WO2010013925A2

WO2010013925A2 - Pharmacological composition for prevention and treatment of respiratory disease containing pyrazolopyrimidinone compound or pharmaceutically acceptable salts thereof

Info

Publication number: WO2010013925A2
Application number: PCT/KR2009/004188
Authority: WO
Inventors: Chan Ho Lee; Seul Min Choi; Dong Hwan Kim; Kyung Koo Kang; Dong Seong Kim; Byoung Ok Ahn; Moohi Yoo
Original assignee: Dong-A Pharmaceutical. Co., Ltd.
Priority date: 2008-07-31
Filing date: 2009-07-28
Publication date: 2010-02-04
Also published as: WO2010013925A3

Abstract

Disclosed herein is composition for prevention and treatment of respiratory disease containing a pyrazolopyrimidinone compound or pharmaceutically acceptable salts thereof as an active ingredient.

Description

Description PHARMACOLOGICAL COMPOSITION FOR PREVENTION

AND TREATMENT OF RESPIRATORY DISEASE CONTAINING PYRAZOLOPYRIMIDINONE COMPOUND OR PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF Technical Field

[1] The present disclosure relates to a pharmacological composition for prevention and treatment of respiratory disease.

[2]

Background Art

[3] Respiratory diseases affect the bronchus and lungs, and include chronic obstructive pulmonary disease, bronchiectasis, bronchial asthma, lung cancer, and bronchial adenoma.

[4] Chronic Obstructive Pulmonary Disease (COPD) is a disease that is characterized by an abnormal inflammatory response in the lungs to inhaled harmful particles and gasses, shows progressive and irreversible restriction of airflow, and includes chronic bronchitis or emphysema. Host factors such as genetic characters and airway hyper- responsiveness and external factors such as smoking, infection, work, and environmental exposure are known to be responsible for the disease. Among these factors, smoking is known to be a high risk factor, and although there are large discrepancies due to different individual genetic dispositions, etc., smoking duration has been reported to have an evident correlation to restricted airflow (MacNee NW, Proc. Am. Thorac. Soc, 2005). The prevalence rate worldwide is estimated at about 5 to 10% in adults (Halbert RJ et al., Chest, 2003; Halbert RJ et al., Eur Respir J. 2006), and in a recent research on South and Central American regions, about 7.8 to 19.7% are reported to be patients with stage 1 of GOLD (Global Initiative for Chronic Obstructive Lung Disease) classification or higher (Menezes et al., Lancet, 2005). As for the domestic prevalence rate, about 7.8% (male 10.9%, female 4.9%) from 18 years to 45 years old and about 17.2% (male 25.8%, female 9.6%) of 45 years or older are reported as GOLD stage 1 or higher. As ages increase, the prevalence rate also increases. In addition, the frequency of COPD exacerbations of smokers, resulting from smoking (known as the riskiest factor for COPD) is 3-fold higher than that of non-smokers (26.9% vs. 8.8%) and it has been revealed that as the smoking level increases, the frequency of the disease proportionally increases (the frequencies of 10 cigarettes or less, 11 to 19 cigarettes, and 20 cigarettes or more are 12.8%, 18.8%, and 35.6%, respectively) (The national COPD Survey Committee, Am J Respir Cirt Care Med, 2003). Cough, sputum, hemoptysis, and airway obstruction are representative symptoms of the disease, whose social and economical costs continuously increase. In the United States, medical care expenditures due directly to COPD in 1993 amounted to about $14.7 billion, and social costs including indirect costs such as labor and productivity reduction from early death were estimated at about $23.9 billion (Sullivan SD et al., Chest, 2000). In 2002, a survey showed that these costs increased to about $32.1 billion (National Heart, Lung, and Blood Institute, 2004). Based on disability-adjusted life years (DALYs), an index indicating when time living in a healthy state is deprived, COPD ranked 12th as a cause in 1990 and is expected to be 5th in 2020, meaning that it is a healthy life-threatening disease.

[5] Drugs currently used in chronic obstructive pulmonary disease may be largely classified into corticosteroids, bronchodilators, and combined therapy. Corticosteroids are used for COPD patients with severe or recurrent symptoms, and prolonged dosage is not recommended because side effects such as muscular weakness, functional reduction, and respiratory failure are caused by the agents. Bronchodilators may be sub-classified into β2 agonists, anticholinergics, and methylxanthines. β2 agonists induce relaxation of airway smooth muscle, may be sub-classified into fast-acting and slow-acting drugs, and have side effects such as tachycardia, tremor, hypokalemia, and tachyphylaxis. General side effects of anticholinergics are dry mouth, and a case of glaucoma caused by use of a mask for inhalation of some drugs has been reported. Xanthine derivatives non- selectively inhibit phosphodiesterase (hereinafter, it may be referred to as PDEs), thus showing bronchiectasis or anti-inflammatory activity, and it is reported that side effects such as headache, insomnia, nausea, heartburn, atrial and ventricular arrhythmia, and grand mal epilepsy may occur dosage-dependently due to the non-selective inhibition activities (Yoo Jee-Hong, Monthly Korean Pharmaceutical Industry News, 2007).

[6] In order to overcome these shortcomings, development of a new therapeutic agent using a selective inhibitor to PDEs is actively underway. It is known that PDEs are a family of enzymes that hydrolyze cyclic nucleotides such as 3', 5'-cyclic adenosine monophosphate (cAMP) or 3', 5'-cyclic guanosine monophosphate (cGMP) and there are 11 families and at least 44 distinct enzymes in humans (Chung KF, Eur J Pharmacol, 2006). Among them, development of a therapeutic agent using a phosphodiesterase type 4 (PDE-4) inhibitor is most actively underway. PDE-4 is present not only in inflammatory cells such as T cells, B cells, eosinophils, and neutrophils, but also in airway smooth muscle cells, and is an enzyme that hydrolyzes cAMP (Houslay MD and Adams DR, Biochem J, 2003). Because cAMP increased in cells inhibits the secretion of pro-inflammatory cytokine, showing anti-inflammatory activities and inducing bronchiectasis through relaxation of airway smooth muscle, PDE-4 has attracted attention as a potential therapeutic agent against diseases like COPD. However, in the case of rolipram, a representative PDE-4 inhibitor, it has side effects such as vomiting and nausea, and second generation drugs which are more selective to PDE-4 have been developed, but it is still difficult to mitigate the expression of side effects.

[7] Phosphodiesterase type 5 (PDE-5) is now receiving attention as a new drug target for airway diseases including COPD. PDE-5 is present not only in endothelial cells, but also in pulmonary vascular smooth muscle, bronchial blood vessel, and airway smooth muscle, and is an enzyme that hydrolyzes cGMP (Sebkhi A, Circulation, 2003; Yanaka N, Eur J Biochem, 1998). In the case of sildenafil, a representative PDE-5 inhibitor, airway hyper-responsiveness and reduction of leukocyte infiltration in bron- choalveolar fluid was induced in a pre-clinical study (Toward TJ, Am J Respir Crit Care Med, 2004), and in a brief clinical experiment on two COPD patients, whereby Forced Expiratory volume in the first second of expiration (FEVl) was increased by 24% and 12%, respectively (Charan NB, Chest, 2001).

[8] The present inventors synthesized a pyrazolopyrimidinone compound of the new

Chemical Formula 1 and reported that the compound could be used as a PDE-5 inhibitor (WO 00/027848, KR Patent No. 0353014). Also, the present inventors have continuously conducted studies on the pyrazolopyrimidinone compound as a PDE-5 inhibitor, confirmed that the pyrazolopyrimidinone compound has excellent effects on prostatic hyperplasia and associated lower urinary tract symptoms (KR Patent Application No. 06-30724 and International Patent Application No. PCT/KR06/1242), portal hypertension (KR Patent Application No. 05-99900 and International Patent Application No. PCT/KR05/3526), and chronic heart failure (KR Patent Application No. 07-56392), and have filed patent applications.

[9] The pyrazolopyrimidinone compound of the present invention remarkably enhanced the selectivity to a conventional PDE-5, thus significantly reducing the side effects, swiftly showed the efficacy of the drug with a 1 hour to peak blood concentration time, and made daily dosage possible due to sustainable duration of drug action resulting from a 12 hour elimination half-life. In addition, the present inventors kept studying tissue distribution of the drug after oral administration and completed the present invention by confirming that the drug has excellent distribution in the pulmonary tissues and has effects on relaxation of airway smooth muscle, alleviation of airway hyper-responsiveness, and inhibition of alveolar damage and inflammatory cell infiltration.

[10] Disclosure of Invention

Technical Problem

[H] One object of the present invention is to provide a pharmacological composition for prevention and treatment of respiratory diseases including airway diseases such as chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and asthma, which has excellent distribution into the lung tissues, effectively induces bron- chodilation without any side effects through selective inhibition of the activity of PDE- 5 present in airway smooth muscle, and alleviates airway hyper-responsiveness.

[12]

Technical Solution [13] The present invention provides a pharmacological compound containing 5- [2-propyloxy-5-( 1 -methyl-2-pyrolidinylethylamidosulphonyl)phenyl] - 1 -methyl-prop yl- 1 ,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one, a pyrazolopyrimidinone compound, represented by the following Chemical Formula 1 or pharmaceutically acceptable salts thereof, as an active ingredient for prevention and treatment of respiratory diseases.

[14] [Chemical Formula 1]

[16]

Advantageous Effects [17] The composition of the present invention effectively induces bronchodilation without any side effects through selective inhibition of the activity of PDE-5, alleviates airway hyper-responsiveness, and inhibits alveolar damage and inflammatory cell infiltration.

[18] In addition, the composition of the present invention may be used in prevention and treatment of airway diseases such as chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and asthma due to rapid expression of the drug efficacy and once a day dosage resulting from more than 12 hour effective duration of the drug efficacy.

[19]

Brief Description of Drawings [20] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[21] FIG. 1 is a graph illustrating an inhibitory action of a histamine-induced contraction reaction after a pre-treatment of the pyrazolopyrimidinone compound of Chemical Formula 1 to an isolated guinea pig trachea.

[22] FIG. 2 is a graph illustrating an evaluation of each inhibitory action of the airway hyper-responsiveness reaction after applying the pyrazolopyrimidinone compound of Chemical Formula 1 and sildenafil to a chronic obstructive pulmonary animal model.

[23] FIG. 3 is a collection of representative photographs by group evaluating alveolar damage after applying the pyrazolopyrimidinone compound of Chemical Formula 1 and sildenafil to a chronic obstructive pulmonary animal model.

[24] FIG. 4 is a collection of representative photographs by group evaluating inflammatory cell infiltration after applying the pyrazolopyrimidinone compound of Chemical Formula 1 and sildenafil to a chronic obstructive pulmonary animal model.

[25]

Best Mode for Carrying out the Invention

[26] The pyrazolopyrimidinone compound of Chemical Formula 1 is a kind of phosphodiesterase type 5 inhibitor. The compound has excellent PDE 5 inhibitory activity and selectivity. It is absorbed fast due to its improved solubility, and has high bioavailability and huge volume of distribution. It is characterized by about a 3-fold longer elimination half- life than those of sildenafil or vardenafil, drugs with the same mechanism.

[27] The pyrazolopyrimidinone compound of Chemical Formula 1 is not a hydrate or solvate, but a white or light-white powder with the melting point of 158-161⁰C and the pKal and pKa2 values of about 6.5 and 12.5, respectively. The compound is insoluble in water, but soluble in acetic acid, methanol, and chloroform.

[28] The pyrazolopyrimidinone compound of Chemical Formula 1 is prepared through a three-step synthetic process and a preparation method of the compound is disclosed in WO 00/027848 and KR Patent No. 0353014. The method is briefly described as follows.

[29] In Step 1,

4-[2-propyloxy-5-(chlorosulfonyl)benzamido]-l-methyl-3-propyl-5-carbamoyl pyrazole is prepared. For preparation, a predetermined amount of 4-[2-propyloxy benzamido]-l-methyl-3-propyl-5-carbamoyl pyrazole is added to a predetermined amount of chlorosulfonic acid cooled at O⁰C. The reaction mixture is stirred, filtered, washed and dried to obtain 4-[2-propyloxy-5-(chlorosulfonyl)benzamido]-l-methyl-3-propyl-5-carbamoyl pyrazole.

[30] In Step 2, 4-[2-propyloxy-5-(l-methyl-2-pyrrolidinylethyl amido- sulfonyl)benzamido]-l-methyl-3-propyl-5-carbamoyl pyrazole is prepared from the pyrazole compound prepared in the above step 1. For preparation, a predetermined amount of 2-(2-aminoethyl)-l-methylpyrrolidine is added at O⁰C to a dichloromethane solution containing a predetermined amount of

4-[2-propyloxy-5-(chlorosulfonyl)benzamido]-l-methyl-3-propyl-5-carbamoyl pyrazole of step 1, followed by stirring. Upon completion of the reaction, the reaction solution is diluted with dichloromethane. The organic layer is washed, dried, concentrated and filtered to obtain 4-[2-propyloxy-5-(l-methyl-2-pyrrolidinylethyl amido- sulfonyl)benzamido]- l-methyl-3-propyl-5-carbamoyl pyrazole.

[31] In step 3, 5-[2-propyloxy-5-(l-methyl-2-pyrrolidinylethyl amido- sulfonyl)phenyl]-l-methyl-3-propyl-l,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one, a pyrazolopyrimidinone compound of the present invention, is prepared from the compound obtained in step 2. For preparation, a predetermined amount of the pyrazole compound synthesized in step 2 is dissolved in t-butanol, to which a predetermined amount of potassium t-butoxide is added, followed by stirring under reflux for a predetermined time. Upon completion of the reaction, the reaction solution is cooled down, diluted, washed and dried. Then, reduced pressure distillation, elimination of a solvent and silica gel column chromatography are performed to obtain a predetermined amount of a novel pyrazolopyrimidinone compound of the invention, represented by Chemical Formula 1.

[32] The pharmaceutically acceptable salts of the present invention include pharmaceutically acceptable salts conventionally used in the pharmaceutical art, for example, sodium salts, magnesium salts, potassium salts, calcium salts, sulfates, bisulfates, hydrochlorides, besylate salts, and camsylate salts.

[33] The composition of the present invention may be used preferably for prevention and treatment of airway diseases among respiratory diseases and more preferably for prevention and treatment of chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and asthma.

[34] The present invention provides a pharmacological composition effective for prevention and treatment of airway diseases such as chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and asthma, which induces effective relaxation of airway smooth muscle, alleviates airway hyper-responsiveness, and have excellent drug distribution into the lung tissue by using a pyrazolopyrimidinone compound which is excellent in selective inhibition activity to PDE-5.

[35] The composition of the present invention prevents and treats airway diseases by bronchodilation through relaxation of smooth muscle, alleviation of airway hyper- responsiveness, and inhibition of alveolar damage and inflammatory cell infiltration, or selective inhibition of phosphodiesterase type 5 (PDE-5) enzyme.

[36] The present invention also provides a method of preventing and treating respiratory diseases including chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and asthma by administering a therapeutically effective amount of a pharmacological compound containing a pyrazolopyrimidinone compound of Chemical Formula 1 to a mammal including human.

[37] The present invention also provides a use of a pyrazolopyrimidinone compound of

Chemical Formula 1 in prevention and treatment of respiratory diseases including airway diseases such as chronic obstructive pulmonary disease, chronic bronchitis, and emphysema. For example, the present invention provides a use of a pyrazolopyrimidinone compound of Chemical Formula 1 in manufacture of a drug or health food for prevention and treatment of respiratory diseases.

[38] When applied in an actual clinical setting, the pharmacological compound of the present invention may be administered in various formulations including oral administration, and inhalation, and in most preferably oral administration. In case of medical preparations, it may be also prepared using commonly used diluents or excipients such as filler, extender, binder, wetting agent, disintegrant, and surfactant.

[39] In addition, the dosage of the pharmacological compound in accordance with the present invention may be varied according to weight, age, gender, health status, diet and excretion rate of patients, time and method of administration, and severity of symptoms, and the dosage of a specific constituent drug is preferably about 25 to about 200 mg once or several times a day to adults.

[40]

Mode for the Invention

[41] Features and advantages of the present invention will be more clearly understood by the following detailed description of the present preferred embodiments by reference to the accompanying drawings. It is first noted that terms or words used herein should be construed as meanings or concepts corresponding with the technical sprit of the present invention, based on the principle that the inventor can appropriately define the concepts of the terms to best describe his own invention. Also, it should be understood that detailed descriptions of well-known functions and structures related to the present invention will be omitted so as not to unnecessarily obscure the important point of the present invention.

[42] In order to determine whether the orally administered pyrazolopyrimidinone compound of Chemical Formula 1 was effectively distributed in the lungs and used as an agent for prevention and treatment of the above-mentioned airway diseases, the compound was orally administered to a rat, followed by evaluation of tissue distributions.

[43] Hereinafter, the present invention will be described in detail with reference to

Examples and Preparation Examples. However, the following Examples and Preparation Examples are only for the understanding of the present invention, and the present invention is not limited to the following Examples and Preparation Examples.

[44] The pyrazolopyrimidinone compound of Chemical Formula 1 used in the following

Examples and Preparation Examples was the compound prepared according to International Publication Patent No. WO 00/027848 by Dong- A Pharmaceutical Co., Ltd.

[45]

[46] <Example 1> Evaluation of distribution of an orally-administered compound in the lungs

[47] In order to evaluate an effective distribution of the pyrazolopyrimidinone compound of Chemical Formula 1 of the present invention in the body, experiments were performed as follows.

[48] The pyrazolopyrimidinone compound of Chemical Formula 1 (purchased from

Dong- A Pharmaceutical Co., Ltd.) and sildenafil were orally administered at a dosage of 30 mg/kg to male white rats (Sprague-Dawley family, body weight: 250 g), respectively and lungs were isolated from each group of the rats after 30 minutes, 2 hours, 6 hours, and 24 hours to evaluate the tissue distributions of the drugs. The concentrations in the lungs were measured using High Performance Liquid Chromatography (HPLC). As a mobile phase, 20 mM potassium phosphate/acetonitrile (72:28, v/v) was used at a flow rate of 1.5 ml/min, and each of the effluent samples was measured using a UV detector set at 292 nm. A reversed-phase column was used and the measurement limitation was 0.1 μg/g.

[49] Table 1

[Table 1]

[Table ]

Evaluation results of tissue distributions after oral administration of the compounds to rats¹⁾

[50] [51] The results showed that the pyrazolopyrimidinone compound of Chemical Formula 1 was distributed in the lungs at high concentrations after 24 hours of drug administration, and the concentrations were much higher than the IC₅₀ (2.9 ng/ml) values of the pyrazolopyrimidinone compound of Chemical Formula 1 to PDE-5. These results mean that the pyrazolopyrimidinone compound of Chemical Formula 1 may exhibit strong PDE-5 inhibitory action in the lungs. That is, it is determined that concentrations of the compound of the present invention in tissues are constantly maintained unlike abrupt reductions in concentrations of other PDE-5 inhibitors in tissues with time, and the compound has a higher tissue affinity in the lungs than those of other conventional PDE-5 inhibitors. Therefore, from the results, it is confirmed that the compound of the present invention is more effective in treatment of pulmonary diseases than other conventional pulmonary disease therapeutics and may be stably used.

[52] [53] <Example 2> Evaluation of trachea inhibitory action of the compound of Chemical Formula 1 on isolated guinea pig tracheas

[54] In order to evaluate an inhibitory action of airway contraction by the pyrazolopyrimidinone compound of Chemical Formula 1 of the present invention, experiments were performed as follows.

[55] Male guinea pigs (Hartley family, body weight: 300 g) were anaesthetized with ether and bled to death to isolate their tracheas. The tracheas were rapidly immersed into Krebs-Henseleit buffer. Connective tissues and adipose tissues were removed from the isolated trachea, cut into about 3-4 mm, and then placed in an organ bath containing Krebs-Henseleit buffer (37⁰C) from which 95% O₂ and 5% CO₂ were released. A prepared ring-shaped organ tissue was connected to a force transducer connected to a polygraph (Grass Instruments) to measure a tension and stabilized under a standard tension of 0.3 g for 1 hour. Subsequently, 10 μM histamine was treated to evaluate only tissues in which contraction reactions were caused.

[56] The pyrazolopyrimidinone compound of Chemical Formula 1 was treated to become solutions at concentrations of 10, 50, and 100 μM, and 10 μM histamine was treated again after 10 minutes to induce a contraction. Evaluation results of the relative percentage of an induced contraction reaction after the pretreatment compared to the contraction reaction prior to the treatment of the compound are shown in Table 2, which FIG. 1 illustrates as a graph (The values as indicated in FIG. 1 are mean+standard deviation and ** means a statistical significance at p<0.01.).

[57] In addition, contraction reaction (%) = (Shrinkage tension after compound treatment/ Shrinkage tension prior to compound treatment) x 100 [58] Table 2 [Table 2] [Table ]

[59] [60] The experimental results showed that concentration-dependent inhibitory actions to organ contraction were observed, meaning that the pyrazolopyrimidinone compound of Chemical Formula 1 of the present invention may be used as a therapeutic agent for chronic obstructive pulmonary disease, chronic bronchitis, and emphysema through an inhibitory action of bronchus contraction.

[61] [62] <Example 3> Evaluation of inhibitory action of airway hyper-responsiveness by the compound of Chemical Formula 1 in a airway disease guinea pig model

[63] In order to evaluate an alleviatory action of airway hyper-responsiveness (AHR) by the pyrazolopyrimidinone compound of Chemical Formula 1 of the present invention, experiments were performed as follows.

[64] For a male guinea pig (Hartley family, body weight: 300 g), 10 μg of ovalbumin and 100 mg of aluminum hydroxide were intraperitoneally administered for sensitization. After 14 days of the sensitization, 100 μg of ovalbumin was inhaled to the guinea pig for 1 hour to cause an airway disease. On the 15th day, airway hyper-responsiveness to histamine (1 mM, inhalation for 20 seconds) was measured using a plethysmography (OCP-3000, Allmedicus Co., Ltd., U.S.A.) for 3 minutes (Toward TJ et al., Am J Respir CrU Care Med, 2004).

[65] On 13th day of the sensitization, the pyrazolopyrimidinone compound (3 and 10 mg/ kg) of Chemical Formula 1 were intraperitoneally administered 30 minutes prior to induction of a secondary ovalbumin and 6 hours after the induction of the secondary ovalbumin, respectively. The number of animals tested is 5 per group and the results are shown in Table 3. The results are notified in the form of an enhanced pause (Penh), an indicator of airway hyper-responsiveness, and the calculation formula is as follows; Penh = ([expiratory time/relaxation time]- l)x (peak expiratory flow/peak respiratory flow).

[66] Table 3 [Table 3] [Table ] Evaluation results of alleviatory action of airway hyper-responsiveness

[67] [68] The experimental results showed that the pyrazolopyrimidinone compound of Chemical Formula 1 shows a concentration-dependent alleviatory action in Penh (enhanced pause), an indicator that shows airway obstruction.

[69] [70] <Example 4> Evaluation of inhibitory action of airway hyper-responsiveness by the compound of Chemical Formula 1 in a chronic obstructive pulmonary disease animal model

[71] In order to evaluate an inhibitory action of airway hyper-responsiveness (AHR) by the pyrazolopyrimidinone compound of Chemical Formula 1 of the present invention, experiments were performed as follows.

[72] Male mice (C57BL/6, body weight: 20-22 g) were exposed to cigarette smoke three times a day and 5 days a week for a total 8 week period; 1 liter of smoke was caused by 1 cigarette (tar 8.5 mg, nicotine 0.9 mg) and the mice were exposed to the smoke for 6 minutes, followed by 1 minute of ventilation. 1 liter of smoke was again caused by 1 cigarette and the mice were exposed to the smoke for 6 minutes, followed by 1 minute of ventilation. Lastly, 1 liter of smoke was caused by 1 cigarette and the mice were exposed to the smoke for 6 minutes. Therefore, animals were exposed to cigarette smoke three times a day (morning, noon, afternoon) and a total of 54 minutes a day (9 cigarettes) (Valenca SS et al., ToxicolPathol, 2004; Valenca SS et al., NitricOxide, 2009)

[73] The exposure was proceeded in a space of 0.025 m3 using a systemic body exposure

(Mauderly JL et al., ExpPathol, 1989). After 3 weeks of exposing the animal to cigarette smoke, a MicroSparyerTM aerosoliser (IA-IC; Penn-Century, Philadelphia, PA, USA) was inserted into the trachea of the animal, and then 50 μl of elastase dissolved in a sterile physiological saline solution was injected through the aerosolier (Pang B et al., Infect Immun, 2008; KinoshitaT etal., Biochem Biophys Res Commun, 2007; Bivas-BenitaMetal., EurJPharmBiopharm, 2005; Hoidal JRetal., Am Rev Respir Dis, 1983). After the total 8 week test, airway hyper-responsiveness was measured using a plethysmography for 3 minutes (Kirschvink N et al., Toxicology, 2005). The results are notified in the form of an enhanced pause (Penh), an indicator of airway hyper-responsiveness, and the calculation formula is as follows; Penh = ([expiratory time/relaxation time]-l)x(peak expiratory flow/peak respiratory flow).

[74] The pyrazolopyrimidinone compound (3 and 10 mg/kg) of Chemical Formula 1 and sildenafil (3 and 10 mg/kg) as a control drug were orally administered for 8 weeks causing disease, and the results are shown in the following [Table 4], which FIG. 2 illustrates as a graph (The values shown in FIG. 2 are mean ± standard error (SEM), and *, +, and # show statistical significances compared to a normal group (Naive), a vehicle control group (Vehicle), and a sildenafil (10 mg/kg) administered group, respectively (P<0.05). In addition, Udenafil in FIG. 2 refers to the pyrazolopyrimidinone compound of Chemical Formula 1 in the specification.

[75] Table 4 [Table 4] [Table ]

[76] [77] Experimental results in FIG. 2 and Table 4 shows that the pyrazolopyrimidinone compound (Udenafil) and sildenafil have a concentration-dependent inhibitory action of airway hyper-responsiveness in Penh, confirming that there was a statistically significant inhibition of airway hyper-responsiveness in the pyrazolopyrimidinone compound (10 mg/kg) administered group, compared to a sildenafil (3 and 10 mg/kg) administered group.

[78] [79] <Example 5> Evaluation of inhibitory action of inflammatory cell infiltration by the compound of Chemical Formula 1 in a chronic obstructive pulmonary disease animal

[80] In order to evaluate an inhibitory action of inflammatory cell infiltration by the pyrazolopyrimidinone compound of Chemical Formula 1 of the present invention, experiments were performed as follows.

[81] Disease causing and compound administration were performed by a method described in <Example 4> and a 22G catheter was inserted into the trachea. Bron- choalveolar lavage fluid (BALF) was isolated with a sterile physiological saline solution, and then a WBC differential count (leukocyte analysis percent) was proceeded using a hematology system (ADVIA90, Bayer HealthCare) (D' hulst AI et al., EurRespirJ, 2005).

[82] The results of 8 -week administration of the pyrazolopyrimidinone compound (3 and 10 mg/kg) and sildenafil (3 and 10 mg/kg) as a control drug are shown in following Table 5. [83] Table 5 [Table 5] [Table ] Evaluation results of inhibitory action of inflammatory cell infiltration

[84] [85] The measurement results of a total number of leukocytes show that there was a statistically significant inhibitory effect in a pyrazolopyrimidinone compound (10 mg/kg) of Chemical Formula 1 administered group, compared to a vehicle control group. A decreasing trend was observed in a sildenafil administered group, but no statistical significance was observed. The measurement results of the rates of neutrophils and eosinophils in BALF shows that inhibitory effects of inflammatory cell infiltration of 45.8% and 59.6%, respectively were significantly observed in a pyrazolopyrimidinone compound of Chemical Formula 1 administered group, compared to a vehicle control group. Through these results, it is confirmed that the pyrazolopyrimidinone compound of Chemical Formula 1 significantly shows an inhibitory effect of inflammatory cell infiltration. [86]

[87] <Example 6> Evaluation of inhibitory actions of alveolar damage and inflammatory cell infiltration by the compound of Chemical Formula 1 through a histological analysis in a chronic obstructive pulmonary disease animal model

[88] In order to evaluate inhibitory actions of alveolar damage and inflammatory cell infiltration by the compound of Chemical Formula 1, experiments were performed as follows.

[89] After disease causing and compound administration were performed by a method described in <Example 4>, 10% formalin was injected through the trachea at a pressure of 25 CmH₂O and maintained for 15 minutes. The trachea was bound to isolate the lung, which was immobilized in a 10% formalin solution for 24 hours (Nakanishi Y et al., Am J Respir Crit Care Med, 2009). A tissue specimen later obtained was stained with H&E staining (tissue staining), then evaluations of peribronchial and perivascular inflammation were performed (McKay A et al., J Immnunol, 2004), and the sized of the alveolar space was measured using a mean linear intercept method (Thurlbeck WM, Am Rev Respir Dis, 1967; Hautamaki RD et al., Science, 1997). The microscope then used was an Axioskop2 plus (ZEISS, Germany), and a 200-fold magnification was used in FIG. 3 and FIG. 4.

[90] The alveolar wall defects were measured using a destructive index and the reference used is as follows; at least two alveolar wall defects (1), at least two intraluminal parenchymal rags in alveolar ducts (2), clearly abnormal morphology (3), classic emphysematous changes (4) (Saetta M et al., Am Rev Respir Dis, 1985; Robbesom AA et al., ModPathol, 2003).

[91] The administration results of the pyrazolopyrimidinone compound (10 mg/kg) of

Chemical Formula 1 and sidenafil (10 mg/kg) as a control drug for 8 weeks are shown in Table 6, FIG. 3, and FIG. 4 (in FIG. 3, A, no treatment group; B, vehicle control group; C, sildenafil (10 mg/kg) administered group; D, pyrazolopyrimidinone compound (10 mg/kg) of Chemical Formula 1 administered group; in FIG. 4, A, no treatment group; B, vehicle control group; C, sildenafil (10 mg/kg) administered group; D, pyrazolopyrimidinone compound (10 mg/kg) of Chemical Formula 1 administered group).

[92] Table 6 [Table 6]

[Table ]

Evaluation results of inhibitory action of alveolar damage

[93] [94] The measurement results of the size of alveolar space using a mean linear intercept method show that there were statistically significant inhibitory effects of alveolar space expansion in a sildenafil (10 mg/kg) administered group and a pyrazolopyrimidinone (10 mg/kg) administered group. The pyrazolopyrimidinone compound of Chemical Formula 1 showed a statistically significant inhibitory effect of damage compared to sildenafil.

[95] In addition, the measurement results of alveolar wall defects through destructive index shows that sildenafil and the pyrazolopyrimidinone compound of Chemical Formula 1 all have inhibitory effects of alveolar wall defects at 42.3% and 49.2%, respectively (Table 6, FIG. 3).

[96] The evaluation results of inflammatory cell infiltration through a histological method as in FIG. 4 shows that sildenafil (10 mg/kg) and the pyrazolopyrimidinone compound (10 mg/kg) of Chemical Formula 1 all have significant inhibitory effects compared to a vehicle control group.

[97] Through these results, it is confirmed that the pyrazolopyrimidinone compound of Chemical Formula 1 has significant inhibitory actions of alveolar damage and inflammatory cell infiltration.

[98] [99] <Preparation Example> Preparation of pharmaceutical formulations for oral admin- istration

[100] 1. Preparation of powder

[101] Pyrazolopyrimidinone compound of Chemical Formula 1 2 g [102] Lactose 1 g

[103] The ingredients were mixed and filled in an airtight bag to prepare a powder. [104]

[105] 2. Preparation of tablet

[106] Pyrazolopyrimidinone compound of Chemical Formula 1 100 mg [107] Corn starch 100 mg [108] Lactose 100 mg [109] Magnesium stearate 2 mg [110] The ingredients were mixed and then compressed by a conventional tablet preparation method to prepare tablets. [I l l]

[112] 3. Preparation of capsule

[113] Pyrazolopyrimidinone compound of Chemical Formula 1 100 mg [114] Corn starch 100 mg [115] Lactose 100 mg [116] Magnesium stearate 100 mg [117] The ingredients were mixed and then filled into gelatin capsules by a conventional tablet preparation method to prepare capsules. [118]

[119] 4. Preparation of inhalant

[120] Pyrazolopyrimidinone compound of Chemical Formula 1 100 mg [121] Yolk lecithin 100 mg [122] α-tocopherol 2 mg [123] Ascorbic acid 3 mg [124] The ingredients were mixed and purified water was added for a 10 ml solution according to a conventional inhalant preparation method to prepare an inhalant. [125]

Industrial Applicability [126] The composition of the present invention effectively induces bronchodilation without any side effects through selective inhibition of the activity of PDE-5, alleviates airway hyper-responsiveness, and inhibits alveolar damage and inflammatory cell infiltration. [127] In addition, the composition of the present invention may be used in prevention and treatment of airway diseases such as chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and asthma due to rapid expression of the drug efficacy and once a day dosage resulting from more than 12 hour effective duration of the drug efficacy. [128] [129] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

Claims [1] 1. A pharmacological composition for prevention and treatment of respiratory disease, the composition comprising: a pyrazolopyrimidinone compound of Chemical Formula 1 or pharmaceutically acceptable salts thereof as an active ingredient

[Chemical Formula 1]

[2] The composition as set forth in claim 1, wherein the respiratory disease is airway disease.

[3] The composition as set forth in claim 1, wherein the respiratory disease is chronic obstructive pulmonary disease, chronic bronchitis, emphysema, or asthma.

[4] The derivative as set forth in claim 2 or claim 3, wherein the prevention and treatment of respiratory disease is achieved by bronchodilation action through relaxation of smooth muscle and alleviation action of airway hyper-responsiveness. [5] The composition as set forth in claim 2 or claim 3, wherein the prevention and treatment of respiratory disease is achieved by selective inhibitory action of phosphodiesterase type 5

(PDE-5) enzyme.

[6] The composition as set forth in claim 2 or claim 3, wherein the pharmacological composition is a pharmacological composition in an orally administered formulation.

[7] The composition as set forth in claim 6, wherein the orally administered formulation is a pharmacological composition for a once-a-day administration. [8] The composition as set forth in any one of claim 1 to claim 3, comprising 25-200 mg of the pyrazolopyrimidinone compound of Chemical Formula 1. [9] The composition as set forth in claim 3, wherein the respiratory disease is asthma.