DC009 for treating acute ischemic stroke

Abstract

The present invention relates to a method for treating acute ischemic stroke in a human comprising administering one or more low doses of DC009. The dosage is about 0.01-0.075 mg / kg / administration, and the low dose administration is safe and effective for treating acute ischemic stroke in human subjects.

Description

[Technical Field]

[0001] The present invention relates to a method for treating acute ischemic stroke in humans using a low dose of 3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-acyl-Lys (Pro-Ala-Lys) (CAS Registry Number (RN): 1639303-73-3). [Background technology]

[0002] Stroke is classically characterized as a neurological deficit resulting from acute focal damage to the central nervous system due to vascular causes. Ischemic stroke accounts for approximately 87% of all strokes, with intracerebral hemorrhage accounting for 10% and subarachnoid hemorrhage for 3%. Every year, 15 million people worldwide suffer a stroke, and in the United States, on average, one person experiences a stroke every 40 seconds. Globally, stroke is the second leading cause of death for people over 60 and a major cause of disability.

[0003] Alteplase (Activase®), a recombinant tissue plasminogen activator (rtPA), was the first drug approved by the U.S. Food and Drug Administration in 1996 for the treatment of ischemic stroke (BLA 103172 / S-1055). Although alteplase has been shown to improve outcomes in subjects with acute ischemic stroke (AIS), its use is limited because it is only approved for administration within 3 hours (in the U.S.) or 4.5 hours after symptom onset, and the risk of symptomatic intracranial hemorrhage is nearly five times higher.

[0004] For the reasons stated above, the use of alteplase is low, and it is administered to only about 5% of stroke patients. Therefore, there is a need to develop treatments that can achieve similar effects to alteplase but with less increased bleeding and / or a longer time window for treatment.

[0005] DC009 is a peptide-tetrahydroisoquinoline complex with the chemical name 3S-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-acyl-Lys(Pro-Ala-Lys) or L-lysine, N6-(L-prolyl-L-alanyl-L-lysyl)-N2-[[(3S)-1,2,3,4-tetrahydro-6,7-dihydroxy-1,1-dimethyl-3-isoquinolinyl]carbonyl](CAS registry number: 1639303-73-3). DC009 is a two-component complex that can be formed by linking a thrombolytic peptide (Pro-Ala-Lys) with a tetrahydroisoquinoline compound having two C1-4 alkyl groups via a lysine linking arm. The structure of DC009 is shown in Figure 1A, and the amide bond between the lysine linkage arm and the Pro-Ala-Lys peptide is shown in Figure 1B.

[0006] Drug development is a stepwise process involving the evaluation of both animal and human efficacy and safety information. The goal of preclinical safety evaluation generally includes characterizing toxic effects, and this information is used to estimate initial safe starting doses and dose ranges for human trials and to identify parameters for clinically monitoring potential side effects. All relevant preclinical data should be considered, including pharmacological doses, the complete toxicological profile of the compound, and information on the pharmacokinetics (absorption, distribution, metabolism, and excretion) of the therapeutic agent.

[0007] The maximum recommended starting dose (MRSD) in first-in-human clinical trials of a new compound in healthy adult subjects should be determined by dividing the human equivalent dose (HED) derived from the no-observed-adverse-effect level (NOAEL) in animals by a safety factor. The commonly used default safety factor is 10, which is a historically accepted value, but it needs to be evaluated based on available information. This is non-binding guidance from the Center for Drug Evaluation and Research (CDER) of the U.S. Food and Drug Administration. However, a safety factor of 10 is not necessarily appropriate in all cases. The safety factor should be increased when there are reasons for increased concern, and decreased when available data providing additional assurance of safety mitigate such concerns (Guidance for Industry "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers", www.fda.gov / media / 72309 / download).

[0008] The development of treatments for acute ischemic stroke (AIS) is a difficult and challenging endeavor due to the complexity of the pathophysiology and clinical aspects of this heterogeneous disease. Activated recombinant tissue plasminogen activator (rtPA) administered within 3 hours after stroke onset is currently the only approved treatment for AIS. Track records for evaluating the use of animal models in the development of AIS treatments are limited. Although many interventions have shown efficacy in AIS animal models, these interventions, primarily neuroprotective agents, have not been shown to improve outcomes of AIS in patients (Fisher, et al, Stroke. Volume 36, Issue 10, 1 October 2005; Pages 2324-2325). The discrepancy in results regarding neuroprotective agents between animal experiments and clinical trials is a major problem. Although many neuroprotective agents have been proven effective in various animal models of ischemic stroke, none have shown efficacy in clinical trials (Xu, et al, Med Sci Monit Basic Res. 2013 Jan 28;19:37-45. doi: 10.12659 / msmbr.883750.)

[0009] There is a need for a method of treating acute ischemic stroke. The method should be effective, with minimal side effects and toxicity. Brief Description of the Drawings

[0010] [Figure 1A] Shows the chemical structure of DC009. [Figure 1B] Shows the chemical structure of DC009 together with details of NH-Lys-Ala-Pro. [Figure 2] Shows a method for evaluating neurological deficit. [Figure 3] Shows blood flow results from a single administration of DC009 in a rat MCAO model. [Figure 4] Shows infarct size results from multiple administrations of DC009 in a rat MCAO model. [Figure 5] Summarizes Examples 1 to 10 (rat model). [Figure 6]This shows the neurological improvement of DC009 in human subjects. [Figure 7] Summarize Examples 11-16 (results or protocols of human clinical trials). [Modes for carrying out the invention]

[0011] Considering the thrombolytic and free radical scavenging effects of DC009 in rats, the inventors have discovered a method for treating acute ischemic stroke in human subjects. The method comprises administering an effective amount of DC009 or a pharmaceutically acceptable salt thereof to a subject in need, wherein the amount of DC009 is effective in treating the disease and is safe at the lowest toxic dose. max The present invention provides an appropriate dosage, which is effective in treating acute ischemic stroke in human subjects, minimizing the risk of bleeding and potentially extending the time window for treatment.

[0012] Based on the preclinical toxicity results of DC009 in miniature pigs conducted by the inventors, the inventors set the plasma exposure limit in miniature pigs at 1177 ng / mL. The inventors then set a safety factor to provide a safety margin to protect human subjects receiving the initial clinical dose. As described in the background art, the default safety factor is 10, but may be adjusted based on available safety data. In the present invention, administration of DC009 to human subjects is considered to be plasma C max The dose is limited to approximately 200 ng / mL or less, preferably approximately 150 ng / mL or less, or approximately 110 ng / mL or less.

[0013] The inventors have found that an effective and safe dose of DC009 for treating acute ischemic stroke in human subjects is a low dose of approximately 0.01–0.075 mg / kg / dose or approximately 0.025–0.05 mg / kg / dose. For example, an effective and safe dose is approximately 0.025 mg / kg / dose or approximately 0.05 mg / kg / dose.

[0014] As used throughout this application, "approximately" refers to ±10% of the listed values.

[0015] The inventors have found that the low dose of DC009 of the present invention is effective in treating acute ischemic stroke, providing low plasma drug levels and reducing potential drug toxicity. The (elimination) half-life of DC009 is very short (half-life < 5 minutes), and DC009 is eliminated from the circulatory system immediately after administration and is barely detectable in human plasma after 30 minutes post-administration. Multiple administrations of DC009 do not cause accumulation of DC009, and the therapeutic effect of DC009 is achieved by exposure to each dose of the drug.

[0016] The pharmaceutically acceptable salts of DC009 (see Figures 1A and 1B) include any pharmaceutically acceptable salt, e.g., hydrochloride, namely L-lysine, N6-(L-prolyl-L-alanyl-L-lysyl)-N2-[[(3S)-1,2,3,4-tetrahydro-6,7-dihydroxy-1,1-dimethyl-3-isoquinolinyl]carbonyl]-hydrochloride (1:3) (CAS Registry No.: 2419930-71-3). The molecular formula of DC009 is C32H51N7O8, and the molecular weight of the free base is 661.8 g / mole.

[0017] The preparation of the DC009 compound is disclosed in Example 63 of U.S. Publication No. 2016-0083423, which is incorporated herein by reference. The present invention uses a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and the DC009 compound or a pharmaceutically acceptable salt thereof.

[0018] A pharmaceutically acceptable carrier, which is an inactive component, can be selected by those skilled in the art according to conventional standards. Examples of pharmaceutically acceptable carriers include physiological saline and electrolyte solutions; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and glucose; pH adjusters and buffers such as hydroxide salts, phosphates, citrates, acetates, and borates; and trolamine; antioxidants such as bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and salts of ascorbyl palmitate, acids, and / or bases; and phospholipids including, but not limited to, lecithin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinosiol. These materials may contain, but are not limited to, surfactants; polysorbates such as poloxamer and poloxamine, polysorbate 80, polysorbate 60, and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives and their salts such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose; polymers of acrylic acid such as carboxypolymethylene gel and hydrophobic modified crosslinked acrylate copolymers; and polysaccharides such as dextran and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers can be preserved from bacterial contamination using well-known preservatives, including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or can be formulated as non-preservative formulations for single or multiple use.

[0019] One of the pharmaceutical compositions for human use is a lyophilized powder containing the hydrochloride salt of DC009 (C32H51N7O8·3HCl), with mannitol as the main excipient. DC009 is further diluted to the appropriate concentration with physiological saline before administration.

[0020] In some embodiments, the dose of DC009 for treating acute ischemic stroke in human subjects is approximately 0.01 to 0.075 mg / kg / dose. In some embodiments, the dose of DC009 for treating acute ischemic stroke in human subjects is approximately 0.025 to 0.05 mg / kg / dose. In one embodiment, the dose of DC009 for treating acute ischemic stroke in human subjects is approximately 0.025 mg / kg / dose. In one embodiment, the dose of DC009 for treating acute ischemic stroke in human subjects is approximately 0.05 mg / kg / dose.

[0021] In some embodiments, the dose is administered at least once daily. In some embodiments, the dose is administered once daily. In some embodiments, the dose is administered twice daily. In some embodiments, the dose is administered three times daily. As used throughout this application, dose refers to the dose of DC009.

[0022] In some embodiments, the dose is administered at dosing intervals of approximately 3 to 12 hours. In some embodiments, the dose is administered at dosing intervals of 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In some embodiments, the dose is administered at dosing intervals of approximately 3, 6, 9, or 12 hours. In some embodiments, the dose is administered at dosing intervals of approximately 3 hours. In some embodiments, the dose is administered at dosing intervals of approximately 6 hours. In some embodiments, the dose is administered at dosing intervals of approximately 12 hours.

[0023] In some embodiments, the dose is administered to the subject for 1 to 6 days. For example, the dose is administered to the subject for 1, 2, 3, 4, 5, or 6 days. In one embodiment, the dose is administered to the subject for 1 day. In one embodiment, the dose is administered to the subject for 2 days. In one embodiment, the dose is administered to the subject for 3 days. In one embodiment, the dose is administered to the subject for 4 days. In one embodiment, the dose is administered to the subject for 5 days. In one embodiment, the dose is administered to the subject for 6 days. In some embodiments, the dose is administered to the subject 1 to 3 times a day for 1 to 6 days. In one embodiment, the dose is administered to the subject 3 times a day for 3 days. In one embodiment, the dose is administered to the subject 2 times a day for 3 days. In one embodiment, the dose is administered to the subject 2 times a day for 6 days. In one embodiment, the dose is administered to the subject 1 time a day for 6 days.

[0024] In one embodiment, the dose is approximately 0.025 mg / kg / dose and is administered as a single dose to the subject. In another embodiment, the dose is approximately 0.05 mg / kg / dose and is administered as a single dose to the subject.

[0025] In one embodiment, the dose is approximately 0.025 mg / kg / dose and is administered to the subject twice daily for 3 days. In one embodiment, the dose is approximately 0.05 mg / kg / dose and is administered to the subject twice daily for 3 days. In one embodiment, the dose is approximately 0.025 mg / kg / dose and is administered to the subject three times daily for 3 days. In one embodiment, the dose is approximately 0.05 mg / kg / dose and is administered to the subject three times daily for 3 days.

[0026] In some embodiments, the compound or pharmaceutical composition is administered to a subject immediately after the onset of acute ischemic stroke, within 1 to 24 hours, or within 3 to 24 hours. In one embodiment, the compound or pharmaceutical composition is administered to a subject immediately after the onset of acute ischemic stroke. In one embodiment, the compound or pharmaceutical composition is administered to a subject within 1 to 24 hours after the onset of acute ischemic stroke. In one embodiment, the compound or pharmaceutical composition is administered to a subject within 3 to 24 hours after the onset of acute ischemic stroke. In some embodiments, the compound or pharmaceutical composition is administered to a subject within 3, 6, 9, or 24 hours after the onset of acute ischemic stroke. In some embodiments, the compound or pharmaceutical composition is administered to a subject within 3 hours after the onset of acute ischemic stroke. In some embodiments, the compound or pharmaceutical composition is administered to a subject within 24 hours after the onset of acute ischemic stroke.

[0027] The National Institute of Stroke Severity Scale (NIHSS) is a predictor for the prognosis of acute ischemic stroke (AIS), and its prediction is time-dependent. In some embodiments, AIS patients suitable for treatment according to the present invention have an NIHSS score of 4 to 30. In some embodiments, AIS patients have an NIHSS score of 4 to 25. In some embodiments, AIS patients have an NIHSS score of 6 to 25. In some embodiments, AIS patients have an NIHSS score of 6 to 12. In some embodiments, AIS patients have an NIHSS score of 13 to 25. In some embodiments, AIS patients have an NIHSS score of 6 or higher. In some embodiments, AIS patients have an NIHSS score of 4 or higher.

[0028] In some embodiments, AIS patients suffer from aortic atherosclerosis.

[0029] In some embodiments, the AIS patient is under 65 years of age. In some embodiments, the AIS patient is under 80 years of age. In some embodiments, the AIS patient is over 65 years of age. In some embodiments, the AIS patient is over 80 years of age.

[0030] In some embodiments, AIS patients have stroke symptoms that develop within 6 hours. In some embodiments, AIS patients have stroke symptoms that develop within 9 hours. In some embodiments, AIS patients have stroke symptoms that develop within 12 hours. In some embodiments, AIS patients have stroke symptoms that develop within 16 hours. In some embodiments, AIS patients have stroke symptoms that develop more than 16 hours.

[0031] In some embodiments, AIS patients have symptomatic intracranial occlusion of the M1 middle cerebral artery. In some embodiments, AIS patients have symptomatic intracranial occlusion of the M2 middle cerebral artery.

[0032] In some embodiments, AIS patients have a mismatch profile on MRI (magnetic resonance imaging) or CTP (computed tomography perfusion) with an ischemic core volume ≤ 70 mL, a mismatch ratio > 1.2, and a mismatch volume ≥ 5 mL. In some embodiments, AIS patients have an ischemic core ≤ 20 mL. In some embodiments, AIS patients have an ischemic core ≤ 30 mL. In some embodiments, AIS patients have an ischemic core ≤ 50 mL.

[0033] In some embodiments, AIS patients have a mismatch ratio > 1.8. In some embodiments, AIS patients have a mismatch volume > 10 mL. In some embodiments, AIS patients have a mismatch volume > 20 mL.

[0034] Intravenous (IV) injection delivers pharmaceutical compositions into the bloodstream, allowing for rapid and maximum absorption. Intravenous therapeutic agents can be administered by push, bolus, or continuous infusion. To achieve this rapid response, an intravenous push (IV push) is administered within 30 seconds. IV pushes are bag-independent. Intravenous bolus (IV bolus) can be easily performed without the need for an infusion pump. Intravenous infusion (IV bolus) takes longer to administer the drug while providing a steady-state concentration of the therapeutic agent.

[0035] In some embodiments, the pharmaceutical composition of the present invention, comprising the DC009 compound, is administered by intravenous infusion. In some embodiments, the pharmaceutical composition of the present invention is administered by intravenous infusion over 5 to 60 minutes. In some embodiments, the pharmaceutical composition of the present invention is administered by intravenous infusion over 15 to 30 minutes. In one embodiment, the pharmaceutical composition is administered by intravenous infusion over 15 minutes. In some embodiments, the pharmaceutical composition is administered by intravenous infusion over 30 minutes.

[0036] In some embodiments, the pharmaceutical composition is administered by intravenous bolus injection.

[0037] In some embodiments, the pharmaceutical composition is administered by intravenous bolus injection followed by intravenous infusion.

[0038] In some embodiments, the pharmaceutical composition was administered to AIS patients via any available vein.

[0039] In one example, the dose is approximately 0.05 mg / kg / dose, and the pharmaceutical composition is administered by intravenous infusion over 30 minutes.

[0040] In other cases, the dose is approximately 0.025 mg / kg / dose, and the pharmaceutical composition is administered by intravenous infusion over 15 minutes.

[0041] In other cases, the dose is approximately 0.025 mg / kg / dose, and the pharmaceutical composition is administered by intravenous infusion over 30 minutes.

[0042] In some embodiments, the pharmaceutical composition is administered simultaneously with aspirin, clopidogrel, apixaban, or dabigatran. In one embodiment, the pharmaceutical composition is administered simultaneously with aspirin. In one embodiment, the pharmaceutical composition is administered simultaneously with clopidogrel. In one embodiment, the pharmaceutical composition is administered simultaneously with apixaban. In one embodiment, the pharmaceutical composition is administered simultaneously with dabigatran.

[0043] In some embodiments, human subjects are administered before, during, or after endovascular thrombectomy. In some embodiments, human subjects are administered before endovascular thrombectomy. In some embodiments, human subjects are administered during endovascular thrombectomy. In some embodiments, human subjects are administered before or after endovascular thrombectomy.

[0044] In some embodiments, both intravascular thrombectomy and DC009 administration are performed within 24 hours of stroke onset.

[0045] In some embodiments, the plasma drug level of human subjects during the treatment period is less than 200 ng / mL. In some embodiments, the plasma drug level of human subjects during the treatment period is less than 150 ng / mL. In some embodiments, the plasma drug level of human subjects during the treatment period is less than 110 ng / mL.

[0046] The efficacy outcome of DC009 can be evaluated by neurological outcomes, such as changes in NIHSS over a specific period after administration. In some embodiments, AIS patients showed a decrease of 4 or more NIHSS from baseline. In some embodiments, AIS patients showed a decrease of 8 or more NIHSS from baseline. In some embodiments, AIS patients achieved an NIHSS ≤ 2 after administration. In some embodiments, AIS patients achieved an NIHSS ≤ 4 after administration.

[0047] The efficacy outcome of DC009 can be evaluated by functional outcomes, such as changes in the modified Rankin scale (mRS) over a specific period after administration. In some embodiments, AIS patients experienced a decrease of 1 or more from baseline in their mRS. In some embodiments, AIS patients achieved an mRS of 0–2 after treatment. In some embodiments, AIS patients achieved an mRS of 0–1 after treatment.

[0048] The efficacy outcome of DC009 can be evaluated by functional outcomes assessed by the Barthel index over a specific period after administration.

[0049] The efficacy outcomes of DC009 can be evaluated by imaging results such as changes in infarct volume from baseline by MRI / CTP and changes in hypoperfusion lesions from baseline by cerebral perfusion imaging MRI / CTP. In some embodiments, AIS patients experienced a 10% reduction in infarct growth. In some embodiments, AIS patients experienced a 20% reduction in infarct growth.

[0050] In some embodiments, AIS patients treated with DC009 achieved >50% reperfusion 10%, 20%, or 30% more often than the untreated group. In some embodiments, AIS patients treated with DC009 achieved >90% reperfusion 10%, 20%, or 30% more often than the untreated group. In some embodiments, AIS patients treated with DC009 achieved recanalization 10%, 20%, or 30% more often than the untreated group.

[0051] In summary, the present invention relates to a method for treating acute ischemic stroke in a human subject, comprising administering the DC009 compound to a human subject requiring treatment for acute ischemic stroke, wherein the dose is approximately 0.01 to 0.075 mg / kg / dose. In one embodiment, the dose is approximately 0.025 to 0.05 mg / kg / dose. In another embodiment, the dose is approximately 0.05 mg / kg / dose.

[0052] In the method of the present invention, the compound is administered at least once a day. In one embodiment, the compound is administered twice a day. In another embodiment, the compound is administered at intervals of about 3 to 12 hours. In yet another embodiment, the compound is administered at least once a day for 2 or 3 days. In yet another embodiment, the compound is administered at least twice a day for 3 days. In yet another embodiment, the compound is administered at intervals of about 12 hours.

[0053] In the method of the present invention, the compound is administered to the subject immediately or within 1 to 24 hours after the onset of acute ischemic stroke.

[0054] In the method of the present invention, the compound is administered by intravenous infusion and / or bolus injection. In one embodiment, the compound is administered by intravenous infusion. In one embodiment, the compound is administered by intravenous infusion over a period of 5 to 60 minutes.

[0055] In the method of the present invention, the compound may be administered simultaneously with aspirin, clopidogrel, apixaban, or dabigatran.

[0056] In the method of the present invention, C in the plasma of the subject after administration max The concentration is less than 200 ng / mL. In some embodiments, the C content in the plasma of subjects after administration is less than 200 ng / mL. max The level is less than 150 ng / mL.

[0057] The following embodiments further illustrate the present invention. These embodiments are intended merely to illustrate the present invention and should not be construed as limiting it. [Examples]

[0058] [List of Abbreviations] [Abbreviation] [Definition] ---------------------------------------- Adverse events (AEs) AIS (Acute Ischemic Stroke) aICH (Asymptomatic Intracranial Hemorrhage) AUC Area under the blood concentration-time curve aPTT (Activated Partial Thromboplastin Time) BID twice a day ECG (Electrocardiogram) C max Maximum plasma concentration C trough Pre-administration trough temperature CDER Center for Drug Evaluation and Research CT (Computed Tomography) CTA (Computed Tomography Angiography) CCA common carotid artery CL: Systemic clearance of plasma concentration ECA external carotid artery EVT Endovascular Thrombectomy HE Hematoxylin and Eosin HED Human Equivalent Dose ICA internal carotid artery IV Intravenous mRS Modified Rankin Scale Modified treatment for mTICI stroke Maximum recommended starting dose for MRSD MRI / CTP (Magnetic Resonance Imaging or Computed Tomography Perfusion) MRA magnetic resonance angiography MCAO (Middle Cerebral Artery Occlusion) MoCA Montreal Cognitive Assessment NIHSS (National Institute of Health Stroke Severity Scale) NOAEL No-observed-adverse-effect level NSS Neurological Severity Score NDS Neurological Deficit Score PK Pharmacokinetics PPA Pterygopalatine Artery PT Prothrombin Time rtPA Recombinant Tissue Plasminogen Activator RT Room Temperature RBC Red Blood Cell RP2D Recommended Phase II Dose rCBF Regional Cerebral Blood Flow SAE Serious Adverse Event SBP / DBP Systolic Blood Pressure / Diastolic Blood Pressure SD rat Sprague-Dawley rat sICH Symptomatic Intracerebral Hemorrhage PIT Photochemically-induced Thrombotic T 1 / 2 Half-life TEAE Treatment-emergent Adverse Event T max Time to Maximum Plasma Concentration TID Three times a day TT Thrombin Time TTC 2,3,5-Triphenyltetrazolium Chloride V ss Steady-state Volume of Distribution

[0059] [Example 1, Embolic Stroke Model in SD Rats] 10% chloral hydrate solution (400 mg / kg) was injected intraperitoneally into male SD rats (240-320 g) for anesthesia. A longitudinal incision approximately 2 cm long was made on the right side near the center of the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated along the medial edge of the sternocleidomastoid muscle. The incision of the internal carotid artery and the proximal end of the common carotid artery were clipped with non-invasive arterial clips. A small incision was made in the external carotid artery, and its distal end was ligated. The arterial clip at the proximal end of the common carotid artery was removed, and 10 μl of blood was collected. After blood collection, the proximal end of the common carotid artery was clipped again with a non-invasive arterial clip. 10 μl of collected blood was placed in a 1 mL EP vial and kept in RT for 30 minutes to allow the blood to coagulate. It was then transferred to a -20°C refrigerator and kept for 1 hour to form a solid coagulation. After 1 hour, the blood clot was removed, 1 mL of saline solution was added, and the clot was broken into relatively uniform microthrombi using a steel spatula. The microthrombi suspension was then transferred to a 1 mL syringe for use. After releasing the clip in the rat's internal carotid artery, the 1 mL thrombi suspension in the syringe was slowly injected into the proximal end of the rat's external carotid artery, and then injected into the rat's brain through the internal carotid artery. Subsequently, the proximal end of the external carotid artery was ligated, and the arterial clips in the internal and common carotid arteries were released to restore blood flow. The common jugular vein was isolated and injected with normal saline solution or the test compound. The vein was ligated. Three drops of penicillin were applied to the wound. The wound was sutured, and we waited for the animal to wake up.

[0060] The degree of neurological impairment in rats was assessed using the Zealonga method 24 hours after awakening. A score of 0 indicated no signs of neurological impairment, a score of 1 indicated inability to extend the forelimb on the uninjured side, a score of 2 indicated walking toward the uninjured side, a score of 3 indicated walking in a circular motion chasing the tail toward the uninjured side, a score of 4 indicated involuntary gait accompanied by impaired consciousness, and a score of 5 indicated death. The assessment results were statistically analyzed, and a t-test was performed.

[0061] After 24 hours of waking, and after assessing the degree of neurological damage using the Zealonga method, the rats were anesthetized with urethane, immediately decapitated, and the brains were extracted. The brain tissue was kept in a -20°C refrigerator for 2 hours, and five coronal sections of approximately 2 mm each were sequentially cut from the anterior frontal lobe. These sections were then placed in a 2% TTC solution and incubated at 37°C for 30 minutes without light. The color change of the brain sections was observed. Normal brain tissue was stained red by TTC, while ischemic brain tissue appeared white. Photographs were taken with a digital camera and processed with image statistical software to calculate the infarct volume of the brain tissue and the area of ​​normal brain tissue in the coronal sections. The ratio of cerebral infarct volume for each group was statistically calculated, and a t-test was performed.

[0062] [Example 2, Middle Cerebral Artery Occlusion (MCAO) Model] [MCAO surgery for Wistar rats] On day 0, rats were anesthetized and blood was collected from the femoral artery to prepare homologous blood clots. A small arterial incision was made using micro-scissors, and then a sterile PE-50 tube (40-50 mm) was inserted along the artery. Arterial blood collected from PE-50 tubes of this length typically formed fewer than 10 individual blood clots suitable for MCAO. The blood clots in the tubes were kept at room temperature for 2 hours, followed by 22 hours at 4°C.

[0063] On day 1, to prepare the embolization, the blood clot was cut into 32 mm long segments along with the PE-50 tube, and the segments were connected using a 3 ml syringe with a 23 G needle filled with saline. The syringe was pushed to forcefully flush the blood clot from the PE-50 tube into a Petri dish filled with saline. The blood clot was then drawn into the PE-10 tube and forcefully flushed out of the PE-10 tube (drawing from the ends of the clot to avoid folding and twisting), repeated 10-15 times each, to flush out most of the captured red blood cells until the clot no longer released any more RBCs. The PE-10 tube was connected to the modified PE-50 catheter, and the blood clot was transferred to the PE-50 catheter. Next, the modified PE-50 catheter was connected to a 100 μl Hamilton microsyringe. The blood clot was now ready for use.

[0064] On day 1, rats were anesthetized. The carotid sheath was bluntly dissected along the common carotid artery (CCA) until the branches of the ECA (ipsilateral external carotid artery), PPA (pterygopalatine artery), and ICA (internal carotid artery) were exposed. The occipital artery and superior thyroid artery (first and second branches of the ECA) were dissected, and the vessels were removed from the ECA using a bipolar microcoagulant. A laser Doppler flow probe was prepared in the head tube. The CCA, PPA, and ICA were temporarily clamped with clips, and 4-0 silk sutures were loosely applied around the ECA trunk near the branches. The clips were slowly released, and the catheter was advanced further into the ICA until resistance was felt, entering the intracranial segment of the ICA. The total length of the advanced catheter was approximately 19–22 mm from the ECA arterial incision site. The catheter was withdrawn 1–2 mm, and 5–10 μl of saline solution was slowly injected into the blood clot at a rate of 10 μl / min. After blood clot injection, the percentage of regional cerebral blood flow (rCBF) decreased to approximately 20-30%. We waited 30 minutes for the blood clot to stabilize in the ICA. The catheter was withdrawn until the tip reached the ECA / ICA bifurcation. The clip was reapplied to temporarily clamp the CCA and ICA, and then the catheter was withdrawn from the arteriotomy site. The arteriotomy site was ligated by tightening a 4-0 silk suture around the ECA trunk. The clip was removed. We waited another 30 minutes to ensure the animal was stable and there were no bleeding problems. The incision was closed and anesthesia was terminated.

[0065] [Neurological function test] Neurological deficits were assessed 24 hours after MCAO surgery. Neurological function was graded on a scale of 0 to 18, with 0 representing normal function and 18 representing the greatest neurological deficit (see Figure 2).

[0066] [TTC staining and calculation of infarct size and swelling rate] Twenty-four hours after induction of MCAO, rats were anesthetized with 5% isoflurane and perfused cardiacly with physiological saline (150 ml). The rats were decapitated and the brain was collected. The collection dish and matrix were rinsed with PBS before use. After taking photographs, the blood clot was removed. The brain was sliced ​​into four 2 mm coronal slices using brain matrix on ice (cutting the brain into eight slices). The brain slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich) in 1X PBS at room temperature (avoiding light) for 20 minutes, and the results were photographically recorded. Next, the size and extent of the infarct were determined using Image J.

[0067] The caudal portion of all TTC sections was scanned with a digital camera, and the images were saved as JPEGs. Non-ischemic and ischemic hemispheric infarct areas were measured using ImageJ (ImageJ, Bethesda, MD) software. All measurements for the eight infarct areas were calculated with a slice distance of 2 mm. The total infarct size (%) for each brain region was calculated using these measurements. The infarct size (%) of each section was calculated using the formula: 100 × [(Volume of healthy hemisphere - Volume of undamaged ipsilateral hemisphere) / Volume of healthy hemisphere]. The total infarct size (%) was calculated by dividing the sum of the 8 sections by 8. The swelling rate (%) of each brain section was calculated using the formula: 100 × [(volume of infarcted hemisphere - volume of healthy hemisphere) / volume of healthy hemisphere]. The total swelling rate (%) was calculated by dividing the sum of the 8 sections by 8.

[0068] [Measurement of cerebral blood flow (rCBF)] To measure rCBF, the temporalis muscle was dissected from the bone. (At this point, especially when using rtPA, bleeding from the skin and muscle must be controlled with a bipolar coagulator.) The bone was thinned 2 mm posteriorly and 6 mm laterally from the bregma using a drill. A laser Doppler flow probe was placed with strong adhesive, and monitoring of regional cerebral blood flow (rCBF) was initiated.

[0069] [Example 3, Photochemically Induced Thrombotic (PIT) Stroke Model] [Photochemically induced middle cerebral artery thrombosis] Under anesthesia, the left middle cerebral artery (MCA) was occluded by a thrombus formed at the light-irradiated site. The left MCA was exposed via a transorbital approach. A longitudinal incision was made between the left orbit and the external auditory canal. The temporalis muscle was reflexed, and a subtemporal craniotomy was performed without resecting the zygomatic arch. A window approximately 3 mm in diameter was made at the base of the skull. The main trunk of the left MCA was visible through the window. The endothelial cells of the MCA were locally damaged by a photochemical reaction between rose bengal and green illumination (λ540 nm, 600,000 lux). Rose bengal (20 mg / kg) was administered intravenously, and then the MCA was exposed to green light for 10 minutes. During the procedure, the animal was placed on a heated pad set to 38°C.

[0070] [Evaluation of rat neurological deficit score] Neurological deficits were scored once 24 hours (±2 hours) after the onset of thromboembolism, and then once daily for the following 5 days, according to the modified method described by Ederson et al. (Stroke, 1986, 17:472-476). A total score of "0" indicates no deficit, while a maximum score of "15" indicates a severe deficit.

[0071] 1) Flexion of the forelimb The rat was lightly grasped by its tail and suspended about 10 cm above the floor, and the flexion of its forelimbs was observed. 0: Both front limbs point towards the floor. 1: There is a slight difference in the extension of the forelimbs. 2: Wrist flexion, shoulder adduction, and elbow extension are mild. 3: There is a severe posture with full flexion of the wrist and elbow, and adduction with internal rotation of the shoulder.

[0072] 2) Flexion of the hind limbs With the rat at rest, I gently pulled its hind legs, with the soles of its feet facing upwards, towards its tail. 0: There is no difference in the retraction response between the hind limbs. 1: The right hind limb's retraction response is weaker than the left hind limb's retraction response. 2: The right hind leg is abnormally extended, but it can be retracted by touching the sole of the foot with a finger. 3: The right hind leg is abnormally extended and cannot be retracted even when the sole of the foot is touched with a finger.

[0073] 3) Rotational behavior I gently grasped the rat's tail and observed its circling behavior while its forelimbs were on the floor. 0: Walking forward. 1: Normally, I walk forward, but I cannot walk to the left. 2: Although I usually walk to the right, I can walk forward. 3: Walking to the right, but unable to walk forward.

[0074] 4) Lateral displacement With the rats at rest, gentle lateral pressure was applied to the back of their shoulders from one direction or the other. 0: Resists sliding equally in both directions. 1: The resistance to lateral pushing to the right is slightly reduced. 2: Resistance to lateral pushing to the right is significantly reduced. 3: Resistance to lateral pushing to the right decreases significantly, causing the rat to fall onto its back.

[0075] 5) General posture We observed typical postures in rats while they were at rest. 0: The general posture after surgery is no different from that of a normal rat. 1: When viewed from above, the rat's left forelimb and hindlimb are visible. 2: The body tilts slightly. 3: The body is noticeably tilted.

[0076] [Brain for hematoxylin and eosin staining] Each rat brain was cut into six coronal blocks using rat brain matrix (2 mm spacing). The brains were fixed overnight in 4% paraformaldehyde at 3.1–6.2°C, stored in phosphate-buffered saline, and sent for histological processing. Each coronal block was embedded in paraffin. One coronal section was taken from each block and stained with hematoxylin and eosin (HE).

[0077] From HE-stained sections, the infarct area (mm) was measured using OsiriX version 8.0.2 (Pixmeo SARL, Bernex, Switzerland). 2 ) was manually marked. Volume of ischemic infarction (mm 3 The total infarct volume was calculated as the sum of the infarct areas in each coronal section (six coronal sections per brain). Infarct volume was calculated for the cerebral cortex and basal ganglia. The total infarct volume was calculated by adding the infarct volumes of the basal ganglia and cerebral cortex.

[0078] [Example 4: Preclinical efficacy of a single dose of DC009 in a rat embolic stroke model (treatment window 3 hours after stroke onset)] The purpose of this study was to evaluate the efficacy of a single dose of DC009 at 0.007 mg / kg three hours after stroke onset in the sprag-dolly rat embolic stroke model described in Example 1.

[0079] Embolistic occlusion of the middle cerebral artery was induced in male Sprague-Dolly rats by introducing a blood clot into the carotid artery. Rats were treated with a medium (saline), rtPA (10 mg / kg, IV bolus injection), or DC009 (0.007 mg / kg, IV bolus injection) 3 hours after stroke onset (n=12-13 for each treatment group). The Neurological Deficiency Scale (NDS) was assessed 24 hours after drug administration (0 = no signs of loss of neurological function; 1 = inability to extend the left forelimb; 2 = walking towards the left side; 3 = walking in a circular motion chasing the tail towards the left side; 4 = involuntary gait with impaired consciousness; 5 = death). After neurological assessment, the animals were killed and coronary brain sections (2 mm thick) were stained with TTC. Infarct volume was quantified using computer-aided image analysis techniques.

[0080] Table 1 below summarizes the NDS and infarct volume of rats 24 hours after stroke onset.

[0081] [Table 1]

[0082] The DC009 treatment group (0.007 mg / kg) showed improvement in neurological behavior scores and a reduction in cerebral infarct volume compared to the saline treatment group. Therefore, the DC009 formulation (0.007 mg / kg), administered as an IV bolus injection 3 hours after stroke onset, was effective in a preclinical rat embolic stroke model.

[0083] [Example 5: Preclinical efficacy of a single dose of DC009 in a rat embolic stroke model (treatment window 6 hours after stroke onset)] The purpose of this study was to evaluate the efficacy of a single dose of DC009 at 0.007 mg / kg six hours after stroke onset in a male Sprague Dolly (SD) rat embolic stroke model described in Example 1.

[0084] Embolistic occlusion of the middle cerebral artery was induced in male SD rats by introducing a blood clot into the carotid artery. A blood clot made from allogeneic whole blood was slowly injected into the proximal end of the external carotid artery of the rats, and then injected through the internal carotid artery. Rats were treated with a medium (physiological saline), rtPA (3 mg / kg, intravenous bolus injection), or DC009 (0.007 mg / kg, intravenous bolus injection) 6 hours after stroke onset (n=11-12 for each treatment group). Neurological deficit scale was assessed on a scale of 6 points 24 hours after drug administration. After neurological assessment, the animals were killed, and coronary brain sections (2 mm thick) were stained with TTC. Infarct volume was quantified using computer-aided image analysis techniques.

[0085] Table 2 below summarizes the NDS and infarct volume of rats 24 hours after stroke onset.

[0086] [Table 2]

[0087] The DC009 treatment group (0.007 mg / kg) showed improvement in neurological behavior scores and a reduction in cerebral infarct volume compared to the saline treatment group. Therefore, the DC009 formulation administered as a single intravenous bolus injection (0.007 mg / kg) 6 hours after stroke onset was effective in a preclinical rat embolic stroke model.

[0088] [Example 6: Preclinical efficacy of multiple doses of DC009 in a rat embolic stroke model (treatment window 24 hours after stroke onset, QD6)] The purpose of this study was to evaluate the efficacy of repeated administration of DC009 at doses of 0.0007 mg / kg, 0.007 mg / kg, and 0.07 mg / kg for 6 consecutive days in the rat embolic stroke model described in Example 1.

[0089] Embolistic occlusion of the middle cerebral artery was induced in male Sprague-Dolly rats by introducing a blood clot into the carotid artery. Rats were treated with DC009 (0.0007 mg / kg, 0.007 mg / kg, and 0.07 mg / kg, intravenous bolus injection) once daily for a further 5 days, either with a medium (saline) or 24 hours after stroke onset (n=9-10 per treatment group). Neurological deficit scales were assessed in rats once daily 24 hours after drug administration and before drug administration. After the final neurological assessment, animals were killed and coronary brain sections (2 mm thick) were stained with TTC. Infarct volume was quantified using computer-aided image analysis techniques. The infarct volume in rats 7 days after stroke onset is summarized in Table 3 below.

[0090] [Table 3]

[0091] Neurological outcomes were also evaluated, but the interpretation of the data is confusing due to differences in baseline scores between treatment groups (data not shown). In conclusion, repeated IV bolus administration of DC009 (0.007 mg / kg and 0.07 mg / kg) for 6 consecutive days was effective in a preclinical rat model of embolic stroke.

[0092] The data from Examples 4-6 indicate that the effective dose of DC009 in the rat embolic stroke model of Example 1 was 0.007-0.07 mg / kg. The therapeutic effect was also demonstrated 24 hours after stroke onset, indicating that this efficacy has potential for clinical application.

[0093] [Example 7: Preclinical efficacy of a single dose of DC009 in a rat MCAO model (treatment window 3 hours after stroke onset, SD)] The purpose of this study was to evaluate the therapeutic effect of a single dose of DC009 at doses of 0.05 mg / kg and 0.005 mg / kg in rats with embolic middle cerebral artery occlusion (MCAO) as described in Example 2.

[0094] Embolistic occlusion of the middle cerebral artery was induced in male Wistar rats by introducing a blood clot into the carotid artery. A blood clot made from allogeneic whole blood was slowly injected into the internal carotid artery. Rats were treated with either rtPA (10 mg / kg, first 10% of the drug administered intravenously as a bolus injection, followed by the remaining drug infusion over 30 minutes) in saline solution, or DC009 (0.05 mg / kg or 0.005 mg / kg, administered intravenously over 15 minutes) 3 hours after stroke onset (n=8-9 per treatment group). Neurological deficits were assessed before treatment, 3 hours after MCAO surgery, and 24 hours after MCAO surgery. The neurological severity score (NSS) was graded on a scale of 0-18, where 0 represents normal and 18 represents severe impairment. Animals with an NSS of 7 or higher were placed into one of the treatment groups 3 hours post-surgery. Next, neurological function of the treated animals was assessed 24 hours post-surgery. After neurological evaluation, the animals were euthanized, and the brain was stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution to quantify the infarct size. The percentage of ischemia and the rate of brain swelling were calculated using ImageJ software.

[0095] [Table 4]

[0096] In the MCAO model, the NSS after treatment with DC009 at a dose level of 0.05 mg / kg 24 hours after stroke onset was significantly lower than the NSS after treatment with physiological saline. DC009 at dose levels of 0.005 mg / kg and 0.05 mg / kg also significantly reduced infarct size compared to rtPA and physiological saline treatment (Table 4).

[0097] [Example 8: Preclinical efficacy of a single dose of DC009 in a rat MCAO model (treatment window 1 hour after stroke onset, SD)] The purpose of this study was to evaluate the therapeutic effect of a single dose of DC009 at doses of 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, and 0.4 mg / kg in rats with embolic middle cerebral artery occlusion (MCAO) as described in Example 2.

[0098] Embolistic occlusion of the middle cerebral artery was induced in male Wistar rats by introducing a blood clot into the carotid artery. A blood clot made from allogeneic whole blood was slowly injected into the internal carotid artery. Rats were treated with either rtPA (10 mg / kg, first 10% of the drug administered intravenously as a bolus injection, followed by the remaining drug infusion over 30 minutes) in saline solution, or DC009 (0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, and 0.4 mg / kg, administered intravenously over 15 minutes) one hour after stroke onset (n=5-8 per treatment group). Blood flow was measured for 3 hours.

[0099] As shown in Figure 3, the data indicate increased blood flow in rtPA and all DC009 treatment groups compared to the saline treatment group (p<0.0001 vs. saline). Low doses of DC009 at 0.05 mg / kg demonstrated excellent efficacy in restoring obstructed blood flow in rats.

[0100] [Example 9: Preclinical efficacy of multiple administrations of DC009 in a rat MCAO model (treatment window 3 hours after stroke onset, twice daily)] The purpose of this study was to evaluate the therapeutic effect of administering DC009 twice daily for one day in embolic MACO rats as described in Example 2.

[0101] A total of 22 rats were treated with saline (n=8), treated with rtPA at 10 mg / kg 3 hours after MCAO surgery (n=8), and treated with DC009 at 0.025 mg / kg 3 and 6 hours after MCAO surgery (n=6). 24 hours after stroke onset, all animals were sacrificed, and brain tissue was cut into eight sections using a brain slicer matrix. The sections were further stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution, and the percentage of ischemic area and brain swelling rate were calculated using ImageJ software. The results showed that administration of DC009 twice daily resulted in significantly smaller infarct size compared to the saline and rtPA-treated groups (Figure 4).

[0102] [Example 10: Preclinical efficacy of a single dose of DC009 in a rat photochemically induced thrombotic stroke model (treatment window 1 hour and 3 hours after stroke onset)] The purpose of this study was to evaluate the efficacy of administering DC009 at a dose of 0.007 mg / kg once daily in the rat photochemically induced thrombotic (PIT) stroke model described in Example 3.

[0103] Rats were treated with either a medium (physiological saline), rtPA (10 mg / kg), DC009 (0.007 mg / kg, 15-minute IV infusion) 1 hour after stroke onset, or DC009 (0.007 mg / kg, 15-minute IV infusion) 3 hours after stroke onset (n=10 for each treatment group). The Neurological Deficiency Scale (NDS) of the rats was evaluated 24 hours after drug administration.

[0104] Twenty-four hours after PIT surgery, the rats were euthanized, and their brains were treated for TTC staining. The infarct volume is summarized in Table 5 below.

[0105] [Table 5]

[0106] Compared to media treatment, either DC009 or rtPA reduced infarction at different administration times after PIT surgery. Early rtPA treatment (1 hour after PIT surgery) reduced total cerebral infarction volume compared to media treatment. DC009 treatment 1 hour after PIT surgery also significantly reduced total cerebral infarction compared to media treatment.

[0107] [Example 11, Study 101: Safety, tolerability, and pharmacokinetics of DC009 formulation in healthy subjects] The primary objective of the Phase I double-blind, randomized, placebo-controlled trial was to assess the safety, tolerability, and pharmacokinetics of a single-dose dose-escalation study of the DC009 formulation administered by 15-minute intravenous infusion in healthy subjects. The secondary objective was to characterize the pharmacodynamics of the single-dose dose-escalation study, investigate the relationship between pharmacokinetics and pharmacodynamics in the single-dose dose-escalation study, and determine the RP2D of the DC009 formulation.

[0108] The subject population consisted of healthy adult men and women aged 18–65 years. Sixteen healthy subjects completed the study. All subjects received a single dose of either the DC009 formulation or a placebo via 15-minute intravenous infusion.

[0109] Cohort 1 (8 subjects) was administered either the DC009 formulation or a placebo at a dose of 0.05 mg / kg, and Cohort 0 (8 subjects) was administered either the DC009 formulation or a placebo at a dose of 0.025 mg / kg. The dosage and design are summarized in Table 6.

[0110] [Table 6]

[0111] [Primary endpoint] • Nature and severity of adverse events (AEs), and the number of subjects experiencing AEs. Changes from baseline in vital signs, electrocardiogram (ECG) results, laboratory findings, plasmin-antiplasmin complex, euglobulin lysis time, platelet aggregation, and physical examination findings. • Pharmacokinetic parameters: Maximum plasma concentration (C) max ), time to reach maximum (peak) plasma concentration (T max ), area under the plasma concentration-time curve (AUC) from 0 hours to the time of the last quantifiable concentration. 0-t ), area under the plasma concentration-time curve (AUC) from 0 hours to the time of the last quantifiable concentration. 0-t ), half-life (T 1 / 2 ), systemic clearance (CL) and distribution volume of drugs from plasma.

[0112] [Secondary endpoint] • Pharmacodynamic effects of DC009 formulation on blood pressure, thrombin time (TT), prothrombin time (PT), euglobulin dissolution time, and activated partial thromboplastin time (aPTT) up to 48 hours after administration. • Relationship between DC009 plasma concentration and selected PD and safety parameters • RP2D (Recommended dose for Phase II clinical trials)

[0113] 〔result〕 The DC009 formulation was safe and well-tolerated in both administration cohorts. No serious adverse events (SAEs) were reported in either cohort. No AEs were reported in Cohort 0. In Cohort 1, TEAEs were reported in two subjects in the 0.05 mg / kg dose group (headache, contact dermatitis) and in one subject in the placebo group (injection site hematoma).

[0114] Safety data collected in this study included vital signs (heart rate, blood pressure, respiratory rate, body temperature), ECG, clinical chemistry, urinalysis, hematology, coagulation parameters, and fecal occult blood testing. None of the measured safety parameters showed clinically significant findings.

[0115] Maximum plasma concentration (C max ), C max Arrival time (T max ), area under the plasma concentration-time curve (AUC) from 0 hours to infinity 0-∞ ), area under the plasma concentration-time curve (AUC) from 0 hours to the time of the last quantifiable concentration. 0-t ), half-life (T 1 / 2 The pharmacokinetic parameters of the DC009 formulation were evaluated by systemic drug clearance (CL) from plasma and steady-state volume of distribution. To quantify DC009 in plasma, single blood samples were collected before administration, at 5, 10, 15 (end of infusion), 20, 25, 30, and 45 minutes, and at 1, 2, 4, 6, 12, 18, and 24 hours after infusion.

[0116] The data in Table 8 shows that DC009 was rapidly removed from the systemic circulatory system and was only measurable in plasma samples within the first 30 minutes after infusion initiation. 1 / 2 The average value was 0.054 hours, ranging from 0.05 to 0.07 hours. max The effects occurred between 6 and 18 minutes. The 0.05 mg / kg dose group showed higher volume of distribution and clearance compared to the 0.025 mg / kg dose group. The data suggest that DC009 exposure from the DC009 formulation was approximately dose-proportional. AUC and C in the 0.05 mg / kg dose group. max The AUC and C of the 0.025 mg / kg dose group are shown. max It was slightly less than twice as much. The dose-normalized geometric mean ratio of the 0.025 mg / kg dose group to the 0.05 mg / kg dose group was AUC 0-inf 1.40, C max The value was 1.46.

[0117] The data shows the mean C for the 0.05 mg / kg group and the 0.025 mg / kg group. max Both values ​​were lower than the safe plasma exposure limit of 110 ng / mL for DC009. Subjects 001-008(C max :152ng / mL) and subjects 001-042(C max The results from (120 ng / mL) showed that DC009 was approximately 150 ng / mL C max This indicates that it was tolerable in humans.

[0118] [Table 7]

[0119] [Example 12, Study 103: Safety, tolerability, and pharmacokinetics of DC009 formulation in healthy subjects] The objective of this Phase I, double-blind, randomized, placebo-controlled trial was to evaluate the safety, tolerability, and pharmacokinetics of multiple doses of the DC009 formulation in healthy adult subjects.

[0120] [Table 8]

[0121] [Primary endpoint] • Nature and severity of AEs, and the number of subjects with AEs • Changes from baseline in vital signs, electrocardiogram (ECG) results, laboratory findings, and physical examination findings. • Pharmacokinetic parameters: Maximum plasma concentration (C) max ), time to reach maximum (peak) plasma concentration (T max ), area under the plasma concentration-time curve (AUC) from 0 hours to infinity 0-∞ ), area under the plasma concentration-time curve (AUC) from 0 hours to the time of the last quantifiable concentration. 0-t ), half-life (T 1 / 2 ), systemic clearance (CL) and distribution volume of drugs from plasma.

[0122] [Secondary endpoint] • Pharmacodynamic effects of DC009 formulation on blood pressure, prothrombin time (PT), and activated partial thromboplastin time (aPTT) up to 24 hours after administration.

[0123] 〔result〕 Maximum plasma concentration (C max ), C max Arrival time (T max ), area under the plasma concentration-time curve (AUC) from 0 hours to infinity 0-∞ ), area under the plasma concentration-time curve (AUC) from 0 hours to the time of the last quantifiable concentration. 0-t ), half-life (T 1 / 2The pharmacokinetic parameters of the DC009 formulation were evaluated by systemic drug clearance (CL) from plasma and steady-state volume of distribution. For the 0.025 mg / kg group, single blood samples were collected before administration and at 5, 10, 15 (end of infusion), 20, 25, 30, 45, and 60 minutes after the first administration (day 1) and after the fifth administration (day 3) to quantify DC009 in plasma. For the 0.05 mg / kg group, single blood samples were collected before administration and at 10, 20, 30 (end of infusion), 35, 40, 45, 50, 60, and 70 minutes after the first administration (day 1) and after the fifth administration (day 3) to quantify DC009 in plasma.

[0124] [Table 9]

[0125] The DC009 formulation was safe and well-tolerated in human subjects when administered twice daily for 3 days at doses of 0.025 mg / kg (15-minute infusion) and 0.05 mg / kg (30-minute infusion). All reported adverse events (AEs) were defined as Grade I and were considered irrelevant or unlikely to be related. Safety risks are well managed.

[0126] T after single and multiple administrations of 0.025 mg / kg (15-minute infusion) and 0.05 mg / kg (30-minute infusion) max and C max The results were similar. AUC of the 0.05 mg / kg group 0-t and AUC 0-∞ This is the AUC of the 0.025 mg / kg group. 0-t and AUC 0-∞ It was higher than the C of both groups. max All of the samples were below the exposure limit of 110 ng / mL.

[0127] No significant effects on blood pressure (SBP / DBP) or coagulation factors (PT / APTT) were observed after administration at doses of 0.025 mg / kg and 0.05 mg / kg.

[0128] As evidenced by the safety results in healthy subjects of the trial, multiple 3-day intravenous infusions of the DC009 formulation were safe and well-tolerated in healthy subjects. max The exposure levels were far below the exposure limits.

[0129] [Example 13, Study 105: Safety, tolerability, and pharmacokinetics of DC009 formulation in healthy subjects] The objective of this Phase I, double-blind, randomized, placebo-controlled trial was to evaluate the safety, tolerability, pharmacokinetics, and drug interactions of multiple doses of the DC009 formulation in healthy adult subjects.

[0130] [Part A] This study will determine the safety, tolerability, and pharmacokinetics (PK) of DC009 administered intravenously at 3-hour intervals (Q3h) within 1 day in healthy subjects, using a 3-day, 3-daily (TID) regimen. Part A is a double-blind, placebo-controlled study that will examine the safety and PK profile of multiple doses of DC009 in healthy subjects.

[0131] [Table 10]

[0132] • Endpoint - Nature and severity of AEs, and the number of subjects with AEs - Changes from baseline in physical examination, vital signs, ECG assessment, oximetry, coagulation, and clinical laboratory tests. - PK parameters of plasma and urine - Effects on systolic and diastolic blood pressure (SBP / DBP), prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT)

[0133] [Part B] An open-label study evaluating the safety and pharmacokinetics of DC009 when administered concurrently with aspirin, clopidogrel, apixaban, or dabigatran.

[0134] [Table 11]

[0135] • Endpoint - Nature and severity of AEs, and the number of subjects with AEs - Changes from baseline in physical examination, vital signs, ECG assessment, oximetry, coagulation, and clinical laboratory tests. - Plasma PK parameters of DC009 - Plasma PK parameters of aspirin, clopidogrel, apixaban, and dabigatran

[0136] 〔result〕 The DC009 formulation was generally safe and well-tolerated, whether administered alone or in combination with aspirin, clopidogrel, apixaban, or dabigatran.

[0137] The reported TEAEs were mild in severity, none were serious, and none led to the exclusion of subjects from the study. The most frequently reported TEAEs in Part A of the study were menstrual irregularities, abdominal pain, oral herpes, and headache, while in Part B, they were abdominal pain and headache. No TEAEs were associated with abnormal laboratory results. No clinically significant changes were reported in laboratory parameters (hematology, clinical chemistry, and urinalysis), vital signs, ECG, or pulse oximetry results in any part of the study.

[0138] When administered simultaneously with apixaban and dabigatran, the DC009 formulation has limited effects on coagulation. When administered simultaneously with aspirin and clopidogrel, the DC009 formulation has limited effects on COL / ADP and COL / EPI.

[0139] For all populations (N=12), the average C of the DC009 formulation was maxThe concentration was 40.14 ng / mL on day 1 and 48.86 ng / mL on day 3. The peak plasma concentration of the DC009 formulation was reached 10-15 minutes after administration, and the DC009 formulation began to be excreted 15 minutes after administration. In the Chinese population (N=6), the mean C of the DC009 formulation was max The average C of the DC009 formulation was 37.30 ng / mL on day 1 and 40.00 ng / mL on day 3. In the non-Asian population (N=6), max The AUC of the DC009 formulation was 42.98 ng / mL on day 1 and 57.72 ng / mL on day 3. 0-last , C trough , and T max The average value was similar across the Chinese and non-Asian populations.

[0140] [Table 12]

[0141] To investigate whether DC009 administration accumulated in multi-dose regimens, potential dose accumulation effects were evaluated by comparing the PK parameters of the first and last doses in multi-day TID regimens of DC009. Comparisons were made when DC009 was administered alone or concurrently with aspirin, clopidogrel, apixaban, or dabigatran. The PK parameters used for comparison included AUC. 0-t and C max This was included. The results showed that 3 days of TID administration of the DC009 formulation resulted in an average C max The value became 48.86 ng / mL, and the maximum C max The level was 95 ng / mL, indicating that this is a safe level of plasma accumulation in the subjects.

[0142] [Example 14, Study 201: Phase II single-dose study in patients with acute ischemic stroke] The objective of this Phase II double-blind, single-dose, randomized, placebo-controlled trial was to evaluate the safety, tolerability, and potential efficacy of DC009 in patients with acute ischemic stroke (AIS). The safety and potential efficacy outcomes of this Phase II trial will guide the design of future trials in patients with acute ischemic stroke. Eligible patients were randomly assigned in a 2:1 ratio to receive either DC009 or placebo via 15-minute intravenous infusion, resulting in approximately 24 evaluable patients. Eligible patients received a single dose of either DC009 or placebo at a dose of 0.025 mg / kg within 24 hours of stroke symptom onset.

[0143] [Table 13]

[0144] The main selection criteria were that subjects a) be between 18 and 90 years of age at the time of selection, b) have an NIHSS score of 4 to 30, and c) receive a clinical diagnosis of AIS within 24 hours of the onset of stroke symptoms.

[0145] Tissue plasminogen activator (alteplase) is the only stroke treatment approved in the US and, according to the label, must be administered within 3 hours of the onset of stroke symptoms. Approved administration time ranges differ in other countries. In the US, there are no approved drug therapies for acute ischemic stroke administered more than 3 hours after the onset of stroke symptoms; therefore, a placebo-controlled trial is ethically acceptable and necessary to objectively evaluate the drug's efficacy. Initial efficacy will be assessed by evaluating infarct volume, incidence of recurrent stroke, neurological outcomes as measured by the NIHSS, and functional independence as measured by the modified Rankin scale (mRS).

[0146] • Primary endpoint - Clinical worsening defined as the occurrence of symptomatic intracranial hemorrhage (sICH) within 36 hours after administration, or an increase of 4 points or more on the NIHSS scale, confirmed by computed tomography or magnetic resonance imaging.

[0147] • Secondary endpoint - Occurrence of symptomatic intracranial hemorrhage (sICH) and asymptomatic intracranial hemorrhage (aICH) within 7 days after administration. - Death due to intracerebral or other serious bleeding complications within 24 hours, 7 days, 30 days, and 90 days after administration. - Number and severity of adverse events within 90 days after administration - Recurrent stroke within 90 days of administration - Functional outcomes such as mRS - Neurological outcomes such as NIHSS after 30 days - Changes in infarct volume in CT or MRI scans 24 hours and 7 days later - Plasma PK parameters of DC009

[0148] 〔result〕 [Baseline characteristics]

[0149] [Table 14]

[0150] [Safety results] No subjects reported developing sICH during the study within 7 days. No subjects died from intracerebral or other major bleeding complications within 90 days. No subjects died for any reason within 30 days.

[0151] On day 30, one subject (14.3%) in the placebo group showed an increase of 4 points or more on the NIHSS, but none was observed in subjects in the DC009 formulation group.

[0152] Serious TEAEs were reported in 4 subjects (25%) in the DC009 formulation group and 2 subjects (25%) in the placebo group, and all were considered unrelated to or unlikely to be related to the study drug. Of these 6 subjects, 2 subjects (1 in each group) died by day 90 due to serious TEAEs.

[0153] With the exception of one subject in the placebo group, at least one TEAE was reported in all subjects in the safety population. The most common TEAEs reported during the study included constipation, reported in 10 subjects (62.5%) in the DC009 formulation group and 4 subjects (50.0%) in the placebo group, and hypertension, reported in 5 subjects (31.3%) in the DC009 formulation group and 2 subjects (25%) in the placebo group. Most of the TEAEs reported during the study were unrelated to or unlikely to be related to the study drug. No subjects reported a TEAE that was clearly related to the study drug. Most of the TEAEs reported during the study were mild or moderate. No subjects discontinued the study drug due to a TEAE.

[0154] No clinically significant trends were observed in clinical laboratory parameters, vital signs, electrocardiogram (ECG), or neurological examinations.

[0155] [Efficacy Results] The proportion of subjects in the DC009 formulation group who reported a reduction of 4 points or more on day 30 (46.7%) was higher than the proportion in the placebo group (14.3%). Furthermore, the baseline NIHSS score in the DC009 formulation group (10.1 [±7.45]) was higher than that in the placebo group (7.3 [±4.77]), suggesting that the baseline NIHSS scores were unbalanced, and that subjects in the DC009 formulation group started with a worse NIHSS score.

[0156] [Table 15]

[0157] Figure 6 illustrates the change in NIHSS scores from baseline in a subgroup of patients with baseline NIHSS ≥ 6. The mean score change in the DC009 group was -4.3 ± 3 points, indicating an improvement in neurological outcomes, while the mean score change in the placebo group was 3.5 ± 2.1 points, indicating a suppression of neurological outcomes. These results suggest that the efficacy of the DC009 formulation may be observed in a subgroup of patients with more severe neurological defects.

[0158] On day 90, an mRS of 0-1 was reported in 3 subjects (21.4%) in the DC009 group and 1 subject (14.3%) in the placebo group. An mRS of 0-2 was reported in 7 subjects (50.0%) in the DC009 group and 3 subjects (57.2%) in the placebo group. The proportion of subjects reporting an mRS of 0-2 was similar in both treatment groups at every visit. At baseline after stroke onset, an mRS of 4-5 was reported in 13 subjects (81.3%) in the DC009 group and 4 subjects (50.0%) in the placebo group. The mRS results are summarized in Table 16.

[0159] [Table 16]

[0160] The results of Trial 201 indicate that the DC009 formulation is safe and well-tolerated in AIS subjects. The DC009 formulation at a dose of 0.025 mg / kg demonstrated efficacy compared to placebo in the treatment of AIS patients.

[0161] [Example 15, Study 202: Phase II multiple-dose study in patients with acute ischemic stroke] The objective of the Phase II double-blind, multi-dose, randomized, placebo-controlled trial was to evaluate the safety, tolerability, and potential efficacy of the DC009 formulation in AIS subjects.

[0162] [Table 17]

[0163] The main selection criteria were that subjects a) be between 18 and 80 years of age at the time of selection, b) have an NIHSS score of 4 to 25, and c) receive a clinical diagnosis of AIS within 24 hours of stroke onset.

[0164] • Safety endpoints - Occurrence of sICH and aICH after administration - Death due to intracerebral or other serious bleeding complications after administration - Occurrence of death after administration - Number and severity of adverse events within 90 days after administration

[0165] • Primary endpoint - Percentage of patients with mRS scores of 0-2 - Percentage of patients whose NIHSS score decreased by 4 points or more after administration, resulting in an NIHSS score of ≤1.

[0166] • Secondary endpoint - Functional outcomes such as changes in the modified Rankin scale (mRS) during a specific period after administration. - Neurological outcomes such as changes in NIHSS compared to baseline during a specific period after administration. - Daily living activities and quality of life as assessed by the Barthel Index during a specific period after administration.

[0167] [Example 16, Study 203: Phase II single-dose and multiple-dose studies in patients with acute ischemic stroke] This study is a Phase II, double-part, double-blind, randomized, placebo-controlled trial evaluating the safety and efficacy of DC009 in AIS subjects undergoing endovascular thrombectomy (EVT).

[0168] The main selection criteria were that subjects a) be between 18 and 90 years of age at selection, b) have an NIHSS score of ≥6, c) be able to receive EVT treatment within 24 hours of stroke symptom onset, d) receive the study drug before EVT and within 24 hours of stroke symptom onset, e) have confirmed symptomatic intracranial occlusion in the M1 middle cerebral artery (MCA) prior to the M2 branch based on magnetic resonance angiography (MRA) / computed tomography (CTA), and f) have a target mismatch profile with an ischemic core volume ≤70 mL and a mismatch ratio >1.2 as measured by MRI (including perfusion) or CTP.

[0169] [Table 18]

[0170] The objective of Part A of the study was to determine the safety and efficacy of a single intravenous dose of DC009 in patients with acute ischemic stroke (AIS) undergoing endovascular thrombectomy (EVT).

[0171] • Primary endpoint - Clinical deterioration defined as the occurrence of sICH within 24 hours after a single dose, or an increase of 4 or more points on the NIHSS, confirmed by magnetic resonance (MR) / computed tomography (CT) imaging.

[0172] • Secondary endpoint (1) Safety Outcomes - Occurrence and death from sICH and aICH - Number and severity of AEs, and number of subjects with AEs (2) Functional Outcomes - Percentage of subjects who demonstrated an independent functional outcome defined as mRS ≤ 2 after administration. - Percentage of subjects who showed excellent functional outcomes, defined as mRS ≤ 1 after administration. - Change from baseline in the proportion of subjects at each grade of the mRS. - Occurrence of recurrent stroke (3) Neurological outcomes - Percentage of subjects with improved neurological outcomes, defined as a decrease / change in NIHSS from baseline. - Percentage of subjects whose NIHSS score was ≤2 after administration (4) Imaging outcomes - Changes in infarct volume from baseline as measured by MRI / CTP - Changes in hypoperfusion lesions from baseline as measured by cerebral perfusion imaging MRI / CPT - Percentage of subjects in whom hypoperfusion lesions decreased by 90% from baseline using brain perfusion imaging MRI / CTP. - Reconnection rate before and after EVT treatment - Percentage of subjects who showed complete recanalization / substantial angiographic reperfusion, defined as modified treatment (mTICI) ≥ 2b for stroke after EVT.

[0173] [Table 19]

[0174] The objective of Part B of the study was to determine the efficacy and safety of multiple doses of the DC009 formulation in AIS subjects undergoing EVT.

[0175] • Primary endpoint - Percentage of subjects with improved neurological outcomes, defined as a decrease of 4 points or more on the NIHSS from baseline.

[0176] • Secondary endpoint (1) Functional outcome Percentage of subjects who demonstrated an independent functional outcome, defined as mRS ≤ 2, after the first dose. - Percentage of subjects who showed excellent functional outcomes, defined as mRS ≤ 1 after administration. - Change from baseline in the proportion of subjects at each grade of the mRS. (2) Neurological outcomes - Percentage of subjects with improved neurological outcomes, defined as a decrease / change in NIHSS from baseline. - Proportion of subjects with NIHSS ≤ 2 after the first administration (3) Occurrence of recurrent stroke (4) Change in cognitive assessment from baseline measured by Montreal Cognitive Assessment (MoCA) (5) Imaging outcomes - Change in infarct volume from baseline measured by MRI / CTP - Change in hypoperfusion lesions from baseline measured by cerebral perfusion imaging MRI / CPT - Proportion of subjects with a 90% reduction in hypoperfusion lesions from baseline measured by cerebral perfusion imaging MRI / CTP - Recanalization rate before EVT, after EVT, 24 hours after EVT, and 7 days after EVT - Proportion of subjects showing complete recanalization / substantial angiographic reperfusion, defined as mTICI ≥ 2b after EVT (6) Safety outcomes - Occurrence of sICH and aICH - Occurrence of death - Number and severity of AEs, and the number of subjects experiencing AEs

[0177] [Example 17, Study 205: Phase II multiple-dose study in patients with acute ischemic stroke] The objective of the Phase II double-blind randomized placebo-controlled trial was to evaluate the safety and efficacy of multiple doses of DC009 formulation in subjects with acute ischemic stroke (AIS).

[0178]

Table 20

[0179] Main inclusion criteria: the subject must (a) be between 18 and 90 years of age at the time of enrollment, (b) have a NIHSS score of 6 to 25, (c) have a target mismatch profile defined as ischemic core volume ≤ 70 mL, mismatch ratio > 1.2, and mismatch volume ≥ 5 mL confirmed by MRI or CTP.

[0180] · Primary endpoint - Percentage of subjects who experienced a treatment-emergent adverse event (TEAE) occurring during treatment, determined to be possibly or probably related to the DC009 preparation, within 90 days after the first administration

[0181] · Secondary endpoints (1) Functional outcomes - Percentage of subjects who achieved mRS ≤ 2 after the first administration - Percentage of subjects who achieved mRS ≤ 1 after the first administration - Change from baseline in the percentage of subjects in each mRS grade after the first administration (2) Neurological outcomes - Percentage of subjects with NIHSS ≤ 4 relative to baseline - Percentage of subjects with improved neurological outcomes, defined as a decrease of ≥4 points in NIHSS or a decrease of 0 to 1 point in NIHSS from baseline after the first administration - Percentage of subjects with NIHSS ≤ 2 and NIHSS ≤ 1 - Change in NIHSS from baseline after the first administration - Occurrence of recurrent stroke - Change in cognitive assessment from baseline measured by the Montreal Cognitive Assessment (MoCA) (c) Imaging outcomes - Change in infarct volume from baseline measured by MRI / CTP - Change in hypoperfusion lesions from baseline measured by cerebral perfusion MRI / CPT - Percentage of subjects with a 90% reduction in hypoperfusion lesions from baseline measured by cerebral perfusion MRI / CTP (d) Safety outcomes - Occurrence of sICH and aICH, and clinical deterioration defined as an increase of ≥4 points in NIHSS and confirmed by nuclear magnetic resonance (MR) / computed tomography (CT) imaging - Occurrence of death from any cause after the first administration - Number and severity of AEs, and the number of subjects experiencing AEs

[0182] [Example 18: Pharmacokinetic study of DC009 after a single intravenous administration in Sprague Dolly rats] The plasma pharmacokinetics of DC009 were investigated in SD rats after a single IV bolus administration. Male SD rats were administered DC009 by intravenous bolus injection at dose volumes of 1 mL / kg at doses of 0.001, 0.01, 0.1, 1, and 10 mg / kg (n=3 per treatment group).

[0183] [Table 21]

[0184] [Table 22]

[0185] Results from the 0.1-10 mg / kg dose group showed that increasing the dose of DC009 resulted in C in SD rats. max and AUC 0-∞ This suggests that the increase was roughly proportional to the dose.

[0186] [Example 19: Pharmaceutical preparation, dosage, and administration (for Examples 11-14)] The preparation of the DC009 compound is disclosed in Example 63 of U.S. Publication No. 2016-0083423.

[0187] DC009 formulation (lyophilized powder for injection) is an injectable product provided as a preservative-free, sterile, lyophilized material in a glass vial sealed with a butyl rubber stopper and a flip-off aluminum crimp seal. Each vial contains DC009 active pharmaceutical ingredient in the form of a lyophilized cake or powder, equivalent to 20 mg of free base. Placebo is formulated to be identical to DC009 formulation in terms of components, composition, and appearance, but does not contain the active compound.

[0188] In the manufacture of the DC009 formulation, first mannitol is dissolved in water for injection (WFI), and then the hydrochloride of DC009 (C 32 H 51Add N7O8·3HCl. After adjusting the pH to 4.5, dilute the solution to the target weight using WFI, check the osmotic pressure of the solution, and then sterilize by sterile filtration using a 0.2 μm PVDF filter. The pre-formulation contains 10 mg / mL DC009 and 3.8 w / w% mannitol. After filling each vial to within ±2% of the target weight, cover the vial halfway with a 20 mm stopper and place it on a tray for the freeze-drying oven. After the freeze-drying cycle is complete, release the vacuum with nitrogen to create an inert headspace for the finished formulation.

[0189] The DC009 formulation is reconstituted to 4 mg / mL with 0.9% physiological saline before use, further diluted to an appropriate concentration with physiological saline, and administered by IV infusion. In Examples 11-14, an appropriate amount of the formulation was diluted to 90 mL, and the infusion volume was 60 mL.

[0190] In Examples 15-17, the pharmaceutical formulation, dosage, and administration of DC009 are the same as described above, except that the amount of DC009 active pharmaceutical ingredient per formulation vial, the dilution factor of physiological saline, and the injection volume may be changed according to convention.

[0191] The present invention, as well as methods and processes for manufacturing and using it, are described in complete, clear, concise, and precise terms so that those skilled in the art can manufacture and use the present invention. The above describes preferred embodiments of the present invention, and it should be understood that modifications can be made without departing from the scope of the invention as defined in the claims. To specifically point out and clearly claim the subject matter of the present invention, the specification is defined by the following claims.

Claims

1. A therapeutic agent for human acute ischemic stroke comprising DC009, characterized in that DC009 is administered in a dose of approximately 0.025 to 0.05 mg / kg / dose. 【Chemistry 1】 A compound of [this].

2. The therapeutic agent according to claim 1, wherein the dose is approximately 0.025 mg / kg / dose.

3. The therapeutic agent according to claim 1, wherein the dose is approximately 0.05 mg / kg / dose.

4. The aforementioned dose is administered at least once a day, according to any one of claims 1 to 3.

5. The therapeutic agent according to claim 4, wherein the aforementioned dose is administered twice a day.

6. The therapeutic agent according to any one of claims 1 to 3, wherein the aforementioned dose is administered at intervals of approximately 3 to 12 hours.

7. The therapeutic agent according to any one of claims 1 to 3, wherein the aforementioned dose is administered at least once a day for two or three days.

8. The therapeutic agent according to claim 3, wherein the aforementioned dose is administered at least twice a day for three days.

9. The therapeutic agent according to claim 8, wherein the aforementioned dose is administered at intervals of approximately 3 to 12 hours.

10. A therapeutic agent according to any one of claims 1 to 3, administered to a subject immediately or within 1 to 24 hours after the onset of acute ischemic stroke.

11. The therapeutic agent according to any one of claims 1 to 3, administered by intravenous infusion and / or bolus injection.

12. A therapeutic agent according to any one of claims 1 to 3, administered by intravenous infusion.

13. The therapeutic agent according to claim 12, which is administered by intravenous infusion over a period of 5 to 60 minutes.

14. A therapeutic agent according to any one of claims 1 to 3, administered simultaneously with aspirin, clopidogrel, apixaban, or dabigatran.

15. C in the plasma of subjects after administration ｍａｘ The therapeutic agent according to any one of claims 1 to 3, wherein the concentration is less than 200 ng / mL.

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