A pharmaceutical composition and uses thereof

Abstract

The present application belongs to the technical field of medicine, and particularly relates to a pharmaceutical composition and use thereof. The pharmaceutical composition composed of a PGI2 receptor agonist compound or a pharmaceutical salt thereof and bosentan has a synergistic effect on inhibiting K+ induced pulmonary artery ring tension increase and / or inhibiting PDGF induced human pulmonary artery smooth muscle cell proliferation. The combined pharmaceutical composition is more helpful for treating the disease of a patient with pulmonary arterial hypertension which cannot be controlled by a single drug. The combined pharmaceutical composition has a stronger synergistic effect on inhibiting K+ induced pulmonary artery ring tension increase and / or inhibiting PDG induced human pulmonary artery smooth muscle cell proliferation than the combined use of the existing drug selexipag and bosentan at a specific ratio and test concentration. Therefore, the combined pharmaceutical composition at a specific ratio and test concentration is more helpful for treating the disease of a patient with moderate to severe pulmonary arterial hypertension which cannot be controlled by the combined use of the existing drug selexipag and bosentan.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application CN202311824286.0, filed on December 27, 2023, and to Chinese patent application CN202410169645.1, filed on February 6, 2024, the full text of which is incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of drug combination therapy, specifically relating to a drug composition and its use for treating pulmonary hypertension. Background Technology

[0004] Pulmonary arterial hypertension (PAH) is a progressive, life-threatening disease characterized by vasoconstriction and vascular remodeling (i.e., intimal hyperplasia, medial hypertrophy, and fibrosis) within the pulmonary arterioles, leading to a gradual increase in mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance (PVR), often resulting in fatal right heart failure. The pathogenesis of PAH is not fully understood. Endothelial damage caused by external stimuli (such as hypoxia or anorexia) or systemic processes (such as connective tissue diseases) can trigger neurohormonal imbalances in susceptible individuals and is considered a focal point or lesion for disease progression. The result is an overproduction of mediators such as endothelin-1, angiotensin II, thromboxane A2, and serotonin, leading to vasoconstriction, smooth muscle cell proliferation, and increased profibrotic and / or prothrombotic effects. Simultaneously, the production of nitric oxide (NO) and prostacyclin (PGI2) is reduced; this imbalance ultimately leads to pulmonary vascular disease.

[0005] The mechanisms that facilitate these pathological effects are complex. This complexity is highlighted by the fact that these mediators operate through numerous overlapping secondary messenger systems. Given this inherent redundancy, many drugs used to treat PAH show short-term efficacy but, in the long run, "fail to meet the standard" in reversing or halting the progression of the disease.

[0006] The redundancy of this pathway justifies the use of combinations of drugs with different mechanisms of action to treat PAH. Combination therapy, involving existing therapies or new drug combinations with improved pharmacodynamic properties, represents a new clinical paradigm for patients with suboptimal responses, further reducing PAH-related morbidity and mortality.

[0007] Therefore, there is a clinical need to find combination drug combinations for the treatment of pulmonary hypertension, so as to provide more and better medication options for patients with different disease conditions. Summary of the Invention

[0008] In view of this, the present invention provides a combination pharmaceutical composition for treating pulmonary hypertension and its application.

[0009] To address the aforementioned technical problems, one object of the present invention is to provide a pharmaceutical composition comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is a PGI2 receptor agonist compound represented by general formula I or a pharmaceutically acceptable salt thereof, and the second active ingredient is bosentan; the compound represented by general formula I or a pharmaceutically acceptable salt thereof is a PGI2 receptor agonist.

[0010]

[0011] In the formula, R1 and R2 are independently selected from H, C1-C3 alkyl or halogen atoms, respectively;

[0012] Z is selected from CR7 or N atoms, wherein R7 is selected from H or C1-C3 alkyl, halogen atom or halo-C1-C3 alkyl;

[0013] R3 is selected from C2-C4 alkyl groups, C 3- C6 monocyclic cycloalkyl;

[0014] R4 is selected from H, C1-C3 alkoxy groups, or 3-6 membered heterocyclic groups;

[0015] R5 represents OH, OR6, or NHSO2R6, where R6 represents C. 1-4 Alkyl or halogen-substituted C 1-4 Alkyl groups; carboxyl groups react with R5 to form acids, esters, or sulfonamides;

[0016] Indicates a single bond or a double bond.

[0017] In one embodiment, the compound of general formula I or a pharmaceutically acceptable salt thereof, wherein Z is an N atom; or Z is CR7, where R7 is selected from H or CH3.

[0018] In one of the implementation schemes, Representing a double bond, R4 is H, and its structural formula is shown in Equation II:

[0019]

[0020] In Formula II, R1 and R2 are selected from H, methane, or F; Z is an N atom, or Z is CR7, and R7 is selected from H or CH3, more preferably H; R3 is selected from isopropyl, ethyl, or cyclopropane, more preferably isopropyl; R5 is selected from OH or NHSO2R6, and R6 is selected from C 1-4 Alkyl or halogen-substituted C 1-4 Alkyl, R6 more preferably methyl.

[0021] In one of the implementation schemes, This represents a single bond, and its structural formula is shown in Equation III:

[0022]

[0023] In Formula III, R4 is selected from H, C1-C3 alkoxy, or 3-6 membered heterocyclic cycloalkanes, preferably H, methoxy, ethoxy, or... More preferably, R4 is H, methoxy, or ethoxy.

[0024] In one embodiment, the compound of general formula I or general formula III or a pharmaceutically acceptable salt thereof is involved, wherein R1 and R2 are selected from H, methane or F; Z is an N atom, or Z is CR7, R7 is selected from H or CH3, more preferably an H atom; R3 is selected from isopropyl, ethyl or cyclopropane, preferably R3 is isopropyl; R5 is preferably OH or NHSO2CH3.

[0025] The compounds of general formula I are selected from, but not limited to, the specific compounds (1)-(15) with the following structural formulas. The code names of each specific compound are also shown in the structural formula. Compounds (1)-(15) are named SYN001, SYN002, SYN003, SYN004, SYN005, SYN006, SYN007, SYN008, SYN013, SYN010, SYN017, SYN044, SYN046, SYN018, and SYN045, respectively.

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] Information regarding the chemical names and preparation methods of the above-mentioned compounds of general formulas I, II, and III and their pharmaceutically usable salts, as well as the specific compounds (1)-(14) and their pharmaceutically usable salts, is disclosed in the Chinese patent application document CN202410701014.X. Information regarding the chemical name and preparation method of the specific compound (15) and its pharmaceutically usable salt is disclosed in the Chinese patent application document CN202410701022.4.

[0035] Through extensive experiments, the inventors discovered that the PGI2 receptor agonist compound or specific compound (1)-(15) shown in general formula I, when used in combination with bosentan, exhibits strong synergistic anti-pulmonary hypertension activity, thereby enhancing the anti-pulmonary hypertension activity.

[0036] The PGI2 receptor agonist compound or specific compound (1)-(15) shown in Formula I, when used in combination with bosentan, has a synergistic effect on inhibiting K+-induced increase in pulmonary artery ring tension and / or inhibiting PDGF-induced proliferation of human pulmonary artery smooth muscle cells. The pharmaceutical composition is more helpful in the treatment of pulmonary hypertension patients whose disease cannot be resolved by monotherapy.

[0037] In some embodiments, the molar ratio of the first active ingredient to the second active ingredient is 1:6-3:1; the preferred molar ratio of the first active ingredient to the second active ingredient is 1:5-2:1; further preferred is a molar ratio of 1:5, 1:1 or 2:1; more preferably a molar ratio of 1:1, at which the combination index (CI) value is relatively lower and the synergistic effect is stronger.

[0038] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (1).

[0039] Furthermore, the molar ratio of the specific compound (1) to bosentan is 1:1-2:1, preferably 1:1 or 2:1.

[0040] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (2).

[0041] Furthermore, the molar ratio of the specific compound (2) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5, and more preferably 1:5.

[0042] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (3).

[0043] Furthermore, the molar ratio of the specific compound (3) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5, and more preferably 1:5.

[0044] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (4).

[0045] Furthermore, the molar ratio of the specific compound (4) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0046] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (5).

[0047] Furthermore, the molar ratio of the specific compound (5) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0048] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (6).

[0049] Furthermore, the molar ratio of the specific compound (6) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0050] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (7).

[0051] Furthermore, the molar ratio of the specific compound (7) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0052] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (8).

[0053] Furthermore, the molar ratio of the specific compound (8) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5, and more preferably 1:5.

[0054] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (9).

[0055] Furthermore, the molar ratio of the specific compound (9) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5, and more preferably 1:1.

[0056] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (10).

[0057] Furthermore, the molar ratio of the specific compound (10) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0058] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (11).

[0059] Furthermore, the molar ratio of the specific compound (11) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0060] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (12).

[0061] Furthermore, the molar ratio of the specific compound (12) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0062] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (13).

[0063] Furthermore, the molar ratio of the specific compound (13) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0064] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (14).

[0065] Furthermore, the molar ratio of the specific compound (14) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5, and more preferably 1:5.

[0066] In some embodiments, the pharmaceutical composition wherein the first active ingredient is a specific compound (15).

[0067] Furthermore, the molar ratio of the specific compound (15) to bosentan is 1:5-2:1, preferably 1:1, 2:1 or 1:5.

[0068] The synergistic effect of the PGI2 receptor agonist compound represented by general formula I or specific compounds (1)-(15) in combination with bosentan at the above-mentioned specific ratio and test concentration is stronger than that of the combination of the existing drug selexipag and bosentan in inhibiting K+-induced increase in pulmonary artery ring tension and / or inhibiting PDGF-induced proliferation of human pulmonary artery smooth muscle cells. Therefore, the combination drug composition has a better anti-pulmonary hypertension effect and is more helpful in the treatment of moderate to severe pulmonary hypertension patients whose condition cannot be controlled by the existing drug combination of selexipag and bosentan.

[0069] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients; and its dosage form may be a tablet, granule, capsule, powder, or injection.

[0070] Another object of the present invention is to provide the use of the above-described pharmaceutical composition in the preparation of a combination medicament for treating pulmonary hypertension. Attached Figure Description

[0071] Figure 1The combined drug index CI dot plot is a comparison of the ring tension inhibition rate of isolated pulmonary artery rings when PGI2 receptor agonist compounds (1)-(15) and selexipag were combined with bosentan (1:1) in Experiment Example 1.

[0072] Figure 2 The CI dot plot is a comparison of the inhibition rates of human pulmonary artery smooth muscle cell proliferation by PGI2 receptor agonist compounds (1)-(15) and selepag in combination with bosentan (2:1) in Experiment Example 2. Detailed Implementation

[0073] The present invention will be further described in detail below through specific embodiments, but this is only for the purpose of helping to understand the present invention and enabling those skilled in the art to implement or use the present invention, and does not constitute any limitation on the present invention.

[0074] Compounds SYN001, SYN002, SYN003, SYN004, SYN005, SYN006, SYN007, SYN008, SYN013, SYN010, SYN017, SYN044, SYN046, and SYN018 were prepared according to the preparation methods of Examples 1-14 in Patent CN202410701014.X, and compound SYN045 was prepared according to the preparation method of Example 1 in Patent CN202410701022.4.

[0075] Purchased commercially available selepag active pharmaceutical ingredient from Cangzhou Enke Pharmaceutical Technology Co., Ltd., batch number 20221231; purchased commercially available bosentan active pharmaceutical ingredient from Patheon Inc., Canada, batch number IP036A01.

[0076] Example

[0077] PGI2 receptor agonist compounds SYN001, SYN002, SYN003, SYN004, SYN005, SYN006, SYN007, SYN008, SYN010, SYN013, SYN017, SYN018, SYN044, SYN045, and SYN046 were formulated with bosentan at molar ratios of 2:1, 1:1, and 1:5, respectively, to prepare combination drug compositions.

[0078] The following experiments were conducted on the isolated pulmonary artery ring tension and the inhibition rate of human pulmonary artery smooth muscle cell proliferation of the above-mentioned multiple combined drug combinations.

[0079] Experimental Example 1: Comparative Study of the Tension of Isolated Pulmonary Artery Rings Treated in Combination with PGI2 Receptor Agonists and Bosentan

[0080] 1. Experimental procedure:

[0081] S1: Rats were anesthetized by intraperitoneal injection of 25% urethane (5 mL / kg). The heart and lungs were separated by thoracotomy. The pulmonary artery vessels were isolated under a microscope, and vascular rings with a length of 2-3 mm were prepared and hung on hooks on tension transducers. The prepared pulmonary artery vascular rings were immersed in incubation solution and finely adjusted to a basic tension of about 0.8-1 g. After stabilization for 1-2 hours, the basic tension was measured.

[0082] S2: Using a high-K+ incubation solution, the tension of the pulmonary artery vascular ring was increased, and the maximum tension induced by high-K+ in the pulmonary artery vascular ring was recorded.

[0083] S3: Then, incubation solutions and the drug combination to be tested were added to multiple incubation tanks respectively. As a comparison, incubation solutions containing single drugs, selexigate, and the combination of selexigate and bosentan were also prepared. The final drug concentrations in the incubation solutions are shown in Table 1. The pulmonary artery vascular rings were added to the incubation solutions containing the drug, and the changes in the tension of the pulmonary artery rings were recorded. The minimum tension after drug action was measured.

[0084] S4: After observing the maximum efficacy of the drug, replace the incubation solution containing the drug with the basic incubation solution to restore the pulmonary artery ring tension to the basic level.

[0085] 2. Calculate the ring tension suppression rate using the following formula:

[0086] .

[0087] The ring tension inhibition rate of the PGI2 receptor agonist compound combined with bosentan (1:1) is shown in Table 1.

[0088] 3. By inputting the concentrations of PGI2 receptor agonist compounds and their corresponding annular tonic inhibition rates, the concentrations of bosentan and their corresponding annular tonic inhibition rates, and the concentrations of PGI2 receptor agonist compounds combined with bosentan and their corresponding annular tonic inhibition rates, the combination index (CI) at each concentration ratio was calculated using Compusym software. A Fa-CI curve was plotted with the annular tonic inhibition rate (Fa) of the PGI2 receptor agonist compound combined with bosentan as the x-axis and the combination index (CI) as the y-axis.

[0089] The results of the combined drug use index (CI) experiment are shown in Table 2, and the Fa-CI curves are shown in [reference needed]. Figure 1 .

[0090] Table 1. Ring tension inhibition rates of different concentrations of single-drug PGI2 receptor agonist compounds, single-drug bosentan, and combinations of PGI2 receptor agonist compounds and bosentan (1:1) in isolated pulmonary artery ring tension comparison experiments.

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Table 2. Combination index (CI) of different concentrations of PGI2 receptor agonist compounds and bosentan in different proportions in the isolated pulmonary artery ring tension comparison test (n=5)

[0100]

[0101]

[0102]

[0103]

[0104] Conclusion: The combination of PGI2 receptor agonist compounds and bosentan at 3 different ratios and 5 concentrations was tested for the inhibition rate of ring tension of isolated pulmonary artery rings by the combination index (CI). When CI was less than 1, the two drugs had a synergistic effect, and the lower the CI value, the stronger the synergistic effect. When CI was equal to 1, the two drugs had an additive effect. When CI was greater than 1, the two drugs had an antagonistic effect.

[0105] At three ratios (referring to a molar ratio of PGI2 receptor agonist compound to bosentan of 2:1, 1:1, and 1:5, the same below) and five concentrations (as shown in Table 2, referring to the five test concentrations corresponding to each PGI2 receptor agonist compound; for example, the concentrations of SYN007 and SYN008 are 1 μM, 2 μM, 4 μM, 8 μM, and 16 μM, respectively, not the bosentan concentration, which is calculated based on the corresponding combination drug ratio, the same below), the combination index (CI) values ​​of SYN007, SYN008, and bosentan were all no higher than 0.7, indicating a synergistic effect. Particularly at the 1:1 ratio of the five concentrations, the CI values ​​were even lower, all no higher than 0.65. In contrast, the comparative studies showed CI values ​​greater than or equal to 1 at both the 2:1 and 1:5 ratios, indicating no synergistic effect, and all CI values ​​were higher than 0.85. At the 1:1 ratio, the CI values ​​were approximately between 0.76 and 0.96. Furthermore, SYN007 and SYN008 showed lower CI values ​​compared to the comparative studies under the same conditions. This suggests that at a molar ratio of 1:5 to 2:1 between the PGI2 receptor agonist compound and bosentan, the synergistic effect of SYN007 and SYN008 in combination with bosentan at concentrations of 1 μM to 16 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the comparative studies.

[0106] At a molar ratio of 2:1 and a concentration of 1.6 μM, the CI value of the combination of SYN001 and bosentan was 0.67, which was significantly lower than the CI value of 0.88 of the control group under the same conditions. This indicates that at a molar ratio of 2:1 for SYN001 and bosentan and a concentration of 1.6 μM, the synergistic effect of SYN001 and bosentan in inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0107] At three different ratios and a concentration of 16 μM, the CI values ​​for the combination of SYN002 and bosentan were 0.53–0.68, significantly lower than the CI values ​​of 0.76–0.89 for the control group under the same conditions. This indicates that at a molar ratio of 1:5–2:1 for SYN002 to bosentan, the synergistic effect of SYN002 at a concentration of 16 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0108] At three different ratios and a concentration of 8 μM, the CI values ​​for the combination of SYN003 and bosentan were 0.62–0.71, significantly lower than the CI values ​​of 0.82–0.95 for the control group under the same conditions. This indicates that at a molar ratio of 1:5–2:1 for SYN003 to bosentan, the synergistic effect of SYN003 at a concentration of 8 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0109] At three different ratios and a concentration of 4 μM, the CI values ​​for the combination of SYN004 and bosentan were 0.70–0.75, significantly lower than the CI values ​​of 0.90–0.96 for the control group under the same conditions. This indicates that at a molar ratio of 1:5–2:1 for SYN004 to bosentan, the synergistic effect of SYN004 at a concentration of 4 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0110] At three ratios and concentrations of 2 μM, 8 μM, and 16 μM, the CI values ​​of the combination of SYN005 and bosentan were 0.47–0.78, significantly lower than the CI values ​​of 0.76–0.97 for the control group under the same conditions. This indicates that at a molar ratio of SYN005 to bosentan of 1:5–2:1, the synergistic effect of SYN005 combined with bosentan at concentrations of 2 μM, 8 μM, and 16 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0111] At three ratios and concentrations of 1 μM and 4 μM, the CI values ​​of the combination of SYN006 and bosentan were 0.64-0.78, significantly lower than the CI values ​​of 0.90-1.02 for the control group under the same conditions. This indicates that at a molar ratio of 1:5 to 2:1, the synergistic effect of SYN006 and bosentan at concentrations of 1 μM and 4 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0112] At three ratios and concentrations of 4 μM, 8 μM, and 16 μM, the CI values ​​for the combination of SYN010 and bosentan were 0.44–0.71, significantly lower than the CI values ​​of 0.76–0.96 for the control group under the same conditions. This indicates that at a molar ratio of 1:5–2:1 for SYN010 to bosentan, the synergistic effect of SYN010 and bosentan at concentrations of 4 μM–16 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0113] At three ratios and concentrations of 1 μM, 2 μM, and 4 μM, the CI value of the combination of SYN013 and bosentan was no higher than 0.8, which was significantly lower than the CI value of 0.9-1.02 of the control group under the same conditions. This indicates that at a molar ratio of SYN013 to bosentan of 1:5-2:1, the synergistic effect of SYN013 and bosentan at concentrations of 1 μM-4 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0114] At three ratios and concentrations of 2μM, 4μM, 8μM, and 16μM, the CI value of the combination of SYN017 and bosentan was no higher than 0.73, which was significantly lower than the CI value of 0.76-0.97 of the control group under the same conditions. This indicates that at a molar ratio of SYN017 to bosentan of 1:5-2:1, the synergistic effect of SYN017 and bosentan at concentrations of 2μM-16μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0115] At three ratios and concentrations of 1 μM, 8 μM, and 16 μM, the CI values ​​for the combination of SYN018 and bosentan were 0.46–0.81, significantly lower than the CI values ​​of 0.76–1.02 for the control group under the same conditions. This indicates that at a molar ratio of 1:5 to 2:1, the synergistic effect of SYN018 and bosentan at concentrations of 1 μM, 8 μM, and 16 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0116] At three ratios and concentrations of 8 μM and 16 μM, the CI values ​​of the combination of SYN044 and bosentan were 0.3-0.62, significantly lower than the CI values ​​of 0.76-0.95 for the control group under the same conditions. This indicates that at a molar ratio of SYN044 to bosentan of 1:5-2:1, the synergistic effect of SYN044 and bosentan at concentrations of 8 μM and 16 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0117] At three different ratios and a concentration of 16 μM, the CI values ​​for the combination of SYN045 and bosentan were 0.53–0.66, significantly lower than the CI values ​​of 0.76–0.89 for the control group under the same conditions. This indicates that at a molar ratio of SYN045 to bosentan of 1:5–2:1, the synergistic effect of SYN045 at a concentration of 16 μM on the inhibition of ring tension in isolated pulmonary artery rings was significantly better than that of the control group.

[0118] At three different ratios and a concentration of 8 μM, the CI values ​​for the combination of SYN046 and bosentan were 0.58–0.67, significantly lower than the CI values ​​of 0.82–0.95 for the control group under the same conditions. This indicates that at a molar ratio of 1:5–2:1 for SYN046 to bosentan, the synergistic effect of SYN046 at a concentration of 8 μM on inhibiting the ring tension of isolated pulmonary artery rings was significantly better than that of the control group.

[0119] The above results indicate that the PGI2 receptor agonist compounds SYN001, SYN002, SYN003, SYN004, SYN005, SYN006, SYN007, SYN008, SYN010, SYN013, SYN017, SYN018, SYN044, SYN045, and SYN046, when used in combination with bosentan at the specific ratios and test concentrations mentioned above, have a synergistic effect in inhibiting the increase in K+-induced pulmonary artery ring tension. Furthermore, the synergistic effect was superior to that of selexipag combined with bosentan. The synergistic effect of SYN007 and SYN008 combined with bosentan was particularly prominent. The synergistic effect was better than that of the control group at 3 ratios and 5 test concentrations. This indicates that the synergistic effect of SYN007 and SYN008 combined with bosentan at a molar ratio of 1:5-2:1 and at a concentration of 1μM-16μM, on the inhibition of human pulmonary artery smooth muscle cell proliferation is very significant.

[0120] Experimental Example 2: Comparative Study on the Inhibition Rate of Human Pulmonary Artery Smooth Muscle Cell Proliferation by Combination of PGI2 Receptor Agonist Compounds and Bosentan

[0121] Evaluating drug efficacy using cell models reveals that excessive proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) are prominent features of vascular remodeling. In PAH, pulmonary vascular smooth muscle cells transform from a contractile phenotype to a proliferative synthetic phenotype, primarily manifested as cell proliferation and hypertrophy. These pathological changes ultimately lead to pulmonary vascular lumen narrowing and decreased wall compliance, thereby promoting the development and progression of PAH. Therefore, PASMCs can be used for drug screening in pulmonary hypertension. The PDGF-induced human pulmonary artery smooth muscle cell proliferation model is a classic model for evaluating pulmonary hypertension therapeutic drugs at the cellular level.

[0122] The experimental drugs, PGI2 receptor agonist compounds SYN001, SYN002, SYN003, SYN004, SYN005, SYN006, SYN007, SYN008, SYN010, SYN013, SYN017, SYN018, SYN044, SYN045, and SYN046, and the control drug, PGI2 receptor agonist selexiparg, were combined with bosentan at molar ratios of 2:1, 1:1, and 1:5, respectively, to conduct a comparative experiment on the inhibition rate of human pulmonary artery smooth muscle cell proliferation, in order to investigate the effect of the combined administration of PGI2 receptor agonist compounds and bosentan on PDGF-induced human pulmonary artery smooth muscle cell proliferation.

[0123] Experimental procedure:

[0124] Control group preparation: Human pulmonary artery smooth muscle cells were digested with 0.25% trypsin solution, seeded at 6×103 / well in 96-well plates (final volume 100μL) and cultured for 24h. Then, they were starved in serum-free Dulbecco's modified eagle medium (DMEM) high glucose medium for 24h. 100μL of DMEM was added again and cultured for 24h to obtain the control group.

[0125] Model group preparation: Human pulmonary artery smooth muscle cells were digested with 0.25% trypsin solution and seeded at 6 × 10³ cells / well in 96-well plates (final volume 100 μL). After culturing for 24 h, the cells were starved in serum-free Dulbecco's modified eagle medium (DMEM) high-glucose medium for 24 h. Then, 100 μL of DMEM containing human platelet-derived growth factor (hPDGF-BB) was added to a final concentration of 20 ng / mL and cultured for 24 h to obtain the model group.

[0126] Preparation of the test group: Human pulmonary artery smooth muscle cells were digested with 0.25% trypsin solution and seeded at 6 × 10³ cells / well in 96-well plates (final volume 100 μL). After culturing for 24 h, the cells were starved in serum-free Dulbecco's modified eagle medium (DMEM) high-glucose medium for 24 h. Human platelet-derived growth factor (hPDGF-BB) was added to DMEM to a final concentration of 20 ng / mL. The pre-prepared single-drug solutions of different concentrations of PGI2 receptor agonist compounds, bosentan single-drug solutions, and combined solutions of PGI2 receptor agonist compounds and bosentan were then serially diluted with DMEM containing hPDGF-BB to the concentrations shown in Tables 3 and 4 to obtain the test solutions. The test solutions were added to the starved cell culture wells at a volume of 100 μL and cultured for 24 h to obtain the test group.

[0127] CellTiter-Glo reagent was added to the culture wells of the test group, control group, and model group. The luminescence value was measured, cell viability was detected, and the cell proliferation inhibition rate was calculated.

[0128] Formula for calculating cell proliferation inhibition rate:

[0129] .

[0130] By inputting the concentrations of PGI2 receptor agonist compounds and their corresponding cell proliferation inhibition rates, the concentrations of bosentan and their corresponding cell proliferation inhibition rates, and the concentrations of PGI2 receptor agonist compounds and bosentan in combination and their corresponding cell proliferation inhibition rates, the combination drug index CI was calculated using Compusym software at each concentration ratio. A Fa-CI curve was plotted with the cell proliferation inhibition rate Fa of the combination of PGI2 receptor agonist compounds and bosentan as the abscissa and the combination drug index CI as the ordinate.

[0131] The experimental results are shown in Tables 3 and 4. Figure 2 .

[0132] Table 3. Cell proliferation inhibition rates of different concentrations of single-drug PGI2 receptor agonist compounds, single-drug bosentan, and combinations of PGI2 receptor agonist compounds and bosentan (2:1) in a comparative study on the inhibition rate of human pulmonary artery smooth muscle cell proliferation.

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] Table 4. Combination index (CI) of different concentrations of PGI2 receptor agonist compounds and bosentan in different proportions during a comparative study on the inhibition rate of human pulmonary artery smooth muscle cell proliferation (n=5).

[0142]

[0143]

[0144]

[0145]

[0146] Conclusion: The combination index (CI) of PGI2 receptor agonist compounds and bosentan was tested at 5 concentrations in 3 ratios to measure the inhibition rate of human pulmonary artery smooth muscle cell proliferation. When CI was less than 1, the two drugs had a synergistic effect, and the lower the CI value, the stronger the synergistic effect. When CI was equal to 1, the two drugs had an additive effect. When CI was greater than 1, the two drugs had an antagonistic effect.

[0147] At three ratios (referring to the molar ratio of PGI2 receptor agonist compound to bosentan being 2:1, 1:1, and 1:5, the same below) and five concentrations (as shown in Table 4, referring to the five test concentrations corresponding to each PGI2 receptor agonist compound, such as SYN005 concentrations of 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM, and 3.2 μM, not bosentan concentrations; the bosentan concentrations were calculated based on the corresponding combination drug ratios, the same below), the combination index CI values ​​of SYN005 and bosentan were all no higher than 0.5, indicating a synergistic effect. The CI values ​​of the comparative samples ranged from 0.74 to 0.97 across the three ratios. Furthermore, SYN005 showed a significantly lower CI value compared to the comparative samples under the same conditions. This indicates that at a molar ratio of SYN005 to bosentan of 1:5 to 2:1, and at concentrations of 0.2 μM to 3.2 μM, the synergistic effect of SYN005 in inhibiting the proliferation of human pulmonary artery smooth muscle cells was significantly better than that of the comparative samples.

[0148] At a 1:1 ratio and a concentration of 0.2 μM, the CI value of the combination of SYN001 and bosentan was 0.74, which was significantly lower than the CI value of 0.93 of the control group under the same conditions. This indicates that at a molar ratio of 1:1 for SYN001 and bosentan, the synergistic effect of the combination of SYN001 and bosentan at a concentration of 0.2 μM on the inhibition of human pulmonary artery smooth muscle cell proliferation was significantly better than that of the control group.

[0149] At a molar ratio of 1:5 and a concentration of 0.2 μM, the CI value of the combination of SYN002 and bosentan was 0.77, which was significantly lower than the CI value of 0.97 of the control group under the same conditions. This indicates that at a molar ratio of 1:5 for SYN002 and bosentan and a concentration of 0.2 μM, the synergistic effect of the combination of SYN002 and bosentan on the inhibition of human pulmonary artery smooth muscle cell proliferation was significantly better than that of the control group.

[0150] At a molar ratio of 1:5 and a concentration of 0.2 μM, the CI value of the combination of SYN003 and bosentan was 0.78, which was significantly lower than the CI value of 0.97 of the control group under the same conditions. This indicates that at a molar ratio of 1:5 for SYN003 and bosentan, the synergistic effect of the combination of SYN003 and bosentan at a concentration of 0.2 μM on the proliferation inhibition of human pulmonary artery smooth muscle cells was significantly better than that of the control group.

[0151] At three different ratios and a concentration of 0.2 μM, the CI values ​​for the combination of SYN004 and bosentan were 0.67–0.71, significantly lower than the CI values ​​of 0.93–0.97 for the control group under the same conditions. This indicates that at a molar ratio of 1:5–2:1 for SYN004 to bosentan, the synergistic effect of the combination of SYN004 and bosentan at a concentration of 0.2 μM on the inhibition of human pulmonary artery smooth muscle cell proliferation is significantly better than that of the control group.

[0152] At three different ratios and a concentration of 0.2 μM, the CI values ​​for the combination of SYN006 and bosentan were 0.73-0.75, significantly lower than the CI values ​​of 0.93-0.97 for the control group under the same conditions. This indicates that at a molar ratio of 1:5 to 2:1, the synergistic effect of SYN006 combined with bosentan at a concentration of 0.2 μM on the inhibition of human pulmonary artery smooth muscle cell proliferation is significantly better than that of the control group.

[0153] At three different ratios and a concentration of 0.2 μM, the CI values ​​for the combination of SYN007 and bosentan were 0.70-0.73, significantly lower than the CI values ​​of 0.93-0.97 for the control group under the same conditions. This indicates that at a molar ratio of 1:5 to 2:1, the synergistic effect of SYN007 combined with bosentan at a concentration of 0.2 μM on the inhibition of human pulmonary artery smooth muscle cell proliferation is significantly better than that of the control group.

[0154] At a molar ratio of 1:5 and a concentration of 0.2 μM, the CI value of the combination of SYN008 and bosentan was 0.81, which was significantly lower than the CI value of 0.97 of the control group under the same conditions. This indicates that at a molar ratio of 1:5 for SYN008 and bosentan and a concentration of 0.2 μM, the synergistic effect of the combination of SYN008 and bosentan on the inhibition of human pulmonary artery smooth muscle cell proliferation was significantly better than that of the control group.

[0155] At three different ratios and a concentration of 0.2 μM, the CI values ​​for the combination of SYN010 and bosentan were 0.72–0.74, significantly lower than the CI values ​​of 0.93–0.97 for the control group under the same conditions. This indicates that at a molar ratio of 1:5–2:1 for SYN010 to bosentan, the synergistic effect of SYN010 at a concentration of 0.2 μM on the inhibition of human pulmonary artery smooth muscle cell proliferation was significantly better than that of the control group.

[0156] At a 1:1 ratio and a concentration of 0.2 μM, the CI value of the combination of SYN013 and bosentan was 0.73, which was significantly lower than the CI value of 0.93 of the control group under the same conditions. This indicates that at a molar ratio of 1:1 for SYN013 and bosentan, the synergistic effect of the combination of SYN013 and bosentan at a concentration of 0.2 μM on the inhibition of human pulmonary artery smooth muscle cell proliferation was significantly better than that of the control group.

[0157] At three different ratios and a concentration of 0.2 μM, the CI values ​​for the combination of SYN017, SYN044, and SYN045 with bosentan ranged from 0.62 to 0.76, significantly lower than the CI values ​​of 0.93 to 0.97 for the control group under the same conditions. This indicates that at a molar ratio of 1:5 to 2:1 between the PGI2 receptor agonist compound and bosentan, the synergistic effect of SYN017, SYN044, and SYN045 in inhibiting the proliferation of human pulmonary artery smooth muscle cells at a concentration of 0.2 μM was significantly better than that in the control group.

[0158] At three ratios and concentrations of 0.2 μM and 1.6 μM, the CI values ​​for the combination of SYN046 and bosentan were 0.62–0.67, significantly lower than the CI values ​​of 0.85–0.97 for the control group under the same conditions. This indicates that at a molar ratio of SYN046 to bosentan of 1:5–2:1, the synergistic effect of SYN046 combined with bosentan at concentrations of 0.2 μM and 1.6 μM on the inhibition of human pulmonary artery smooth muscle cell proliferation was significantly better than that of the control group.

[0159] At a molar ratio of 1:5 and a concentration of 0.4 μM, the CI value of the combination of SYN018 and bosentan was 0.76, which was significantly lower than the CI value of 0.92 of the control group under the same conditions. This indicates that at a molar ratio of 1:5 for SYN018 and bosentan and a concentration of 0.4 μM, the synergistic effect of the combination of SYN018 and bosentan on the inhibition of human pulmonary artery smooth muscle cell proliferation was significantly better than that of the control group.

[0160] The above results indicate that the PGI2 receptor agonist compounds SYN001, SYN002, SYN003, SYN004, SYN005, SYN006, SYN007, SYN008, SYN010, SYN013, SYN017, SYN018, SYN044, SYN045, and SYN046, when used in combination with bosentan at the specific ratios and test concentrations mentioned above, exhibit a synergistic effect on inhibiting PDGF-induced proliferation of human pulmonary artery smooth muscle cells. This synergistic effect is superior to that of selexipag combined with bosentan. The synergistic effect of SYN005 combined with bosentan is particularly pronounced, showing better synergistic effects than the control group at all three ratios and five test concentrations. This demonstrates that the synergistic effect of SYN005 combined with bosentan at molar ratios of 1:5-2:1 and SYN005 concentrations of 0.2 μM-3.2 μM on inhibiting the proliferation of human pulmonary artery smooth muscle cells is very significant.

[0161] In summary, the PGI2 receptor agonist compounds SYN001, SYN002, SYN003, SYN004, SYN005, SYN006, SYN007, SYN008, SYN010, SYN013, SYN017, SYN018, SYN044, SYN045, and SYN046, when used in combination with bosentan at the specific ratios and test concentrations described above, exhibit a synergistic effect in inhibiting K+-induced pulmonary artery ring tension increase and / or inhibiting PDGF-induced human pulmonary artery smooth muscle cell proliferation. Furthermore, this synergistic effect is superior to that of selexipag combined with bosentan.

[0162] Among them, SYN007 and SYN008, when used in combination with bosentan, showed a more pronounced synergistic effect in inhibiting K+-induced increases in pulmonary artery ring tension, with superior synergistic effects compared to the control group at all three ratios and five tested concentrations. Meanwhile, SYN005, when used in combination with bosentan, showed a more pronounced synergistic effect in inhibiting PDGF-induced proliferation of human pulmonary artery smooth muscle cells, with superior synergistic effects compared to the control group at all three ratios and five tested concentrations. The aforementioned PGI2 receptor agonist compounds, when used in combination with bosentan, show broad application prospects in the treatment of pulmonary hypertension.

[0163] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It includes a first active ingredient and a second active ingredient, wherein the first active ingredient is a PGI2 receptor agonist compound represented by general formula II or III or a pharmaceutically acceptable salt thereof, and the second active ingredient is bosentan; In general formulas II and III, R1 and R2 are selected from H, methyl, or F; Z represents an N atom, or Z represents CR7 and R7 represents H; R3 is isopropyl; R5 is selected from OH or NHSO2CH3; In general formula III, R4 is selected from H, methoxy, ethoxy, or ; The molar ratio of the first active ingredient to the second active ingredient is 1:6-3:

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The first active ingredient is selected from specific compounds (1)-(15) with the following structural formulas: 。 3. The pharmaceutical composition according to any one of claims 1-2, characterized in that, The molar ratio of the first active ingredient to the second active ingredient is 1:5-2:

1.

4. The pharmaceutical composition according to claim 3, characterized in that, The molar ratio of the first active ingredient to the second active ingredient is 1:5, 1:1, or 2:

1.

5. The pharmaceutical composition according to claim 4, characterized in that, The first active ingredient is a specific compound (5), wherein the molar ratio of the specific compound (5) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (7), wherein the molar ratio of the specific compound (7) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (10), wherein the molar ratio of the specific compound (10) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (11), wherein the molar ratio of the specific compound (11) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (12), wherein the molar ratio of the specific compound (12) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (13), wherein the molar ratio of the specific compound (13) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (15), wherein the molar ratio of the specific compound (15) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (6), wherein the molar ratio of the specific compound (6) to bosentan is 1:1, 2:1, or 1:5; or The first active ingredient is a specific compound (4), and the molar ratio of the specific compound (4) to bosentan is 1:1, 2:1 or 1:

5.

6. The pharmaceutical composition according to claim 4, characterized in that, The first active ingredient is a specific compound (8), wherein the molar ratio of the specific compound (8) to bosentan is 1:5; or The first active ingredient is a specific compound (14), wherein the molar ratio of the specific compound (14) to bosentan is 1:5; or The first active ingredient is a specific compound (9), wherein the molar ratio of the specific compound (9) to bosentan is 1:1; or The first active ingredient is a specific compound (3), wherein the molar ratio of the specific compound (3) to bosentan is 1:5; or The first active ingredient is a specific compound (2), wherein the molar ratio of the specific compound (2) to bosentan is 1:5; or The first active ingredient is a specific compound (1), and the molar ratio of the specific compound (1) to bosentan is 1:1 or 2:

1.

7. The pharmaceutical composition according to any one of claims 1-2 and 4-6, characterized in that, It also includes acceptable carriers and / or excipients, and the pharmaceutical composition is in the form of tablets, granules, capsules, powders, or injections.

8. Use of a pharmaceutical composition according to any one of claims 1-7 in the preparation of a combination medicament for treating pulmonary arterial hypertension.

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