplished by intratumoral injection or peritumoral injection of an aqueous botulinum toxin solution. For example, the formulation can be administered subcutaneously or into a soft tissue, eg, intramuscularly. The composition can inhibit tumor growth or inhibit tumor development at a location away from the location by intratumoral or peripheral administration at a specific location, and the composition can exhibit systemic effects when administered locally.

In one aspect, a therapeutically effective amount of botulinum toxin is about 0.01 U/kg to about 100 U/kg, about 0.1 U/kg to about 100 U/kg, about 0.2 U/kg to about 100 U/kg, about 0.2 U/kg kg to about 50 U/kg, from about 0.2 U/kg to about 30 U/kg, from about 0.2 U/kg to about 10 U/kg, from about 0.2 U/kg to about 1 U/kg. In another aspect, based on an adult weighing 60 kg, from about 1 U to about 10,000 U, from about 1 U to about 5,000 U, from about 1 U to about 2,500 U, from about 1 U to about 1,000 U, from about 1 U to about 500 U, about 1 U to about 300 U, about 1 U to about 200 U, about 10 U to about 200 U, about 10 U to about 100 U, about 10 U to about 50 U.

As used herein, the term "unit", "unit(s)", or "U" refers to the LD50 dose, defined as the amount of botulinum toxin that killed 50% of mice receiving a botulinum toxin injection. , are used interchangeably within a product.

In one aspect, the botulinum toxin may be administered in single or multiple treatment sessions. Dosages may be administered as single or divided injections at the site of injection. In multiple treatment sessions, botulinum toxin may be administered at intervals of up to 6 months. In the multiple treatment session, the administration interval of the botulinum toxin includes the first treatment and the second treatment, and the dose of the second treatment may be less than, more than, or the same as the dose of the first treatment.

The dose, administration time, administration method, administration period or interval, etc. of the botulinum toxin may be appropriately selected and used by a person skilled in the art according to the patient's weight, age, sex, health condition, severity of disease, and the like.

In one embodiment, botulinum toxin or a composition comprising the same can be used in subjects with increased exosome secretion.

In one aspect, a method of treating an exosome-mediated disease may comprise the steps of:

1) quantifying the exosomes secreted from the biological sample obtained from the subject; and,

2) administering a therapeutically effective amount of a botulinum toxin or a composition comprising the same to a subject with increased exosome secretion.

The secreted exosomes can be separated and quantified from a biological sample of a subject, for example, blood according to a conventional method known in the art, and there is no particular limitation on the type of sample, separation and quantification method.

The subject to which the botulinum toxin or a composition comprising the same is administered may include, without limitation, a human or an animal, for example, a human, a pig, a dog, a cat, a cow, a horse, a rat, and the like.

In one aspect, botulinum toxin or a composition comprising the same may be administered in combination with one or more other drugs or therapies. As used herein, "combination administration" refers to any form of two or more different therapeutic agents that allow a second therapeutic agent to be administered while the previously administered therapeutic agent is still effective in the body. For example, two therapeutic agents are simultaneously effective in a subject, which may include a synergistic effect of the two therapeutic agents. The different therapeutic agents may be administered simultaneously or sequentially in the same formulation or in separate formulations.

In an embodiment, botulinum toxin or a composition comprising the same may be administered in combination with other therapeutic agents, for example, anticancer agents, antiviral agents, cytokines, or immune agents.

The botulinum toxin or a composition comprising the same may be administered in combination with a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosourases, antimetabolites, anticancer drugs, plant-derived alkaloids, topoisomerase inhibitors, hormonal drugs, hormone antagonists, leukopenia (neutropenia) therapeutic drugs, thrombocytopenia. therapeutic drugs, antiemetics, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, other immunotherapeutic drugs, and other anticancer drugs. Exemplary cytotoxic agents that may be administered in combination include anti-microtubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, and may interfere with signal transduction pathways. agents that promote apoptosis, proteosome inhibitors, and radiation (local or systemic irradiation). Non-limiting examples of additional therapeutic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.

The botulinum toxin or a composition comprising the same may be administered in combination with an immunotherapeutic agent. In one embodiment, the immunotherapeutic agent may use any substance or therapy known in the art, and may include cytokines, cancer vaccines, oncolytic viruses, monoclonal antibodies, non-cytokine adjuvants, immune cells (T cells, NK cells, dendritic cells, B cells, etc.), and immune checkpoint inhibitors. In an embodiment, the immunotherapeutic agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. For example, an immune checkpoint inhibitor may be administered to enhance the proliferative, migratory, persistence and/or cytotoxic activity of CD8+ T cells in a subject, and particularly tumor invasiveness of CD8+ T cells in a subject. Typically, checkpoint inhibitors are activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PD-1), or the killer cell immunoglobulin-like receptor (KIR) family. antagonists that block immunosuppressive receptors expressed by NK cells, such as various members of It is a blocking antagonist. For example, an immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof. Specifically, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, an anti-IDO1 antibody. antibody, anti-TIGIT antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-BTLA antibody, and anti-B7H6 antibody, and antigen-binding fragment thereof. More specifically, immune checkpoint inhibitors include Ipilimumab (Yervoy®, BMS/Ono), Tremelimumab (AstraZeneca), atezolizumab (Tecentric®, Roche), and nivolumab (nivolumab) (Opdivo®, BMS/Ono), Pembrolizumab (Keytruda®, MSD), Avelumab (Bavencio®, Pfizer/Merck Germany), Durvalumab ( Imfinzi®, AstraZeneca/MedImmune), and antigen-binding fragments thereof may be at least one selected from, but not limited to.

The other therapeutic agent may include a CTLA-4 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, or an EGFR antagonist.

According to the present disclosure, a botulinum toxin or a composition comprising the same may be useful as a therapeutic agent for an exosome-mediated disease, for example, a cancer, for example, a metastatic cancer, for example, a solid cancer by inhibiting exosome secretion.

1 is a graph showing tumor growth over time after administration of botulinum toxin in a mouse malignant melanoma transplantation model.

2 is a graph showing the number of lung metastatic lesions by dose after administration of botulinum toxin in a mouse malignant melanoma transplant model.

3A is a graph showing CCL2 levels in blood for each dose administered after administration of botulinum toxin in a mouse malignant melanoma transplantation model.

Figure 3b is a graph showing the level of IL-6 in the blood for each dose administered after botulinum toxin administration in a mouse malignant melanoma transplantation model.

FIG. 3c is a graph showing TNF levels in blood for each dose administered after administration of botulinum toxin in a mouse malignant melanoma transplantation model.

4 is a graph showing tumor growth over time after administration of botulinum toxin in a mouse mammary gland tumor transplantation model.

5 is a graph showing the number of exosomes in blood for each dose administered after botulinum toxin administration in a mouse mammary gland tumor transplantation model.

FIG. 6A is a graph showing the growth inhibitory effect of each concentration of botulinum toxin in a mouse malignant melanoma cell line (B16-F10).

6B is a graph showing the growth inhibitory effect of each concentration of botulinum toxin in a human lung cancer cell line (A549).

FIG. 6c is a graph showing the growth inhibitory effect of each concentration of botulinum toxin in a human pancreatic cancer cell line (SNU324).

6D is a graph showing the growth inhibitory effect of each concentration of botulinum toxin in a human pancreatic cancer cell line (Capan-2).

7 is a graph showing the effect of reducing exosomes by treatment with botulinum toxin in mouse melanoma.

8 is a graph showing the effect of reducing exosomes by treatment with botulinum toxin in a human pancreatic cancer cell line.

9A is a graph showing the growth inhibitory effect of each concentration of botulinum toxin in a human brain cancer cell line (U87MG).

9B is a graph showing the growth inhibitory effect of each concentration of botulinum toxin in a human prostate cancer cell line (PC3).

Hereinafter, the present disclosure will be described in more detail with reference to examples, but this is only for explaining the present disclosure and is not intended to limit the scope in any way.

Example 1: Anticancer efficacy test of botulinum toxin in mouse malignant melanoma transplantation model

Male 6-week-old C57BL/6 (place of purchase: Orient Bio) mice were purchased, acclimatized and quarantined for 1 week, and 7-week-old mice were used for the experiment. For feed, sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and negative water was fed freely by autoclaving tap water at high temperature. During the acclimatization, quarantine, and experiment period, the mouse was set at a temperature of 23±3℃, relative humidity of 55±15%, lighting time of 12 hours (8am to 8pm), ventilation frequency of 15 times/hour, and illumination intensity of 150~300 Lux. Raised under specific-pathogen-free conditions. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

A B16-F10 mouse malignant melanoma cell line (KCLB No: 80008) was obtained from the Korean Cell Line Bank (KCLB). B16-F10 cells were cultured in DMEM medium (CAT No: 11965-092, Gibco) containing 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco). ℃, 5% CO ₂ Conditions were cultured in an incubator. ^{0.1 ml of 5×10 5} cells were transplanted into the right flank of anesthetized C57BL/6 mice using a syringe.

As a vehicle, sterile physiological saline (Lot No: M8T7AF3, Daehan Pharmaceutical Co., Ltd.) used for the preparation of botulinum toxin was used, and as a botulinum toxin, 100 units Coretox (Lot No: NSA19007A, Medytox) was used. As shown in Table 1 below, each test substance was prepared and administered to each group.

army	number of animals	substance to be administered	Volume	route of administration	Dosing frequency
One	10	vehicle	1 ml/kg	within the tumor	Once (8 days after tumor transplantation)
2	10	botulinum toxin	1.5 units/kg	within the tumor	Once (8 days after tumor transplantation)
3	10	botulinum toxin	5 units/kg	within the tumor	Once (8 days after tumor transplantation)
4	8 *	botulinum toxin	15 units/kg	within the tumor	Once (8 days after tumor transplantation)
5	10	botulinum toxin	50 units/kg	within the tumor	Once (8 days after tumor transplantation)

^* Excluding 2 mice (injuries around tumors caused by fighting between individuals)

On the 8th day after transplantation of malignant melanoma in mice, the body weight and tumor size were measured, and mice were randomly assigned to each group at an average size of about 55 mm 3 without any difference between the test groups. On the 8th day after tumor transplantation, vehicle and botulinum toxin were administered intratumorally according to the composition of the test group based on body weight. Tumor size was measured twice a week after tumor transplantation. The tumor size was measured with a vernier caliper and the tumor volume was calculated using the following equation using the measurements.

Tumor volume (mm3)=(W ² ×L)/2

W (mm) = width (short axis of tumor), L (mm) = length of tumor (long axis of tumor)

Tumor growth inhibition (TGI; tumor growth inhibition) (%) = (1-Mean tumor size _{test group} / Mean tumor size _vehicle )×100

On the 19th day after tumor transplantation, before the tumor size reached 2000 mm 3 , blood was collected from surviving individuals using an EDTA tube, and all animals were euthanized. For lung metastasis analysis, after lung tissue removal, the weight was measured and the number of lung metastatic lesions was measured. Blood inflammatory cytokines CCL2 (CC Motif Chemokine Ligand 2), IL-6 (interleukin 6), and TNF (tumor necrosis factor) were analyzed using the BD CBA mouse inflammation kit (Cat No: 552364, Lot No: 0031162, BD). measured. Graphpad Prism (version 7.05, GraphPad Software Inc, CA, USA) was used for graph presentation, and SPSS software (version 25.0, SPSS Inc., IL, USA) was used for statistical analysis. For parametric data, a two-tailed t -test was performed, and for non-parametric data, statistical analysis was performed by Mann-Whitney test at a significance level of p = 0.05.

The measurement results of the tumor growth inhibition rate are shown in Table 2 and FIG. 1 .

army	substance to be administered	Volume	Average tumor size 1) (average ㎣±SEM)	TGI (%) 1)
One	vehicle	1 ml/kg	1705±269	0
2	botulinum toxin	1.5 units/kg	1150±266	33
3	botulinum toxin	5 units/kg	1336±208	22
4	botulinum toxin	15 units/kg	1195±255	30
5	botulinum toxin	50 units/kg	682±135 **	60

¹⁾ Results on day 18 after tumor transplantation, ^** p <0.01, vs. vehicle. Two -sided t -test.

As shown in Table 2 and FIG. 1 , on the 18th day after mouse malignant melanoma transplantation, intratumoral administration of 1.5 units/kg botulinum toxin alone showed an average tumor size of 1150 mm 3 and a tumor growth inhibition rate of 33%, and 5 units In the /kg administration group, the average 1336 mm3, 22%, in the 15 units/kg administration group, the average 1195 mm3, 30%, and 50 units/kg administration group showed an average 682 mm3 and 60% tumor growth inhibition rate.

The results of measuring the number of lung metastatic lesions are shown in FIG. 2 . As shown in FIG. 2 , the number of lung metastatic lesions was significantly reduced in the 1.5, 15, and 50 units/kg administration groups compared to the vehicle administration group.

As a result of analyzing inflammatory cytokines in the blood, CCL2 was decreased in the 15 units/kg administration group compared to the vehicle administration group ( FIG. 3a ), and TNF was significantly reduced in the 5 and 50 units/kg administration group ( FIG. 3b ). It was confirmed that IL-6 in the blood was also reduced by administration of botulinum toxin ( FIG. 3C ).

Example 2: Anticancer efficacy test of botulinum toxin in mouse mammary tumor transplantation model

After purchasing a 6-week-old female BALB/C mouse (Orient Bio), acclimatization and quarantine for 1 week, 7-week-old mice were used for the experiment. Feed, drinking water, and breeding conditions are the same as in Example 1. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

The 4T1 mouse mammary carcinoma cell line (ATCC® CRL-2539™) was obtained from the American Type Culture Collection (ATCC). 4T1 cells were cultured in RPMI-1640 medium (CAT No: LM011-51, Welgene) containing 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco) 37 ℃, 5% CO ₂ Conditions were cultured in an incubator. ^{Using a syringe, 0.05 ml of 5×10 5} cells were transplanted into the fat layer in the fourth right breast of anesthetized female BALB/C mice.

As a vehicle, sterile physiological saline (Lot No: M8T7AF3, Daehan Pharmaceutical Co., Ltd.) used for botulinum toxin preparation was used, and 100 units Coretox (Lot No: NSA19007A, Medytox) was used as the botulinum toxin. As shown in Table 3, each test substance was prepared and administered to each group.

army	number of animals	substance to be administered	Volume	route of administration	Dosing frequency
One	10	vehicle	1 ml/kg	within the tumor	Once (5 days after tumor transplantation)
2	10	botulinum toxin	1.5 units/kg	within the tumor	Once (5 days after tumor transplantation)
3	10	botulinum toxin	5 units/kg	within the tumor	Once (5 days after tumor transplantation)
4	10	botulinum toxin	15 units/kg	within the tumor	Once (5 days after tumor transplantation)
5	10	botulinum toxin	50 units/kg	within the tumor	Once (5 days after tumor transplantation)

On the 5th day after transplantation of mouse mammary gland carcinoma, the body weight and tumor size were measured, and the mice were randomly assigned to each group at an average size of about 69 mm 3 without any difference between the test groups. On the 5th day after tumor transplantation, vehicle and botulinum toxin were administered intratumorally according to the composition of the test group based on body weight. Tumor size was measured twice a week after tumor transplantation. Tumor size was measured in the same manner as in Example 1. On day 21 after tumor transplantation, before the tumor size reached 1000 mm 3 , blood was collected from surviving individuals and all animals were euthanized. Exosomes in blood were isolated using Exoquick exosome precipitation solution (Cat No: EXOQ20A-1, Lot No: 200107-001, System Biosciences), and ExoELISA-ULTRA Complete Kit (Cat No: EXELULTRA-CD63-1, Lot No: 200226-002, System Biosciences). Statistical processing was performed in the same manner as in Example 1.

The measurement results of the tumor growth inhibition rate are shown in Table 4 and FIG. 4 .

army	substance to be administered	Volume	Average tumor size 1) (average ㎣±SEM)	TGI (%)1)
One	vehicle	1 ml/kg	989±82	0
2	botulinum toxin	1.5 units/kg	916±59	7
3	botulinum toxin	5 units/kg	773±89	22
4	botulinum toxin	15 units/kg	776±37 *	22
5	botulinum toxin	50 units/kg	833±57	16

¹⁾ Results on day 21 after tumor transplantation, ^* p <0.05, vs. vehicle. Two -sided t -test.

As shown in Table 4 and FIG. 4, on the 21st day after mouse mammary tumor transplantation, 1.5 units/kg botulinum toxin alone intratumoral administration showed an average tumor size of 916 mm 3 and a tumor growth inhibition rate of 7%, and 5 units/kg In the administration group, an average of 773 mm 3 and 22%, in the 15 units/kg administration group, an average of 776 mm 3 and 22%, and in the 50 units/kg administration group, an average 833 mm 3 and 16% tumor growth inhibition rate were shown.

The results of measuring the number of exosomes in the blood are shown in FIG. 5 . As shown in FIG. 5 , the number of exosomes in the blood was significantly reduced in the 15 and 50 units/kg botulinum toxin administration group compared to the vehicle group.

Example 3: Botulinum toxin tumor cell line proliferation inhibitory effect test

B16-F10 mouse malignant melanoma cell lines (KCLB No: 80008), A549 human lung cancer cell line (KCLB No: 10185), SNU324 (KCLB No: 00324), and Capan-2 (KCLB No: 30080) human pancreatic cancer cell lines were obtained from the Korea Cell Line Bank. (KCLB: Korean Cell Line Bank) was obtained. B16-F10 cells were cultured in DMEM medium (CAT No: 11965-092, Gibco) containing 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco). ℃, 5% CO ₂ Conditions were cultured in an incubator. A549 cell line 10%, 1% penicillin-streptomycin containing RPMI1640 (CAT No: LM011-51, Welgene Inc) medium SNU324 and Capan-2 cell line 10% FBS, 25 mM HEPES (CAT No: 15630-080, Gibco) It was cultured in an incubator at 37 °C, 5% CO _{2 conditions in RPMI1640 containing medium.} This experiment using a human tumor cell line was performed after being exempted from deliberation (IRB-2020-002) by the Institutional Bioethics Committee of Medytox Co., Ltd.

100 units Coretox (Lot No: NSA19007A or NSA19004, Medytox) was used as a botulinum toxin, and doxorubicin (LOT No: DROTK-GP, TCI) was used as a positive control material.

To investigate the effect of botulinum toxin on tumor cell proliferation, CCK8 proliferation assay (Cell Counting Kit-8, CK04, Dojindo) was used. ^{Specifically, cells were seeded at a rate of 5×10 3} cells/0.1 ml per well in a 96-well plate (Cat No: 167008, Thermo Fisher) and cultured in an incubator for 24 hours. After 24 hours, after removing the medium from each well, botulinum toxin was diluted in the medium used for tumor cell line culture at a concentration of 0.016-250 U/ml, and treated with 10 μg/ml doxorubicin. After removing the medium from each well 6 hours after the initial treatment time, the same test substance was re-treated into each well. It was cultured in an incubator for 24 hours from the time of initial botulinum toxin treatment. After incubation for a total of 24 hours, each well was treated with 10 μl of CCK-8 reagent, and after additional incubation for 3 hours, absorbance was measured at 450 nm using SpectraMax i3 (Model No: 10192-220, Molecular Devices). The proliferation rate was compared. The growth rate was calculated using the following equation.

Growth rate (%) =

(Ab: blank, Ac: negative control)

For each concentration, 4 wells of a 96-well plate were used, and the average value thereof was shown as a result, and the experiment was repeated for a total of 3 plates. Statistical analysis was performed in the same manner as in Example 1.

As a result of the experiment, it was found that the growth of tumor cells was significantly inhibited by botulinum toxin. In the mouse melanoma cell line (B16-F10), growth was significantly inhibited in the 0.4, 2, and 50 U/ml botulinum toxin treatment group (FIG. 6a), and in the human lung cancer cell line (A549), 2, 50, and 250 U/ml In the botulinum toxin treatment group (Fig. 6b), in the human pancreatic cancer (SNU324) cell line, in the 0.08-50 U/ml treatment group (Fig. 6c), in the human pancreatic cancer (Capan-2) cell line, in the 0.4-50 U/ml botulinum toxin treatment group In (Fig. 6d) a significant growth inhibitory effect was observed.

Example 4: Exosome reduction effect test of botulinum toxin in mouse melanoma cell line

A B16-F10 mouse malignant melanoma cell line (KCLB No: 80008) was obtained from the Korean Cell Line Bank (KCLB). B16-F10 cells were cultured in DMEM medium (CAT No: 11965-092) containing 10% exosome-depleted FBS (CAT No: A2720801, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco). , Gibco) in 37 ℃, 5% CO ₂ Conditions were cultured in an incubator.

100 units Coretox (Lot No: NSA19004, Medytox) was used as a botulinum toxin.

In a 6-well plate (Cat No: 140675, Thermo Fisher), 8×10 ⁵ cells/2 ㎖ per well were seeded into 6 wells in total and cultured for 24 hours in an incubator. After 24 hours, the medium from each well was removed, and botulinum toxin was diluted in the medium used for tumor cell line culture at a concentration of 10 U/ml, and the same amount of medium was treated for the control group. After 6 hours from the initial treatment time, the medium from each well was removed, and the same test substance was re-treated into each well. It was cultured in an incubator for 24 hours from the time of initial botulinum toxin treatment. After culturing for 24 hours, exosomes were measured for each well of the medium. The exosomes in the medium were isolated using ExoQuick-TC exosome precipitation solution (Cat No: EXOTC50A-1, Lot No: 191231-002, System Biosciences).

Measurements were made using the ExoELISA-ULTRA Complete Kit (Cat No: EXEL-ULTRA-CD63-1, Lot No: 200226-002, System Biosciences).

The results are shown in FIG. 7 . As shown in FIG. 7 , it was confirmed that 10 U/ml botulinum toxin had an effect of reducing exosomes in mouse melanoma.

Example 5: Testing the effect of botulinum toxin on reducing exosomes in human pancreatic cancer cell lines

The SNU324 human pancreatic cancer cell line (KCLB No: 00324) was obtained from the Korean Cell Line Bank (KCLB). SNU324 cells were cultured in RPMI-1640 medium (CAT No: LM011-51) containing 10% exosome-free FBS (CAT No: A2720801, Gibco) and 10% FBS, 25 mM HEPES (CAT No: 15630-080, Gibco). , Gibco) in 37 ℃, 5% CO ₂ Conditions were cultured in an incubator. This experiment using a human tumor cell line was conducted after being exempted from deliberation by the Institutional Bioethics Committee of Medytox Co., Ltd. (IRB-2020-002).

As the botulinum toxin, 100 units Coretox (Lot No: NSA19004, Medytox) was used.

In a 6-well plate (Cat No: 140675, Thermo Fisher), 1.2×10 ⁶ cells/2 ㎖ per well were seeded in a total of 6 wells and cultured for 24 hours in an incubator. After 24 hours, the medium from each well was removed, and botulinum toxin was diluted in the medium used for tumor cell line culture at a concentration of 10 U/ml, and the same amount of medium was treated for the control group. After 6 hours from the initial treatment time, the medium from each well was removed, and the same test substance was re-treated into each well. It was cultured in an incubator for 24 hours from the time of initial botulinum toxin treatment. After culturing for 24 hours, exosomes were measured for each well of the medium. The exosomes in the medium were isolated using ExoQuick-TC exosome precipitation solution (Cat No: EXOTC50A-1, Lot No: 191231-002, System Biosciences), and ExoELISA-ULTRA Complete Kit (Cat No: EXEL-ULTRA-CD63-1, Lot No: 200226-002, System Biosciences).

The results are shown in FIG. 8 . The measured number of exosomes was calculated using a two-sided t-test between the test group and the control group ( ^* p<0.05) As shown in FIG. 8, the effect of reducing exosomes in human pancreatic cancer cell lines by 10 U/ml botulinum toxin was it was confirmed that there is

Example 6: Test of botulinum toxin tumor cell line proliferation inhibitory effect

Human brain cancer cell lines U87MG (KCLB No: 30014) and PC3 (KCLB No: 21435) human prostate cancer cell lines were obtained from Korean Cell Line Bank (KCLB). U87MG cell line is DMEM (CAT No: LM007-01, Welgene) and PC3 cell line is RPMI-1640 (CAT No: LM011-01, Welgene) in 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin- Streptomycin (CAT No: 15140-122, Gibco) in a medium containing 37 ℃, 5% CO ₂ Conditions were cultured in an incubator. This experiment using a human tumor cell line was performed after being exempted from deliberation by the Institutional Bioethics Committee (IRB-2020-015) of Medytox Co., Ltd.

As the botulinum toxin, 100 units Coretox (Lot No: NSA19007A or NSA19004, Medytox) was used, and doxorubicin (LOT No: DROTKGP, TCI) was used as a positive control.

To investigate the effect of botulinum toxin on tumor cell proliferation, CCK8 proliferation assay (Cell Counting Kit-8, CK04, Dojindo) was used. Cells were seeded in a 96-well plate (Cat No: 167008, Thermo Fisher) at an amount of 5×10 ³ cells/0.1 ml per well, and cultured in an incubator for 24 hours. After 24 hours, after removing the medium from each well, botulinum toxin was diluted in the medium used for tumor cell line culture at a concentration of 0.016-50 U/ml, or treated with 10 μg/ml doxorubicin. After 6 hours from the initial treatment time, the medium was removed from each well, and the same test substance was re-treated in each well. It was cultured in an incubator for 24 hours from the time of initial botulinum toxin treatment.

After incubation for a total of 24 hours, each well was treated with 10 μl of CCK-8 reagent, and after additional incubation for 3 hours, absorbance was measured at 450 nm using SpectraMax i3 (Model No: 10192-220, Molecular Devices). The proliferation rates were compared. The growth rate was calculated using the following equation.

Growth rate (%) =

(Ab: blank, Ac: negative control)

For each concentration, 4 wells of a 96-well plate were used, and the average value thereof was shown as a result, and the experiment was repeated for a total of 3 plates. Graphpad Prism (version 7.05, GraphPad Software Inc, CA, USA) was used for graph presentation and was used for statistical analysis by SPSS software (version 25.0, SPSS Inc, IL, USA) or Excel (2013, MS, USA). For parametric data, a two-tailed t -test was performed, and for nonparametric data, statistical analysis was performed using Mann-Whitney test at a significance level of p = 0.05.

The measured tumor growth rate was calculated using a two-tailed t -test between the test group and the control group ^(* p <0.05, ^** p <0.01, ^*** p <0.001) of human brain tumor and prostate cancer cells by botulinum toxin. It was found that growth was significantly inhibited. A significant growth inhibitory effect was observed in the human brain tumor cell line (U87MG) in the 0.016 ~ 50 U / ml treatment group (Fig. 9a), and in the human prostate cancer (PC3) cell line in the 0.4 ~ 50 U / ml treatment group (Fig. 9b). .

Claims (15)

1. A composition for treating exosome-mediated diseases, comprising botulinum toxin as an active ingredient.
2. The composition of claim 1 , wherein the exosome-mediated disease is cancer.
3. The composition of claim 2, wherein the cancer is metastatic cancer.
4. The composition according to claim 2 or 3, wherein the cancer is a solid tumor.
5. The composition according to claim 4, wherein the solid cancer is at least one selected from the group consisting of melanoma, breast cancer, lung cancer, pancreatic cancer, brain tumor and prostate cancer.
6. A composition according to any one of claims 1 to 5 for use in subjects with increased exosome secretion.
7. 7. The composition of any one of claims 1-6, wherein the botulinum toxin is serotype A, B, C, D, E, F, G, H, or a combination thereof.
8. 8. The composition according to any one of claims 1 to 7, further comprising a pharmaceutically acceptable excipient or additive.
9. The composition of claim 8, wherein the pharmaceutically acceptable excipient or additive is a stabilizer, an ionic compound, a surfactant, a buffer, a lyophilization protectant, or a combination thereof.
10. 10. The composition of claim 9, wherein the pharmaceutically acceptable excipient or additive is an amino acid, a salt, a buffer, a nonionic surfactant, a sugar, a sugar alcohol, or a combination thereof.
11. 11. The composition according to any one of claims 8 to 10, which does not contain albumin or animal-derived components or polysaccharides.
12. 12. The composition according to any one of claims 8 to 11, in the form of a lyophilized powder, liquid, or prefilled syringe formulation.
13. 13. A composition according to any one of claims 8 to 12 for topical administration.
14. 14. The composition of claim 13 for intratumoral or peritumoral administration.
15. 15. The composition according to any one of claims 8 to 14, comprising 50 to 200 units of botulinum toxin.

Images