KR20220041744A - Formulation for nasal or oral inhalation product containing nafamostat or camostat - Google Patents

Abstract

The present invention relates to an inhalable formulation comprising nafamostat, camostat or salts thereof. The inhalable formulation can be inhaled through the nose and thus is excellent for preventing or treating a primary nasal infection by a virus, is in a simple form which can easily be self-administered by a patient and can be applied to a patient with an advanced disease via a respirator. Therefore, the present invention can be utilized in technology for preventing or treating coronavirus- or influenza virus-related diseases. Also, the inhalable formulation delivers a drug through inhalation unlike an existing oral or injection administration method, and further comprises a substrate which works competitively on esterase and salts which delay elution, and thus inhibits in vivo degradation of nafamostat or camostat to maintain a good residue amount in the applied parts.

Description

Formulation for nasal or oral inhalation product containing nafamostat or camostat}

It relates to a formulation for inhalation comprising nafamostat or camostat, wherein the formulation for inhalation may be inhaled through the oral cavity or nasal cavity.

Nafamostat was synthesized in the 1990s (Nakayama et al. Synthesis and Structure-Activity Study of Protease Inhibitors. V. Chemical Modification of 6-Amidino-2-naphthyl 4-Guanidinobenzoate. Chem. Pharm. Bull. (1993) 41(1):117) It is a serine protease inhibitor and relieves symptoms of acute pancreatitis and when using an extracorporeal dialysis machine and extracorporeal membrane oxygenation (ECMO) in dialysis patients It has been approved and used in Korea and Japan as an injection for the purpose of preventing blood clotting.

On the other hand, in order for the corona virus to invade human cells, the spike protein of the virus must first bind to ACE2 (angiotensin converting enzyme 2), which is exposed on the outer surface of human cells. TMPRSS2 (transmembrane protease serine subtype 2), a serine proteolytic enzyme present on the outer surface of the cell, must operate to cut a part of the dendritic protein bound to ACE2 (M. Hoffmann et al. SARS-CoV-2 Cell Entry) Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. (2020) Cell 181:271).

In 2016, research results have already been reported that nafamostat inhibits cellular infection of the MERS virus (MERS, Middle East Respiratory Syndrome) by blocking the proteolytic action of TMPRSS2, which is essential for the corona virus to invade human cells. (M. Yamamoto et al. Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay. Antimicrob. Agents Chemother. (2016) 60(11) ):6532), in May 2020, a study result from Pasteur Institute in Korea was reported that nafamostat was the best in inhibiting lung cell infection of coronavirus among 24 drugs that are expected to have antiviral effects (M Ko et al. Comparative analysis of antiviral efficacy of FDA-approved drugs against SARS-CoV-2 in human lung cells: Nafamostat is the most potent antiviral drug candidate.BioRxiv. (2020) https://doi.org/10.1101/ 2020.05.2.090035), and the supporting effect of nafamostat was announced by a German research team (M. Hoffmann et al. Nafamostat Mesylate Blocks Activation of SARS-CoV-2: New Treatment Option for COVID-19. Antimicrob. Agents). Chemother. (2020) 64(6):1), June 2020 The effect of nafamostat in inhibiting cell invasion of coronavirus is about 10 times better than that of camostat, a similar drug The results of the study were published one after another by Japanese researchers (M. Yamamoto et al. The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner. Viruses (2020) 12(6):629). The geometric mean value of the reported IC ₅₀ of nafamostat for inhibiting COVID-19 cell invasion is 2.26 nM (=1.22 ng/mL).

Serine proteolytic enzyme TMPRSS2 is not only SARS-CoV-2, the causative virus of COVID-19, but also SRAS-CoV, the causative virus of severe acute respiratory syndrome (SARS) that was prevalent in 2003, and the Middle East, which became a problem in Korea in 2015. Several coronaviruses such as MERS-CoV of Respiratory Syndrome (MERS) and H1N1 influenza virus of the 2009 H1N1 influenza virus are being used as a means of invading human cells. Therefore, an inhalable formulation containing nafamostat or camostat can be a useful countermeasure not only for COVID-19, but also for a new coronavirus infection that may recur in the future and an influenza virus infection that may occur again in the future. In addition, since TMPRSS2, the target of nafamostat, is an enzyme of the human body, not a virus, the possibility of loss of efficacy due to mutation of an RNA virus with rapid mutation is less than that of therapeutics or vaccines that target viral proteins. there is.

Based on these research results, clinical trials are being attempted to use natamostat “injection” as a treatment for the COVID-19 virus at home and abroad.

However, since nafamostat has an ester bond in the middle of the molecular structure as shown in Formula 1 below, it is very easily degraded by carboxylesterase, which is distributed in a large amount in various human tissues. In this case, the half-life in the blood is very short, around 5 minutes, and the degradation product has no inhibitory effect on TMPRSS2 (H. Nakae et al. Pharmacokinetics of Nafamostat Mesilate During Continuous Hemodiafiltration with a Polyacrylonitrile Membrane. Ther. Apher. Dial. (2003) 7( 5):483, Y. Cao et al. A method for quantifying the unstable and highly polar drug nafamostat mesilate in human plasma with optimized solid-phase extraction and ESI-MS detection: more accurate evaluation for pharmacokinetic study. Anal. Bioanal. Chem (2008) 391:1063). In addition, both the guanidine group (pKa: about 13) and the amidan group (pKa: about 12) present on both sides of the molecule are protonated in body fluid (pH 7.4), and the positively charged band is an ionic material, so ) is difficult to pass through.

[Formula 1]

Napamostat's short half-life and difficult passage through biological membranes make it an advantage as an injection for the purpose of preventing blood coagulation in an extracorporeal dialysis machine for dialysis patients, but it is a serious disadvantage as a treatment for COVID-19. In order to block the disease progression of the Corona-19 virus, the cells in the oral cavity, nasal cavity, trachea, and lung cavity are located at the location where the virus proliferates and destroys cells by primary invasion and re-invades into other cells. Napamostat has to reach the side that comes into contact with the air, the outer surface of the cell where TMPRSS2, which is the point of action of napamostat, is present). In addition, it effectively avoids esterases present in lung tissue (D. Wang et al. Human carboxylesterases: a comprehensive review. Acta Pharm. Sin. B (2018) 8(5):699) and re- It is difficult to pass through the cell membrane and reach the outer surface of the alveolar cells (the lung cavity). Therefore, although a very good effect was shown in the cell experiment in which the chemical was mixed with the culture medium, it is difficult to reach the site of action (lung cavity) with intravenous injection therapy of the human body, and thus the desired effect cannot be obtained.

When infected with COVID-19, 20-30% of patients pass asymptomatic, and even with symptoms, most of them have only mild symptoms such as fever, cough, sputum, but some patients transition to severe pneumonia, and the fatality rate is low. about a few percent. ACE2 and TMPRSS2, which are used to invade cells of the Corona-19 virus, are human enzymes, not viral proteins, and are highly expressed on the outer surface of respiratory epithelial cells, making them a virus target (H. Xu et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int. J. Oral Sci. (2020) 12(8):1, W. Sungnak et al. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. (2020) 26:681).

In the nasal passages and organs of the human body, ciliated cells and secretory cells in which ACE2 and TMPRSS2, which are used as a means of invasion of the Corona 19 virus, are simultaneously expressed are arranged in large quantities. In the alveoli, ACE2 and TMPRSS2 are co-expressed in alveoar epithelial type II (W. Sungnak et al. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. (2020) 26:681). Therefore, the corona-19 virus that invades via droplets first infects the cells in the nasal cavity that are easily contacted, and the virus particles proliferated in the infected cells destroy the cells and spread out, passing-infecting other cells, causing the disease to progress. do. If the number of viruses is too large or the patient's immunity is pushed back in the fight against the virus in the process of passing infection along the airways, the infection progresses to the patient's alveoli and becomes a serious disease.

When the Corona-19 virus invades, 1) the symptoms similar to general upper respiratory tract infections such as cough, sputum, and fever appear first in the case of symptoms, and 2) non-Indian swab (runny nose) even when asymptomatic as well as symptomatic ) and the fact that the viral genetic material appears in the oral diaphragm (sputum), 3), and the fact that when it progresses to a severe condition over time, it leads to a sharp decrease in blood oxygen saturation, the virus first infects the upper respiratory tract, such as the nasal cavity, rather than the lungs Infection appears to pass through the trachea and spread down the lower respiratory tract through the trachea, and when it reaches the alveoli, it can be seen as rapidly progressing to severe disease. Therefore, it effectively reaches napamostat, which blocks the re-infection of the corona-19 virus, in the nasal cavity, lower respiratory tract, and the outer surface of the alveoli of mild patients who develop cold-like symptoms due to upper respiratory tract infection, and ester decomposes the reached napamostat If it can partially protect against enzymatic hydrolysis, it is judged that it will be able to effectively block the progression of the COVID-19 disease.

Meanwhile, nafamostat mesylate is dissolved in pure water or a solution containing no ionic substances such as 0.5% mannitol or dextrose solution to about 25 mg/mL. However, in 0.9% sodium chloride isotonic solution, a semi-solid reversible fine dispersion is formed, which lowers the solubility, and in a solution containing divalent and trivalent anions such as sulfate ion and phosphate ion, a precipitate is formed and the solubility is further lowered. Therefore, the dry powder inhalant containing these anions lowers solubility as partial precipitation is generated immediately upon dissolution under the influence of surrounding anions. In addition, due to the delay in dissolution, the continuity of the antiviral effect can be expected. However, the solubility of the precipitate precipitated in a solution containing anions in body fluids must be higher than the effective concentration capable of suppressing the Corona-19 virus.

The present inventors focused on this point, "Napamostat containing a non-toxic substance that can inhibit the hydrolysis of nafamostat and a salt that can lower toxicity and expect durability by delaying the dissolution of nafamostat" Through inhalation Invented a method for direct administration to the nasal cavity, trachea, and lung cavity. In the case of intravenous injection of nafamostat, it is degraded by esterases present in the body and has a very short half-life of about 5 minutes in the blood. of carboxylesterases (CES1 and CES2) in human lung. Biochemical pharmacol. (2018) 150:64) A substance that competitively binds to namostat and inhibits hydrolysis of nafamostat by an esterase enzyme and a dissolution delaying agent added When nafamostat in powder form is inhaled and administered directly to the nasal cavity, trachea, and lung cavity, it was confirmed that it exhibits a much superior effect in the treatment of COVID-19, thereby completing the present invention.

One object of the present invention is to provide a formulation for inhalation for delivery of nafamostat, camostat, or a salt thereof.

Another object of the present invention is to provide a preparation for preventing or treating a coronavirus or influenza virus-related disease comprising the preparation for inhalation.

Another object of the present invention is to provide an inhaler filled with the above formulation.

In order to achieve the above object,

The present invention provides a formulation for inhalation comprising nafamostat, camostat, or a salt thereof.

In addition, the present invention provides a preparation for preventing or treating a corona virus or influenza virus-related disease, comprising the preparation for inhalation.

In addition, the present invention provides an inhaler filled with the formulation for inhalation or the formulation.

A formulation for inhalation containing nafamostat, camostat, or a salt thereof can be inhaled through the nose, so it is possible to excellently prevent or treat a primary infection in the nasal cavity caused by a virus, and it is a simple product that the patient can administer without difficulty by himself/herself. It can be used as a ventilator for severe patients, so it can be usefully used for the prevention or treatment of coronavirus or influenza virus-related diseases. In addition, the formulation for inhalation directly delivers the drug to the site of action through inhalation, unlike the conventional oral injection administration method, and further includes a substrate that competitively acts on an esterase and a dissolution delaying agent that delays the dissolution of the drug By doing so, it excellently inhibits the decomposition of nafamostat or camostat in the body, thereby lowering the risk of toxicity, and at the same time exhibiting a lasting effect, thereby exhibiting an excellent residual amount within the site of action.

1 is a graph showing the analysis of the amount of nafamostat present in bronchoalveolar lavage fluid according to the administration method (intravenous injection, intratracheal injection) and the presence or absence of an ester degradation inhibitor.
2 is a graph showing the analysis of the amount of nafamostat present in lung tissue according to the administration method (intravenous injection, intratracheal injection) and the presence or absence of an ester degradation inhibitor.
Figure 3 is a graph showing the analysis of the concentration trend of nafamostat in the blood according to the administration method (intravenous injection, intratracheal injection) and the presence or absence of an ester degradation inhibitor.
4 is a graph showing the stability of nafamostat in bronchoalveolar lavage (BALF) according to the ratio of the ester inhibitor, lecithin, analyzed over time.
5 shows the results of scanning electron microscopy (SEM) measurement of the prepared dry powder formulations for inhalation.
6 is a graph showing the degree of reaching the powder in each stage of the respiratory tract as an RS01 inhaler and an Anderson cascade impactor by putting the prepared dry powder formulation for inhalation into a capsule.
Figure 7 is a graph showing the measurement of the nafamostat dissolution pattern of the dry powder according to the presence or absence of the ester inhibitor lecithin.
8 is a napamostat solution and dry powder not containing lecithin and dry powder containing lecithin administered to the organs of rats by intratracheal instillation or intratracheal powder injection (insufflation), The residual amount in bronchial lavage fluid and lung tissue according to time was measured and presented as a graph.

Hereinafter, the present invention will be described in detail.

The present invention provides a formulation for inhalation comprising nafamostat, camostat, or a salt thereof.

In this case, the salt may be nafamostat mesylate or camostat mesylate.

The preparation for inhalation further comprises a pharmaceutically acceptable esterase substrate, thereby inhibiting the degradation of nafamostat, Camostat or salt thereof by the esterase enzyme, thereby inhibiting the degradation of the nasal cavity and organs in the body. , it is possible to excellently increase the residual amount of nafamostat, camostat, or a salt thereof in the lung cavity and lung tissue. The substrate of the esterase means that it is degraded by the esterase competitively with the napamostat, camostat or salt thereof. A substrate of an esterase that is competitively bound to an esterase and degraded by an esterase, such as namostat, may also be used as an esterase inhibitor in the specification of the present invention.

The esterase substrate contains one or more ester bonds in the structure, so that it can be degraded by competing with each other as a substrate for nafamostat, kamostat, or a salt thereof and a carboxyesterase in the body. There is no limitation as long as it is an acceptable substance, but lecithin, phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, DPPG), glycerin fatty acid ester, poly glycerin fatty acid, polyoxyethylene glycol fatty acid ), sucrose fatty acid or sorbitan fatty acid.

In this case, the weight ratio of the napamostat, camostat, or salts thereof and the esterase substrate may be appropriately adopted and used by those skilled in the art, for example, 0.05:10 to 10:0.05, 0.05:5.0 to 5.0 :0.05, 0.05:1.0 to 1.0:0.05, 0.1:1.0 to 1.0:0.1, or 0.5:1.0 to 1.0:0.5.

The preparation for inhalation includes a salt having monovalent, divalent, or trivalent anions as pharmaceutically acceptable substances, and the dissolution of the drug is delayed by these anions, so that the nasal cavity, trachea, lung cavity, and lung tissue By lowering the instantaneous concentration in the back, it is possible to lower the possibility of toxicity and indicate the continuity of the drug effect. The salts may be used as a term for a dissolution retardant in the specification of the present invention.

The dissolution delaying agent is not limited as long as it is a pharmaceutically acceptable salt with high solubility in water, but sodium chloride (NaCl), potassium chloride (KCl), sodium nitrate (NaNO ₃ ), potassium nitrate (KNO ₃ ), sodium sulfate (Na ₂ SO ₄ ), potassium sulfate (K ₂ SO ₄ ), sodium monohydrogen phosphate (Na ₂ HPO ₄ ), potassium monohydrogen phosphate (K ₂ HPO ₄ ), sodium phosphate (NaH ₂ PO ₄ ), or potassium phosphate (KH) ₂ PO ₄ ).

At this time, the weight ratio of the napamostat, camostat or salts thereof and the dissolution delaying agent may be appropriately selected by a person skilled in the art, for example, 0.1:10 to 10:0.1, 0.1:1.0 to 1.0:0.1 or 0.5 :1.0 to 1.0:0.5.

The formulation for inhalation may further include a pharmaceutically acceptable carrier. The carrier (or filler) may be used without limitation as long as it is a pharmaceutically acceptable carrier, for example, mannitol, polyvinyl pyrrolidone, trehalose, glycine ), sorbitol, polyethylene glycol, propylene glycol, triethyl citrate, lactose, maltose, xylitol, Lactitol, leucine, methionine, starch or diethyl phthalate may be used.

The carrier may be used as an ultrafine powder having a size that can reach the alveoli with a particle size of preferably 1 to 5 μm in average diameter, or 20 to 200 μm in size and does not reach the alveoli, but does not reach the alveoli. It can be used as a fine powder for the purpose of increasing acidity.

In this case, the weight ratio of the napamostat, camostat, or salts thereof and the carrier may be appropriately adopted and used by those skilled in the art, for example, 0.05:10 to 10:0.05, 0.05:5.0 to 5.0:0.05, 0.05:1.0 to 1.0:0.05, 0.1:1.0 to 1.0:0.1, or 0.5:1.0 to 1.0:0.5.

The preparation for inhalation may be in a liquid or solid state that can be micronized to be inhaled through the nose or mouth. Preferably, it may be a solid that can be micronized so that it can be inhaled through the nose. For example, it may be an inhalation aerosol, or a powder for inhalation. In this case, when the formulation for inhalation is in a solid state, the particle size may be 0.5 to 10 μm. Preferably, it may be 1.0 to 5 μm. The solid formulation for inhalation may be prepared by a known method without limitation, for example, it may be prepared by an air jet mill, a spray dryer, or a freeze drying method. .

The formulation for inhalation may further include a pharmaceutically acceptable additive. In this case, the additive may be, for example, a diluent or a surfactant. For example, polyethylene glycol, sodium lauryl sulfate, stearic acid, magnesium stearate, etc. can be appropriately adopted by those skilled in the art as needed.

The formulation for inhalation may be delivered to the nasal cavity, trachea and lung cavity through inhalation. In this case, the inhalation may be through the nose (nasal) or the oral cavity. Preferably, it can be inhaled through the nose (nasal). On the other hand, inhalation through the nasal cavity (or used as the term 'nasal') refers to a drug intended to be deposited in the nasal cavity by spraying liquid or dry powder into aerosol particles and then reaching the trachea and lung cavity. On the other hand, oral inhalation is a formulation that administers liquid or dry powder in the form of inhalation through the mouth rather than the nose, and the target sites are the trachea, bronchi, and lung cavity. or not done The preparation for inhalation can be delivered to both the nasal cavity, the airway and the lung cavity by inhalation through the nose.

In particular, in the case of COVID-19 patients, it is important to apply all drugs to the trachea, bronchi, and lungs, including the nasal cavity, through the nose. This is because only when the drug is applied including the nasal passages, virus amplification in the nasal passages can be prevented, and passage infection leading to the lower respiratory tract can be blocked to effectively suppress the transition to severe disease. If a simple dry powder form of nasal inhalant that can be administered without difficulty by patients is commercialized, it will completely defeat COVID-19, which is driving the world into disaster, or at least dramatically reduce the power of the disease to the level of a common cold. can weaken it. On the other hand, an aerosol formulation applicable to a ventilator can also be used for severe patients who have difficulty breathing on their own.

When the formulation for inhalation is inhaled through the nasal cavity, it may reach the nasal cavity at a rate of 20 to 60%, the trachea 10 to 40%, and the lung cavity at a rate of 10 to 60%.

In addition, the present invention provides a preparation for preventing or treating a corona virus or influenza virus-related disease, comprising the preparation for inhalation.

The coronavirus-related disease is, for example, severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), or coronavirus disease 2019 (COVID-19) ) can be

The influenza virus-related disease may be, for example, avian influenza, swine influenza, novel flu, influenza, cold, sore throat, bronchitis or pneumonia.

In addition, the present invention provides an inhaler filled with the agent for inhalation or the agent for preventing or treating a virus or influenza virus-related disease.

Hereinafter, Examples and Experimental Examples of the present invention will be specifically illustrated and described below. However, the Examples and Experimental Examples to be described below are only illustrative of a part of the present invention, and are not limited thereto.

<Example 1> Preparation of Napamostat formulation for inhalation in solution form-1

In order to prepare a formulation containing nafamostat for inhalation, a sterile formulation of a solution containing napamostat mesylate 25 mg/mL and 0.5% mannitol was prepared.

<Example 2> Preparation of napamostat formulation for inhalation in solution form-2

In order to prepare a formulation containing nafamostat for inhalation, nafamostat mesylate 25 mg/mL, lecithin 25 mg/mL, and lipophilic solution containing 0.5% mannitol A sterile formulation was prepared. Mannitol plays a role of complementing osmotic pressure, and lecithin plays a role in inhibiting hydrolysis of nafamostat by competitively binding to esterases present in the respiratory lumen.

<Comparative Example 1> Preparation of nafamostat formulation for intravenous injection

In order to prepare a formulation containing nafamostat for intravenous injection, a sterile formulation of nafamostat mesylate 25 mg/mL and 0.5% mannitol containing 0.5% mannitol was prepared.

<Experimental Example 1> Analysis of napamostat abundance in the target site

In order to compare the disappearance trend in the lungs of the formulations containing nafamostat according to Examples 1, 2 and Comparative Example 1, bronchoalveolar lavage fluid (BALF) and lung tissue (lung) In order to analyze the trend of nafamostat abundance, the following experiment was conducted.

1-1. Analysis of napamostat abundance in bronchoalveolar lavage fluid (BALF)

45 9-week-old male SD-rat rats were divided into 3 groups of 15 rats, and 100 μL of the formulation according to Comparative Example 1 was administered to the first group through the tail vein of rats (2.5 mg nafamostat/rat, IV), To the second group, the formulation according to Example 1 and to the third group, 100 μL of the formulation according to Example 2 containing lecithin was administered directly to the respiratory tract by intratracheal instillation (2.5 mg nafamostat/rat). Then, after 10, 30, and 60 minutes of drug administration, 5 animals are euthanized each, and 5 mL of distilled water is injected into the lung cavity through the trachea using a syringe and retrieved 5 times to obtain bronchoalveolar lavage fluid (BALF). Then, centrifuged to precipitate the remaining cells, 100 μL of acetonitrile was added to 100 μL of the supernatant, centrifuged again to precipitate the protein, and nafamostat in the supernatant was analyzed and shown in FIG. 1 .

1-2. Analysis of napamostat abundance in lung tissue

On the other hand, considering the characteristics of napamostat that a reversible fine semi-solid dispersion is generated even when in contact with physiological saline, it is not thought that napamostat in the lung cavity will be completely extracted by washing with distilled water, and a part of it is bronchial alveolar lavage. It is presumed that it is adsorbed to the later lung tissue and remains. After bronchoalveolar lavage, the lung tissue is immediately homogenized by adding 10 mL of distilled water, then centrifuged to precipitate the solid, and 100 μL of acetonitrile is added to 100 μL of the supernatant, and then centrifuged again to precipitate the protein. The analysis of napamostat in the supernatant is shown in FIG. 2 .

As a result, after injecting the formulations according to Examples 1 and 2 into the trachea, the amount of drug present in the lung cavity (bronchial alveolar lavage) and lung tissue was administered intravenously with the formulation according to Comparative Example 1 at all time points after administration. It can be seen that it is much larger than the case of injection. In the case of intravenous injection, 30 minutes after administration, the amount of napamostat in the lung cavity and lung was lowered below the quantitation limit of the HPLC UV detector, but in the case of intratracheal injection, it was confirmed that a lot of napamostat remained. there was.

In particular, when the formulation according to Example 2 containing an ester degradation inhibitor is intratracheally injected, nafamostat of about 26 μg/rat in the bronchoalveolar lavage solution and about 36 μg/rat in lung tissue remains even at 60 minutes of administration. Therefore, it can be seen that the concentration of the drug in the lung cavity is continuously maintained compared to intratracheal injection and intravenous injection of the formulations of Examples 1 and 2 in which the residual amount was not detected at the same time period. Through this, it can be seen that the inhalation administration of nafamostat with an ester degradation inhibitor is significantly superior to other methods in order to suppress the respiratory passage infection of the Corona-19 virus.

<Experimental Example 2> Analysis of blood concentration trend of nafamostat

In order to analyze the blood concentration trend of the preparations containing nafamostat according to Examples 1, 2, and Comparative Example 1, an experiment was performed as follows.

15 9-week-old male SD-rat rats were divided into 3 groups of 5 rats, and 100 μL of the formulation according to Comparative Example 1 was administered to group 4-1 through the tail vein of rats (2.5 mg nafamostat/rat, IV). ), the formulation according to Example 1 to group 4-2, and the formulation according to Example 2 to which lecithin was added to group 4-3 by 100 μL of intratracheal injection (2.5 mg nafamostat/rat, intratracheal instillation) After administering directly to the respiratory tract, blood is collected by orbital blood sampling at 1, 5, 10, 30, and 60 minutes after administration, respectively, and centrifuged to precipitate blood cells, and then add acetonitrile to 100 μL of supernatant. After adding 100 μL and centrifuging again to precipitate the protein, napamostat in the supernatant was analyzed by HPLC and shown in FIG. 3 .

As a result, it was confirmed that the preparation according to Comparative Example 1 rapidly decreased the blood concentration of nafamostat after intravenous injection (half-life of α-phase about 5 to 6 minutes). As can be seen in FIG. 3 , in the case of intratracheal injection, in Examples 1 and 2, the blood concentration rises as it is absorbed into the blood from the respiratory tissue, and after 5 minutes of administration, it is almost the same as the blood concentration after intravenous injection. there is. Afterwards, until 60 minutes, all were maintained higher than the blood concentration after intravenous injection, so that the area under the curve (AUC) was 423±31 μg·min/mL compared to the intravenous injection, the formulation according to Example 1 was administered to the organ. In the case of intratracheal injection, it was 641±91 μg·min/mL, and when the formulation according to Example 2 was intratracheally injected, it was 656±68 μg·min/mL. It can be seen that the blood concentration AUC is about 1.5 times greater than that of intravenous injection. In the formulation according to Example 2 containing the ester degradation inhibitor, the initial blood concentration was slightly lower than the formulation according to Example 1 without the ester degradation inhibitor, but the concentration after 30 minutes was high. This is because the effective residual amount of nafamostat in the lung cavity, which is the target site of nafamostat, is higher than that of the formulation according to Example 1 due to the nafamostat degradation inhibitory effect of lecithin, an ester degradation inhibitor.

Currently, there have been no reports of nafamostat oral or inhaled, and only an injection for instillation for alleviation of acute pancreatitis and prevention of blood coagulation when using an extracorporeal dialysis machine or an extracorporeal membrane oxygenator (ECMO) is approved. Injections are also being used in an attempt to reposition the drug to the COVID-19 virus. As a currently approved clinical trial method, nafamostat is administered at a dose of 0.1 to 0.2 mg/kg/hr considering the severity of the patient. Instillation” (CRIS registration number KCT0005003). For a patient weighing 60 kg, this corresponds to administration of 12 to 24 mg for 2 hours. and highly polar drug nafamostat mesilate in human plasma with optimized solid-phase extraction and ESI-MS detection: more accurate evaluation for pharmacokinetic study. Anal. Bioanal. Chem. (2008) 391:1063), Nafamostat 10-20 When mg was instilled into a human over 2 hours, the steady-state blood concentration was measured to be about 10-35 ng/mL. However, although the blood concentrations at 30 and 60 minutes after the intravenous injection of FIG. 3 were 2.48 μg/mL and 1.52 μg/mL, which are tens to hundreds of times higher than the steady state blood concentration of the instillation in Cao's report, FIG. 1 and As shown in FIG. 2 , no drug was detected in the bronchoalveolar lavage fluid and lung tissue at 30 and 60 minutes, that is, the target site of nafamostat.

<Experimental Example 3> Ex vivo stability of nafamostat in bronchoalveolar lavage according to lecithin ratio

In order to confirm the stability and persistence of nafamostat in the trachea and lung cavity according to the content of the esterase inhibitor (lecithin), the bronchoalveolar lavage fluid (BALF) obtained by sacrificing rats (male SD, 8 weeks of age) , Napamostat alone or nafamostat: lecithin 2:1, 1:1, 1:2 ratio of mixed solution was mixed and stored at 37 degrees Celsius at 37 degrees Celsius while analyzing the residual amount of nafamostat over 3 days shown in

In the absence of lecithin, nafamostat is very quickly lost in the bronchoalveolar lavage fluid, but the higher the lecithin ratio, the higher the residual amount. It was confirmed that nafamostat could remain at a concentration several thousand times higher than that of 2.26 nM (=1.22 ng/mL), which is the Corona-19 virus inhibition IC ₅₀ value in the hyperalveolar fluid.

<Experimental Example 4> Effect of dissolution delaying agent on solubility in physiological saline

Physiological saline (0.9% sodium chloride isotonic solution) was selected as a solvent equivalent to the body fluid of the living body, and the solubility of the precipitate produced by nafamostat mesylate and a dissolution delaying agent in physiological saline was measured. Napamostat mesylate dissolves up to about 25 mg/mL in pure water or a solution that does not contain ionic substances such as 0.5% mannitol or dextrose solution, but a semi-solid reversible precipitate is formed in 0.9% sodium chloride isotonic solution. and the solubility is lowered.

When nafamostat mesylate is dissolved in physiological saline, it is stably dissolved up to a concentration of 5 mg/mL. However, when nafamostat mesylate is dissolved to a concentration of 6 to 10 mg/mL, it is completely dissolved at the beginning of shaking, but if left unattended, a semi-solid fine dispersion is formed. It shows a property of reversibly forming a fine dispersion. After repeating this process, the solution in the dissolved state was analyzed by HPLC, and it was confirmed that the semi-solid precipitate was also nafamostat and not a decomposed or changed product.

In order to check the effect of phosphate contact, 5.0 mg of nafamostat mesylate was added to 1.0 mL of 1X-PBS (phosphate buffered saline) and shaken to form a fibrous precipitate. After separating the precipitate from the supernatant by centrifugation, in order to completely remove the residual PBS and see the solubility of the precipitate in physiological saline, add 1.0 mL of physiological saline to the precipitate from which the supernatant has been removed, shake for 30 minutes, and leave for 30 minutes Then, the process of removing the supernatant by centrifugation was repeated 5 times, and then the concentration of nafamostat present in the supernatant solution 6, 7, and 8 was analyzed by HPLC, and the results are shown in Table 1 below. it was

In order to confirm the effect of contact with sulfate, 5 mg of nafamostat mesylate was added to 1.0 mL of a 100 mM sodium sulfate (Na ₂ SO ₄ ) solution and shaken, and a crystalline precipitate was formed. After separating the precipitate from the supernatant by centrifugation, in order to completely remove the residual PBS and see the solubility of the precipitate in physiological saline, add 1.0 mL of physiological saline to the precipitate from which the supernatant has been removed, shake for 30 minutes, and leave for 30 minutes Then, the process of removing the supernatant by centrifugation was repeated 5 times, and the concentration of nafamostat present in the supernatant solution 6, 7, and 8 was analyzed by HPLC, and the results are shown in Table 1 below. it was

treatment method Solubility in physiological saline Napamostat mesylate 3.22 mg/mL (9.3 mM) Precipitation fished by PBS contact 188 ± 38 μg/mL (541 ± 37 μM) Precipitation produced by sodium sulfate contact 87 ± 13 μg/mL (251 ± 37 μM)

Compared to the solubility of nafamostat in physiological saline, the solubility of the precipitate generated by the phosphate contact was reduced to about 1/20, and the solubility of the precipitate generated by the sulfate contact was reduced to about 1/40. It was confirmed that the concentration is more than 40,000 times higher than the effective concentration (ED50) of 2.2 nM for the Corona-19 virus of START, and when nafamostat mesylate is in contact with phosphate or sulfate, a reversible precipitation is generated and the dissolution is delayed. It was confirmed that there is

<Example 3> Preparation of formulation for inhalation in dry powder form

A solution of napamostat, an ester degradation inhibitor (lecithin) and a carrier (mannitol) was prepared according to Example 2, and nafamostat: mannitol (1: 100), nafamostat: lecithin: mannitol using a spray dryer A fine dry powder for inhalation was prepared in a ratio of (1:1:100), nafamostat:lecithin:mannitol (1:1:50), and nafamostat:lecithin:mannitol (1:1:10). Ratios in parentheses are by weight. A photograph of the prepared sample observed with a scanning electron microscope is shown in FIG. 5 .

<Example 4> Preparation of capsules filled with dry powder containing napamostat

The four types of dry powders prepared in Example 3 were filled in capsules to prepare capsules filled with dry powders containing napamostat.

<Experimental Example 5> Aerodynamic evaluation of formulations for inhalation

The sample powder prepared according to Example 3 was filled into capsules, and aerodynamic inhalation behavior was evaluated using RS01 inhaler and Anderson cascade impactor (ACI), and the ratio collected at each stage of ACI was presented in FIG. 6 .

Emitted dose (ED) in the capsule of each sample measured by ACI, fine particle fraction (FPF), mass median aerodynamic diameter (MMAD) , geometric standard deviation (GSD) is shown in Table 2 below.

Napamostat
:Mannitol
(1:100)
dry powder Napamostat
: Lecithin : Mannitol
(1:1:100)
dry powder Napamostat
: Lecithin : Mannitol
(1:1:50)
dry powder Napamostat
: Lecithin : Mannitol
(1:1:10)
dry powder Discharge rate (ED, %) 93.7±2.0 98.6±0.8 99.7±0.4 100.0±0.0 Ultrafine powder fraction (FPF, %) 75.1±5.3 50.8±3.5 24.4±1.9 19.9±0.6 Mass Median Aerodynamic Diameter
(MMAD, μm) 2.1±0.6 3.5±0.3 6.8±0.8 6.4±0.7 geometric standard deviation
(GSD) 2.6±0.3 3.0±0.2 4.2±0.3 3.9±0.3

As the content of mannitol as a carrier increases, the mass median aerodynamic diameter (MMAD) decreases, the ratio of ultrafine powder (FPF) increases, and at the same time, the distribution rate of the lower stage evaluated by ACI increases. This superiority was confirmed. Meanwhile, the scanning electron microscope photograph of FIG. 5 also shows that the higher the ratio of mannitol, the more clean spherical particles are formed. When lecithin is included, it can be seen that the composition of nafamostat:lecithin:mannitol (1:1:100) is suitable for the discharge rate and aerodynamic behavior required for this inhalant and is an excellent composition.

<Experimental Example 6> In vitro dissolution evaluation of inhalation formulations, in vivo respiratory residual evaluation

Among the samples prepared in Example 3, nafamostat: lecithin: mannitol (1:1: 100) having an excellent appearance and showing excellent aerodynamic behavior in Experimental Example 5 was selected and lecithin-free nafamostat: mannitol (1) :100) The in vitro dissolution rate with dry powder was compared and presented in FIG. 7 .

The dissolution test used the Franz diffusion cell system. A cellulose membrane filter (0.45 μm) was attached to a receiver containing 12 mL of distilled water maintaining a sink condition of 37 ± 1 °C, and 1 mg of sample was applied to the membrane. After spreading evenly on the top, 200 μL of the solution in the receptor was taken for each hour, and the content of nafamostat was analyzed by HPLC. Referring to FIG. 7 , it can be seen that the formulation additionally containing a substrate for an esterase such as lecithin has a slower dissolution rate than the formulation not containing the substrate, which is advantageous in terms of drug efficacy.

<Experimental Example 7> In vitro dissolution evaluation of inhalation formulations, in vivo respiratory residual evaluation

In order to compare and confirm the behavior of the dry powder sample prepared in Example 3 in the organs of rats, the nafamostat solution of Example 1 was administered by intratracheal instillation (2.5 mg nafamostat/rat). :Mannitol (1:100) dry powder and nafamostat:lecithin:mannitol (1:1:100) dry powder were administered to rats by intratracheal insufflation (2.5mg nafamostat/rat) after administration to rats, Fig. 8 shows the residual amount of napamostat up to 60 minutes by collecting bronchial lavage (BALF) and lung tissue from sequentially euthanized rats.

According to FIG. 8 , it can be seen that the dry powder additionally containing a substrate for an esterase, such as lecithin, has an effective residual amount over time compared to a formulation not containing a substrate, which is advantageous in continuity of drug efficacy.

As mentioned above, although the present invention has been described in detail through preferred examples and experimental examples, the scope of the present invention is not limited to specific examples and should be construed according to the appended claims. In addition, those skilled in the art will understand that many modifications and variations are possible without departing from the scope of the present invention.

Claims (21)

A formulation for inhalation comprising nafamostat, Camostat or a salt thereof.
According to claim 1,
A formulation for inhalation, characterized in that it further comprises a pharmaceutically acceptable esterase substrate.
3. The method of claim 2,
The substrate of the ester-degrading enzyme is characterized in that it is degraded by the ester-degrading enzyme competitively with the napamostat, camostat or salt thereof, the formulation for inhalation.
3. The method of claim 2,
The substrate of the esterase is,
Lecithin, phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (1,2-dipalmitoyl-sn) -glycero-3-phosphoglycerol, DPPG), glycerin fatty acid ester, poly glycerin fatty acid, polyoxyethylene glycol fatty acid, sucrose fatty acid ester A formulation for inhalation, characterized in that at least one selected from the group consisting of fatty acid) and sorbitan fatty acid.
According to claim 1,
The formulation for inhalation further comprises a pharmaceutically acceptable dissolution delaying agent, wherein the dissolution delaying agent is a salt having a monovalent, divalent, or trivalent anion.
6. The method of claim 5,
The dissolution retardant is sodium chloride (NaCl), potassium chloride (KCl), sodium nitrate (NaNO ₃ ), potassium nitrate (KNO ₃ ), sodium sulfate (Na ₂ SO ₄ ), potassium sulfate (K ₂ SO ₄ ), sodium monohydrogen phosphate (Na ₂ HPO ₄ ), potassium monohydrogen phosphate (K ₂ HPO ₄ ), sodium phosphate (NaH ₂ PO ₄ ), and potassium phosphate (KH ₂ PO ₄ ) Inhalation, characterized in that at least one selected from the group consisting of For formulations.
6. The method of claim 5,
A formulation for inhalation, characterized in that the weight ratio of nafamostat, camostat or a salt thereof and the dissolution delaying agent is 0.1:10 to 10:0.1.
According to claim 1,
The formulation for inhalation, characterized in that it further comprises a pharmaceutically acceptable carrier.
9. The method of claim 8,
The carrier is mannitol, polyvinyl pyrrolidone, trehalose, glycine, sorbitol, polyethylene glycol, propylene glycol , triethyl citrate, lactose, maltose, xylitol, lactitol, leucine, methionine, starch, and diethyl A formulation for inhalation, characterized in that at least one selected from the group consisting of phthalate (diethyl phthalate).
According to claim 1,
The formulation for inhalation, characterized in that in a liquid or solid form that can be micronized to be inhalable, the formulation for inhalation.
11. The method of claim 10,
The solid formulation for inhalation, characterized in that the particle size of 0.5 to 10 μm, formulation for inhalation.
11. The method of claim 10,
The solid formulation for inhalation, characterized in that it is prepared through an air jet mill, a spray dryer, or freeze drying (freeze dry), the formulation for inhalation.
According to claim 1,
The inhalation formulation further comprises a pharmaceutically acceptable additive.
According to claim 1,
The formulation for inhalation, characterized in that delivered to the nasal cavity, airway or lung cavity through inhalation, formulation for inhalation.
According to claim 1,
The salt is nafamostat mesylate or camostat mesylate, characterized in that the formulation for inhalation.
9. The method of claim 8,
A formulation for inhalation, characterized in that the weight ratio of nafamostat, camostat or a salt thereof and the carrier is 0.05:10 to 10:0.05.
3. The method of claim 2,
A formulation for inhalation, characterized in that the weight ratio of nafamostat, camostat or a salt thereof to the substrate of the esterase is 0.05:10 to 10:0.05.
A preparation for the prevention or treatment of coronavirus or influenza virus-related diseases, comprising the preparation for inhalation of claim 1.
19. The method of claim 18,
The corona virus-related disease is selected from the group consisting of severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS) and coronavirus infection-19 (coronavirus deisease 2019, COVID-19) A formulation characterized in that it is at least one type.
19. The method of claim 18,
The influenza virus-related disease is characterized in that at least one selected from the group consisting of avian influenza, swine influenza, novel flu, flu, cold, sore throat, bronchitis and pneumonia, formulation.
An inhaler filled with the formulation of claim 1 or 18.

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