WO2023232126A1 - Use of sesquiterpene polyketone compound for preventing and treating pulmonary arterial hypertension - Google Patents

Abstract

The present invention relates to use of a sesquiterpene polyketone compound and a salt thereof for preventing and treating pulmonary arterial hypertension. A biological activity experiment shows that the compound can reverse pulmonary vasculature reconstruction and is used for treating pulmonary arterial hypertension.

Description

Use of a sesquiterpene polyketide compound for the prevention and treatment of pulmonary arterial hypertension

Cross-references to related applications

This application claims the rights and interests of Chinese application number 2022106262318 submitted on June 2, 2022. Said application number 2022106262318 is hereby incorporated by reference in its entirety.

Technical field

The invention belongs to the field of natural medicine, and specifically relates to the use of a sesquiterpene polyketide compound for preventing and treating pulmonary arterial hypertension.

Background technique

Pulmonary hypertension (PH) is a large group of malignant pulmonary vascular diseases characterized by increased pulmonary artery pressure, with or without pulmonary arteriole lesions. The prognosis is similar to that of many advanced cancers, and its main feature is progressive pulmonary vascular resistance. Elevated, eventually leading to right heart failure and death ^[1] . PH is a major global health problem. The latest data shows that the prevalence of PH is about 1% of the global population and can reach 10% among people over 65 years old ^[2] . Its causes are complex and its treatment is difficult. The main reason for the long-term slow development of this field. Pulmonary vascular remodeling is the most important pathological feature of PH ^[3] . Currently, targeted drugs for the clinical treatment of PH mainly exert the effect of pulmonary vasodilation and cannot effectively reverse pulmonary vascular remodeling. They can improve the quality of life of patients to a certain extent. , but the prognosis of patients is still very poor, with a 3-year survival period of approximately 68-70% ^[4] . At present, PH is still a progressive and fatal disease. The fundamental reason why it is difficult to treat is that it cannot effectively reverse pulmonary vascular remodeling. Therefore, it is urgent to develop related drugs that can effectively reverse pulmonary vascular remodeling.

Contents of the invention

The object of the present invention is to provide a sesquiterpene polyketide compound for use in preventing and treating pulmonary arterial hypertension. Specifically, the inventor discovered and isolated and identified a sesquiterpene polyketide compound from a strain of Lactobacillus fungus, which has a significant effect in treating pulmonary arterial hypertension.

A first aspect of the present invention provides a sesquiterpene polyketide compound or a pharmaceutically acceptable salt thereof for use in the treatment of pulmonary arterial hypertension. The sesquiterpene polyketide compound is represented by formula (I):

Further, the pharmaceutically acceptable salt is a salt formed by a compound of formula (I) and an organic base or an inorganic base.

Furthermore, the salt formed is sodium salt, potassium salt, calcium salt, iron salt, magnesium salt, zinc salt, aluminum salt, barium salt or ammonium salt.

A second aspect of the present invention provides a method for preparing the above-mentioned compound, which includes: fermenting a microorganism that produces a sesquiterpene polyketide compound represented by formula (I) and then separating it by chromatography.

Further, the microorganism is a fungus of the genus Lactobacillus.

Further optionally, the fungus of the genus Lactobacillus is ZLW0801-19, and its preservation number is CGMCC No. 19039.

The third aspect of the present invention provides a pharmaceutical composition for treating pulmonary arterial hypertension, including the above-mentioned sesquiterpene polyketide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Further optionally, the salt is any one of sodium salt, potassium salt, calcium salt, iron salt, magnesium salt, zinc salt, aluminum salt, barium salt and ammonium salt.

Further optionally, in one embodiment, the amount of the active ingredient (i.e., the compound of the present invention) contained in the pharmaceutical composition can be specifically applied according to the patient's condition and the situation diagnosed by the doctor. The amount or concentration of the active compound is within a relatively small range. Adjusted within a wide range, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 1-90% by weight of the composition.

Further optionally, pharmaceutically acceptable carriers include diluents, lubricants, binders, disintegrants, stabilizers, solvents, etc. The diluents of the present invention include but are not limited to starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include but are not limited to magnesium stearate, stearic acid, and sodium chloride. , sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include but are not limited to water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, seaweed sodium bicarbonate, polyvinylpyrrolidone, etc.; the disintegrants include but are not limited to starch effervescent mixture, namely sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the stabilizers include but are not limited to polysaccharides Such as acacia gum, agar, alginic acid, Cellulose ether, carboxymethyl chitosan, etc.; the solvent includes but is not limited to water, balanced salt solution, etc.

Further optionally, the pharmaceutical composition is an oral preparation or injection; preferably, the oral preparation includes but is not limited to ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dropping pills, powders, oral liquids or Any one of emulsions; preferably, the injection is selected from any one of small water injections, infusions or freeze-dried powder injections.

beneficial effects

The compound of formula (I) of the present invention is derived from microbial fermentation and is convenient for large-scale fermentation and industrial preparation; activity testing has found for the first time that the compound can reverse pulmonary vascular remodeling and has a significant effect in treating pulmonary arterial hypertension.

Description of the drawings

Figure 1 shows that the compound of formula (I) improves the weight loss (Figure 1A) and the increase in right ventricular systolic pressure (RVSP) in mice with hypoxia-induced pulmonary hypertension (Figure 1B); the lung tissue pathological analysis results show that the formula Compound (I) reduced the percentage of media thickness of pulmonary arterioles at all levels and improved the degree of remodeling of pulmonary arterioles at all levels (Figure 1C).

[Correction 07.08.2023 under Rule 91]
Figure 2 shows that: the compound of formula (I) has no significant improvement effect on the weight loss of MCT-PH rats (Figure 2A); the compound of formula (I) improves the increase in RVSP in MCT-induced pulmonary hypertension rats (Figure 2B) and The percentage of media thickness (%MT) of pulmonary arterioles at all levels increased (Figure 2C).

Detailed ways

The present invention will be further illustrated below. It should be noted that the examples illustrate some preparation or use methods, however, it is to be understood that these examples do not limit the present invention. The protection scope of the present invention shall be determined by what is described in the appended claims.

In the following examples, the mass spectrometer is an LCQ-Advantage mass spectrometer produced by Finnigan Company in the United States. The superconducting NMR instrument was Bruker AV-400. Silica gel GF254 for thin layer chromatography and silica gel for column chromatography (200-300 mesh) are both products of Qingdao Ocean Chemical Factory. The reversed-phase ODS filler 50μm is a product of Japan YMC Company. The medium and low pressure liquid chromatograph is a product of Shanghai Lisui Electronic Technology Co., Ltd. The chromatographic column used for liquid phase separation is Phenomenex Gemini C18 column (10.0×250mm, 5μm). Methanol used for liquid chromatography was of chromatographic grade, water was double distilled water, and other reagents were of analytical grade.

The animal experimental data shown in the examples are expressed as mean ± SEM. To compare the differences between two groups of data, use Two-tailed Student’s t test for analysis; to compare the differences between more than 2 groups of data, use One-way Anova for analysis. Statistical differences are marked as follows: * represents P<0.05, ** represents P<0.01, *** represents P<0.001, ns represents not significant difference. In the data analysis of pulmonary vascular remodeling (%MT), ** represents P<0.01, *** represents P<0.001 (compared with the unmodeled group); ### represents P<0.001 (compared with the modeled group) compared to).

Example 1 Large-scale fermentation of the fungus ZLW0801-19 and its sample pretreatment method

(1) The fungal strain ZLW0801-19 of the genus ZLW0801-19 was cultured on potato dextrose agar (PDA) slant at 25°C for 5 days. After activation by PDA slope, inoculate into 4 Erlenmeyer flasks (250 mL) containing PDB medium to prepare seed liquid. Each Erlenmeyer flask contains 100 mL of potato dextrose (PDB) medium. The rotation speed is 200 rpm and cultured at 25°C for 5 days to prepare seeds. liquid. Fermentation was carried out in 24 Erlenmeyer flasks (500mL), each containing 70g of rice. Distilled water (105mL) was first added to each Erlenmeyer flask, the rice was soaked overnight and then autoclaved at 120°C. 30 minutes. After cooling to room temperature, 5 mL of seed solution was inoculated into each Erlenmeyer flask and cultured at room temperature in the dark for 51 days.

(2) Add the fermentation product to ethyl acetate for soaking and extraction three times, and concentrate the extract to dryness under reduced pressure to obtain a crude extract (81.7g).

Example 2 Preparation of compound of formula (I)

In Example 1, the crude extract of ethyl acetate (81.7g) was subjected to silica gel column chromatography, using cyclohexane-ethyl acetate (100:0, 98:2, 95:5, 90:10, 80:20, 70:30, 50:50, 0:100, v/v) and methanol were eluted, and each gradient elution volume was 6L to obtain 7 fraction samples (F1-F7). Fraction F6 was subjected to medium and low pressure liquid phase ODS column chromatography, and was eluted with methanol-water (60:40, 70:30, 80:20, 90:10, 100:0, v/v) in sequence, with each gradient elution volume 2.5 L, 7 fraction samples (F6.1-F6.7) were obtained. Fraction F6.4 was subjected to medium and low pressure liquid phase ODS column chromatography, using methanol-water (70:30, v/v) to elute, with an elution volume of 0.7L, and 5 fraction samples (F6.4.1-F6.4.5) were obtained. . Fraction F6.4.2 (1630 mg) was prepared by reverse-phase preparative HPLC (Phenomenex, Packed C18 column), and methanol-water (70:30, v/v) with a flow rate of 8 mL/min was used for elution to obtain formula (I ) compound (t _R :35.2min, 820mg).

The physical and chemical constants are as follows:

Compound of formula (I): light yellow solid; [α] ² _D ⁹ +36.0 (c 1.0, CH ₃ OH); UV (CH ₃ OH) λ _max (logε) 206 (4.43), 225 (4.35), 240 ( 4.18),299(4.31)nm; IR(KBr)ν _max 3146,2969,2929,2881,1621,1625,1448,1258cm ^-1 ; ESI-MS(positive)m/z 389[M+H] ⁺ , 411[M+Na] ⁺ ；ESI-MS(negative)m/z 387[M–H] ^– ,775[2M–H] ^– ；HR-ESI-MS(positive)m/z 389.2331[M+H] ⁺ (calcd.for C ₂₃ H ₃₃ O ₅ ,389.2328), the molecular formula of the compound is determined to be C ₂₃ H ₃₂ O ₅ ; ¹ H and ¹³ C NMR are shown in Table 1.

Table 1 ¹³ C NMR and ¹ H NMR data and assignments of compounds of formula (I)

The data recorded in DMSO-d ₆ ( ¹ H NMR for 300MHz, ¹³ C NMR for 75MHz)

Example 3 Compounds of formula (I) have a protective effect on hypoxic pulmonary hypertension model (mouse)

The hypoxic pulmonary hypertension mouse model was established using the normal pressure continuous hypoxia method. C57BL/6 male mice aged 8 weeks and weighing 20-25g were selected and used for experiments after adapting to the environment for 3 days. The mice were exposed to a hypoxic chamber for 4 weeks, respectively, where the _O2 concentration was maintained at 10% and the _CO2 concentration was maintained below 5%. Open the cabin door every 2 days to clean, replace the bedding and add rat food and drinking water. The water vapor in the cabin is absorbed with anhydrous calcium chloride. Give 12 hours of light/12 hours of darkness every day. The temperature inside the cabin is maintained at 20-25°C. Except for inhaling air, other conditions of the normoxia control group were the same as those of the hypoxia group.

Male C57BL/6 mice were divided into 3 groups, and each group was treated as follows: Normoxic group (N4W): raised in a normoxic environment for 4 weeks, injected with solvent intraperitoneally, 2 times/week, 8 times in total, n=8; low Oxygen group (H4W): reared in a hypoxic environment for 4 weeks, injected with solvent intraperitoneally, 2 times/week, 8 times in total, n=8; hypoxia + formula (I) compound treatment group (H4W+Formula I): hypoxic The animals were reared under environmental conditions for 4 weeks, and 20 mg/kg compound of formula (I) was injected intraperitoneally once, 2 times/week, a total of 8 times, n=9.

The changes in mouse body weight, right ventricular systolic pressure (RVSP, reflecting the mouse's pulmonary artery pressure), and pulmonary vascular remodeling (the thickness of the media in the blood vessel wall as a percentage of the outer diameter of the blood vessel, %MT) were observed.

The specific results are shown in Figure 1: The compound of formula (I) can improve the effect of hypoxia on the body weight of mice (Figure 1A); after being exposed to a hypoxic environment, compared with the normoxic group, mice in the hypoxic group had significant RVSP. After intraperitoneal injection of the compound of formula (I), RVSP decreased in the hypoxia group (Figure 1B); compared with the normoxia group, the percentage of media thickness (%MT) of the pulmonary arterioles at all levels in mice in the hypoxia group was significantly Increased, the media was significantly thickened, that is, obvious hypoxic pulmonary vascular remodeling occurred, and the degree of remodeling of pulmonary arterioles at all levels was improved after treatment with the compound of formula (I) (Figure 1C). The above results suggest that the compound of formula (I) can reverse the severity of pulmonary vascular remodeling and prevent the formation of hypoxia-related pulmonary hypertension.

Example 4 Compounds of formula (I) have a protective effect on monocrotaline (MCT)-induced pulmonary hypertension model (rat)

The pulmonary hypertension rat model was prepared by a one-time intraperitoneal injection of MCT (60 mg/kg).

Male SD rats were divided into 3 groups, and each group was treated as follows: Control group (Control): Intraperitoneal injection of solvent, 2 times/week, 8 times in total, n=3; MCT group (MCT): From the 1st day of MCT injection At the beginning, the solvent was injected intraperitoneally, 2 times/week, 8 times in total, n=4; MCT+Formula I compound treatment group (MCT+Formula I): Starting from the 1st day of MCT injection, 20 mg/kg Formula I was injected intraperitoneally. ) compound/time, 2 times/week, 8 times in total, n=4.

The changes in the rats' body weight, right ventricular systolic pressure (RVSP, reflecting the rat's pulmonary artery pressure), and pulmonary vascular remodeling (the thickness of the media in the blood vessel wall as a percentage of the outer diameter of the blood vessel, %MT) were observed.

The specific results are shown in Figure 2: The compound of formula (I) has no significant improvement effect on the weight loss of MCT-PH rats (Figure 1A); but compared with the control group, the RVSP of rats in the MCT group was significantly increased, and intraperitoneal injection (I) Compound RVSP was lower than that in the MCT group (Figure 2B); compared with the control group, the percentage of media thickness (%MT) of the pulmonary arterioles of rats in the MCT group at all levels was significantly increased, and the media was significantly thickened, that is, Obvious pulmonary vascular remodeling occurred, and the degree of remodeling of pulmonary arterioles at all levels was improved after treatment with the compound of formula (I) (Figure 2C). The above results suggest that the compound of formula (I) can reverse the severity of pulmonary vascular remodeling and prevent the formation of MCT-induced pulmonary hypertension.

The results of Examples 3 and 4 illustrate that the compound of formula (I) has a protective effect on the animal model of pulmonary arterial hypertension and can be used for the treatment of pulmonary arterial hypertension.

references:

[1] Galie, N., et al., 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). Eur Heart J, 2016.37(1):67-119.

[2]Hoeper, M.M., et al., A global view of pulmonary hypertension. Lancet Respir Med, 2016.4(4):306-22.

[3]Tuder,R.M.,Pulmonary vascular remodeling in pulmonary hypertension.Cell Tissue Res,2017.367(3):643-649.

[4]Farber, HW, et al., Five-Year outcomes of patients enrolled in the REVEAL Registry. Chest, 2015.148(4):1043-1054.

Claims (10)

A sesquiterpene polyketide compound or a pharmaceutically acceptable salt thereof is used for treating pulmonary arterial hypertension, characterized in that the sesquiterpene polyketide compound is represented by formula (I):
The use according to claim 1, characterized in that the pharmaceutically acceptable salt is a salt formed by the compound of formula (I) and an organic base or an inorganic base. The compound of formula (I) according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that the salt formed is a sodium salt, a potassium salt, a calcium salt, an iron salt, a magnesium salt, a zinc salt, Aluminum salt, barium salt, or ammonium salt. The method for preparing the sesquiterpene polyketide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that it includes: producing a sesquiterpene polyketide represented by formula (I) The compound is obtained by microorganisms after fermentation and then separated by chromatography. The preparation method according to claim 4, characterized in that the microorganism is a fungus of the genus Lactobacillus. The preparation method according to claim 5, characterized in that the fungus of the genus Lactobacillus is ZLW0801-19, and its preservation number is CGMCC No. 19039. A pharmaceutical composition for treating pulmonary arterial hypertension, characterized by comprising the sesquiterpene polyketide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 7, wherein the content of the sesquiterpene polyketide compound or its pharmaceutically acceptable salt is 1-90% by weight of the composition. The pharmaceutical composition according to claims 7-8, characterized in that the pharmaceutical composition is an oral preparation or an injection. The pharmaceutical composition according to claim 9, wherein the oral preparation is selected from ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dropping pills, powders, oral liquids or emulsions, and the injection is Small water injection, infusion solution or freeze-dried powder injection.

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