CN116421602B

CN116421602B - New application of flubendazole or derivative thereof

Info

Publication number: CN116421602B
Application number: CN202310459743.4A
Authority: CN
Inventors: 贾志龙; 杨晶; 宋欣雨
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2023-04-25
Filing date: 2023-04-25
Publication date: 2024-01-30
Anticipated expiration: 2043-04-25
Also published as: CN116421602A

Abstract

The present invention relates to a novel use of flubendazole or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof. The invention provides the use of flubendazole or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof in the manufacture of a medicament for the treatment or prevention of a altitude stress-related disease. According to the embodiment of the invention, the detection of the flubendazole based on the cell hypoxia experiment and the animal hypoxia simulation experiment proves that the flubendazole can be used for preventing acute altitude reaction and has important significance for guaranteeing non-altitude local crowd to go to altitude areas.

Description

New application of flubendazole or derivative thereof

Technical Field

The invention relates to the field of medicines, in particular to a novel application of flubendazole or a derivative thereof, and more particularly relates to a novel application of flubendazole or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof.

Background

Acute altitude stress (Acute Mountain Sickness, AMS) refers to a series of acute low-pressure hypoxia stress reactions that occur after personnel residing in non-altitude areas enter the altitude area (generally considered to be above 2500 m in altitude) due to the lack of established habitual adaptation mechanisms to the altitude hypoxic environment. The hypoxia symptoms may occur 1-3 days or even hours after the initial altitude. It is counted that 25% -43% of travelers can develop acute mountain sickness at altitude of 2,000-4,300 m. Acute altitude reaction is a common disease in high altitude areas, and if the common disease cannot be timely controlled, the common disease can be developed into altitude pulmonary edema, altitude cerebral edema, altitude pulmonary arterial hypertension and the like, so that physical health of tourists and residents in the altitude areas is seriously endangered, and economic development and safe work in the altitude areas are affected.

However, current means of treating or preventing acute altitude stress remain to be improved.

Disclosure of Invention

The present invention aims to solve, at least to some extent, one of the above technical problems or at least to provide a useful commercial choice. To this end, an object of the present invention is to propose a means which can be effectively used for the treatment or prevention of diseases associated with altitude stress.

The present invention has been completed based on the following findings by the inventors:

acute altitude stress seriously affects activities such as travel, learning, task execution, work and life of non-altitude people in altitude areas. However, current drugs do not provide a good control of acute altitude stress, and there is a lack of alternatives to the treatment of acute altitude stress.

Currently, acetazolamide is the first line drug approved by the FDA in the united states to prevent acute altitude stress. The acetazolamide is most effective when the AMS appears to be light in early stages, and the prophylactic dose is 125mg and the therapeutic dose is 250mg for 12 hours. Acetazolamide acts primarily in the kidneys, causing bicarbonate diuresis and metabolic acidosis. This kidney effect counteracts respiratory alkalis caused by hyperventilation due to altitude and increases central chemoreceptor sensitivity, the net effect of which is increased ventilation in hypoxic environments. For a high altitude mission scenario of several days, acetazolamide has the additional advantage of promoting hypoxia respiratory accommodation, forming plateau Xi Fu enhancing cognitive and physical activity phenotypes. However, because it is difficult to penetrate the blood brain barrier faster with oral acetazolamide administration, the dosage is generally increased when treating acute altitude stress, resulting in an increased probability of adverse reactions. In addition, in recent years, there has been some controversy about whether or not the physical ability of the plateau worker is affected after taking acetazolamide. The national drug administration has not approved acetazolamide for the control of acute altitude sickness.

The inventor of the invention screens and identifies a drug flubendazole capable of preventing acute altitude reaction on the basis of proteome research and cell and animal model experiments of crowd queues before and after altitude, and finds that the effect of the flubendazole is superior to that of the traditional acute altitude reaction first-line drug acetazolamide on the basis of evaluation of cell activity, cell proliferation, cell cycle, stem-wet ratio of rat brain and the like, therefore, the invention provides a new alternative scheme for the treatment of acute altitude reaction on the basis of cell hypoxia experiments and animal experiments to verify that the flubendazole can be used for preventing acute altitude reaction, has important significance for guaranteeing work, travel and the like of non-altitude crowd to altitude areas.

Flubendazole (also called flubendazole) having a structural formula shown in the specification

Fluorobendazole is a novel broad-spectrum highly effective anthelmintic agent, a member of the benzoxazole class in which benzoyl is substituted with p-fluorobenzoyl, formula C ₁₆ H ₁₂ FN ₃ O ₃ Molecular weight 313.28 is an agonist of the autophagy-related protein ATG4B (Zhang L, guo M, li J, zheng Y, zhang S, xie T, liu B.systems biology-based discovery of a potential Atg B agonist (Flubendazole) that induces autophagy in breast cancer biosystem.2015 nov;11 (11): 2860-6.). Veterinarians are used to protect dogs and cats from endoparasites and helminths and are also used by humans to treat helminth infections. Its mechanism of action is to cause disintegration of the parasite's gut endothelial cells and to lyse the cells so as to die. The composition is clinically used for treating infection of echinococci, cysticercus, roundworm, whipworm, hookworm, pinworm, trichostrongylodes, cercosis sinensis, clonorchis sinensis, posttestosterone fluke and dysmorphism fluke. And can be used for treating skin larva migration caused by hook and ascariasis. Has good therapeutic effect on scabies. Is commonly used for the treatment of single infections and parasitic mixed infections.

The flubendazole exerts an anticancer effect by inhibiting mechanisms such as microtubule function (Michaelis M, agha B, rothweiler F,n, voges Y, mittelbronn M, starzetz T, harter PN, abhari BA, fulda S, westermann F, riecken K, spek S, langer K, wiese M, dirks WG, zehner R, cinctl J, wass MN, cinctl J Jr.identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen. Sci Rep.2015Feb 3; 5:8202.). Fluobendazole induces p 53-mediated apoptosis, blocking the cell cycle at G2/M (Zhou X, liu J, zhang J, wei Y, li H. Flubendazole inhibits glioma proliferation by G/M cell cycle arrest and pro-apoptosis cell Death discover.2018Feb 14; 4:18.). Flurobendazole can be induced by targeting EVA1A in breast cancerMitochondrial dysfunction and DRP1-mediated autophagy are mediated, thereby exerting antitumor effects (Zhen Y, yuan Z, zhang J, chen Y, fu Y, liu Y, fu L, zhang L, zhou XL. Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1Ain disease cancer cell de ath Dis.2022Apr 19;13 (4): 375.). Scientists at the university of new south wilms found that flubendazole was effective in inhibiting the growth and metastasis of human tumors in the mouse body. The antitumor effects of the drug flubendazole include mainly that it inhibits the activity of a cell surface protein named apoptosis protein-1 (PD-1) (Zhou X, zou L, chen W, yang T, luo J, wu K, shu F, tan X, yang Y, cen S, li C, mao X.Flubendazole, FDA-approved anthelmintic, elicits valid antitumor effects by targeting P53 and promoting ferroptosis in castration-resistant prostate cancer Pharmacol Res.2021Feb; 164:105305.).

Thus, in one aspect of the invention, the invention provides the use of flubendazole, or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof, in the manufacture of a medicament for the prevention or treatment of a altitude-response related disease.

According to an embodiment of the invention, the altitude-response related disease is selected from at least one of acute altitude response, altitude pulmonary edema, altitude cerebral edema, and altitude pulmonary arterial hypertension.

According to an embodiment of the invention, the medicament is suitable for use in adults or children.

According to an embodiment of the invention, the medicament is for oral administration.

According to an embodiment of the invention, the dosage form of the medicament is selected from a tablet, a capsule, an oral liquid, a granule or a sustained and controlled release preparation.

In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising: flubendazole or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof as an active ingredient, and optionally pharmaceutically acceptable excipients or carriers.

According to an embodiment of the invention, the pharmaceutical composition is suitable for adults or children.

According to an embodiment of the invention, the pharmaceutical composition is for oral administration.

According to an embodiment of the present invention, the dosage form of the pharmaceutical composition is selected from a tablet, a capsule, an oral liquid, a granule or a sustained and controlled release preparation.

In a further aspect of the invention, the invention provides the use of the pharmaceutical composition as described above for the preparation of a medicament for the prevention or treatment of diseases associated with altitude stress.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram showing the results of CCK-8-based cell activity assay according to an embodiment of the present invention, wherein p represents 0.05 or less, p represents 0.01 or less, p represents 0.001 or less, p represents 0.0001 or less, and ns represents p >0.05;

fig. 2 is a schematic diagram of early apoptosis evaluation experiment results based on a flow cytometer according to an embodiment of the present invention, wherein p represents p value less than or equal to 0.05, p represents p value less than or equal to 0.01, p represents p value less than or equal to 0.001, p represents p value less than or equal to 0.0001, and ns represents p value >0.05;

fig. 3 is a schematic diagram of the results of a flow cytometer based late apoptosis evaluation experiment according to an embodiment of the present invention, wherein p represents 0.05 or less, p represents 0.01 or less, p represents 0.001 or less, p represents 0.0001 or less, and ns represents p >0.05 or less;

fig. 4 is a schematic diagram of the results of an experiment for evaluating total apoptosis based on a flow cytometer according to an embodiment of the present invention, wherein p represents 0.05 or less, p represents 0.01 or less, p represents 0.001 or less, p represents 0.0001 or less, and ns represents p >0.05 or less;

FIG. 5 is a schematic diagram showing the results of a flow cytometer based evaluation experiment for the living cell rate, wherein p represents 0.05 or less, p represents 0.01 or less, p represents 0.001 or less, p represents 0.0001 or less, and ns represents p >0.05;

fig. 6 is a schematic diagram of experimental results of early analysis of cell synthesis based on a flow cytometer according to an embodiment of the present invention, wherein p represents 0.05 or less, p represents 0.01 or less, p represents 0.001 or less, p represents 0.0001 or less, and ns represents p >0.05 or less;

fig. 7 is a schematic diagram of experimental results of flow cytometry-based cell synthesis phase analysis according to an embodiment of the present invention, wherein p represents 0.05 or less, p represents 0.01 or less, p represents 0.001 or less, p represents 0.0001 or less, and ns represents p >0.05 or less;

fig. 8 is a schematic diagram of experimental results of a flow cytometer based cell post-synthesis analysis according to an embodiment of the present invention, wherein p represents p value less than or equal to 0.05, p represents p value less than or equal to 0.01, p represents p value less than or equal to 0.001, p represents p value less than or equal to 0.0001, and ns represents p value >0.05;

fig. 9 shows the effect of a test agent on water content of brain tissue, wherein p represents 0.05 or less, p represents 0.01 or less, p represents 0.001 or less, p represents 0.0001 or less, and ns represents p >0.05 or less, according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

The inventor firstly identifies key protein expression profile characteristics of acute altitude reaction based on a proteomics means of an antibody proximity connection technology PEA, identifies an acute altitude reaction candidate drug of flubendazole based on an expression profile connection diagram strategy, and proposes that constructing a hypoxia cell model by utilizing rat myocardial H9c2 cells evaluates the effect of the flubendazole on cell activity, cell proliferation and cell cycle, constructing a rat hypoxia animal model evaluates the effect of the brain on protecting hypoxia injury, and the effect is superior to that of first-line drug of acetazolamide for preventing and treating acute altitude reaction.

The term "altitude-reaction-related disease" as used herein refers to various discomfort caused by exposure of a human body to a low-pressure and low-oxygen environment, such as symptoms associated with a human body entering an altitude of 2000 meters, for example, an altitude above 3000 meters. Common symptoms are headache, insomnia, anorexia, tiredness, dyspnea, etc.

According to an embodiment of the invention, the altitude-response related disease comprises at least one of acute altitude response, altitude pulmonary edema, altitude cerebral edema, and altitude pulmonary arterial hypertension. According to an embodiment of the present invention, flubendazole can be used for treating or preventing acute altitude stress, thereby preventing the occurrence of altitude pulmonary edema, altitude cerebral edema, altitude pulmonary arterial hypertension, etc. by controlling altitude stress in time.

Compared with flubendazole, the existing medicament dexamethasone for treating or preventing acute altitude reaction is taken as a corticosteroid, and is not suitable for basic diseases such as endocrine disturbance, peptic ulcer, upper gastrointestinal hemorrhage, certain infections (such as amoeba disease and acanthocell nematode disease) and the like, and attention is paid to the problem of combining antiepileptic medicaments, alcohol, aspirin and nonsteroidal anti-inflammatory medicaments. Dexamethasone causes adrenal suppression and is not suitable for long-term use. Rapid withdrawal within 24 hours may cause recurrence and exacerbation of AMS, requiring a slow withdrawal of 1 week when the time of use reaches 10 d. Dexamethasone has hypothalamic-pituitary-adrenal axis inhibitory toxicity and has greater adverse effects including gastrointestinal reactions and hyperglycemia over long periods of time, and the risk of developing depression following sudden withdrawal is also increased. Therefore, the dosage should be gradually reduced when the medicine is cut off, the medicine cannot be cut off suddenly, and the medicine cannot be used as a medicine for preventing and treating the AMS of children. In addition, because it is difficult to penetrate the blood brain barrier faster with oral acetazolamide administration, the dosage is generally increased when treating acute altitude stress, resulting in an increased probability of adverse reactions. Therefore, the flubendazole provided by the invention can be effectively administered orally, and is not only suitable for adults and children, but also can be used for children.

The medicament of the invention is preferably prepared into a dosage form of an oral preparation. According to embodiments of the present invention, oral dosage forms of the drug include, but are not limited to, tablets, capsules, gelatin pills, oral liquids, granules or sustained and controlled release formulations. The shape of the oral preparation is not particularly limited, and may be any of a circle, a capsule, a ring (doughnut), a rectangle, and the like.

When the oral preparation is a solid preparation, for example, a tablet, a capsule, a powder, a granule, a lozenge, or the like can be mentioned.

The solid formulation may be coated with a coating agent and may have indicia and letters for identification and further score lines for separation. The coating is carried out with the addition of conventional coating media and film forming agents (commonly referred to collectively as coating materials) familiar to those skilled in the art. The coating may be performed using, for example, a sugar coated substrate, a water-soluble film coated substrate, an enteric film coated substrate, a slow release film coated substrate, or the like. For sugar coated substrates, a combination of sucrose and one or more selected from the group consisting of: talc, precipitated calcium carbonate, gelatin, acacia, pullulan, carnauba wax, and the like. For the water-soluble film-coated substrate, for example, a cellulose polymer such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, methyl hydroxyethyl cellulose, or the like can be used; synthetic polymers such as polyvinyl acetal diethylaminoethyl ester, aminoalkyl methacrylate copolymer E [ Eudragit E (trade name) ], polyvinyl pyrrolidone, and the like; polysaccharides such as pullulan and the like. For the enteric film-coated substrate, for example, a cellulose polymer such as hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethylcellulose, cellulose acetate phthalate, or the like can be used; acrylic polymers such as methacrylic copolymer L [ Eudragit L (trade name) ], methacrylic copolymer LD [ Eudragit L-30D55 (trade name) ], methacrylic copolymer S [ Eudragit S (trade name) ] and the like; naturally occurring substances, such as shellac and the like; etc. For the sustained-release film-coated substrate, for example, a cellulose polymer such as ethyl cellulose, cellulose acetate, etc. can be used; acrylic polymers such as aminoalkyl methacrylate copolymer RS [ Eudragit RS (trade name) ], ethyl acrylate-methyl methacrylate copolymer suspension [ Eudragit NE (trade name) ] and the like. Two or more of the above coating bases may be mixed in a suitable ratio. Furthermore, coating additives may be used in coating. For the coating additive, for example, a photo masking agent and/or a coloring agent such as titanium oxide, talc, iron oxide, etc. may be used; plasticizers such as polyethylene glycol, triethyl citrate, castor oil, polysorbate, and the like; organic acids such as citric acid, tartaric acid, malic acid, ascorbic acid, and the like.

Solid formulations may be formulated for immediate release (i.e., immediate release) and/or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

When the solid preparation is a tablet, any pharmaceutically acceptable excipient commonly used for preparing solid preparations can be used. Tablets may be prepared by compression or molding, optionally with one or more physiologically acceptable/pharmaceutically acceptable excipients. Compressed tablets may also be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or capsule, optionally mixed with a binder, lubricant, filler, solubilizer or disintegrant. Shaped tablets may be prepared by shaping a mixture of the moistened powdered compound and an inert liquid dispersion medium in a suitable machine. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. The formulation of the tablets is described in "Pharmaceutical Dosage Forms: tablets, vol.1 ", by H.Lieberman and L.Lachman, marcel Dekker, N.Y., 1980.

When the solid formulation is a capsule, any conventional encapsulation is suitable, for example using the carriers mentioned above in a hard gelatin capsule. When the composition is in the form of a soft gelatin capsule, any physiologically acceptable/pharmaceutically acceptable excipient commonly used to prepare dispersions or suspensions may be considered and incorporated into the soft gelatin capsule.

The pharmaceutical formulation may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy, whereby a unit dosage may be administered to a subject. Preferably, the pharmaceutical composition is in unit dosage form, e.g., a solid formulation in unit dosage form (e.g., a tablet, powder, dry suspension, granule or capsule).

The term "pharmaceutically acceptable auxiliary materials or carriers" as used herein refers to the general term for all medicinal materials except the main drug in the formulation, which is added to the formulation for the purpose of solving the problems of moldability, effectiveness, stability and safety of the formulation when the formulation is formulated. Generally, solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retarders, and the like.

According to embodiments of the present invention, the dosage forms of the pharmaceutical composition include, but are not limited to, tablets, capsules, gelatin pills, powders, granules, oral liquids or sustained and controlled release preparations.

Examples

The present invention is illustrated below by way of examples, but it should not be construed that the scope of the inventive subject matter is limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention. The compounds or reagents used in the following examples are commercially available or may be prepared by conventional methods known to those skilled in the art; the laboratory apparatus used is commercially available.

General procedure

Experimental materials

Rats: 18 SD rats, male, 7-8 weeks of age, 240-260g were housed in SPF-class animal houses under the following conditions: 12h of light and shade, 55% of humidity and 2222 ℃ of temperature, and animals can drink water and eat freely. After 7 days of environmental adaptive feeding, animal model production was started.

Cell lines: the rat myocardial H9c2 cell line was purchased from Shanghai China academy of sciences cell bank.

Culture conditions: culturing the cells in DMEM medium containing 10% high quality foetal calf serum and 1% penicillin-streptomycin double antibody, changing liquid for 1 time for 2-3d, and passaging at 80% confluence of cells at a ratio of 1:3, and culturing at 37deg.C and 5% CO ₂ Culturing in a saturated humidity incubator.

The main reagents are shown in Table 1

TABLE 1

Preparation of main reagents

PBS phosphate buffer: PBS powder with ddH added per bag ₂ O is fixed to 1000mL, pH7.2 is adjusted, the temperature is 121 ℃, the time is 30 minutes, the autoclave is sterilized, and the preservation is carried out at 4 ℃.

Cell cryopreservation solution: before use, 10% DMSO is added into the fetal calf serum, and the mixture is uniformly mixed and stored in a refrigerator at 4 ℃.

Rat myocardial H9c2 cell growth medium: before use, 10% fetal bovine serum is added according to the DMEM culture medium as required, and finally, a double-antibody stock solution (penicillin + streptomycin) is added according to 1% volume fraction, so that the final concentration of penicillin and streptomycin is 100U/mL and 100ug/mL respectively, and the obtained mixture is stored in a refrigerator at 4 ℃.

Preparing the medicine: mother liquor of each drug was prepared by adding DMSO as shown in table 2, and then diluted to the corresponding concentration according to experimental requirements. Wherein, the administration concentration of the flubendazole is 6nM.

TABLE 2

Identification of acute altitude reaction queue and protein expression profile and candidate drug screening

The inventors first established an acute altitude stress queue to collect pre-plateau and on-plateau blood samples. Meanwhile, whether there is an acute altitude reaction is evaluated on the plateau based on the easy lake score of the acute altitude reaction path. For this cohort, 20 pre-plateau and on-plateau plasma samples of 10 persons with acute altitude reactions were selected for analysis using 12 panels of antibody-based proximal extension experiments (Proximity Extension Assay) proteomics technology, protein expression profiles of 20 samples were identified, 49 differential proteins were identified based on statistical tests, including 8 downregulated proteins (CA 2, CA1, PFKM, MCP-1, RBKS, MYOC, EN-RAGE and TN-R) and 41 upregulated proteins (WFIKN 1, VSIG4, TM, TFF3, TCN2, STC1, SPOCK1, SPARC, SELE, SCGB A1, SCF, SCARA5, RET, REN, PROK1, PLA2G7, PAPA, NRP2, MMP-3, MEGF9, MATN3, LEPR, ISLR2, INHBC, IL-27, IL-18R1, GPNMB, GFRA2, GDF-15, FGF-23, FCN2, XIN, CST6, STN1, CD99, CD 6, ASGR 4, and ADA 1, and ADA 4. Based on the above-mentioned differential proteins, the inventors proposed to use, as a candidate drug, flubendazole (Flubendazole) or the like.

In the cell-based example, the following experimental groupings will be employed, respectively:

a: normal cardiomyocyte group (Ctrl)

B: hypoxia model set (Hypoxia)

C: acetazolamide + Hypoxia model group (Hypoxia + acetozolamide)

D: fluobendazole plus Hypoxia model group (Hypoxia plus Flubendazole)

In animal-based examples, the following experimental groupings will be employed, respectively:

a: normal group (Ctrl)

B: hypoxia model set (Hypoxia)

C: acetazolamide + Hypoxia model group (Hypoxia + acetozolamide)

D: fluobendazole plus Hypoxia model group (Hypoxia plus Flubendazole)

Test drug injection and animal model construction: after 1 hour of intraperitoneal injection of test drugs (DMSO as a control, acetazolamide as a control, and flubendazole as a control, rats as a B, C group and a D group were placed in a simulated plateau environment hypoxia chamber, and the mixture was raised to a simulated altitude 6500m level at a constant speed of 10m per second, and taken out of the warehouse after the end of 72 hours. The water was changed every 24 hours. Wherein, the administration concentration of the group C acetazolamide is 40mg/kg, and the papers Pichon A, connes P, quidu P, marchant D, brunet J, levy BI, vilar J, safeukui I, cymbalista F, maigin M, richanet JP, favret F.Acetazolamide and chronic hypoxia: effects on haemorheology and pulmonary haemody dynamics.Eur Respir J.2012Dec are referred to; 40 (6) 1401-9; the concentration of flubendazole in group D was 10mg/kg, as described in paper Maki J, yanagisawa T.Studies on anthelmintic effects of flubendazole and mebendazole on the rat lungworm Angiostrongylus cantonensis in mice and rates.J Parasitol.1986Aug;72 (4):512-6..

Statistical analysis

Statistical analysis was performed using either a one-sided t-test or Wilcoxon rank sum test (R language v4.2.1, stats package v 4.2.1).

EXAMPLE 1CCK8 detection of proliferation Effect of test agent on H9c2 cells

In this example, well-grown H9c2 cells were collected in the same centrifuge tube, diluted and mixed with a medium containing 1% fetal bovine serum to prepare 1.5X10 ⁴ After each ml, 200. Mu.l of cells were plated per well, i.e.3X 10 cells per well ³ Separately, the normal group and the hypoxia group are respectively paved in 2 96-well plates and placed in a incubator for culture. After 24h of incubation, the medium was aspirated, 200. Mu.L of DMEM medium containing 10% fetal bovine serum was added to the normal cardiomyocyte group (ctrl), 200. Mu.L of DMEM medium containing 10% fetal bovine serum was added to the hypoxia model group (hypoxia), and 200. Mu.L of medium containing the corresponding drug was added to the hypoxia + drug group (hypoxia + corresponding drug), respectively. Culturing in incubator for 24 hr, changing to DMEM medium containing 10% fetal calf serum, and culturing in 1%O ₂ +5％CO ₂ +94％N ₂ Incubation was performed in incubator for 1d, and a blank group was set.

After the end of the incubation, 10. Mu.L of CCK-8 solution was added to each well. The culture plate is placed in an incubator for incubation for 2-4 h. Absorbance values at 450nm were measured with a microplate reader. Cell viability of each experimental group was calculated according to the following formula, cell viability (%) = (OD experimental group-OD blank)/(OD control group-OD blank) ×100%

As shown in the figure 1, the cell activity analysis experiment based on the CCK-8 kit shows that the cell activity is reduced due to hypoxia, the acetazolamide can improve the cell activity in the hypoxia environment, the flubendazole can obviously improve the cell activity in the hypoxia environment, and the flubendazole is superior to the acetazolamide in the aspect of improving the cell activity.

Example 2 flow assay of the Effect of test Agents on H9c2 apoptosis

The procedure was substantially as in example 1, except that after the completion of the culture, each group of cell culture solutions was aspirated into 15ml centrifuge tubes, the adherent cells were washed once with PBS, and an appropriate amount of pancreatin was added to digest the cells. Incubating for 2-5min at room temperature, and removing pancreatin cell digestive juice. The collected cell culture broth was added, mixed slightly, transferred into a centrifuge tube, centrifuged at 1000g for 5min, the supernatant was discarded, the cells were collected, gently resuspended in PBS and counted. 5-10 ten thousand resuspended cells were taken in a 1.5ml EP tube, centrifuged at 1000g for 5min, the supernatant discarded, and 195. Mu.l Annexin V-FITC conjugate was added to gently resuspend the cells. Mu.l Annexin V-FITC was added and gently mixed. Incubate at room temperature for 10min in the dark. Centrifuge at 1000rpm for 5min, discard supernatant, add 190. Mu.l Annexin V-FITC conjugate and gently resuspend cells. Add 10 μl propidium iodide staining solution, mix gently, and place in ice bath in the dark. And then carrying out flow cytometry detection, wherein Annexin V-FITC is green fluorescence, and PI is red fluorescence.

The result of the early apoptosis evaluation experiment based on the flow cytometry is shown in fig. 2, and the result shows that the early apoptosis of cells is inhibited by hypoxia based on the detection of the apoptosis experiment of the flow cytometry, the acetazolamide can obviously cause the early apoptosis of the cells in the hypoxia environment, the early apoptosis proportion of the flubendazole can be obviously restored to approach to the normal myocardial group, and the flubendazole and the acetazolamide have no statistically significant difference in the aspect of early apoptosis of the cells.

The results of the flow cytometry-based late apoptosis evaluation experiment are shown in fig. 3, and the results show that the late apoptosis of cells is caused by the hypoxia condition, the late apoptosis of cells in the hypoxia environment can be obviously inhibited by the acetazolamide and the flubendazole, and the statistically significant difference between the flubendazole and the acetazolamide is avoided in the aspect of inhibiting the late apoptosis of cells.

The total apoptosis evaluation experiment result based on the flow cytometry is shown in fig. 4, and the result shows that from the aspect of total apoptosis rate, the total apoptosis of cells is obviously promoted by hypoxia, the total apoptosis rate of cells in a hypoxia environment can be obviously inhibited by the acetazolamide, the total apoptosis rate of cells in the hypoxia environment can be obviously inhibited by the flubendazole, and the flubendazole is superior to the acetazolamide in the aspect of inhibiting the total apoptosis of cells.

The results of the living cell rate evaluation experiment based on the flow cytometry are shown in fig. 5, and the results show that the living cell rate is remarkably reduced by hypoxia based on the living cell rate evaluation of the flow cytometry, the living cell rate in the hypoxia environment can be remarkably improved by the acetazolamide, the living cell rate in the hypoxia environment can be remarkably improved by the flubendazole, and in terms of the living cell rate, the flubendazole is superior to the acetazolamide.

Example 3 flow assay of the Effect of test Agents on H9c2 cell cycle

Substantially the same as in example 1, except that the cells of each group after the treatment were digested with pancreatin, centrifuged at 2000rpm for 5 minutes, the cell pellet was collected, and the supernatant was discarded; washing the cells with pre-chilled PBS 2 times, and collecting 1-5×10 cells ⁵ A cell;

cell fixation: adding into 1mL of 70% ethanol precooled in ice bath, lightly blowing and mixing, and fixing at 4 ℃ for more than 4 hours.

Centrifugation at 1000rpm for 5min precipitated cells, washing 1 with about 3mL of ice-bath pre-chilled PBS, carefully pipetting the supernatant, and about 50. Mu.L of PBS may remain to avoid pipetting away the cells. The bottom of the centrifugal tube is lightly flicked to disperse cells properly and avoid cell agglomeration.

Preparing propidium iodide staining solution: for 12 samples, 6mL staining buffer, 300. Mu.L propidium iodide staining solution (20X), 120. Mu.L RNase A (50X), final volume of 6.42mL.

Dyeing: 0.5mL of propidium iodide staining solution is added to each tube of cell sample, and cell precipitation is slowly and fully resuspended, and the cell precipitation is subjected to light-shielding warm bath at 37 ℃ for 30min. And can be stored at 4 ℃ or in an ice bath in a dark place. After the dyeing is finished, the flow detection is preferably finished within 24 hours.

And (3) flow detection: red fluorescence was detected with a flow cytometer at excitation wavelength 488nm, while light scattering was detected.

The results of the flow cytometer-based early cell synthesis analysis experiment are shown in fig. 6, and the results show that the flow cytometer-based cell cycle detection experiment shows that hypoxia significantly improves the ratio of early cell synthesis (G0/G1) cells, but that acetazolamide and flubendazole can reduce the ratio of early cell synthesis in a hypoxic environment, and that flubendazole is superior to acetazolamide in reducing the ratio of early cell synthesis.

The results of the flow cytometer-based cell synthesis phase analysis experiment are shown in fig. 7, and the results show that the flow cytometer-based cell cycle detection experiment shows that hypoxia significantly reduces the ratio of cells in the cell synthesis phase (S), but that the acetazolamide and the flubendazole can significantly improve the cell synthesis phase cell ratio in the hypoxia environment, and that the flubendazole and the acetazolamide have no statistically significant difference in improving the cell synthesis phase cell ratio.

The results of the flow cytometer based cell synthesis later analysis experiments are shown in fig. 8, and the results show that the flow cytometer based cell cycle detection experiments show that hypoxia does not significantly change the ratio of cells in the late cell synthesis (G2/M), but that acetazolamide significantly reduces the ratio of cells in the late cell synthesis in the hypoxic environment, and that flubendazole does not reduce the ratio of cells in the late cell synthesis in the hypoxic environment. In terms of cell ratio in the late stage of cell synthesis, flubendazole is superior to acetazolamide.

Example 4 evaluation of the Effect of the test agent on the Water content of brain tissue

After 72 hours of hypoxia, the rats are rapidly sacrificed by a cervical fracture method, brain tissues (including brain, midbrain and cerebellum; not including olfactory bulb) are taken, large blood stains are washed by ice salt water, water on the surface of the tissues is carefully sucked by water-absorbing paper, and the wet weight is rapidly weighed by an electronic balance (W1); baking at 55deg.C for 72 hr to constant weight, and weighing dry weight (W2).

Dry-wet ratio calculation of brain tissue: W2/W1.

The effect of the drug on the water content of brain tissue is shown in fig. 9, and the result shows that hypoxia can significantly reduce the dry-wet ratio of brain, acetazolamide can promote the reduction of the dry-wet ratio caused by hypoxia, and flubendazole can significantly promote the reduction of the dry-wet ratio caused by hypoxia, namely, the occurrence of cerebral edema is significantly prevented.

In conclusion, the invention is based on the verification of the hypoxia cell model and the hypoxia animal model, and the effect of the flubendazole on protecting hypoxia injury in the aspects of cell activity, cell proliferation, cell cycle, brain stem humidity ratio and the like is superior to that of the first-line medicine acetazolamide for preventing and treating acute altitude stress. Thus, flubendazole can be used as a medicament for preventing acute altitude stress.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.

Claims

1. Use of flubendazole or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of a altitude-response related disease, which is a variety of discomfort caused by exposure of a human body to a low-pressure hypoxic environment, selected from at least one of altitude pulmonary edema, altitude cerebral edema, acute altitude response, and altitude pulmonary arterial hypertension.

2. The use according to claim 1, wherein the medicament is suitable for use in adults or children.

3. The use according to claim 1, wherein the medicament is suitable for oral administration.

4. The use according to claim 1, wherein the pharmaceutical dosage form is selected from the group consisting of tablets, capsules, powders, granules, oral liquids and sustained release formulations.