Composition for the prevention and / or improvement of peripheral neuropathy

Abstract

The inventors' primary objective was to provide a new technology for preventing and / or improving peripheral neuropathy. [Solution] The present inventors have found that peripheral neuropathy may be prevented and / or ameliorated by nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof.

Description

Technical Field

[0001] The present disclosure relates to a composition for preventing and / or improving peripheral neuropathy, a method for preventing and / or improving peripheral neuropathy, and the like.

Background Art

[0002] Peripheral neuropathy is known as one of the side effects of anticancer agent treatment (Non-Patent Document 1). Peripheral neuropathy can cause various symptoms such as muscle weakness, muscle atrophy, paresthesia such as numbness, pain, and dulled sensation in the hands and feet, dizziness, and sweating abnormalities. Peripheral neuropathy can significantly reduce the quality of life (QOL) of patients, and in some cases, even force the interruption of anticancer agent treatment. Therefore, preventing and / or improving peripheral neuropathy is very important from the viewpoints of improving QOL and continuing anticancer agent treatment. In addition, it is known that peripheral neuropathy can be caused by various factors other than anticancer agent treatment, such as drug administration, diabetes, and excessive alcohol intake.

[0003] There are currently several options for the current treatment of peripheral neuropathy. First, gabapentin and pregabalin are used as drug therapies. These are considered effective for the management of neuropathic pain, but are not always effective for all patients. In addition, the selective serotonin-norepinephrine reuptake inhibitor (SNRI) duloxetine may also be used for the treatment of neuropathic pain. Furthermore, vitamin B group, especially vitamin B1, B6, B 12 is said to have a neuroprotective effect and is used adjunctively in some patients. Non-drug therapies for peripheral neuropathy include physical therapy and occupational therapy, which help maintain muscle strength and balance and contribute to pain management. In addition, electrostimulation therapy aims to promote the regeneration of peripheral nerves and reduce pain. Acupuncture therapy has also been shown to be effective in reducing neuropathic pain in some studies. Furthermore, improvement of lifestyle is also important. Moderate exercise is said to be useful for the management of neuropathic pain, and a balanced diet including antioxidants and anti-inflammatory foods is recommended.

Prior Art Documents

[0004] [Non-Patent Document 1] Argyriou, Andreas A., Panagiotis Polychronopoulos, Gregoris Iconomou, Angelos Koutras, Thomas Makatsoris, Miltiadis K. Gerolymos, Philippos Gourzis, Konstantinos Assimakopoulos, Haralabos P. Kalofonos, and Elisabeth Chroni. 2007. “Incidence and Characteristics of Peripheral Neuropathy during Oxaliplatin-Based Chemotherapy for Metastatic Colon Cancer.” Acta Oncologica 46 (8): 1131-37. doi:10.1080 / 02841860701355055. [Overview of the project] [Problems that the invention aims to solve]

[0005] However, current treatments for peripheral neuropathy are mainly symptomatic and do not address the underlying causes. Therefore, their effectiveness is limited. Thus, the inventors of this invention aimed to provide a new technology for preventing and / or improving peripheral neuropathy. [Means for solving the problem]

[0006] The inventors have found that peripheral neuropathy may be prevented and / or ameliorated by nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof. Further improvements have led to the completion of this disclosure.

[0007] This disclosure includes, for example, the following subjects: Section 1. A composition for the prevention and / or improvement of peripheral neuropathy, comprising nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof. Section 2. The composition according to item 1, wherein the peripheral neuropathy is drug-induced peripheral neuropathy. Section 3. The composition according to item 2, wherein the drug is an anticancer agent. Section 4. The composition according to item 2 or 3, wherein the drug is oxaliplatin. Section 5. A composition according to any one of claims 1 to 4, comprising nafamostat mesylate or a solvate thereof. Section 6. Nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, for use in the prevention and / or improvement (especially treatment) of peripheral neuropathy. Section 7. Nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, for use as described in item 6, wherein the peripheral neuropathy is drug-induced peripheral neuropathy. Section 8. Nafamostat or a pharmaceutically acceptable salt thereof for use as described in paragraph 7, wherein the drug is an anticancer agent. Section 9. Nafamostat or a pharmaceutically acceptable salt thereof for use as described in item 7 or 8, wherein the drug is oxaliplatin. Section 10. Nafamostat mesylate, nafamostat or a pharmaceutically acceptable salt thereof for use as described in any one of items 6 to 9. Section 11. A method for the prevention and / or improvement of peripheral neuropathy, comprising administering a therapeutically effective dose of nafamostat or a pharmaceutically acceptable salt thereof to a subject in need. Section 12. The method according to item 11, wherein the peripheral neuropathy is drug-induced peripheral neuropathy. Item 13. The method according to item 12, wherein the drug is an anticancer agent. Item 14. The method according to item 12 or 13, wherein the drug is oxaliplatin. Item 15. The method according to any one of items 11 to 14, wherein the nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof is nafamostat mesylate or a solvate thereof. Item 16. Use of nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof for the manufacture of a composition for preventing and / or improving peripheral neuropathy. Item 17. The use according to item 16, wherein the peripheral neuropathy is drug-induced peripheral neuropathy. Item 18. The use according to item 17, wherein the drug is an anticancer agent. Item 19. The use according to item 17 or 18, wherein the drug is oxaliplatin. Item 20. The use according to any one of items 16 to 19, wherein the nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof is nafamostat mesylate or a solvate thereof.

Advantages of the Invention

[0010] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, compositions for the prevention and / or improvement of peripheral neuropathy, and encompasses everything disclosed herein and recognizable to those skilled in the art.

[0011] The compositions for the prevention and / or improvement of peripheral neuropathy included in this disclosure contain nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof. Hereinafter, such compositions included in this disclosure may be referred to as "the compositions of this disclosure."

[0012] 1. Compositions of the Disclosure The compositions for the prevention and / or improvement of peripheral neuropathy included in this disclosure contain nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof. Hereinafter, such compositions included in this disclosure may be referred to as "the compositions of this disclosure." The structure of nafamostat is shown below. [ka]

[0013] Examples of nafamostat salts include salts with inorganic or organic acids. Examples of such acids include mesylic acid, hydrochloric acid, phosphoric acid, sulfuric acid, orotic acid, chondroitin sulfate, hydrobromic acid, nitric acid, perchloric acid, oxalic acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, succinic acid, tartaric acid, acetic acid, formic acid, benzoic acid, malonic acid, citric acid, trifluoroacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluene-p-sulfonic acid, and naphthalene-2-sulfonic acid. While not particularly limited, in the art of this disclosure, it is particularly preferable to use nafamostat mesylate or its solvate.

[0014] Nafamostat or a pharmaceutically acceptable salt thereof may be in the form of a solvate. Examples of solvates include those with water, methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, or dimethoxyethane.

[0015] Furthermore, in the technology of this disclosure, any prodrug of nafamostat may be used instead of nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0016] In the technology of this disclosure, nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, may be mixed with a pharmaceutically acceptable base, carrier, excipient, diluent, solubilizer, emulsifier, preservative, pH adjuster, adjuvant, chelating agent, etc. These components may be used individually or in combination of two or more.

[0017] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, as well as benzoic acid, sodium benzoate, phenoxyethanol, and alkyldiaminoethylglycine hydrochloride. Preservatives can be used individually or in combination of two or more.

[0018] Examples of pH adjusting agents include citric acid, phosphoric acid, malic acid, pyrophosphate, lactic acid, tartaric acid, glycerophosphate, acetic acid, nitric acid, and chemically possible salts thereof, as well as sodium hydroxide and potassium hydroxide. pH adjusting agents can be used alone or in combination of two or more.

[0019] The method for preparing the compositions of this disclosure is not particularly limited insofar as the effects of this disclosure are achieved. For example, they can be prepared according to methods known in the art, and more specifically, they can be prepared by mixing, for example, nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, and other components with sterile distilled water.

[0020] 2.Applications The technology disclosed herein is suitably used for the prevention, improvement, and treatment of peripheral neuropathy. Here, the term "prevention of peripheral neuropathy" in this disclosure includes preventing the onset of peripheral neuropathy by applying the technology to the subject before they become aware of the symptoms of peripheral neuropathy or before they are diagnosed with peripheral neuropathy. Furthermore, the terms "improvement of peripheral neuropathy" or "treatment of peripheral neuropathy" in this disclosure include reducing peripheral neuropathy and suppressing its worsening by applying the technology to the subject who is aware of the symptoms of peripheral neuropathy and / or has been diagnosed with peripheral neuropathy.

[0021] Herein, “peripheral neuropathy” refers to dysfunction of one or more peripheral nerves (parts of the nervous system distal to nerve roots and nerve plexuses). The symptoms of peripheral neuropathy are diverse and may appear individually or in combination in patients, including varying degrees of muscle weakness, muscle atrophy, sensory abnormalities such as numbness, pain, and decreased sensation in the hands and feet, dizziness, and abnormal sweating. Based on the estimated mechanisms described later, it is reasonably predicted that peripheral neuropathy can be fundamentally prevented and / or improved by the technology of this disclosure. Therefore, any of the diverse symptoms of peripheral neuropathy described above can be prevented and / or improved by the technology of this disclosure. Although not particularly limited, the technology of this disclosure can effectively prevent and / or improve sensory abnormalities (especially pain, particularly allodynia). In this disclosure, "allodynia" refers to a sensory abnormality in which non-noxious stimuli such as contact that would not normally be perceived as painful, mild pressure, cold, or heat are perceived as painful. In animal experiments, allodynia is evaluated by the von Frey test (allodynia to mechanical stimuli), the acetone test (allodynia to cold stimuli), the hot plate test (allodynia to heat stimuli), etc.

[0022] Peripheral neuropathy is known to be caused by a variety of factors, including chemotherapy, drug administration, diabetes, and excessive alcohol consumption. The causes of peripheral neuropathy to which the technology of this disclosure applies are not particularly limited. As will be described later, it is presumed that in the technology of this disclosure, nafamostat or its salts, or their solvates, exert a preventive and / or ameliorative effect on peripheral neuropathy by protecting and / or repairing the glycocalyx covering the vascular endothelium. Based on this presumed mechanism, it is reasonably predicted that the technology of this disclosure will exert a preventive and / or ameliorative effect on peripheral neuropathy regardless of the cause, such as chemotherapy, drug administration, diabetes, and excessive alcohol consumption.

[0023] While not particularly limited, the technology of this disclosure is preferably applied to peripheral neuropathy caused by drug administration or diabetes (i.e., drug-induced peripheral neuropathy), and is particularly preferably applied to peripheral neuropathy caused by the administration of anticancer drugs.

[0024] It is known that peripheral neuropathy occurs relatively frequently with several types of anticancer drug treatments. Specifically, cytotoxic anticancer drugs such as paclitaxel, docetaxel, cabazitaxel, vinorelbine, vincristine, vinblastine, vindesine, eribulin, oxaliplatin, carboplatin, cisplatin, and nelarabine; molecularly targeted anticancer drugs such as bortezomib, ixavomib, trastuzumab emtansine, brentuximab vedotin, lorlatinib, entrectinib, pemigatinib, polatuzumab vedotin, and enfortumab vedotin; cancer immunotherapy drugs such as nivolumab, pembrolizumab, and ipilimumab; and drugs such as thalidomide, lenalidomide, pomalidomide, and darinaparsin are known to cause peripheral neuropathy relatively frequently. The technology of this disclosure can be suitably used for peripheral neuropathy caused by at least one selected from the group consisting of the aforementioned drugs. The technology of this disclosure is not particularly limited, but is preferably applied to peripheral neuropathy caused by administration of cytotoxic anticancer agents, more preferably to peripheral neuropathy caused by administration of vincristine and / or oxaliplatin, and particularly preferably to peripheral neuropathy caused by administration of oxaliplatin (oxaliplatin-induced peripheral neuropathy (OIPN)).

[0025] Furthermore, the technology of this disclosure can be used to protect and / or repair vascular endothelial glycocalyx in subjects with and / or at high risk of developing peripheral neuropathy. More specifically, it can be used to protect and / or repair vascular endothelial glycocalyx in subjects receiving or about to receive anticancer drug treatment. The vascular endothelial glycocalyx is preferably capillary endothelial glycocalyx. The aforementioned anticancer agents include, for example, cytotoxic anticancer agents such as paclitaxel, docetaxel, cabazitaxel, vinorelbine, vincristine, vinblastine, vindesine, eribulin, oxaliplatin, carboplatin, cisplatin, and nelarabine; bortezomib, ixavomib, trastuzumab emtansine, brentuximab vedotin, lorlatinib, entrectinib, pemigatinib, polatuzumab vedotin, and The anticancer agent may be at least one selected from the group consisting of molecularly targeted anticancer agents such as enfortumab vedotin; cancer immunotherapy agents such as nivolumab, pembrolizumab, and ipilimumab; and thalidomide, lenalidomide, pomalidomide, and darinaparsin, and is preferably a cytotoxic anticancer agent, more preferably vincristine and / or oxaliplatin, and particularly preferably oxaliplatin.

[0026] In this disclosure, the term vascular endothelial glycocalyx refers to a structure attached to the surface of vascular endothelial cells, composed of glycolipids, proteoglycans, glycoproteins, and polysaccharides. More specifically, vascular endothelial glycocalyx is known to be mainly composed of glycosaminoglycans such as heparan sulfate, chondroitin sulfate, and hyaluronic acid, as well as core proteins such as syndecane.

[0027] The technologies described herein are applicable to, for example, humans and other mammals (e.g., rats, mice, rabbits, cattle, pigs, dogs, cats, sheep, monkeys, etc.), with humans being particularly preferred.

[0028] The method of applying the technology of this disclosure is not particularly limited as long as the desired effect is achieved. For example, nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, may be administered intravenously, intra-arterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, etc., by injection or infusion, or directly by injection into a lesion such as cancer, or by perfusion via a catheter. Although not particularly limited, it is preferable to administer nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, intravenously by injection or infusion.

[0029] There are no particular limitations on the frequency of application of the technology disclosed herein. For example, it may be applied once or more times a day, once or more times a week, once or more times a month, or once or more times a year. Furthermore, there are no particular limitations on the duration of application of the technology disclosed herein, as long as the effects of the disclosure are achieved.

[0030] Furthermore, the dose of nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, is not particularly limited insofar as the effects of the present disclosure are achieved. For example, it may be 0.1 to 100 mg / kg, preferably 1 to 100 mg / kg, more preferably 5 to 80 mg / kg, even more preferably 10 to 50 mg / kg, and particularly preferably 20 to 40 mg / kg. The upper or lower limit of the above range may be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg / kg.

[0031] The frequency of application, duration of application, and dosage may be appropriately adjusted by those skilled in the art depending on the patient's condition and the course of treatment.

[0032] When the technology of this disclosure is used to prevent and / or improve peripheral neuropathy caused by drug administration, nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, may be administered to the subject before, simultaneously with, or after the administration of the drug. While not particularly limited, it is preferable that nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof, be administered to the subject before the administration of the drug.

[0033] 3. Estimation Mechanism While we do not wish to be bound by any theory, in the technology of this disclosure, it is presumed that nafamostat or its pharmaceutically acceptable salts, or their solvates, exert a preventive and / or ameliorative effect on peripheral neuropathy by protecting and / or repairing the glycocalyx covering the vascular endothelium. More specifically, by protecting and / or repairing the vascular endothelial glycocalyx, the blood-nerve barrier may be maintained and / or improved, and increased vascular permeability may be suppressed. As a result, it is presumed that the leakage of cytotoxic substances (e.g., drugs) from blood vessels into nerve fibers is suppressed, thereby exerting a preventive and / or ameliorative effect on peripheral neuropathy.

[0034] In this specification, the term “comprising” includes not only “containing” but also “essentially consisting of” and “consisting of.” Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0035] Furthermore, the various characteristics (properties, structure, function, etc.) described in each embodiment of this disclosure above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0036] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below. In these embodiments, nafamostat mesylate may be simply referred to as "nafamostat."

[0037] 1. Materials and Methods 1-1. Mouse Male C57BL / 6NCr Slc mice were obtained from CLEA Japan at 7 weeks of age and acclimatized for one week before the start of the experiment. The mice were housed at 22±1℃ with a normal light-dark cycle and fed with normal feed (CE-2, CLEA Japan) and autoclaved tap water. All behavioral experiments were conducted in the same room, in a randomized order before and after drug administration. Body weight (g) was recorded every 7 or 14 days, including on the day of administration and immediately before euthanasia. Mice were examined daily for abnormal clinical symptoms such as follicular formation, hind limb weakness, gait disturbances, and gastrointestinal disorders.

[0038] 1-2. Administration of oxaliplatin and / or nafamostat mesylate to tumor-bearing mouse models. Eight-week-old male C57BL / 6NCr Slc mice (n = 10 per group) were used. Each mouse was given MC38 mouse colorectal cancer cells (1 × 10⁶). 6 The drug (individual doses / mouse) was subcutaneously inoculated into the right flank and dorsal region. The tumors were measured with calipers every 1-2 weeks, and their volume (mm³) was measured. 3 The length was calculated using the formula π / 6 × length × width × thickness. The subcutaneous tumor was approximately 700 mm. 3 At this point, mice were randomly assigned to one of four groups: control vehicle (G1), oxaliplatin vehicle (G2), control-nafamostat mesylate (G3), and oxaliplatin-nafamostat mesylate (G4).

[0039] Since oxaliplatin and nafamostat mesilate are water-soluble drugs, a 5% glucose solution was used as the vehicle. Mice in the control group (G1) were administered a 5% glucose solution throughout the study. Mice in the oxaliplatin group (G2) were administered oxaliplatin (5 mg / kg / mouse) dissolved in a 5% glucose solution twice a week for 5 weeks. Mice in the nafamostat mesilate group (G3) were administered nafamostat mesilate (30 mg / kg / day) dissolved in a 5% glucose solution daily. Mice in the oxaliplatin + nafamostat mesilate group (G4) were administered oxaliplatin (5 mg / kg / mouse) and nafamostat mesilate (30 mg / kg / mouse / day) as shown in Figure 5A. This was repeated weekly until the endpoint.

[0040] 1-3. Assessment of allodynia in response to mechanical stimulation (von Frey test) Allodynia to mechanical stimulation was assessed using the von Frey test. Specifically, mice were first placed in a plexiglass chamber on an elevated mesh and allowed to adapt for at least 30 minutes. Then, using a von Frey filament, a mechanical force was applied to the ventral surface of the middle of the hind limb, increasing until the mouse retracted its hind limb. The mechanical force (g) at which the mouse retracted its hind limb was recorded as the paw withdrawal threshold.

[0041] 1-4. Evaluation of allodynia in response to cold stimulation (acetone test) Allodynia in response to cold stimuli was evaluated using the acetone test. Specifically, 50 μL of acetone was dropped onto the sole of the metapod of the hind limb of a mouse placed on a 25 × 25 cm plexiglass plate, and the number of nociceptive behaviors (shaking, lifting, licking, guarding, biting) exhibited by the hind limb was recorded for one minute to obtain an aversive response score.

[0042] 1-5. Tissue preparation The chests of anesthetized mice were exposed, and the inferior vena cava was incised. Perfusion irrigation was performed using an intravenous drip infusion of equal volumes of cold 1M PBS and 4% paraformaldehyde solution. The tissue was separated and divided into small sections, and paraffin-embedded sections and frozen sections were prepared. The paraffin-embedded sections and frozen sections were cut to thicknesses of 3 μm and 5 μm, respectively, and stained with hematoxylin and eosin (H&E) using conventional methods.

[0043] 1-6. Histological analysis and immunohistochemical staining After injecting DyLight488-Tomato lectin (50 μg / 100 μL dH2O, VECTOR) or FITC-dextran (40,000 kDa, 200 μg / 100 μL dH2O, Sigma) into the jugular vein for 15 minutes, the hind limbs and tumor-containing tissues of mice were excised. The excised tissues were fixed overnight with 4% paraformaldehyde at 4°C, incubated with 30% sucrose for cryoprotection (2-3 days at 4°C), embedded in an optimal cutting temperature compound for frozen tissue section preparation, and frozen in liquid nitrogen. The specimens were cut into 5 μm coronal sections using a cryostat (Leica), and each section was stored at -80°C.

[0044] For immunofluorescence staining, sections were incubated overnight at 4°C with rat anti-CD31 antibody (dilution 1:50, Dianova), followed by incubation at 37°C for 60 minutes with anti-rat Alexa594 labeled secondary antibody (dilution 1:250, Abcam). Slides were then stained with 4',6-diamidino-2-phenylindole (DAPI), mounted in Vector fluorescent hard mounts (Vector Laboratories), and observed. Images were acquired using an Olympus FV10i confocal laser scanning microscope (Olympus).

[0045] For immunohistochemical staining, nonspecific antibody binding was blocked with 2% normal bovine serum for 40 minutes. Sections were then incubated overnight at 4°C with rabbit anti-S100 antibody (dilution 1:500, Abcam) or rat anti-CD31 antibody (dilution 1:50, Dianova). Subsequently, sections were incubated with peroxidase-labeled anti-rabbit antibody or anti-rat antibody (Histofine Simplestain Max PO (R); Nichirei) at 37°C for 60 minutes. The immunoreaction was visualized using 3,3-diaminobenzidine tetrahydrochloride (Sigma), and sections were counterstained with hematoxylin.

[0046] 1-7. Immunohistochemical staining and quantification of intraepidermal nerve fibers Frozen sections of mouse hind limbs were subjected to immunohistochemical staining for PGP9.5. Sections were blocked with 2% normal bovine serum. Sections were incubated overnight at 4°C with rabbit anti-PGP9.5 antibody (dilution 1:200, Abcam, ab108986), followed by incubation with peroxidase-labeled anti-rabbit antibody (Histofine Simplestain Max PO (R); Nichirei) at 37°C for 60 minutes. The immunoreaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride (Sigma). Sections were counterstained with hematoxylin. Intraepidermal nerve fibers in each leg section were counted blindly at 40x magnification, and fiber density was determined at fibers / mm². 2 The calculation was performed as follows. For each group with n=5, the average fiber density was calculated.

[0047] 1-8.Platinum concentration measurement Tumors and dorsal root ganglia (DRGs) were excised from mice (n = 4 in each group) and stored at -80°C until analysis. After homogenization of the tissue, the total platinum concentration, a surrogate marker for oxaliplatin concentration, was measured by ICP-MS (7,700x, Agilent Technology).

[0048] 1-9. Scanning Electron Microscope (SEM) Observation After incising the right atrial adnexa of anesthetized mice, a lanthanum-containing alkaline solution was injected at a steady rate of 1 mL / min using a perfusion pump. Tissue was removed from the mice, fixed using conventional methods, and then its three-dimensional microstructure was observed using a scanning electron microscope.

[0049] 1-10. Cell culture and in vitro vascular permeability assays Primary human cutaneous microvascular endothelial cells (adults) were purchased from Lonza (catalog number: CC-2543). The cells were cultured in EBM-2 Basal Medium (CC-3156, Lonza) supplemented with EGMTM-2MV Microvascular Endothelial Cell Growth Medium Single Quots (CC-4147, Lonza). All cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

[0050] The vascular permeability assay was performed using the following procedure. First, primary human cutaneous microvascular endothelial cells were seeded into a collagen-precoated semipermeable insert and cultured until confluence. The cells were treated with either vesicles, oxaliplatin, or nafamostat mesylate for 24 hours. After 24 hours, the treatment solution was removed. FITC-dextran was added to the semipermeable insert coated with human cutaneous microvascular endothelial cells and incubated at room temperature for 60 minutes. Subsequently, 100 μL of medium was collected from the receiver tray and transferred to a 24-well plate, and the emission at 535 nm with excitation light at 485 nm was analyzed using a fluorescence plate reader.

[0051] 1-11. Administration of vincristine and / or nafamostat mesylate to mice Seven-week-old male C57BL6 / J mice were used (n=8 in each group). Mice were randomly assigned to one of four groups: an untreated control group (G1), a vincristine group (G2), a nafamostat mesilate group (G3), and a vincristine + nafamostat mesilate group (G4). Nafamostat mesilate and vincristine sulfate (hereinafter simply referred to as "vincristine") were purchased from Wako. Vincristine (0.01 mg / mL, dissolved in 0.9% physiological saline, 0.1 mg / kg / day) and nafamostat mesilate (0.02 mg / mL, dissolved in 5% glucose aqueous solution, 0.2 mg / kg / day) were administered daily by intraperitoneal injection until tissue dissection.

[0052] 1-12. Evaluation of allodynia in response to thermal stimulation (hot plate test) Allodynia in response to thermal stimulation was evaluated using a hot plate test. Specifically, mice were placed on a heated surface maintained at 55±1°C, and transparent plastic cylinders were placed around the mice to prevent them from moving off the surface. When the surface heat became uncomfortable, the mice would lift and lick their hind limbs, so the time from when the mouse was placed on the heated surface until it licked its hind limbs was manually recorded using a stopwatch. After lifting and licking its hind limbs, the mouse was immediately removed from the device. Some mice may jump or vocalize; in these cases, the mouse was immediately removed from the device, and the time (hot plate reaction time, hot plate latency) was recorded. A cutoff time (usually 30 seconds) was set to minimize the risk of tissue damage to the mice due to prolonged exposure to the heated surface.

[0053] 1-13. Diabetic model mice Male C57BLKS / J Iar- +leprdb / leprdb (db / db) mice purchased from SLC Japan were used as a type 2 diabetes model mouse.

[0054] 2.Results 2-1. Observation of peripheral tissues Peripheral nerves are surrounded by three types of connective tissue: the endoneurium, perineurium, and epineurium. In peripheral nerves, individual nerve bundles consisting of unmyelinated and myelinated axons, along with small intraneural blood vessels, are covered by the perineurium, forming the intraneural microenvironment (Figure 1A). These structures were observed in peripheral tissues of both mice and humans (Figure 1B).

[0055] Capillaries are covered with endothelial cells and normally lack a window structure, instead possessing an endothelial glycocalyx layer (Figure 1C). The blood-nerve barrier, composed of endothelial cells and glycocalyx, is the boundary separating peripheral nerve axons from blood flow, preventing the transfer of substances from plasma to nerve fibers. It was hypothesized that a decrease in vascular endothelial glycocalyx disrupts the blood-nerve barrier, allowing mononuclear cells and soluble factors to leak out of the blood vessels and transfer to nerve fibers, potentially leading to peripheral neuropathy.

[0056] 2-2. Nafamostat suppression of oxaliplatin-induced peripheral neuropathy 2-2-1. Occurrence of peripheral neuropathy due to oxaliplatin administration The inventors hypothesized that exposure to oxaliplatin damages the vascular endothelial glycocalyx and disrupts the blood-nerve barrier, thereby causing oxaliplatin-induced peripheral neuropathy, and they investigated this hypothesis. Specifically, mice were administered oxaliplatin (30 mg / kg / mouse) intraperitoneally twice a week for 5 weeks, and the von Frey test was performed at 0, 2, 9, 16, 23, and 30 days after the start of oxaliplatin administration (Figure 2A).

[0057] As shown in Figure 2B, at 0, 2, 16, 23, and 30 days after the start of oxaliplatin administration, mice in the oxaliplatin-treated group showed a significantly lower hindlimb withdrawal threshold compared to untreated control mice. In other words, mice in the oxaliplatin-treated group withdrew their legs in response to weaker mechanical stimuli. These results clearly indicate that oxaliplatin administration caused allodynia in the mice. Allodynia is one of the typical symptoms of peripheral neuropathy.

[0058] After the oxaliplatin administration trial, peripheral nerves were observed under a light microscope after being stained with hematoxylin and eosin (H&E) or immunohistochemically with the neuronal cell marker S100 (Figure 2C). In Figure 2C, the arrows indicate the perineurium. As shown in Figure 2C, the subcutaneous nerves surrounding the perineurium of the hind limbs of mice administered oxaliplatin were slightly edematous compared to untreated control mice. This result suggests that the mice in the oxaliplatin-administered group developed peripheral neuropathy.

[0059] To clarify the microenvironment of the perineurium, including endothelial cells and vascular endothelial glycocalyx, lanthanum-stained tissue was observed using a scanning electron microscope (SEM) (Figure 3A). The arrowhead at the bottom of Figure 3A indicates the moss-like vascular endothelial glycocalyx. As shown in Figure 3A, in the hind limbs of control mice, peripheral nerves and capillaries were fused together and extended as a single unit. On the other hand, in the hind limbs of mice administered oxaliplatin, edema was observed around the capillaries, and the peripheral nerves were frayed and fragmented. Furthermore, as shown in the magnified image of the capillary lumen at the bottom of Figure 3A, vascular endothelial glycocalyx was densely and abundantly present in control mice, but it was almost completely absent in oxaliplatin-administered mice.

[0060] These results suggest that a decrease in vascular endothelial glycocalyx at the blood-nerve barrier, accompanied by a defect in endoneurotic capillaries, may be an early stage of oxaliplatin-induced peripheral neuropathy (Figure 3B).

[0061] 2-2-2. Drug administration studies in tumor-bearing mouse models 2-2-2-1. Preliminary Examination The inventors investigated whether peripheral neuropathy is suppressed by administration of nafamostat mesilate. Specifically, mice were administered different doses of nafamostat mesilate (0, 3, 10, and 30 mg / kg / day) along with a fixed amount of oxaliplatin, and the von Frey test was performed (Figure 4). As shown in Figure 4, nafamostat mesilate suppressed allodynia caused by oxaliplatin administration in a dose-dependent manner. No bleeding tendencies or deaths were observed even in mice administered the highest dose of nafamostat mesilate (30 mg / kg / day) in this test. Therefore, the dose of nafamostat mesilate was set to 30 mg / kg / day, and subsequent tests were conducted according to the schedule shown in Figure 5A.

[0062] 2-2-2-2. Weight measurement To investigate whether drug administration affects mouse body weight, the body weight of mice in the control group (G1), oxaliplatin group (G2), nafamostat mesylate group (G3), and oxaliplatin + nafamostat mesylate group (G4) was measured every one or two weeks (Figure 5B). As shown in Figure 5B, there was no significant interaction between body weight at time (weeks 0, 1, 3, 5, 7, and 9) and treatment (control, oxaliplatin, nafamostat mesylate, and oxaliplatin + nafamostat mesylate).

[0063] 2-2-2-3. Tumor Volume Measurement To investigate the effects of oxaliplatin and / or nafamostat mesilate on tumor progression, tumor volume was measured in each group. Significant interactions were observed between time (weeks 0, 1, 3, 5, 7, and 9) and treatment (control, oxaliplatin, nafamostat mesilate, and oxaliplatin + nafamostat mesilate) with respect to tumor volume (Figure 5C). Tumor volume in the control group (G1) and the nafamostat mesilate group (G3) increased approximately ninefold compared to baseline. In the oxaliplatin group (G2) and the oxaliplatin + nafamostat mesilate group (G4), tumor volume increased approximately fourfold compared to baseline. Tumor growth at the endpoint was suppressed more effectively in the oxaliplatin group (G2) and the oxaliplatin + nafamostat mesilate group (G4) than in the control group (G1).

[0064] 2-2-2-4. Assessment of allodynia in response to mechanical stimulation The von Frey trial was conducted to investigate the effect of nafamostat mesilate on allodynia in response to mechanical stimulation (Figure 5A). A significant interaction was observed between time (weeks 0, 1, 3, 5, 7, and 9) and treatment (control, oxaliplatin, nafamostat mesilate, oxaliplatin + nafamostat mesylate) regarding the hindlimb pull-out threshold (Figure 5D). In the oxaliplatin group (G2), the hindlimb pull-out threshold decreased over time. On the other hand, at the endpoint, the oxaliplatin + nafamostat mesilate group (G4) showed a hindlimb pull-out threshold similar to that of the control group (G1) and the nafamostat mesilate group (G3).

[0065] 2-2-2-5. Evaluation of allodynia in response to cold stimuli To investigate the effect of nafamostat mesylate on allodynia in response to cold stimuli, an acetone test was conducted. Regarding aversion response scores, an interaction trend was observed between time (days 0, 5, 11, 17, 23, and 26) and treatment (control, oxaliplatin, oxaliplatin + nafamostat mesylate) (Figure 6). In the oxaliplatin group, aversion response scores to cold stimuli were significantly higher than in the control group on days 17, 23, and 26. On the other hand, no significant difference was observed between the oxaliplatin + nafamostat mesylate group and the control group (Figure 6).

[0066] 2-2-2-6. Evaluation of oxaliplatin uptake in tumor tissue To evaluate the uptake of oxaliplatin in tumor tissue, total platinum concentration was measured as a surrogate marker for oxaliplatin concentration in the control group (G1), the oxaliplatin group (G2), and the oxaliplatin + nafamostat mesylate group (G4). There was no significant difference in oxaliplatin concentration in tumor tissue between the oxaliplatin group (G2) and the oxaliplatin + nafamostat mesylate group (G4) (Figure 5E). These results suggest that nafamostat mesylate does not inhibit oxaliplatin delivery to tumor tissue.

[0067] 2-2-2-7. Evaluation of oxaliplatin uptake within the dorsal root ganglia Oxaliplatin-induced peripheral neuropathy is known to involve both the dorsal root ganglion (DRG) and peripheral nerves. To evaluate whether oxaliplatin strongly accumulates in the DRG, the total platinum concentration in the DRG was measured in the control group (G1), the oxaliplatin group (G2), and the oxaliplatin + nafamostat mesylate group (G4) (Figure 7). The oxaliplatin + nafamostat mesylate group (G4) tended to have a higher oxaliplatin concentration in the DRG compared to the oxaliplatin group (G2), but the difference between the groups was not statistically significant. These results suggest that nafamostat mesylate does not inhibit the accumulation of oxaliplatin in the DRG.

[0068] 2-2-2-8. Evaluation of intraepidermal nerve fibers Allodynia in response to mechanical stimulation is known to be closely associated with the loss of intra-epidermal nerve fibers (IENFs). IENF density was assessed in hindlimb samples dissected at the endpoint for each group of mice. IENF density was evaluated by immunohistochemical staining to detect the neuronal marker PGP9.5.

[0069] In the control group (G1), IENF was uniformly and abundantly distributed. On the other hand, in the oxaliplatin group (G2), almost no IENF was observed (Figure 8A). The oxaliplatin group (G2) had a significantly lower IENF density compared to the control group (G1) and the oxaliplatin + nafamostat mesylate group (G4) (Figure 8B). The IENF density in the oxaliplatin + nafamostat mesylate group (G4) was significantly higher than that of the oxaliplatin group (G2) (Figure 8B). These results suggest that nafamostat mesylate suppressed the reduction of IENF caused by oxaliplatin.

[0070] 2-2-2-9. Evaluation of peripheral foot vascular permeability To investigate peripheral foot vascular permeability in the control group (G1), oxaliplatin group (G2), and oxaliplatin + nafamostat mesylate group (G4), FITC-dextran was intravenously injected, and capillaries were observed using immunofluorescence staining for the endothelial cell marker CD31.

[0071] In the oxaliplatin group (G2), FITC-dextran leakage from capillaries was increased compared to the untreated control group (G1) (Figure 8C). While the capillary boundaries in the control group (G1) were clearly defined, the vascular boundaries in the oxaliplatin group (G2) were blurred and indistinct (Figure 8C). These results suggest that oxaliplatin damages capillary endothelial cells, increasing vascular permeability and causing extravasation. Furthermore, extravasation of FITC-dextran was significantly suppressed in the hind limbs of mice in the oxaliplatin + nafamostat mesylate group (G4) compared to the G2 group (P < 0.001) (Figure 8D).

[0072] 2-2-2-10. Evaluation of vascular endothelial glycocalyx The endothelial glycocalyx of hindlimb capillaries was analyzed in the control group (G1), the oxaliplatin group (G2), and the oxaliplatin + nafamostat mesylate group (G4). Specifically, DyLight 488-labeled Lycopersicon esculentum (Tomato) lectin was intravenously injected, and the fluorescence intensity of the capillaries was evaluated by CD31 immunofluorescence staining (Figure 9A).

[0073] Expression of DyLight 488-labeled tomato lectin on the surface of endothelial cells in hindlimb capillaries was lower in the oxaliplatin group (G2) compared to the control group (G1) and the oxaliplatin + nafamostat mesylate group (G4) (Figure 9A). Furthermore, quantitative analysis revealed that the fluorescence intensity and thickness of the vascular endothelial glycocalyx were significantly reduced in the oxaliplatin group (G2) compared to the control group (G1) and the oxaliplatin + nafamostat mesylate group (G4) (Figure 9B).

[0074] Furthermore, the inventors obtained three-dimensional scanning electron microscope images of hind limb capillaries in the control group (G1), the oxaliplatin group (G2), and the oxaliplatin + nafamostat mesylate group (G4) (Figure 10A). In the control group (G1) and the oxaliplatin + nafamostat mesylate group (G4), peripheral nerves and capillaries were adhered and merged. On the other hand, in the oxaliplatin group (G2), edema was observed around the capillaries, and the peripheral nerves were frayed and shattered. In the control group (G1) and the oxaliplatin + nafamostat mesylate group (G4), it was confirmed that endothelial glycocalyx covered the entire surface of the capillary lumen. On the other hand, in the oxaliplatin group (G2), vascular endothelial glycocalyx was hardly observed.

[0075] Furthermore, quantitative analysis of immunohistochemical staining with anti-S100 antibody showed that the area of ​​extravascular edema in the control group (G1) and the oxaliplatin + nafamostat mesylate group (G4) was significantly smaller than that in the oxaliplatin group (G2) (Figure 10B).

[0076] 2-2-3. Vascular permeability assay in cultured human dermal microvascular endothelial cells To evaluate the direct effect of nafamostat mesylate on oxaliplatin-induced endothelial cell damage, a vascular permeability assay was performed in cultured human cutaneous microvascular endothelial cells (Figure 11). As shown in Figure 11, nafamostat mesylate suppressed oxaliplatin-induced increased permeability in vitro. This result suggests that nafamostat mesylate directly suppresses oxaliplatin-induced increased vascular permeability in human cutaneous endothelial cells.

[0077] 2-2-4.Summary The above studies revealed that oxaliplatin mesylate induces endothelial glycocalyx damage in capillaries and increases vascular permeability. Furthermore, it was suggested that nafamostat mesylate suppresses increased vascular permeability by protecting and / or repairing the vascular endothelial glycocalyx, thereby preventing nerve fiber damage by inhibiting oxaliplatin exposure to nerve fibers and suppressing oxaliplatin-induced peripheral neuropathy. At the same time, it was shown that nafamostat mesylate does not inhibit the antitumor effect of oxaliplatin.

[0078] 2-3. Suppression of vincristine-induced peripheral neuropathy by nafamostat From Test 2-2, it was suggested that nafamostat mesylate suppresses oxaliplatin-induced peripheral neuropathy by protecting and / or repairing the vascular endothelial glycocalyx. Based on this proposed mechanism, the inventors hypothesized that peripheral neuropathy caused by drugs other than oxaliplatin would also be suppressed by nafamostat mesylate, and they tested this hypothesis.

[0079] Specifically, the von Frey test, which assesses allodynia in response to mechanical stimulation, was performed on day 0 and day 22 of treatment for mice in each of the following groups: the untreated control group (G1), the vincristine group (G2), the nafamostat mesylate group (G3), and the vincristine + nafamostat mesylate group (G4) (Figure 12A). In addition, the hot plate test, which assesses allodynia in response to thermal stimulation, was performed on day 0 and day 23 of treatment for mice in each group (Figure 12B).

[0080] As shown in Figures 12A and 12B, significant differences were observed in the results of both the von Frey test and the hot plate test between the control group (G1) and the vincristine group (G2), the vincristine group (G2) and the nafamostat mesylate group (G3), and the vincristine group (G2) and the vincristine + nafamostat mesylate group (G4). More specifically, the vincristine group (G2) had a significantly lower hind limb pull-out threshold and a shorter hot plate reaction time than the control group (G1). In other words, vincristine administration caused allodynia in response to mechanical and thermal stimuli. On the other hand, the vincristine + nafamostat mesylate group (G4) had a significantly higher hind limb pull-out threshold and a longer hot plate reaction time than the vincristine group (G2). In other words, nafamostat mesylate administration suppressed vincristine-induced allodynia.

[0081] Furthermore, the inventors observed the endothelial glycocalyx of hind limb capillaries in the control group (G1), the vincristine group (G2), and the vincristine + nafamostat mesylate group (G4) using a transmission electron microscope. In the control group (G1) and the vincristine + nafamostat mesylate group (G4), numerous moss-like endothelial glycocalyx covered the endothelium of the capillary lumen, but in the vincristine group (G2), almost no vascular endothelial glycocalyx was observed. When the percentage of endothelial glycocalyx covering the vascular lumen was calculated by image analysis, the percentage in the control group (G1) (33.53±7.06%) and the vincristine + nafamostat mesylate group (G4) (36.12±7.06%) was significantly higher than the percentage in the vincristine group (G2) (3.73±7.06%) (Figure 13). There was no significant difference between G1 and G4 in the proportion of endothelial glycocalyx covering the vascular lumen.

[0082] This study suggests that nafamostat also suppresses vincristine-induced peripheral neuropathy by protecting and / or repairing the vascular endothelial glycocalyx.

[0083] 2-4. Suppression of vascular endothelial glycocalyx damage in diabetic model mice by nafamostat mesylate. Peripheral neuropathy is known to occur due to a variety of factors, including drug administration and diabetes. The inventors investigated the possibility of suppressing peripheral neuropathy occurring in diabetic patients by administering nafamostat mesylate.

[0084] Specifically, skin flaps were surgically created in diabetic model mice, and then nafamostat mesylate (30 mg / kg / day) or an equivalent volume of 5% glucose aqueous solution was administered intraperitoneally once daily for one week. After one week, the mice were dissected, and the capillary endothelial glycocalyx in the skin flaps was observed using a scanning electron microscope (Figure 14). As shown in Figure 14, in mice administered nafamostat mesylate, numerous moss-like endothelial glycocalyx covered the endothelium of the capillary lumen, while in mice administered glucose (Saline), almost no vascular endothelial glycocalyx was observed. This study suggests that nafamostat mesilate suppresses endothelial glycocalyx damage in diabetic patients. This suggests that nafamostat mesilate may be able to suppress peripheral neuropathy in diabetic patients.

Claims

1. A composition for the prevention and / or improvement of peripheral neuropathy, comprising nafamostat or a pharmaceutically acceptable salt thereof, or a solvate thereof.

2. The composition according to claim 1, wherein the peripheral neuropathy is drug-induced peripheral neuropathy.

3. The composition according to claim 2, wherein the drug is an anticancer agent.

4. The composition according to claim 2, wherein the drug is oxaliplatin.

5. A composition according to any one of claims 1 to 4, comprising nafamostat mesylate or a solvate thereof.

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