CN116528843A

CN116528843A - Stroke treatment

Info

Publication number: CN116528843A
Application number: CN202180079969.0A
Authority: CN
Inventors: D·比尔德; D·因格贝尔; O·乌尊; F·博贝
Original assignee: Oxford University Innovation Ltd; Harvard College
Current assignee: Oxford University Innovation Ltd; Harvard College
Priority date: 2020-09-29
Filing date: 2021-09-28
Publication date: 2023-08-01
Also published as: GB202107137D0

Abstract

The present invention relates to a shear activated nanotherapeutic agent (SA-NT) for treating stroke by increasing blood supply to the brain via a side branch vessel, wherein the SA-NT comprises an aggregate comprising a plurality of nanoparticles, the aggregate further comprising one or more vasodilators or pharmaceutically acceptable salts thereof; wherein the aggregate is configured to disaggregate above a predetermined shear stress.

Description

Stroke treatment

Technical Field

The present invention relates to the treatment of ischemic stroke by increasing blood flow in side branch vessels supplying the penumbra.

Background

Stroke is a major health problem worldwide. Recent estimates (Feigin VL et al Global and regional burden of stroke during 1990-2010:findings from the Global Burden of Disease Study 2010.Lancet.2014;383 (9913): 245-254) indicate a global prevalence of 5 strokes per 1000 people per year, equivalent to 3300 tens of thousands of people surviving after a stroke. In 2010, 590 ten thousand people died from a stroke, which resulted in a loss of over 1.02 hundred million disability-adjusted life year (DALY) years of life, equivalent to the sum of premature death and loss of healthy life due to disability. In recent years, little progress has been made in stroke treatment regimens or in reducing stroke-related mortality/morbidity.

Stroke occurs when a thrombus or clot is present to impede blood flow to the brain. The consequent decrease in blood supply leads to brain cell damage and death. Ischemic stroke occurs when the cerebral blood supply is completely cut off or severely reduced by a thrombus. This is in contrast to hemorrhagic strokes, which occur when blood in an artery flows into the brain due to a vascular injury or rupture.

Currently, there is no specific therapy in ischemic state other than prevention of ischemic injury by thrombectomy or thrombolysis. Minimizing the time to reduce the brain's loss of adequate blood supply due to thrombus or clots is critical to rescue of ischemic tissue.

The reduction in blood flow and cell death rate after stroke is not consistent. In the central region (ischemic core), the blood flow is severely reduced, leading to rapid tissue death. Around the core region there may be enough residual perfusion to survive the brain tissue for a limited period of time. If the occluded artery is opened fast enough, tissue in the surrounding area (ischemic penumbra) can be saved. After the ischemic stroke episode, the semi-dark band acquires its residual perfusion from adjacent non-occluded arterial regions through the pial collateral (or bypass) blood vessels (also referred to herein as "collateral" or "collateral blood vessels") on the brain surface. Clinical studies have shown that the greater the flow through these vessels, the greater the penumbra (the smaller the core area of cell death), the better the prognosis for stroke patients. For example, vagal et al (Stroke (2018) 49 (9): 2102-2107) report that Stroke patients with weaker side branches have lower penumbra relief rates than Stroke patients with stronger side branches. Wufuer et al (Exp Ther Med.2018, 1, 15 (1): 707-718) reported an improved prognosis for stroke patients with good collateral circulation after thrombolytic treatment, while Leng et al (J Neurol Neurosurg Psychiatry.2016, 5; 87 (5): 537-44) reported an improved prognosis for stroke patients with good collateral circulation after intravascular (thrombectomy) treatment.

Therapies that maintain or enhance blood flow through side branch vessels may allow cells in the ischemic penumbra to survive longer, thereby extending the chance that an occluded cerebral artery may be opened. Although the importance of collateral blood vessel status in human stroke outcomes has been demonstrated, few such therapies are currently being investigated for collateral blood vessels. Therefore, side branch therapy is an emerging field.

Previously suggested side branch treatment strategies include interventions that increase side branch perfusion, for example by increasing blood pressure or by vasodilation. These methods are primarily around enhancing the driving pressure of blood on the brain or dilating side branch vessels. Although many of these interventions have been shown to improve collateral circulation in animal models of stroke (e.g., induced hypertension, partial aortic occlusion, inhaled nitric oxide and sphenopalatine ganglion stimulation), their ultimate clinical utility is limited due to problems of invasiveness, side effects, and need for specialized equipment. For example, increasing blood pressure can easily lead to bleeding or vascular rupture in already compromised patients. Furthermore, vasodilation therapy may dilate the vascular bed in the brain and other parts of the body. This results in vessel theft (i.e., expansion of the peripheral vascular network "steals" blood flow from another area (e.g., the brain)), resulting in a decrease in systemic perfusion pressure, ultimately worsening stroke results.

For example, recent studies have shown (back et al, the Lancet, volume 393, 10175, pages 1009-1020) that transdermal (i.e., systemic) delivery of nitroglycerin, an effective vasodilator, does not improve The functional outcome of patients with hyperacute stroke.

Thus, there is a need for further therapies to improve the prognosis of stroke patients.

Disclosure of Invention

The present invention describes a strategy for selectively dilating a side branch vessel while reducing or avoiding the deleterious side effects associated with systemic vasodilation during ischemic stroke. Accordingly, the present invention relates to a shear activated nanotherapeutic agent (shear-activated nanotherapeutic, SA-NT) for treating stroke by increasing blood supply to the brain via a side branch vessel, wherein the SA-NT comprises an aggregate comprising a plurality of nanoparticles (nanoparticle aggregate/NPA), the nanoparticle aggregate further comprising one or more vasodilators or pharmaceutically acceptable salts thereof; wherein the nanoparticle aggregates are configured to deagglomerate above a predetermined shear stress.

The invention also relates to a composition for treating stroke by increasing the blood supply to the brain via a side branch vessel, wherein the composition comprises such SA-NT in combination with one or more pharmaceutically acceptable excipients, carriers and/or diluents.

The invention also relates to a method of treating stroke by increasing the blood supply to the brain via a side branch vessel, comprising administering such SA-NT or such a composition to a patient in need thereof.

The invention also relates to the use of such SA-NTs or such compositions for the preparation of a medicament for the treatment of stroke by increasing the blood supply to the brain via side branch vessels.

The invention also relates to a SA-NT comprising an aggregate comprising a plurality of nanoparticles; wherein the aggregate is configured to disaggregate above a predetermined shear stress; and wherein the aggregate further comprises from about 0.1% to about 20% by weight of one or more vasodilators. Preferably, the aggregate comprises from about 0.4% to about 2.5% by weight of nitroglycerin, or a pharmaceutically acceptable salt thereof, as one or more vasodilators.

The use of SA-NT as a side-branch therapeutic agent (therapeutic) has a number of significant advantages over previously tested side-branch therapeutic agents, which may improve clinical utility. For example, SA-NTs are readily administered directly into the blood stream, such as intravenously. No specialized equipment is required and the treatment is non-invasive. The treatment may be performed at or before the arrival at the hospital, for example by a caregiver coping with emergency situations. Thus, this is an improved method of increasing blood flow to the brain after a stroke without reducing systemic blood pressure.

Selective treatment by increasing the blood flow to the side branch vessels, by side branch vessel dilation, is also advantageous over previous treatments for causing occlusion of the stroke itself. In particular, drug administration to the occlusion itself is delayed until after the stroke nature is diagnosed. Thus, the present invention, which is specifically directed to a side branch vessel, also has advantages over such treatment because it can be administered immediately after a stroke as an emergency treatment. This maximizes the survival of brain tissue during the period between the onset of stroke and the administration of further treatment.

In addition, SA-NT is activated only at the side branch vascular site where vasodilators are released. This minimizes systemic exposure of the vasodilator and increases the vasodilating effect of the vasodilator.

Drawings

FIG. 1 shows the effect of injection of SA-NT comprising nitroglycerin-containing nanoparticle aggregates (NG-NPA) and blank NPA on side branch perfusion in a rat model of stroke.

Figure 2 shows the mean change in collateral perfusion during treatment with NG-NPA and blank NPA in a rat model of stroke.

Figure 3 shows a comparison of the peak collateral perfusion obtained using NG-NPA with increasing doses of "free" nitroglycerin (i.e. soluble nitroglycerin without NPA) in a rat model of stroke.

FIG. 4 shows collateral perfusion and mean arterial pressure following NG-NPA infusion administered at a rate of 2.75 μg/kg/min in a rat model of stroke.

FIG. 5 shows collateral perfusion and mean arterial pressure following NG-NPA infusion administered at a rate of 4.15 μg/kg/min in a rat model of stroke.

FIG. 6 shows the change in collateral perfusion over time following NG-NPA infusion administered to the first group and blank NPA infusion administered to the second group at a rate of 4 μg/kg/min in a rat model of stroke.

Figure 7 shows the change in perfusion of the contralateral/control hemispheres over time following NG-NPA infusion administered to the first group and blank NPA infusion administered to the second group at a rate of 4 μg/kg/min in a rat model of stroke.

FIG. 8 shows the mean arterial pressure over time following NG-NPA infusion administered to the first group and blank NPA infusion administered to the second group at a rate of 4 μg/kg/min in a rat model of stroke.

FIG. 9 shows infarct size after treatment with NG-NPA and blank NPA in a rat model of stroke.

Fig. 10 shows the relationship between collateral perfusion and infarct volume in a rat model of stroke (including NG-NPA group and blank NPA group).

FIG. 11 shows the change in collateral perfusion over time following administration of free nitroglycerin (GTN) infusion to the first group and saline infusion to the second group at a rate of 4 μg/kg/min in a rat model of stroke.

Fig. 12 shows the change in perfusion of the contralateral/control hemispheres over time following administration of free nitroglycerin (GTN) infusion to the first group and saline infusion to the second group at a rate of 4 μg/kg/min in a rat model of stroke.

FIG. 13 shows the mean arterial pressure over time following infusion of free nitroglycerin (GTN) to the first group and saline to the second group at a rate of 4. Mu.g/kg/min in a rat model of stroke.

FIG. 14 shows the change in collateral perfusion over time following administration of free nitroglycerin (GTN) infusion to the first group and saline infusion to the second group at a rate of 40 μg/kg/min in a rat model of stroke.

Fig. 15 shows the change in perfusion of the contralateral/control hemispheres over time following administration of free nitroglycerin (GTN) infusion to the first group and saline infusion to the second group at a rate of 40 μg/kg/min in a rat model of stroke.

FIG. 16 shows the mean arterial pressure change over time following administration of free nitroglycerin (GTN) infusion to the first group and saline infusion to the second group at a rate of 40 μg/kg/min in a rat model of stroke.

Detailed Description

SA-NTs comprise aggregates of similar size to natural platelets, but are made into aggregates of smaller nanoparticles. These aggregates remain intact when flowing in blood under normal physiological flow conditions. However, when exposed to levels above normal [ ] >100dyne/cm ² ) The aggregates break apart into individual nanoparticles upon shear stress. These nanoparticles experience lower drag forces and adhere more effectively to the vessel surface through which they flow than larger aggregated particles. Thus, any therapeutic agent carried by the nanoparticle may be selectively delivered to the depolymerised sites in the localized high shear stress region.

Previously, SA-NTs have been used to deliver thrombolytic tissue plasminogen activator (tissue plasminogen activator, t-PA) in thromboembolic diseases such as pulmonary embolism (see, e.g., FIG. 3 of Korin et al (science.2012, month 8, 10 days; 337 (6095): 738-42)). However, the disclosure of Korin is limited to release of therapeutic agents in occluded blood vessels. Targeting non-occluded vessels that experience abnormally high shear stresses due to abnormally high flow rates has not previously been considered.

During stroke, the blood flow velocity in the side branch vessels is extremely high due to the large pressure difference between the two vascular regions (healthy vascular tissue and penumbra tissue) of the side branch vascular connection. This significantly increases the shear stress independent of changes in vessel diameter (see, e.g., beard, d.j. Et al (2015), "Intracranial Pressure Elevation Reduces Flow through Collateral Vessels and the Penetrating Arterioles they Supply, a Possible Explanation for 'Collateral Failure' and Infarct Expansion after Ischemic strain. Journal of Cerebral Blood Flow & Metabolism",35 (5), 861-872).

Although the side branch vessels are not occluded after stroke, the inventors have found that selective delivery of these vessels can be achieved by administration of SA-NTs. As shown in the examples and corresponding figures 1-5, such SA-NTs provide a controlled and effective method of artificially enhancing the perfusion of a side branch vessel after a stroke. This increases blood flow to the penumbra cells and increases brain survival in stroke patients where blood clots remain in place.

The present invention provides an easy to use therapy that can be used in centers where clot retrieval (clot retrieval) therapy is not available (e.g., in smaller emergency treatment centers). This new therapy expands the chance that the penumbra cells can survive. Not only does this directly contribute to increased recovery of brain tissue following a stroke, but it can also provide additional time for the patient to transport to a larger specialty center for clot retrieval, for example, maximizing the likelihood that the patient will obtain favorable clinical results.

Nanoparticles

The SA-NTs described herein comprise aggregates of nanoparticles. In the present invention, the size of the nanoparticles is on the order of about 1nm to about 1000 nm. Typically, the average diameter of the nanoparticles is from about 50nm to about 500nm, preferably from about 70nm to about 400nm, preferably from about 90nm to about 300nm, preferably from about 100nm to about 240nm.

Nanoparticles suitable for drug delivery are well known in the art. Thus, nanoparticles for use in the SA-NTs of the present invention may be included in, for example, korin et al (science.2012, 8/10; 337 (6095): 738-42); U.S. Pat. nos. 6,645,517, 5,543,158, 7,348,026, 7,265,090, 7,541,046, 5,578,325, 7,371,738, 7,651,770, 9,801,189, 7,329,638, 7,601,331, 5,962,566; U.S. patent application publication nos. us2006/0280798, 2005/0281884, 2003/0223938, 2004/0001872, 2008/0019908, 2007/0269380, 2007/0264199, 2008/0138404, 2005/0003014, 2006/012767, 2006/0078218, 2007/024359, 2005/0058603, 2007/0053870, 2006/0105049, 2007/0224277, 2003/0147966, 2003/0082237, 2006/0226525, 2006/02323883, 2008/0193547, 2007/0292524, 2007/0014804, 2004/0219221, 0193787, 2004/0081688, 2008/0095856, 2006/01345209, 2006/01345, 2004/024383 and WO 024383 are all described herein and incorporated by reference herein in their entirety. Examples of nanoparticles particularly suitable for use in the present invention are those described by Korin et al (science.2012, 8, 10; 337 (6095): 738-42) and WO 2013/185032, the contents of which are incorporated herein by reference.

For example, the types of nanoparticles that can be used to form the aggregates described herein can be: (1) Nanoparticles formed from a polymer or other material, wherein one or more vasodilators absorb/adsorb the polymer or other material or form a coating on the nanoparticle core; (2) Nanoparticles formed from a core formed from one or more vasodilators, the core being coated with a polymer or other material; (3) Nanoparticles formed of a polymer or other material covalently linked to one or more vasodilators; (4) Nanoparticles formed from one or more vasodilators and other molecules; (5) Nanoparticles formed to comprise a substantially uniform mixture of one or more vasodilators and nanoparticles or other non-drug substance components; (6) Nanoparticles of pure drug or drug mixture with a coating on the core of one or more vasodilators; (7) Nanoparticles without any associated vasodilators; (8) Nanoparticles consisting entirely of one or more vasodilators; (9) Nanoparticles impregnated with one or more vasodilators in the nanoparticles; and (10) nanoparticles having one or more vasodilators adsorbed on the nanoparticles.

The nanoparticles typically comprise one or more biocompatible polymers. The biocompatible polymer may be biodegradable or non-biodegradable, but is preferably biodegradable. In some embodiments, the biocompatible polymer is a copolymer of polylactic acid and polyglycolic acid, poly (glycerol sebacate) (PGS), poly (aziridine) (poly (ethylenimine)), pluronic (Poloxamers) 407,188, hyaluronic acid (Hyaluron), heparin, agarose, or Pullulan (Pullulan). In some embodiments, the polymer is a fumaric acid/sebacic acid copolymer.

The average molecular weight of the polymer used may be from about 20000Da to about 500000Da.

Any method known in the art may be used to prepare the nanoparticles for SA-NTs of the present invention. For example, vaporization methods (e.g., free jet expansion method, laser vaporization method, spark erosion method, electro-explosion method, and chemical vapor deposition method), physical methods involving mechanical milling (e.g., pearl milling technology (the pearl milling technology) developed by Elan Nanosystems), and interfacial deposition after solvent replacement may be used.

Preferably, the nanoparticles used in the SA-NTs of the present invention comprise copolymers of polylactic acid and polyglycolic acid (also known as PLGA). When the nanoparticles used in the SA-NT of the invention comprise PLGA, the ratio of lactide to glycolide in PLGA is preferably about 10:90 to about 90:10, preferably about 25:75 to about 75:25, most preferably about 50:50.

When the nanoparticles comprise PLGA, the nanoparticles may be formed as perfluorobutane polymer microspheres. For example, the nanoparticle used in the SA-NTs of the present invention may be HDDS obtained from Acusperee ^TM (Hydrophobic Drug Delivery System ) nanoparticles. These nanoparticles are made by creating an emulsion containing PLGA, phospholipid and pore former. This emulsion is further processed by spray drying to produce porous microspheres containing gas having a honeycomb-like structure.

The nanoparticles of the present invention may be manufactured, for example, according to the methods described in examples 1 and 2 a.

Nanoparticle aggregates

The aggregates used in the SA-NTs of the present invention comprise a plurality of constituent nanoparticles. Typically, the aggregate comprises from about 2 to about 10000 nanoparticles, preferably from about 3 to about 8000 nanoparticles, preferably from about 7 to about 6000 nanoparticles, preferably from about 10 to about 6000 nanoparticles and preferably from about 20 to about 4000 nanoparticles.

In the present invention, aggregates are micrometer-sized aggregates, i.e., on the order of about 0.1 μm to about 1000 μm in size. In general, the aggregate is similar in size to natural platelets. The aggregate preferably has an average diameter of about 0.5 μm to about 10 μm, preferably about 1 μm to about 7 μm, preferably about 1.4 μm to about 4.5 μm, preferably about 1.8 μm to about 3.5 μm, and preferably about 2 μm to about 3 μm.

Nanoparticles may be bound together to form aggregates by one or more types of intermolecular (i.e., non-covalent) forces and/or covalent bonds. For example, the nanoparticles may be bound together by one or more of electrostatic interactions, dipole-dipole interactions, van der waals forces, hydrogen bonding, and/or covalent bonding. Cleavable linkers may be used when the nanoparticles are bound together by covalent bonds to form aggregates. Any cleavable linking agent known in the art or as defined herein may be used.

The binding strength between nanoparticles can be modulated by increasing or decreasing the extent of interaction between adjacent nanoparticles. For example, the strength of nanoparticle binding may be increased by modifying the surface of the nanoparticle to include one or more positively charged groups and one or more negatively charged groups, thereby increasing the degree of electrostatic interaction between adjacent nanoparticles. Alternatively, the strength of nanoparticle binding may be reduced, for example, by modifying the surface of the nanoparticle to remove electronegative moieties from the surface of the nanoparticle. This will reduce the strength of dipole-dipole and/or hydrogen bonding between adjacent nanoparticles.

Nanoparticle formation of nanoparticle aggregates can be induced by a variety of methods available and well known in the art. Many hydrophobic nanoparticles, such as PLGA-based nanoparticles, can self-aggregate in aqueous solutions (see, e.g., C.E.Astete et al, J.biomater.Sci, polymer ed.17:247 (2006)). Alternatively, the concentrated solution of nanoparticles may be spray dried to form aggregates (see, e.g., sung et al, pharm. Res.26:1847 (2009) and Tsapis et al, proc. Natl. Acad. Sci. USA,99:12001 (2002)). Other methods of forming aggregates include, but are not limited to, w/o/w emulsion methods and simple solvent displacement methods.

Nanoparticle aggregates described herein disaggregate under shear stress conditions. Shear stress is the frictional drag exerted by the blood flow in dynes/cm ² (dynes/cm ² ). As blood flows through a blood vessel, blood adjacent the vessel wall tends to adhere to the vessel wall, resulting in reduced blood flow and velocity gradients. The blood flow velocity in the center of the blood vessel is higher than the blood flow velocity at the edge of the blood vessel. The difference in blood flow velocity results in shear stress being applied to cells and particles in the blood. The shear stress increases with decreasing distance to the vessel wall. Shear stress generated by flowing blood is also applied to molecules or aggregates present in the blood. The shear stress experienced by an aggregate is a function of the size of the aggregate-the larger the aggregate, the greater the shear stress applied thereto.

Under normal physiological conditions, shear stress in the brain and peripheral vascular system is tightly controlled at about 15-30dynes/cm ² Between them. After ischemic stroke, the shear stress in the side branch vessels supplying ischemic penumbra exceeds this range, and may, for example, exceed 100dynes/cm ² . The shear stress of the collateral blood flow was measured using Beard, d.j.et al, (2015), "Intracranial Pressure Elevation Reduces Flow through Collateral Vessels and the Penetrating Arterioles they Supply, a Possible Explanation for 'Collateral Failure' and Infarct Expansion after Ischemic strain.journal of Cerebral Blood Flow&Metabolism ",35 (5), 861-872, calculated using the equation τ = γ x η, where τ is the shear stress, γ is the shear rate, η is the viscosity. The shear rate was calculated as 8× (blood flow velocity)/(blood vessel diameter)). Published blood viscosity (3 cP) values were used.

The aggregates of SA-NTs of the present invention are configured to deagglomerate into their component nanoparticles above a predetermined shear stress. Preferably, the aggregate is configured to disaggregate as the aggregate flows through areas of increased shear stress in the blood vessel (i.e., areas of higher than normal physiological levels of shear stress in the blood vessel). Preferably, the aggregates of SA-NTs of the present invention are configured to break down into their component nanoparticles in the collateral blood vessels supplying the penumbra after ischemic stroke.

Preferably, the aggregates are configured to be at a distance of greater than about 30dynes/cm ² Preferably greater than about 50dynes/cm ² Preferably greater than about 75dynes/cm ² Most preferably greater than about 100dynes/cm ² Is depolymerized at a predetermined shear stress. Optionally, the aggregate may be configured to be at greater than about 125dynes/cm ² Optionally greater than about 150dynes/cm ² Is depolymerized at the shear stress of (c). In general, deagglomeration of the aggregates into constituent nanoparticles occurs when the aggregates are subjected to shear stresses sufficient to overcome intermolecular forces (e.g., electrostatic interactions, dipole-dipole interactions, van der Waals forces, and/or hydrogen bonds) that hold the nanoparticles together.

The deagglomeration of the aggregates may be partial or complete. When depolymerization is partial depolymerization, the aggregate may depolymerize at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% upon exposure to a predetermined high shear stress. Alternatively, the aggregates may undergo complete deagglomeration (i.e., they may completely deagglomerate into their component nanoparticles) when exposed to a predetermined high shear stress.

Suitable nanoparticles and aggregates of these nanoparticles for use in the present invention are described in Korin et al (science.2012, 8, 10; 337 (6095): 738-42) and WO 2013/185032, the contents of which are incorporated herein by reference.

Vasodilators

The SA-NT according to the invention is used for selectively delivering vasodilators/drugs to side branch vessels supplying ischemic penumbra. Vasodilators/drugs are compounds that cause vasodilation. As used herein, the terms vasodilator and vasodilator are used interchangeably.

Any vasodilator capable of providing side branch vasodilation may be used. Preferably, the one or more vasodilators are selected from the group consisting of nitrates, angiotensin II receptor blockers (ARB, also known as sartan), calcium Channel Blockers (CCB), selective alpha blockers, β1 agonists, β2 agonists, β agonists, ET1 receptor antagonists, phosphodiesterase 5 inhibitors or agonists of the Small (SK) and medium (IK) calcium activated potassium channels. More preferably, the one or more vasodilators are selected from the group consisting of nitrates, angiotensin II receptor blockers (ARBs, also known as sartan), calcium Channel Blockers (CCBs), selective alpha blockers or beta 1 agonists. Most preferably, the one or more vasodilators are selected from the group consisting of nitrate, angiotensin II receptor blockers (ARB, also known as sartan) or Calcium Channel Blockers (CCB).

When the one or more vasodilators are nitrates, the nitrate is preferably Nitroglycerin (NG), nicorandil (Nicorandil), moxidecylamine (Molsidomine)/3-morpholinoglycoaniline hydrochloride (3-Morpholinosydnonimine hydrochloride), nitroprusside Lu Ganna (Sodium Nitropruside), isosorbide mononitrate (Isosorbide Mono-nitate) or Isosorbide dinitrate (Isosorbide di-nitate); more preferably Nitroglycerin (NG), nicorandil, moxidecylamine/3-morpholinoglycosylanilide hydrochloride, or sodium nitroprusside; and most preferably Nitroglycerin (NG).

When the one or more vasodilators are angiotensin II receptor blockers (ARB, also known as sartan), the ARB is preferably Candesartan (Candesartan), valsartan (Valsartan), iroxortan (Iosartan), eprosartan (Eprosartan), olmesartan (Olmesartan), telmisartan (telmesartan) or Irbesartan; more preferably candesartan, valsartan, iroxortan, eprosartan or olmesartan; and most preferably candesartan or valsartan.

When the one or more vasodilators are Calcium Channel Blockers (CCBs), the CCBs are preferably Nimodipine (Nimodipine), lercanidipine (Lercanidipine), nifedipine (Nifedipine), felodipine (Felodipine), nicardipine (nicodipine), verapamil (Verapamil), diltiazem (Diltiazem), amlodipine (amiodipine) or Clevidipine (Clevidipine); more preferably nimodipine, lercanidipine, nifedipine, felodipine, nicardipine or verapamil; more preferably nimodipine, lercanidipine, nifedipine or felodipine; and most preferably nimodipine.

When the one or more vasodilators are selective alpha blockers, the selective alpha blocker is preferably Doxazosin (Doxazosin) (mesylate), prazosin (Prazosin) or Terazosin (Terazosin), most preferably Doxazosin (mesylate).

When the one or more vasodilators are β1 agonists, the β1 agonist is preferably Dobutamine (Dobutamine) or Dopamine (Dopamine).

When the one or more vasodilators are β2 agonists, the β2 agonist is preferably Eformoterol (Eformoterol), indacaterol (Indacaterol), salbutamol (Salbutamol), salmeterol (Salmeterol) or terbutaline (Turbutaline).

When the one or more vasodilators are beta-agonists, the beta-agonist is preferably Isoprenaline (Isoprenaline).

When the one or more vasodilators are ET1 receptor antagonists, the ET1 receptor antagonist is preferably Ambrisentan (Ambrisentan), bosentan (Bosentan) or BQ123, and preferably BQ123.

When the one or more vasodilators are phosphodiesterase 5 inhibitors, the phosphodiesterase 5 inhibitor is preferably Sildenafil (Sildenafil), tadalafil (Tadalafil) or papaverine (papaverine).

When the one or more vasodilators are agonists of the Small (SK) and medium (IK) calcium-activated potassium channels, the agonists of the Small (SK) and medium (IK) calcium-activated potassium channels are preferably NS309, EBIO, SKA-31, SKA-121 or SKA-111, most preferably NS309.

When the one or more vasodilators are not one of the above listed classes, the one or more vasodilators are preferably Diazoxide (Diazoxide), hydralazine (Hydralazine), methyldopa (Methyldopa) or Minoxidil (Minoxidil).

When multiple vasodilators are used in the SA-NTs of the present invention, these may be from the same class of vasodilators, or from multiple different classes of vasodilators (including classes not discussed herein).

Most preferably, the one or more vasodilators are nitroglycerin or nimodipine or a combination thereof. In one embodiment, the one or more vasodilators are nitroglycerin. In another embodiment, the one or more vasodilators are nimodipine.

The vasodilators may be used alone or a combination of two or more different vasodilators may be used. When two or more vasodilators are used, these may be provided on the same nanoparticle or on different nanoparticles that are combined together as a single aggregate. Furthermore, when two or more vasodilators are used, they may be provided on the same aggregate or on different aggregates.

Vasodilators may be provided in the form of pharmaceutically acceptable salts. As used herein, reference to a vasodilator or vasodilator drug includes reference to a pharmaceutically acceptable salt unless otherwise indicated.

One or more vasodilators may be coupled to the nanoparticle or nanoparticle aggregate before or after the nanoparticle aggregate into the SA-NT aggregate. The one or more vasodilators may be coupled to the nanoparticle or nanoparticle aggregate in any manner known in the art.

As used herein, the phrase "coupled to" with respect to a vasodilator and a nanoparticle or nanoparticle aggregate refers to the vasodilator entangled, entrapped, blended, encapsulated, bound to a surface, or otherwise associated with the aggregate or nanoparticle component of the aggregate.

In some embodiments, one or more vasodilators are encapsulated within the aggregate or nanoparticle component of the aggregate. In some embodiments, one or more vasodilators are absorbed or adsorbed on the surface of the aggregate. Thus, one or more vasodilators may be associated with the outer surface of the aggregate. This may occur, for example, when the nanoparticles on the outer surface of the aggregate are coupled to one or more vasodilators after the nanoparticle aggregate is formed.

In some embodiments, one or more vasodilators are absorbed or adsorbed on the surface of at least a plurality of nanoparticle components of the aggregate. This may occur, for example, when the nanoparticle is coupled to one or more vasodilators prior to formation of the nanoparticle aggregate.

In some embodiments, one or more vasodilators are covalently coupled to the aggregate or nanoparticle component of the aggregate. Covalent coupling between molecules of one or more vasodilators and the aggregate or nanoparticle component of the aggregate may be mediated by a linker. Without limitation, any conjugation chemistry known in the art for binding together two molecules or different portions of one molecule can be used to attach the vasodilator to the nanoparticle or nanoparticle aggregate. Exemplary linkers and/or functional groups for attaching the vasodilator to the nanoparticle or aggregate include, but are not limited to, polyethylene glycol (PEG, NH) ₂ PEGx-COOH, which may have PEG spacer arms of various lengths X, 1 of<X<100, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K, etc.), maleimide linkers, PAS (PASylation), HES (HESylation), bis (sulfosuccinimide) octadiester linkers, DNA linkers, peptide linkers, silane linkers, polysaccharide linkers, hydrolyzable linkers, etc. PEG is the preferred linker. For example, binding of SA-NT aggregates to a therapeutic agent may be achieved by PEG linkers, as described in WO 2013/185032, the contents of which are incorporated herein by reference.

In some embodiments, the linker comprises at least one cleavable linking group, i.e., the linker is a cleavable linker as defined herein.

In some embodiments, one or more vasodilators are non-covalently coupled to the aggregate or nanoparticle component of the aggregate. Non-covalent coupling between molecules of one or more vasodilators and the aggregate or with the nanoparticle component of the aggregate may be based on ionic interactions, van der Waals interactions, dipole-dipole interactions, hydrogen bonding, electrostatic interactions, and/or shape recognition interactions.

There is no need to couple one or more vasodilators to the nanoparticles prior to forming the nanoparticle aggregates. For example, preformed nanoparticles may be aggregated in the presence of one or more vasodilators. Without wishing to be bound by theory, one or more vasodilators may be present in the space (or cavity) within the aggregate, i.e. one or more vasodilators may be encapsulated within the aggregate.

In the SA-NT of the present invention, the nanoparticle aggregates may contain one or more vasodilators in a wide weight range. For example, the nanoparticle aggregate may comprise from about 0.1% to about 20% by weight of one or more vasodilators. Preferably, the aggregate comprises from about 0.2% to about 10%, preferably from about 0.3% to about 5%, preferably from about 0.4% to about 2.5%, preferably from about 0.8% to about 1.6%, preferably from about 1% to 1.4% by weight of one or more vasodilators.

When nitroglycerin (or a pharmaceutically acceptable salt thereof) is intended to be used as one or more vasodilators, the nanoparticle aggregate of SA-NTs preferably comprises about 0.4% to about 2.5%, preferably about 0.8% to about 1.6%, preferably about 1% to about 1.4% by weight nitroglycerin or a pharmaceutically acceptable salt thereof.

When nitroglycerin is intended to be used as one or more vasodilators, nanoparticle aggregates comprising nitroglycerin may be prepared according to the method described in example 2a, for example.

One or more vasodilators may be released when the nanoparticle and/or aggregate adheres to the vascular surface. As described above, smaller nanoparticles experience lower drag forces than larger aggregated particles. This means that smaller nanoparticles adhere more effectively to the surface of the blood vessel through which they flow than larger aggregated particles. The rate of release of one or more vasodilators to the vascular surface is generally greater for smaller nanoparticles than for larger aggregated particles due to the more efficient adhesion of the smaller nanoparticles.

Thus, when the nanoparticles are deagglomerated, one or more vasodilators are typically released at a higher rate and/or in greater amounts than when the nanoparticles are aggregated. Thus, the SA-NTs described herein are capable of selectively delivering one or more vasodilators to the surface of a blood vessel having high shear stress (i.e., a side branch vessel supplying a penumbra) following an ischemic stroke.

The examples described below relate to the use of SA-NTs of the present invention to deliver vasodilators to side branch vessels in the brain. Figures 1 and 2 (based on example 3 a) show that a substantial increase in side branch perfusion was observed when the nitroglycerin-containing nanoparticle aggregates were administered, as compared to no change in side branch perfusion when the vasodilator-free nanoparticle aggregates ("blank-NPA") were administered.

As noted above, back et al (The Lancet, volume 393, 10175, pages 1009-1020) have recently shown that transdermal (i.e., systemic) delivery of nitroglycerin, an effective vasodilator, does not alter The functional outcome of patients with hyperacute stroke. This finding is supported by example 3b and fig. 3 herein, which reports that delivery of "free" nitroglycerin to rodents (i.e., selective delivery to side branch vessels without using SA-NT) only causes a modest increase in side branch perfusion peaks.

In contrast, figures 1-5 show that delivery of nitroglycerin using SA-NTs according to the present invention, respectively, results in a significant increase in side branch perfusion. Thus, the selective delivery of one or more vasodilators to a side branch vessel using the SA-NTs of the present invention provides an effective method of improving the clinical outcome of patients suffering from acute stroke.

Application of

Administration of SA-NTs of the invention may be used to increase blood flow to the brain following a stroke. Generally, SA-NT increases blood flow in the penumbra after stroke. Typically, SA-NTs dilate a side branch vessel, which is typically a selective dilation of the side branch vessel, such that the side branch vessel dilates to a greater extent than other arteries. Thus, in general, administration of SA-NT results in an increase in perfusion through the side branch vessel, which may be a selective increase in perfusion through the side branch vessel, i.e., minimal or no change in mean arterial perfusion. Preferably, the mean arterial perfusion changes less than about 25%, preferably less than about 20%, and preferably less than about 15% of the pre-administration baseline after administration of the SA-NTs of the invention.

The SA-NTs of the present invention may be administered by any method known in the art. Preferably, the nanoparticle is administered intravenously or intra-arterially, most preferably intravenously.

When the SA-NT of the present invention is administered intravenously, the administration may be continuous infusion (infusion). Preferably, the infusate is delivered to the patient until the occlusion is removed and blood flow to the brain is restored. For example, the infusate may be delivered for a period of up to about 500 minutes, optionally up to about 400 minutes, optionally up to about 300 minutes, optionally up to about 200 minutes, and optionally up to about 100 minutes.

When the SA-NTs of the invention are administered intravenously, the administration may alternatively be as a bolus, i.e., as a single discrete dose administered to the patient over a short period of time. Multiple boluses may be administered to the patient during the course of treatment, i.e., from the administration of the first bolus to the initiation of reperfusion. One or more bolus doses may be combined with continuous infusion. In general, vasodilators with longer half-lives may be administered by a regimen that includes a bolus dose, while vasodilators with shorter half-lives may be administered by a regimen that includes continuous infusion.

For example, nitroglycerin has a short half-life, while nimodipine has a longer half-life. Thus, when the one or more vasodilators comprise nitroglycerin, the SA-NT is preferably administered as a continuous infusion (e.g., as shown in example 3d and as shown in FIGS. 4 and 5). Conversely, when the one or more vasodilators comprise nimodipine, the SA-NT is preferably administered by a regimen that includes bolus injection.

The SA-NTs of the present invention may be administered at any point in time after a suspected ischemic stroke, for example, after one or more stroke symptoms selected from the group consisting of sudden numbness or weakness in the face, arms or legs, sudden mental confusion (fusion), difficulty speaking or understanding speech, sudden difficulty seeing by one or both eyes, sudden difficulty walking, dizziness, loss of balance or lack of coordination, sudden severe headache, complete paralysis of one side of the body, difficulty swallowing (dysphagia), and loss of consciousness are observed.

It is preferred that the SA-NTs of the present invention be administered to the patient first as soon as possible after the onset of stroke symptoms. For example, SA-NTs may be administered as emergency medications, such as by medical personnel (e.g., ambulance caregivers) responding to emergency calls.

In the case of administration as an emergency drug, the SA-NTs of the present invention are preferably administered to a patient within about 4 hours of the onset of stroke symptoms, preferably within about 3 hours of the onset of stroke symptoms, preferably within about 2 hours of the onset of stroke symptoms, more preferably within about 1 hour of the onset of stroke symptoms.

The invention is particularly useful for treating ischemic stroke. However, prior to diagnosing the type of stroke, SA-NT may be administered to any patient exhibiting symptoms of acute stroke. For example, administration may be performed without the need for imaging studies to determine the nature of the stroke suffered by the patient. This is particularly advantageous as it can be applied in an emergency situation. Without wishing to be bound by theory, it may have a detrimental effect if typical ischemic stroke treatments are administered to patients suffering from hemorrhagic stroke. However, acute hemorrhagic stroke is not associated with an increase in vascular shear stress. Thus, SA-NTs according to the present invention generally do not depolymerize or selectively deliver vasodilators to the blood vessels of patients suffering from hemorrhagic stroke. Thus, the SA-NTs of the present invention may be administered in an emergency to any patient exhibiting symptoms of acute stroke while avoiding the potentially detrimental consequences associated with many existing stroke therapies for hemorrhagic stroke patients.

In contrast to the present invention, treatment of an occlusion (e.g., administration of a clot disrupting drug such as t-PA, or thrombectomy) for causing a stroke is typically deferred until after diagnostic imaging has been performed. The potential risk of such treatment means that the nature of the stroke needs to be determined prior to treatment. This may delay treatment and brain tissue may be lost during the delay. Thus, the ability to treat patients in emergency situations using the present invention is advantageous compared to treatments directed to the occlusion itself.

Dosage of

The aim of the invention is to increase the side branch perfusion as much as possible due to SA-NT while minimizing the variation in mean arterial blood pressure. The particular dose of vasodilator required to achieve this will vary depending on a number of factors, including the efficacy of the particular vasodilator. It has been shown that for an exemplary vasodilator, the dose required to deliver the vasodilator using the SA-NT of the present invention is about 1/70 of the typical injection dose of "free" vasodilator (i.e., not delivered as part of the SA-NT) required to increase cerebral blood flow in rats that did not experience a stroke (as described in Hoffman et al, stoke, volume 13, phase 2, 1982). In particular, FIGS. 4 and 5 show that by delivering nitroglycerin at concentrations of 2.75 μg/kg/min and 4.15 μg/kg/min, cerebral collateral perfusion of rats can be significantly increased.

Because of the selective delivery of one or more vasodilators to the side branch vessels by the SA-NTs of the present invention, it is preferred to administer a dose of about 0.1% to about 10%, preferably about 0.5% to about 8%, preferably about 1% to about 7%, preferably about 1.5% to 6%, preferably about 2% to about 5%, preferably about 2.5% to about 4.5%, most preferably about 3% to about 4% of the recommended dose of "free" vasodilator.

When nitroglycerin is used as the vasodilator or vasodilators, the patient is preferably administered a dose of about 0.001 μg/kg/min to about 8 μg/kg/min, preferably about 0.01 μg/kg/min to about 2 μg/kg/min, preferably about 0.05 μg/kg/min to about 1 μg/kg/min, preferably about 0.1 μg/kg/min to about 0.75 μg/kg/min, most preferably about 0.15 μg/kg/min to about 0.4 μg/kg/min.

Combination therapy

The SA-NTs of the present invention may be administered to a patient in combination (but typically delivered alone) with a treatment for or intended to remove an obstruction in the blood stream (i.e., a treatment for or intended to initiate reperfusion, such as thrombolytic treatment and/or thrombectomy). This may occur simultaneously, for example when SA-NT is administered to a patient at reperfusion, or separately; for example, when SA-NT is administered after stroke ischemia but before reperfusion, and then treatment to initiate reperfusion is started (during administration of SA-NT of the invention, or after such administration has ceased). Preferably, the SA-NT is administered to the patient in combination with thrombolytic therapy and/or thrombectomy.

When a treatment for or intended to initiate reperfusion is used in combination with SA-NT, the treatment for or intended to initiate reperfusion may be administered substantially simultaneously with SA-NT of the present invention. Alternatively, the treatment for or intended to initiate reperfusion may be administered at a different time than the SA-NT of the invention. Typically, the treatment for or intended to initiate reperfusion is administered within about 5 hours of initiating administration of the SA-NT, optionally within about 4 hours, optionally within about 3 hours, optionally within about 2 hours, optionally within about 1 hour, optionally within about 30 minutes, and optionally within about 10 minutes of initiating administration of the SA-NT. Typically, the treatment for or intended to initiate reperfusion is administered about 10 minutes after administration of the SA-NT from the beginning, optionally about 30 minutes, optionally about 1 hour, optionally about 2 hours, optionally about 3 hours, optionally about 4 hours, and optionally about 5 hours after administration of the SA-NT from the beginning. Preferably, the thrombolytic therapy is selected from the group consisting of applying a blood diluent or applying a lysing agent to the patient. Suitable blood diluents may be selected from Coumadin ^TM (warfarin); pradaxa ^TM (dabigatran); xaleito ^TM (rivaroxaban) and Eliquis ^TM (apixaban), fondaparinux (Fondaparinux), plain heparin, low molecular weight heparin (including but not limited to enoxaparin and ditalaparin), thrombolytic agents (including but not limited to Streptokinase (SK)), urokinase, lanoteplase, reteplase, staphylokinase (Staphylokinase), tenecteplase (teneplase) and Alteplase (Alteplase) or antiplatelet agents (e.g., aspirin, clopidogrel or ticagrelor).

The invention may also be used in combination with the delivery of one or more neuroprotective agents. The combination of the selective application of vasodilator therapy and the administration of one or more neuroprotective agents may enhance the efficacy of the one or more neuroprotective agents by increasing the rate of delivery of the one or more neuroprotective agents to the brain via the side branch vessels.

Preferred classes of neuroprotective agents that can be used in combination with the SA-NTs of the present invention include free radical scavengers, ion channel interaction/excitotoxicity blocking therapies, and immunomodulation/anti-inflammatory therapies (see, e.g., rajah et al, "Experimental neuroprotection in ischemic stroke: a Concise review". Neurosurg focus.2017, month 4; 42 (4)). Preferred neuroprotective agents that can be used in combination with the SA-NTs of the present invention are NXY-059, NA-1, interleukin-1 receptor antagonist and uric acid. These preferred neuroprotective agents are known in the art to be well tolerated in patients with clinical stroke.

One or more neuroprotective agents may be combined with the vasodilators in the SA-NTs of the present invention, or they may be delivered alone (i.e., not as part of the SA-NT).

When one or more neuroprotective agents are combined with a vasodilator in an SA-NT, the one or more neuroprotective agents may be provided on the same nanoparticle as the vasodilator or, or on a different nanoparticle than the vasodilator that is combined together as a single aggregate. Similarly, when neuroprotective agents and vasodilators are used, they may be provided in the same aggregate or in different aggregates.

When one or more neuroprotective agents are used in combination with a SA-NT but delivered alone, the one or more neuroprotective agents may be administered substantially simultaneously with the SA-NT of the present invention. Alternatively, one or more neuroprotective agents may be administered within about 10 minutes of administration of the SA-NT, optionally within about 30 minutes of delivery of the SA-NT of the invention, optionally within about 1 hour, optionally within about 2 hours, and optionally within about 3 hours. Alternatively, one or more neuroprotective agents may be administered prior to administration of the SA-NTs of the present invention, for example, within about 10 minutes, optionally within about 30 minutes, optionally within about 1 hour, optionally within about 2 hours, and optionally within about 3 hours prior to delivery of the SA-NTs.

In the present invention, SA-NTs may be used in combination with therapies (e.g., thrombolysis and/or thrombectomy) and neuroprotection therapies (e.g., administration of one or more neuroprotective drugs) for or intended to remove blood flow obstruction.

Composition and method for producing the same

The invention also relates to a composition for treating stroke, wherein the composition comprises SA-NT as defined herein in combination with one or more pharmaceutically acceptable excipients, carriers or diluents.

Suitable excipients, carriers and diluents can be found in standard pharmaceutical textbooks. See, e.g., handbook for Pharmaceutical Additives, 3 rd edition (editions: m.ash and i.ash), 2007 (Synapse Information Resources, inc., endicott, new york, usa) and Remington: the Science and Practice of Pharmacy, 21 st edition (editions: d.b. troy) 2006 (Lippincott, williams and Wilkins, philadelphia, usa).

Excipients for use in the compositions of the present invention include, but are not limited to, microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine, and may be used with various disintegrants such as starches (preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, as well as with granulating binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia.

The drug carrier comprises a sterile aqueous medium, various nontoxic organic solvents and the like. Pharmaceutically acceptable carriers include gums, starches, sugars, cellulosic materials and mixtures thereof. The formulation may also be administered by means of, for example, intravenous, arterial or intramuscular injection of a liquid formulation.

Furthermore, as used herein, a "pharmaceutically acceptable carrier" is well known to those skilled in the art and includes, but is not limited to, 0.01-0.1M, preferably 0.05M phosphate buffer or 0.9% saline. In addition, such pharmaceutically acceptable carriers can be aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including saline and buffered media. Other examples of substances that may be used as pharmaceutically acceptable carriers include: (1) saccharides such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) Cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) Lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) Polyols such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) a pH buffer solution; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL and LDL; (22) a C2-C12 alcohol, such as ethanol; and (23) other non-toxic compatible substances for use in pharmaceutical formulations. Wetting agents, colorants, mold release agents, coating agents, sweeteners, flavoring agents, preservatives and antioxidants may also be present in the formulation.

Pharmaceutically acceptable parenteral vehicles (vehicles) include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements, such as ringer's dextrose-based supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, complexing agents, inert gases and the like.

Pharmaceutically acceptable carriers for controlled or sustained release compositions that can be administered according to the present invention include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). The invention also includes particulate compositions coated with a polymer (e.g., poloxamer or poloxamer) and a compound coupled to an antibody directed against a tissue-specific receptor, ligand or antigen, or a ligand coupled to a tissue-specific receptor.

Pharmaceutically acceptable carriers include compounds modified by covalent attachment of water-Soluble polymers (e.g., polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline), which are known to exhibit significantly longer half-lives in blood after intravenous injection than the corresponding unmodified compounds (Abuchowski and Davis, solution Polymer-Enzyme products, enzymes as Drugs, hicenberg and Roberts, eds., wiley-Interscience, new York, n.y., pp 367-383). Such modifications may also increase the solubility of the compound in aqueous solutions, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity can be achieved by administering such polymer-compound inducers less frequently or at lower doses than the unmodified compound.

As used herein, the terms "drug," "drug substance," "active pharmaceutical ingredient," and the like refer to a compound that can be used to treat a patient in need of treatment.

As used herein, the term "excipient" refers to any substance that can affect the bioavailability of a drug but is otherwise pharmacologically inactive.

As used herein, the term "pharmaceutically acceptable" refers to those substances (specie) which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit-risk ratio, and are effective for their intended use.

As used herein, the term "pharmaceutically acceptable salts" are those salts that can be administered to humans and/or animals as a component of a drug or pharmaceutical composition, and which retain at least some of the biological activity of the free compound (neutral compound or non-salt compound) after administration. Salts of the desired basic compounds may be prepared by methods known to those skilled in the art by treating the compounds with an acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, sulfonic acid, and salicylic acid. Salts of basic compounds with amino acids, such as aspartate and glutamate, may also be prepared. Salts of the desired acidic compounds may be prepared by methods known to those skilled in the art by treating the compounds with a base. Examples of inorganic salts of acid compounds include, but are not limited to, alkali metal and alkaline earth metal salts, such as sodium, potassium, magnesium and calcium salts; an ammonium salt; and aluminum salts. Examples of organic salts of acid compounds include, but are not limited to, procaine, dibenzylamine, N-ethylpiperidine, N-dibenzylethylenediamine, and triethylamine salts. Salts of acidic compounds with amino acids, such as lysine salts, may also be prepared. Other salts particularly suitable for use in pharmaceutical formulations are described in Berge s.m. et al, "Pharmaceutical Salts", j.pharm.sci.1977, month 1; 66 (1):1-19.

The term "pharmaceutical composition" as used herein refers to a combination of one or more pharmaceutical substances and one or more excipients.

The term "patient" as used herein refers to a human or non-human mammal. Examples of non-human mammals include domestic animals such as sheep, horses, cattle, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses.

The term "body" as used herein refers to the body of a patient as defined above.

As used herein, the term "therapeutically effective amount" of a drug refers to an amount of the drug or composition that is effective to treat a patient and thereby produce a desired therapeutic or ameliorating effect. The therapeutically effective amount may depend on the weight and age of the patient, the route of administration, and the like.

As used herein, the term "treating" refers to reversing, alleviating, inhibiting the progression of, or preventing a disorder, disease, or condition to which the term applies; or reverse, alleviate, inhibit the progression of, or prevent one or more symptoms of the disorder, disease, or condition.

As used herein, the term "treatment" refers to "treatment" behavior as defined above.

As used herein, the term "thrombolysis" refers to the removal of an obstruction of blood flow, such as a blood clot, using a chemical method, such as by using a thrombolytic agent.

As used herein, the term "thrombectomy" refers to the use of mechanical means to remove obstructions of blood flow, such as blood clots.

As used herein, the term "bolus" refers to a single administration of a relatively large discrete dose of a drug that is administered intravenously rapidly.

As used herein, the term "continuous infusion" refers to continuous intravenous administration of a drug over a set period of time.

As used herein, the term "biocompatible" refers to exhibiting substantially no cytotoxicity or immunogenicity upon contact with body fluids or tissues.

The term "polymer" as used herein refers to oligomers, co-oligomers, polymers, and copolymers, such as random blocks, multiblocks, star, graft, gradient copolymers, and combinations thereof.

As used herein, the term "biocompatible polymer" refers to a polymer that is non-toxic, chemically inert, and substantially non-immunogenic, and substantially insoluble in blood when used in a subject. The biocompatible polymer may be non-biodegradable or preferably biodegradable. Preferably, the biocompatible polymer is also non-inflammatory when used in situ.

As used herein, the term "cleavable linking group" is a chemical group that is stable under one set of conditions, but cleaves under a different set of conditions to release two moieties that are bound together by a linker. Cleavable linking groups are sensitive to cleavage agents, e.g., hydrolysis, pH, high shear stress, redox potential, temperature, radiation, ultrasoundThe presence of a physiological or degrading molecule (e.g., an enzyme or chemical agent), etc. Exemplary cleavable linking groups include, but are not limited to, hydrolyzable linkers, redox cleavable linking groups (e.g., -S-S-and-C (R)) ₂ -S-S-, wherein R is H or C ₁ -C ₆ Alkyl, and at least one R is C ₁ -C ₆ Alkyl radicals, e.g. CH ₃ Or CH (CH) ₂ CH ₃ ) The method comprises the steps of carrying out a first treatment on the surface of the Phosphate-based cleavable linking groups (e.g., -O-P (O) (OR) -O-, -O-P (S) (SR) -O-, -S-P (O) (OR) -O-, -O-P (O) (OR) -S-, -S-P (O) (OR) -S-, -O-P (S) -O-, -O-P (O) (R) -O-, -O-P (S) (R) -O-, -S-P (O) (R) -O-, -S-P (S) (R) -O-, -S-P (O) (R) -S-, -O-P (S) (OH) -O-, -O-P (S) (SH) -O-, -S-P (O) (OH) -O-, -O-P (O) (OH) -S-, -S-P (O) (OH) -S-, -O-P (S) (OH) -S-, -S-P (S) (OH) -O-, -O-P (O) (H) -O-, -O-P (S) (H) -O-, -S-P (O) (H) -O-, -S-P (S) (H) -O-, -S-P (O) (H) -S-and-O-P (S) (H) -S-, wherein R is optionally substituted straight or branched C ₁ -C ₁₀ An alkyl group); acid cleavable linking groups (e.g., hydrazones, esters, and esters of amino acids, -c=nn-and-OC (O) -); an ester-based cleavable linking group (e.g., -C (O) O-); peptide-based cleavable linkers (e.g., linkers cleaved by enzymes in the cell (e.g., peptidases and proteases), e.g., -NHCHR ^A C(O)NHCHR ^B C (O) -, wherein R ^A And R is ^B R groups of two adjacent amino acids). The peptide-based cleavable linking group comprises two or more amino acids. The peptide-based cleavage bond comprises an amino acid sequence that is a substrate for a peptidase or protease.

As used herein, "nitroglycerin" or "NG" is known and equivalent to "glycerol trinitrate" or "GTN".

As used herein, the term "nitroglycerin nanoparticle aggregate" or "NG-NPA" refers to SA-NTs according to the present invention comprising nitroglycerin as a vasodilator.

The term "comprising" as used herein means "consisting at least in part of …". In interpreting each statement in this specification that includes the term "comprising," other features may be present in addition to or in place of the term. Related terms such as "comprise" and "comprise" should be interpreted in the same manner.

Examples

EXAMPLE 1 nanoparticle aggregate preparation

Nanoparticles (NPs) were prepared from PLGA (50:50, 17kDa, acid termination; lakeshore Biomaterials, AL) using a simple solvent displacement method. Fluorescent hydrophobic dye coumarin-6 was included in the nanoparticles for visualization and quantification in this study.

1mg/ml of polymer and 0.1wt.% coumarin were dissolved in dimethyl sulfoxide (DMSO, sigma, MO), dialyzed against water at room temperature, and nanoparticles formed by solvent displacement and subsequent self-assembly in aqueous solution.

Preparation of NP aggregates: PLGA NP was centrifuged and concentrated to a suspension in 10mg/mL water and 1mg/mL L-leucine (Spectrum Chemicals & Laboratory Products, CA) was added. NP aggregates were prepared by spray drying technique using a mobile mini spray dryer (Niro, inc.; columbia, MD). The aqueous leucine-NP suspension was injected separately from the organic phase (ethanol) in a 1.5:1 ratio and mixed in-line immediately before atomization. The inlet temperature was 80℃and the liquid feed rate was 50ml/min; the gas flow rate was set at 25g/min and the nozzle pressure at 40psi. The spray-dried powder was collected in a vessel at the outlet of the cyclone. The SA-NT suspension is formed by reconstituting the powder in water at the desired concentration. Filtering the aggregate suspension through a 20 μm filter to filter out any oversized aggregates; centrifugation (2000 g for 5 min) followed by washing was also used to remove individual unbound NPs. Dynamic light scattering (Dynamic Light Scattering, DLS) was used to determine the size of NPs in dilute solutions, using a zeta particle size analyzer (Malvern Instruments, UK), operated with HeNe laser, 173 ° back scatter detector. Samples were prepared at a concentration of 1mg/mL in PBS buffer at pH 7.4.

EXAMPLE 2a preparation of nitroglycerin nanoparticles (NG-NP)

Nanoparticles (NPs) were prepared from PLGA (50:50, acid terminated; GMP grade PLGA poly (D, L-lactide-co-glycolide) from Durect Lactol) using a single emulsion solvent evaporation method. A1% (w/v) solution of polyvinyl alcohol (PVA, sigma-Aldrich) that was sterilized by filtration was prepared in milli-Q water and used as the aqueous phase. 50mg/ml PLGA polymer dissolved in methylene chloride (Sigma-Aldrich) was used as the organic phase. 5mg/ml nitroglycerin (American Regent-nitroglycerin injection, USP grade) solution was transferred to the organic phase. The organic phase containing the PLGA and nitroglycerin solution was mixed in a beaker containing an aqueous phase containing 50ml PVA. The solution mixture was sonicated for 1.5 minutes at an intensity of 40AMP using a probe sonicator (Q-sonic model Q700) in a cold room at 4 ℃. Stock solutions were dialyzed against Milli-Q water overnight using a 100kD CE tube (Spectrum-Labs). The sample concentration was determined by dry weight analysis (three 0.5ml suspensions were dried in an oven at 120 ℃ and the average was used to calculate the total nanoparticle yield, >90 wt%). Dynamic Light Scattering (DLS) was used to characterize the size distribution of the NPs formed. The Z-average size of NPs was found to be 185.6nm with a standard deviation of 75.18. The mode average size of NPs was found to be 210.8nm.

Example 2b preparation of nitroglycerin nanoparticle aggregates (NG-NPA)

The NG-NP suspension was diluted to 5mg/mL by adding Milli-Q water. 2mg/mL L-leucine (Spectrum Chemicals & Laboratory Products, CA) was used as the aqueous phase. NG nanoparticle aggregates prepared by spray drying techniques were sterilized by autoclaving using a bench top Buchi 290 spray dryer (Buchi-290, switzerland) unit. The aqueous leucine-NP suspension was combined with an organic phase (ethanol): the NG-NP suspension mixtures were injected separately in a 1.5:1 ratio and mixed in-line immediately before atomization. The inlet temperature was 110℃and the liquid feed rate was 6ml/min; the suction pressure was 60mbar. The spray-dried powder was collected in a vessel at the outlet of the cyclone and stored in a dryer at-20 ℃. The nitroglycerin loading in the nanoparticle aggregates was 1.2 wt% as measured by high pressure liquid chromatography (high-pressure liquid chromatography, HPLC). For size measurement, samples were prepared at a concentration of 1mg/mL in PBS buffer at pH 7.4 and measured using a Markov (Malvern) laser diffractometer. The median size of the nanoparticle aggregates was found to be 2.5 μm.

Example 3-use of nitroglycerin-containing nanoparticles to increase side branch perfusion in rodents

Animal experiments were performed on male Spontaneous Hypertensive Rats (SHR) weighing 300-350g (Harlan, binder, UK). All animals were placed in a 12 hour light/dark cycle and food and water were freely available prior to the experiment. All procedures were in compliance with the animal (scientific procedure) laws (Animal (Scientific Procedures) Act 1986) (UK) and national institutes of health, guidelines for laboratory animal care and use (National Institures of Health guidelines for care and use of laboratory animals) and were approved by the animal ethics committee of the oxford university (University of Oxford Animal Ethics Committee, the Home Office (UK)) of the department of internal administration (UK).

Anesthesia and monitoring

Rats were treated with 5% isoflurane at O ₂ /N ₂ (1:3) and maintained with 1% to 2% isoflurane. The core temperature was maintained at 37 ℃ by thermocouple rectal probe and heating plate (Harvard Apparatus, UK). The incision site was shaved, cleaned and subcutaneously injected with 2mg/kg of 0.05% Bupivacaine (Aspen, UK). Continuous arterial pressure monitoring was performed using femoral artery lines.

Experimental stroke model

Rats were occluded for middle cerebral artery (MCAo/stroke) using a silicone head lumen internal thread occlusion method (as shown by sprat et al (2006): "Modification of the method of thread manufacture improves stroke induction rate and reduces mortality after thread-occlusion of the middle cerebral artery in young or aged rates." J Neurosci Methods), using a 4-0 monofilament occlusion wire with a silicone tip of 4mm length x 0.35mm diameter. The filaments were advanced to the right external carotid stump and up the internal carotid artery to occlude the origin of the right MCA.

Measuring core and side branch blood flow changes using a multi-point dual laser Doppler probe

Laser doppler flowmeters (Laser Doppler flowmetry, LDF) (Oxford Optronix, oxford, UK) are used to measure Cerebral Blood Flow (CBF) changes in MCA and side branch arterial areas using dual probes. The head of the animal is fixed in the stereotactic frame by the ear rod. Probe 1 was placed at +4mm outside the midline and-2 mm posterior to bregma to measure changes in core MCA CBF. The probe 2 was placed at +3mm outside the midline and +2mm anterior to bregma to measure the change in CBF supplied by the side branch vessels in the boundary region between the Anterior Cerebral Artery (ACA) and the Middle Cerebral Artery (MCA) perfusion region. In probe 1, LDF signal was reduced by >70% from baseline, confirming middle cerebral artery occlusion.

Drug administration

Example 3a

The femoral vein was inserted with a 2-French silicone tube prior to MCAo. 30 minutes after MCAo, animals received either a random intravenous bolus followed by infusion of blank nanoparticle aggregates (NPA control, 1mg in saline, n=6) or a intravenous bolus followed by intravenous infusion of nitroglycerin nanoparticle aggregates (NG-NPA, 12.5 μg nitroglycerin in saline of 1mg NPA, n=6), i.v. Side branch perfusion was measured as% change from baseline before injection. The variation of side branch perfusion is shown in figure 1. The average side branch perfusion comparisons of blank NPA and NG-NPA are shown in FIG. 2.

The blank NPA has no obvious effect on the side branch circulatory perfusion. NG-NPA administration significantly increased collateral blood perfusion 4 minutes after the start of administration, on average 40% above baseline (fig. 1). NG-NPA administration also increased mean side branch perfusion during treatment (fig. 2).

Example 3b

The femoral vein was inserted with a 2-French silicone tube prior to MCAo. One Spontaneous Hypertensive Rat (SHR) received an intravenous bolus of free nitroglycerin (NG, 12.5, 25, 50 and 100 μg) at a dose of more than 1 minute beginning 30 minutes after MCAo, which was not packaged in NPA. There was a NG washout period of 5 half-lives between each dose administration. Side branch perfusion was measured as% change from baseline before injection. The results are shown in FIG. 3.

The peak collateral blood flow increase of the NG-NPA group was 2.5 times higher than the equivalent dose of nitroglycerin not packaged into NPA. Furthermore, increasing the dose of "free" nitroglycerin did not additionally increase side branch perfusion (fig. 3).

Example 3c

The femoral artery and vein were cannulated with 2-French silicone tubing prior to MCAo. Mean arterial pressure was measured continuously through the femoral catheter. Side branch perfusion was measured as% change from baseline before injection. One SHR began to receive intravenous infusion of NG-NPA 30 minutes after MCAo (NG dose of 2mg NPA saline, 2 ml/hr = 2.75 μg/kg/min with 25 μg nitroglycerin). The variation of SHR collateral perfusion and mean arterial pressure is shown in figure 4.

The other SHR began to receive intravenous NG-NPA 30 minutes after MCAo (NG dose of 2mg NPA saline containing 25 μg nitroglycerin, 3 ml/hr = 4.15 μg/kg/min). The variation of SHR collateral perfusion and mean arterial pressure is shown in figure 5.

Example 4-use of nitroglycerin-containing nanoparticles to increase side branch perfusion in rodents

Animal experiments were performed on male Spontaneous Hypertension Rats (SHR) (ARC, perth, australia) weighing 280-310 g. All animals were placed in a 12 hour light/dark cycle and food and water were freely available prior to the experiment. The experiment was approved by the university of newcastle (University of Newcastle) animal care and ethics committee (Animal Care and Ethics Committee) (protocol # a-2020-003) and met the requirements of the national institutes of animal care and use behavior rules (Australian Code of Practice for the Care and Use of Animals for Scientific Purposes).

Anesthesia and monitoring

Rats were treated with 5% isoflurane (in O ₂ /N ₂ (1:3) and maintained with 1% to 2% isoflurane. Core temperature by thermocouple rectal probe and heating plateMaintained at 37 ℃. The incision site was shaved, cleaned, and subcutaneously injected with 2mg/kg of 0.05% bupivacaine. Continuous arterial pressure monitoring was performed using femoral artery lines.

Experimental stroke model

Rats were subjected to middle cerebral artery occlusion (MCAo/stroke) using a silicone head end lumen internal thread occlusion method (as described by sprat et al (2006): "Modification of the method of thread manufacture improves stroke induction rate and reduces mortality after thread-occlusion of the middle cerebral artery in young or aged rates." J Neurosci Methods "), using a 4-0 monofilament occlusion wire having a silicone tip of 4mm length x 0.35mm diameter. The filaments were advanced to the right external carotid stump and up the internal carotid artery to occlude the right MCA's beginning.

Side branch blood flow in the stroke (right) hemisphere and side branch blood in the control (left) hemisphere were measured using laser speckle contrast imaging Flow change

Laser speckle contrast imaging (Laser speckle contrast imaging) by bilaterally thinning the cranial window (RWD Life Sciences) for measuring Cerebral Blood Flow (CBF) changes in the right (stroke) side collateral artery region of the brain and the left (control/contralateral) equivalent region of the brain. The head of the animal is fixed in the stereotactic frame by the ear rod. A cranium window (4 mm x 4 mm) was created bilaterally starting 1mm posterior to the bregma and 1mm lateral to the midline. The region of interest on the laser speckle imaging software was placed 2mm behind the bregma and 3mm outside the right midline to measure the change in CBF supplied by the branch vessels inside the border region between the Anterior Cerebral Artery (ACA) and the Middle Cerebral Artery (MCA) perfusion regions. Another region of interest was placed in the equivalent area of the left-hand (control/contralateral) hemisphere (i.e., 2mm posterior to bregma, 3mm lateral to the midline).

Drug administration

The femoral artery and vein were cannulated prior to MCAo. Mean arterial pressure was measured continuously through the femoral catheter. The% change in collateral and control hemispheric perfusion from the pre-injection baseline was measured.

Starting 25 minutes after MCAo, animals received either intravenous infusion of blank nanoparticle aggregates (NPA control, 4mg in 2mL saline, n=7), or infusion of nitroglycerin nanoparticle aggregates (NG-NPA, 50 μg nitroglycerin in 4mg of NPA in 2mL saline, 2.8-3 mL/hr=4 μg/kg/min, n=7). Infusion was continued for 45 minutes until reperfusion was induced by retraction of the occlusion line. Animals were recovered 24 hours after surgery, at which time they were euthanized and analyzed for final stroke (infarct) size of their brains.

Results

NG-NPA administration significantly increased side branch blood perfusion, while blank NPA did not significantly alter side branch perfusion (fig. 6). NG-NPA had no effect on perfusion in the corresponding region of the control/contralateral hemisphere (fig. 7). NG-NPA did not significantly lower blood pressure (fig. 8). NG-NPA treatment significantly reduced the size of stroke at 24 hours (fig. 9). When the two groups were combined, there was a significant negative correlation between the extent of change in side branch perfusion and infarct volume (fig. 10). This inverse relationship between collateral perfusion and infarct volume demonstrates an improved protective effect of collateral perfusion on stroke outcome. These results demonstrate that NG-NPA enhances collateral perfusion, specifically targets collateral (contralateral/control hemispheric perfusion and no change in blood pressure) and improves stroke outcome (outome).

EXAMPLE 5 Effect of free nitroglycerin on rodent perfusion and blood pressure

Animal experiments were performed on male Spontaneous Hypertension Rats (SHR) (ARC, perth, australia) weighing 280-310 g. All animals were placed in a 12 hour light/dark cycle and food and water were freely available prior to the experiment. The experiments were approved by the university of newcastle animal care and ethics committee (protocol # a-2020-003) and met the requirements of the national institutional animal care and use behavior rules of australia.

Anesthesia and monitoring

Experimental stroke model

Laser speckle contrast imaging (RWD Life Sciences) by bilaterally thinning the cranial window was used to measure Cerebral Blood Flow (CBF) changes in the right (stroke) side collateral artery region of the brain and the left (control/contralateral) equivalent region of the brain. The head of the animal is fixed in the stereotactic frame by the ear rod. A cranium window (4 mm x 4 mm) was created bilaterally starting 1mm posterior to the bregma and 1mm lateral to the midline. The region of interest on the laser speckle imaging software was placed 2mm behind the bregma and 3mm outside the right midline to measure the change in CBF supplied by the branch vessels inside the border region between the Anterior Cerebral Artery (ACA) and the MCA perfusion region. Another region of interest is placed in the equivalent region of interest of the left-hand (control/contralateral) hemisphere.

Drug administration

4 μg/kg/min nitroglycerin (GTN)

Starting 25 minutes after MCAo, animals received either intravenous infusion of normal saline (control, 0.3ml/hr, n=6) or infusion of free nitroglycerin (free-GTN, 0.25 μg/μl in normal saline, 300 μl/h=4 μg/kg/min, n=6) at random. Infusion was continued for 45 minutes until reperfusion was induced by retraction of the occlusion line.

free-GTN administration at 4 μg/kg/min did not alter side branch blood perfusion (FIG. 11). free-GTN at 4 μg/kg/min had no significant effect on perfusion in the corresponding region of the control/contralateral hemisphere (FIG. 12). free-GTN reduced blood pressure by 4 μg/kg/min (FIG. 13).

These results demonstrate that GTN is administered as a free drug, has no effect on side branch perfusion, but can lead to hypotension.

40 μg/kg/min nitroglycerin (GTN)

Starting 25 minutes after MCAo, animals received intravenous infusion of free nitroglycerin (free-GTN, 2.5 μg/μl in physiological saline, 300 μl/h=40 μg/kg/min, n=4). Infusion was continued for 45 minutes until reperfusion was induced by retraction of the occlusion line. Vehicle control (normal saline) was identical to the control group used in the 4 μg/kg/min study (n=6).

free-GTN administration at 40 μg/kg/min did not alter collateral blood perfusion (FIG. 14). free-GTN at 40 μg/kg/min had no significant effect on perfusion in the corresponding region of the control/contralateral hemisphere (FIG. 15). free-GTN at 40 μg/kg/min significantly reduced blood pressure (FIG. 16).

These results demonstrate that GTN administered as free drug has no effect on side branch perfusion but causes dose-dependent hypotension at a dose equivalent to 10 times the dose in NG-NPA. These results underscore that simply increasing the dose of free GTN from 4 μg/kg/min to 40 μg/kg/min did not enhance collateral perfusion, likely as a result of hypotension due to the expansion of systemic blood vessels by free GTN. Thus, it was not possible to determine the equivalent dose of free drug required to cause the same collateral enhancement as 4 μg/kg/min GTN (packaged into NG-NPA), and thus it was not possible to determine the fold enhancement of collateral enhancement by NG-NPA.

In combination with the results of example 4, these results demonstrate that NG-NPA can change GTN from ineffective to extremely effective side branch enhancement therapy, as it is selectively delivered to side branch vessels and without systemic hypotension.

While specific embodiments of the invention have been described above, it should be understood that the invention may be practiced otherwise than as described. The above description is intended to be illustrative, and not restrictive. It will therefore be apparent to those skilled in the art that modifications may be made to the invention as described without departing from the scope of the claims set out below.

Claims

1. A shear activated nanotherapeutic agent (SA-NT) for treating stroke by increasing blood supply to the brain via a side branch vessel, wherein the SA-NT comprises an aggregate comprising a plurality of nanoparticles, the aggregate further comprising one or more vasodilators or pharmaceutically acceptable salts thereof; wherein the aggregates are configured to disaggregate above a predetermined shear stress.

2. The shear activated nanotherapeutic agent for use according to claim 1, wherein the one or more vasodilators are selected from the group consisting of: nitrate, angiotensin II receptor blockers, calcium channel blockers, selective alpha blockers, beta 1 agonists, beta 2 agonists, beta agonists, ET1 receptor antagonists, phosphodiesterase 5 inhibitors or agonists of the Small (SK) and medium (IK) calcium-activated potassium channels.

3. The shear activated nanotherapeutic agent for use according to claim 1 or claim 2, wherein the one or more vasodilators are selected from the group consisting of: nitrate, angiotensin II receptor blockers or calcium channel blockers.

4. The shear activated nanotherapeutic agent for use according to any one of claims 1-3, wherein the one or more vasodilators comprise nitroglycerin.

5. The shear activated nanotherapeutic agent for use according to any one of claims 1 to 4, wherein the one or more vasodilators comprise nimodipine.

6. The shear activated nanotherapeutic agent for use of any one of claims 1-5, wherein the SA-NT is configured to be at greater than about 100dynes/cm ² Releasing its constituent nanoparticles at the shear stress.

7. The shear activated nanotherapeutic agent for use according to any one of claims 1 to 6, wherein the nanoparticle comprises a copolymer of polylactic acid and polyglycolic acid.

8. The shear activated nanotherapeutic agent for use according to any one of claims 1 to 7, wherein the one or more vasodilators or pharmaceutically acceptable salts thereof are encapsulated in, adsorbed on the surface of, or covalently linked to the nanoparticle.

9. The shear activated nanotherapeutic agent for use according to any one of claims 1 to 8, wherein the one or more vasodilators or pharmaceutically acceptable salts thereof are released at a higher rate and/or in a higher amount when the nanoparticles are deagglomerated than when the nanoparticles are aggregated.

10. A composition for treating stroke by increasing blood supply to the brain via a side branch vessel, wherein the composition comprises a shear activated nanotherapeutic agent (SA-NT) as defined in any of claims 1 to 9 in combination with one or more pharmaceutically acceptable excipients, carriers and/or diluents.

11. The shear activated nanotherapeutic for use according to any one of claims 1 to 9 or the composition for use according to claim 10, wherein the SA-NT or the composition is administered intravenously.

12. The shear activated nanotherapeutic agent or composition for use according to any one of claims 1 to 11, wherein the SA-NT or the composition is administered as a continuous infusion.

13. The shear activated nanotherapeutic agent or composition for use according to any one of claims 1 to 11, wherein the SA-NT or the composition is administered as a bolus.

14. The shear activated nanotherapeutic agent or composition for use according to any one of claims 1 to 13, wherein the stroke is ischemic stroke.

15. The shear activated nanotherapeutic agent or composition for use according to any one of claims 1 to 14, wherein the treatment increases blood flow to the penumbra.

16. The shear activated nanotherapeutic agent or composition for use according to any one of claims 1 to 15, wherein the SA-NT or the composition is administered in combination with neuroprotective therapy.

17. The shear activated nanotherapeutic agent or composition for use according to any one of claims 1 to 16, wherein the SA-NT or the composition is administered in combination with one or more additional therapies selected from thrombolytic therapy and thrombolytic therapy; wherein the additional therapy is not administered as part of SA-NT.

18. A method of treating stroke by increasing blood supply to the brain via a side branch vessel, comprising administering to a patient in need thereof a shear activated nanotherapeutic agent (SA-NT) as defined in any of claims 1 to 9 or a composition as defined in claim 10.

19. Use of a shear activated nanotherapeutic agent (SA-NT) as defined in any of claims 1 to 9 or a composition as defined in claim 10 for the manufacture of a medicament for treating stroke by increasing the blood supply to the brain via a side branch vessel.

20. A shear activated nanotherapeutic agent (SA-NT) comprising an aggregate comprising a plurality of nanoparticles, wherein the aggregate is configured to disaggregate above and below a predetermined shear stress; and wherein the aggregate further comprises from about 0.4% to about 2.5% by weight of nitroglycerin or a pharmaceutically acceptable salt thereof.

21. The shear activated nanotherapeutic agent of claim 20, wherein the aggregate comprises about 0.8% to about 1.6% by weight of nitroglycerin or a pharmaceutically acceptable salt thereof.

22. The shear activated nanotherapeutic agent of claim 20 or 21, wherein the aggregate comprises about 1% to about 1.4% by weight of nitroglycerin or a pharmaceutically acceptable salt thereof.

23. The shear-activated nanotherapeutic agent of any one of claims 20-22, wherein the SA-NT is configured to be at greater than about 100dynes/cm ² Releasing its constituent nanoparticles at the shear stress.

24. The shear activated nanotherapeutic agent for use according to any one of claims 20 to 23, wherein the aggregate further comprises nimodipine.

25. The shear activated nanotherapeutic agent of any one of claims 20-24, wherein the nanoparticle comprises a copolymer of polylactic acid and polyglycolic acid.