Application of phosphodiesterase 4 inhibitor

Abstract

The invention relates to a medicament and a method for treating or preventing vascular diseases. Specifically, the invention relates to a method for treating or preventing vascular endothelial dysfunction related diseases by down-regulating phosphodiesterase 4, and application of a phosphodiesterase 4 inhibitor in preparation of drugs for treating or preventing vascular endothelial dysfunction related diseases.

Description

[0001] This application is a divisional application of the invention patent application filed on April 28, 2020, with application number 202010348196.9 and invention title "Use of Phosphodiesterase 4 Inhibitor". Technical Field

[0002] This invention relates to pharmaceuticals and methods for treating or preventing vascular diseases. Specifically, this invention relates to methods for treating or preventing diseases related to vascular endothelial dysfunction by downregulating phosphodiesterase 4 (PDE4), and the use of PDE4 inhibitors in the preparation of pharmaceuticals for treating or preventing diseases related to vascular endothelial dysfunction. Background Technology

[0003] Vascular endothelial cells participate in a series of processes, including promoting vasodilation, inhibiting smooth muscle proliferation, and suppressing vascular endothelial inflammation, in order to maintain vascular homeostasis. Endothelial dysfunction significantly increases the risk of cardiovascular disease.

[0004] Currently, all drugs that improve vascular endothelial function in clinical practice can cause certain adverse drug reactions in patients. For example, long-term use of statins can cause myalgia, making it difficult for patients to tolerate them long-term; biguanides can cause gastrointestinal disorders or occasionally lactic acidosis; and thiazolidinediones can cause serious side effects such as fluid retention, weight gain, and liver dysfunction. Therefore, it is essential to seek safe and effective treatments for diseases related to vascular endothelial dysfunction.

[0005] Phosphodiesterase 4 (PDE4) is a cAMP-metabolizing enzyme that plays a crucial role in regulating inflammation. Inhibition of PDE4, particularly its B isoform (PDE4B), is used to suppress inflammation (Komatsu et al., Nature Communications. 2013; 4:1684). PDE4 inhibitors have been investigated as anti-inflammatory agents due to their ability to inhibit excessive neutrophil activation (Suzuki et al., Acta pharmaceutica (Zagreb, Croatia). 2015; 65:191-197).

[0006] Although existing technologies have reported that phosphodiesterase 4 has a variety of biological activities, there has been no report to date that phosphodiesterase 4 inhibitors can directly alleviate vascular endothelial dysfunction or improve vascular endothelial function and their mechanism of action. Summary of the Invention

[0007] In one aspect, the present invention provides a method for treating or preventing vascular endothelial dysfunction-related diseases in a subject, comprising administering to the subject a pharmaceutical composition containing an effective amount of a phosphodiesterase 4 (PDE4) inhibitor, said inhibitor inhibiting the expression and / or activity of PDE4.

[0008] In another aspect, the present invention relates to the use of a PDE4 inhibitor in the preparation of a pharmaceutical composition for the treatment or prevention of diseases related to vascular endothelial dysfunction, wherein the inhibitor inhibits the expression and / or activity of PDE4.

[0009] In another aspect, the present invention provides a pharmaceutical composition containing an effective amount of a PDE4 inhibitor for treating or preventing diseases related to vascular endothelial dysfunction in subjects, wherein the inhibitor inhibits the expression and / or activity of PDE4.

[0010] In an embodiment of the present invention, the PDE4 inhibitor is a selective PDE4 inhibitor.

[0011] In specific embodiments of the aspects described in this invention, the selective PDE4 inhibitor is selected from piracetam, cyclophosphamide, AN-2728, AN-2898, CBS-3595, ELB-353, KF-66490, K-34, LAS-37779, IBBB-211913, AWD-12-281, cipanthenine, sillolast, roflumilast, BAY19-8004 and SCH-351591, AN-6415, indus-82010, TPI-PD3, ELB-353, CC-11050, GSK-256066, omepiracetam, OX-914, tetostrept, MEM-1414 and RPL-554 and their derivatives, solvates, polymorphs, prodrugs, active metabolites and / or pharmaceutically acceptable salts of these compounds.

[0012] In one specific embodiment of this invention, the selective PDE4 inhibitor is piracetam. In another specific embodiment of this invention, the selective PDE4 inhibitor is cyclophosphamide. In yet another specific embodiment of this invention, the selective PDE4 inhibitor is roflumilast.

[0013] In embodiments of the present invention, selective PDE4 inhibitors can act directly on vascular endothelial cells.

[0014] In embodiments of the present invention, selective PDE4 inhibitors can suppress the interaction between inflammatory cells and vascular endothelial cells. Therefore, in some embodiments, the vascular endothelial dysfunction-related disease is a disease characterized by or mediated by the interaction between inflammatory cells and vascular endothelial cells.

[0015] In embodiments of the present invention, the disease associated with endothelial dysfunction is atherosclerosis. In some embodiments, the atherosclerosis is characterized by or mediated by endothelial dysfunction. In some embodiments, the atherosclerosis is characterized by or mediated by interactions between inflammatory cells and endothelial cells. In some embodiments, the atherosclerosis is characterized by or mediated by endothelium-dependent vasomotor dysfunction.

[0016] In another embodiment of this invention, the diseases associated with vascular endothelial dysfunction are myocardial ischemia, ischemia-reperfusion injury, hypertension, aneurysm, post-myocardial infarction heart failure, restenosis after angioplasty, arteritis, diabetes, obesity, metabolic syndrome, or any combination thereof. In some embodiments, the above-mentioned diseases are characterized by or mediated by vascular endothelial dysfunction. In some embodiments, the above-mentioned diseases are characterized by or mediated by interactions between inflammatory cells and vascular endothelial cells. In some embodiments, the above-mentioned diseases are characterized by or mediated by endothelium-dependent vasomotor impairment.

[0017] In one specific embodiment of the aspect of the invention, the subject suffers from atherosclerosis and further suffers from hyperlipidemia, diabetes, obesity, metabolic syndrome, or other metabolic diseases. In another specific embodiment of the aspect of the invention, the subject suffers from atherosclerosis and further suffers from myocardial ischemia, myocardial ischemia-reperfusion injury, myocardial infarction, aneurysm, post-myocardial infarction heart failure, restenosis after angioplasty, arteritis, hypertension, or any combination thereof.

[0018] In another embodiment of the aspect described in this invention, vascular endothelial dysfunction-related diseases are tissue ischemia-reperfusion injuries at any site.

[0019] In embodiments of the present invention, the pharmaceutical composition is administered as tablets, capsules, powders, pills, granules, injections, or emulsions.

[0020] In embodiments of the present invention, the pharmaceutical composition is administered to the subject by oral, sublingual, inhalation, intravenous, arterial, intramuscular, subcutaneous, intraperitoneal, or intracoronary administration.

[0021] In another aspect, the present invention provides a percutaneous endovascular angioplasty device comprising a balloon or stent, wherein the device contains or is coated with a PDE4 inhibitor.

[0022] In an embodiment of the present invention, the PDE4 inhibitor is a selective PDE4 inhibitor.

[0023] In specific embodiments of the aspects described in this invention, the selective PDE4 inhibitor is selected from piracetam, cyclophosphamide, AN-2728, AN-2898, CBS-3595, ELB-353, KF-66490, K-34, LAS-37779, IBBB-211913, AWD-12-281, cipanthenine, sillolast, roflumilast, BAY19-8004 and SCH-351591, AN-6415, indus-82010, TPI-PD3, ELB-353, CC-11050, GSK-256066, omepiracetam, OX-914, tetostrept, MEM-1414 and RPL-554 and their derivatives, solvates, polymorphs, prodrugs, active metabolites and / or pharmaceutically acceptable salts of these compounds. Attached Figure Description

[0024] Figure 1 Representative slices showing the expression of PDE4B in injured mouse and human endothelial cells, Bar = 50 μm. Figure 1 A shows immunofluorescence staining of PDE4B (red) and vWF (endothelial cell marker, green) in sections of normal mouse hearts and hearts 24 hours after IR (irradiation). Figure 1 B shows immunofluorescence staining of cardiac sections from patients with ventricular aneurysms.

[0025] Figure 2 The graph shows the expression of PDE4B in the heart 24 hours after IR (irritation). The left panel shows the relative protein expression levels of PDE4B in normal mouse hearts and IR-damaged hearts; the right panel shows the relative mRNA levels of all four PDE4 isoforms.

[0026] Figure 3 The results showed that PDE4B deficiency can alleviate myocardial ischemia-reperfusion injury in mice. Figure 3 Photograph A shows representative images of TTC-stained cross sections of Evans blue-perfused hearts in the control and PDE4BKO groups. Figure 3 B and Figure 3 C shows the infarct area (IS) and risk area (AAR) quantified in PDE4B KO and control mice 24 hours after I / R, respectively. Figure 3 D shows a representative echocardiogram. Figure 3 E-3G displays the shortening fraction (FS%), ejection fraction (EF%), and area change fraction (FAC%), respectively. Figure 3Unpaired Student's t-tests were performed on B, C, E, F, and G (n=10). *P<0.05, **P<0.01, and NS indicate non-significant.

[0027] Figure 4 The results showed that PDE4B deficiency helps improve microvascular blood flow recovery in MI / R, Bar = 50 μm. Figure 4 A shows representative blood flow images at each different time point. Figure 4 B shows the relative change in blood flow (percentage change relative to baseline). Figure 4 C is a representative photograph of immunofluorescence staining of myeloperoxidase (MPO, green) and DAPI (nuclear staining, blue) on frozen sections of the heart, and quantification of MPO-positive cells in ischemic areas. Figure 4 B performs a Bonferroni test using two-way ANOVA (n=7). Figure 4 C performed an unpaired Student's t-test (n=6), *P<0.05, **P<0.01.

[0028] Figure 5 This study demonstrates that PDE4B deficiency improves blood flow recovery after lower limb ischemia-reperfusion. Figure 5 A shows representative perfusion images at each time point. Figure 5 B records the changes in microvascular blood flow relative to baseline at specified time points before and after arterial ligation release. Figure 5 B performed a two-way ANOVA using the Bonferroni test (n = 11, 12). At baseline before ischemia, there was no difference in tissue perfusion between the control and PDE4B KO mice (89.16 ± 3.14 vs 94.52 ± 2.77 PU, p = 0.237, unpaired Student's t-test).

[0029] Figure 6 The study showed that PDE4B deficiency reduced neutrophil-endothelial (EC) cell interactions and inhibited the production of the pro-inflammatory factor IL-1β induced by LPS and ATP. Figure 6 A shows representative photographs of cell adhesion under each treatment condition. Figure 6 B shows the fold change in the number of adherent cells in the presence of C5a relative to the unstimulated condition, with cell genotype markers below each column. Figure 6 C shows the IL-1β level as detected by ELISA 6 hours after neutrophils were co-stimulated with LPS and ATP. All results were from at least 3 independent datasets. Figure 6 B performs a one-way ANOVA with Tukey's multiple comparison test (n = 10-12). Figure 6C performed an unpaired Student's t-test (n=6). Results are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001.

[0030] Figure 7 Mice derived from PDE4B knockout bone marrow cells showed a significant reduction in myocardial ischemia-reperfusion injury. Figure 7 A shows a schematic diagram of generating chimeric mice through bone marrow transplantation. Figure 7 B shows the percentage of lymphocytes (LYM), neutrophils (NE), and monocytes (MON) among white blood cells after bone marrow transplantation. Figure 7 C and Figure 7 D, from left to right, shows the infarct area (IS) and risk area (AAR) quantified after I / R following wild-type bone marrow transplantation into wild-type mice, wild-type bone marrow transplantation into PDE4B KO mice, PDE4B KO bone marrow transplantation into wild-type mice, and PDE4B KO bone marrow transplantation into PDE4B KO mice.

[0031] Figure 8 The changes in lower limb microvascular blood flow relative to baseline at specified I / R time points are shown after wild-type bone marrow transplantation into wild-type mice, wild-type bone marrow transplantation into PDE4B KO mice, PDE4BKO bone marrow transplantation into wild-type mice, and PDE4B KO bone marrow transplantation into PDE4B KO mice.

[0032] Figure 9 The study showed that piracetam (PICL) reduces neutrophil-endothelial cell interactions, improves cardiac microcirculation, and prevents MI / R injury. Figure 9 A shows representative photographs of TTC-stained cross sections of Evans blue-perfused hearts in the carrier injection group and the 10 mg / kg piracetam injection group. Figure 9 B and Figure 9 C represents the quantitative infarct area and the risk area, respectively. Figure 9 D-9G shows representative echocardiograms, fractional shortening (FS%), fractional ejection (EF%), and fractional area change (FAC%). Figure 9 H shows representative perfusion images. Figure 9 I displays the statistics of the perfusion results. Figure 9 J showed the detection of neutrophils (CD11b) in ischemic cardiac tissue by flow cytometry. + Ly6G + ), and quantitatively counted cells and CD11b + The percentage of neutrophils in the cells. Figure 9 K shows immunofluorescence staining of cardiac sections for MPO and quantification of the number of MPO-positive cells. Figure 9L and M represent cell adhesion with and without C5a under various concentrations of pyramistase treatment conditions. Figure 9 N shows the production of IL-1β by neutrophils induced by LPS and ATP under treatment with various concentrations of piracetam. Figure 9 Perform a one-way ANOVA Dunn multiple comparison test on B and C (n = 10, 9, 11). Figure 9 Unpaired Student's t-tests were performed on E, F, and G (n = 9, 8). Figure 9 K was subjected to an unpaired Student's t-test (n=6). Figure 9 I performed a Bonferroni test on two-way ANOVA (n=8). Figure 9 J performed a one-way ANOVA with Tukey's multiple comparison test (n = 9-11). *P < 0.05, **P < 0.01, NS indicates non-significant.

[0033] Figure 10 It was shown that piramistera improved lower limb reperfusion injury after I / R. Figure 10 A shows representative blood flow images at each time point. Figure 10 B shows the microvascular blood flow recorded relative to baseline at specified time points before and after arterial ligation. Figure 10 B performs a Bonferroni test using two-way ANOVA (n=10).

[0034] Figure 11 The expression of PDE4 in endothelial cells (ECs) and neutrophils is shown under LPS plus ATP stimulation. Figure 11 A shows the expression of four PDE4 isoforms in endothelial cells (ECs). Figure 11 B shows the expression of PDE4 in neutrophils 6 hours after LPS plus ATP treatment.

[0035] Figure 12 Demonstrates the use of ApoE SA / SA A mouse model was used to study the effects of the PDE4 inhibitor leupram on atherosclerotic lesions. Figure 12 AB represents the statistical analysis of staining of the thoracic aorta (**P<0.01). Figure 12 CD represents the statistical analysis of staining of the coronary arteries (*P<0.05).

[0036] Figure 13 This demonstrates the effect of piracetam on vascular tone. Figure 13 A represents the effect of adding acetylcholine (ACh) and piracetam on vasodilation. Figure 13 B represents the effect of adding sodium nitroprusside (SNP) and piracetam on vasodilation.

[0037] Figure 14 Roflumilast was shown to reduce myocardial ischemia-reperfusion injury in mice. Figure 14 A and Figure 14 B shows the statistical results of infarct area (A) and risk area (B) quantified by TTC staining of the heart perfused with Evans blue 24 hours after I / R in mice of the carrier injection group and the 1 mg / kg roflumilast injection group. Figure 14 Unpaired Student's t-tests were performed on A and B respectively (n=5,6). The results are expressed as mean ± SEM. *P<0.05, NS indicates non-significant. Detailed Implementation

[0038] The inventors have surprisingly discovered that PDE4 expression is significantly increased in the vascular endothelium of mouse ischemic-reperfusion (IR) hearts and in the vascular endothelium of human aneurysm patients. Upregulation of PDE4 expression leads to increased infiltration of cardiac tissue by bone marrow-derived leukocytes (primarily neutrophils) and the production of various pro-inflammatory cytokines, thereby causing cardiac remodeling and dysfunction. The inventors have also found that inhibiting PDE4 activity reduces the incidence of atherosclerosis and promotes vasodilation.

[0039] In one aspect, the present invention provides a method for treating or preventing vascular endothelial dysfunction-related diseases in a subject, comprising administering to the subject a pharmaceutical composition containing an effective amount of a phosphodiesterase 4 (PDE4) inhibitor, said inhibitor inhibiting the expression and / or activity of PDE4.

[0040] In another aspect, the present invention relates to the use of a PDE4 inhibitor in the preparation of a pharmaceutical composition for the treatment or prevention of diseases related to vascular endothelial dysfunction, wherein the inhibitor inhibits the expression and / or activity of PDE4.

[0041] In another aspect, the present invention provides a pharmaceutical composition containing an effective amount of a PDE4 inhibitor for treating or preventing diseases related to vascular endothelial dysfunction in subjects, wherein the inhibitor inhibits the expression and / or activity of PDE4.

[0042] As used in this article, "vascular endothelium" usually refers to the single layer of squamous epithelium lining the inner surface of blood vessels, also known as endothelial cells. During inflammation, they highly express adhesion molecules, interacting with adhesion molecules on the surface of leukocytes in the bloodstream, thereby mediating leukocyte passage through the vessel wall. Endothelial cells, or vascular endothelium, are a thin layer of specialized epithelial cells, composed of a single layer of squamous cells. They form the inner wall of blood vessels, serving as the interface between the blood within the vessel lumen and the rest of the vessel wall (single layer of squamous epithelium). Endothelial cells are also a type of non-professional antigen-presenting cells; they engulf foreign bodies, bacteria, necrotic and senescent tissue, and participate in collective immune activities, including: vasoconstriction and vasodilation, thereby controlling blood pressure; coagulation (thrombosis and fibrinolysis); atherosclerosis; angiogenesis; inflammation and swelling (such as edema); endothelial cells also control some substances, such as the entry and exit of leukocytes from blood vessels.

[0043] As used in this article, the term "endothelial dysfunction" is clinically assessed as endothelium-dependent vasomotor impairment, such as an imbalance between vasodilation and vasoconstriction, representing a physiological dysfunction of endothelial cells that prevents them from performing their normal biochemical functions. Normal endothelial cells are involved in mediating processes such as coagulation, platelet adhesion, immune function, volume control, and electrolyte contents within and outside the blood vessels. Endothelial dysfunction is associated with pro-inflammatory, pro-oxidative, and procoagulant changes in the arterial wall, as well as increased vessel wall thickness and collagen content. Endothelial dysfunction is considered a significant event in the development and progression of atherosclerosis and arterial stiffness, often preceding clinically apparent vascular complications. Endothelial dysfunction has prognostic significance in detecting vascular disease and predicting adverse vascular events. Risk factors for atherosclerosis and vascular disease / events are associated with endothelial dysfunction. Endothelial injury also leads to acute kidney injury and / or chronic or progressive kidney injury, such as tubulointerstitial fibrosis, glomerulonephritis, micro or macroalbuminuria, nephropathy and / or chronic kidney disease or kidney failure. There is evidence that oxidative stress not only causes endothelial dysfunction or damage but also contributes to vascular disease.

[0044] "Diseases related to vascular endothelial dysfunction" refer to diseases that are associated with "vascular endothelial dysfunction", including but not limited to atherosclerosis, hyperlipidemia, diabetes, obesity, metabolic syndrome or other metabolic diseases, myocardial ischemia, myocardial ischemia-reperfusion injury, myocardial infarction, aneurysm, heart failure after myocardial infarction, restenosis after angioplasty, arteritis, hypertension or any combination thereof.

[0045] As used herein, the term "object" refers to a mammal, preferably a primate. Alternatively, as used herein, the term "object" refers to a human being. In embodiments of the invention, the terms "object" and "patient" may be used interchangeably.

[0046] As used herein, the term "effective amount" or "therapeutic effective amount" refers to the amount of a substance, compound, material, drug, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount required to prevent, cure, improve, block, or partially block the symptoms of a disease or condition. The actual dose of the drug of the present invention administered to a patient may be determined based on the following physical and physiological factors: weight, sex, severity of symptoms, type of disease being treated, previous or current treatment interventions, the patient's unknown etiology, time of administration, excretion rate of the specific compound, and route of administration. In any case, the concentration of the active ingredient in the composition and the appropriate dose for the individual subject will be determined by the medical personnel responsible for administration.

[0047] As used herein, the term “inhibitor” refers to any substance capable of reducing or eliminating the expression and / or activity of PDE4, including but not limited to antagonistic antibodies or antibody fragments, interfering RNA molecules, double-stranded RNA (dsRNA), small molecules or nucleic acid molecules or expression vectors that inhibit PDE4 expression.

[0048] In an embodiment of the present invention, the PDE4 inhibitor is a selective PDE4 inhibitor.

[0049] As used herein, the terms “selectivity” and “specificity” are used interchangeably when referring to inhibitors, meaning that the inhibitor has an inhibitory effect only on the target, or that it has a higher inhibitory effect on the target than on other compounds or molecules.

[0050] Selective PDE4 inhibitors that can be used in this invention include, but are not limited to, piracetam, cyclophosphamide, AN-2728, AN-2898, CBS-3595, ELB-353, KF-66490, K-34, LAS-37779, IBBB-211913, AWD-12-281, cipanthenine, sillolast, roflumilast, BAY19-8004 and SCH-351591, AN-6415, indus-82010, TPI-PD3, ELB-353, CC-11050, GSK-256066, omepilast, OX-914, tetostrept, MEM-1414 and RPL-554 and their derivatives, solvates, polymorphs, prodrugs, active metabolites and / or pharmaceutically acceptable salts of these compounds.

[0051] In one specific embodiment of the present invention, the selective PDE4 inhibitor is piracetam. In another specific embodiment of the present invention, the selective PDE4 inhibitor is cyclophosphamide. In yet another specific embodiment of the present invention, the selective PDE4 inhibitor is roflumilast.

[0052] In embodiments of the present invention, selective PDE4 inhibitors can act directly on vascular endothelial cells. In embodiments of the present invention, selective PDE4 inhibitors can directly alleviate vascular endothelial dysfunction.

[0053] In embodiments of the invention, selective PDE4 inhibitors can suppress the interaction between inflammatory cells and vascular endothelial cells. Therefore, in some embodiments, the vascular endothelial dysfunction-related disease is a disease characterized by or mediated by the interaction between inflammatory cells and vascular endothelial cells.

[0054] As used herein, the term "inflammatory cells" refers to cells involved in the inflammatory response, including leukocytes. Leukocytes are a type of immune cell in the human body, which can be divided into three main categories based on their morphology, function, and origin: granulocytes, monocytes, and lymphocytes. Granulocytes can be further divided into neutrophils, eosinophils, and basophils based on the staining properties of the granules in their cytoplasm. In a specific embodiment of this invention, "inflammatory cells" refers to "neutrophils."

[0055] In embodiments of the present invention, the disease associated with vascular endothelial dysfunction is atherosclerosis. In some embodiments, the atherosclerosis is characterized by or mediated by vascular endothelial dysfunction. In some embodiments, the atherosclerosis is characterized by or mediated by interactions between inflammatory cells and vascular endothelial cells. In some embodiments, the atherosclerosis is characterized by or mediated by endothelium-dependent vasomotor impairment. Atherosclerosis is an inflammatory disease of the vascular endothelium caused by a variety of factors, including hypertension. Further development of atherosclerosis can obstruct the arterial lumen, leading to life-threatening cardiovascular diseases such as myocardial ischemia, myocardial ischemia-reperfusion injury, myocardial infarction, aneurysm, post-myocardial infarction heart failure, and restenosis after angioplasty.

[0056] In embodiments of the present invention, the diseases related to vascular endothelial dysfunction are myocardial ischemia, myocardial ischemia-reperfusion injury, myocardial infarction, aneurysm, post-myocardial infarction heart failure, restenosis after angioplasty, arteritis, hypertension, or any combination thereof. In some embodiments, the above-mentioned diseases are characterized by or mediated by vascular endothelial dysfunction. In some embodiments, the above-mentioned diseases are characterized by or mediated by interactions between inflammatory cells and vascular endothelial cells. In some embodiments, the above-mentioned diseases are characterized by or mediated by endothelium-dependent vasomotor impairment.

[0057] In an embodiment of the present invention, the subject suffers from atherosclerosis.

[0058] In one specific embodiment of the invention, the subject suffers from atherosclerosis, and / or further suffers from hyperlipidemia, diabetes, obesity, metabolic syndrome or other metabolic diseases.

[0059] In another specific embodiment of the invention, the subject suffers from atherosclerosis, and / or further suffers from myocardial ischemia, myocardial ischemia-reperfusion injury, myocardial infarction, aneurysm, post-myocardial infarction heart failure, restenosis after angioplasty, arteritis, hypertension, or any combination thereof.

[0060] In another embodiment of the invention, vascular endothelial dysfunction-related diseases are tissue ischemia-reperfusion injuries at any site.

[0061] In embodiments of the present invention, the pharmaceutical composition of the present invention is administered in the form of tablets, capsules, powders, pills, granules, injections, or emulsions.

[0062] In another embodiment, the pharmaceutical composition of the present invention is administered to the subject by oral, sublingual, inhalation, intravenous, arterial, intramuscular, subcutaneous, intraperitoneal, or intracoronary administration. In one specific embodiment, the PDE4 inhibitor of the present invention is used for intracoronary administration.

[0063] The pharmaceutical compositions of the present invention can be used as is, or in some embodiments, in combination with a suitable drug carrier, loading agent, or excipient containing an inert component. The pharmaceutical compositions of the present invention also refer to any product resulting directly or indirectly from a combination, complexation, or aggregation of any two or more components, or from the dissociation of one or more components, or from other types of reactions or interactions of one or more components. Typically, pharmaceutical compositions are prepared by uniformly and tightly mixing the active ingredient with a liquid carrier, loading agent, or excipient, or finely pulverized solid carrier, loading agent, or excipient, or both, and then, if necessary, shaping the product into the desired formulation. The pharmaceutical composition contains sufficient active ingredient to produce the desired effect; in the present invention, the active ingredient is a PDE4 inhibitor, specifically a selective PDE4 inhibitor.

[0064] As used in this article, "pharmaceutically acceptable carrier" means that the carrier, delivery unit, or excipient must be compatible with the other components of the formulation and harmless to its recipients. This includes any and all solvents, dispersion media, antioxidants, salts, coatings, surfactants, preservatives (e.g., methylparaben or propylparaben, sorbic acid, antibacterial agents, antifungal agents), isotonic agents, solution blocking agents (e.g., paraffin wax), adsorbents (e.g., kaolin, bentonite), drug stabilizers (e.g., sodium lauryl sulfate), gels, binders (e.g., syrups, gum arabic, gelatin, sorbitol, astragalus gum, polyvinylpyrrolidone, carboxymethyl cellulose, alginate), excipients (e.g., lactose, polyethylene glycol), disintegrants (e.g., agar, etc.). Starch, lactose, calcium phosphate, calcium carbonate, alginic acid, sorbitol, glycine), wetting agents (e.g., hexadecyl alcohol, glyceryl monostearate), lubricants, absorption enhancers (e.g., quaternary ammonium salts), edible oils (e.g., almond oil, coconut oil, oily esters or propylene glycol), sweeteners, flavoring agents, coloring agents, fillers (e.g., starch, lactose, sucrose, glucose, mannitol, silicic acid), tableting lubricants (e.g., magnesium stearate, starch, glucose, lactose, chalk), inhalation carriers (e.g., hydrocarbon propellants), buffers, or substances like these and combinations thereof.

[0065] The preparation of suitable pharmaceutical compositions is known to those skilled in the art based on the present disclosure and is exemplified in Remington: The Science and Practice of Pharmacy, 21st edition, 2005, which is incorporated herein by reference. Furthermore, for human use, it should be understood that the preparation should also meet the standards for sterility, pyrogenicity, overall safety, and purity required by the drug regulatory authority.

[0066] Based on common knowledge in the art, those skilled in the art are familiar with suitable carriers and excipients for desired pharmaceutical compositions, such as antioxidants, dispersants, emulsifiers, preservatives, solubilizers, colorants, complexing agents, flavoring agents, buffers, viscosity modifiers, surfactants, adjuvants, binders, lubricants, carriers, diluents, stabilizers, or penetration enhancers. By appropriately selecting adjuvants and / or excipients, a precisely suitable dosage form (e.g., sustained-release or enteric-coated form) for the active compound and / or the desired therapeutic effect can be achieved.

[0067] The pharmaceutical compositions of the present invention are administered to subjects in need at different frequencies, which may vary depending on the severity of the disease related to vascular endothelial dysfunction. Those skilled in the art can readily determine the frequency of administration based on the route of administration and the patient's clinical condition. The frequency range may be from three times daily to once every two or three weeks, for example, three times daily, twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once every two weeks, or once every three weeks.

[0068] The PDE4 inhibitors of the present invention, such as piramylast, leupram, and roflumilast, can be administered in single or multiple doses at dose levels of 0.5 to 100 mg / kg (body weight), 1 to 50 mg / kg (body weight), 5 to 20 mg / kg (body weight), or 5 to 10 mg / kg (body weight).

[0069] The PDE4 inhibitors of the present invention, such as piramipexole, rofloxacin, and roflumilast, are administered daily at doses of approximately 1 mg / kg to approximately 20 mg / kg (body weight), approximately 2 mg / kg to approximately 20 mg / kg (body weight), approximately 3 mg / kg to approximately 20 mg / kg (body weight), approximately 4 mg / kg to approximately 20 mg / kg (body weight), approximately 5 mg / kg to approximately 20 mg / kg (body weight), approximately 6 mg / kg to approximately 20 mg / kg (body weight), approximately 7 mg / kg to approximately 20 mg / kg (body weight), approximately 8 mg / kg to approximately 20 mg / kg (body weight), and approximately 2 mg / kg to approximately 20 mg / kg (body weight). 0 mg / kg (body weight), about 9 mg / kg (body weight) to about 20 mg / kg (body weight), about 10 mg / kg (body weight) to about 20 mg / kg (body weight), about 11 mg / kg (body weight) or more, about 12 mg / kg (body weight) or more, about 13 mg / kg (body weight) or more, about 14 mg / kg (body weight) or more, about 15 mg / kg (body weight) or more, about 16 mg / kg (body weight) or more, about 17 mg / kg (body weight) or more, about 18 mg / kg (body weight) or more, about 19 mg / kg (body weight) or more, or about 20 mg / kg (body weight) or more. In some embodiments, the PDE4 inhibitors of the present invention, such as piramylast, rolipram, and roflumilast, are administered daily at a dose of about 10.0 mg / kg (body weight). In some embodiments, the pharmaceutical composition is administered daily subcutaneously at a dose of about 5.0 mg / kg (body weight).

[0070] In some embodiments, the unit dosage form of the pharmaceutical composition of the present invention may contain 0.1 to 10 mg of the active compound, for example, about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg, 7.5 mg, 8.0 mg, 8.5 mg, 9.0 mg, 9.5 mg, and 10 mg of the active compound.

[0071] The pharmaceutical compositions of the present invention may be in the form of, for example, tablets, coated tablets, capsules, microcapsules, suppositories, films, emulsions, suspensions, gels, or solutions. In some embodiments, the pharmaceutical composition described herein comprises about 0.05 to about 99% by weight (based on the weight percentage of the total formulation) of the active compound. In other embodiments, the pharmaceutical composition described herein comprises about 0.10 to about 70% by weight of the active compound. In other embodiments, the pharmaceutical composition described herein comprises about 0.10 to about 50% by weight of the active compound. By appropriately selecting adjuvants and / or excipients, a precisely suitable dosage form (e.g., delayed-release or enteric-coated form) for the active compound and / or desired efficacy can be achieved.

[0072] In embodiments of the present invention, a pharmaceutical composition is provided for treating vascular endothelial dysfunction-related diseases, comprising a PDE4 inhibitor and other conventional clinical medications for treating cardiovascular diseases. In specific embodiments, the pharmaceutical composition of the present invention prevents leukocyte adhesion and infiltration of endothelial cells. In specific embodiments, the pharmaceutical composition of the present invention reduces the occurrence of atherosclerosis and plaque formation. In specific embodiments, the pharmaceutical composition of the present invention promotes vasodilation.

[0073] In another embodiment, the present invention also provides a pharmaceutical composition for the prevention or treatment of tissue ischemia-reperfusion injury at any site of a subject, comprising a PDE4 inhibitor and other conventional pharmaceuticals.

[0074] In another aspect, the present invention provides a percutaneous endovascular angioplasty device comprising a balloon or stent, wherein the device contains or is coated with a PDE4 inhibitor.

[0075] In an embodiment of the present invention, the PDE4 inhibitor is a selective PDE4 inhibitor.

[0076] In specific embodiments of the aspects described in this invention, the selective PDE4 inhibitor is selected from piracetam, cyclophosphamide, AN-2728, AN-2898, CBS-3595, ELB-353, KF-66490, K-34, LAS-37779, IBBB-211913, AWD-12-281, cipanthenine, sillolast, roflumilast, BAY19-8004 and SCH-351591, AN-6415, indus-82010, TPI-PD3, ELB-353, CC-11050, GSK-256066, omepiracetam, OX-914, tetostrept, MEM-1414 and RPL-554 and their derivatives, solvates, polymorphs, prodrugs, active metabolites and / or pharmaceutically acceptable salts of these compounds.

[0077] Example

[0078] The embodiments described herein are for illustrative purposes and are not intended to limit the scope of the invention. Those skilled in the art can modify the specific steps according to the spirit and teachings of the invention. Unless otherwise specified or clearly apparent from the content, any features described relating to some embodiments may be used in conjunction with any other embodiments.

[0079] Example 1: Materials and Methods

[0080] animal

[0081] PDE4 subtype B knockout (PDE4B KO) mice were established and backcrossed with C57BL / 6J mice more than 10 times. Knockout (KO) mice and littermate controls were generated by crossing heterozygous mice (or homozygous KO mice). Since no phenotypic differences were detected between heterozygous and wild-type littermates, these control mice were pooled for further analysis. C57BL / 6J wild-type (WT, Ptges+ / +) or Ptges+ / - mice derived from Ptges- / - crosses with Ptges+ / + (or + / -) mice were used as controls for PDE4B KO mice.

[0082] The PDE4 inhibitor piramist (PICL-H: 10 mg / kg; PICL-L: 5 mg / kg) or its carrier was administered intraperitoneally to male C57BL / 6J mice (8-12 weeks old) after left coronary artery ligation and 12 hours after reperfusion. Mice were sacrificed 24 hours after reperfusion for sample collection.

[0083] All animal protocols followed the guidance of the Animal Management and Experimentation Committee of the Laboratory Animal Center, Fuwai Hospital, National Center for Cardiovascular Diseases, China.

[0084] Human ventricular aneurysm samples

[0085] Human ventricular aneurysm samples were obtained from five patients with ventricular aneurysms treated at Fuwai Hospital. Samples were collected during repair surgery, fixed overnight in 10% formalin buffer, and further processed for paraffin embedding and tissue sectioning. Sample collection was approved by the Medical Ethics Committee of Fuwai Hospital, and written informed consent was obtained from each patient.

[0086] Statistical analysis

[0087] Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, California, USA). Student's t-test (two-tailed, unpaired) was used for comparisons between two groups. Two-way ANOVA was used for comparisons involving time. One-way ANOVA was used for comparisons between multiple groups. Post-hoc tests were performed after two-way or one-way ANOVA, and Bonferroni correction was applied unless otherwise specified. Data are expressed as mean ± SEM. P-values ​​<0.05 were considered significant and marked with an asterisk (*P<0.05; **P<0.01; ***P<0.001).

[0088] Mouse model of myocardial ischemia / reperfusion (I / R)

[0089] As described by Gao et al. (Circ Res. 2010; 107:1445-1453), myocardial I / R injury was induced using a non-ventilated method. In short, mice were anesthetized with 3% isoflurane by inhalation, followed by maintenance anesthesia with 1.5-2% isoflurane. The mice were placed in a supine position. The skin of the left chest was incised, and the pectoral muscles were simply dissected. Then, the thoracic cavity was quickly exposed via a left 4th intercostal thoracotomy. After opening the pericardium, the mouse heart was exposed and sutured with 7-0 silk sutures from the origin. The left anterior descending (LAD) coronary artery was ligated with a slipknot. Successful ligation was confirmed by the simultaneous occurrence of whitening of the anterior wall of the left ventricle and ST segment elevation on the electrocardiogram (ECG). The heart was then quickly returned to the pleural cavity, air was manually expelled, and the pleural cavity was closed with 4-0 sutures. The inner end of the slipknot suture was cut as short as possible, leaving the other end, approximately 0.8 cm long, outside the pleural cavity. Anesthesia was then discontinued, and the animal was allowed to recover. After 30 minutes of local ischemia, the mouse was re-anesthetized, and the slipknot was loosened by smoothly pulling the long end of the suture until it was felt to be completely released, at which point myocardial reperfusion began. Twenty-four hours after I / R, cardiac function and ventricular structure were assessed using echocardiography (VisualSonics VeVo 2100 imaging system) by evaluating ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular area change fraction (FAC), mitral valve ratio of peak left ventricular posterior wall thickness (E / A), left ventricular anterior wall thickness (LVAW), left ventricular posterior wall thickness (LVPW), left ventricular volume, and left ventricular mass. Mortality was similar across groups, approximately 20%. Twenty-four hours after I / R, the left aorta (LAD) was reoccluded at the previous position, and 2% Evans blue dye (Sigma, Darmstadt, Germany) was injected into the heart chambers via the ascending aorta. Mice were then euthanized, their hearts were collected, and rinsed with PBS. The hearts were then frozen at -80°C for 30 minutes and transversely sliced ​​into six pieces below the ligation suture. Sections were incubated with 1% triphenyltetrazolium chloride (TTC, Amresco, America) in a dark room at 37°C for 10 minutes, then fixed with formalin for 2 hours. Images were taken using a stereomicroscope (Zeiss, Germany). The ischemic area, infarcted tissue, and left ventricular area were measured and calculated using Image-Pro Plus 6.0 software (Media Cybernetics).

[0090] Detection of myocardial microcirculation perfusion during I / R

[0091] Mice were anesthetized with sodium pentobarbital, intubated, and ventilated with a positive pressure ventilator. After removing the pectoral muscles, the thoracic cavity was opened via a thoracotomy at the 4th intercostal space on both sides and in the middle of the thorax. The heart was then fully exposed by cutting 2-4 ribs on each side and removing the anterior chest wall. The LAD coronary artery was ligated 2-3 mm from its origin using 7-0 silk sutures to induce myocardial ischemia. After 30 minutes, the ligation was removed to allow reperfusion of the ischemic myocardium. Cardiac blood flow was measured at baseline (before ligation), 0 and 30 minutes after ligation, and 0, 10, 20, and 30 minutes after reperfusion using a laser Doppler flowmeter (PeriCam PSI System, Perimed, Sweden). Left ventricular epicardial blood flow was determined and analyzed blinded.

[0092] Immediately after ligation, male C57BL / 6J mice (8-12 weeks old) were administered intraperitoneally the PDE4 inhibitor piramistase (PICL-H: 10 mg / kg) or its carrier.

[0093] Detection of microcirculatory response in hind limb I / R

[0094] Mice were anesthetized with sodium pentobarbital, and the left femoral artery was bluntly dissected. Hindlimb ischemia (HLI) was induced by ligation of the proximal portion of the femoral artery bifurcation, followed by reperfusion after 10 minutes by suture release. Distal hindlimb blood flow during I / R was monitored immediately before and after ligation using laser Doppler flowmetry at different time points (baseline; 0 and 10 minutes after ligation; 0, 5, 10, and 20 minutes after reperfusion).

[0095] Neutrophil adhesion under in vivo microscopy

[0096] Neutrophil adhesion in vivo was monitored by examining the femoral artery using in vivo microscopy. Mice were intravenously injected with rhodamine 6G (Sigma, Darmstadt, Germany; 50 mg / kg in saline solution) before femoral artery ligation. In vivo imaging and video recording of neutrophil adhesion were performed using a Nikon microscope (E400; green fluorescence excitation) connected to a Nikon camera (S1TC01M). Offline analysis of time-lapse videos was performed, counting the number of rolling neutrophils for 1 minute at different time points (before ligation; 2, 5, 10, 20, and 30 minutes after reperfusion).

[0097] Bone marrow transplant

[0098] Recipient mice (WT or PDE4BKO) were irradiated with a sublethal dose of 10 Gy (Gammacell 40 137Cs γ-irradiation source) for 10–12 minutes. Each recipient received 5 × 10 Gy via retrobulbar intravenous injection. 6 WT- or KO- bone marrow cells were used for reconstruction. Mice were then allowed to recover for approximately 5 weeks, including 1–2 weeks of antibiotic treatment (1–2 mg / ml gentamicin or 1.1 mg / ml neomycin and 1000 μg / ml polymyxin B in drinking water). I / R injury studies were then conducted on chimeric bone marrow mice.

[0099] Flow cytometry analysis

[0100] Hearts were isolated 24 hours after MI / R, and approximately 20 mg of cardiac tissue was excised from the apex. The tissue fragments were then digested for 7 minutes at 37°C using collagenase (0.895 mg / ml, type II, Sigma, USA) and protease (0.5 mg / ml, type XIV, Sigma, USA). The digestion mixture was filtered through a 74 μm filter and centrifuged at 200 g for 5 minutes at 4°C. The cell pellet was resuspended in 100 μl of FACS buffer, with 50 μl stained on ice with an antibody mixture (PE Rat Anti-Mouse CD45, FITC Rat Anti-Mouse CD11b, APC-Cy7 Rat Anti-Mouse Ly-6G; BD Biosciences; New York, USA) and incubated in the dark for 30 minutes. Each sample was then added to 250 μl of FACS buffer, filtered through a 74 μm nylon membrane, and analyzed by flow cytometry at a constant flow rate and fixed collection time.

[0101] Immunofluorescence staining

[0102] Immunofluorescence staining was performed according to the same protocol described by Hao et al. (Circulation. 2017; 135:1253-1264). Briefly, paraffin sections (5 μm) were deparaffinized, rehydrated, and antigen retrieval was performed using EDTA antigen retrieval water (pH 9.0; ZSGB-BIO, Beijing, China). Frozen sections of the heart (10 μm) were continuously collected at 200 μm intervals along the ligation line, for a total of four levels. The frozen sections were fixed with 95% ethanol for 15 minutes. After blocking and membrane rupture with normal goat serum containing 0.3% Triton X-100 at room temperature for 90 minutes, the samples were incubated overnight with primary antibody at 4°C, followed by incubation at room temperature for 1 hour with secondary antibody conjugated with Alexa Fluor-488 or Alexa Fluor-594. The sections were then mounted with VectaShield media containing DAPI stained cell nuclei and imaged using a Zeiss microscopy system (AXI0; Zeiss) or a laser scanning confocal microscopy system (SP8; Leica). To identify positive cells, images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The primary antibodies and dilutions used in the study were as follows: mouse monoclonal anti-PDE4B antibody (1:100; Novus, Littleton, CO, USA), rabbit anti-myeloperoxidase (MPO) antibody (1:50; Abcam, Cambridge, UK), and goat polyclonal anti-Von Willebrand factor (vWF) antibody (1:100; Abcam, Cambridge, UK, used for delineating endothelial cells).

[0103] Cell Research

[0104] Endothelial cells: As previously described (Hao et al., Cell metabolism. 2011; 13:592-600), mouse aortic endothelial cells (MAECs) were isolated. In short, the aorta was harvested and cut into 1-2 mm sections. 2 Sections were prepared. The luminal side of the aortic segment was attached to a culture dish and cultured for 5 to 7 days in DMEM medium containing 20% ​​fetal bovine serum (FBS) and 100 μg / mL endothelial cell (EC) growth supplement to allow EC growth. The ECs were then passaged and cultured. MAECs from passages 2 to 6 were used in this study.

[0105] Myeloid cells: Mice were intraperitoneally injected with 4% Brewer modified thioethanolamine medium (BD, Franklin Lakes, NJ, USA) (1 mL / mouse). Five hours later, the peritoneal fluid was washed with PBS containing 0.1% BSA for collection and then centrifuged (800 rpm, 3 min). The collected leukocytes, which were mainly myeloid cells, were used for cell adhesion studies.

[0106] Endothelial cell-neutrophil adhesion assay: For the endothelial cell-neutrophil adhesion assay, MAECs were seeded into 96-well flat-bottom plates and incubated with the specified drug (dissolved in DMSO) for 2 hours. Next, complement factor 5a (C5a) dissolved in PBS was added to a final concentration of 100 nM and incubated for 30 minutes. Neutrophils collected from mouse peritoneum were prepared in RPMI medium containing 10% FBS. When EC preparation was complete, the medium containing the specified drug was then filled with neutrophils (5 × 10⁶ cells per well). 4 The ECs and neutrophils were then co-cultured for 30 minutes. Subsequently, the cells were washed once with RPMI medium containing 6G rhodamine (200 μg / mL; Sigma, Darmstadt, Germany), followed by three washes with fresh 1640 medium. The fluorescence signal was finally detected using a microplate reader (excitation wavelength 560 nm; emission wavelength 560 nm; Infinite M200; Tecan, Hombrechtikon, Switzerland).

[0107] ELISA analysis: All neutrophils (5 × 10⁶ cells) collected from the peritoneum of mice were stimulated with LPS (1 μg / mL; E. coli, Sigma, Darmstadt, Germany) and ATP (5 mM; Sigma, Darmstadt, Germany). 5 ( / well) for 6 hours. Then collect the supernatant by centrifugation (3000 rpm, 5 min). IL-1β was measured in the supernatant using a commercially available ELISA kit (R&D Systems, Minnesota, USA) according to the manufacturer's protocol. OD was measured at 450 nm using a microplate reader (Infinite M200; Tecan, Hombrechtikon, Switzerland). Data analysis was performed once the signal stabilized and was normalized to an unstimulated wild-type control.

[0108] Hyperlipidemia and hypertension model: using ApoE SA / SAMice (see WO 2018 / 196874 for specific construction methods) were fed a high-fat diet (HFD: containing 21% fat and 0.2% cholesterol). To induce hypertension, doxycycline (Dox) (1 mg / ml) was added to the mice's drinking water, i.e., the mice were fed HFD+DOX at the same time.

[0109] Vascular tension detection: Mice were euthanized with CO2. The mouse hearts were placed in Krebs buffer solution (118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM glucose). A segment of the coronary artery, starting from the aortic segment, was gently dissected under a stereomicroscope and cut into approximately 2 mm segments (the coronary artery varies considerably among different mice; some can be separated into two segments, while others can only be separated into one). The blood vessels were fixed to the probe of a sample loading chamber of a Multi Myograph (620M, Danish, Myo Technology A / S, Aarhus, Denmark) using two tungsten wires. 5 ml of Krebs buffer solution was added to each reaction chamber, and the mixture was incubated at 37°C with a binary gas flow (95% O2 - 5% CO2). After equilibration at 0 initial tension for 30 min, an initial tension of approximately 1 mN was applied. After equilibration for 10 min, stimulation was performed with 60 mM KCl buffer. To determine if PDE4 was endothelial-dependent, stimulation was initiated with 50 nM U46619, followed by a gradient of the PDE4 inhibitor piracetam (10 nM-10 μM), and three washes were performed. The mixture was then incubated with 100 μM L-NAMEKrebs buffer for 15 min, followed by stimulation with 50 nM U46619, then a gradient of the PDE4 inhibitor piracetam (10 nM-10 μM), and finally, a gradient of sodium nitroprusside (10 nM-10 μM) was applied for relaxation.

[0110] Example 2: Endothelial PDE4B expression in ischemic human and mouse hearts

[0111] First, immunofluorescence staining of PDE4B (red), vWF (endothelial cell marker, green), and cell nucleus (DAPI, blue) detected low expression levels of PDE4B in healthy mice. Figure 1 A). However, in injured hearts undergoing 24 hours of ischemia-reperfusion, PDE4B expression was upregulated and primarily observed in endothelial cells. Figure 1 A). Furthermore, PDE4B expression was also observed in the vascular endothelium of the damaged area in hearts from patients with ventricular aneurysms. Figure 1 B). In addition, such as Figure 2 As shown in the left column, PDE4B protein expression is elevated in MI / R injury; Figure 2 In the right column, among the four PDE4 subtypes in MI / R injury, the mRNA expression of PDE4A, PDE4B, and PDE4D was all upregulated. Therefore, PDE4 expression in the vascular endothelium of the subjects is significantly increased in diseases related to vascular endothelial dysfunction.

[0112] Example 3: PDE4B deficiency can alleviate myocardial ischemia-reperfusion injury in mice.

[0113] Previous data indicated that phosphodiesterase-4 plays a crucial role in the inflammatory response. Therefore, the inventors investigated the potential role of PDE4B in in vivo MI / R. First, ischemia was induced by ligation of the left anterior descending coronary artery in PDE4B KO mice and their littermate controls. After 30 minutes of ischemia, the ligation was released, followed by 24 hours of reperfusion. The same MI / R protocol was used in subsequent embodiments of this application. The absence of PDE4B significantly reduced the percentage of infarct area (IS) in the area of ​​risk (AAR), with similar AAR values ​​across groups (…). Figure 3 AC). Next, echocardiography showed that, compared with littermate controls, PDE4B KO mice significantly improved MI / R-induced cardiac systolic dysfunction, as reflected in left ventricular ejection fraction (EF%), fractional shortening (FS%), and fractional area change (FAC%). Figure 3 DG). Compared with the control, PDE4BKO mice that did not undergo myocardial ischemia-reperfusion showed no difference in ultrasound parameters (DG). Figure 3 These results indicate that the loss of PDE4B activity in endothelial cells has a protective effect against MI / R injury.

[0114] Example 4: PDE4B deficiency facilitates microvascular blood flow recovery during MI / R

[0115] One of the key determinants of myocardial ischemia / reperfusion injury is microvascular dysfunction (Prasad et al., Circulation. 2009; 120:2105-2112; Gutterman et al., Circ Res. 2016; 118:157-172; Pries and Reglin, Eur Heart J. 2017; 38:478-488). In a mouse model of myocardial ischemia / reperfusion, blood flow in mice undergoing myocardial ischemia-reperfusion was monitored in a blinded manner at specified time points. The monitored cardiac area was the same between the control and PDE4B KO groups. Laser Doppler imaging showed that PDE4B deficiency significantly improved cardiac perfusion recovery after restoration of coronary blood flow (PDE4B KO). Figure 4 A and B). Compared with control mice, immunofluorescence staining also showed significantly less aggregation of myeloperoxidase (MPO)-positive cells in ischemic heart tissue of PDE4B KO mice. Figure 4C), while MPO is mainly found in neutrophils, indicating that the accumulation of neutrophils in PDE4B KO mice is significantly reduced.

[0116] Furthermore, using another lower limb ischemia-reperfusion mouse model, the inventors demonstrated that PDE4B deficiency significantly promoted the restoration of blood flow throughout the lower limb after femoral artery ischemia injury, as assessed in vivo using laser Doppler flowmeter. Figure 5 ).

[0117] In summary, these results indicate that inhibiting PDE4, especially its B subtype, can reduce vascular inflammation, improve cardiac microcirculation, and limit MI / R injury.

[0118] Example 5: PDE4B deficiency reduces neutrophil-endothelial cell interactions and inhibits LPS and ATP-induced [interactions]. Production of the pro-inflammatory factor IL-1β

[0119] In this invention, neutrophils are incubated on a monolayer of mouse aortic endothelial cells, and the adhesion of cells is quantitatively measured. For example... Figure 6 As shown in A and 6B, neutrophil-endothelial cell adhesion was significantly reduced in PDE4B-deficient endothelial cells and neutrophils, particularly in the presence of C5a, which stimulates neutrophil adhesion. The adhesion was lowest when both neutrophils and endothelial cells were PDE4B-deficient. Even when only endothelial cells were PDE4B-deficient, adhesion was significantly lower than when only neutrophils were PDE4B-deficient. These results indicate that PDE4B deficiency in endothelial cells has a greater effect on alleviating adhesion than PDE4B deficiency in neutrophils, and that the absence of PDE4B in neutrophils and endothelial cells has a potential synergistic effect, significantly reducing their interaction and neutrophil adhesion and infiltration into endothelial cells.

[0120] Furthermore, compared with control mice, IL-1β secretion from neutrophils derived from PDE4B KO mice co-stimulated with LPS and ATP was significantly reduced, with IL-1β concentrations in the supernatant being 530.1±36.84 pg / mL and 342.7±32.19 pg / mL, respectively. Figure 6 C).

[0121] Therefore, these data demonstrate that PDE4B deficiency, especially PDE4B deficiency in endothelial cells, reduces neutrophil-endothelial cell interactions, thereby improving the inflammatory condition.

[0122] Example 6: Prevention of myocardial ischemia-reperfusion injury in mice with PDE4B-deficient bone marrow cells

[0123] To directly test whether PDE4B bone marrow cells affect cardiac injury (IR), chimeric mice were generated through bone marrow cell (BM) transplantation, including wild-type bone marrow transplanted into wild-type mice, wild-type bone marrow transplanted into PDE4B KO mice, PDE4B KO bone marrow transplanted into wild-type mice, and PDE4B KO bone marrow transplanted into PDE4B KO mice. Figure 7 A). Statistical analysis of lymphocytes (LYM), neutrophils (NE), and monocytes (MON) after bone marrow transplantation showed no significant changes in the number of lymphocytes, neutrophils, and monocytes produced by the various transplantation strategies mentioned above. Figure 7 B). WT mice reconstituted with PDE4B KO BM showed a significantly reduced infarct area ( Figure 7 C). Reconstruction of PDE4B KO mice with PDE4B KO BM significantly reduced these pathological changes in MI / R injury. Figure 7 C). These findings suggest that the loss of PDE4B in bone marrow cells helps improve myocardial ischemia-reperfusion injury, and that the loss of PDE4B in bone marrow cells and all mouse cells (including vascular endothelial cells) can further improve myocardial ischemia-reperfusion injury.

[0124] Furthermore, using another mouse model of hindlimb ischemia-reperfusion, the inventors further verified that PDE4B deficiency in bone marrow cells and PDE4B deficiency in all cells significantly promoted the restoration of lower limb blood flow after femoral artery blood flow restoration, as measured in vivo using a laser Doppler flowmeter. Figure 8 ).

[0125] Example 7: Piramysterol (PICL) reduces neutrophil-endothelial cell interaction and improves cardiac microcirculation Ring and prevent MI / R damage

[0126] It is worth noting that PDE4 inhibitors are considered promising new drugs for the treatment of chronic inflammatory diseases due to their anti-inflammatory effects. This invention further investigated the effects of piramisterone on vascular endothelial cells. C57BL / 6J mice underwent MI / R surgery. Before reperfusion and 12 hours after reperfusion following ligation of the left anterior descending coronary artery, intraperitoneal injections of a carrier, 5 mg / kg (PICL-L), or 10 mg / kg (PICL-H) of piramisterone were administered. Piramisterone significantly reduced the infarct area in a concentration-dependent manner. Figure 9 AC), and improved cardiac function after MI / R ( Figure 9 DG). In another group of mice, myocardial microcirculation perfusion was measured during MI / R, and piramylast significantly improved cardiac perfusion recovery after MI / R by laser Doppler imaging. Figure 9 H and 9I).

[0127] Flow cytometry analysis showed that, compared with the control, neutrophils (CD11b) in the damaged heart... + Ly6G + neutrophil infiltration was significantly elevated in MI / R injury, while piramister treatment significantly reduced the magnitude of the increase in neutrophil infiltration (absolute count: 738±91 vs. 459±69; CD11b + Percentage of neutrophils in cells: 50.1±1.7% vs. 42.9±2.4%; Figure 9 J). Immunofluorescence staining also showed less aggregation of myeloperoxidase (MPO)-positive cells (i.e., neutrophils) in damaged cardiac tissue treated with piracetam. Figure 9 K).

[0128] Furthermore, under LPS plus ATP stimulation, PDE4, especially PDE4B, was increased in both endothelial cells (ECs) and neutrophils. Figure 11 Treatment with piramister significantly inhibited neutrophil-endothelial cell adhesion in a concentration-dependent manner, with adhesion decreasing as piramister concentration increased. Figure 9 L and Figure 9 M). Treatment with piracetam in a concentration-dependent manner significantly reduced IL-1β production in LPS- and ATP-stimulated neutrophils. Figure 9 The results (N) indicate that in an inflammatory environment, piramisterase can reduce the production of pro-inflammatory factors, thereby improving the progression of inflammation. Furthermore, piramisterase significantly improved hindlimb perfusion after I / R (inflammation / reperfusion). Figure 10 ).

[0129] Example 8: Cefoperazone reduces the incidence of atherosclerosis

[0130] ApoE was used in a hyperlipidemic and hypertensive mouse model constructed using methods such as WO 2018 / 196874. SA / SA In mice, hyperlipidemia and hypertension were induced, and the effects of the PDE4 selective inhibitor rolipram on atherosclerotic lesions were investigated. (ApoE) SA / SA Mice were fed a high-fat diet (HFD: containing 21% fat and 0.2% cholesterol). To induce hypertension, doxycycline (Dox) (1 mg / ml) was added to the mice's drinking water, i.e., the mice were fed HFD+DOX. The mice were also injected intraperitoneally daily with either the carrier or 10 mg / kg of cyclophosphamide. After 4 weeks, the mice were harvested, and the thoracic aorta and coronary arteries were subjected to Oil Red chromatographic staining and statistical analysis.

[0131] Oil red pathological staining of the thoracic aorta ( Figure 12A) It can be observed that the plaque area in the rolipram group mice (n=10) is significantly smaller than that in the vector control group (n=8), and Figure 12 B's statistics also showed that the percentage of plaques in mice treated with leupram was significantly lower than that in the control group.

[0132] Similarly, oil red pathological staining was performed on heart sections ( Figure 13 A) It can be found that the area of ​​cardiac vascular plaques in the rolipram group mice (n=9) was significantly smaller than that in the control group (n=8), and Figure 13 The statistics from B also showed that the absolute count of plaque area in mice treated with leupram was significantly smaller than that in the control group.

[0133] Therefore, these data show that selectively inhibiting PDE4 activity helps reduce the formation of atherosclerotic plaques and the occurrence of atherosclerosis.

[0134] Example 9: Piramysterol mediates vasodilation via endothelial cells

[0135] Mice were sacrificed with CO2, and the coronary artery segments of the heart were treated with a carrier, piclamilast, acetylcholine (Ach), and sodium nitroprusside (SNP) at the concentrations shown in the figure, respectively, to study the effect of piclamilast on vasodilation.

[0136] Acetylcholine (ACh) acts on vascular endothelial cells, stimulating them to produce NO. NO is a vasodilator that acts on vascular smooth muscle, causing it to relax and thus dilate blood vessels, lowering blood pressure. Acetylcholine cannot directly act on smooth muscle cells to cause relaxation; therefore, the vasodilation observed through the action of acetylcholine can indirectly reflect the function of the vascular endothelium. In this embodiment, the carrier is used as a negative control, and acetylcholine is used as a positive control. Figure 13 As can be seen from A, the PDE4 inhibitor piracetam can stimulate the dilation of coronary arteries in mice, and the level of change is similar to that of the conventional diastolic agent acetylcholine.

[0137] Sodium nitroprusside (SNP) can directly act on vascular smooth muscle, causing it to dilate and thus lowering blood pressure. Therefore, the vasodilation observed through the action of sodium nitroprusside can indirectly reflect the function of vascular smooth muscle. In this embodiment, the endothelial nitric oxide synthase inhibitor L-NAME was added to investigate whether piracetam can directly act on vascular smooth muscle to cause vasodilation. Figure 13 As shown in Figure B, the addition of piracetam did not cause vasodilation, while the addition of SNP caused significant vasodilation. Therefore... Figure 13 B shows that the diastolic relaxation induced by piramister is endothelial-dependent.

[0138] The above results indicate that the PDE4 inhibitor of the present invention can act directly on vascular endothelial cells and promote vasodilation in an endothelial-dependent manner, thereby alleviating vascular endothelial dysfunction.

[0139] Example 10: Roflumilast alleviates myocardial ischemia-reperfusion injury in mice

[0140] This invention further investigated the effects of roflumilast on vascular endothelial cells. Similar to Example 7, C57BL / 6J mice underwent MI / R surgery. Before reperfusion and 12 hours after reperfusion following ligation of the left anterior descending coronary artery, the mice were injected intraperitoneally with a carrier and 1 mg / kg roflumilast, respectively. Figure 14 It can be seen that roflumilast significantly reduced the infarct area in mice.

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Claims

1. Use of a selective inhibitor of phosphodiesterase 4B (PDE4B) in the preparation of a pharmaceutical composition for the treatment or prevention of myocardial ischemia-reperfusion injury in a subject, wherein the inhibitor inhibits the expression and / or activity of PDE4B.

2. The use of claim 1, wherein the PDE4B selective inhibitor acts directly on vascular endothelial cells.

3. The use of claim 2, wherein the vascular endothelial cells express PDE4B.

4. The use of claim 2, wherein the PDE4B selective inhibitor causes vasodilation.

5. The use of claim 1, wherein the PDE4B selective inhibitor inhibits the interaction between inflammatory cells and vascular endothelial cells.

6. The use of claim 1, wherein the pharmaceutical composition is administered in the form of tablets, capsules, powders, pills, granules, injections, or emulsions.

7. The use of claim 1, wherein the pharmaceutical composition is administered to the subject by oral, sublingual, inhalation, intravenous, arterial, intramuscular, subcutaneous, intraperitoneal, or intracoronary administration.

8. A percutaneous endovascular angioplasty device comprising a balloon or stent, wherein the device contains or is coated with a PDE4 inhibitor.

9. The apparatus of claim 8, wherein the PDE4 inhibitor is a selective PDE4 inhibitor.

10. The apparatus of claim 8 or 9, wherein the selective PDE4 inhibitor is selected from piracetam, cyclophosphamide, AN-2728, AN-2898, CBS-3595, ELB-353, KF-66490, K-34, LAS-37779, IBBB-211913, AWD-12-281, cipanthenine, sillolast, roflumilast, BAY19-8004 and SCH-351591, AN-6415, indus-82010, TPI-PD3, ELB-353, CC-11050, GSK-256066, omepilast, OX-914, tetostrept, MEM-1414 and RPL-554 and their derivatives, solvates, polymorphs, prodrugs, active metabolites and / or pharmaceutically acceptable salts of these compounds.

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