Application of PSFL-1411 in preparation of medicine for treating refractory chronic cough

Abstract

The invention discloses an application of PSFL-1411 in preparation of a medicine for treating intractable chronic cough. In the application, a new application of the PSFL-1411 is provided, and the PSFL-1411 can be used as a high-specificity and high-selectivity P2X3 inhibitor and can be used as an effective new medicine for treating refractory chronic cough; when the PSFL-1411 provided by the invention is used for treating refractory chronic cough, compared with the existing medicine AF-219, the PSFL-1411 shows a better cough relieving effect, the cough frequency can be reduced by at least 70%, and the side effect of taste reaction disorder or loss can be avoided.

Description

Technical Field

[0001] The embodiments of the present invention relate to biomedicine, and in particular to the use of PSFL-1411 in preparing a medicine for treating refractory chronic cough. Background Art

[0002] Coughs can be divided into three types according to their duration: acute (lasting less than 3 weeks), subacute (lasting 3 to 8 weeks) and chronic (lasting more than 8 weeks). Among them, the causes of acute and subacute coughs are relatively clear. Generally, on the basis of treating the primary disease, appropriate antitussive drugs are selected to achieve better clinical results. Among chronic coughs, some patients cannot be cured of their cough symptoms despite diagnosis and treatment using the recognized cough diagnosis and treatment guidelines. This situation is called persistent or unexplained chronic cough. Because these patients have received long-term special treatments without success, it is also called refractory chronic cough.

[0003] Although patients with refractory chronic cough are diagnosed and treated according to the recognized cough diagnosis and treatment guidelines, their symptoms are still difficult to be effectively relieved. Long-term persistent cough can lead to complications such as lack of sleep, headache, hoarseness, fractures, as well as social and psychological complications such as anxiety, anger, difficulty speaking, and social exclusion. It can be seen that refractory chronic cough seriously affects human health and quality of life.

[0004] There are no existing cough suppressants specifically for refractory chronic cough, and these drugs have very limited effects on relieving the symptoms of refractory chronic cough. In the treatment of cough, the first thing is to find the cause of the cough as much as possible and perform specific symptomatic treatment. The non-specific cough suppressant dextromethorphan can relieve chronic cough, but due to its central opioid receptor mechanism of action, it has certain side effects and huge risks. Therefore, refractory chronic cough has a large number of patients and a lack of treatment strategies. The development of new drugs for the treatment of refractory chronic cough can meet the clinical needs of the majority of patients.

[0005] Purinergic P2X receptors are ATP-gated non-selective cation channels, widely expressed in the nervous, immune, respiratory, cardiovascular, skeletal, gastrointestinal and endocrine systems; P2X3 is one of the P2X family subtypes, which is distributed in large quantities in dorsal root ganglia, spinal cord and brain neurons in the form of homotrimers (P2X3) and heterotrimers (P2X2 / 3). P2X3 receptors are believed to be associated with refractory chronic cough. Clinical studies have shown that overexpression of P2X3 receptors in airway vagal afferent nerves triggers sensory neuron hypersensitivity, which in turn leads to chronic cough. Elevated respiratory ATP levels under pathological conditions aggravate this phenomenon. The clinical results of the P2X3 receptor inhibitor AF-219 showed that AF-219 significantly reduced the cough frequency of patients with refractory chronic cough, becoming the first new target drug for the treatment of refractory chronic cough that has passed conceptual verification. P2X3 receptors are also the only new target for the treatment of refractory chronic cough that has passed conceptual verification in more than 60 years.

[0006] Although AF-219 has a good therapeutic effect on refractory chronic cough, the compound has clear adverse reactions. AF-219 can reduce the frequency of coughing in patients by 50% to 75%, but at the same time, 58.0% and 68.6% of the 45mg BID (twice a day) dose groups experienced taste-related adverse drug events; higher doses, such as 200 to 600mg BID, will cause about 100% of patients to have varying degrees of taste disorders. This side effect greatly limits the clinical application of AF-219. Therefore, the field urgently needs to develop drugs with low side effects for the treatment of refractory chronic cough. Summary of the invention

[0007] The purpose of the present invention is to provide a new use of PSFL-1411.

[0008] Another object of the present invention is to provide a method for treating refractory chronic cough.

[0009] In order to solve the above technical problems, the first aspect of the present invention provides the use of PSFL-1411, wherein the structure of PSFL-1411 is shown in the following formula I:

[0010]

[0011] The use is selected from at least one of the following:

[0012] (i) as a P2X3 receptor inhibitor;

[0013] (ii) non-therapeutic inhibition of P2X3 receptor activation in vitro;

[0014] (iii) preparing drugs for preventing and / or treating diseases associated with P2X3 receptor activation; and

[0015] (iv) preventing and / or treating diseases related to excessive activation of P2X3 receptors.

[0016] In some preferred embodiments, the disease associated with P2X3 receptor activation is refractory chronic cough.

[0017] In some preferred embodiments, the use is selected from at least one of the following:

[0018] (i) as a P2X3 receptor inhibitor;

[0019] (ii) non-therapeutic inhibition of P2X3 receptor activation in vitro;

[0020] (iii) preparing a medicament for preventing and / or treating refractory chronic cough; and

[0021] (iv) Prevention and / or treatment of refractory chronic cough.

[0022] In some preferred embodiments, the inhibition is selective inhibition.

[0023] In some preferred embodiments, the PSFL-1411 inhibits the activation of the hP2X3 receptor, and preferably, the PSFL-1411 selectively inhibits the activation of the hP2X3 receptor.

[0024] In some preferred embodiments, the PSFL-1411 selectively inhibits the activation of the hP2X3 receptor without affecting the activation of other subtypes of the P2X receptor family.

[0025] In some preferred embodiments, the PSFL-1411 selectively inhibits the activation of the hP2X3 receptor without affecting the activation of hP2X1, hP2X2, hP2X4, hP2X5, and hP2X7.

[0026] In some preferred embodiments, the PSFL-1411 reduces the response level of the hP2X3 receptor to ATP activation by at least 10 times, more preferably at least 20 times, more preferably at least 30 times, more preferably at least 40 times, more preferably at least 50 times, more preferably at least 60 times, more preferably at least 70 times, more preferably at least 80 times, more preferably at least 90 times, more preferably at least 100 times.

[0027] In some preferred embodiments, the PSFL-1411 reduces the cough frequency of patients with refractory chronic cough by at least 70%, more preferably at least 80%, more preferably at least 85%, and more preferably at least 90%.

[0028] In some preferred embodiments, the PSFL-1411 prevents and / or treats refractory chronic cough by inhibiting the activation of P2X3 receptors.

[0029] The second aspect of the present invention provides a method for preventing and / or treating a disease associated with excessive activation of P2X3 (preferably refractory chronic cough), the method comprising the steps of:

[0030] A therapeutically effective amount of PSFL-1411 is administered to the subject.

[0031] In some preferred embodiments, the therapeutically effective amount is not less than 0.1 mg / kg, preferably not less than 1 mg / kg, and more preferably not less than 5 mg / kg.

[0032] In some preferred embodiments, the therapeutically effective amount is 1-200 mg / kg, such as 15 mg / kg.

[0033] In some preferred embodiments, the administration frequency of PSFL-1411 is 1-3 times a day, for example, twice a day.

[0034] In some preferred embodiments, the PSFL-1411 is administered by intragastric administration.

[0035] The third aspect of the present invention provides a method for non-therapeutic inhibition of P2X3 receptor activation in cells expressing P2X3 receptors in vitro, the method comprising the steps of:

[0036] The cells expressing the P2X3 receptor are cultured in a culture system containing PSFL-1411.

[0037] Compared with the prior art, the present invention has at least the following advantages:

[0038] (1) The present invention provides a new use of PSFL-1411 as a highly specific and selective P2X3 receptor inhibitor, and can be used as an effective new drug for treating refractory chronic cough;

[0039] (2) When used to treat refractory chronic cough, PSFL-1411 provided by the present invention has a better antitussive effect than the existing drug AF-219, can reduce the cough frequency by at least 70%, and can avoid the side effects of taste response disorder or loss;

[0040] (3) The PSFL-1411 provided by the present invention can be used as a highly selective P2X3 inhibitor. At a concentration of 10 μM, PSFL-1411 selectively and almost completely inhibits the hP2X3 receptor in the P2X receptor family subtype, and has no inhibitory effect on the hP2X1, hP2X2, hP2X4, hP2X5, and hP2X7 receptors. The selectivity for the hP2X2 / 3 receptor is more than 100 times.

[0041] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0043] Figure 1A This is a diagram of an animal experiment model for ammonia-induced coughing according to an embodiment of the present invention;

[0044] Figure 1B This is a statistical graph of the ammonia-induced cough animal experiment according to the embodiment of the present invention, the ordinate represents the cough frequency, the abscissa represents the intragastric injection of CMC-Na, 15 mg / kg AF-219, and 15 mg / kg PSFL-1411, bid represents twice a day (the value is mean ± standard deviation, n>10), and the Bonferroni test of one-way ANOVA is performed with CMC-Na. *** represents P < 0.001, **** represents P < 0.0001;

[0045] Figure 2A This is a diagram of an animal experiment on the taste preference of the sweetener acesulfame potassium according to an embodiment of the present invention;

[0046] Figure 2B This is a drinking water statistics chart of the taste preference animal experiment of the sweetener acesulfame potassium according to the embodiment of the present invention. The ordinate represents the proportion of drinking acesulfame potassium, and the abscissa represents the intragastric injection of CMC-Na, 5 mg / kg AF-219, and 15 mg / kg PSFL-1411 (values ​​are mean ± standard deviation, n=8-10), bid means twice a day, and a one-way ANOVA Bonferroni test is performed with CMC-Na. * represents P < 0.05, ns represents no difference. ;

[0047] Figure 2C This is a diagram of an animal experiment on the taste preference of bitter agent quinine according to an embodiment of the present invention;

[0048] Figure 2DThis is a drinking water statistics chart of the bitter agent quinine taste preference animal experiment according to the embodiment of the present invention. The ordinate represents the proportion of drinking quinine, and the abscissa represents the intragastric injection of CMC-Na, 5 mg / kg AF-219, and 15 mg / kg PSFL-1411 (values ​​are mean ± standard deviation, n=8-10), bid means twice a day, and a one-way ANOVA Bonferroni test is performed with CMC-Na. * represents P < 0.05, ns represents no difference;

[0049] Figure 3A This is a representative current diagram of the inhibitory effect of PSFL-1411 on ATP current on various subtypes of hP2X receptors according to an embodiment of the present invention;

[0050] Figure 3B is a fitting diagram of the inhibitory effect of PSFL-1411 on each subtype according to the embodiment of the present invention (normalized current is I0 / I control , values ​​are mean ± standard deviation, n ≥ 3, fitted using Hill1 equation), where the IC of PSFL-1411 on hP2X3 receptor 50 The value was 1.28±0.17μM. DETAILED DESCRIPTION

[0051] The inventors found in the study that AF-219 has poor specificity and selectivity, and is effective not only for P2X3 homotrimers associated with refractory chronic cough, but also for P2X2 / 3 heterotrimers associated with taste signal transduction. This may be the reason for its large side effects when used to treat refractory chronic cough. In view of this, the inventors found that PSFL-1411 can be used as a highly specific and highly selective P2X3 receptor inhibitor, selectively inhibiting hP2X3 receptors, while having no effect on other subtypes of the P2X receptor family. Animal experiments have shown that PSFL-1411 can more significantly inhibit the cough frequency of patients with refractory chronic cough and has no effect on taste.

[0052] Compound

[0053] The compound "PSFL-1411" used in the present invention has a structure as shown in the following formula I, has a CAS number of 421580-64-5, and can be obtained through commercial channels.

[0054]

[0055] Uses of Compounds

[0056] The present invention relates to the use of PSFL-1411 for: (i) as a P2X3 receptor inhibitor; (ii) non-therapeutic inhibition of P2X3 receptor activation in vitro; (iii) preparation of drugs for preventing and / or treating diseases related to P2X3 receptor activation; and / or (iv) prevention and / or treatment of diseases related to excessive activation of P2X3 receptors.

[0057] The PSFL-1411 of the present invention is used as a P2X3 receptor inhibitor with high specificity and selectivity. PSFL-1411 can inhibit the activation of the hP2X3 receptor, preferably selectively inhibit the activation of the hP2X3 receptor. Selectively inhibiting the activation of the hP2X3 receptor means that PSFL-1411 selectively activates the hP2X3 receptor without affecting the activation of other subtypes of the P2X receptor family, which include but are not limited to hP2X1, hP2X2, hP2X4, hP2X5, and hP2X7. It can be considered that the administration of PSFL-1411 has no promoting or inhibiting effect on the activation of other subtypes of the P2X receptor family.

[0058] In some embodiments of the present invention, PSFL-1411 is used as a P2X3 receptor inhibitor to reduce the response level of hP2X3 to ATP activation by at least 10 times, more preferably at least 20 times, more preferably at least 30 times, more preferably at least 40 times, more preferably at least 50 times, more preferably at least 60 times, more preferably at least 70 times, more preferably at least 80 times, more preferably at least 90 times, more preferably at least 100 times. In other embodiments, compared with the activation of other subtypes of the P2X receptor family, PSFL-1411 reduces the response level of hP2X3 receptor to ATP activation by at least 10 times, more preferably at least 20 times, more preferably at least 30 times, more preferably at least 40 times, more preferably at least 50 times, more preferably at least 60 times, more preferably at least 70 times, more preferably at least 80 times, more preferably at least 90 times, more preferably at least 100 times. In a specific embodiment, the selectivity of PSFL-1411 for hP2X2 / 3 receptor is not less than 100 times. In certain embodiments, PSFL-1411 has an inhibitory effect on hP2X2 / 3 receptors that is no less than 100-fold greater than that on other subtypes of the P2X receptor family.

[0059] As used herein, the term "P2X receptor" refers to purinergic P2X receptors, which are ATP-gated non-selective cation channels, widely expressed in the nervous, immune, respiratory, cardiovascular, skeletal, gastrointestinal and endocrine systems, and involved in a variety of physiological and pathological processes, such as synaptic transmission, immune response, blood pressure regulation and inflammatory response, etc. The term "P2X3 receptor" is one of the subtypes of the P2X receptor family, which is distributed in large quantities in dorsal root ganglia, spinal cord and brain neurons in the form of homotrimers (P2X3) and heterotrimers (P2X2 / 3).

[0060] In the present invention, PSFL-1411 can theoretically prevent and / or treat any disease associated with P2X3 receptor activation, such as cardiovascular disease, diabetic pathological pain, neuropathic pain, epilepsy, urinary incontinence, Parkinson's disease, inflammatory pain, cancer pain, visceral pain or refractory chronic cough. In a preferred embodiment of the present invention, the disease associated with P2X3 receptor activation is refractory chronic cough.

[0061] In some embodiments of the present invention, PSFL-1411 reduces the cough frequency of patients with refractory chronic cough by at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%.

[0062] As used herein, the term "refractory chronic cough" refers to a chronic cough with a duration of cough > 8 weeks, and the cause of which remains unknown after recommended standard examinations and treatments; or a chronic cough that cannot be relieved after empirical treatment of known causes of chronic cough; or a chronic cough with partial examination evidence of the cause of chronic cough, but poor treatment effect, and persistent cough. All conditions that meet the definition of refractory chronic cough in China's "Guidelines for the Diagnosis and Treatment of Cough (2015)" should fall within the scope of the indications of the compounds of the present invention.

[0063] As used herein, the term "inhibitor" refers to a substance that blocks or reduces the rate of a biochemical reaction, and its blocking or inhibitory effect is produced by at least one of the following: 1) irreversible inhibition, which refers to an inhibitory effect that completely destroys the structure and function of the receptor; 2) reversible inhibition, which refers to an inhibitory effect in which the structure or function of the receptor can be restored after the inhibitor is removed; 3) competitive inhibition, which refers to the inhibitor having a similar structure to a biological molecule that binds to the receptor in the pathway, and therefore competitively binds to the receptor to produce an inhibitory effect; 4) non-competitive inhibition, which refers to the inhibitor and a biological molecule that binds to the receptor in the pathway acting on different sites of the receptor respectively, causing receptor inactivation and producing an inhibitory effect; 5) anti-competitive inhibition, which refers to the inhibitor producing an inhibitory effect by binding to a complex formed by the receptor and other biological molecules.

[0064] As used herein, the term "activation" of P2X3 receptors refers to the fact that P2X3 receptors act as non-selective ligand-gated cation channels that are activated by ATP and open to allow Na + , K + and Ca 2+ The "overactivation" of P2X3 receptors refers to the large amount of ATP released under pathological conditions such as damage or inflammation, which causes the P2X3 receptors on neurons to be rapidly activated, causing a large amount of cations to pass through the ion channels, keeping the current in the ion channels high, thereby causing sensory neuron hypersensitivity, etc.

[0065] Treatment

[0066] The present invention also relates to a method for preventing and / or treating refractory chronic cough, comprising the step of administering a therapeutically effective amount of PSFL-1411 to a subject.

[0067] As used herein, the term "subject" is defined herein to include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a specific embodiment, the subject is a human.

[0068] As used herein, the term "prevention" refers to preventing the onset, recurrence or spread of a disease or condition, or one or more symptoms associated with the disease or condition. In one embodiment, such symptoms are known to those skilled in the art to be associated with the disease or condition to be prevented. In a specific embodiment, the term refers to administering the compounds provided herein with or without other additional active agents prior to the onset of symptoms to a patient at risk of developing a disease or disorder described herein. The term encompasses the inhibition and reduction of symptoms of a particular disease.

[0069] As used herein, the term "treat" refers to eradicating or ameliorating a disease or condition, or one or more symptoms associated with the disease or condition. In one embodiment, such symptoms are known to those skilled in the art to be associated with the disease or condition to be treated. In specific embodiments, the term refers to minimizing the spread or worsening of a disease or condition by administering one or more preventive or therapeutic agents to a subject suffering from the disease or condition. In some embodiments, the term refers to administering a compound of the invention after the onset of symptoms of a particular disease, with or without other additional active agents.

[0070] As used herein, a "therapeutically effective amount" of a compound refers to the amount of the compound sufficient to provide a therapeutic effect in the treatment or control of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with the disease or disorder. The therapeutically effective amount of a compound refers to the amount of a therapeutic agent that can provide a therapeutic effect in the treatment or control of a disease or disorder when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. In some embodiments of the invention, the therapeutically effective amount is not less than 0.1 mg / kg, more preferably not less than 1 mg / kg. In some embodiments of the invention, the therapeutically effective amount is 1-200 mg / kg. In some embodiments, the therapeutically effective amount is 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, or 200 mg / kg.

[0071] As used herein, the "dosing frequency", "application frequency" and "dosing frequency" of a compound are used interchangeably and refer to the frequency and number of times a drug is used over a period of time. Those skilled in the art can increase or decrease the frequency of administration, increase or decrease the number of administrations according to the actual therapeutic effect of a particular subject during a certain treatment period. In some embodiments of the present invention, the administration frequency of the compound is once a day, twice a day, three times a day, once every two days, once every three days, once a week, once every two weeks, or once a month.

[0072] As used herein, the "administration method" and "administration route" of a compound are used interchangeably and refer to the method and route by which a compound enters the human body. The administration method may be, without limitation, oral administration, intravenous injection (intravenous injection), intramuscular injection (intramuscular injection), subcutaneous injection (subcutaneous injection), sublingual administration (sublingual), rectal irrigation (rectal administration), eye drops, nasal spray, oral spray (inhalant), topical or systemic administration through the skin. In a preferred embodiment of the present invention, the administration method of the compound is oral administration or intravenous injection.

[0073] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0074] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0075] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0076] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0077] Example 1

[0078] In this example, an ammonia-induced cough model was established.

[0079] Male C57BL / 6 mice (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., 20-24 g, 2 months old) were raised in this laboratory for 3-7 days to adapt to the experimental environment. They were kept in experimental mouse cages with independent ventilation systems at a temperature of about 23°C and had free access to water and food.

[0080] During the experiment, the mice were placed in a 1L beaker for 7-8 minutes, then sealed with plastic wrap, and then 1% ammonia water was sprayed into the beaker with an ultrasonic nebulizer. The maximum spray intensity lasted for 10s, and the beaker mouth was pressed with a lid, and then the cough latency within 5min (recording the start to the first cough) and the number of coughs within 5min were recorded, and the coughing of the mice was recorded with a video. Mice with a coughing frequency of 10-30 times were screened out, and then randomly divided into 3 groups, and administered for 7 days respectively. The positive control group was gavaged with AF-219 (suspended in 0.5% CMC-Na solution) at 15mg / kg (twice a day), the PSFL-1411 group was gavaged with PSFL-1411 (suspended in 0.5% CMC-Na solution) at 15mg / kg (twice a day), and the model group was gavaged with an equal volume of 0.5% CMC-Na solution. The changes in the number of coughs before and after administration were compared under the same experimental conditions.

[0081] Figure 2 shows the antitussive effect of PSFL-1411. The experimental results show that AF-219 (positive control, 15 mg / kg, twice a day) and PSFL1411 (15 mg / kg, twice a day) can significantly reduce the number of coughs in mice, with AF-219 reducing the cough frequency by 55.97% and PSFL1411 reducing the cough frequency by 77.22%.

[0082] Example 2

[0083] In this example, a double-bottle experiment was used to determine the effects of different doses of drugs on taste perception of male C57BL / 6 mice (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., 20-24 g, 2 months old).

[0084] (1) Evaluating the taste disturbance side effects of PSFL-1411 through sweetener experiments

[0085] The animals were randomly divided into 3 groups, each with ≥10 mice: blank group (water and acesulfame solution were used as drinking conditions after saline administration), PSFL-1411 group (water and acesulfame solution were used as drinking conditions after mouse drug administration), and positive control group (water and acesulfame solution were used as drinking conditions after AF-219 administration). One mouse per cage was housed in ventilated cages with a 12 / 12 hour light / dark cycle, fed with standard feed, two bottles of drinking water, and adapted for 5 days (23±2℃, relative humidity 50-60%). After the mice were adapted, they were given drugs, and then one of the two bottles of drinking water was replaced with acesulfame solution. The positions of the two bottles were also swapped every 24 hours. After 5 days of continuous administration (BID dose), the consumption mass of each bottle was measured.

[0086] (2) Evaluation of the taste disturbance side effects of PSFL-1411 through bittering agent experiments

[0087] The animals were randomly divided into 3 groups, each with ≥10 mice: blank group (water and quinine solution were used as drinking conditions after saline administration), PSFL-1411 group (water and quinine solution were used as drinking conditions after mouse drug administration), and positive control group (water and quinine solution were used as drinking conditions after AF-219 administration). One mouse per cage was housed in ventilated cages with a 12 / 12 hour light / dark cycle, fed with standard feed, double bottles of drinking water, and adapted for 5 days (23±2℃, relative humidity 50-60%). After the mice were adapted, they were given drugs, and then one of the double bottles of drinking water was replaced with quinine solution. The positions of the two bottles were also swapped every 24 hours. After 5 days of continuous administration (BID dose), the consumption mass of each bottle was measured.

[0088] Figure 3AThis is the effect of PSFL-1411 on the taste of experimental animals. As can be seen from the figure: compared with the blank group of mice, AF-219 significantly reduced the amount of sweetener acesulfame potassium solution that mice drank and significantly increased the amount of bitter agent quinine solution that mice drank. At a dose of 5 mg / kg, obvious side effects of taste disorders appeared, indicating that the taste of mice was damaged and they could not normally distinguish between sweet and bitter tastes. However, PSFL-1411 did not change the amount of sweetener acesulfame potassium solution and bitter agent quinine solution that mice drank at a dose of 15 mg / kg, indicating that the taste of mice was not damaged and they could still distinguish tastes normally.

[0089] Example 3

[0090] In this example, the inhibitory effect of PSFL-1411 on P2X3 receptors was determined by whole-cell patch clamp. The currents of hP2X1, hP2X3, hP2X4, hP2X5 and hP2X2 / 3 were recorded using nystatin (Sangon Biotech) perforated patch to avoid current drops during multiple applications of ATP. The intracellular solution of nystatin (0.15 mg / mL) perforated cells contained (in mM) 75K2SO4, 55KCl2, 5MgSO4 and 10HEPES (pH 7.4). The currents of P2X2 and P2X7 receptors were recorded using a conventional whole-cell patch configuration. After transfection for 24-48 hours, HEK293 cells were recorded at room temperature (25 ± 2 ° C) using an Axopatch 200B amplifier (Molecular Devices, USA) with a holding potential of -60 mV. Current data were sampled at 10 kHz, filtered at 2 kHz, and analyzed using PCLAMP 10 (Molecular Devices, USA). HEK293 cells were bathed in a standard extracellular solution containing (mM) 2CaCl2, 1MgCl2, 10HEPES, 150NaCl, 5KCl, and 10 glucose (pH 7.4, Tris base). When using perforated patch clamp recording, the interval between each administration was 7 min. For hP2X2 and hP2X7, traditional whole-cell recording was used, and the internal solution consisted of (mM) 120KCl, 30NaCl, 0.5CaCl2, 1MgCl2, 10HEPES, and 5EGTA (pH 7.4, Tris base), and the interval between each administration was 2-3 min.

[0091] Figure 3BThe figure shows the inhibition rate of PSFL-1411 on various subtypes of P2X receptors. As shown in the figure, the compound PSFL-1411, as a P2X3 receptor modulator, almost completely inhibits hP2X3 at a concentration of 10 μM, but has no inhibitory effect on hP2X1, hP2X4, hP2X5, and hP2X7; the selectivity of PSFL-1411 for hP2X2 / 3 is more than 100 times, indicating that PSFL-1411 is a compound with high specificity and selectivity for targeting P2X3.

[0092] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A use of PSFL-1411, characterized in that: The structure of PSFL-1411 is shown in Formula I below: The use is selected from at least one of the following: (i) as a P2X3 receptor inhibitor; (ii) non-therapeutic inhibition of P2X3 receptor activation in vitro; (iii) preparing drugs for preventing and / or treating diseases associated with P2X3 receptor activation; and (iv) preventing and / or treating diseases related to excessive activation of P2X3 receptors.

2. The use according to claim 1, characterized in that The disease associated with P2X3 receptor activation is refractory chronic cough.

3. The use according to claim 1, characterized in that The PSFL-1411 inhibits the activation of the hP2X3 receptor.

4. The use according to claim 1, characterized in that The PSFL-1411 selectively inhibits the activation of the hP2X3 receptor without affecting the activation of other subtypes of the P2X receptor family.

5. The use according to claim 1, characterized in that: Other subtypes of the P2X receptor family include hP2X1, hP2X2, hP2X4, hP2X5, and hP2X7.

6. The use according to claim 2, characterized in that: PSFL-1411 prevents and / or treats refractory chronic cough by inhibiting overactivation of P2X3 receptors.

7. The use according to claim 2, characterized in that: PSFL-1411 reduced cough frequency by at least 70% in patients with refractory chronic cough.

8. A method for preventing and / or treating a disease associated with P2X3 receptor activation (preferably refractory chronic cough), characterized in that: The method comprises the steps of: A therapeutically effective amount of PSFL-1411 is administered to the subject.

9. The method according to claim 8, characterized in that The therapeutically effective amount is not less than 0.1 mg / kg.

10. A method for non-therapeutic inhibition of P2X3 receptor activation in vitro in cells expressing P2X3 receptors, characterized in that: The method comprises the steps of: The cells expressing the P2X3 receptor are cultured in a culture system containing PSFL-1411.

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