Application of xipapia isonicotinic acid in the preparation of drugs for preventing and or treating pulmonary hypertension

Abstract

The present invention relates to the technical field of novel pharmacological effects of isonicotinoic acid xipapia, and specifically discloses the use of isonicotinoic acid xipapia in the preparation of a drug for the prevention and / or treatment of pulmonary arterial hypertension. Pulmonary arterial hypertension is caused by abnormal proliferation of pulmonary arterial smooth muscle, which can lead to right heart failure and high disability and mortality rates. The beneficial effects of the present invention are: isonicotinoic acid xipapia prevents pulmonary arterial hypertension and / or prevents the progression of pulmonary arterial hypertension symptoms by inhibiting progressive increases in pulmonary arterial pressure, right ventricular hypertrophy, or pulmonary vascular remodeling. It can also inhibit the activation of RhoA kinase by increasing the expression of p27kip1, one of the main downstream factors of RhoA kinase, thereby inhibiting pulmonary arterial smooth muscle proliferation, inhibiting pulmonary arterial remodeling, reducing pulmonary arterial pressure, and reducing right ventricular systolic pressure, thereby achieving a therapeutic effect on pulmonary arterial hypertension. Therefore, isonicotinoic acid xipapia has good application and development prospects in the prevention and / or treatment of pulmonary arterial hypertension.

Description

Technical Field

[0001] The present invention relates to the technical field of new pharmacological effects of xipapia isonicotinic acid, and particularly to application of xipapia isonicotinic acid in the preparation of a drug for preventing and or treating pulmonary hypertension. Background Art

[0002] Pulmonary arterial hypertension (PAH) is a cardiovascular disease defined as a pathophysiological state in which pulmonary artery pressure rises above a certain threshold. PAH is primarily caused by a variety of heart, lung, or pulmonary vascular diseases. For example, in cases of pulmonary artery and pulmonary capillary stenosis caused by thickening of the pulmonary artery wall, pulmonary vascular resistance increases, requiring the right ventricle to exert greater force to propel blood through the pulmonary circulation, leading to changes in hemodynamics and pathophysiological states, and ultimately leading to right heart failure. In clinical practice, PAH is a common and frequently occurring disease with high disability and mortality rates, and deserves high attention.

[0003] Pulmonary artery smooth muscle cells (PASMCs) are an important factor in determining the thickness of the pulmonary artery wall. Abnormal proliferation of PASMCs will lead to thickening of the pulmonary artery wall, thereby causing PAH.

[0004] Currently, the main therapeutic agents for PAH include epoprostenol and its derivatives, endothelin receptor antagonists, and calcium channel blockers. Phosphodiesterase-5 (PDE-5) inhibitors have received considerable research attention. These drugs selectively dilate blood vessels by increasing intracellular cyclic guanosine monophosphate (cGMP) concentrations, thereby reducing the abnormal proliferation of PASMCs. These drugs can improve the quality of life of PAH patients to a certain extent, but they have drawbacks such as high cost, numerous adverse reactions, and inability to reverse disease progression. Furthermore, there is currently a lack of drugs that target vascular remodeling and right ventricular hypertrophy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a use of Hipapia isonicotinic acid in the preparation of a drug for preventing and / or treating PAH. A pulmonary smooth muscle cell (PASMC) survival rate assay (MTT assay) is used to provide cultured abnormally proliferating pulmonary smooth muscle cells (PASMCs) with different concentration gradients of Hipapia isonicotinic acid (hereinafter referred to as Hpapia), and the cell survival rate is measured to determine the inhibitory effect of Hpapia on the abnormal proliferation of PASMCs. A PAH model is established using SD rats. Fasudil (an existing drug that dilates blood vessels by blocking the final stage of vasoconstriction, myosin light chain phosphorylation, to achieve the therapeutic effect of PAH) and Hpapia are administered with corresponding dosing regimens. At the end of the experiment, the animals' right ventricular systolic pressure and pulmonary artery pressure are measured. The animals' hearts and lungs are processed using Masson staining to observe pathological changes in the pulmonary blood vessels to determine the preventive and / or therapeutic effect of Hpapia on PAH.

[0006] The chemical name of the isonicotinic acid Xipapiamide is 2-(2-{[3-(3-hydroxypiperidin-1-yl)propyl]amino}pyrimidin-4-yl)isonicotinic acid, and the molecular formula is C 18 H 23 N5O3, has the following structure:

[0007]

[0008] The present invention is achieved through the following technical solutions.

[0009] The present invention provides a medicine, characterized by comprising Hipapia isonicotinic acid (hereinafter referred to as Hpapia), for treating PAH.

[0010] As a further improvement of the present invention, the administration method of the drug includes at least one of intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual administration, intralesional or intracerebral delivery, and spray administration.

[0011] As a further improvement of the present invention, the drug includes a preparation made of Hpapia and a pharmaceutically acceptable carrier or excipient, and the dosage form of the preparation is selected from one of injection, capsule, tablet, granule, suspension, emulsion, spray, powder, liposome, oral solution, and pill.

[0012] The present invention provides use of the above-mentioned drug containing Hpapia in preventing and / or treating PAH.

[0013] As a further improvement of the present invention, PAH is caused by abnormal proliferation of pulmonary arterial smooth muscle. When PASMCs transform from a quiescent contractile phenotype to a proliferative synthetic phenotype, they undergo excessive proliferation and apoptosis. This abnormal proliferation directly leads to thickening of the pulmonary artery wall. Simultaneously, PASMCs also undergo a certain degree of migration, which, combined with their abnormal proliferation, causes pulmonary vascular remodeling. In both cases, pulmonary vascular resistance increases. Increased pulmonary vascular resistance requires greater force from the right ventricle to propel blood through the pulmonary circulation, leading to the development of PAH.

[0014] As a further improvement of the present invention, the symptoms of PAH include at least one of progressive increase in pulmonary artery pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Such manifestations will become the main basis for determining the prevention and / or treatment of PAH.

[0015] As a further improvement of the present invention, Hpapia is a RhoA kinase (ROCK) inhibitor that inhibits abnormal proliferation of pulmonary arterial smooth muscle by increasing the expression of p27kip1, a downstream factor of ROCK, thereby achieving targeted prevention and / or treatment of PAH.

[0016] The RhoA / Rho kinase (ROCK) signaling pathway plays a key role in the pathogenesis of human PAH by controlling cell proliferation, apoptosis, and contraction by affecting its downstream factors, such as Bcl-2 and Bax, which are related to cell proliferation and apoptosis.

[0017] RhoA kinase (ROCK) is a serine / threonine kinase that, along with Rho proteins, regulates smooth muscle cell adhesion, migration, apoptosis, proliferation, and differentiation, ultimately modulating endothelial barrier function. Key downstream factors of ROCK include p27kip1, a cell cycle-dependent protein kinase inhibitor that blocks the transition from the G1 phase of the cell cycle to the mitotic phase (S phase). It is a crucial regulator of smooth muscle cell proliferation. Activation of ROCK in the pulmonary vasculature inhibits p27kip1 expression, leading to abnormal proliferation of pulmonary vascular smooth muscle cells.

[0018] ROCK inhibitors can target and inhibit ROCK activation, increase the expression of p27kip1, inhibit PASMCs proliferation, inhibit pulmonary artery remodeling, improve pulmonary blood flow distribution, reduce pulmonary artery pressure, reduce right ventricular systolic pressure, and improve endothelial cell dysfunction and inflammatory cell migration.

[0019] As a further improvement of the present invention, the drug prevents PAH and / or stops the progression of PAH symptoms by inhibiting the progressive increase of pulmonary artery pressure, or inhibiting right ventricular hypertrophy, or inhibiting pulmonary vascular remodeling.

[0020] As a further improvement of the present invention, the drug achieves PAH treatment by inhibiting abnormal proliferation of pulmonary arterial smooth muscle, and the therapeutic effects include thinning of the pulmonary vascular wall and enlargement of the lumen diameter.

[0021] As a further improvement of the present invention, the single dosage of Xipapia isonicotinate for treating PAH in adults is 0.24 to 0.80 mg / kg / d.

[0022] The determination of the single dose for adults in the above scheme is based on the calculation method of body surface area between humans and animals in pharmacological experiments. Specifically, the formula for converting the clinical equivalent dose from animals to adults is: rat dose / human dose = rat specific surface area / human specific surface area. After adjustment, it can be obtained: human dose = rat dose / (rat specific surface area / human specific surface area). Generally speaking, the average body surface area of ​​a 70kg adult is 1.73m 2 The body surface area of ​​a 200g rat is approximately 305cm 2 , the specific surface area of ​​humans = 1.73 / 70 = 0.024, the specific surface area of ​​rats = 305 / 200 / 10 = 0.152. In the embodiments of the present invention, the preferred range of the rat dosage is 1.5 to 5 mg / kg / d. According to the above formula, the preferred range of the single-dose dosage for adults can be calculated to be 0.24 to 0.80 mg / kg / d.

[0023] The beneficial effects of the present invention include:

[0024] (1) Xipapillary isonicotinic acid prevents PAH and / or stops the progression of PAH symptoms by inhibiting the progressive increase of pulmonary artery pressure, right ventricular hypertrophy, or pulmonary vascular remodeling;

[0025] (2) Xipapillary isonicotinic acid inhibits the abnormal proliferation of PASMCs, causing the pulmonary vascular wall to thin and the lumen diameter to increase, thereby achieving the treatment of PAH;

[0026] (3) Isonicotinate Xipapillary inhibits the activation of ROCK and the proliferation of PASMCs, thereby preventing and / or treating PAH. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings to help understand the objects and advantages of the present invention, wherein:

[0028] Figure 1 This is the structural diagram of xipapia isonicotinic acid;

[0029] Figure 2 This is a graph showing the inhibitory effect of various concentrations of xipapia isonicotinic acid on the proliferation of rat smooth muscle cells in Example 1;

[0030] Figure 3This is a graph showing the effect of xipapia isonicotinic acid on hemodynamics in rats with pulmonary hypertension in Example 2;

[0031] Figure 4 This is the Masson staining result of the heart of the pulmonary hypertension rat in Example 2;

[0032] Figure 5 This is the HE staining result of pulmonary blood vessels in rats with pulmonary hypertension in Example 2. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and implementation examples.

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention, rather than to represent all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] (1) Pulmonary smooth muscle cells (PASMCs) survival rate detection (MTT method) experiment

[0037] 1. Take the 2nd to 3rd generation PASMCs cells with good growth conditions, add trypsin to digest for 30 seconds, add appropriate amount of fetal bovine serum under sterile conditions to terminate the digestion, and gently pipette to make a cell suspension.

[0038] 2. Place the cell suspension in a centrifuge, set the centrifuge speed to 1000r / min, and centrifuge for 5 minutes.

[0039] 3. After centrifugation and cell counting, PASMCs were evenly seeded into 96-well culture plates (100 μl / well, approximately 5×10 cells per well). 3 The four wells of the 96-well plate were filled with phosphate buffered saline (PBS) to avoid edge effects.

[0040] 4. Place the plated PASMCs in an incubator at 37°C and 5% CO2 for 24 hours. After the cells have attached, discard the supernatant.

[0041] 5. Except for the control group, 100 μl of platelet-derived growth factor BB (PDGF-BB) was added to each well to a final concentration of 20 ng / ml. An equal amount of serum-free culture medium was added to the control group.

[0042] 6. Add H. papillomavirus (H. papillomavirus) at different concentrations (1, 2, 4, 6, 8, 16, and 32 μM) to each well, with six replicate wells for each concentration. After administration, place the cells in an incubator and continue culturing for 24 hours.

[0043] 7. After the incubation period, discard the drugs from each well, wash twice with phosphate buffered saline (PBS), replace each well with 100 μl serum-free DMEM / F12 medium, add 10 μl 5 mg / ml MTT-PBS solution, and continue incubation for 4 hours before terminating the culture.

[0044] 8. Discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well and shake for 10 minutes. Measure the absorbance OD value (A) at 490 nm on a microplate reader. Calculate the inhibition rate based on the absorbance value of each well. The experimental results are as follows: Figure 2 shown.

[0045] Microscopic observations revealed that untreated PASMCs had a low density, large intercellular spaces, and a uniform distribution. However, after stimulation with PDGF-BB, the cell density increased significantly, the intercellular spaces decreased, and the distribution became very compact, indicating a trend of abnormal proliferation. Cell survival rates were significantly higher than those in the normal group, demonstrating the success of the model. Following administration of different concentrations of Hpapia, cell survival rates decreased to varying degrees, demonstrating that Hpapia effectively inhibits the abnormal proliferation of PASMCs.

[0046] Example 2

[0047] 1. 60 SD rats were randomly divided into 4 groups, 15 in each group. The rats were weighed, recorded, and labeled as normal group, MCT group, Hpapia treatment group, and Fasudil group.

[0048] 2. Except for the normal group, rats in all other groups received a single intraperitoneal injection of 0.5 mL of 2% MCT solution (55 mg / kg). Two hours after modeling, rats in each group received an equal volume of saline and Hpapia (1.5 mg / kg / d) intraperitoneally, and Fasudil (5 mg / kg / d) was administered orally starting on day 1 and continuing for 21 days.

[0049] 3. On the 21st day after modeling, the rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital and fixed on the operating table. A longitudinal incision was made on the right side of the neck, the right external jugular vein was bluntly separated, the distal end was ligated, and about 1 / 3 of the external jugular vein was cut at the proximal end. A pre-filled heparin catheter was slowly inserted into the right atrium through the superior vena cava and then into the right ventricle. The pressure transducer was connected to the biological signal acquisition system, and according to the waveform changes on the display, the pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) were recorded after confirming that the catheter had entered the right ventricle and pulmonary artery.

[0050] 4. After the data is stable for 10 to 15 minutes, save it, remove the catheter, and ligate the right external jugular vein. Figure 3 As shown, the results showed that after modeling, the RVSP and mPAP of rats in each group increased significantly compared with the normal group, and both pressures decreased after drug treatment.

[0051] In the Fasudil group and after modeling, RVSP and mPAP in all groups of rats significantly increased compared to the normal control group. Both pressures decreased after treatment, with the decrease being particularly pronounced in the positive drug Fasudil and Hpapia groups, indicating that Hpapia can effectively improve hemodynamic changes in PAH rats. The decrease was particularly pronounced in the high-dose group, indicating that Hpapia can effectively improve hemodynamic changes in PAH rats.

[0052] 5. Sacrifice the rats, quickly remove the lung tissue and heart, and rinse with ice-cold physiological saline to obtain bloodless lung tissue and heart. Isolate the right upper lobe of the lung and soak it in 4% paraformaldehyde. Put cotton in the bottle containing the specimen to completely immerse the lung tissue in paraformaldehyde. After fixing at room temperature for 24 hours, perform Masson staining on the heart and HE staining on the lung tissue. The specific staining results are shown below. Figure 4 and Figure 5 shown.

[0053] Masson staining results Figure 4 The normal group showed neatly arranged cardiomyocytes, uniform in size, with normal intercellular matrix and no damage. However, the model group showed uneven cardiomyocytes, irregular arrangement, enlarged intercellular matrix, and significant myocardial fiber rupture, indicating some damage to the heart tissue. Following treatment, both the Fasudil and Hpapia groups showed some improvement, with Hpapia showing more pronounced improvement. This treatment significantly improved PAH-induced lung and heart tissue damage in rats, demonstrating that Hpapia has a beneficial effect on the hearts of PAH rats.

[0054] HE staining results Figure 5 Compared to the normal control group, the model group showed significant thickening of the pulmonary vascular wall, narrowing of the lumen, and significant cell proliferation and migration, indicating a degree of pulmonary vascular remodeling. In contrast, the Hpapia and Fasudil groups showed some thinning of the pulmonary vascular wall and an increase in lumen diameter. The Hpapia group showed greater improvement, with significant thinning of the pulmonary vascular wall and a marked decrease in cell migration and proliferation. This suggests that Hpapia can effectively ameliorate pulmonary vascular remodeling in PAH rats.

[0055] Finally, it should be noted that the above implementation cases are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above implementation cases, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above implementation cases, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the implementation cases of the present invention.

Claims

1. Use of Xipapiia isonicotinic acid in the preparation of a medicament for treating pulmonary hypertension, characterized in that: The chemical name of the isonicotinic acid Xipapiamide is 2-(2-{[3-(3-hydroxypiperidin-1-yl)propyl]amino}pyrimidin-4-yl)isonicotinic acid, and the molecular formula is C 18 H 23 N5O3, has the following structure: 。 2. The use according to claim 1, characterized in that The pulmonary hypertension is caused by abnormal proliferation of pulmonary arterial smooth muscle.

3. The use according to claim 1, characterized in that The drug achieves the treatment of pulmonary hypertension by inhibiting the abnormal proliferation of pulmonary artery smooth muscle.

4. The use according to any one of claims 1 to 3, characterized in that The single dosage of Xipapia isonicotinic acid for treating pulmonary arterial hypertension in adults is 0.24-0.80 mg / kg / d.

5. The use according to any one of claims 1 to 3, characterized in that The administration method of the drug includes at least one of intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual administration, and spray administration.

6. The use according to any one of claims 1 to 3, characterized in that The drug comprises a preparation made from the xipapia isonicotinic acid and a pharmaceutically acceptable carrier, and the dosage form of the preparation is selected from one of injection, capsule, tablet, granule, suspension, emulsion, spray, powder, liposome, oral solution, and dripping pill.

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