阅读说明：本技术 通过激活Notch信号传导治疗血管阻塞 (Treatment of vascular obstruction by activation of Notch signaling ) 是由 王蓉 于 2019-07-25 设计创作，主要内容包括：本发明提供了用于治疗血流或循环不足的各种病症的新疗法。本文公开的新发明基于以下发现：动脉血管中Notch信号传导的增加对血流或循环和组织再生以及在动脉闭塞、收缩或其他血流减少后降低组织损伤具有有益作用。动脉中Notch信号传导的增加促进有益作用,包括急性血管扩张和/或动脉生成以及副动脉生长,并改进缺血或其他循环条件降低后的恢复。本发明还提供了用于将Notch激活剂递送至血管的医疗装置。(The present invention provides novel therapies for treating various conditions of inadequate blood flow or circulation. The new invention disclosed herein is based on the following findings: an increase in Notch signaling in arterial blood vessels has beneficial effects on blood flow or circulation and tissue regeneration as well as reducing tissue damage following arterial occlusion, constriction or other reduction in blood flow. An increase in Notch signaling in arteries promotes beneficial effects, including acute vasodilation and/or arteriogenesis and arteriolar growth, and improves recovery following ischemia or a decrease in other circulatory conditions. The invention also provides medical devices for delivering Notch activators to blood vessels.)

1. A Notch activator for use in treating a vascular disease, wherein the vascular disease comprises a disease, disorder or condition in which blood flow in one or more blood vessels is reduced, impeded or blocked.

2. The Notch activator of claim 1, wherein the vascular disease is an ischemic disease.

3. The Notch activator of claim 2, wherein the ischemic disease is cardiac ischemia.

4. The Notch activator of claim 3, wherein the cardiac ischemia comprises stable angina, unstable angina, acute coronary syndrome, angina, myocardial infarction, and atherosclerosis-induced ischemia.

5. The Notch activator of claim 2, wherein the ischemic disease is cerebral ischemia.

6. The Notch activator of claim 5, wherein the cerebral ischemia is acute ischemic stroke, transient ischemic attack, micro-infarction, vascular dementia, or cerebrovascular disease.

7. The Notch activator of claim 2, wherein the ischemic disease is limb ischemia.

8. The Notch activator of claim 2, wherein the ischemic disease is carotid artery disease.

9. The Notch activator of claim 1, wherein the vascular disease is selected from the group consisting of: peripheral arterial disease, renal insufficiency or kidney disease, including reduced blood flow; vascular disease of the eye; a decrease in spleen blood flow; mesenteric artery occlusion; intestinal infarction; small vessel disease; and reduced blood flow in blood vessels due to atherosclerosis or diabetes.

10. The Notch activator of any one of claims 1-9, wherein the Notch activator is a Notch1 activator.

11. The Notch activator of any one of claims 1-9, wherein the Notch activator is a Notch4 activator.

12. The Notch activator of any one of claims 1-9, wherein the Notch activator is a Notch ligand.

13. The Notch activator of any one of claims 1-9, wherein the Notch ligand is selected from the group consisting of: delta-like ligand 1, delta-like ligand 2, delta-like 3, delta-like ligand 4, Jagged-1, Jagged-2, and the aforementioned Notch activating variants.

14. The Notch activator of any one of claims 1-9, wherein the Notch activator is a Notch intracellular domain.

15. The Notch activator of any one of claims 1-9, wherein the Notch intracellular domain is selected from the group consisting of: notch1, Notch2, Notch3 and Notch4 intracellular domains.

16. The Notch activator of any one of claims 1-9, wherein the Notch activator is selected from the group consisting of: n-methylhemeanthidine chloride, valproic acid, resveratrol, triacetyl resveratrol, phenethylisothiocyanate and Yhhu-3792.

17. The Notch activator of any one of claims 1-9, wherein the Notch activator comprises a nucleic acid construct encoding a Notch activator or a downstream effector of Notch signaling.

18. A drug delivery device, wherein the drug delivery device is functionalized with one or more Notch activators or coated with a composition that elutes the one or more Notch activators when exposed to physiological conditions.

19. The drug delivery device of claim 18, wherein the one or more Notch activators comprise a Notch1 activator.

20. The drug delivery device of claim 18, wherein the one or more Notch activators comprise a Notch4 activator.

21. The drug delivery device of claim 18, wherein the one or more Notch activators comprise a Notch ligand.

22. The drug delivery device of claim 19, wherein the Notch ligand is selected from the group consisting of: delta-like ligand 1, delta-like ligand 2, delta-like 3, delta-like ligand 4, Jagged-1, Jagged-2, and the aforementioned Notch activating variants.

23. The drug delivery device of claim 18, wherein the one or more Notch activators comprise a Notch intracellular domain.

24. The drug delivery device of claim 23, wherein the Notch intracellular domain is selected from the group consisting of: notch1, Notch2, Notch3 and Notch4 intracellular domains.

25. The drug delivery device of claim 18, wherein the one or more Notch activators are selected from the group consisting of: n-methylhemeanthidine chloride, valproic acid, resveratrol, triacetyl resveratrol, phenethylisothiocyanate and Yhhu-3792.

26. The drug delivery device of claim 18, wherein the one or more Notch activators comprise a nucleic acid construct encoding a Notch activator or a downstream effector of Notch signaling.

27. The drug delivery device of any one of claims 18-26, wherein the device is a drug eluting balloon.

28. The drug delivery device of any one of claims 18-26, wherein the device is a stent.

29. Red blood cells functionalized with one or more Notch activators.

30. A method of treating a vascular disease in a subject in need of treatment, wherein the vascular disease comprises a disease, disorder or condition in which blood flow in one or more blood vessels is reduced, impeded or blocked, the method comprising:

administering to the subject a pharmaceutically effective amount of a Notch activator.

31. The method of treating a vascular disease as in claim 30, wherein said vascular disease is an ischemic disease.

32. A method of treating a vascular disease as in claim 31, wherein said ischemic disease is cardiac ischemia.

33. The method of treating vascular disease of claim 32, wherein said cardiac ischemia comprises stable angina, unstable angina, acute coronary syndrome, angina, myocardial infarction, and atherosclerosis-induced ischemia.

34. A method of treating a vascular disease as in claim 31, wherein said ischemic disease is cerebral ischemia.

35. The method of treating a vascular disease as in claim 34, wherein said cerebral ischemia is acute ischemic stroke, transient ischemic attack, micro-infarction, vascular dementia or cerebrovascular disease.

36. A method of treating a vascular disorder as claimed in claim 31, wherein said ischemic disorder is limb ischemia.

37. A method of treating a vascular disease as in claim 31, wherein said ischemic disease is carotid artery disease.

38. The method of treating a vascular disease as in claim 30, wherein said vascular disease is selected from the group consisting of: peripheral arterial disease, renal insufficiency or kidney disease, including reduced blood flow; vascular disease of the eye; a decrease in spleen blood flow; mesenteric artery occlusion; intestinal infarction; small vessel disease; and reduced blood flow in blood vessels due to atherosclerosis or diabetes.

39. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is a Notch1 activator.

40. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is a Notch4 activator.

41. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is a Notch ligand.

42. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is selected from the group consisting of: delta-like ligand 1, delta-like ligand 2, delta-like 3, delta-like ligand 4, Jagged-1, Jagged-2, and the aforementioned Notch activating variants.

43. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is a Notch intracellular domain.

44. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is selected from the group consisting of: notch1, Notch2, Notch3 and Notch4 intracellular domains.

45. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is selected from the group consisting of: n-methylhemeanthidine chloride, valproic acid, resveratrol, triacetyl resveratrol, phenethylisothiocyanate and Yhhu-3792.

46. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator comprises a nucleic acid construct encoding a Notch activator or a downstream effector of Notch signaling.

47. The method of treating a vascular disorder according to any of claims 31-38, wherein said treatment is prophylactic treatment.

48. The method of treating a vascular disease as in any of claims 31-38, wherein the treatment is administered intravenously.

49. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is administered by a drug eluting balloon.

50. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is administered on a stent.

51. The method of treating a vascular disease of any of claims 31-38, wherein the Notch activator is a red blood cell, a platelet, or a synthetic mimetic thereof functionalized with a Notch activator.

Background

Stenosis, constriction, obstruction, and/or occlusion can occur in blood vessels that traverse the circulatory system of the body, including in large or small arteries, arterioles, capillaries, venules, and veins. These vasoconstriction and obstruction can occur as a result of atherosclerosis, thrombosis, clotting, embolism, neoplasm or other obstruction, as well as other vascular abnormalities, reducing blood supply to a particular tissue or organ, resulting in poor circulation, hypoperfusion, ischemia and/or infarction. When constriction or obstruction occurs, diseases that require restoration of blood flow can result, including arterial occlusive disease, peripheral artery disease, critical limb ischemia, claudication, carotid artery disease, stroke, mini-stroke, cerebrovascular disease, heart disease, coronary artery disease, and other vascular ischemic diseases. Small strokes, micro-infarcts or hypoperfusion in the brain can impair organ function, leading to or contributing to conditions such as dementia, alzheimer's disease or other cognitive function decline.

Constriction and/or blockage of arteries affects up to 35% of americans. With the increase in the population of diabetes, hypertension and aging at risk for obstructive conditions, the incidence of these diseases may increase unless effective preventive and therapeutic strategies are developed.

In the treatment of vessel occlusion, constriction, hypoperfusion or other insufficiency of circulation, the goal is to restore normal blood flow and improve circulation. Currently, the treatment options for these conditions are primarily surgical procedures aimed at removing or opening the occluded arterial segment. For example, current treatments for critical limb ischemia include invasive surgical procedures such as surgical revascularization, percutaneous angioplasty, or stent placement. However, these procedures inherently damage the blood vessel. In addition, many patients may not be suitable for performing these procedures, while others may require multiple iterations of the procedure. In the case of preventive care, options include changing lifestyle, such as smoking cessation, increasing physical exercise, and weight loss, but these options have only variable success because they require patient compliance, they act indirectly on the vasculature, and they do not change genetics, age, gender, environment, or other non-lifestyle risk factors.

Ischemic stroke affects millions of people worldwide and is a major cause of death in many countries. Stroke can be treated by thrombolytic drugs such as Tissue Plasminogen Activator (TPA), neuroprotective drugs, and surgery. Despite the therapeutic utility of these treatments, the mortality, morbidity, and costs associated with this condition remain high, and there remains a need for improved therapies for the prevention and treatment of stroke.

There are many other medical conditions associated with insufficient blood flow, including renal insufficiency or disease (including that due to atherosclerosis or diabetes) which encompasses reduced blood flow, ocular diseases involving reduced blood flow (including that due to atherosclerosis or diabetes), and spleen infarction due to obstruction of blood flow.

Accordingly, there is a need in the art for new prophylactic and therapeutic options to address numerous diseases and conditions in which reduced blood flow is involved that do not meet the medical needs.

Summary of The Invention

In a first aspect, the scope of the present invention encompasses novel treatments for improving blood flow or circulation to treat various conditions of insufficient blood flow or circulation. The new invention disclosed herein is based on the following findings: an increase in Notch signaling in arterial blood vessels has beneficial effects on blood flow or circulation and tissue regeneration as well as reducing tissue damage following arterial occlusion, constriction or other reduction in blood flow. The increase in Notch signaling in arteries promotes beneficial effects, including acute vasodilation and/or arteriogenesis and collateral artery growth, where small collateral blood vessels are remodeled into the ductal arteries surrounding the occlusion, which is essential for restoring perfusion to ischemic tissue. The increase in Notch signaling in the artery enhances the conductance and reduces the resistance of the vessel. The increase in Notch signaling in arteries can also act through other mechanisms, resulting in improved blood flow or circulation. Thus, increasing Notch signaling in arterial blood vessels can be useful in the treatment of a number of diseases and disorders, including vascular occlusion, constriction, poor blood flow, or poor circulation.

In one aspect, the scope of the invention encompasses the treatment of disorders characterized by loss or reduction of blood flow or circulation, such as those caused by occlusion or constriction of blood vessels, by administering agents that increase Notch signaling in arterial blood vessels.

In another aspect, the scope of the invention encompasses the treatment of a disorder characterized by insufficient blood or circulation, such as ischemia, by administering an agent that increases Notch signaling in arterial blood vessels.

In yet another aspect, the scope of the invention encompasses prophylactic treatment of a subject at risk of vascular occlusion or impaired circulation by administering an agent that increases Notch signaling in arterial blood vessels.

The scope of the present invention also encompasses novel medical devices for delivering Notch activators to blood vessels.

Drawings

FIGS. 1A and 1B. Bmx-CreERT 2-mediated activation of Notch signaling through Notch1 endodomain (ICD) expression promotes neurological function recovery and reduces infarct volume in a distal middle cerebral artery occlusion (dMCAO) mouse model. FIG. 1A: after dmCAO-induced stroke, Bmx-CreERT 2-mediated activation of Notch1 signaling improves neurological recovery. Neurological function was assessed by modified Bederson ratings, elevated body swing tests, ladder tests and adhesion tests. BMX-Notch1 ICD (Bmx-CreERT 2; ROSA: LNL: tTA; TRE-Notch1 ICD) mice after dMCAO surgery exhibited better recovery of neurological function compared to control mice. Triangle: BMX-Notch1 ICD, circle: and (6) comparison. Control, n-12, BMX-Notch1 ICD; n is 13. P <0.05, P <0.01, P < 0.001. FIG. 1B: quantification of infarct volume compared to control mice showed significantly less infarct volume in BMX-Notch1 ICD mice (p < 0.01). Each n is 6.

FIGS. 2A and 2B. Bmx-CreERT2 activation of Notch signaling by Notch4 expression promotes neurological function recovery and reduces infarct volume in a distal middle cerebral artery occlusion (dMCAO) mouse model. FIG. 2A: activation of Notch4 following dMCAO improved neurological recovery. Neurological function was assessed by modified Bederson ratings, elevated body swing tests, ladder tests and adhesion tests. Triangle: BMX-Notch4, circle: and (6) comparison. FIG. 2B: quantification of infarct volume showed significantly less infarct volume in BMX-Notch4 mice compared to control mice (p < 0.01). Each n is 9.

Fig. 3A and 3B. Activation of post-stroke Notch signaling initiated by expression of Notch1 ICD promotes neurological recovery and reduction in infarct volume following ischemic injury. FIG. 3A: Bmx-CreERT2 activation of Notch1 after ischemic injury improves neurological recovery. After dMCAO, neurological function was assessed by modified Bederson grade, elevated body swing test, ladder test and adhesion test. Triangle: BMX-Notch1 ICD, circle: and (6) comparison. FIG. 3B: quantification of infarct volume by HE staining showed significantly less infarct volume in BMX-Notch1 ICD mice compared to control mice. Control, n-7, BMX-Notch1 ICD; n is 9.

Fig. 4A and 4B. In the initiation by Notch4 expression following ischemic injuryActivation of postwind Notch signaling promotes recovery of neural function and reduction of infarct volume. FIG. 4A: arterial activation of Notch4 after ischemic injury improved neurological recovery. After dMCAO, neurological function was assessed by modified Bederson grade, elevated body swing test, ladder test and adhesion test. Triangle: BMX-Notch4, circle: and (6) comparison. FIG. 4B: quantification of infarct volume by HE staining showed BMX-Notch4 in comparison to control mice^*Infarct volume in mice was significantly less. Control, n-8, BMX-Notch4^*；n＝7。

Fig. 5A and 5B. Arterial expression of Notchl ICD promotes growth of side branches and promotes restoration of cerebral blood flow. Figure 5A depicts an experimental protocol that begins Notch1 ICD expression 14 days before and shuts down Notch1 ICD 14 days after dMCAO. FIG. 5B: arteriolar diameter, velocity and flux during the time of the experiment, the vertical line indicates when the Notchl ICD expression was switched off. Triangle: BMX-Notch1 ICD, circle: and (6) comparison.

Fig. 6A, 6B and 6C. Treatment with Notch activating ligand DLL4 can improve growth of the posterior collateral branch of dMCAO and retain cerebral blood flow, as well as improve neurological function. Fig. 6A depicts an experimental time course in which recombinant DLL4 was administered one day before and seven days after the induction of a dmco stroke. FIG. 6B: arteriolar diameter, velocity and flux, the area between the vertical lines indicates the time of DLL4 administration. Triangle: rDLL4, circle: and (6) comparison. Control, n-5, treated; n is 5.^*P<0.05,^**P<0.01. Fig. 6C depicts the results of a step test on mice subjected to behavioral testing 15 to 17 days after experimental stroke, with the score being the average of the two measurements.

FIGS. 7A, 7B and 7C. Recovery in limb ischemia mouse model was improved by expression of Notch 4. Notch4 induction started 14 days before EFAO. Figure 7A, BMX-Notch4 mice significantly more blood flow within 5 weeks after EFAO. Foot perfusion, expressed as the ratio of the left foot (ischemic) to the right foot (control), was measured by Laser Doppler Perfusion Imaging (LDPI). Triangle: BMX-Notch4, circle: and (6) comparison. Data are mean ± SEM. P <0.01, p < 0.001. FIG. 7B: comparison of collateral artery diameter in left and right paw of BMX-Notch4 with control and Notch4 expressing mice 21 days after EFAO. Figure 7C, rate of change of EFAO posterior collateral artery diameter. For each n 8, P < 0.05.

Fig. 8. Figure 8 depicts quantification of muscle necrosis at day 7 after EFAO two weeks after Notch4 expression, indicating a statistically significant reduction in necrosis in Notch4 expressing mutants.

Fig. 9. Persistent improvement in foot perfusion in mice expressing Notch4 by day 14 post EFAO. Notch4 expression was initiated in the left hind limb of the mutant mice 14 days before EFAO. Foot perfusion was measured in the treated left foot and untreated right foot before and after EFAO. The ratio of perfusion values in the treated and untreated feet was tracked over time. On day 14 post EFAO, Notch4 expression was turned off. Square: BMX-Notch4 mice, circle: and (6) comparison.

Fig. 10. Significant improvement in foot perfusion was observed in mice expressing Notch4 after EFAO. Notch4 expression was induced by tamoxifen administration, followed immediately by EFAO induction in the left hind limb, and one day after EAFO (arrows). Foot perfusion was measured in the left foot operated and right foot not operated before and after EFAO. Fig. 10 depicts the ratio of left and right perfusion values for treated and untreated feet as a function of time. Square: BMX-Notch4, circle: and (6) comparison.

Detailed Description

The scope of the present invention encompasses the treatment of vascular disease by administering agents that activate Notch signaling in the blood vessels. In one embodiment, the scope of the invention encompasses agents that activate Notch signaling in blood vessels for use in the treatment of vascular disease. In related embodiments, the scope of the invention encompasses methods of treating vascular disease in a subject in need thereof by administering a pharmaceutically effective amount of an agent that activates Notch signaling in blood vessels. In related embodiments, the scope of the invention encompasses methods for the preparation of a medicament for the treatment of a vascular occlusion or constriction disorder using an agent that activates Notch signaling in blood vessels. Various features of the invention are described next.

And (6) treating. Various embodiments of the present invention relate to the treatment of vascular diseases as defined below. As used herein, "treatment" shall encompass any prophylactic or therapeutic treatment. In a first aspect, "treating" would encompass eliciting a therapeutic effect against one or more vascular diseases, including for example: inhibiting processes underlying vascular disease; ameliorating the symptoms of vascular disease; slowing the progression of vascular disease; reducing the severity of vascular disease; and cure vascular diseases.

In some embodiments, "treatment" as used herein shall encompass prophylactic treatment, e.g., treatment to effect one or more of the following: preventing the onset of vascular disease; delay, slow or arrest the progression of vascular disease; improving vascular circulation or health; reducing the risk of developing vascular disease; slowing or arresting the progression of ischemic disease; and increasing or restoring Notch signaling in blood vessels.

In some embodiments, "treating" or "treatment" as used herein refers to achieving one or more physiological, physical, functional, therapeutic, or performance results. For example, the treatment may encompass: increasing Notch signaling in one or more blood vessels; improving blood flow or circulation through one or more blood vessels; promoting arteriogenesis; dilating one or more blood vessels; enhancing electrical conductance of one or more blood vessels; reducing the resistance of one or more blood vessels; and improving vascular tone of one or more blood vessels.

The treatment of the invention may achieve a local effect, for example improving blood flow at an ischemic site or organ, or may achieve a systemic effect, for example improving the general circulation of the whole body.

Vascular diseases. Various embodiments of the present invention relate to the treatment of vascular disease. As used herein, "vascular disease" may include any disease, disorder, or condition, including acute and chronic diseases, in which blood flow in one or more blood vessels is reduced, impeded, or blocked. Vascular disease may include diseases that constitute a reduction, obstruction, or blockage of blood flow. Vascular disease may also include diseases that cause or reduce, impede or block blood flow.

In one aspect, the vascular disorder is ischemia. In one embodiment, the ischemia is cardiac ischemia, also known as coronary artery disease, also known as ischemic heart disease. Cardiac ischemia encompasses stable angina, unstable angina, acute coronary syndrome, angina, myocardial infarction, and atherosclerosis-induced ischemia.

In one aspect, the vascular disease is cerebral ischemia. In one embodiment, the cerebral ischemia is acute ischemic stroke. In one embodiment, the cerebral ischemia is a transient ischemic attack or a stroke. In one embodiment, the cerebral ischemia is or micro-infarction. In one embodiment, the cerebral ischemia is vascular dementia. In one embodiment, the vascular disease is cerebrovascular disease. In one embodiment, the vascular disease is a disease that encompasses a reduction in circulation in the brain, including, for example, dementia, alzheimer's disease, or other decline in cognitive function.

In one embodiment, the vascular disorder is limb ischemia. In one embodiment, the limb ischemia is critical limb ischemia.

In one embodiment, the vascular disease is intestinal ischemia.

In one embodiment, the vascular disease is carotid artery disease.

In one embodiment, the vascular disease is peripheral arterial disease. In one embodiment, the peripheral arterial disease is critical limb ischemia. In one embodiment, the peripheral arterial disease is claudication.

In one embodiment, the vascular disease is renal insufficiency or kidney disease, including that due to atherosclerosis or diabetes, which encompasses reduced blood flow.

In one embodiment, the vascular disease is an ocular disease involving reduced blood flow, including due to atherosclerosis or diabetes.

In one embodiment, the vascular disease involves reduced blood flow in the spleen, including infarction of the spleen due to obstruction of blood flow.

In one embodiment, the vascular disease is mesenteric artery occlusion or ileus.

In one embodiment, the vascular disease is a smoke disease.

In one embodiment, the vascular disease is a cerebral autosomal dominant hereditary arteriopathy with subcortical infarction and white matter encephalopathy, sometimes referred to as cadisil.

In one embodiment, the vascular disease is Alagille syndrome or Alagille-Watson syndrome, including genetic or spontaneous forms thereof, such as that found in the liver, heart, kidney, and other systems of the body. ALGS is caused by loss of function mutations in JAG1 or NOTCH 2.

In one embodiment, the vascular disease is small vessel disease, small vessel infarction, or leukosis.

In one embodiment, vascular disease is a need to establish or improve blood flow after receiving transplanted tissue (e.g., kidney, liver, lung, heart, cell graft).

In one embodiment, vascular disease is a need to establish or improve blood flow in a region of regenerating tissue.

The method of the invention is particularly suitable for treating vascular obstructions or constrictions in blood vessels. In one embodiment, the vascular disease is a disease that encompasses reduced circulation in arterioles or arterioles, where it can also cause poor circulation and impair organ function. In other embodiments, the vascular disease is manifested in veins, capillaries, or transplanted blood vessels.

In one embodiment, the vascular disease is arterial occlusive disease of any part of the body. In one embodiment, the vascular disease is hypoperfusion. In one embodiment, the vascular disease is atherosclerosis. In one embodiment, the vascular disease is thrombosis. In one embodiment, the vascular disease is the formation or persistent presence of a clot. In one embodiment, the vascular disease is embolism. In one embodiment, the vascular disease is pulmonary embolism. In one embodiment, the vascular disease is a neoplasm or other obstruction of a blood vessel.

A subject. The methods of the invention are applied to treating a subject. The subject will be a subject in need of treatment for a vascular disease, e.g., a subject suffering from or at risk of a vascular disease. The subject can be any animal, e.g., a human subject, a non-human primate, a mouse, a rat, other rodents, dogs, cats, cows, pigs, horses, or any other animal species. In one embodiment, the subject is a human patient. In one embodiment, the subject is a veterinary subject. In one embodiment, the subject is a test animal.

In one embodiment, the subject is a subject at risk for vascular disease. In one embodiment, the subject at risk for vascular disease is an elderly subject. For example, for a human subject, an elderly subject may be at least 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 80 years old, or older. In other embodiments, the subject at risk for a vascular disease is a smoker or a ex-smoker. In other embodiments, the subject at risk for a vascular disease is a subject with a medical or family history of vascular disease. In other embodiments, the subject at risk for a vascular disease is an overweight or obese person, for example, with a BMI greater than 25 or greater than 30. In one embodiment, the subject at risk is a subject with diabetes. In one embodiment, the subject at risk is a subject with hypertension.

Notch signaling is activated. The scope of the present invention encompasses the use of agents that are activators of Notch signaling. Notch receptors are single-pass transmembrane receptor proteins. Mammalian Notch receptors encompass Notch1, Notch2, Notch3 and Notch4, wherein Notch1 and Notch4 are expressed in arterial endothelial cells.

Notch pathways mediate intercellular signaling, by which a series of cell fate decisions in neuronal, vascular, cardiac, immune, endocrine development and other processes are regulated. In Notch signaling, cell surface Notch receptors interact with transmembrane ligands present on the surface of adjacent cells. Ligand binding then results in a series of proteolytic cleavages of the Notch receptor, releasing the Notch intracellular domain (ICD) from the membrane. ICDs are translocated into the nucleus where they enter a complex with mammalian nuclear proteins (CSL protein CBF-1/RBPJ) to regulate transcription of various target genes. Notch activates classical signaling through RBPJ. Notch also activates non-classical signaling through other downstream effectors.

As used herein, "activation of Notch" or "activation of Notch", referred to herein as "activation of Notch signaling", encompasses the induction, increase or upregulation of any Notch signaling activity or effect (classical or non-classical) known in the art. Activation of Notch signaling as referred to herein includes enhancement of existing endogenous Notch activity, restoration of normal endogenous Notch activity, and enhancement and increase of Notch activity in arterial vessels. Notch signaling may encompass, for example: cleaving Notch to release ICD, translocation of ICD to nucleus, increasing TNF- α ADAM metalloprotease converting enzyme (TACE) activity; increasing ubiquitination of the cleaved Notch extracellular domain by Mib, increasing γ -secretase mediated release of the Notch intracellular domain, enhancing formation or activity of the CBF1/Su (H)/Lang-1 transcription factor complex; and/or a Notch downstream gene that modulates consistent with internal Notch activity (either by ICD or by an agent that bypasses and mimics one or more of the effects of ICD). Downstream genes include Deltex-1, Deltex-2, Deltex-3, Deltex repressor (SuDx), Numb and its isoforms, Numb-associated kinase (NAK), Notchless, Disheveled (Dsh), emb5, border genes (e.g., radial, Lunatic, and Manic), PON, LNX, Disabled, Numblike, Nur77, NFkB2, Mirror, Warthog, Engrailed-1 and Engrailed-2, Lip-1 and its homologs, polypeptides involved in the Ras/MAPK cascade regulated by Deltex, Epherin-B2, Myc, p21, and HES family members, ephrin-B2, Eph-B4, connexin 40, FBW7, and other Notch target genes. Activation of Notch signaling ultimately leads to changes (up-regulation or down-regulation) in gene expression of downstream target genes.

Notch activation would further encompass, for example, an increase in Notch activity in the target cell; increased expression of a Notch gene or protein, a Notch ligand gene or protein, a Notch positive regulatory gene or protein, a Notch signaling mediating gene or protein, a Notch signaling regulatory gene or protein, or a downstream target of Notch in a target cell; up-regulation of Notch signaling; increased expression or activity of downstream substances or effectors of Notch activation; regardless of the mode of action, inhibition of a negative regulator of Notch signaling or any other increase in Notch signaling occurs in a beneficial manner in the target cell.

As used herein, Notch activation will encompass any activation of the Notch signaling pathway, including pathways mediated by Notch1, Notch2, Notch3, and Notch4 through classical or non-classical signaling. In a major embodiment, since Notch1 and Notch4 are subtypes expressed predominantly in arterial endothelial cells, Notch activation will refer to Notch1 and/or Notch4 activation.

As used herein, Notch activation will encompass activation of Notch signaling in any cell type of the body. In a main embodiment, Notch signaling activation will refer to activation of the Notch signaling pathway in vascular cells, including vascular endothelium or smooth muscle cells. In a main embodiment, Notch signaling activation will refer to activation of the Notch signaling pathway in arterial cells (e.g., arterial endothelial cells). Without being bound by any particular theory, it is believed that Notch activation in vascular endothelial cells is primarily responsible for the therapeutic effects described herein. However, it is to be understood that the therapeutic effect of Notch activation may result from Notch activation in other cell types (e.g., smooth muscle cells or blood cells) or in other regions of the body, and the scope of the present invention is not limited to the therapeutic effect of Notch activation in arterial endothelial cells.

A Notch activator. The scope of the invention encompasses administration of Notch activators. Notch activators include compositions of any substance that increases Notch signaling in cells of the body (e.g., vascular endothelial cells, such as arterial endothelial cells). The Notch activator can comprise any agent having Notch activating activity, including, for example, antibodies, small molecules, peptides and proteins, and nucleic acids.

In one embodiment, the Notch activator is a small molecule. Exemplary small molecule Notch agonists include N-methylhemeanthidine chloride, valproic acid, resveratrol, triacetyl resveratrol, phenethylisothiocyanate and Yhhu-3792, as well as the aforementioned Notch activating chemical analogs and variants.

In one embodiment, the Notch activator is a peptide or protein. In a first embodiment, the peptide or protein is a Notch ligand, i.e., a substance that is expressed on the cell surface in vivo and interacts with Notch and activates Notch signaling upon binding to the Notch extracellular domain. Notch ligands include any mammalian Notch ligand, such as Delta-like ligands, including Delta-like ligand 1(DLL1), Delta-like ligand 2(DLL2), Delta-like 3(DLL3), Delta-like ligand 4(DLL 4). Notch ligands also include the mammalian Jagged ligands Jagged-1 and Jagged-2. The Notch ligands may further comprise non-mammalian Notch ligands or variants thereof, for example, Delta proteins, Serrate family proteins (including Serrate-1 and Serrate-2), and LAG-2.

The Notch ligand may comprise a variant of a known Notch ligand, e.g., a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a Notch ligand, while maintaining or enhancing Notch activating activity. Notch ligand variants may also comprise truncations of the wild type protein. For example, in one embodiment, the Notch activating ligand variant comprises a highly conserved Jagged 1 delta/Serrate/lang-2 (DSL) domain. In another embodiment, the Notch activating ligand variant comprises amino acid residue 188-204 of human Jagged 1, which has been shown to have Notch activating activity, for example as described in Kannan et al (2013) Notch activation inhibitors AML growth and preservation a potential therapeutic approach.J Exp Med 210: 321-337. Wild-type DLL ligands contain eight epidermal growth factor-like (EGF-like) sequence repeats, while wild-type Jagged ligands contain twelve EGF-like repeat sequences. The Notch ligand variants of the invention may comprise variants of DLL or Jagged with altered EGF-like sequence repeat numbers compared to the wild-type sequence. Furthermore, Notch ligand variants may comprise hybrid proteins consisting of subunits of two or more ligand types, e.g. as in tvariakhina et al, The ectomians defined ligand function in vivo and selectivity of DLL1 and DLL4 aware Notch1 and Notch2 in vitro life 2018; 7 e 40045. For example, in one embodiment, the Notch ligand variant comprises N-terminal MNNL and DSL domains and adjacent EGF repeats 1-3 of DLL1, DLL2, DLL3, DLL4, Jagged 1, or Jagged 2. In another embodiment, the Notch Ligand variant comprises a DLL4 mutant comprising a proline substituted arginine at position 257, such as Liu et al, Identification of Domains for effective Notch Signaling Activity in Immobilized Notch Ligand Proteins, J Cell Biochem 2017 Apr; 118(4) 785 and 796.

In one embodiment, the peptide or protein activator of Notch is a Notch ligand mimetic, including engineered variants and de novo synthesized molecules, comprising amino acid sequences, peptides and proteins having Notch activating activity. For example, engineered variants may comprise the ligand binding domain of a Notch ligand and other active domains thereof, e.g., altered to have increased activity.

In one embodiment, the peptide or protein activator of Notch is Notch ICD, e.g., Notch1, Notch2, Notch3 or Notch4 ICD. ICDs can be fused or conjugated to vectors for intramembranous or transmembrane transport, such as lipid vectors or peptide substances that facilitate transmembrane transport. For example, ICDs may be fused to trans-Transcriptional Activator (TAT) peptides, osmoxins, cholesterol-dependent hemolysins, or other cell penetrating peptides.

In one embodiment, the Notch activating peptide or protein comprises an antibody or antigen binding fragment thereof, wherein the antibody binds to Notch and initiates ICD cleavage and/or Notch activation. For example, in one embodiment, the Notch activator comprises antibody A13, as described in Li et al, Modulation of Notch Signaling by Antibodies specificity for the excellular Negative Regulatory Region of NOTCCH 3, J Bio Chem 283:8046-8054, 2008.

In one embodiment, the Notch activator comprises a lipid. Jagged and DLL ligands comprise lipid binding motifs, where binding of certain lipids can modulate Notch activation. In one embodiment, the lipid having Notch activating activity is a lipid that binds to Notch or a Notch ligand. For example, in one embodiment, the Notch activating lipid comprises sphingosine-1-phosphate (S1P). Similarly, the S1P receptor 3 protein may be used.

In one embodiment, the Notch activator is a nucleic acid, such as a genetic construct, that is delivered to and expressed by a target cell. In one embodiment, the Notch activator is a nucleic acid construct encoding a Notch activator protein or a downstream effector of Notch signaling. In one embodiment, the construct encodes a Notch receptor (e.g., Notch1, Notch2, Notch3, or Notch 4). In one embodiment, the Notch receptor is Notch4, which as used herein represents a Notch Flk1/int-3 allele that is a gain of function Notch4 mutant known in the art that produces a Notch4 protein in which upon expression the intracellular domain is constitutively cleaved. In one embodiment, the construct encodes a Notch intracellular domain, e.g., lacks an extracellular domain, e.g., a Notch1 or Notch4 intracellular domain. In one embodiment, the construct encodes a Notch ligand, e.g., DLL1, DLL2, DLL3, DLL4, Jagged 1, Jagged 2, or a variant of a Notch ligand.

The Notch activating gene construct may comprise any type of expression vector, including, for example, gene constructs delivered by gene therapy techniques: viral vectors (e.g., adenovirus or adeno-associated virus, lentivirus), clustered regularly interspaced short palindromic repeat-associated nuclease system (CRISPR/Cas) -type constructs, CRISPRa, nanoparticle-mediated gene delivery (e.g., dendrimers, lipids, chitosan gene delivery particles, etc.), or any other gene therapy construct known in the art. The genetic construct may further comprise a high level of a constitutive promoter for expression of Notch activators or an inducible promoter for controlled expression of genes in the Notch signaling pathway. The promoter may be a tissue-specific promoter, for example, a human artery-specific promoter, such as fms-like tyrosine kinase-1 (FLT-1), intercellular adhesion molecule-2 (ICAM-2) and von Willebrand factor (vWF) promoters, DLL4, Notch1, Notch4, connexin 40, connexin 43, connexin 37. In murine subjects, promoters such as BMX may be used.

In one embodiment, the Notch activator is an inhibitor of a negative regulator of Notch, such as inhibitors of ubiquitin ligases RNF8, NUMB, SEL-10 and FBW 7. The inhibitor may be a small molecule, peptide, amino acid, or other substance that down-regulates or inhibits a negative regulator of Notch activation, and wherein inhibiting the negative regulator causes induction or increase of Notch signaling.

In one embodiment, the Notch activator is an RNA that affects Notch activation, such as a microRNA, RNAi construct, short hairpin RNA, or other RNA sequence that can increase Notch signaling. For example, the RNA can comprise microRNA or other RNA that inhibits the expression or activity of a negative regulator of Notch signaling. The RNA construct can comprise a transient expression vector for expression of Notch activator protein.

Delivery of the nucleic acid construct to the target cell (e.g., endothelial cells of an artery) can be accomplished by any means known in the art. For example, delivery can be achieved by viral gene vectors, electroporation, bio-ammunition delivery systems, microinjection, ultrasound, hydrodynamic delivery, liposome delivery, polymer or protein based cationic agents (e.g., polyethyleneimine, polylysine), injection systems, and DNA delivery dendrimers. Gene delivery can be systemic (e.g., intravenous), or local, e.g., by local injection, through a catheter (e.g., a drug eluting balloon catheter), or through a drug eluting implant (e.g., a stent). Liposomal delivery systems may also be used. [00] For example, by delivering the transgene to be expressed into vascular tissue. Methods for targeted delivery to blood vessels can be adapted according to methods known in the art, for example, those described in U.S. patent application publication No. 20050053590 to Meininger, PCT International patent application publication No. 2002042426 to Yu et al, U.S. patent application publication No. 20090209630 to Carrier system for specific tissue Gene delivery, and Coleman et al, entitled "Gene delivery methods and methods for therapeutic of chemical conditions".

In one embodiment, the Notch activator is an herbal or plant based composition. For example, lilium Raphiolepis (Zephyranthes Candida) extract has the Notch activating molecule N-methylhemeanthidine chloride, as described in Ye et al, Small molecule activation of NOTCH signaling inhibition of tissue culture muscle Scientific Reports volume 6, Article number: 26510.

Constructs, formulations and administration of Notch activators. The Notch activators of the present invention can be configured and formulated to enhance efficacy and can be combined with devices or other agents.

As known in the art, the Notch activators of the present invention can be administered in combination with pharmaceutically acceptable excipients, carriers, diluents, release formulations, and other drug delivery vehicles.

The Notch activators of the present invention can include a targeting moiety, which is a composition of matter that targets the Notch activator to the target vascular cell. In some embodiments, the methods of the invention are applied in any arterial vessel, including arteries and arterioles. In one embodiment, the methods of the invention comprise administering an agent that selectively or preferentially increases Notch signaling in arterial cells, including arterial endothelial cells. Selectively or preferentially increasing Notch signaling in arterial cells may encompass enhanced Notch signaling that targets arterial cells, is not important or present in non-arterial vessels and other non-target tissues, or has a greater amplitude or duration in arterial cells than in non-arterial vessels.

The target vascular cells may further include other vascular cells in other vascular types (e.g., veins), including endothelial cells, smooth muscle cells, and blood cells. For example, targeting moieties that bind to ligands that occur during stress or inflammation can be used to target Notch activators to diseased or damaged endothelial cells. Exemplary targeting moieties include, for example, leukocyte adhesion molecules, LDL, and derivatized phospholipids. Other ligands present in blood vessels (e.g., arterial blood vessels) can be used to target Notch activators to relevant cell types.

In some embodiments, the Notch activator is delivered alone substantially in buffer, saline or water without excipients. Advantageously, as described in the examples section, intravenously delivered Notch activators appear to activate Notch primarily in the arterial endothelium, without the presence of specific targeting moieties.

In some embodiments, the Notch activator consists of a lipidAnd (4) body delivery. Liposome delivery systems are suitable for the delivery of nucleic acids, peptides and other agents. Arterial Delivery of agents is described, for example, in Hwang et al, stimulating Cerebral Blood Flow Through lipid Delivery of antigenic Peptides¹⁸F-FDG PET Imaging in Ischemic Stroke Treatment J Nucl Med.2015；56:1106-1111。

In some embodiments, methods of microbubble delivery known in the art, including ultrasound-mediated microbubble delivery, can be utilized for site-specific delivery.

In some embodiments, the Notch activator is delivered from a nanoparticle-loaded membrane, such as a membrane wrapped around a target vessel, as known in the art.

In some embodiments, the Notch activator is delivered by a drug-antibody conjugation technique, wherein the Notch activator is conjugated to an antibody or antigen-binding fragment thereof (e.g., a binding fragment generated by phage display), wherein the antibody or antigen-binding fragment selectively binds to a ligand present in an artery or other target cell type. For example, the targeting moiety may be directed to CD34, adhesion molecule 1, and other arterial ligands, e.g., ligands found in damaged blood vessels (e.g., cross-linked fibrin).

In one embodiment, the Notch activator comprises a Notch activating composition chemically conjugated to a magnetizable microparticle or nanoparticle (e.g., iron oxide, iron oxide) for use in magnetically targeted drug delivery methods known in the art.

In some embodiments, the targeting moiety and the Notch activating composition are both proteins or peptides. In this embodiment, the Notch activator comprises a fusion protein comprising a targeting moiety and a Notch activator protein or peptide.

In one embodiment, the Notch activators of the present invention are coated, conjugated, or otherwise present on the device. As is known in the art, the device may be delivered to the target site by transcatheter delivery.

In one embodiment, the device can be coated with a formulation of Notch activator mixed with a polymeric material for timed release elution of the agent, as is known in the art. Exemplary drug eluting polymers may include materials known in the art, such as polyurethane, polyclonal, polymethylmethacrylate, polyvinyl alcohol, and polyethylene. In another embodiment, the Notch activator is conjugated directly to the surface of the device.

For example, in one embodiment, the device may comprise an implant. In one embodiment, the implant may comprise a stent. The stent may be a metal stent or a polymer stent, such as a biodegradable or absorbable polymer stent.

In one embodiment, the device may comprise a drug-coated balloon as known in the art. Drug-coated balloons include polymeric balloons that are deployed from a catheter, such as, once positioned via radiographic imaging, the balloon is inflated so that it contacts the vessel wall and exposes the vessel wall to agents coated on the outside of the balloon, typically in a carrier or vehicle such as polysorbate, sorbitol, iopromide, trihexyl butyryl citrate (BTHC), glyoxylic acid, and laccaic acid. The balloon may be placed for a period of time to effectively transfer to the arterial wall.

In some embodiments, the Notch activator is formulated in or on a drug delivery particle composition (e.g., microspheres, nanospheres, nanoparticles, vesicles, synthetic exosomes, and other drug delivery particles).

In one embodiment, the Notch activator is conjugated to a red blood cell, a platelet, or a synthetic mimetic of a red blood cell or a platelet. Methods for modifying red blood cells are known in the art and include, for example, those described in Shi et al, Engineered red blood cells as carriers for system delivery of a wide array of functional probes, PNAS 201428: 10131-. Similarly, functional platelets can be used, for example as reviewed by Lu et al, Platlet for Drug Delivery 2014, Curr OpBiotech 58: 81-91.

The Notch activator can be administered in combination with any other therapeutic composition, such as: thrombolytic agents, such as tissue plasminogen activator; a neuroprotective agent; hypotensor; an anticonvulsant; an adrenergic receptor antagonist; and other drugs used to treat ischemia or other vascular diseases.

Administration of Notch activators. In the treatment of the present invention, a Notch activator is delivered to one or more sites in the body in a pharmaceutically effective amount to treat a selected vascular disorder. A pharmaceutically effective amount is any amount sufficient to induce a measurable therapeutic effect or a measurable Notch signaling activity.

The method and timing of administration will be selected based on various factors: the vascular disease in question; the development and status of vascular disease; a condition of the subject; physical and pharmacological properties of the selected Notch activators; and a delivery mode.

For example, local or systemic delivery of Notch activators may be performed. In one embodiment, for example, systemic administration to the circulatory system is achieved. In the case of prophylactic treatment, e.g. to enhance arterial elasticity in a subject at risk of a vascular disease, e.g. prophylactic treatment of ischemia or another vascular disease in an elderly subject, systemic administration may be desirable. Systemic delivery may also be possible when the Notch activator has minimal or acceptable off-target effects. In other cases, local delivery is preferred, for example, when treating acute injury, occlusion, or other local conditions, such as where a catheter or other device has difficulty accessing a stenotic region.

For example, in the case of prophylactic treatment to create or maintain a reduced risk of vascular disease, or in the treatment of chronic disease, administration of the agent may be chronic. Administration may be short-term, for example in the treatment of acute vascular diseases such as ischemia.

In a main embodiment, considering that the target cell is a vascular cell, the administration of the Notch activator will be performed by intravenous route. In alternative embodiments, administration may be oral, topical, by intraperitoneal injection, or compatible with the Notch activator of choice.

In a first embodiment, the Notch activator is administered systemically by intravenous injection. In this case, the agent is readily exposed to target endothelial cells of the circulatory system. This route of administration advantageously enables delivery to vessels deep within the body, or otherwise which are not readily accessible by surgical intervention.

In alternative embodiments, the Notch activator is delivered locally, for example by injection into a diseased vessel. For example, the Notch activator can be delivered to a portion of the vessel immediately upstream and/or downstream of or within the diseased area, such as an occluded portion or infarct.

In one embodiment, the Notch activator is delivered locally to the diseased vessel by a catheter or similar device introduced into the vessel. The Notch activator can flow or partition from the catheter or can be present on the surface of the catheter or in a drug eluting structure (e.g., a balloon) delivered by the catheter to the diseased site.

In another embodiment, the Notch activator is administered in combination with a surgical implant. For example, in one embodiment, the Notch activator is administered on a stent or other implant, wherein the implant is coated with the Notch activator in a drug eluting delivery vehicle or formulation.

Appropriate dosages and treatment regimens will vary and may be selected by those skilled in the art depending on the patient, disease, agent, and delivery factors. An exemplary dose of 1 nanomole to 100 micromole can be administered, such as a dose of 1 to 1000 micrograms of agent per kilogram of body weight per day, such as a dose of about 50 to 100 micrograms of agent per kilogram of body weight per day. The dose may be administered, for example, in the case of a drug eluting pump or device, multiple times per day, daily, every other day, or weekly or continuously. The dose may be administered for several days, weeks, months or longer.

Detailed description of the preferred embodiments

Example 1. mice with inducible arterial Notch signaling. To investigate the role of Notch signaling in the ischemic mouse model, constitutively active forms of Notch1 and Notch4, denoted Notch1 ICD and Notch4, respectively, were used. Notch1 ICD comprises a truncated Notch1 receptor intracellular domain (ICD) without a transmembrane or extracellular domain, functionally replicates the cleaved Notch intracellular domain, and is constitutively active when expressed. The Notch4 × mutant contains a truncated Notch4 receptor with transmembrane and intracellular domains but no extracellular domain and is constitutively cleaved into Notch4 ICD. Thus, Notch4 was functionally constitutively active when expressed. Thus, Notch1 ICD or Notch4 increased Notch signaling when expressed in cells.

Expression of these two mutant Notch genes was placed under the tetracycline (Tet) response element (TRE) activated by Tet transactivator (tTA), TRE-Notch1 ICD and TRE-Notch4 in the tetracycline (Tet) -OFF gene expression system. tTA is expressed upon Cre activation in the ROSA: LNL: tTA allele. Thus, the expression of Notch1 ICD and Notch4 was in Bmx (PAC) -CreER^T2Under the control of the transgene. Bmx (PAC) -CreER^T2Active primarily in arterial endothelial cells, where reporter studies also indicated some sporadic detectable expression in the cerebral veins and capillaries, some sporadic detectable expression in the limb veins, and no substantial expression detected in the limb capillaries. This construct was coupled to a tamoxifen inducible system and a tetracycline (tet) -OFF gene expression system. Administration of tamoxifen activates bmx (pac) -CreER^T2And results in tTA expression. In the absence of tetracycline, tTA is active (and thus the gene is expressed as ON). In this system, induction of transgene expression is controlled only by tamoxifen administration if the animal is not treated with tetracycline or a derivative thereof. For gene induction, tamoxifen was administered to adult mice two consecutive days. [ tamoxifen injection was injected once daily for two days by IP. In certain treatments, injections were administered on days 14 and 13 prior to dmao or EFAO. In other treatments, induction of Notch signaling begins at the time of the occlusion event (dmaco or EFAO), administered on days 0 (day of occlusion) and 1 following dmaco or EFAO.

Carrying Bmx (PAC) -CreER^T2The mouse of (1); ROSA, LNL, tTA; the mice of TRE-Notch1 ICD are referred to herein as BMX-Notch1 ICD mice, and carry Bmx (PAC) -CreER^T2(ii) a ROSA, LNL, tTA; the TRE-Notch4 mice are referred to herein as BMX-Notch4 mice. Control mice harbored Bmx (PAC) -CreER^T2And/or ROSA LNL tTA construct but no Notch.

In mice expressing Notch4, studies of Notch4 staining in limbs showed that Notch4 co-localized with vascular endothelial cells, but could not exclude expression of Notch4 in other cell types.

The detection of membrane-targeted green fluorescent protein produced by Rosa26-mT/mG reporter gene shows that Bmx (PAC) -CreER^T2Activity is mainly present in arteries, strong in arterial Endothelial Cells (EC), weak and sporadic in venous cells, and absent in capillaries of the hind limb. Analysis of multiple tissues showed that in all cases Bmx (PAC) -CreER^T2The activity is mainly present in arterial cells. Furthermore, Bmx (PAC) -CreER^T2Activity is also present to a lesser extent in the veins and lymphoid ECs of the ear, lymphoid ECs of the mesentery and venous ECs of the cremaster muscle. None of the tissues showed significant Bmx (PAC) -CreER in the capillary bed^T2And (4) expressing.

Example 2 arterial activation by Notch signaling expressed by the intracellular domain (ICD) of Notch1 promotes recovery in a mouse model of ischemic stroke. As known in the art, Notch1 ICD expression was induced in BMX-Notch1 ICD mice by administration of tamoxifen 14 days and 13 days prior to experimental stroke induced by the dMCAO process. Control and BMX-Notch1 mice were subjected to the dMCAO program. Nerve function was tested before and after dMCAO. Arterial activation of Notch1 improved neurological recovery after dMCAO. Neurological functions were assessed (fig. 1A), including by modified Bederson ratings, elevated body swing tests, ladder tests, and adhesion tests. BMX-Notch1 ICD mice exhibited better recovery of neurological function after dMCAO surgery compared to control mice. H & E staining showed fewer infarcts in BMX-Notch1 ICD mice compared to control mice. Quantification of infarct volume compared to control mice showed significantly less infarct volume (p <0.01) in BMX-Notch1 ICD mice (fig. 1B).

Furthermore, the angio-casting of the middle cerebral artery of BMX-Notch1 ICD mice [ removal of all pia (pia) ] showed more pronounced remodeling of the collateral (anterior cerebral artery-middle cerebral artery anastomosis) compared to control mice. Although collateral remodeling was observed ipsilateral to dMCAO in both BMX-Notch1 ICD and control mice, the degree of remodeling of vessel number, diameter and tortuosity was more pronounced in BMX-Notch1 ICD mice. The magnification clearly shows that pia mater collateral is more pronounced and tortuous in mice expressing BMX-Notch1 ICD compared to control mice.

Similar results were observed in arterial activation of Notch4 signaling. Notch4 expression promoted neurological recovery and reduced infarct volume in the dMCAO model mice. Arterial activation of Notch4 improved neurological recovery after dMCAO. Neurological functions were assessed (fig. 2A), including by modified Bederson ratings, elevated body swing tests, ladder tests, and adhesion tests. H & E staining showed fewer infarcts in BMX-Notch4 mice compared to control mice. Quantification of infarct volume compared to control mice showed significantly less infarct volume in BMX-Notch4 mice (p <0.01) (fig. 2B).

Vascular remodelling of arteries in BMX-Notch4 mice showed more pronounced collateral remodelling compared to control mice. The magnification clearly shows that the medial side branch is more pronounced and tortuous in BMX-Notch4 mice compared to control mice.

These results indicate that induction of Notch signaling can greatly improve recovery in injured mice, with greater restoration of blood flow and reduced infarct volume, before and after acute ischemic stroke. The data indicate a prophylactic as well as a therapeutic effect.

Example 3 activation of Notch signaling following ischemia promotes recovery of nerve function, reduction of infarct volume and increased collateral artery growth.

Previous results indicate that activation of Notch signaling before and after injury can achieve improved results in the brain following ischemic injury. To demonstrate that this effect can be applied in a therapeutic setting following injury, experiments were performed in which Notch activation was induced following ischemic injury. In these experiments, control and mutant Notch mice were subjected to dMCAO and then induced Notch ICD expression by tamoxifen administration. To determine activation of Notch signaling after stroke onset, tamoxifen (2 mg/body, ip) was injected two consecutive days after the dMCAO surgery.

Arterial activation of Notch1 after ischemic injury improved neurological recovery. Neurological function was assessed before and after the dcao ischemic injury (fig. 3A), including by modified Bederson grade, elevated body swing test, ladder test and adhesion test. BMX-Notch1 ICD mice showed significantly better recovery of neurological function compared to control mice. Since Notch1 signaling was gradually obtained after stroke, the difference between the two groups became significant starting 7 days after stroke. H & E staining showed fewer infarcts in BMX-Notch1 ICD mice (in which upregulation of Notch1 signaling was induced following stroke) compared to the control group. Quantification of infarct volume as determined by H & E staining showed significantly less infarct volume in BMX-Notchl ICD mice compared to control mice (fig. 3B).

Images of arterial angioplasty indicate more pronounced collateral remodeling in BMX-Notch1 ICD mice when Notch1 ICD is expressed after experimental stroke compared to control mice. The images clearly show that the side branches of BMX-Notch1 ICD mice are more pronounced and tortuous than control mice.

Similarly, post-ischemic Notch4 signaling activation initiated after ischemic injury promotes neurological recovery, reduction in infarct volume, and improvement in collateral artery growth. Neurological functions were assessed (fig. 4A), including by modified Bederson grade, elevated body swing test, ladder test and adhesion test before and after MCAO. Arterial activation of Notch4 was induced after ischemic injury by tamoxifen injection. A significant improvement in neurological recovery was observed in BMX-Notch4 mice. Since activation of Notch4 signaling was gradually obtained after stroke, the difference between the two groups became significant starting 7 days after stroke. HE staining showed fewer infarcts in BMX-Notch4 mice (where up-regulated Notch4 signaling was obtained after stroke) compared to control mice. Quantification of infarct volume determined by H & E staining showed significantly less infarct volume in BMX-Notch4 mice (fig. 4B).

Example 4 transient Notch signaling induces a long lasting effect on post-ischemic recovery. To further investigate the effect of Notch signal upregulation after ischemic brain injury, experiments were performed as summarized in fig. 5A. Notch signaling was induced in BMX-Notch1 mice by administration of tamoxifen 14 days prior to dMCAO. Notch1 ICD expression was turned OFF by Tet-OFF expression constructs and doxycycline administration 14 days after dMCAO.

Despite the turning off of Notchl ICD, arterial expression of Notchl ICD promotes collateral growth and preserves cerebral blood flow.

Collateral artery diameter, velocity and flux were improved in BMX-Notch1 ICD mice compared to control mice (fig. 5B). Significant improvements in vessel size and function persist after closure of Notch1 ICD, suggesting that Notch activation produces a sustained therapeutic effect over the post-injury period.

Similarly, despite the shut-down of Notch4 expression 14 days after dMCAO, the more pronounced collateral remodeling shown by the angiocasts of BMX-Notch4 mice was maintained compared to control mice. The magnification clearly shows that more pronounced and tortuous collateral branches in BMX-Notch4 mice are maintained for a long period of time after Notch4 expression is turned off compared to control mice.

Example 5 therapeutic Effect of Notch signaling activation by administration of Notch ligands.

The foregoing experiments demonstrate the therapeutic role of Notch signaling in ischemic brain injury through the genetic pathway. To further demonstrate the therapeutic effect of upregulation of Notch signaling following ischemic brain injury, wild type mice (C57BL/6J line) were administered recombinant mouse DLL4 daily for eight days from the previous day of dMCAO, at a dose of 1 microgram per gram of body weight for the first three doses, and thereafter 0.8 microgram daily, as summarized in fig. 6A. Collateral artery size and function were assessed 7 and 14 days after injury. In mice treated with rDLL4, collateral artery diameter, velocity and flux (fig. 6B) were significantly improved on day 7, at which time administration of rDLL4 was discontinued. On day 14, significant improvement in treated mice was sustained. Nerve function was higher in treated mice as assessed by the ladder test on days 15 and 17 (fig. 6C).

These results indicate that exogenous application of Notch signaling activators can have a substantial and long-lasting therapeutic effect in treating ischemic injury.

Example 6 upregulation of Notch signaling improves recovery in acute limb ischemia models.

To investigate the therapeutic potential of arterial Notch signaling in acute limb ischemia, BMX-Notch4 mice and control mice were subjected to Experimental Femoral Artery Occlusion (EFAO) in the left hind limb. Notch4 expression was induced in BMX-Notch4 mice by administration of tamoxifen 14 days prior to FFAO. After EFAO, foot blood flow and hind limb artery diameter were measured for both the operated and non-operated limbs. Figure 7A depicts improved blood flow in the ischemic paw of mice expressing Notch 4. Figure 7B depicts improved recovery of hind limb artery diameter in BMX-Notch4 mice, while no off-target effect was observed in the non-operated limb. Fig. 7C depicts the ratio of collateral artery diameter change between the operated and non-operated limb after EFAO. Figure 8 depicts a significant reduction in muscle necrosis in BMX-Notch4 mice at day 7 post EFAO. [00] In a related experiment, the sustained effect of Notch4 activation was studied in an acute limb ischemia model by subjecting one limb of BMX-Notch4 mice and control mice to EFAO. At 13 and 14 days prior to the EFAO process, BMX Notch4 mice were injected with tamoxifen to induce Notch4 expression. Fourteen days after EFAO, Notch4 expression was turned off by doxycycline administration (in food). Several days after doxycycline administration, Notch4 expression may decrease. Foot perfusion in both the surgical and non-surgical limbs is measured by laser doppler perfusion imaging or LDPI, as known in the art, before and after surgical injury. The ratio of foot perfusion for the operated limb to the non-operated limb provides a measure of foot blood flow recovery. Paw blood flow in the ischemic limb was greater in BMX-Notch4 mice compared to control mice, becoming significantly improved by day 7, and so throughout the remaining 56 days of the measurement (figure 9). Although Notch4 expression was shut off at 14 days post-surgery, significant improvement in blood flow continued throughout the measurement after Notch4 was shut off.

In a separate experiment, the therapeutic effect of Notch activation was studied in an acute limb ischemia model by subjecting one limb of BMX-Notch4 mice and control mice to EFAO. Immediately following the EFAO procedure, tamoxifen administration was performed to induce Notch4 expression in BMX-Notch4 mice. Foot perfusion in the surgical and non-surgical limbs is measured by (laser doppler perfusion imaging or LDPI as known in the art) before and after surgical injury. The ratio of foot perfusion in the surgical to non-surgical limb provides a measure of foot blood flow recovery. As shown in figure 10, by day 21, significant improvement in EFAO hindfoot perfusion was observed in mice expressing Notch 4. These improvements were sustained throughout the measurement period on day 63.

These results indicate that the prophylactic and therapeutic effects of Notch signaling activation in the brain following ischemic injury are also applicable to other ischemic sites, and that the effects may function prophylactically, therapeutically, and be long lasting, remaining well-sustained after cessation of Notch4 activation.

Exemplary embodiments

In one embodiment, the scope of the present invention encompasses a Notch activator for the treatment of a vascular disease, wherein the vascular disease is an ischemic disease, wherein the ischemic disease may be cardiac ischemia, cerebral ischemia, limb ischemia, or carotid artery disease.

In one embodiment, the scope of the present invention encompasses a Notch activator for the treatment of a vascular disease, wherein the vascular disease is ischemic disease of the brain, wherein the ischemic disease may be acute ischemic stroke, transient ischemic stroke, micro-infarction, vascular dementia or cerebrovascular disease.

In one embodiment, the scope of the present invention encompasses Notch activators for the treatment of vascular diseases, wherein the vascular disease is selected from the group consisting of: peripheral arterial disease, renal insufficiency or kidney disease, including reduced blood flow; vascular disease of the eye; a decrease in spleen blood flow; mesenteric artery occlusion; intestinal infarction; small vessel disease; and reduced blood flow in blood vessels due to atherosclerosis or diabetes.

In one embodiment, the scope of the present invention encompasses a Notch activator for the treatment of vascular disease, wherein the Notch activator is a Notch ligand. In one embodiment, the Notch ligand is selected from the group consisting of: delta-like ligand 1, delta-like ligand 2, delta-like 3, delta-like ligand 4, Jagged-1, Jagged-2, and the aforementioned Notch activating variants.

In one embodiment, the scope of the present invention encompasses a Notch activator for the treatment of vascular disease, wherein the Notch activator is a Notch intracellular domain. In one embodiment, the Notch intracellular domain is selected from the group consisting of: notch1, Notch2, Notch3 and Notch4 intracellular domains and the aforementioned Notch activating variants.

In one embodiment, the scope of the present invention encompasses a Notch activator for the treatment of vascular disease, wherein the Notch activator is selected from the group consisting of: n-methylhemeanthidine chloride, valproic acid, resveratrol, triacetyl resveratrol, phenethylisothiocyanate and Yhhu-3792.

In one embodiment, the scope of the present invention encompasses a Notch activator for use in the treatment of a vascular disease, wherein the vascular disease is an ischemic disease, wherein the ischemic disease is selected from the group consisting of: cardiac ischemia, cerebral ischemia, limb ischemia, or carotid artery disease, wherein the cerebral ischemia disorder is selected from the group consisting of: acute ischemic stroke, transient ischemic attack, micro-infarction, vascular dementia or cerebrovascular disease; wherein the Notch activator is a Notch ligand; wherein the Notch ligand is selected from the group consisting of: delta-like ligand 1, delta-like ligand 2, delta-like 3, delta-like ligand 4, Jagged-1, Jagged-2, and the aforementioned Notch activating variants.

In one embodiment, the scope of the present invention encompasses drug delivery devices functionalized with or coated with one or more Notch activators or compositions that elute the one or more Notch activators when exposed to physiological conditions; wherein the Notch activator is selected from the group consisting of: a Notch ligand, a Notch intracellular domain, a small molecule activator of Notch and an antibody, wherein the Notch ligand is selected from the group consisting of: delta-like ligand 1, delta-like ligand 2, delta-like 3, delta-like ligand 4, Jagged-1, Jagged-2, and the aforementioned Notch activating variants; wherein the device may be a drug eluting balloon or stent.

In one embodiment, the scope of the present invention encompasses red blood cells functionalized with one or more Notch activators. In one embodiment, the Notch activator is a Notch ligand. In one embodiment, the Notch ligand is selected from the group consisting of: delta-like ligand 1, delta-like ligand 2, delta-like 3, delta-like ligand 4, Jagged-1, Jagged-2, and the aforementioned Notch activating variants.

All patents, patent applications, and publications cited in this specification are herein incorporated by reference to the same extent as if each individual patent application or publication were specifically and individually indicated to be incorporated by reference. The disclosed embodiments are provided for purposes of illustration and not limitation. Although the present invention has been described with reference to the described embodiments thereof, those skilled in the art will understand that changes may be made in the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.