HK1204965B - Methods for treating a stroke-related sensorimotor impairment using aminopyridines - Google Patents

Abstract

Disclosed herein are methods and compositions related to therapeutic use of aminopyridines in patients with a stroke-related impairment, in particular, in patients with a stroke-related sensorimotor impairment.

Description

Methods for treating stroke-related sensorimotor impairment using aminopyridines

1. Field of the invention

The present invention relates to stroke-related injuries, and in particular, to the treatment of sensorimotor injuries.

2. Background of the invention

Central Nervous System (CNS) injury is a serious health problem. CNS injuries are generally not completely cured, leaving the subject with a degree of permanent dysfunction. The remaining dysfunctions may include motor, sensory, cognitive, emotional, and autonomic abnormalities.

Important types of CNS injury include brain injury following stroke. Stroke is the third leading cause of death in western countries and is the leading cause of disability. Thus, stroke poses a significant socio-economic burden. The pathology of stroke can be ischemic or hemorrhagic, with ischemic being the primary condition of stroke. Ischemic stroke can be caused by clots that form elsewhere in the body and travel to the brain through the blood stream (embolic stroke) or by blood clots that form inside the cerebral arteries (thrombotic stroke). After massive cell death in the center of rapid infarction due to the lack of glucose and oxygen, the infarct zone expands for several days, which is caused by secondary mechanisms such as glutamate excitotoxicity, apoptotic mechanisms and free radical production. Following nerve injury (e.g., an ischemic event), animal and human function may recover days, weeks, and months without any therapeutic intervention. However, often this recovery is only partial, and animals and humans suffer long-term or permanent disabilities, which may include motor, sensory, and cognitive deficits. Motor, sensory, and cognitive impairment due to stroke can have a significant impact on daily activities and quality of life. Stroke survivors often leave permanent neurological deficits, with an estimated 15-30% of stroke survivors becoming life-long disabilities (Roger et al, Circulation 2012; 125:22-e 220).

Risk factors that increase the likelihood of an individual suffering from a stroke are well known. These include, and are not limited to, risk factors that do not vary: advanced age, genetics, race, sex, previous stroke or cardiac history; and risk factors that can be varied, treated and controlled: hypertension, smoking, diabetes, carotid or other artery disease, atrial fibrillation, other heart disease, sickle cell disease, high blood cholesterol, poor diet and physical inactivity and obesity.

To date, direct drug therapy for ischemic stroke has been limited to drugs administered in the acute phase following stroke. The acute phase ranges from the time of onset of the injury (e.g., stroke) to about 6 hours after the injury. Currently, there is no drug therapy for hemorrhagic stroke.

In addition to tissue plasminogen activator ("tPA") and certain mechanical clot retrieval tools suitable for acute use (see Eesa et al, 2011, Expert Rev Neurother.11(8):1125-1139), there is currently no approved therapy in the United states for stroke treatment. After available treatment, patients often maintain a level of dysfunction that may at most slightly improve endogenously over about 60 days and then improve very slightly over a period of up to one year or more. This recovery can only be improved by physical therapy. Unfortunately, many patients leave permanent disability and find the hope for improvement overwhelming.

Treatment of acute stroke is accompanied by restoration of blood flow in the occluded vessel by time-sensitive use of thrombolytic agents (specifically tPA). tPA ruptures blood clots in the arteries supplying blood to the brain, thereby facilitating restoration of blood flow and oxygenation to the brain. However, only a small percentage of stroke patients receive successful tPA therapy: currently, the FDA is only approved for tPA use within 3 hours of the onset of stroke symptoms, and it is only given to about 3% of stroke individuals. Many patients are not candidates for tPA therapy, they do not arrive in the hospital in a timely manner to receive tPA, or they have multiple small infarcts over time and are not treatable with tPA. Furthermore, even those patients successfully treated with tPA often have some degree of brain cell damage.

tPA is a serine protease that converts plasminogen to cytosolic. The cytosol then breaks down the fibrin, which is a component of the clot that occludes the cerebral vessels and causes stroke. Ideally, tPA is administered within the first 3 hours after an occlusion, but may not be administered by some clinicians until 6 hours after the occlusion. Unfortunately, the vast majority of patients who experience stroke cannot reach the hospital in a timely manner to consider such therapy. For those patients who arrive at the hospital within an effective time window, tPA is administered in an attempt to reverse blood flow blockage, restore cerebral oxygenation, and limit the extent of brain structure loss. However, there are some obvious contraindications that limit the use of tPA. Patients receiving tPA after 3 hours have an increased risk of severe bleeding, thereby reducing the effectiveness of tPA. For these reasons, tPA is restricted to administration during the acute phase to achieve any therapeutic efficacy.

To date, there are no other FDA approved drugs for stroke therapy. Current experimental therapies, such as arterial delivery of prourokinase, are being investigated for possible ways of disrupting clots and restoring blood flow. However, the scientific literature has described agents that have proven beneficial for protecting brain substance and restoring function in experimental animal models of stroke. Most of these agents focus on reducing acute cell death, inflammation and apoptosis and therefore must be delivered within hours (some up to 24 hours) after an ischemic event.

Some organizations also recommend aspirin (ASA) when an individual has symptoms of stroke. Some other antiplatelet therapies are used to help reduce the likelihood of stroke.

To date, it has become generally accepted that there is an urgent need for treatment for Stroke (Abe et al, 2008, J Cerebblood Flow Meteb. Jul23, Epub ahead of print; Sun et al, 2008, Stroke Jul10, Epubahead of print; Dohare et al, 2008, Behav Brain Res.193(2): 289-97; Belayev et al, 2001, Stroke32(2): 553-60). With few exceptions, for example, with the exception of glial growth factor 2(GGF2) (see Iaci et al, 2010, Neuropharmacology59: 640-.

After an acute occlusion, there is usually a local brain substance destruction zone, which is surrounded by an ischemic shadow, and which will die within hours if circulation is not restored. In experimental models, the death time of the ischemic shadow can be extended by several hours by neuroprotective agents, such as NMDA antagonists, calcium channel blockers, radical scavengers and trapping agents, anti-apoptotic agents, caspase inhibitors, parp inhibitors, and the like. However, after 24 to 48 hours, the hope of protecting cells from necrotic Death is less, and when apoptotic Death persists for several days, the therapeutic window for anti-apoptotic therapy has not been demonstrated to be broader than that of acute protective therapy (Schulz et al, 1998, Cell Death Differ.5(10): 847-57; Komjati et al, 2004, Int J Mol Med.13(3): 373-82).

Often, permanent sensorimotor deficiency in individuals who survive a stroke is only partially addressed by rehabilitation with physical therapy. In addition, there is little concern about pharmacological intervention to treat the potential for permanent functional deficits in these patients. This may be due to commonly adhered beliefs: it is not possible to replace the nerve cells and circulation lost due to stroke by anything.

Potassium channel blockers

An exemplary property of certain aminopyridines is that they are potassium channel blockers. 4-aminopyridine (4-AP) is an example of an aminopyridine having such potassium channel blocking properties. At the 4-AP plasma concentrations obtained in clinical studies, which are typically <1 micromolar (94ng/mL-1), the potassium channel blocking activity of 4-AP appears to be selective for certain types of these channels. Interestingly, at high concentrations (e.g., millimolar concentrations), 4-AP is a broad spectrum blocker of potassium channels. The clinical neurological effects of 4-AP are consistent with the molecular mechanisms of potassium channel blockade.

In addition to controlled or sustained release formulations, studies have been conducted with 4-aminopyridines (dalvapyridine, aminopyridine) using intravenous (i.v.) administration and Immediate Release (IR) oral capsule formulations. Administration of IR capsules resulted in a rapid and short time peak of 4-aminopyridine in plasma. Early pharmacokinetic studies were conducted using an orally administered Immediate Release (IR) formulation consisting of 4-aminopyridine powder in gelatin-based capsules or oral solution. Administration results in rapidly changing 4-aminopyridine plasma levels that are not well tolerated. Then, sustained release matrix tablets (known as aminopyridine-SR orAcordia Therapeutics, Hawthorne, NY). The sustained release matrix tablets show improved stability and a suitable pharmacokinetic profile for twice daily dose administration. In (for example) us patent 5370879, us patent 5540938; us patent 8007826; and U.S. patent publication No. US2005-0228030 describe sustained release compositions of 4-aminopyridine and related uses of these compositions. For example, suitable formulations, Methods of production, pharmacokinetic characteristics of extended Release Aminopyridine Compositions, and Methods of treating various neurological disorders are also described in U.S. patent No.8007826 entitled "extended Release Aminopyridine Compositions" issued at 30.8.2011 and U.S. patent publication No. 2005-8002230 entitled "Methods of using extended Release Aminopyridine Compositions" (published at 13.2005); the contents of each of the above patents are incorporated herein by reference in their entirety.

The compound 4-aminopyridine is approved by the U.S. food and drug administration as a potassium (K +) channel blocker for the treatment of MS patients. As shown in fig. 1, daltepridine (Dalfampridine) is the U.S. adopted name (USAN) for the chemical 4-aminopyridine (4AP), having a molecular formula of C5H6N2 and a molecular weight of 94.1; this compound was previously known under the USAN name aminopyridine (which remains the international generic name). Throughout the specification, the terms "dalvapyridine", "aminopyridine" and "4-aminopyridine" will be used to refer to the active drug substance.

International publication No. WO 89/09600 discloses the use of a combination of a potassium channel blocker and a choline or choline source to treat certain diseases, including "post-stroke or post-toxic syndrome affecting memory or cognition" (see page 6).

There is a long-felt unmet need in the art for effective treatment of stroke-induced damage, e.g., sensorimotor damage. In particular, such therapy is needed in delayed, non-acute situations beyond hours, days or weeks following acute injury. In addition to treatment in the acute phase, there remains a significant unmet medical need for therapies that can be delivered in the chronic phase that will improve sensory, motor, cognitive, emotional, or autonomic nerve function.

3. Summary of the invention

Provided herein are methods of treating a patient having a stroke by administering a therapeutically effective amount of an aminopyridine or a pharmaceutically acceptable salt thereof. In certain embodiments, disclosed herein is the treatment of stroke-related injury in a patient having a stroke. In particular, disclosed herein are treatments that result in an improvement in one or more sensorimotor impairments associated with or caused by stroke. In particular, the use of aminopyridines in these treatments is disclosed herein. In one embodiment, one or more aminopyridines are used in the methods disclosed herein. In one embodiment, the aminopyridine is a monoaminopyridine or a diaminopyridine. In some embodiments, the monoaminopyridine is 3-aminopyridine or 4-aminopyridine. In one embodiment, the di-aminopyridine is 3, 4-diaminopyridine.

In certain embodiments, the patient treated according to the methods described herein is a mammal. In a preferred embodiment, the patient treated according to the methods described herein is a human. In certain embodiments, a stroke treated according to the present invention is an ischemic stroke. The sub-classes of ischemic stroke that can be treated according to the present invention include without limitation, atherosclerosis (embolism/thrombus), cardiac cerebral embolism (cardiac cerebral embolism stroke), small vessel occlusion (lacunar stroke), and other strokes of established or indeterminate etiology. In certain embodiments, stroke treated according to the present invention is associated with a non-atherosclerotic vascular disease, hypercoagulable state, or hematological disorder. In another embodiment, the stroke treated according to the present invention is a hemorrhagic stroke. The subclasses of hemorrhagic stroke that can be treated according to the invention include, without limitation, subarachnoid hemorrhage and intracerebral hemorrhage.

In some embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in a sustained release composition. In other embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in an immediate release composition. In certain embodiments, the methods according to the present invention comprise administering the aminopyridine or a pharmaceutically acceptable salt thereof once daily, twice daily, or three times daily. In a specific embodiment, the aminopyridine (e.g., 4-AP) or a pharmaceutically acceptable salt thereof is in a sustained release composition and is administered once or twice daily, preferably orally. In another specific embodiment, the aminopyridine (e.g., 4-AP) or a pharmaceutically acceptable salt thereof is in an immediate release composition and is administered three or more times per day, preferably orally.

In a particular embodiment, the aminopyridine itself, rather than a pharmaceutically acceptable salt thereof, is used in any of the methods described herein.

In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, is administered to the patient orally, intravenously, intramuscularly, or subcutaneously. In one embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to the patient orally. In some embodiments, where the aminopyridine or a pharmaceutically acceptable salt thereof is administered orally, it is formulated in the form of a tablet, pill, or capsule. In one embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to the patient intravenously.

In a specific embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, is orally administered to the patient twice daily (i.e., b.i.d.) in a sustained release composition. In certain embodiments, twice daily administration comprises administering aminopyridine or a pharmaceutically acceptable salt thereof every 12 hours. In a specific embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof in a sustained release composition provides a T in humans of about 2 hours to about 6 hours_max. In another specific embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or a salt thereof, is administered orally to the patient once daily in a sustained release composition.

In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in an amount in the range of about 4mg to about 17.5mg, or in the range of 4mg to 17.5mg (e.g., about 4,5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, or 17.5mg), in particular embodiments once or twice daily, and preferably in a sustained release composition. In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in an amount in the range of about 8mg to about 30mg, or in the range of 8mg to 30mg (e.g., about 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30mg), in particular embodiments once or twice daily, and preferably in a sustained release composition. In some embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in an amount in the range of 4mg to 40 mg. In some embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof in the sustained release composition is administered twice per day in an amount between about 5mg to 15mg, 5mg and 10mg, 5mg to 7.5mg, or 7.5mg to 10mg, or the aminopyridine or a pharmaceutically acceptable salt thereof in the sustained release composition is administered twice per day in an amount between about 5mg to 15mg, 5mg and 10mg, 5mg to 7.5mg, or 7.5mg to 10 mg. In one embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof in a sustained release composition is administered (preferably) twice daily at a dose of 5 mg. In another embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof in a sustained release composition is administered (preferably) twice daily at a dose of 10 mg. In another embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof in a sustained release composition is administered (preferably) at a dose of 10mg once daily. In some of these embodiments, the aminopyridine is 4-aminopyridine. In other embodiments, about 8mg to 30mg, 8mg to 20mg, 10mg to 15mg, or between 10mg to 20mg of the aminopyridine or a pharmaceutically acceptable salt thereof is administered once daily (e.g., in a sustained release composition).

Any of the dosages and dosage regimens described in this patent application may be used as therapeutically effective amounts of the aminopyridine or a pharmaceutically acceptable salt thereof used in the methods of the invention.

In some embodiments, the methods of the invention comprise administering an aminopyridine, or a pharmaceutically acceptable salt thereof, in an early chronic stage and/or a stable chronic stage following stroke. In other embodiments, the methods of the invention comprise administering an aminopyridine or a pharmaceutically acceptable salt thereof in the acute phase following a stroke. In certain embodiments, treatment is initiated at the acute phase following stroke and continued at the early chronic phase and/or the stable chronic phase. In some embodiments, treatment is initiated in the early chronic phase and continued in the stable chronic phase. In one embodiment, treatment is initiated during a stable chronic phase. In particular embodiments, treatment is initiated after a stroke when spontaneous recovery of sensorimotor function is expected or observed in the patient. In other specific embodiments, treatment is initiated after a stroke when little measurable spontaneous recovery or improvement in sensorimotor performance is expected or observed in the patient. In some embodiments, treatment is initiated after a stroke when spontaneous recovery of sensorimotor performance is expected or observed in the patient and continued for any period of time outside of this phase (e.g., for 6 months, 1 year, 5 years, 10 years, 20 years outside of this phase, or for a lifetime for the treated patient).

In certain embodiments, treatment according to the invention is 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days after stroke; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, or 12 weeks; or 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12 months later. In certain embodiments, treatment according to the invention lasts for more than 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, or 12 weeks from the start of treatment; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, or 12 months; or 1, 2,3, 4,5, 6, 7,8, 9, or 10 years. In some embodiments, treatment according to the present invention is initiated at any time after a stroke.

In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 4,5, 6, 7,8, 9, 10, 11 or 12 months from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins more than 4,5, 6, 7,8, 9, 10, 11 or 12 months from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins at least 8 weeks from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins at least 4 weeks from the time the patient had a stroke. In another embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins at least 1 week from the time the patient had a stroke. In another embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins 2 to 7 days from the time the patient had a stroke. In some embodiments, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks from the time the patient has a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 4 months from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 6 months from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 8 months from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 12 months from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins more than 4 months from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins more than 6 months from the time the patient had a stroke.

In a specific embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, begins at least 3, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 48 hours from the time the patient has a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 6 hours from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 12 hours from the time the patient had a stroke. In another embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 24 hours from the time the patient had a stroke. In another embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins at least 48 hours from the time the patient had a stroke. In one embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins more than 6 hours from the time the patient had a stroke. In another embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins more than 24 hours from the time the patient had a stroke.

In another embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably a therapeutically effective amount of the aminopyridine or salt, begins immediately after the stroke or within 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, or 48 hours from the time the patient had a stroke. In a specific embodiment, the step of administering the aminopyridine or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount of the aminopyridine or salt, is initiated within 1 day or within 2 days from the time the patient had a stroke. In certain embodiments, treatment according to the invention begins immediately after a stroke or within 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after a stroke and continues for at least 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, or 12 weeks from the beginning of treatment; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, or 12 months; or 1, 2,3, 4,5, 6, 7,8, 9, or 10 years.

In certain embodiments, provided herein are methods of treating one or more sensorimotor impairments in a patient having a stroke. In certain embodiments, provided herein are methods of treating one or more motor or sensory impairments in a patient having a stroke. Sensorimotor impairment treated according to the methods described herein include, without limitation: motor ataxia, general body control impairment (global body control impairment), impairment of coordination or balance, impairment of somatosensory perception, impairment of endurance, impairment of hand function, loss or impairment of fine hand coordination, hyperreflexia, impairment of grip strength, impairment of hand strength, impairment of manual dexterity, muscle weakness, impairment of muscle tone, impairment of range of motion, stiffness, impairment/weakness of strength, tremor, impairment of limb function, impairment of upper limb function, impairment of lower limb muscle strength, impairment of walking (e.g. reduced walking speed or abnormal gait), impairment of speech (e.g. dysarthria), impairment of jaw function, impairment of chewing and impairment of jaw articulation.

In one embodiment, the sensorimotor impairment treated in accordance with the present invention is a walking impairment, such as a decrease in walking speed. In one embodiment, the sensorimotor impairment treated according to the methods described herein is proprioceptive impairment. In other embodiments, the sensorimotor impairment treated according to the invention is systemic control or somatosensory impairment. In another embodiment, the sensorimotor impairment treated according to the invention is an impairment of limb function (e.g., impairment of lower limb function, impairment of lower limb muscle strength, or impairment of upper limb function). In one embodiment, the sensorimotor impairment treated according to the invention is a lower limb functional and/or lower limb muscle impairment. In one embodiment, the sensorimotor impairment treated in accordance with the present invention is an impairment of upper limb function. In one embodiment, the sensorimotor impairment treated according to the invention is a tonic injury of the upper limbs. In another embodiment, the sensorimotor impairment treated according to the invention is impairment of hand function, impairment of fine hand coordination, or impairment of grip strength. In another embodiment, the sensorimotor impairment treated in accordance with the present invention is hand force impairment. In another embodiment, the sensorimotor impairment treated according to the invention is impairment of manual dexterity in a specific embodiment, the sensorimotor impairment treated according to the invention is impairment of oral motor function. In a specific embodiment, the sensorimotor impairment treated according to the invention is speech impairment (e.g., dysphonia, psychogenic apraxia, or dysphonia). In a specific embodiment, the sensorimotor impairment treated in accordance with the present invention is chewing and/or swallowing impairment (e.g., dysphagia). In a specific embodiment, the sensorimotor impairment treated according to the invention is facial paralysis. In a specific embodiment, the sensorimotor impairment treated according to the invention is paralysis of the limbs. In a specific embodiment, the sensorimotor impairment treated according to the invention is hand paralysis. In one embodiment, the sensorimotor impairment treated according to the invention is a balance impairment. In one embodiment, the sensorimotor impairment treated according to the invention is sensory impairment. In some embodiments, the sensorimotor impairment treated according to the invention is visual impairment, such as a sensation of visual function and/or ocular motor impairment.

In particular embodiments, treatment according to the invention is effective to treat one or more symptoms of stroke-associated sensorimotor impairment (e.g., ameliorate, lessen the severity of, or shorten the duration of the symptoms). In some embodiments, treatment according to the invention is effective to treat one or more symptoms of stroke-associated motor impairment (e.g., ameliorate, lessen the severity of, or shorten the duration of the symptoms). In some embodiments, treatment according to the invention is effective to treat (e.g., ameliorate, improve, reduce the severity of or shorten the duration of) one or more symptoms of stroke-related sensory impairment. In some embodiments, treatment according to the present invention restores one or more of the motor, sensory, or sensorimotor impairment caused by stroke. In certain embodiments, methods of assessing the level of motor, sensory, or sensorimotor impairment after (or before and after) repeated administration of aminopyridine are also provided. The method may be any method described herein or known in the art for assessing motor, sensory or sensorimotor function.

In some embodiments, a therapeutically effective amount of an aminopyridine or a pharmaceutically acceptable salt thereof used in the methods described herein is such that a C of at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng/ml is obtained in a human_minssOr average C_minss. In one embodiment, the therapeutically effective amount of the aminopyridine or a pharmaceutically acceptable salt thereof is such that a C in the range of about 12ng/ml to 20ng/ml is obtained in a human_minssOr average C_minss. In some of these embodiments, the aminopyridine is 4-aminopyridine.

In particular embodiments, any of the methods, dosages, and dosage regimens described in the present patent application can be used to treat patients with post-stroke stable motor deficits.

3.1 terminology

In order to provide a clear and consistent understanding of the present specification and claims, the following definitions are provided:

as used herein, the term "about" includes the addition or subtraction of a specified value by 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15% of the specified value. In one embodiment, "about" represents 98-102% of the specified value. In one embodiment, "about" represents 95-105% of the specified value. However, specifically, a "about" value for a particular ng/ml includes plus or minus 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 ng/ml. The meaning of the term "about" will be clear based on the context in which it appears.

As used herein, C if no liquids are mentioned or not otherwise shown in context_minss、C_maxss、C_avssValues are typically referred to as plasma.

The term "improvement" with respect to injury means a change in a parameter in the therapeutic direction. As used herein, "improving" also includes stability of a parameter that would otherwise deteriorate or move in a non-therapeutic direction.

By "pharmaceutically acceptable" is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not prohibited by the regulatory agencies, such as the food and drug administration or the european drug administration, for human or veterinary administration (as the case may be).

With respect to aminopyridines, the term "pharmaceutically acceptable salt" as used herein refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic acids or bases, or organic acids or bases). In one embodiment, the pharmaceutically acceptable salts are prepared from pharmaceutically acceptable non-toxic acids, which may be inorganic or organic. In one embodiment, non-toxic acids include, but are not limited to, inorganic and organic acids such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, vinylsulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. In one embodiment, the non-toxic acid is hydrochloric acid. Suitable pharmaceutically acceptable Salts will be apparent to those skilled in the art and include those described in S.M. Barge et al, "Pharmaceutical Salts," 1977, J.pharm.Sci.66:1-19, which is incorporated herein by reference in its entirety.

As used herein, the term "steady state" refers to a system in which one or more properties do not change over time or "steady state" refers to a system in which one or more properties change over time within a limited range. Generally, steady state is a more general case than dynamic equilibrium. If the system is in steady state, the recently observed behavior of the system will generally persist into the future. In many systems, steady state cannot be achieved until a certain time has elapsed after the system has started or started. This initial condition is typically identified as a transient, drip period, start-up or warm-up period.

As used herein, "stroke" may also be referred to as a "brain attack". A stroke occurs when blood flow to the brain region is blocked, and thus the supply of oxygen and nutrients to brain cells is interrupted, resulting in the death of a portion of the cells. There are two main types of stroke: ischemic and hemorrhagic. Ischemic stroke is caused by an occlusion of blood flow to the brain (ischemia) and is almost always caused by a blood clot that blocks a blood vessel, while hemorrhagic stroke is caused by bleeding from a ruptured blood vessel (hemorrhage).

Other terms and/or abbreviations are provided below:

abbreviations or terms of art	Explanation of the invention
		b.i.d.(bid)	Twice daily
cm	Centimeter
		Cmax	Maximum measured plasma concentration
Cmaxss	Maximum measured plasma concentration at steady state
		Cmin	Minimum measured plasma concentration
Cminss	Minimum measured plasma concentration at steady state
		CNS	Central nervous system
Davalpyridine	Aminopyridine, 4-aminopyridine
		DAP	Bis-aminopyridines
Aminopyridines	Davalpyridine, 4-aminopyridine
		g,kg,mg,μg,ng	G, kg, mg, microgram, ng
GLP	Good laboratory practice
		h,hr	Hour(s)
HPLC	High performance liquid chromatography
		IR	Quick release
IV, i.v. or IV	Intravenous administration of drugs
		K+	Ionic potassium
L,mL	Liter, ml
		LEMMT	Lower limb manual muscle strength test
LCMS,LC/MS/MS	Liquid chromatography/Mass Spectrometry
		MCAO	Middle cerebral artery occlusion
Min	Minute (min)
		mM,μM	Millimole, micromole
MS	Multiple sclerosis
		MSWS-12	12-item multiple sclerosis walk table
NF	National prescription set
		p.o.	Is administered orally
q.d.(qd)	Once a day
		SR	Sustained release
SS	Steady state
		T25FW	Timing 25 feet walk
t.i.d.(tid)	Three times a day
		Tmax	Time to maximum measured plasma concentration after dose administration
USP	United states pharmacopoeia
		WS	Walking speed
3AP or 3-AP	3-aminopyridines
		4AP or 4-AP	4-aminopyridines
3,4DAP or 3,4-DAP	3, 4-di-aminopyridines

4. Description of the drawings

FIG. 1 shows information related to 4-aminopyridine.

Figure 2 is a diagram showing a schedule of dose administration and behavioral testing.

Figure 3 shows the results of the forelimb placement test: the X-axis represents the number of days after a stroke event (i.e., days after MCAO). The Y-axis represents the behavioral score (0 to 12, where 0 is normal function and 12 represents maximum injury). The figure shows the average behavioral score ("D" ═ day) for animals in each test group (i.e. groups 1-3) at D-1, D1, D7, D14, D21, D28, D30, D32, D42, D44, D46, D56, D58, D60 as described in the examples.

Fig. 4A-4D show the results of the hindlimb placement test: the X-axis represents the number of days after a stroke event (i.e. days after MCAO). The Y-axis represents the behavioral score (0 to 6, where 0 is normal function and 6 represents maximum injury). Figure 4A shows the average behavioral scores of animals in each test group (i.e., groups 1-3) at D-1, D1, D7, D14, D21, D28, D30, D32, D42, D44, D46, D56, D58, D60 as described in the examples. Figure 4B shows the average behavioral scores for animals in group 1 at D-1, D1, D7, D14, D21, D28, D30, D32, D42, D44, D46, D56, D58, D60 as described in the examples. Figure 4C shows the average behavioral scores for animals in group 2 at D-1, D1, D7, D14, D21, D28, D30, D32, D42, D44, D46, D56, D58, D60 as described in the examples. Figure 4D shows the average behavioral scores for animals in group 3 at D-1, D1, D7, D14, D21, D28, D30, D32, D42, D44, D46, D56, D58, D60 as described in the examples.

Fig. 5 shows the results of the body swing test: the X-axis represents the number of days after a stroke event (i.e., days after MCAO). The Y-axis represents the behavioral score. The figure shows the average behavioral scores of animals in each test group (i.e., groups 1-3) at D-1, D1, D7, D14, D21, D28, D30, D32, D42, D44, D46, D56, D58, D60 as described in the examples.

Figure 6 shows the average body weight (g) of animals in each test group (i.e. groups 1-3) at a certain number of days (i.e. D-1, D1, D7, D14, D21, D28, D30, D32, D42, D44, D46, D56, D58, D60) after the stroke event (i.e. MCAO).

Fig. 7 shows the results of the cylinder test: the X-axis represents the number of days after a stroke event (i.e., days after MCAO). The Y-axis represents the behavioral score. The graph shows the average behavioral scores of animals in each test group (i.e., groups 1-3) at day-1 (pre-operative), day 7, day 21, day 30, day 32, day 44, day 46, day 58, day 60 as described in the examples.

Figure 8 shows the total movement score of the animals subjected to the cylinder test: the X-axis represents the number of days after a stroke event (i.e., days after MCAO). The Y-axis represents the behavioral score. The graph shows the average behavioral scores for animals in each test group (i.e., groups 1-3) at day-1 (pre-operative), day 7, day 21, day 30, day 32, day 44, day 46, day 58, day 60.

Figure 9 shows the mean infarct volume (%) of animals in groups 1, 2 and 3 after MCAO.

Figure 10 shows the study design of the clinical protocol described in example 16.

Figure 11 shows the results of the forelimb placement test: the X-axis represents the number of days after a stroke event (i.e., days after MCAO). The Y-axis represents the behavioral score (0 to 12, where 0 is normal function and 12 represents maximum injury). The figure shows the average behavioral score ("D" ═ day) of animals in each test group (i.e. vehicle and 4-AP) as described in example 17. Data are presented as mean ± SEM. X ═ p<0.05；

Figure 12 shows the results of the hindlimb placement test: the X-axis represents the number of days after a stroke event (i.e., days after MCAO). The Y-axis represents the behavioral score (0 to 6, where 0 is normal function and 6 represents maximum injury). The figure showsThe average behavioral score ("D" ═ day) of animals in each test group (i.e. vehicle and 4-AP) is shown as described in example 17. Data are presented as mean ± SEM. X ═ p<0.05；

Fig. 13 shows the results of the body swing test: the X-axis represents the number of days after a stroke event (i.e., days after MCAO). The Y-axis represents the behavioral score. The figure shows the average behavioral score ("D" ═ day) of animals in each test group (i.e. vehicle and 4-AP) as described in example 17. Data are presented as mean ± SEM. X ═ p<0.05；

5. Detailed description of the invention

As a sequela of stroke, individuals suffer from neurological damage and thus often leave behind some degree of motor, sensory or sensorimotor damage. Experimental treatments focus on protecting neurons from death during and shortly after ischemia. The FDA has no approved drugs other than time-limited tPA administration to restore function in humans after stroke, TIA, or multiple infarction syndrome.

The present invention provides treatment of patients suffering from stroke, and in some embodiments, treatment of patients suffering from nerve damage due to stroke. In particular, the present invention provides for the treatment of patients who have left a degree of motor, sensory or sensorimotor impairment after a stroke. This damage can range from very slight to severe and incapacitating. This damage may be due to loss of neurons and myelin following ischemic disease periods due to ischemic events or due to inflammation and immune responses. Such damage may be due to loss or damage to neurons or myelin in brain regions that regulate sensorimotor function (e.g., cortical, subcortical, non-cortical) resulting from stroke. For example, such damage may be due to loss or damage to neurons or myelin in the motor region of the cortex, the sensory cortex, or the somatosensory cortex, or in the sensory-motor cortex or in the cortical region responsible for sensorimotor function. In some embodiments, a patient treated according to the methods described herein has ischemic stroke. In one embodiment, a patient treated according to the methods described herein has a stroke in the middle cerebral artery (e.g., caused by occlusion of the middle cerebral artery). In other embodiments, a patient treated according to the methods described herein has a hemorrhagic stroke. In some embodiments, a patient treated according to the methods described herein has a stable or chronic sensorimotor deficiency due to a stroke (such as a hemorrhagic stroke or an ischemic stroke, e.g., a middle cerebral artery stroke). In one embodiment, a patient treated according to the methods described herein does not have multiple sclerosis.

Disclosed herein is the use of aminopyridine (e.g., 4-AP or 3,4-DAP) or a pharmaceutically acceptable salt thereof in the treatment of stroke-associated neuronal loss or damage, particularly in brain regions that modulate sensorimotor function (e.g., cortical, subcortical, non-cortical). In particular, disclosed herein are 4-AP and other aminopyridines or pharmaceutically acceptable salts thereof useful in restoring sensory motor function loss following a stroke event. As described herein, in a preferred embodiment, an aminopyridine (e.g., 4-AP) or a pharmaceutically acceptable salt thereof is administered to an individual who has exhibited a loss of sensorimotor performance associated with or following a stroke event. In certain embodiments, described herein is the use of an aminopyridine (e.g., 4-AP or 3,4-DAP), or a pharmaceutically acceptable salt thereof, in the treatment of stroke-associated neurological impairment. In some of these embodiments, treatment of a patient with an effective amount of an aminopyridine restores or ameliorates neurological impairment due to stroke.

In particular embodiments, the injury treated according to the methods described herein does not affect memory or cognition. In other specific embodiments, the composition comprising an aminopyridine to be administered to a patient according to the invention is free of choline, a source of choline, a precursor of acetylcholine, or a precursor of choline.

Patients or subjects treated by the methods described herein include, but are not limited to, humans and non-human vertebrates, such as wild animals, domestic animals and farm animals. In certain embodiments, the patient treated according to the invention is a mammal, e.g., a human, a cow, a dog, a cat, a goat, a sheep, a horse, or a pig. In a preferred embodiment, the patient is a human.

As described herein, the present inventors have shown that aminopyridines, and in particular, 4-aminopyridine, are effective in restoring neurological function following occlusion of the middle cerebral artery in rats as a model of human ischemic stroke. The studies described in examples 2 and 17 used the rat permanent Middle Cerebral Artery Occlusion (MCAO) model of stroke to evaluate the effect of 4-aminopyridine on the restoration of endogenous sensory motor function to stabilization. As described herein, the present inventors have surprisingly found that 4-aminopyridines are effective for treating sensorimotor impairment following ischemic events. The data obtained by the inventors and described herein show efficacy even when dose administration is initiated in a chronic period following an ischemic event (e.g., 4 or 8 weeks following an ischemic event). Thus, in certain embodiments, described herein are methods of treating stroke-associated sensorimotor impairment in a patient using an aminopyridine, e.g., 4-aminopyridine, or a pharmaceutically acceptable salt thereof. In particular embodiments, the treatment is during the early chronic phase and/or the stable chronic phase following stroke. In some embodiments, described herein is treatment of a patient at 1, 2,3, 4,5, 6, 7,8 weeks after stroke; 1. 2,3, 4,5 and 6 months; or 1, 2,3, 4,5, 10, 15, 20 years or later, or at any time according to the methods disclosed herein. In other embodiments, 1, 2, 4,6, 8, 10, 12, 14, 16, 18, 20, or 22 hours after stroke is described herein; or within or after 1, 2,3, 4,5, 6, 7,8, 9, or 10 days of treatment of the patient according to the methods disclosed herein.

5.1 AminopyrazolePyridines and compositions comprising aminopyridines

The structure of aminopyridines is well known in the art. As shown in U.S. patent No.5952357, mono-or diaminopyridines have the following structure:

wherein x is 1 or 2.

Aminopyridines having the above formula (wherein x is 1) are, for example, 2-aminopyridine, 3-aminopyridine and 4-aminopyridine. Aminopyridine compounds having the above formula (wherein x is 2) are, for example, 2, 3-diaminopyridine; 2, 5-diaminopyridine; 2, 6-diaminopyridine; 3, 4-diaminopyridine; 4, 5-diaminopyridine and 4, 6-diaminopyridine.

In one embodiment, the aminopyridine is a mono-or diaminopyridine-in one embodiment, the monoaminopyridine is a 3-aminopyridine or a 4-aminopyridine. In one embodiment, the di-aminopyridine is 3, 4-diaminopyridine.

As will be appreciated, in any or all of the methods of treatment discussed herein, a pharmaceutically acceptable salt of an aminopyridine may be used instead of or in addition to the aminopyridine. Thus, in a specific embodiment, a pharmaceutically acceptable salt of an aminopyridine (i.e., any pharmaceutically acceptable salt of any of the aminopyridine compounds listed above) is used in the methods provided herein for treating stroke-related injury, e.g., sensorimotor injury. These salts may be prepared, for example, in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. In some embodiments, salts of mono-or di-aminopyridines are used in the methods described herein. In another embodiment, a salt of 3-aminopyridine or 4-aminopyridine is used. In another embodiment, a salt of 3, 4-diaminopyridine is used. In some embodiments, pharmaceutically acceptable salts of aminopyridines are prepared using acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, vinylsulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric, and p-toluenesulfonic acids. In one embodiment, as used herein, 1 equivalent of an aminopyridine may form an acidic salt with less than 1 equivalent or with 1 equivalent or more than 1 equivalent of an acid. In one embodiment, as used herein, an aminopyridine may form a dihydrochloride salt in one embodiment, as used herein, an aminopyridine may form a phosphate salt. For additional descriptions of pharmaceutically acceptable Salts that can be used in the methods described herein, see, e.g., S.M. Barge et al, "Pharmaceutical Salts," 1977, J.pharm.Sci.66:1-19, which is incorporated herein by reference in its entirety.

In a preferred embodiment, the aminopyridine itself, rather than a pharmaceutically acceptable salt thereof, is used in any of the methods of treating stroke-related injuries described herein.

Preferred aminopyridines or pharmaceutically acceptable salts thereof for use according to the invention are compounds that specifically inhibit potassium channels. These compounds preferably exhibit a spectrum or type of selective inhibition of neuronal potassium channels relative to other tissues, similar to the inhibition spectrum of 4-aminopyridine or 3, 4-diaminopyridine, or exhibit a spectrum of selective inhibition of neuronal potassium channels relative to other tissues, similar to the inhibition spectrum common to 3, 4-diaminopyridine and 4-diaminopyridine. Preferred aminopyridines include, without limitation, 4-aminopyridine and 3, 4-diaminopyridine.

The aminopyridine or a pharmaceutically acceptable salt thereof for use according to the invention may be in a slow or immediate release composition. In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof for use according to the invention is formulated for oral, subcutaneous, intramuscular or intravenous administration.

In particular embodiments, the extended release composition of the aminopyridine or a pharmaceutically acceptable salt thereof results in the release of the aminopyridine or a pharmaceutically acceptable salt thereof from the dosage formulation at an extended release rate to maintain therapeutically beneficial blood levels for a period of at least about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours, or more than 18 hours, or more than 24 hours, or more than 30 hours. Preferably, the amount of aminopyridine or a pharmaceutically acceptable salt thereof in an oral dosage formulation according to an embodiment of the invention is established at therapeutically useful plasma or CNS concentrations by t.i.d., b.i.d., or q.d. administration of the pharmaceutical composition. The terms "sustained release" and "extended release" are generally synonymous unless the context clearly dictates otherwise.

In certain embodiments, the therapeutically effective amount of an aminopyridine or a pharmaceutically acceptable salt thereof is between 4mg and 17.5mg (e.g., 4,5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, or 17.5mg), or in the range of 4 to 40mg, and in particular embodiments, it is administered once daily or twice daily, preferably in a sustained release composition. In a specific embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in a sustained release composition. In other specific embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in an immediate release composition. In certain embodiments, the therapeutically effective amount of the 4-aminopyridine or a pharmaceutically acceptable salt thereof is between 4mg and 17.5mg (e.g., 4,5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17 or 17.5mg), or in the range of 4 to 40mg, and in particular embodiments it is administered once daily or twice daily, preferably in a sustained release composition. In one embodiment, twice daily administration is administration of aminopyridine or a pharmaceutically acceptable salt thereof every 12 hours.

In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in an amount ranging from 4mg to 17.5mg (e.g., 4,5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, or 17.5mg), or 4mg to 17.5mg, or 4mg to 40mg, once or twice daily, and preferably in a sustained release composition. In a specific embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in a sustained release composition. In other specific embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered in an immediate release composition. In certain embodiments, an amount of 4-aminopyridine or a pharmaceutically acceptable salt thereof is administered in a range of 4mg to 17.5mg (e.g., 4,5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, or 17.5mg), or 4mg to 17.5mg, or 4mg to 40mg, once or twice daily, and preferably in a sustained release composition. In one embodiment, twice daily administration is administration of aminopyridine or a pharmaceutically acceptable salt thereof every 12 hours.

In particular embodiments of any of the methods of treatment described herein, the aminopyridine (e.g., 4-aminopyridine) is administered twice daily in a sustained release composition in an amount in the range of 4 to 17.5mg (e.g., 4,5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, or 17.5mg) or once daily in a sustained release composition in an amount in the range of 8 to 40mg (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mg).

In one embodiment, a method according to the present invention is provided wherein the therapeutically effective amount of an aminopyridine (e.g., 3, 4-diaminopyridine, 4-aminopyridine, etc.) or a pharmaceutically acceptable salt thereof is 10mg twice daily in a sustained release composition.

In another embodiment, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 5mg twice daily in a sustained release composition. In another embodiment, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 7.5mg twice daily in a sustained release composition. In another embodiment, a method is provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 10mg twice daily in a sustained release composition. In another embodiment, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 12.5mg twice daily in a sustained release composition. In another embodiment, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 15mg twice daily in a sustained release composition. In another embodiment, a method is provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 17.5mg twice daily in a sustained release composition.

In some embodiments, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 20mg once daily in a sustained release composition. In another embodiment, a method is provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is 8, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27.5, 30, or 35 mg once daily in a sustained release composition.

In another embodiment, the method according to the invention comprises administering a therapeutically effective amount of 4-aminopyridine, or a pharmaceutically acceptable salt thereof, in a sustained release composition in a total amount of 8, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27.5, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 milligrams per day. Exemplary embodiments include twice daily administration, wherein 15mg is administered in the morning in a sustained release composition; and 10mg in the sustained release composition at night. Exemplary embodiments include twice daily administration, wherein 12.5 milligrams are administered in the sustained release composition in the morning; and 7.5mg in the sustained release composition at night. Another exemplary embodiment includes administration of a total daily amount in the once daily composition.

In another embodiment, the method according to the invention comprises administering a therapeutically effective amount of 4-aminopyridine, or a pharmaceutically acceptable salt thereof, in an immediate release composition in a total amount of 8, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27.5, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 milligrams per day. In some embodiments, the immediate release composition comprising the aminopyridine or a pharmaceutically acceptable salt thereof is administered 3 or more times per day (e.g., 4,5, or 6 times per day).

In certain embodiments, an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is formulated into Sustained Release (SR) or Extended Release (ER) matrix tablets of varying strengths (e.g., 4 to 40mg, with 5-, 7.5-, 10-, 12.5-, 15-, and 17.5 being preferred in the present invention), one embodiment of 4-aminopyridine-SR being 10mg, which is preferably administered twice daily in doses, other dosage administration regimens are also within the scope of the invention; accordingly, other amounts of active ingredient in a sustained release formulation are also encompassed within the scope of the invention.

In other embodiments, the sustained release formulation used in the methods described herein is 4-aminopyridine-SR or(Acordia Therapeutics, Hawthorne, NY), or in U.S. Pat. No.5,370,879, U.S. Pat. No.5,540,938; us patent 8,007,826; or sustained release compositions of 4-aminopyridine as described in U.S. patent publication US2005-0228030 (the contents of each of the above patents are incorporated herein by reference in their entirety).

In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof may be present in a pharmaceutical composition, such as a tablet, chewable tablet, pill, capsule, microcapsule, solution, suspension, parenteral solution, lozenge, powder, granule, lozenge, syrup, suppository, injection or blister pack. The compositions may be formulated in dosage units (which may be single tablets or capsules or appropriate amounts of liquid) containing a daily dose, a semi-daily dose or an appropriate fraction of a daily dose. In one embodiment, the solution is prepared from a water soluble salt (e.g., a hydrochloride salt). In general, all compositions are prepared according to methods known in pharmaceutical chemistry. Capsules may be prepared by mixing the aminopyridine or a pharmaceutically acceptable salt thereof with a suitable carrier or diluent and filling the appropriate amount of the mixture into capsules. Commonly used carriers and diluents include, but are not limited to, inert powdered materials such as various different types of starch, powdered cellulose, particularly crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, granulated flour and similar edible powders.

Suitable formulations may be prepared by methods which generally employ conventional organic or inorganic additives, such as one or more of the following: excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, or calcium carbonate), binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, or starch), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate, or calcium citrate), lubricants (e.g., magnesium stearate, light anhydrous silicic acid, talc, or sodium lauryl sulfate), flavoring agents (e.g., citric acid, menthol, glycine, or orange-flavored powder), preservatives (e.g., sodium benzoate, sodium bisulfite, methyl hydroxybenzoate, or propyl hydroxybenzoate), stabilizers (e.g., citric acid, sodium citrate, or acetic acid), Suspending agents (e.g., methylcellulose, polyvinylpyrrolidone or aluminum stearate), dispersing agents (e.g., hydroxypropylmethylcellulose), diluents (e.g., water) and base waxes (base wax) (e.g., cocoa butter, white petrolatum or polyethylene glycol). In some embodiments, suitable formulations of aminopyridines or pharmaceutically acceptable salts thereof may be prepared using one, two, three or more of the following additives: colloidal silicon dioxide, hydroxypropyl methylcellulose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, and titanium dioxide.

In one embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof used in the methods of the invention is formulated as a tablet. Tablets may be prepared by direct extrusion, by wet granulation, or by dry granulation. In certain embodiments, their formulations incorporate diluents, binders, lubricants, and disintegrants as well as the compounds. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. In one embodiment, the pharmaceutical composition is lactose-free. Typical tablet binders are substances such as starch, gelatin and sugars (e.g. lactose, fructose, glucose, etc.). Natural and synthetic gums are also suitable, including acacia, alginate, methylcellulose, polyvinylpyrrolidone, and the like. Polyethylene glycol, ethyl cellulose and waxes may also be used as binders. In certain embodiments, the following excipients may be included in the tablet: hydroxypropyl methylcellulose, USP; microcrystalline cellulose, USP; colloidal silica, NF; magnesium stearate, USP; and/or white opadry white.

Pharmaceutical compositions for use in the methods described herein can be as described, for example, in U.S. patent application publication No.2005/0276851, published at 12/15/2005 and U.S. patent application publication No.2005/0228030, published at 10/13/2005, the contents of each of which are incorporated herein by reference in their entirety. The aminopyridines according to the present invention may exist in unsolvated form as well as in solvated form with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to unsolvated forms for the purposes of the present invention.

In another embodiment, a method according to the invention is described, wherein the therapeutically effective amount of an aminopyridineFor example, 4-aminopyridine, or a pharmaceutically acceptable salt thereof, achieves at least or in excess of: 5.6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng/mL of C_minss. In another embodiment, methods are described wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof achieves at least or more than: 5. an average C of 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20ng/ml_minss. In some embodiments, methods are described wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof achieves an average C of about 20ng/ml_minssWhich includes an average lower limit value of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng/ml and an average upper limit value of 20, 21, 22, 23, 24, 25, 26, or 27 ng/ml. In one embodiment, an amount of a drug (e.g., a dose amount) is administered to an individual patient, wherein the dose amount corresponds to a dose that achieves at least or exceeds: 10. 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20ng/ml of average C_minssThe amount of (c). Fluid or tissue level (e.g., C) in a reference population_minss、C_maxss、C_avss) May be referred to as standard values. In another embodiment, methods are described wherein the therapeutically effective amount of the aminopyridine or a pharmaceutically acceptable salt thereof achieves a C ranging between about 5 to 25ng/ml, 10 to 18ng/ml, 13 to 15ng/ml, or 15 to 30ng/ml_minss. In another embodiment, methods are described wherein the therapeutically effective amount of the aminopyridine or a pharmaceutically acceptable salt thereof achieves a C of about 20ng/ml_minss. In another embodiment, methods are described wherein the therapeutically effective amount of the aminopyridine or a pharmaceutically acceptable salt thereof achieves a C of about 20ng/ml_minss(ii) a In certain embodiments, about 20ng/ml of C_minssIncluding lower values of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20g/ml and upper values of 20, 21, 22, 23, 24, 25, 26, or 27 ng/ml.

In an alternative embodiment, a method of treating stroke-related motor, sensory, or sensorimotor impairment in a patient is provided comprising administering to the patient a therapeutically effective amount of a compound of formula (I)Administering a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof to the patient to obtain a C in a range of 5 to 25ng/ml, 10 to 20ng/ml, 15 to 30ng/ml, or 12 to 20ng/ml_minss. In another embodiment, a method of treating stroke-related motor, sensory, or sensorimotor impairment in a patient comprises administering to the patient a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof to achieve a C in a range of at least 12ng/ml to 15ng/ml_minss. In another embodiment, a method of treating stroke-related motor, sensory, or sensorimotor impairment in a patient comprises administering to the patient a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof to achieve a C in a range of at least 13ng/ml to 15ng/ml_minss. In one embodiment, an amount of a drug (e.g., a dose amount) is administered to an individual patient, wherein the dose amount corresponds to a dose that achieves at least or exceeds: 5. an average C of 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20ng/ml_minssThe dosage of (a); plasma levels (e.g., C) in a reference population_minss、C_maxss、C_avss) May be referred to as standard values. In one embodiment, the method according to the invention comprises administering to a patient a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof to obtain a C of at least 11 or 12ng/ml_minss。

In certain embodiments, there is provided a method according to the invention, wherein the therapeutically effective amount of the aminopyridine or a pharmaceutically acceptable salt thereof achieves a T in the patient of about 2 hours to about 6 hours_max. In some of these embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof in a sustained release composition is administered (e.g., once daily, twice daily, or three times daily). In one of these embodiments, the aminopyridine is 4-aminopyridine. The therapeutically effective amount of the 4-aminopyridine can be any amount disclosed herein. In one embodiment, the patient is a human. In some embodiments, once daily administration, twice daily administration, or daily administration in a sustained release compositionAchieving a T of about 2 hours to about 6 hours in a human with three therapeutically effective amounts of 4-aminopyridine_max。

In another embodiment, a method according to the invention is provided, wherein the therapeutically effective amount of an aminopyridine, e.g., 4-aminopyridine, or a pharmaceutically acceptable salt thereof, achieves the following values, or less than the following values: 60. 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20ng/ml of C_maxss. In another embodiment, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof achieves the following values, or less than the following values: 50. 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20ng/ml of average C_maxss. In one embodiment, an amount of a drug (e.g., a dose amount) is administered to an individual patient, wherein the dose amount corresponds to obtaining the following value, or less than the following value, when administered to a standard or reference population: 50. 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20ng/ml of average C_maxssThe amount of (c). Fluid or tissue level (e.g., C) in a reference population_minss、C_maxss、C_avss) May be referred to as standard values. In another embodiment, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof achieves a C in a range of about 15 to 30ng/ml, 25 to 35ng/ml, 25 to 40ng/ml, or 35 to 55ng/ml_maxss. In another embodiment, methods are provided wherein the therapeutically effective amount of the aminopyridine or a pharmaceutically acceptable salt thereof achieves a C of about 30ng/ml_maxss. In another embodiment, methods are provided wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is achieved including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, b,26. A lower limit value of 27, 28, 29 or 30ng/ml and an upper limit value of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60ng/ml_maxss。

In another embodiment, methods are described wherein the therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof achieves the following values or less: 50. 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20ng/ml of average C_maxss. In one embodiment, an amount of a drug (e.g., a dose amount) is administered to an individual patient, wherein the dose amount corresponds to obtaining the following value, or less than the following value, when administered to a standard or reference population: 50. 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20ng/ml of average C_maxssThe dosage of (a); plasma levels (e.g., C) in a reference population_minss、C_maxss、C_avss) May be referred to as standard values.

In another embodiment, a unit dose composition substantially as described herein is used.

The actual dose of the aminopyridine, a pharmaceutically acceptable salt thereof, or a composition comprising the aminopyridine administered to a subject can be determined by physical or physiological factors such as age, sex, body weight, severity of the condition, type of disease to be treated, previous or concurrent therapeutic intervention, the subject's underlying disease, and the route of administration. These factors are readily determined by the skilled artisan. The physician responsible for administration will typically determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject. The dosage may be adjusted by the individual physician if any complications or changes occur in the patient's state.

Dosage unit forms, as used herein, refer to physically discrete units suitable for use as unitary dosages for the subject to be treated; each unit containing a predetermined amount of a therapeutic compound calculated to produce a desired therapeutic effect in association with a desired pharmaceutical carrier. The specification for the dosage unit forms of the invention is dictated by and directly dependent on (a) the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of preparing therapeutic compounds for use in the treatment of the selected condition in a patient. The unit dosage form may be a tablet or blister pack. In certain administration protocols, a patient may use more than one single unit at a time, for example, using two tablets contained in separate blisters of a blister pack.

Optical isomers-diastereoisomers-geometric isomers-tautomers: the compounds described herein may contain asymmetric centers and thus may exist as enantiomers. When the compounds according to the invention have two or more asymmetric centers, they may additionally be present as non-corresponding isomers. The present invention includes all such possible stereoisomers as substantially pure resolved enantiomers, racemic mixtures thereof, and mixtures of non-corresponding isomers. The chemical formula shown has no defined stereochemistry at certain positions. The present invention includes all stereoisomers of these formulae and their pharmaceutically acceptable salts. Diastereomeric pairs of enantiomers may be separated, for example, by fractional crystallization from a suitable solvent, and the enantiomeric pairs thus obtained may be separated into the individual stereoisomers by conventional methods, for example, by using an optically active acid or base as resolving agent, or on a chiral HPLC column. Furthermore, any enantiomer or diastereomer of the compound represented by the general formula may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.

The aminopyridine of the present invention or a pharmaceutically acceptable salt thereof is administered at a therapeutically effective dose sufficient to treat stroke-related damage in a patient. In certain embodiments, the treatment reduces the amount of injury symptoms in the patient by at least about 10%, more preferably 20%, more preferably at least about 40%, more preferably at least about 60%, and more preferably at least about 80% relative to an untreated subject. Preferably, such a percentage change quantification is applied to sensorimotor performance determinations that provide a measure of the results in a continuous linear scale, such as T25FW or the like. Other sensorimotor performance tests will not be expressed as a percentage change, but will be predicted by appropriate statistical comparisons to result in significant changes. These tests include semi-quantitative measurements that assign values to the ability to perform certain skills. In some embodiments, treatment according to the invention results in a statistically significant improvement in stroke-associated sensorimotor impairment as compared to a control (e.g., as measured by the ability of the patient to perform certain tasks or skills). Such a control may be the ability of the patient to perform the task or skill being evaluated prior to the initiation of treatment.

5.2 sensorimotor impairment and results of aminopyridine administration according to the invention

The present invention provides methods of treating neural damage caused by stroke in a mammal, and in particular, methods of treating stroke-associated sensorimotor impairment. In some embodiments, a patient treated according to the methods described herein suffers from (e.g., has been diagnosed with or exhibits one or more symptoms of) sensorimotor impairment. In certain embodiments, a patient treated according to the methods described herein has damage due to neuronal damage (e.g., neuronal loss or demyelination) in the cortical or other region of the brain responsible for or involved in sensory motor function. A preferred embodiment of the present invention relates to a method of treating impaired sensorimotor performance caused by stroke using 4-aminopyridine. Such treatment may be by administration of any of the dosages and dosage administration regimens described in the present application.

Sensorimotor impairment or sensorimotor performance impairment treated according to the present invention includes, without limitation: motor ataxia, global body control impairment (global body control impairment), impairment of coordination or balance, impairment of somatosensory perception, impairment of proprioception, impairment of gait, impairment of responsiveness, impairment of mobility, impairment of endurance, impairment of hand function, loss or impairment of fine hand coordination, hyperreflexia, impairment of hand strength, impairment of manual dexterity, impairment of grip strength, muscle weakness, impairment of muscle tone, impairment of range of motion, rigidity, impairment/weakness of strength, tremor, impairment of limb function, impairment of upper limb function, impairment of lower limb muscle strength, impairment of walking (e.g. reduced walking speed), impairment of speech (e.g. dysarthria), impairment of jaw function, impairment of chewing or impairment of jaw articulation. In one embodiment, the sensorimotor impairment treated according to the methods described herein is proprioceptive impairment. In one embodiment, the sensorimotor impairment treated according to the methods described herein is impaired oral motor function. In particular embodiments, the sensorimotor impairment treated according to the methods described herein is speech impairment (e.g., dysphonia, psychogenic apraxia, or dysphonia), or chewing and/or swallowing impairment (e.g., dysphagia). In one embodiment, the sensorimotor impairment treated according to the methods described herein is visual impairment, such as a sensation of visual function and/or ocular motor impairment. In other specific embodiments, the sensorimotor impairment treated according to the methods described herein is walking speed impairment, manual agility impairment, hand strength impairment, or upper limb rigidity. In some embodiments, the sensorimotor impairment treated according to the methods described herein is impairment of motor and/or sensory function as measured using the Fugl-Meyer evaluation. In specific embodiments, the sensorimotor impairment treated according to the methods described herein is motor function impairment, balance impairment, sensory impairment, or joint function impairment. In a specific embodiment, the sensorimotor impairment treated according to the methods described herein is facial paralysis, limb paralysis, or hand paralysis.

In certain embodiments, the sensorimotor impairment treated according to the present invention includes, but is not limited to, walking impairment, limb functional impairment, lower limb muscle impairment, muscle tone impairment, stiffness, upper limb functional impairment, hand functional impairment, fine hand coordination impairment, grip impairment, balance or coordination impairment, general control impairment, jaw functional impairment, chewing impairment, or jaw occlusion impairment.

In one embodiment, the sensorimotor impairment treated according to the invention is a lower limb functional and/or lower limb muscle impairment. In one embodiment, the sensorimotor impairment treated in accordance with the present invention is impaired motor function of the lower extremities. In one embodiment, the sensorimotor impairment treated in accordance with the present invention is walking impairment (e.g., decreased walking speed). In one embodiment, the sensorimotor impairment treated in accordance with the present invention is an impairment of upper limb function (e.g., upper limb motor function). In one embodiment, the sensorimotor impairment treated in accordance with the present invention is paralysis of the limbs. In one embodiment, the sensorimotor impairment treated in accordance with the present invention is myotonic impairment or spasticity (e.g., upper limb spasticity). In one embodiment, the sensorimotor impairment treated according to the invention is a balance or coordination impairment. In one embodiment, the sensorimotor impairment treated according to the invention is sensory impairment. In one embodiment, the sensorimotor impairment treated in accordance with the present invention is impairment of oral motor function. In particular embodiments, the impairment of oral motor function is impairment of muscle strength of the lips and/or tongue (e.g., in dysphonia). In another specific embodiment, the impairment of motor function of the oral cavity is an impairment of the muscular coordination of the lips and/or tongue (as in psychogenic apraxia). In another specific embodiment, the impaired motor function of the oral cavity is an impairment of the muscular strength involved in breathing. In one embodiment, the sensorimotor impairment treated according to the present invention is speech impairment (e.g., dysphonia, psychogenic apraxia, dysphonia). In some embodiments, the sensorimotor impairment treated according to the invention is sensorimotor abnormalities of the facial, tongue and/or glossopharyngeal muscles. In one embodiment, the sensorimotor impairment treated in accordance with the present invention is chewing and/or swallowing impairment (e.g., dysphagia). In one embodiment, the sensorimotor impairment treated according to the invention is impairment of jaw function or jaw occlusion. In one embodiment, the sensorimotor impairment treated in accordance with the present invention is facial paralysis. In one embodiment, the sensorimotor impairment treated according to the invention is impairment of hand function, impairment of hand coordination (e.g., impairment of fine hand coordination), impairment of grip strength, impairment of manual dexterity, or hand paralysis. In some embodiments, the sensorimotor impairment treated according to the invention is visual impairment or abnormality. The visual impairment treated according to the methods described herein may be a sensory of visual function and/or an impairment of ocular movement. In one embodiment, the visual impairment treated according to the methods described herein is a sensory impairment of visual function. In one embodiment, the ocular damage treated according to the methods described herein is ocular motor impairment of visual function.

In one embodiment, administration of the aminopyridine restores one or more sensorimotor functions. This is manifested or measured as, for example, an improvement in walking ability, balance, standing ability, hand strength, dexterity, reaction ability, an answer to a measure of quality of life accepted in the art, or any other sensory motor function improvement described herein or known in the art.

In certain embodiments, treating a patient by administering an amount of an aminopyridine or a pharmaceutically acceptable salt thereof is effective to ameliorate or prevent stroke-associated sensorimotor impairment. In one embodiment, treating a patient by administering an amount of an aminopyridine or a pharmaceutically acceptable salt thereof is effective to prevent the onset of symptoms of sensorimotor impairment. In other embodiments, treating a patient by administering an amount of an aminopyridine or a pharmaceutically acceptable salt thereof is effective to reduce the symptoms (e.g., reduce the severity) of stroke-associated sensorimotor impairment. In other embodiments, treating a patient by administering an amount of an aminopyridine or a pharmaceutically acceptable salt thereof is effective to reduce the duration of stroke-associated sensorimotor impairment. In particular embodiments, treating a patient by administering an amount of an aminopyridine or a pharmaceutically acceptable salt thereof is effective to eliminate stroke-associated sensorimotor impairment and/or to restore sensorimotor performance impaired by stroke. In certain embodiments, administration of an aminopyridine or a pharmaceutically acceptable salt thereof is effective for restoring sensorimotor function damaged by stroke. In some of these embodiments, the stroke is an ischemic stroke. In one embodiment, the stroke is a middle cerebral artery stroke (e.g., caused by a middle cerebral artery occlusion). In other embodiments, the stroke is a hemorrhagic stroke.

In another embodiment, a method of maintaining an improvement in perceived motor function in a patient, wherein such function is impaired by stroke, is provided comprising: after achieving such an improvement in impaired sensorimotor performance in the patient during the prior administration of 4-aminopyridine, a therapeutically effective amount of an aminopyridine (e.g., 3, 4-diaminopyridine, 4-aminopyridine, etc.) or a pharmaceutically acceptable salt thereof is administered to the patient.

In one embodiment, a method of maintaining an improvement in sensorimotor performance in a patient suffering from stroke-related impairment of such function comprises administering to the patient a therapeutically effective amount of an aminopyridine or a pharmaceutically acceptable salt thereof for an extended period of time. In another embodiment, a method of achieving sustained improvement in a patient suffering from stroke-associated sensorimotor impairment comprises continuously administering to the patient a therapeutically effective amount of an aminopyridine (e.g., 3, 4-diaminopyridine, 4-aminopyridine, etc.) or a pharmaceutically acceptable salt thereof, for an extended period of time.

In particular embodiments, the improvement in patients experiencing stroke-related sensorimotor impairment is at least or exceeds: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months; or 1, 2,3, 4,5, 6, or greater than 5 years of treatment.

Sensorimotor performance can be assessed using any method known in the art, including impaired sensorimotor performance and improved sensorimotor performance. For example, evaluation tests may include, without limitation, timed 25 foot walking for lower limb function (T25FW), 2 minute walking, 6 minute walking (6MW), Box & Block test, 6 point step test, manual muscle strength test, LEMMT for upper limb function, Ashworth score, modified Ashworth scale, grip strength test, 9-hole stick test, finger fine motion, rapid finger alternation, functional system score for sensory function, and nose and heel-to-shin test for motor ataxia (heel-to-shin test). Specifically, T25W may be used to measure walking, LEMMT may be used to measure lower limb muscle strength, and the modified Ashworth scale may be used to measure rigidity. Upper limb function assessment accepted in the art includes, without limitation, performance scales-self-reported measurements, hand-held muscle strength assays, and upper limb indices (UEIs). Other evaluation tests that may be used to measure sensorimotor performance include, but are not limited to, the Berg Balance Scale (BBS), the Kela coordination test, the postural stability test, the timed 10-meter walking test, the shoulder traction test, the grip strength, the maximum isometric contraction force of the knee extensor, the muscle endurance test, the passive straightening and leg-raising test, TEMPA (elderly upper limb performance test), the Jebsen-Taylor hand function test, the arm, shoulder and hand Disability (DASH) questionnaire, and the hand function measurement-36 (MAM-36). Another evaluation test that can be used to measure sensorimotor performance is the Fugl-Meyer evaluation. In some embodiments, the Fugl-Meyer evaluation may be used to measure motor function (e.g., lower limb motor function and/or upper limb motor function), balance, sensation, and/or joint functionality. In a specific embodiment, the Fugl-Meyer evaluation is used to measure lower limb motor function, upper limb motor function and/or sensation. These evaluations can be performed before and after administration of the aminopyridine or a pharmaceutically acceptable salt thereof to the patient according to the methods disclosed herein. For example, sensorimotor function of a patient having stroke-related impairment of aminopyridine can be assessed prior to and/or after administration of the aminopyridine, e.g., at 1, 2,3, 4,5, 6, 7,8, 9, 10 days from the start of treatment according to the methods described herein; 1. 2,3, 4,5, 6, 7,8 weeks; 1. 2,3, 4,5, 6, 7,8, 9, 10 months; or 1, 2,3, 4,5 years old or later.

In particular embodiments, the treatment outcome of stroke-related sensorimotor impairment is determined and detected at any one, two, three, four, five or more or each of the following time points, and/or at a time point later than any one of the following time points: 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days after initiation of treatment with the aminopyridine or a pharmaceutically acceptable salt thereof; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, and 66 months; 5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, and 6.5 years.

5.3 modes of administration of aminopyridines

In some embodiments, the method according to the present invention comprises administering the aminopyridine or a pharmaceutically acceptable salt thereof once daily, twice daily, or three times daily. In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered orally. In other embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered intravenously. In other embodiments, for example, the aminopyridine or a pharmaceutically acceptable salt thereof is administered intramuscularly or subcutaneously.

In certain embodiments, the methods of the present invention comprise administering an aminopyridine or a pharmaceutically acceptable salt thereof during the acute phase following a stroke. The acute phase following a stroke is characterized by continued damage to brain tissue (e.g., enlargement of ischemic lesions) following the stroke. For example, during the acute phase, sustained damage to brain tissue can occur in the ischemic shadow region surrounding the central region where initial damage due to a stroke has occurred. Such damage may include cell death, for example, due to hypoxia. Typically, the acute phase continues from the onset of stroke to about 6 hours after stroke. In some embodiments, treatment according to the present invention comprises administering an aminopyridine or a pharmaceutically acceptable salt thereof to a patient during a post-stroke period in which brain tissue damage persists. In one embodiment, such treatment is during the post-stroke period when the ischemic lesion is still expanding. For example, a patient may be treated according to the invention during an acute phase within 1, 2,3, 4,5 or 6 hours after a stroke.

In some embodiments, the methods of the invention comprise administering an aminopyridine or a pharmaceutically acceptable salt thereof in an early chronic phase after stroke. After the acute phase following stroke, there is a spontaneous recovery phase of neurological function-the early chronic phase-which can last weeks (e.g., up to 4,5, or 6 weeks) in rodent species and months (e.g., up to 4,5, 6, 7,8, 10, 11, or 12 months) in humans. The early chronic phase is characterized by impaired neurological function from stroke, and in particular, by an ongoing, sustained endogenous restoration of sensory motor function. In some embodiments, treatment according to the present invention comprises administering an aminopyridine or a pharmaceutically acceptable salt thereof to a patient during a post-stroke period in which spontaneous or endogenous recovery of neurological function, e.g., sensorimotor function, is observed. For example, the stroke may be 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours after the stroke; 1. 2,3, 4,5, 6, 7,8, 9, 10 days; 1. human patients are treated according to the invention during the early chronic phase at or after 2,3, 4,5, 6 weeks or 1, 2,3 or 4 months and before 4,5, 6, 7,8, 9, 10, 11, 12 months or 1 year after stroke.

In other embodiments, the methods of the invention comprise administering an aminopyridine or a pharmaceutically acceptable salt thereof in a stable chronic period following a stroke. The stable chronic phase following stroke is characterized by little or no measurable spontaneous or endogenous improvement in neurological function (specifically sensory motor function) that is impaired by stroke. Typically, in rodent species, a stable chronic phase is reached 4-6 weeks after stroke; and in human species, 4-8 months after stroke (and, sometimes, 1 year later). The stable chronic phase is usually manifested as stable lifelong disability, and in particular, stable lifelong sensorimotor impairment, which does not improve measurably in the absence of treatment. In certain embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is effective for ameliorating stroke-associated sensorimotor impairment in a patient when administered in a stable chronic phase following a stroke. In some embodiments, treatment according to the invention comprises administering an aminopyridine or a pharmaceutically acceptable salt thereof to a patient during post-stroke with little or no measurable spontaneous or endogenous improvement in neurological function, e.g., sensorimotor function. For example, the stroke may be 4,5, 6, 7,8, 9, 10, 11, 12 months after stroke; 1. human patients are treated according to the invention for a stable chronic period 2,3, 4,5, 7, 10, 12, 15, 20 years or later or at any time.

In one embodiment of the invention, treatment is initiated after the acute phase following a stroke. In one embodiment of the invention, treatment is initiated and continued during the acute phase following stroke. In one embodiment, treatment is initiated after the early chronic phase following stroke. In another embodiment, treatment is initiated in the early chronic phase after stroke and continued thereafter. In another embodiment, treatment is initiated during a stable chronic period following stroke.

The therapeutic benefits of aminopyridines (e.g., 4-aminopyridine) or pharmaceutically acceptable salts thereof can be achieved by administering a therapeutically effective amount to a mammal. In certain embodiments, treatment is performed 1 hour, 2 hours, 6 hours, 8 hours, 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, before, during, after, or after 4 weeks after stroke. In certain embodiments, treatment is performed at or after 3 hours, 6 hours, 8 hours, 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks after stroke. In one embodiment, the treatment is performed 6 hours or later after the stroke. In one embodiment, the treatment is performed 24 hours or later after the stroke. In one embodiment, the treatment is performed 7 days (1 week) or later after the stroke. In one embodiment, treatment is performed 14 days (2 weeks) or later after stroke. In one embodiment, the treatment is performed 1 month or later after the stroke. In one embodiment, the treatment is performed 4 months or later after the stroke. In one embodiment, the treatment is performed 6 months or later after the stroke. In one embodiment, the treatment is performed 8 months or later after the stroke. In one embodiment, the treatment is performed 12 months or later after the stroke. In a specific embodiment, the methods described herein comprise administering an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof to a mammal, wherein administration is within an early chronic period and/or a stable chronic period following an ischemic event in the mammal for at least 2,3, 4, 7, or 10 days after the ischemic event and in a therapeutically effective amount sufficient to promote improvement in sensorimotor function. In certain embodiments, treatment according to the present invention is performed at any time after a stroke. In a specific embodiment, the methods of the invention comprise administering an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof to a mammal in an amount sufficient to promote an improvement in sensorimotor performance, wherein the administration is at any time after stroke.

In certain embodiments, the invention encompasses treatment of a stroke from 1, 2, or 3 and up to and including 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14 days after the stroke; 1 or more than 1 week post stroke, 2 or more than 2 weeks post stroke; 3 or more than 3 weeks after stroke; 4 weeks or more after stroke; 1 month or more than 1 month after stroke; 2 months or more after stroke; 3 months or more after stroke; 4 months or more after stroke; 5 months or more than 5 months after stroke; administration of an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof to a mammal is initiated 6 months or more after a stroke. In certain embodiments, an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof is administered to the patient at or after 1, 2,3, 4,5, 6, 7, or 8 weeks after the stroke.

According to another aspect of the invention, there is provided a method of promoting an improvement in neurological function, e.g., sensorimotor function, in a mammal during a period other than the acute phase following an ischemic event. In particular embodiments, treatment according to the invention may begin within the acute phase, but includes at least one, two, three, four, five, six or more treatments outside the acute phase.

In certain embodiments, the administering step begins within the following time after the stroke: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7,8, 9, 10, 12, 15, 20, 25, or 30 years later. In other embodiments, the administering step begins after the following time after the stroke: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7,8, 9, 10, 12, 15, 20, 25, or 30 years later.

In some embodiments of the invention, a method of treating stroke-related sensorimotor impairment in a patient: comprising administering to said patient a therapeutically effective amount of an aminopyridine (e.g., 3, 4-diaminopyridine, 4-aminopyridine, etc.) or a pharmaceutically acceptable salt thereof, for a period of time. In certain embodiments, the administering step begins within the following time after the stroke event: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7,8, 9, 10, 12, 15, 20, 25, or 30 years later. In other embodiments above, the administering step continues for at least or more than: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, and 66 months; a period of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, or 35 years.

In some embodiments, the treatment regimen (specific dose and frequency of administration, which may be selected from any of the doses and frequencies described herein) is stable over a period of time, e.g., at least 4 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, or at least 6 months.

In a specific embodiment, the invention encompasses a method of effectively treating stroke-associated sensorimotor impairment in a patient during a short-term, initial or non-chronic period comprising administering a therapeutically effective amount of an aminopyridine (e.g., 3, 4-diaminopyridine, 4-aminopyridine, etc.) or a pharmaceutically acceptable salt thereof to the patient. In certain embodiments provided herein, the patient is treated with the aminopyridine or a pharmaceutically acceptable salt thereof for 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 days; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 weeks; 1. a period of 2,3 or 4 months. It is understood that continuation outside of this time period may be possible and still be within the scope of the present invention.

In another embodiment, the invention encompasses a method of effectively treating stroke-related sensorimotor impairment in a patient during the early chronic phase and/or the stable chronic phase, comprising administering to the patient a therapeutically effective amount of an aminopyridine (e.g., 3, 4-diaminopyridine, 4-aminopyridine, etc.) or a pharmaceutically acceptable salt thereof, for an extended period of time. In another embodiment, the invention includes a method of chronically treating stroke-related sensorimotor impairment comprising: administering a therapeutically effective amount of an aminopyridine (e.g., 3, 4-diaminopyridine, 4-aminopyridine, etc.) or a pharmaceutically acceptable salt thereof to the patient for an extended period of time. In some embodiments, the extended period of time is at least or exceeds: 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months; or 1, 2,3, 4,5, 6, 7,8, 9, 10, or more than 10 years.

In certain embodiments, a therapeutically effective amount of an aminopyridine, e.g., 4-aminopyridine, or a pharmaceutically acceptable salt thereof, is administered intravenously during the acute phase following a stroke. In some embodiments, a therapeutically effective amount of an aminopyridine, e.g., 4-aminopyridine, or a pharmaceutically acceptable salt thereof, is administered intravenously within 1, 2,3, 4,5, 6, 7 days or 1, 2,3, 4, 8 weeks after a stroke. Intravenous administration may occur once daily, twice daily, three times daily, once every two days, once every three days, or once weekly. In one embodiment, the patient is treated with a single intravenous administration of an aminopyridine or a pharmaceutically acceptable salt thereof.

In another embodiment, a therapeutically effective amount of an aminopyridine, e.g., 4-aminopyridine, or a pharmaceutically acceptable salt thereof, is administered orally in the acute, early chronic, and/or stable chronic phase following a stroke. In a specific embodiment, a therapeutically effective amount of an aminopyridine, e.g., 4-aminopyridine, or a pharmaceutically acceptable salt thereof, is administered orally only in the early chronic phase and/or the stable chronic phase following a stroke. Oral administration may occur once daily, twice daily, three times daily, or more than three times daily in an immediate release composition or in a sustained release composition.

Administration of the aminopyridine compounds can be accomplished by a variety of techniques as described herein. The compound may be administered, for example, by administering the compound into or onto a target tissue; administration of an aminopyridine or a pharmaceutically acceptable salt thereof according to the invention is carried out by, for example, intravenous (e.g., parenteral) or oral (e.g., enteral) or topical (e.g., transdermal, patch, suppository) administration or inhalation (e.g., transmucosal) whereby the compound reaches the target tissue to provide the compound systemically to the patient. Administration of the aminopyridine or a pharmaceutically acceptable salt thereof to the patient can be carried out by the patient himself or by a care-giver, such as a medical professional; including swallowing in a patient or application to a patient, etc., where the compound may exert its effect.

In one embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof is administered topically, i.e., directly by a non-systemic route at or near the site of the affliction, condition or sensation of pain.

In certain embodiments, the patient is treated intravenously with an aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof at a dose of 0.01 to 1.0mg/kg once daily, twice daily, once every other day, or once weekly starting from 1, 2,3, 6, 7,8, 9, 10, 11, 12, 14, 16, 18, 20, 22 hours or 1, 2,3, 4,5, 6, or 7 days after the stroke for more than 1, 2,3, 4,5, 6, 7,8, 9, or 10 days, more than 1, 2,3, 4,5, 6, 7, or 8 weeks, or more than 1, 2,3, 4,5, 6, 7, or 8 months (or between 1 day and 5 days, between 2 days and 10 days, between 10 days and 1 month, between 10 days and 6 months, or between 10 days and 1 year).

Alternatively, the patient is orally treated with an amount of aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof once daily or twice daily for more than 5, 10, 15, 20 days, more than 1, 2,3, 5, 6, or 7 days after stroke (or after 1, 2,3, 4,5, 6, or 7 days), or after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks, or after 1 month, 2 months, 3 months, 4 months, 6 months, 8 months, 10 months, or 12 months, or after stroke, the patient is treated with an amount of aminopyridine (e.g., 4-aminopyridine) or a pharmaceutically acceptable salt thereof ranging from 4mg to 17.5mg (e.g., 4,5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17mg) once daily or twice daily for more than 5, 10, 15, 20 days, more than 1, 2,3, 4,5, 6, 7,8 weeks, 3,4, or 5 days after stroke, or 1, 2 days, 6. 9 months, or more than 1, 2,3, 4,5, 10, 15, 20 years (or between 10 days and 3 months, between 10 days and 6 months, between 10 days and 1 year, between 3 months and 1 year, between 6 months and 5 years, or between 1 year and 50 years). In some embodiments, the patient is treated with 5mg, 7.5mg, 10mg, or 12.5mg 4-aminopyridine twice daily beginning 4 weeks after the stroke (or 4 weeks after the stroke). In other embodiments, treatment of the patient with 5mg, 7.5mg, 10mg, or 12.5mg 4-aminopyridine twice daily is initiated 4 months after the stroke (or 4 months after the stroke). In other embodiments, the patient is initially treated with 8mg, 10mg, 12mg, 12.5mg, 15mg, 20mg or 25mg of 4-aminopyridine once daily at 4,5, 6, 7,8 weeks, or 3,4, 5, 6, 7 or 8 months or later after the stroke.

5.4 combination therapy

The compositions and methods described herein may be used in the context of some therapeutic or prophylactic applications. To improve the efficacy of treatment with aminopyridines or to increase the protection of another therapy (the second therapy), it may be desirable to combine these compositions and methods with other agents and methods that are effective in the treatment of stroke-related diseases and pathologies (e.g., sensorimotor impairment).

Thus, in particular embodiments, an aminopyridine or a pharmaceutically acceptable salt thereof may be combined with one or more other agents and/or physical or occupational therapy for the treatment of stroke-related injury (e.g., sensorimotor injury). In some embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to the patient together with or sequentially with one or more other drugs or therapies. For example, the aminopyridine or a pharmaceutically acceptable salt thereof may be administered to the patient at the same time, before, or after administration of another drug effective for stroke-related injuries. Such other drugs may be, for example, cholinesterase inhibitors, such as donepezil, rivastigmine or galantamine, or immunomodulators, such as interferons. In a specific embodiment, the combination of the aminopyridine or a pharmaceutically acceptable salt thereof and one, two or more other drugs is a fixed dose combination. For example, the aminopyridine or a pharmaceutically acceptable salt thereof and one or more other drugs (such as any of those described above) can be formulated as a composition, such as a pill, tablet or capsule. In other embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to a patient having a stroke along with (e.g., simultaneously, prior to, or after) physical therapy, occupational therapy, or language correction, or the like. In some embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to a patient using an orthopedic brace, standing stand, or other orthopedic device, such as a walking aid (walking walker), or a communication aid, such as a computer with an attached speech synthesizer. In particular embodiments, the aminopyridine (or a salt thereof) and the other drug or therapy are administered at the same physician's follow-up, or within 1, 2,3, 4,5, 6, or 12 hours or within 1, 2,3, 4,5, 6, or 7 days of each other.

Various combinations may be used; for example, the aminopyridine or a pharmaceutically acceptable salt thereof is "a" and the second therapy (e.g., cholinesterase inhibitors such as donepezil, rivastigmine and galantamine, and immunomodulators such as interferons and the like) is "B", a non-limiting combination cycle comprising:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

administration of the compositions of the invention to a subject will be in accordance with the general procedures for administration described herein, and will also follow the general procedures for administration of the particular second therapy, taking into account the toxicity, if any, of the treatment. It is expected that the treatment cycle will repeat as necessary. It is also contemplated that a variety of standard therapies may be used in conjunction with the therapy.

In some embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to the patient with occupational or physical therapy. In other embodiments, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to the patient after the patient has undergone occupational or physical therapy following a stroke. In another embodiment, the aminopyridine or a pharmaceutically acceptable salt thereof is administered to a patient who has not undergone occupational or physical therapy. In one embodiment, patients treated according to the methods described herein do not receive occupational therapy or physical therapy together. In another embodiment, a patient treated according to the methods described herein is not undergoing occupational or physical therapy following a stroke. In certain embodiments, the treatment according to the invention (with or without occupational therapy or physical therapy) is more effective than occupational therapy or physical therapy alone.

5.5 kits

Kits include exemplary embodiments of the invention. The kit may include an outer receptacle or container configured to hold one or more inner receptacles/containers, vessels, and/or instructions. A vessel according to the invention may comprise an item for administering a drug, such as a patch, an inhalation device, a liquid container cup, a syringe or a needle. Compositions containing an aminopyridine or a pharmaceutically acceptable salt thereof may be contained within a reservoir of the present invention. The reservoirs of the present invention may contain a sufficient amount of an aminopyridine or a pharmaceutically acceptable salt thereof for multiple doses, or may be in unit or single dose form. In certain embodiments, the kit comprises a composition comprising an aminopyridine or a pharmaceutically acceptable salt thereof in the form of a tablet, a pill, a blister pack, or a capsule.

The kits of the invention generally include instructions for administration according to the invention. The instructions may include treating one or more of the following diseases: motor ataxia, general body control impairment (global body control impairment), impairment of coordination or balance, impairment of somatosensory perception, impairment of endurance, impairment of hand function, loss or impairment of fine hand coordination, hyperreflexia, impairment of grip strength, muscle weakness, impairment of muscle tone, impairment of range of motion, stiffness, impairment/weakness of strength, tremor, impairment of limb function, impairment of upper limb function, impairment of lower limb muscle strength, impairment of walking (e.g. reduced walking speed), dysphonia, impairment of jaw function, impairment of chewing or impairment of jaw articulation. Any administration forms described or supported herein may form part of the specification.

In one embodiment, the instructions show that the aminopyridine or a pharmaceutically acceptable salt thereof is administered twice daily. In one embodiment, the instructions show that the aminopyridine or a pharmaceutically acceptable salt thereof is administered once daily. In one embodiment, the instructions show that the composition comprising an aminopyridine or a pharmaceutically acceptable salt thereof is administered once or more than once during the acute phase following a stroke. In one embodiment, the instructions show that the composition is administered one or more times during the early chronic stage and/or the stable chronic stage following a stroke.

The instructions may be affixed to any container/receptacle of the present invention. In one embodiment, the instructions show administering an aminopyridine or a pharmaceutically acceptable salt thereof to achieve or achieve a therapeutic range according to the invention. The instructions may be affixed to any of the containers/receptacles of the present invention or may be a separate sheet of paper within the container or receptacle of the present invention. Alternatively, the instructions may be printed, embossed, or formed as part of the reservoir of the present invention. Alternatively, the instructions may be printed onto the material and the material packaged in the receptacle or container of the kit of the invention. In one embodiment, the kit has an outer receptacle, such as a box, with a container, such as a bottle; and instructions are provided on and/or within the outer receptacle and/or the bottle. The kit may also include instructions for using the kit components and using any other reagents not contained within the kit; it is contemplated that these reagents are embodiments of the kits of the present invention. According to the present invention, the kit is not limited to the specific items identified above and may include any agent used directly or indirectly in a therapeutic search.

5.6 other embodiments:

embodiments of the invention include methods of effectively treating stroke-related sensorimotor impairment in a patient over an extended or prolonged or delayed or protracted or sustained period of time; this is also referred to as "permanent" treatment or "permanent" treatment method; this is also referred to as "continuous" treatment or "continuous" treatment methods. Another embodiment of the invention is directed to a method of maintaining an improvement in stroke-related sensorimotor impairment in a patient comprising administering to the patient a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) during an adjacent or consecutive or previous administration of the aminopyridine after previously achieving an improvement in stroke-related sensorimotor impairment in the patient. Any of these methods comprise administering to the patient an extended, prolonged, protracted, sustained, or prolonged (as used herein, extended, prolonged, protracted, sustained, or prolonged is synonymous unless the context clearly dictates otherwise) period of a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine). In certain embodiments, the extended, delayed, protracted, or chronic or sustained period of time is at least or exceeds: 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months; or 1, 2,3, 4,5, 6 years or more than 5 years. In certain embodiments, the extended, prolonged, protracted, chronic, or sustained period of time is the lifetime of the patient. These methods may also include administering a therapeutic level (e.g., C) according to the invention_minssOr average C_minss) Or range (e.g. C)_minssRange or average C_minssReference range of values) is administered.

In one embodiment, an amount of a drug (e.g., a dose amount) is administered to an individual patient, wherein the dose amount corresponds to a dose that achieves at least or exceeds: 6.7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20ng/ml of average C_minssThe dosage of (a).

In certain embodiments, the therapeutically effective amount of 4-aminopyridine is 10mg in a sustained release composition administered twice daily. The method of administration may also include administering a therapeutic level (e.g., C) according to the invention_minss) Or range (e.g. C)_minssRange) 4-aminopyridine.

Another embodiment of the invention relates to a method of maintaining an improvement in sensorimotor performance, e.g., general control, coordination, balance, somatosensory, stamina, hand function, fine hand coordination, grip strength, muscle tone, range of motion, strength, limb function, upper limb function, lower limb muscle strength, walking (e.g., walking speed), dysphonia, jaw function, chewing, or jaw occlusion, in a patient having a stroke-related impairment of one of these sensorimotor functions, comprising administering to the patient a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) over an extended period of time. In certain embodiments, the extended, prolonged, protracted, sustained, or extended period of time is at least or exceeds: 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months; or 1, 2,3, 4,5, 6 years or more than 5 years. In certain embodiments, the extended, prolonged, protracted, chronic, or sustained period of time is the lifetime of the patient. Such maintenance may be relatively stable, wherein there is a substantially uniform percent improvement relative to a reference or standard population, or may be relatively different, wherein there is a fluctuation in percent improvement relative to a reference or standard population; when the maintenance is relatively altered, this may include a worse time period than is possible for the reference or standard population of subject patients.

Other embodiments of the invention relate to methods of achieving a sustained or relatively sustained improvement in any one or more signs or symptoms of stroke, such as any one or more sensorimotor impairments caused by or associated with stroke, comprising continuing to administer a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) to the patient for an extended period of time. Relative to a control or standard amount or value, it is understood that there is sometimes a progressive decline in sensorimotor performance in the patient following stroke, and an improvement or relative improvement may be suitably considered in terms of the decline in function that accompanies the intrinsic development of stroke-related sensorimotor pathology. In certain embodiments, the sustained improvement occurs for an extended period of time, such as at least or exceeding: 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months; or 1, 2,3, 4,5, 6 years or more than 5 years. In certain embodiments, the extended period of time is the lifetime of the patient. In certain embodiments, the therapeutically effective amount of 4-aminopyridine is 10mg in a sustained release composition. In certain embodiments, the sustained release composition may be administered twice daily. In certain embodiments, the sustained release composition may be administered once daily. These methods may also include administering a therapeutic level (e.g., C) according to the invention_minss) Or range (e.g. C)_minssRange) is administered. This sustained improvement may be a relative increase, where there is a growing increase in percent improvement relative to the reference or standard population, or the improvement may be a relative difference, where there is a fluctuation in percent improvement relative to the reference or standard population, such that there is a greater propensity than the standard group does; when the improvement is relatively altered, this may include a worse time period than is possible for the reference or standard population of subject patients.

In certain embodiments, a therapeutically effective amount of an aminopyridine (e.g., 4-aminopyridine) is a stable or constant or consistent or fixed or unmodified dosage administration regimen comprising a therapeutically effective amount of the aminopyridine administered in a uniform manner (e.g., in a specific number of milligrams or a specific number of milligrams per day, e.g., in the morning the dosage may be higher and in the evening the dosage may be lower, or vice versa) and on a uniform schedule (e.g., twice per day), wherein the amount or schedule of the dosage in the stable or constant or consistent or fixed dosage administration regimen is unchanged. As used herein, the terms "stable" or "constant" or "consistent" or "unchanged" or "immobile" or "unchanged" are synonymous, unless the context clearly dictates otherwise. It is understood that occasional patient non-compliance or deviations from a stable, constant, consistent, unchanging, motionless or unchanging course of treatment, for example, are within the definition of such treatment. In certain embodiments, titration (whether increase or decrease) of a dose (e.g., milligram amount) of aminopyridine does not occur during the entire stable dose administration regimen.

Embodiments of the invention also relate to methods of treating or ameliorating stroke-associated sensorimotor impairment in a patient comprising administering to the patient an amount or range of 4-aminopyridine such that a minimum steady-state concentration (C) in a range of at least 5ng/ml to 20ng/ml, 10ng/ml to 20ng/ml, or 12ng/ml to 20ng/ml is obtained_minss) Or obtaining C in the range of 20ng/ml_minss. Embodiments of the invention also relate to methods of treating or ameliorating stroke-associated sensorimotor impairment in a patient comprising administering to the patient an amount or range of 4-aminopyridine such that a mean minimum steady-state concentration (mean C) in a range of at least 7ng/ml to 20ng/ml or 12ng/ml to 20ng/ml is obtained_minss) Or obtaining an average C in the range of 20ng/ml_minss. In certain embodiments, C in the range of 20ng/ml_minssAbout 20ng/ml of C is realized_minss. In other embodiments, a C of about 20ng/ml is obtained_minss(ii) a In certain embodiments, C in the range of 20ng/ml_minssIncluding lower values of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng/ml and upper values of 20, 21, 22, 23, 24, 25, 26, or 27 ng/ml. In certain embodimentsObtaining a C in the range of at least 12ng/ml to 15ng/ml_minss. In certain embodiments, a C in the range of at least 13ng/ml to 15ng/ml is obtained_minss. In certain embodiments, a C in the range of at least 15ng/ml to 25ng/ml is obtained_minss. In certain embodiments, a C of at least or in excess of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25ng/ml is obtained_minss. In other embodiments, an average C of about 20ng/ml is obtained_minss(ii) a In certain embodiments, the average C in the range of 20ng/ml_minssIncluding an average lower limit of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng/ml and an average upper limit of 20, 21, 22, 23, 24, 25, 26, or 27 ng/ml. In certain embodiments, an average C in the range of at least 12ng/ml to 15ng/ml is obtained_minss. In certain embodiments, an average C in the range of at least 13ng/ml to 15ng/ml is obtained_minss. In certain embodiments, an average C in the range of at least 15ng/ml to 25ng/ml is obtained_minss. In certain embodiments, an average C of at least or in excess of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25ng/ml is obtained_minss。

Alternatively, a method according to the invention (e.g., a method of treating stroke-related sensorimotor impairment, or a method of ameliorating symptoms of stroke-related sensorimotor impairment in a patient, or a method of obtaining therapeutically effective levels of aminopyridine in a patient having stroke-related sensorimotor impairment) comprises administering aminopyridine (e.g., 4-aminopyridine) to the patient to obtain a C in the range of 5-12ng/ml_minss(ii) a C in the range of 10-20ng/ml is obtained_minss(ii) a C in the range of 15-25ng/ml is obtained_minss(ii) a C in the range of 15-30ng/ml is obtained_minss(ii) a C in the range of 17-23ng/ml is obtained_minss(ii) a C in the range of 18-22ng/ml is obtained_minss(ii) a Or to obtain C in the range of 19-21ng/ml_minss. In a specific embodiment, C_minssWherein the lower limit is selected from 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20ng/ml and the upper limit is selected from 20, 21, 22, 23, 24, 25. In the range of 26 or 27ng/ml, it is to be understood that this means that any particular combination is considered without limitation, for example, the following ranges: 16-23ng/ml, 12-24ng/ml, 13-27ng/ml and the like.

In certain embodiments, a therapeutically effective amount of an aminopyridine (e.g., 4-AP) is administered to achieve C for an extended period of time_minssOr average C_minss(or respective ranges thereof) for at least or more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months; or 1, 2,3, 4,5, 6 years or more than 5 years. In certain embodiments, the extended period of time is the lifetime of the patient.

Other embodiments of the invention are methods of treating stroke-related sensorimotor impairment, or symptoms thereof, comprising administering to the patient a therapeutically effective amount of 4-aminopyridine to achieve a mean plasma concentration of about 13ng/mL to about 15ng/mL and a mean maximum plasma concentration of no greater than about 15 ng/mL.

In certain embodiments, described herein are methods of treating stroke-associated sensorimotor impairment, or symptoms thereof, comprising administering to the patient a therapeutically effective amount of 4-aminopyridine to achieve a mean steady state plasma concentration (Cmax) of about 15ng/ml to about 27ng/ml_avss). In some embodiments, described herein are methods of treating stroke-associated sensorimotor impairment, or symptoms thereof, comprising administering to the patient a therapeutically effective amount of 4-aminopyridine to achieve a mean steady state plasma concentration (Cmax) of about 20ng/ml to about 40ng/ml_avss). In one embodiment, described herein is a method of treating stroke-associated sensorimotor impairment, or a symptom thereof, comprising administering to the patient a therapeutically effective amount of 4-aminopyridine to achieve a mean steady state plasma concentration (Cmax) of about 10ng/ml to about 20ng/ml_avss). In another embodiment, described herein is a method of treating stroke-related sensorimotor impairment or symptoms thereof comprising administering to the patient a therapeutically effective amount of 4-aminopyridine to achieve a mean steady state plasma concentration of about 5ng/ml to about 15ng/ml(C_avss)。

In certain embodiments of the methods of the invention, patients having or suspected of having stroke-associated sensorimotor impairment and not having or not suspected of having multiple sclerosis are identified and treated according to the methods described herein.

In certain embodiments, the improvement in stroke-related sensorimotor impairment may be at least about (or more than) 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%. In certain embodiments, the improvement may be at least about (or more than) 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30%. In certain embodiments, the improvement may be at least about 20%. In certain embodiments, the improvement may be at least about 25%. In certain embodiments, the improvement may be at least about (or more than) 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40%. In certain embodiments, the improvement may be at least about 40%. In certain embodiments, the improvement may be at least about 45%. In certain embodiments, the improvement may be at least about (or more than) 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50%. In certain embodiments, the improvement may be at least about 50%. In certain embodiments, the improvement may be at least about or greater than 55%. In certain embodiments, the improvement may be at least about 60%. In certain embodiments, the improvement may be at least about or greater than 65%. In certain embodiments, the improvement may be at least about or greater than 70%. In certain embodiments, the improvement may be at least about or greater than 75%. In certain embodiments, the improvement may be at least about or greater than 80%. In certain embodiments, the improvement may be at least about or greater than 85%. In certain embodiments, the improvement may be at least about or greater than 90%. In certain embodiments, the improvement may be at least about or greater than 95%. In certain embodiments, the improvement may be at least about 100%. In certain embodiments, the improvement may be greater than about 100%. In certain embodiments, the improvement may be greater than about 150%. In certain embodiments, the improvement may be more than about 200%. In certain embodiments, the improvement may be greater than about 250%. In certain embodiments, the improvement may be more than about 300%. In certain embodiments, the improvement may be: 4-100%, 4-20%, 5-20%, 6-20%, 7-20%, 8-20%, 9-20%, 10-30%, 10-60%, 20-30%, 20-40%, 20-50%, 20-60%, 20-100%, 30-100%, 50-100%, 30-150%, 50-150%, 100-200%, 50-250%, 100-250% or 100-300% such percentage change quantification is preferably applied to sensorimotor performance assays that provide a measure of results in a continuous linear scale, such as T25FW and the like.

Embodiments of the present invention also relate to methods of monotonically improving stroke-associated sensorimotor impairment in a patient, comprising administering a therapeutically effective amount of an aminopyridine (e.g., 4-AP) to the patient for an extended period of time. In certain embodiments, the extended period of time is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months; or 1, 2,3, 4,5, 6 years or more than 5 years. In certain embodiments, the extended period of time is the lifetime of the patient. As used herein, a monotonic increase in a parameter is a sustained increase without any decrease from baseline (i.e., prior to treatment with aminopyridine).

Various parameters known as quality of life or daily activities per day are known in the art. These parameters can be measured to assess the improvement in the condition (e.g., sensorimotor performance) of a patient having a stroke following a period of treatment according to the present invention. These include, for example, the impact of injury on daily life:

● walking between rooms in own home

● go to bathroom

● shower

● Care children

● safe road crossing

● remain employed

● Purchase groceries

● making method

● go upstairs

● exercising

● participate in the social event.

In some embodiments, the method according to the invention enables a subject to achieve any of the above activities that they have previously been unable to achieve. In particular embodiments, the method according to the invention enables subjects to better perform any of the above activities that they were previously limited in their ability to perform.

In certain embodiments, the methods according to the present invention allow for the maintenance of an improvement in the symptoms, parameters, characteristics, values, findings or performance of stroke-related sensorimotor impairment by administering to the patient a therapeutically effective amount of aminopyridine after previously achieving an improvement in such symptoms, parameters, characteristics, values, findings or performance that the aminopyridine was previously effective against. The previous period of efficacy may be 10, 11, 12, 13, 14, 15, 16, 17, or 18 weeks; 3. 4,5, 6, 7,8, 9, 10, 11, 12, or 13 months; 1. 2,3, 4,5, 6, 7,8, 9, 10 years or more than 10 years.

6.Examples

6.1Example 1: rodent model for apoplexy

Male rats (about 300-400g, Sprague-Dawley) were operated to cause ischemic brain injury. Animals were orally administered a single dose of 4-AP (0.1, 0.3, and 1mg/kg) starting 1 day, 10 days, and 4 weeks after ischemic injury, or daily for 2 weeks. Neurological behavior was evaluated after dose administration (for a single treatment) or the last dose administration (for multiple treatments) by performing the forelimb placement test, hindlimb placement test, body swing test, cylinder test and activity box test at 4 hours [ equal to 1 hour after Cmax (about 3 hours) ]. Neurological function was also assessed after a period of drug withdrawal. In addition, small blood samples (100 μ L) were also collected from the lateral tail vein at various time points after the vehicle or 4-AP dose to establish plasma concentrations of 4-AP using the HPLC-MS/MS method. This blood sampling enables the determination of plasma exposure when the animal is evaluating neurological improvement.

At the end of the experiment, animals were deeply anesthetized with pentobarbital and transfused with PBS and paraformaldehyde for cerebral infarction volume measurements, and neuronal damage was assessed with H & E staining and Luxol fast blue staining, respectively.

Table 1 shows a summary of treatment groups and endpoints.

TABLE 1 summary of treatment groups and endpoints

At the completion of the study, neurological function post-infarction was measured for neurological function improvement, relative infarction reduction, relative myelination and/or axonal survival (as described in table 1).

6.2Example 2: effect of oral administration of 4-AP: function was restored in rats after MCA occlusion (MCAO). Blindness method and carrier Control double crossover study.

In rats with stable motor deficits, the ability of 4-AP to promote functional sensorimotor improvement following ischemic stroke was evaluated at a time distant from their ischemic event. The animal model mimics the situation in human ischemic stroke and is produced by Middle Cerebral Artery Occlusion (MCAO), which results in a large infarct in the cerebral cortex and striatum, including the corticospinal tract (white matter).

In particular, the MCAO model in Sprague Dawley rats used in the following experiments mimics the situation in human ischemic stroke. In this model, focal cerebral infarction was caused by permanent occlusion of the proximal right Middle Cerebral Artery (MCA) using the method of modified Tamura et al (No To Shinkei 1986; 38: 747-51). Briefly, the temporal muscle is divided in half and folded back through an incision made halfway between the eye and the ear canal. The proximal MCA was exposed by a subtopic craniectomy without removing the zygomatic arch and without transecting the facial nerve. The artery is then occluded and transected by a micro bipolar coagulation from the immediate vicinity of the olfactory tract to the sub-cerebral vein.

The MCAO model described in this example results in the production of a pattern of recovery similar in many respects to the pattern of typical human neurological recovery following stroke. After MCAO there was a rapid and complete loss of sensorimotor function on postoperative day 1 as measured by specific tactile, proprioceptive and sensory tests (forelimb and hindlimb placement and body roll symmetry). This is followed by a relatively rapid partial recovery period within the first few weeks. In the MCAO model, recovery begins to reach a plateau by 4 weeks after MCAO, and the sensory-motor function is still measurable at that time. Similar but slower types of recovery occur in humans during the first months after stroke (see Cramer, Ann Neurol 2008; 63: 272-87).

Design of experiments

In this experiment, Sprague Dawley rats were anesthetized, operated to cause Middle Cerebral Artery Occlusion (MCAO), treated with and without 4-aminopyridine and behavioral assessments were performed as described below. Treatment was started at 4 weeks post stroke.

Animals: 45 male Sprague Dawley rats, 300-275 g (obtained from Charles river laboratories, coming to the laboratory 7-10 days before surgery, weighing 250-275g) were used. Animals were randomly assigned to treatment groups.

The terms: the terminology used for study days is as follows: day 0 is the day of MCAO and the subsequent days are numbered sequentially (day 1, day 2, day 3, etc.); day-1 represents the day before MCAO.

Grouping details: the amount of time required for some procedures in this study required the 3 treatment groups (listed below) to be divided into 8 working groups (as written in the schedule, see below). The 6 animals received stroke surgery daily. If the animal died within the 8 day surgical period of the study, it was replaced with a backup. If not, the animal is not replaced. Most animal deaths (< 5% of total) occurred within a period of time from immediately to 7 days post op.

Anesthesia: 1-3% isoflurane in N₂O:O₂(2: 1). In the presence of a catalyst in N₂O:O₂Anesthesia is induced in the inhalation chamber with 2-3% isoflurane in (2:1) and 1-1.5% isoflurane is maintained through the mask. Sufficient depth of anesthesia was assessed by the lack of retraction of the hind limb pinch and loss of blink reflex. Once anesthetized, animals received cefazolin sodium (40mg/kg, i.p.) and buprenorphine (0.1mg/kg, s.c.). Cefazolin was used as a prophylactic antibiotic for this procedure (as it ensured a negligible infection rate). An ophthalmic veterinary ointment, Lacrilube, was applied to the eyes.

And (3) surgical procedures: small focal strokes (infarcts) are caused on the right side of the brain surface (cerebral cortex) by Middle Cerebral Artery Occlusion (MCAO). Hair was shaved on the right side of the head with an electric hair cutter (approximately 3 x 5cm slices between the eyes and ears). The area was carefully cleaned with sterile liquid soap. Using sterile techniques, an incision is made intermediate the eye and the eardrum tract. The temporal muscle was separated, cut in half and folded back. The small bone window was removed by drill and forceps (temporosubcraniectomy) to expose the Middle Cerebral Artery (MCA). Care was taken not to remove the zygomatic arch or transect the facial nerve, otherwise the animal's ability to chew would be impaired post-operatively. Using a dissecting microscope, the dura mater was dissected and the MCA was electrocoagulated from immediately adjacent the olfactory tract to the sub-cerebral vein (taking care not to rupture the vein) using micro-bipolar electrocautery. Then, the MCA was transected. The temporal muscle is then repositioned and the incision is closed subcutaneously by suturing. The skin incision was closed with surgical staples (2-3 were required). The body temperature was maintained at 37.0 + -1 deg.C throughout the surgery using a self-adjusting heating plate attached to the anal thermometer.

And (3) monitoring after operation: after surgery, the animals remained on the hot plate until they awakened from anesthesia. They are then returned to the clean rearing cage. They are often observed on the day of MCAO surgery (day 0) and at least once daily thereafter.

Feeding, surgery and injection schedules: unless severe aggressiveness is exhibited or the companion in the cage dies, 2-3 animals are placed in each cage before and after surgery. Animals were housed for 7 days prior to surgery. Cefazolin sodium was administered i.p. (40mg/kg) immediately before surgery. Buprenorphine was administered (0.1mg/kg) immediately s.c. prior to surgery.

Dose administration and treatment: rats were treated according to the dose administration schedule as shown in tables 2A, 2B, 3 and figure 2, where each phase was a period of two weeks. A solution of 4-AP was used in this experiment. Dose administration was started 4 weeks after the ischemic event. 4-aminopyridine was dissolved in water for injection (WFI, Cellgro) and sterile filtered. 4-aminopyridine was delivered by gavage at 2mL/kg at final concentrations of 0.315mg/mL or 1.0mg/mL, resulting in final doses of 0.63mg/kg and 2mg/kg, respectively. The vehicle control treatment was WFI delivered at 2mL/kg by gavage. The study was divided into three treatment periods (1-3) where each randomized animal cohort received a different dose level during each treatment period. Starting on day 30 after MCAO (day 30, beginning of phase 1), animals received a gavage dose of the administered solution (2mL/kg) every approximately 12 hours, for a total of 5 doses. The same schedule was repeated with different treatments on days 44 and 58 for the phase 2 and 3 studies, respectively. Animals were not treated for 10 days between each phase (washout phase).

Table 2A:

treatment ID	Treatment (dose TBD)
		V	Carrier (Water)
L	Low 4-AP (0.63mg/kg, b.i.d, p.o)
		H	High 4-AP (2.0mg/kg, b.i.d, p.o)

Table 2B:

group (n 15)	Stage 1 treatment	Stage 2 treatment	Stage 3 treatment
				1	H	L	V
2	L	V	H
				3	V	H	L

Treatment groups: dose administration was started 4 weeks after MCAO surgery. During these 4 weeks, behavioral assessments as defined below were performed weekly. Two dose levels of 4-AP plus vehicle control were evaluated, with treatment starting 4 weeks after the ischemic event. All dose administrations were performed by gavage, with a volume not exceeding 2 mL/kg. Animals were given a first dose and behavioral assessments were made starting 60 minutes after dose administration. The animals then receive a second dose at the appropriate time of day and are thereafter administered twice daily (preferably every 12 hours) for a further 2 days (3 days for a total dose, 5 doses). Animals were subjected to behavioral assessment as defined below 1 hour after the 5 th dose. After final behavioral assessment, withdrawal was performed for 10 or 11 days, behavior was again assessed, and then animals were again challenged with cross-treatment (stage 2 of table 2B) as described in tables 2A, 2B and 3, followed by the same behavioral testing and dose administration regimen. Also, the cross-treatment was repeated 1 more times (stage 3 of table 2B) (see fig. 2).

Behavior test details: behavioral assessments were performed by evaluators whose treatment assignment was unknown. Using limb placement and body swing behavior testing, blind evaluations of sensorimotor function were performed immediately prior to MCAO surgery, 24 hours after MCAO surgery, and weekly thereafter, up to phase 1 of dose administration. As described above, the behavior assessment time is accurately made as a function of the dose administration time. Animals were tested 1 hour after the 1 st and 5 th doses of each phase (i.e., days 30 and 32 of phase 1, days 44 and 46 of phase two; and days 58 and 60 of phase three); animals were also tested on days 42 and 56 of the washout period. Animals were given a first dose, behavioral assessment commenced 60 minutes later, and blood was collected 90 minutes after dose administration. The animals then receive a second dose at the appropriate time of day and are thereafter administered twice daily (preferably every 12 hours) for a further 2 days (3 days for a total dose, 5 doses). Animals were behavioral tested 1 hour after the 5 th dose. After final behavioral assessment, withdrawal was performed for 10 or 11 days, behavior was again assessed, and animals were then challenged again with cross-treatment as described in tables 2A, 2B, 3 and figure 2, followed by the same behavioral testing and dose administration regimen.

Placing the limbs: evaluation was performed on day-1 (before operation), day 1, day 7, day 14, day 21, day 28, day 30, day 32, day 42, day 44, day 46, day 56, day 58, and day 60. The limb placement test is divided into forelimb and hindlimb tests. For the forelimb placement test, the examiner holds the rat close to the table and scores the rat's ability to place forelimbs on the table in response to beard, visual, tactile or proprioceptive stimuli. Similarly, for the hindlimb placement test, the examiner evaluated the rat's ability to place the hindlimb on a table in response to tactile and proprioceptive stimuli. These tests collectively reflect the function and recovery of the sensorimotor system (DeRyck et al Brain Res 1992; 573: 44-60). Separate scoring scores were obtained for each sensory input modality and added to give a total score (0 normal, 12 maximal injury for forelimb placement test; 0 normal, 6 maximal injury for hindlimb placement test). Scores are given in 0.5 point increments (see below). Generally, limb placement behavior recovers slowly and steadily during the first month after a stroke.

Forelimb placement test (0-12):

setting beard (0-2);

visual placement (Forward (0-2), lateral (0-2))

Tactile placement (Back (0-2), side (0-2))

Proprioceptive placement (0-2).

Hindlimb placement test (0-6):

tactile placement (Back (0-2), side (0-2))

Proprioceptive placement (0-2).

For each subtest, animals were scored as follows:

0.0 ═ instant reaction

0.5 ═ in 2 seconds

1.0 ═ 2-3 seconds reaction

1.5 ═ 3 seconds reaction

2.0-No reaction

Body swinging: evaluation was performed on day-1 (before operation), day 1, day 7, day 14, day 21, day 28, day 30, day 32, day 42, day 44, day 46, day 56, day 58, and day 60. Body swing is evaluated by counting head movements to one side or the other when suspended by the tail. For this test, the rat was held about 1 inch from its tail root. It was then raised 1 inch above the table surface. The rat is held in the vertical axis, which is defined as being biased to the left or right by no more than 10 °. The swing was recorded each time the rat moved its head off the vertical axis to either side. Before attempting another swing, the rat must return to the vertical position for the next swing to be counted. The total of 30 swings were counted. This test reflects the symmetry of striatal function (Borlongan et al, J.Neurosci1995; 15:5372-8), and normal rats usually swing equally to either side. Following focal ischemia, the rats tended to swing to the contralateral side (in this case the left side). Body swing scores are expressed as a percentage of the right side relative to total swing. Within the first month after stroke, there is a spontaneous partial recovery of the body swing score (up to 50%). The body swing test is performed simultaneously with the limb placement test.

And (3) cylinder testing: evaluation was performed on day-1 (before operation), day 7, day 21, day 30, day 32, day 44, day 46, day 58, and day 60. This test evaluates the asymmetry of limb use. The rats were placed in a transparent cylinder (20 cm diameter, 30cm height) for 3-6 minutes. A mirror is placed behind the cylinder to determine forelimb movement when the animal turns away from the camera. The degree of forelimb use asymmetry exhibited by the animal is determined by counting the number of times the left or right forelimb contacts the wall during hind leg stance. Simultaneous use of the left and right forelimbs when contacting the wall during hind leg stance was also scored. During the test period, the counts resulted in a total of 20 forelimb placements. Data are expressed in terms of observation of wall motion as a percentage of intact and/or injured forelimbs relative to the total number of limb uses.

Blood sampling: after completion of the on-drug behavior evaluation (days 30, 32, 44, 46, 58 and 60), approximately 300 μ l of blood sample was collected at 90 minutes after the dose was administered to evaluate the 4-AP plasma level at that time. Blood was collected from the saphenous vein of each animal. Blood was collected into K3EDTA tubes and centrifuged at 10000rpm for 10 minutes at 4 ℃. Plasma was obtained, frozen and stored at about-80 ℃. Samples were analyzed for 4-AP levels. 4-AP concentration was determined using a validated liquid chromatography tandem mass spectrometry detection method in electrospray positive ion mode.

Euthanasia and post-mortem treatments: infarct volume analysis. On day 63, after the last behavioral assessment, rats were deeply anesthetized with ketamine/xylazine (100mg/kg ketamine, 10mg/kg xylazine, i.p.) and perfused transcardially with physiological saline (with 2 units/ml heparin) followed by 4% paraformaldehyde or formalin. Brains were harvested and processed for histological evaluation. A subset of 10 brains in each group was treated for infarct volume measurement (H & E staining).

Infarct measurement: brains were embedded in paraffin and 5 micron thick coronal sections were cut using a microtome. Sections were stained with hematoxylin and eosin (H & E) using standard methods. Seven coronal sections of each brain (+ 4.7, +2.7, +0.7, -1.3, -3.3, -5.3 and-7.3, respectively, compared to the pro-halogen) were photographed by a digital camera and infarct size on each section was determined by NIH Image (Image J) using an "indirect method" (intact contralateral [ left ] hemispherical area-ipsilateral [ right ] hemispherical intact area) to correct cerebral edema. The infarct size of each slice is then added and multiplied by the slice thickness (distance between slices) to give the total infarct volume, which can be expressed as a percentage of the intact contralateral hemisphere volume.

Regulatory compliance: the study was conducted in a non-GLP environment, in an AAALAC certified institution and according to standard good scientific principles and practices.

Quality Assurance (QA): the collected data was validated by a second scientist who was not involved in the data set collection by the present laboratory during the study. The verification is recorded in the raw data and saved with the data package of the study. When the study is complete, the entire data package (all raw data, measurements, notebooks, and calculations) is validated and reviewed for final reporting.

Statistical method

The statistical method comprises the following steps: the change from the baseline behavior values was calculated for each treatment group in each session. Baseline was defined as the behavioral value measured when the animals were not receiving treatment before the start of the dose administration period (days 28, 42 and 56 for phases 1, 2 and 3, respectively). Mean behavioral parameter data for each complete dose administration period were subjected to analysis of variance (ANOVA). The data were also subjected to mixed model analysis using the differences in the least squares of the averaging method, using SAS pairwise comparisons between each pair of treatments to examine dose, order, residual effects and experimental period as covariates. Values with p <0.005 were considered statistically significant.

Within each period, baseline was defined as the measurement on the first day of the period, which was day 28, day 42 and day 56. The change from baseline was taken as the difference from baseline on other days of measurement. For each subject, the mean change from baseline was calculated by adding the two changes from baseline values over each period (e.g., day 30 and day 32 of period 1) and taking 2.

Descriptive statistics (mean and standard deviation) are provided in excel pages for different behavioral endpoints. The change from baseline at different stages was calculated for each treatment group (N15), regardless of the number of days in which the measurements were taken during that period. During one phase, one-way ANOVA with treatment as the only covariate was used to compare the mean values under different treatments. The null hypothesis is the same average under different treatments. Statistical significance was strongly indicated when p-values were <0.01 (for body swing) and <0.0001 (for hind and forelimb). At 99% confidence level, the null hypothesis is negated, thus, concluding: the three dose levels showed significantly different treatment effects on the muscle function studied.

Two sets of mixed models have been used to further investigate other effects on the results. In the first set of mixed models, the result variable is the average change measured after two baselines over a period. The fixed impact includes covariates: "dose", "order", "residual effect (co)" and "phase". "dose" refers to three treatments, and "order" refers to the order of treatment assigned to each group (i.e., "high-low-vector"). "co" is the residual effect, which is defined as the dose from the previous phase, with the residual effect of phase 1 set to 0. There is only one random influence, id, which is the subject id nested in the order. In the second set of mixed models, the resulting variable is the original change from baseline over the course of time (no mean was calculated for the two post-baseline measurements). The number of days was added to the model as a fixed impact, such that the fixed impact includes covariates: "dose", "order", "residual effect (co)", "phase" and "day". "days" are the days on which the measurements were made, which are nested in the middle of the day. The remaining fixed influences are the same as those in the hybrid model described above. There is only one random influence, id, which is the subject id nested in the order. In both hybrid models, a two-part output is provided from the SAS. The first part is "type 3 test of fixed influence". By statistically significant P-values (< ═ 0.05), the following conclusions can be drawn: the effect predicts the outcome significantly. The second part is the "least squares difference of means" where a pair-wise comparison is made between each pair of treatments (i.e., high versus low). A p-value of less than or equal to 0.05 advocates the statistical significance of the differences in results under different treatments.

For forelimb function, the first model indicated that phase, dose and residual effects were significant effects, not sequential. High doses significantly improved forelimb function compared to low doses (p ═ 0.0334) and vehicle (p ═ 0.001); whereas the low dose showed no statistically significant improvement compared to the 0.05 level of vehicle. In the second mixed model, days showed another significant effect, and all three treatments showed significant differences from each other (i.e., high versus low, high versus vehicle, and low versus vehicle), p < 0.0001. Overall, for forelimb function, both models showed that phase, dose and residual effects were significantly affected, but not in sequence. Both models show that high doses will significantly improve forelimb function compared to low doses and vehicle. From the smaller p-value, the second hybrid model appears to be more sensitive in detecting treatment effects.

The same analytical procedure was applied to each outcome measurement, which included forelimb, hindlimb and body swing.

For hindlimb function, the first model indicated that the duration and dose were significantly affected, but the order or residual effect was not significant. The high dose significantly improved hindlimb function compared to vehicle (p <0.0001), the low dose also significantly improved hindlimb function compared to vehicle (p ═ 0.0027), whereas the high dose did not show statistical significance compared to the low dose, at a level of 0.05. In the second mixture model, all effects showed significant effects except for order, and all three treatments showed significant differences from each other (i.e., high versus low, high versus vehicle, and low versus vehicle), p < 0.0001. Overall, both models showed that period and dose were significantly affected for hindlimb function. Both models also showed that high and low doses would significantly improve hindlimb function compared to vehicle. From the smaller p-value, the second hybrid model appears to be more sensitive in detecting treatment effects.

For body swing function, the first model indicates that only dose is a significant effect. The high dose significantly improved the body rocking function compared to vehicle (p ═ 0.0131), the low dose also significantly improved the body rocking function compared to vehicle (p ═ 0.033), while the high dose did not show statistical significance compared to the low dose, at a level of 0.05. In the second hybrid model, the phase shows another significant effect. High doses significantly improved body rocking function compared to low doses (p ═ 0.006) and vehicle (p < 0.0001); whereas the low dose showed no statistically significant improvement compared to vehicle, at a level of 0.05. Overall, for body swing function, both models show that dose is a significant effect.

The mixed model is the change from baseline-dose, order, residual effect, phase, days with id nested in order as a random effect for each phase, the baseline is defined as day 28, day 46 and day 56 respectively-dose has three stable levels, high, low and vehicle-seq (order) has three fixed values as three different dose administration orders, i.e. "hlv", "lvh" and "vhl", co (residual effect) is defined as the dose of the previous phase with the residual effect of phase 1 set to 0-id is the rat id from the data representing statistical significance according to the bilateral test based on α -0.05.

Results

Table 3 shows the allocation of animals between treatment groups. Tables 4-6 show the total score of the forelimb placement test in groups 1-3, respectively. Tables 7-9 show the total score of the hindlimb placement test in groups 1-3, respectively. Tables 10-12 show the overall scores for the body swing tests in groups 1-3, respectively. Tables 13-15 show the body weights of the animals in groups 1-3, respectively, at different time points. Tables 16-18 show the total scores (% bulk asymmetry) of the cylinder tests in groups 1-3, respectively. Tables 19-21 show the total exercise scores for the cylinder tests in groups 1-3, respectively. Table 3: assignment of animals in treatment groups

Table 4: forelimb placement summary of group 1

Table 5: forelimb placement summary of group 2

Table 6: forelimb placement total score for group 3

Table 7: total hind limb placement score for group 1

Table 8: total hind limb placement score for group 2

Table 9: total hind limb placement score for group 3

Table 10: body swing test of group 1 (% Right swing)

Table 11: body swing test of group 2 (% right side swing)

Table 12: body swing test of group 3 (% right side swing)

Table 13: body weight (g) of group 1

Table 14: body weight (g) of group 2

Table 15: body weight (g) of group 3

Table 16: cylinder test score for group 1 (Total asymmetry (%))

Table 17: cylinder test score for group 2 (Total asymmetry (%))

Table 18: cylinder test score for group 3 (Total asymmetry (%))

Table 19: cylinder test of group 1 (Total motion)

Table 20: cylinder test of group 2 (Total motion)

Table 21: cylinder test of group 3 (Total motion)

MCAO resulted in a large acute loss of sensorimotor function, with partial recovery of sensorimotor function in all animals by the end of week 4 pretreatment and a plateau phase of stable deficits.

All groups (1-3) showed a typical recovery response to MCAO-induced ischemia with a normal score of 0 just before surgery (day-1) and complete loss of function within 24 hours after occlusion (day 1) (forelimb, score 12; hindlimb, 6). During the subsequent 4-week untreated period, the forelimb and hindlimb scores improved to about 5.5 and 3, respectively, and a restored plateau level was reached (fig. 3 and 4). In the body swing test, animals showed less than 5% swing to the right on the day after surgery and returned to about 25% swing to the right at the end of the 4-week untreated period (fig. 5). Although not significant, baseline behavioral measures slightly improved relative to pretreatment levels during the drug-free period between stage 1 and stage 2 and between stage 2 and stage 3. This may be due to slow continuous endogenous recovery, training for repeated behavioral assessments, and possible treatment residual effects.

At the end of the study, all animals received each treatment. Rats were administered 4-AP twice daily (in this study and in the study provided in example 17). This regimen did not maintain long-term plasma levels of the compound due to a clearance half-life of 1-1.5 hours (Hayes et al, j. clin. pharmacol.2003; 43:379-85), but it did allow repeated exposure in animals daily. Behavioral assessments were performed 1 hour after dose administration to ensure adequate exposure during the assessment period, and the three day interval of each dose administration period may help the animal adapt to the pressure of oral feeding prior to performing the behavioral assessments. 30 minutes after performance evaluation was complete, blood was drawn to confirm dose-related daltepyridine levels in the animals (Table 22). It should be noted that it is not possible to equate the doses used herein or the plasma concentrations obtained with the concentrations expected in patients treated with sustained release formulations of the drug, where their pharmacokinetics are very different. There is also a delay in the peak concentration measured in cerebrospinal fluid compared to blood, which is about 1 hour in human subjects (Donovan et al, Spinal Cord 2000; 38: 7-15). Thus, the concentration of 4-AP achieved in the central nervous system for a given plasma level after feeding is likely to be much lower than the transient plasma peak, compared to similar concentrations maintained over a long period of time.

Forelimb placement tests show the effect of treatment on forelimb function. Figure 3 shows that 4 weeks after ischemic brain injury, treatment with low or high dose 4-aminopyridine was effective in improving forelimb function in rats. Figure 3 also shows that this effect is dose-responsive. It is also reversible as the effect diminishes after withdrawal.

Hindlimb placement tests show the effect of treatment on hindlimb function. Figures 4A-D show that treatment with low or high dose 4-aminopyridine was effective to improve hindlimb function in rats 4 weeks after ischemic brain injury. Fig. 4A-D also show that the effect is reversible. Notably, this effect was dose-responsive since treatment with high doses resulted in improved behavioral scores relative to treatment with low doses or vehicle controls.

The body swing test shows the effect of treatment on systemic control. Figure 5 shows that treatment with low or high dose 4-aminopyridine was effective in improving the percentage of right-bias relative to total swing in rats and, therefore, in improving one of the symptoms of ischemic stroke. Thus, figure 5 shows that 4-aminopyridine is effective in improving systemic control in rats. Figure 5 also shows that this effect is reversible and dose dependent.

Animals of group 1 (figures 3-5) that received 2mg/kg 4-aminopyridine during the first dose administration period showed significant improvement in forelimb, hindlimb and body swing scores compared to the pre-treatment baseline score (day 28 versus day 32; p-value < 0.05). Between dose administration phases 1 and 2 (washout phase, days 33-42), the effect on limb placement returned to near baseline levels. Animals in group 1 received 0.63mg/kg of 4-aminopyridine during the 2 nd dose administration period. All behavioral scores improved significantly (day 42 versus day 46; p <0.05) compared to the scores in the washout period immediately prior to dose administration, although they did not achieve the same degree of improvement as the first high dose administration period. During the washout period between stages 2 and 3 (days 47-56), the behavioral scores dropped to a level similar to the baseline scores (day 56). Animals in this group received vehicle during the third dose administration period and no change in behavioral score was found (at day 56) compared to the day immediately prior to dose administration.

Animals of group 2 (figures 3-5) that received 0.63mg/kg 4-aminopyridine during the first dose administration period showed significantly improved behavioral scores in all measurements compared to the pre-treatment baseline score (day 28 versus day 32; p-value < 0.05). Between dose administration phases 1 and 2, when the animals did not receive the drug, the effect on behaviour decreased to a level similar to that at the previous dose administration (day 42). Animals in this group received vehicle during dose administration 2, and showed no change in the behavioral test score. They remained at baseline functional levels during the washout period between stages 2 and 3 (days 47-56). Animals in this group received 2mg/kg of 4-aminopyridine at dose administration 3 and all behavioral test scores were significantly improved compared to the previous baseline score (day 56 versus day 60; p-value < 0.05).

Animals in group 3 (fig. 3-5) had similar results to those observed in groups 1 and 2 at different treatment periods. These animals received the vector at stage 1. During the washout period between stage 1 and 2, there was no change in the behavioral score and the animals remained at this functional level. Treatment with 4-aminopyridine at 2mg/kg in phase 2 and 0.63mg/kg in phase 3 resulted in a significant improvement in limb placement compared to withdrawal evaluation immediately prior to each phase (day 42 versus day 46, and day 56 versus day 60, respectively; p-value < 0.05). During the high dose treatment period in phase 2, the body swing score improved (day 42 versus day 46; p <0.05), but was unchanged for the low dose treatment in phase 3. Return to baseline behavior (day 56) during the washout period between stages 2 and 3.

In summary, all animals responded similarly to their respective treatments regardless of their order of treatment. In all cases, the highest dose within any dose administration period resulted in a significant improvement (p-value <0.05) compared to vehicle and low doses, and low doses were statistically better and tended to be significant compared to vehicle, depending on the statistical model used (ANOVA or mixed model analysis, see statistical methods section above).

In addition to the pre-treatment weekly assessments, two assessments were performed during any given dose administration period (after the 1 st and 5 th doses). A slight improvement between these scores was noted (e.g., group 3, between day 30 and day 32, when animals received vehicle treatment). This may be due to adaptation to oral feeding pressure or may be an indication of learning response due to animal familiarity and prediction of testing. This effect was not observed in the study provided in example 17 (see control panel, day 56, etc.), where animals were tested 1 time over a 3 day dose administration period, respectively. Since the baseline was still slightly improved and all possible dosing sequences were not tested, it was not possible to determine that such 4-AP exposure would predispose the animal to a greater or lesser response when 4-AP is administered in the subsequent phase. To eliminate this possible residual effect due to differences in dose order, the study provided in example 17 was designed as a dose escalation study without a washout period.

The cylinder test shows the effect of treatment on systemic control aspects such as body symmetry and coordination. Figure 7 shows that treatment with 4-aminopyridine is effective in ameliorating the asymmetry in limb use resulting from stroke by showing an increase in the percentage of injured forelimb use relative to total limb use in rats. Thus, figure 7 shows that 4-aminopyridine is effective in improving body symmetry and coordination in rats. Figure 7 also shows that this effect is reversible and dose dependent.

Figure 9 shows that no difference in infarct volume was observed between groups 1-3. In particular, the mean infarct volume (% of the contralateral hemisphere) was the same between any of these groups. Mean infarct volume (%) was 45.0 (+ -1.8) in group 1, 41.4 (+ -2.3) in group 2, and 39.0 (+ -3.3) in group 3.

4-aminopyridine plasma levels: when the animal is treated with the vehicle, the level of 4-aminopyridine in the drawn blood sample is below the lower limit of quantitation for this method. When the animals received 4-aminopyridine, samples were drawn to confirm that exposure at the time of the behavioral test correlated appropriately with dose level. The 4-aminopyridine plasma levels are shown in table 22.

Table 22: plasma levels of 4-aminopyridine.

SE, standard deviation

Lower limit of quantitation (<1.0ng/mL)

The data show that 4-aminopyridine treatment resulted in significant improvement in forelimb, hindlimb and body swing function during each individual treatment period and the total treatment period. Furthermore, several cross-statistical models used by the inventors showed that on the same basis, high doses were significantly better compared to vehicle controls and low doses (p <0.0001 for limb placement test, p <0.001 for body swing). The low dose showed a strong trend or reached a significant improvement compared to the vehicle control. In addition, the score during the second evaluation was significantly better than the first drug evaluation over the dose administration period. Thus, this example shows reversible and dose-dependent improvement in forelimb and hindlimb sensorimotor performance when 4-AP is at detectable plasma levels in animals. The body swing test data also showed a dose-dependent effect on the recovery of body state function. This may be an indication of an effect on tracts in the striatum or possibly an effect on subcortical white matter regions. In addition, this example shows a clear and dose-dependent response to treatment with 4-AP within each group and between groups at each phase.

In addition, the results in FIGS. 3-8 show that continued treatment with 4-aminopyridine can produce further improvements in sensorimotor performance outcomes. Specifically, on average, the behavioral score after administration of multiple doses of 4-aminopyridine was improved relative to the behavioral score after a single dose of 4-aminopyridine.

These results show that treatment with 4-aminopyridine is effective for improving sensorimotor function in mammals suffering from stroke-related impairment of these functions. These results also indicate an improvement in stroke-related sensorimotor impairment when treatment is initiated in the chronic phase with stable motor deficits following a stroke event. Based on this data, one can conclude that: 4-aminopyridine significantly ameliorated chronic sensorimotor deficits following stroke.

6.3Example 3: treatment of ischemic stroke

Patients entering a medical facility have signs and symptoms of ischemic stroke. The patient's blood circulation is reestablished with tPA or other therapies to restore blood flow. Although blood flow has been restored, some level of brain damage has occurred. 3 days after the stroke, the patient was evaluated neurologically and shown to have measurable sensorimotor deficiency. After day 2 and after day 3, the patient was treated intravenously with 4-aminopyridine starting on day 4 at doses between 0.01 and 1.0mg/kg for 10 days to 3 months. Sensorimotor function was evaluated during and after treatment.

6.4Example 4: treatment of stroke and resulting right hand paralysis

Patients entering the emergency department had a right hand paralysis. After evaluation and imaging, the patient is determined to have ischemic stroke. According to approved procedures, patients receive tPA and blood flow is restored through the thrombus. However, after 1 week of tPA treatment, the patient had residual right hand paralysis as measured by standard neurological measurements of hand motor activity. 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 or 6.5 years or more, the patient begins treatment with 4-aminopyridine (0.01 to 1.0mg/kg, IV) once a week for 4 weeks. Improvements in hand function are measured periodically by a neurologist or other physician using standard neurological tests, including ergometers and other strength tests. Sensorimotor function of the right hand was evaluated during and after treatment.

6.5Example 5: treatment of ischemic stroke

Patients entering a medical facility have signs and symptoms of ischemic stroke. They were found to suffer from left-sided paralysis. Patients fail to arrive in time for revascularization therapy. Through clinical evaluation, some brain damage was found to have occurred. 3 days after the stroke, the patient was evaluated neurologically and shown to have measurable sensorimotor deficiency. 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 or 6.5 years or more, the patient being initially treated intravenously with 4-aminopyridine at a dose of between 0.01 and 1.0mg/kg per dose on a daily basis for 4 weeks; thereafter, they received weekly doses for 6 months. They also received physical therapy. Left-sided sensorimotor function was evaluated during (e.g., after 2 weeks) and after treatment.

6.6Example 6: treatment of ischemic stroke

Patients entering emergency have left-handed paralysis. Patients report that the problem with their hands started "one week ago". After evaluation and imaging, the patient is determined to have ischemic stroke. The patient did not receive tPA. Patients were found to have residual left-handed paralysis by neurological examination as measured by standard neurological measurements of hand motor activity; patients also suffer from sensory deficits. Patients refused to participate in physical or occupational therapy. 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 or 6.5 years or more, the patient begins treatment with 4-aminopyridine (0.01 to 1.0mg/kg, IV) once a week for 12 weeks. Improvements in hand function are measured periodically by a neurologist or other physician using standard neurological tests, including ergometers and other strength tests. Left-handed sensorimotor performance was evaluated during (e.g., after 2 weeks of) and after treatment.

6.7Example 7: treatment of hemorrhagic stroke

Patients entering a medical facility have signs and symptoms consistent with ischemic stroke or cerebral hemorrhage. The patient is stabilized. By neurological evaluation, it was found that some level of brain damage occurred. 1 week after stroke, the patient was again evaluated neurologically and shown to have measurable sensorimotor deficiency. 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 or 6.5 years or more, the patient begins intravenous treatment with 4-aminopyridine at a dose of between 0.01 and 1.0mg/kg per day for 10 days, then administers the dose weekly for 2 months, at which point all treatments are discontinued. Sensorimotor function is evaluated (e.g., 1, 2,3, 4,5, 6 weeks and/or 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, or 12 months after stroke and/or from the start of therapy).

6.8Example 8: 4-aminopyridine treatment of stroke-related injuries, including treatment in the chronic phase

For the comprehensive test, inclusion criteria included: adults, both male and female, have clinical signs of nerve damage.

Indications to be investigated:

ischemic stroke with thrombolytic agents is characterized by the use of thrombolytic agents,

ischemic stroke without the use of thrombolytic agents,

hemorrhagic stroke.

Dose range to be studied:

the dosage is 0.001mg/kg to 10.0 mg/kg.

Dose frequency to be studied:

daily life

Every other day

Every 4 days

1 time per week

Every 1 week for 1 time

1 time per month.

Mixed periodic scheme:

once daily for 1 or 2 weeks, then weekly, biweekly or 1 monthly for the remainder of the study.

1 every other day for 1 or 2 weeks, and thereafter 1 time per week, two weeks or month. Initiation of the treatment to be studied:

1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more.

Duration of treatment to be studied:

treatment is for 1, 2, 4, 10, 30 weeks.

Treating for 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12 months

The treatment lasts for 1, 2,3, 4,5, 6, 7,8, 9 and 10 years.

The function to be studied:

the function of the hand movement is realized,

the function of the facial movement is that,

the survival rate of the plants is higher than the standard,

and returning to the working position.

Recovery was measured by standard neurological measurements.

As a result: sensorimotor performance was evaluated for patients treated with 4-aminopyridine and placebo using methods known in the art by treatment as described above, and test results were compared.

In an alternative embodiment, combinations of less than all of the above parameters are investigated.

6.9Example 9: 4-aminopyridine treatment of ischemic stroke of unilateral hand weakness and/or paralysis (without blood use) Suppository dissolving)

Inclusion criteria included: adults, both male and female, have been based on signs of stroke due to loss of consciousness, disorientation, dysphasia, facial or limb paralysis. Ischemic stroke was confirmed by radiographic imaging.

Patients were selected for those with unilateral hand weakness and/or paralysis and for any reason were not tPA (or other thrombolytic agent) candidates or had not previously received tPA. Consent is obtained from the patient and/or someone authorized to sign for the patient.

1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; for 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more, patients were recruited and randomly assigned to receive 4-aminopyridine or placebo and treatment commenced as soon as they entered a medical facility (including a hospital or doctor's office), diagnosed, and imaged.

For this trial, treatment was initiated between 1 hour and 7 days post injury. Treatment lasted 3 months, with doses administered every other day for 1 week, then once per week for the remainder of the treatment period. A dose of 0.0001mg/kg to 1.0mg/kg is administered to the patient intravenously, intramuscularly or subcutaneously.

Recovery was measured by standard neurological measurements of sensory motor activity of the hand every other week during the study.

As a result: patients treated with 4-aminopyridine and placebo were evaluated for hand function by treatment as described above using methods known in the art, and test results were compared.

6.10Example 10: 4-aminopyridine treatment of stroke with unilateral facial nerve palsy without thrombolytic agents

The patients were selected for unilateral facial paralysis and were not or could not receive a thrombolytic agent. Function was assessed by methods known in the art every other week over a 3 month dose administration period. Consent is obtained from the patient and/or someone authorized to sign for the patient.

Patients were recruited and randomly assigned to receive 4-aminopyridine or placebo. 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; treatment is initiated 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more. Treatment lasted 3 months, with doses administered every other day for 1 week, then once per week for the remainder of the treatment period. A dose of 0.0001 to 1.0mg/kg is administered to the patient intravenously, intramuscularly or subcutaneously.

As a result: patients treated with 4-aminopyridine and patients treated with placebo were evaluated for facial movement by treatment as described above; and the test results are compared.

6.11 implementationExample 11: 4-aminopyridine treatment of ischemic stroke (with thrombolysis)

Inclusion criteria included: adults, both male and female, have been based on signs of stroke due to loss of consciousness, disorientation, dysphasia, facial or limb paralysis. Ischemic stroke was confirmed by radiographic imaging.

The patient is selected for those patients with unilateral hand weakness and/or paralysis and who have been treated with tPA or other thrombolytic agents. Consent is obtained from the patient and/or someone authorized to sign for the patient.

Patients were enrolled and randomized to receive 4-aminopyridine or placebo as soon as they entered the medical facility, diagnosed and completed imaging.

1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; treatment is initiated 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more. Treatment lasted 3 months, with doses administered every other day for 1 week, then once per week for the remainder of the treatment period. A dose of 0.0001mg/kg to 1.0mg/kg is administered to the patient intravenously, intramuscularly or subcutaneously.

Recovery was measured by standard neurological measurements of sensory motor activity of the hand every other week during the study.

As a result: hand function was measured by methods known in the art for patients treated with 4-aminopyridine by treatment as described above and compared to patients treated with placebo.

6.12Example 12: 4-aminopyridine treatment of dysphonia stroke without thrombolytic agents

The patient is selected for those patients who have difficulty speaking and do not receive or cannot receive the thrombolytic agent. Function was assessed by methods known in the art every other week over a 3 month dose administration period. Consent is obtained from the patient and/or someone authorized to sign for the patient.

Patients were recruited and randomly assigned to receive 4-aminopyridine or placebo. 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; treatment is initiated 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more. Treatment lasted 3 months, with doses administered every other day for 1 week, then once per week for the remainder of the treatment period. A dose of 0.0001 to 1.0mg/kg is administered to the patient intravenously, intramuscularly or subcutaneously.

As a result: patients treated with 4-aminopyridine were evaluated for speech impairment due to dysphonia using methods known in the art by treatment as described above, and compared to patients treated with placebo.

6.13Example 13: 4-aminopyridine treatment of dysphonia patients (with thrombolytic agents)

Inclusion criteria included: adults, both male and female, have been based on signs of stroke due to loss of consciousness, disorientation, dysphasia, facial or limb paralysis. Ischemic stroke was confirmed by radiographic imaging. The patient selects those patients who have difficulty speaking and who do receive thrombolytic agents.

1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; treatment is initiated 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more. Treatment lasted 3 months, with doses administered every other day for 1 week, then once per week for the remainder of the treatment period. Administering to the patient from 0.0001 to 1.0mg/kg intravenously, intramuscularly, or subcutaneously.

Function was assessed by methods known in the art every other week over a 3 month dose administration period.

As a result: the speech impairment due to dysphonia in patients treated with 4-aminopyridine and placebo was evaluated and compared by treatment as described above.

6.14 implementationExample 14: 4-aminopyridine treatment of patients with hemorrhagic stroke

Inclusion criteria included: adults, both male and female, have been based on signs of stroke due to loss of consciousness, disorientation, dysphasia, facial or limb paralysis. Hemorrhagic stroke was confirmed by radiographic imaging. Consent is obtained from the patient and/or someone authorized to sign for the patient.

The patient is selected from those with unilateral hand weakness.

Patients were enrolled and randomized to receive 4-aminopyridine or placebo as soon as they entered the medical facility (including the hospital or doctor's office), diagnosed, imaged and given consent.

1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; treatment is initiated 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more. Treatment lasted 3 months, with doses administered every other day for 1 week, then once per week for the remainder of the treatment period. A dose of 0.0001mg/kg to 1.0mg/kg is administered to the patient intravenously, intramuscularly or subcutaneously.

Recovery was measured by standard neurological measurements of sensory motor activity of the hand every other week during the study.

As a result: hand function was measured by methods known in the art for patients treated with 4-aminopyridine by treatment as described above and compared to patients treated with placebo.

6.15Example 15: 4-aminopyridine treatment of patients with hemorrhagic stroke

In humans, the effect of 4AP administered as a single dose or as multiple daily doses at a dose level of 0.05 to 0.1 on functional outcome following thrombotic stroke was evaluated using the following protocol.

The purpose is as follows: the efficacy and safety of oral or intravenous administration of 4AP in chronic ischemic stroke was evaluated.

Designing: multicenter, randomized, double-blind, placebo-controlled safety and efficacy studies

And (3) inclusion standard: patients with ischemic stroke and limb weakness and fully independent of functionality prior to stroke

Exclusion criteria: patients with severe disease and with a life expectancy of less than 6 months with known severe kidney disease, presently known alcohol abuse or abuse of illegal drugs or their dependence. Patients with hemorrhagic stroke were excluded.

The patients participated in: 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the stroke event; 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks; 2.3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, 60, or 66 months; patients are randomized to receive a single dose of 4AP or placebo, or a 2 week daily dose of 4AP or placebo beginning at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5 years or more. Blood samples were taken at various time points after 4AP treatment to determine plasma drug concentrations approximately at the time of evaluation of sensorimotor function outcomes.

The main results are: overall recovery and sensorimotor function recovery at 2 days (single dose administration) or 2 weeks (multiple dose administration) post-treatment were measured by modifying the Rankin scale and NIH scale.

Sensorimotor function in stroke patients after single or multiple administrations was measured and compared to baseline function and placebo treatment by 4-AP treatment.

6.16Example 16: study of 10mg Davalpyridine sustained Release tablets in Chronic Defect Subjects after ischemic Stroke

6.16.1 abbreviation list

The following abbreviations and nomenclature are used in this study protocol:

abbreviations or terms of art	Explanation of the invention
		ADL	Activities of daily living
AE	Adverse events
		AST	Aspartate aminotransferase
BDI	Baishi depression scale
		BMI	Body mass index
BUN	Urea nitrogen of blood
		C	Degree centigrade
CFB	Change from baseline
		CGI	Clinician global impression
CP	Cerebral palsy
		CRF	Case report form
EOS	Onset of early epilepsy
		ER	Sustained release
F	Degree of Fahrenheit
		FAP	Total analysis population
FIM	Function independent measurement
		FMA	Fugl-Meyer evaluation

Abbreviations or terms of art	Explanation of the invention
		GCP	Good clinical practice
HDPE	High density polyethylene
		HEENT	Head, ear, eye, nose and throat
ICH	International conference of coordination
		IEC	Independent ethical committee
IND	Novel experimental medicine
		INN	International non-patent name
IRB	Institutional review board
		LOS	Late stage epilepsia
LEMMT	Lower extremity manual muscle testing
		mg	Milligrams of
MRI	Magnetic resonance imaging
		MS	Multiple sclerosis
MSWS-12	12-item multiple sclerosis walk table
		PPP	Population according to a protocol
RBC	Red blood cell
		SAE	Serious adverse events
SGI	Overall impression of the subjects
		SSRI	Serotonin reuptake inhibitor
T25FW	Timing 25 feet walk
		TMS	Transcranial magnetic stimulation
UPT	Urine pregnancy test
		US	United states of America
USAN	Names adopted in the United states
		UTI	Urinary tract infection
WBC	White blood cell

6.16.2 research object

The effect of 10mg of dalvapyridine-ER (i.e., 10mg of 4-aminopyridine in a sustained release formulation) administered twice daily at approximately 12 hours intervals on the following clinical functions was examined:

● Walking speed measured by timed 25 foot walk test (T25FW)

● hand agility measurement through Block and Box testing

● measurement of hand strength by grip and pinch tests

● measurement of motor and sensory function by Fugl-Meyer evaluation (FMA)

● optionally, measuring upper limb rigidity by Disability Assessment Scale (DAS)

● measurement of assistance required to perform Activities of Daily Living (ADL) by a Function Independent Measurement (FIM) scale

● Subject Global Impression (SGI) Scale

● Clinician Global Impression (CGI) Scale

● optionally, measuring depression by the white Depression Scale (BDI) (to exclude major depression)

6.16.3 research project

This is a study of 10mg of dalvapyridine-ER taken twice a day with an interval of about 12 hours in subjects with chronic stable sensorimotor deficits after ischemic stroke. This study was designed as a double-blind, placebo-controlled, 2-phase crossover study. The study will be conducted at multiple locations, scheduled for 66 subjects. Study time of 8 weeks included a 2-week screening period, a 2-week first treatment, a 1-week washout period, a 2-week second treatment, and a follow-up phone follow-up after 1-week treatment. Adverse events will be monitored throughout the study. In addition, a simple physical examination and vital sign measurements will be made during each study period to assess possible changes from baseline. A set of functional and subjective clinical assessments will also be administered as described below.

After informed consent was obtained from the subjects, eligibility was determined at the screening visit (visit 1) by a medical history check, previous brain imaging results, SMA-12 chemistry tests including creatinine clearance estimation, urinalysis and Urine Pregnancy Test (UPT) of women with the possibility of delivery. The white's depression scale (BDI) will be implemented to exclude major depression. In the screening, walking speed (T25FW), hand agility (block and box test), hand strength (grip and pinch test), upper and lower limb motor Function (FMA), upper limb motor rigidity (DAS) and auxiliary (FIM) measurements required to perform activities of daily living will also be performed. The screening period (days-14 to-1) will be completed at visit 2, which marks the beginning of the eligible subjects 1 period.

At visit 2 (day 1), subjects will be enrolled at a 2: the ratio of 1 was randomly assigned to one of two blind processing sequences: placebo followed by dabigatran-ER (sequence A) or dabigatran-ER followed by placebo (sequence B). The same clinical evaluations conducted in the previous visit will be conducted. Subjects were given their assigned test treatments for 1 week and then discharged home. They will be instructed to take the 1 st dose at the evening and about 1 dose every 12 hours until the morning of the next visit. During this time, they will be visited twice more at 1 week intervals: visit 3 (day 8) and visit 4 (day 15). In each of these visits, a similar set of clinical assessments will be made, with the addition of a Subject Global Impression (SGI) and a Clinician Global Impression (CGI). At each visit, the detailed evaluation schedule is seen in table 23. At visit 3, a new experimental product will be dispensed upon completion of the study procedure.

At visit 4, at the completion of the study program (end of phase 1), a1 week washout period will begin and all subjects will take placebo. Subjects will be provided with a single blind placebo and discharged home, instructing them to take the 1 st dose at the evening, and about 1 dose every 12 hours until the morning of the next visit.

Visit 5 (day 22) marked the beginning of phase 2 for both groups. During this visit, subjects will have a new set of clinical assessments, after which they will begin cross-treatment according to the order established for each group. During this time, at two additional weekly visits: a similar set of evaluations was performed for visit 6 (day 29) and visit 7 (day 36). In addition to visit 7 at the end of phase 2, at each visit, at the completion of the study procedure, the test product will be dispensed.

The phone tracks visits, visit 8 (day 43), which will occur after 1 week to evaluate adverse events. After tracking the call, study participation will be completed.

Subjects will be indicated to have a dose administration schedule for the test product taken at the morning dose 2 hours prior to the start of the scheduled study evaluation during the treatment evaluation visit (visits 3,4, 6 and 7) to correspond to approximately the peak plasma concentration of daltepid-ER.

To monitor the compliance of the treatment, blood samples will be obtained to determine plasma study drug concentrations at all clinical visits following the screening visit.

The study design is shown diagrammatically in fig. 10. Table 23 provides a by-visit schedule for the study program and a description of the program is seen below.

Table 23: evaluation schedule

¹All measurements will be taken before the start of treatment 1 on day 1 and the start of cross-treatment on day 22.

²Complete body, height and weight examinations were performed only at screening; a simple physical examination is performed at a subsequent visit.

³All samples were taken after the clinical functional assessment had been performed.

⁴If the result is positive, confirmation is requiredCulturing

⁵Test each hand, test the dominant hand first

⁶Three tests were performed for each hand, grip test, finger pinch, key pinch and palm test

⁷Test product was dispensed for 1 week and subjects were instructed to take 1 dose about every 12 hours at home, starting at the night of visit

⁸Phone tracking access to evaluate adverse events

6.16.4 selection and withdrawal of subjects

(a) Inclusion criteria

Subjects can be included in the study if they meet all of the following criteria:

1. history of stable sensorimotor deficiency due to ischemic stroke, as confirmed by an evaluator through previous imaging findings (MRI/CT scan)

2. More than or equal to 6 months after stroke

3. Including males and females aged 18 to 85 years

4. Body Mass Index (BMI) of 18.0-35kg/m²Is selected within the range of

5. Ampyra, Davapridine, aminopyridine or 4-aminopyridine (4AP) have not been used previously

6. Has sufficient walking ability to complete T25FW at screening visit and all other visits as needed

7. Fugl-Meyer score of lower limb movement is less than or equal to 27

8. Ability to perform all required research procedures

9. Sufficient cognitive ability to provide informed consent, as determined by the rater.

10. Stable concomitant drug therapy regimens within 4 week screening visit.

(b) Exclusion criteria

Subjects who met any of the following exclusion criteria were not eligible for participation in the study:

1. sexually active women with childbearing potential who are not surgically sterilized, have less than two years post-menopausal, or have not used an effective method of birth control

2. Pregnant or lactating

3. History of epilepsy, except simple fever epilepsy

4. Moderate or severe renal injury, as defined by creatinine clearance ≦ 50 mL/min calculated using the Cockcroft-Gault equation

5. Evidence of active Urinary Tract Infection (UTI) at or 4 weeks prior to screening visit

6. Prescribed drug treatment regimens or therapies initiated within 4 weeks prior to the screening visit, and/or concomitant drug treatment regimens or concomitant therapies expected to change over the course of the study

7. Baclofen or tizanidine starting within 4 weeks prior to the screening visit or any change in dosage regimen within 4 weeks prior to the screening visit

8. Serotonin Reuptake Inhibitors (SSRIs) beginning within 3 months prior to the screening visit, or any change in dosage regimen within 3 months prior to the screening visit

9. Botulinum toxin use within 2 months prior to screening visit

10. History of drug abuse or alcohol abuse over the past year

11. Over the past 6 months, either limb was subjected to an orthopaedic surgical procedure

12. The subjects had abnormal laboratory values that, at the discretion of the evaluator, were clinically significant and likely affected the ability of the subjects to safely complete the study

13. Unstable angina, uncontrolled hypertension or any other significant cardiovascular abnormality as deemed by the evaluator

14. Major depression as indicated by a score of ≧ 30 on the Bull's Depression Scale (BDI)

15. It will interfere with the performance of the study or the interpretation of the results of the study, depending on any other medical condition the evaluator judges

16. The experimental intervention test was performed within 4 weeks prior to the screening visit

17. Diagnosis of multiple sclerosis

(c) Subject withdrawal criteria

Exit criteria are optional, including one or more of the following reasons:

● the subject experiences an adverse event (e.g., seizure)

● pregnancy

● Subjects were not matched to the protocol

● failure of the subject to follow

● the subject is alcoholizing or abusing a drug or is no longer meeting another eligibility criterion

6.16.5 treatment of a subject

(a) The treatment to be applied

Each subject will receive 28 doses of (a)10mg of daltepyridine-ER, and 42 doses of (B) placebo (including 14 doses during the placebo washout period). The tablets will be taken with water at home. The treatment order will be determined as described in section (b) below.

Test products will be dispensed to subjects after evaluation is complete at visits 2,3, 4,5 and 6. The subject will be instructed to take the 1 st dose at the visit evening and, after about 12 hours, the next dose in the following morning. Subjects will be instructed to continue dosing every 12 hours at as consistent a time as possible. Subjects will be informed that they cannot compensate for the missed dose.

The last dose dispensed each time will be the dose taken in the morning of the next scheduled visit. The subject will be indicated to have access to the treatment assessment: visit 3,4, 6 and 7 sessions, a dose administration schedule for the morning dose of the test product taken 2 hours prior to the start of the scheduled study evaluation.

(b) Method of assigning subjects to treatment groups

At visit 1, according to the random scheme generated before the study began, the data was written at 2: a ratio of 1 randomly assigned subjects to one of two blind treatment sequences (a or B).

A: placebo followed by Davalpyridine-ER

B: Davalopyridine-ER followed by placebo

(c) Blind method

Drug administration will be double blind, indicating that the treatment sequence is unknown to both the subject and the point of care personnel.

The washout period will be single blind, meaning that the test point personnel will know that placebo is being administered during this period, but the subject is unaware.

(d) Therapeutic compliance

The subject will be encouraged to take all prescribed doses. Treatment compliance will be monitored by a list of drug tablet counts in the returned vials and by obtaining blood samples to determine plasma daltepyridine concentration for each treatment session. Any reason for the mismatch will be noted.

(e) Prior and concomitant medications

For the purpose of maintaining stable symptoms, during the study and also for some time before the study, the following drugs were excluded:

● beginning less than 4 weeks prior to a screening visit or varying doses of baclofen or tizanidine

● SSRI with onset or dose change less than 3 months prior to screening visit

● botulinum toxin administered less than 2 months prior to screening visit

● other prescribed drugs (or therapies) that begin or change less than 4 weeks prior to the screening visit

In addition to safety as required for the subject, concomitant therapy will not be altered during the study.

6.16.6 description of the test products

Active agent(s): commercial drugs will be used. AMPYRA (dalvapyridine) sustained release tablets are white to off-white, biconvex, ovoid, coated films, non-scored tablets with flat edges having an "a 10" notch on one side containing 10mg of dalvapyridine. Inactive ingredients include colloidal silicon dioxide, hydroxypropyl methylcellulose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, and titanium dioxide.

Placebo: the placebo tablets have the same physical form as the AMPYRA tablets and contain the same inactive ingredients.

6.16.7 research procedure

The following section describes the baseline and clinical function measurements that will be obtained in this study. A detailed program schedule for the study visits is provided below and summarized in table 23.

Prior to participating in any study procedure, subjects had to provide written informed consent.

(a) Plasma Davalpyridine concentration

After all clinical functional evaluations are completed, blood samples will be obtained for determination of plasma daltepyridine concentrations. The purpose of these measurements is to evaluate treatment compliance. A minimum of 7mL of whole blood was collected into appropriately labeled heparin tubes and kept at low temperature (i.e., on wet ice) until centrifugation. Immediately after collection, the tubes were centrifuged at low speed and approximately 3mL of plasma was transferred from each sample to the labeled tubes. Plasma was stored at-20 ℃ until required for transport to a central laboratory. At this point, frozen plasma samples were collected together and sent to a designated central laboratory by express overnight on dry ice in a warm-keeping container.

(b) Clinical evaluation

Timing 25 feet walking (T25FW)

The T25FW test is a quantitative measure of walking function. Subjects were instructed to walk as quickly as possible from one end of a clearly marked, unobstructed 25 foot course to the other. T25FW (Fischer J et al, National Multiple Sclerosis society.2001; 1-410) will be performed according to the detailed instructions provided in the "Administration and Scoring Manual" published by the National Multiple sciences society. The subject will stand with their toe on the marked departure line and begin timing with any portion of the subject's foot exceeding the line. The timing is ended when any part of the subject's foot exceeds the marked finish line. Using the stopwatch provided for this study, time was recorded in seconds and rounded to the nearest tenth of a second. The task was performed again by letting the subject walk back from the same distance with a rest period of up to 5 minutes allowed between the two trials. The subject may use a suitable aid if desired. Subjects were asked to maintain their normal activities and were unable to practice or practice the measurements in order to unfairly improve their performance scores between visits. For T25FW, the same test room and the same designated area were used as much as possible at each evaluation. The possibility of external disturbances is kept to a minimum as possible.

Standard data on walking speed is available (Bohannon R., Age and ageng.1997; 26: 15-19). For subjects ≧ 18 years and <20 years of age, standard data for the 20-30 year age group (20s decade group) will be used.

Box and block testing

The box and block test (Mathiowetz V et al, Am J OccupTher. 1985; 36(6):386-391) has been used as an effective and reliable hand agility measurement. Subjects were instructed to quickly pick up the blocks from one side of the box, one at a time, and transport each block through the spacer to the other side of the box and throw the block down. This test was originally developed to evaluate the rough dexterity of the hands of adults with cerebral palsy. Data is available for normal adults (Bohannon R., Agean Ageing. 1997; 26: 15-19). For subjects aged 18 and <20 years, standard data for the 20-24 year age group will be used.

The building block and box test is carried out before the squeezing test and the grip test to reduce the fatigue influence to the maximum extent. Dominant and non-dominant hands will be tested, starting with the dominant hand.

Hand strength by grip and pinch tests

The grip strength test (Mathiowetz V, et al, Arch Phys Med Rehabil.1985; 66:69-72) is used as a simple, effective and reliable measure to identify hand strength problems, to detect changes that may be caused by occupational therapy programs, the course of disease or injury, or to show the relationship between patient strength and the general population. Hand strength was measured using a dynamometer.

The pinch test (Mathiowetz V, et al, Arch Phys Med Rehabil.1985; 66:69-72) is used as a simple, effective and reliable measure to identify pinch force problems, to detect changes that may be caused by occupational therapy programs, the course of disease or injury, or to show the relationship between patient force and the general population. It has three parts, finger-tip kneading, key kneading and palm kneading. The pinch strength was measured using a finger-pinch meter.

Three trials were performed for grip strength test, finger pinch, key pinch and palm test of each hand at each measurement.

Standard data for grip and pinch tests are available in adults (Mathiowetz V, et al, Arch PhysMed Rehabil.1985; 66: 69-72). For subjects aged 18 and <20 years, standard data for the 20-24 year age group will be used.

Fugl-Meyer evaluation (FMA)

FMA is a performance-based impairment assessment designed to assess motor function, balance, sensation, and joint function in patients with hemiparalysis after stroke (Fugl-Meyer AR et al, Scand J Rehabil Med.1975; 7(1): 13-31). For this study, the areas of upper limb (UE) motor function, lower Limb (LE) motor function and sensation will be evaluated (see table 24).

Table 24: Fugl-Meyer evaluation score table

Items were scored on a 3-point scale of severity. The total score for each region may be determined separately, and the total UE and LE scores may be combined to yield a total motion score.

Disability evaluation scale (DAS)

Optionally, a DAS is performed.

DAS was developed to evaluate the impairment in 4 functional regions affected in post-stroke tonic upper limb patients in general: personal hygiene, dressing, pain and limb position. The clinician will score the level of injury in the subject in each of these areas using a 4-point scale ranging from "non-incapacitated" to "severely incapacitated". Evaluation of 4 functional areas will be performed according to the following guidelines (Brashear A et al, ArchPhys Med Rehabil.2002; 83(10): 1349-54):

sanitation:the grader will evaluate the extent of maceration, ulceration and/or palm infection in the patient's daily life; palm and hand cleanliness; the cleanliness is easy; ease of nail trimming; and the degree of interference caused by hygiene-related disability.

Dressing:the grader will evaluate the difficulty or ease with which the patient can wear clothes (e.g., shirt, jacket, gloves) in his or her daily routine and the degree of interference caused by the inability to wear the clothes.

The position of the limb:the grader will evaluate the amount of upper limb abnormal position.

Pain:the grader will evaluate the intensity of pain or discomfort associated with upper limb rigidity.

Each of the 4 functional regions will be scored using the following scale: 0 ═ failure; 1 ═ mild disability (evident, but not significantly interfering with normal activities); 2-moderate disability (normal activity requires increased effort and assistance); severe disability (restricted normal activity).

Measurement of Functional Independence (FIM)

The FIM scale is a widely used disability assessment that measures the degree of assistance an individual requires to perform Activities of Daily Living (ADL). It consists of 18 items: 13 are in the physical domain and 5 are in the cognitive domain. The scoring was based on direct observation by the clinician, with each item being scored on a 7-part scale ranging from "fully assisted" to "fully independent". The evaluated ranges were: eating, dressing, bathing, upper body dressing, lower body dressing, toileting, bladder control, bowel control, bed to chair movement, toilet transfer, shower transfer, mobility (walking or wheelchair level), going up and down stairs, cognitive understanding, expression, social interaction, problem solving, memory.

The scoring criteria are as follows (see the rehabilitation measures database web page):

7	is completely independent
		6	Condition independent
5	Supervision or assistance
		4	Minimal contact assistance (the patient can be 75% or more on the task)
3	Moderate assistance (patients can perform 50% to 74% of the tasks)
		2	Maximum assistance (patient can do 25% to 49% of the task)
1	Complete assistance

Subject Global Impression (SGI)

SGI is a common measure of treatment response and uses a 7-part scale ranging from "poor" to "good" by querying the subject to score the effect of the drug on their physical fitness in the last week of the trial.

The subject is given a form to complete, which shows: "how we want to know what you feel about the effect of the study medication on your physical well-being". During the last 7 days, how do you feel for the effect of the study drug. The subjects gave the following choice for the response: "extremely bad", "most disappointing", "neutral/mixed", "most satisfactory", "good" and "very good". The subject was asked to interpret the response on his own.

Clinician Global Impression (CGI)

CGI is a common measure of treatment response that queries clinicians to provide an overall impression of changes in neurological and general health status of a subject after treatment as compared to baseline subject condition (and not as compared to a previous visit). CGI is scored according to a 7-part scale ranging from "much improved" to "much worse".

The clinician is issued a form to complete, the form illustrating: "generally, how will you score the subject's current neurological status with respect to their screening visit, taking into account the subject's symptoms and other neurological functions? Please consider only neurological changes, regardless of other factors. The clinician gives the following choices for response: "much improved", "some improved", "no change", "some deteriorated", "much deteriorated", and "much deteriorated". The clinician is asked to interpret any indication of change, if possible.

Baishi Depression Scale (BDI)

BDI (see table 25) is a widely used self-reporting depression questionnaire that measures the severity of depressive symptoms. Each of the 21 items was scored on a 4-degree scale ranging from minimal to severe. Subjects with major depression as indicated by a score at screening of ≧ 30 were excluded from the study. BDI will also be implemented at other visits as one of the clinical evaluations.

Table 25: baishi Depression Scale (BDI)

(c) Study sequence

The following section describes the evaluations to be performed at each clinical visit during the study.

Visit 1, day-14 to day-1 (screening visit)

The evaluator will evaluate the eligibility of the study after performing the following procedure. These procedures will be completed within 14 days prior to random access. They will proceed in the following order.

● obtaining informed consent for signature

● complete medical history including demographic information

● examination of previous and concomitant medication

● Subjects completed BDI

● complete the full body examination

● routine sitting posture vital sign measurements are made, including height and weight.

● calculating BMI

● implementation of T25FW

● implementation of the building blocks and Box test (dominant hand and non-dominant hand, dominant hand first)

● grip strength tests, finger pinch, key pinch and palm pinch tests were performed. These tests will be performed three times per hand.

● implementing FMA, DAS and FIM

● blood and urine samples were collected for laboratory evaluation (SMA-12, calculated creatinine clearance, urinalysis and urine pregnancy test for women with possibility of delivery)

● checking for adverse events

● if the subject is eligible, the subject is scheduled to return to the test center within 14 days.

Visit 2, day 1 (random visit, beginning of phase 1)

The following evaluations and procedures will be performed in the following order:

● carrying out simple physical examination

● performing sitting posture vital sign measurement

● implementation of T25FW

● implementation of the building blocks and Box test (dominant hand and non-dominant hand, dominant hand first)

● grip strength tests, finger pinch, key pinch and palm pinch tests were performed. These tests will be performed three times per hand.

● implementing FMA, DAS and FIM

● Subjects completed BDI

● obtaining blood samples for plasma Davalpyridine concentration

● urine pregnancy test of women with possibility of delivery

● examination for adverse events and concomitant medications

● are randomly assigned to one of two processing sequences

● dispensed 1 week double blind trial product indicating that the 1 st dose was taken the evening. See section 6.16.5(a) for additional instructions regarding subject dosage regimens.

● the subjects were discharged from the hospital and scheduled for the next visit within 1 week on a day and time (+ -1 day).

Visit 3, 8 days

The following evaluations and procedures will be performed in the following order:

● carrying out simple physical examination

● performing sitting posture vital sign measurement

● Subjects completed SGI

● implementation of T25FW

● implementation of the building blocks and Box test (dominant hand and non-dominant hand, dominant hand first)

● grip strength tests, finger pinch, key pinch and palm pinch tests were performed. These tests will be performed three times per hand.

● implementing FMA, DAS and FIM

● complete CGI

● obtaining blood samples for plasma Davalpyridine concentration

● examination for adverse events and concomitant medications

● collecting the last visited test product and performing drug accountability

● dispensed a new 1 week double blind trial product that was dispensed, indicating that the dose was taken about 12 hours after the last 1 dose. See section 6.16.5(a) for additional instructions regarding subject dosage regimens.

● the subjects were discharged from the hospital and scheduled for the next visit within 1 week on a day and time (+ -1 day).

Visit 4, day 15 (end of 1, beginning of washout period)

The following evaluations and procedures will be performed in the following order:

● carrying out simple physical examination

● performing sitting posture vital sign measurement

● Subjects completed SGI

● implementation of T25FW

● implementation of the building blocks and Box test (dominant hand and non-dominant hand, dominant hand first)

● grip strength tests, finger pinch, key pinch and palm pinch tests were performed. These tests will be performed three times per hand.

● implementing FMA, DAS and FIM

● complete CGI

● Subjects completed BDI

● blood samples for plasma Davalpyridine concentration and SMA-12

● examination for adverse events and concomitant medications

● collecting the last visited test product and performing drug accountability

● A1 week single-blind placebo was dispensed indicating that the 1 st dose was taken the evening. See section 6.16.5(a) for additional instructions regarding subject dosage regimens.

● the subjects were discharged from the hospital and scheduled for the next visit within 1 week on a day and time (+ -1 day).

Visit 5, day 22 (end of washout period, beginning of period 2)

The following evaluations and procedures will be performed in the following order:

● carrying out simple physical examination

● performing sitting posture vital sign measurement

● Subjects completed SGI

● implementation of T25FW

● implementation of the building blocks and Box test (dominant hand and non-dominant hand, dominant hand first)

● grip strength tests, finger pinch, key pinch and palm pinch tests were performed. These tests will be performed three times per hand.

● implementing FMA, DAS and FIM

● complete CGI

● Subjects completed BDI

● obtaining blood samples for plasma Davalpyridine concentration

● examination for adverse events and concomitant medications

● collecting the last visited test product and performing drug accountability

● Cross-treatments were dispensed for 1 week indicating the 1 st dose to be taken the evening. See section 6.16.5(a) for additional instructions regarding subject dosage regimens.

● the subjects were discharged from the hospital and scheduled for the next visit within 1 week on a day and time (+ -1 day).

Visit 6, day 29

The following evaluations and procedures will be performed in the following order:

● carrying out simple physical examination

● performing sitting posture vital sign measurement

● Subjects completed SGI

● implementation of T25FW

● implementation of the building blocks and Box test (dominant hand and non-dominant hand, dominant hand first)

● grip strength tests, finger pinch, key pinch and palm pinch tests were performed. These tests will be performed three times per hand.

● implementing FMA, DAS and FIM

● complete CGI

● obtaining blood samples for plasma Davalpyridine concentration

● examination for adverse events and concomitant medications

● collecting the last visited test product and performing drug accountability

● A new 1 week cross-treatment medication was dispensed indicating that the dose was taken about 12 hours after the last 1 dose in the evening. See section 6.16.5(a) for additional instructions regarding subject dosage regimens.

● the subjects were discharged from the hospital and scheduled for the next visit within 1 week on a day and time (+ -1 day).

Visit 7, day 36 (end of 2 period)

The following evaluations and procedures will be performed in the following order:

● carrying out simple physical examination

● performing sitting posture vital sign measurement

● Subjects completed SGI

● implementation of T25FW

● implementation of the building blocks and Box test (dominant hand and non-dominant hand, dominant hand first)

● grip strength tests, finger pinch, key pinch and palm pinch tests were performed. These tests will be performed three times per hand.

● implementing FMA, DAS and FIM

● complete CGI

● Subjects completed BDI

● blood samples for plasma Davalpyridine concentration and SMA-12

● examination for adverse events and concomitant medications

● collect the last visited test product and perform drug accountability. The test product is no longer dispensed.

● the subjects were discharged from the hospital and scheduled for the next visit (telephone visit) within 1 week on a day and time (+ -1 day).

Telephone tracking, day 43. + -.1

The research point will make a follow-up phone call to check for any adverse events or changes in medication. Tracking calls may be performed up to 2 days around day 43 due to weekends/holidays.

● evaluation of end condition

6.16.8 statistical results

(a) Statistical efficacy

Sixty-six (66) subjects will be enrolled at a rate of 2: the ratio of 1 is randomly assigned to one of two treatment sequences: placebo followed by dabigatran-ER (sequence A) or dabigatran-ER followed by placebo (sequence B). This sample volume will provide sufficient planning estimates to facilitate future study design.

(b) Derived endpoint and data processing

The baseline for analysis will be defined as the last missed assessment before the 1 st dose of double-blind drug.

Based on the date of informed consent, the age and time from ischemic stroke will be calculated

The age will be calculated as: age ═ date informed consent-date of birth ]/365.25, rounded to the previous integer.

The number of days from ischemic stroke will be calculated as: day as date of informed consent-stroke date

The derivation of the baseline measurements will be performed in accordance with the derivation listed in the section entitled "derived variables and data processing" below.

(c) Analysis of functional evaluation

The computational details of all applicable variable derivations in this section can be found in the section entitled "derivations variables and data processing" below.

Functional evaluation

In this study, the function of the study was evaluated as:

● Walking speed measured by timed 25 foot walk test (T25FW)

● hand agility measurement through Block and Box testing

● measurement of hand strength by grip and pinch tests

● Total motor function score and Single movement score on Fugl-Meyer evaluation (FMA):

-upper limb function

Lower limb function

Upper limb stiffness measured by Disability Assessment Scale (DAS)

● measurement of assistance required to perform Activities of Daily Living (ADL) by a Function Independent Measurement (FIM) scale

● Subject Global Impression (SGI) Scale

● Clinician Global Impression (CGI) Scale

● Depression measured by the white Depression Scale (BDI)

Derived variables and data processing

The baseline for analysis will be defined as the last missed assessment before the 1 st dose of double-blind drug.

Walking speed

At each visit, two T25FW test trials will be performed. The walking speed (feet/second) for a single trial was derived by multiplying the inverse of the time (seconds) to complete walking by 25 (feet). The walking speed for a particular study visit was derived by calculating the average of the walking speeds of trial 1 and trial 2 for that study visit. If one of the trials is missed, then the walking speed for that visit will be that of the missed trial.

Grip and pinch test

Three trials will be performed for grip and pinch tests, respectively, at each visit. Responses at a particular study visit are the average of three trials of a particular test. The grip test and pinch test will be summarized with the dominant hand and the non-dominant hand, respectively.

Building block and box testing

The response to the block and box test was the number of blocks transported to the other side of the compartment in 60 seconds. No derivation is required for the reaction variables. The block and box tests will be summarized with dominant and non-dominant hands, respectively.

Fugl-Meyer evaluation (FMA)

FMA is a measure of motor and sensory impairment of the upper and lower limbs and comprises 155 items out of 5 fields. The score for each domain may be determined by adding the scores of the related terms. The overall FMA score may be generated by adding the fractional scores of the various domains. The FMA overall score and the fractional scores for each domain will be summarized separately.

Disability evaluation scale (DAS)

DAS was used to evaluate the damage in the 4 functional areas that are usually affected in patients with stiff upper limbs after stroke. 4 functional regions will be analyzed separately. No derivation is required for the reaction variables.

Function Independent Measurement (FIM) scale

The FIM scale is an assessment of physical and cognitive disability and comprises 18 items. For each visit, the reaction on the FIM scale is the sum of the individual reactions to the 18 items. The total score may range between 18 (lowest functional level) to 126 (highest functional level).

Subject Global Impression (SGI) Scale

For each visit, the response on SGI is the subject's score for the last week of the test product on his physical fitness. No derivation is required for the reaction variables.

Clinician Global Impression (CGI) scale

For each visit, the response on CGI is an overall impression of the clinician's change in neurological condition and general health condition of the subject after treatment with the test product as compared to baseline. No derivation is required for the reaction variables.

Baishi Depression Scale (BDI)

BDI is a self-reported 21-item depression questionnaire that measures the severity of depressive symptoms. For each access, the reaction on the BDI is the sum of the individual reactions to the 21 items. The total score may range from 0 to 63.

Statistical method

Analysis will be performed to determine the effect of dalvapyridine-ER on functional assessment. For each functional assessment, in addition to SGI and CGI, the change from baseline in the same subject will be calculated for each treatment period.

Stage 1: visit 4 evaluation-visit 2 evaluation

Stage 2: visit 7 rating-visit 5 rating

For SGI and CGI, visit 4 rating and visit 7 rating will be used in the analysis.

For any functional rating, if the visit 4 rating is missed, then the visit 3 rating estimate will be used. If an access 7 rating is missed, then an access 6 rating evaluation will be used.

For all clinical measurements except SGI and CGI, the following two types of analysis will be performed. Type 1 will be based on the use of 44 subjects randomized to order a, the difference in change from baseline among subjects for phase 1 (placebo) versus phase 2 (da vap-ER). The type 2 analysis that will compare changes from baseline between two treatments using the paired t-test will be based on comparisons of changes from baseline between treatment groups within only 1 phase. Changes from baseline between the two treatments will be compared using the two-sample t-test

For SGI and CGI, two types of analysis will be performed as well. However, the SGI and CGI results of visit 4 and visit 7 will be used for analysis rather than based on changes from baseline. Statistical methods as described above will also be used for the analysis of SGIs and CGIs.

6.16.9 reference to example 16

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●Adams HP,Del Zoppo G,Alberts M,et al.Guidelines for the earlymanagement of adults with ischemic stroke:A guideline from the American HeartAssociation/American Sroke Association Stroke Council,Clinical CardiologyCouncil,Cardiovascular Radiology and Intervention Council,and theAtherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes inresearch Interdisciplinary Working Groups.Stroke2007；38:1655-1711.

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●Goodman AD,Brown TR,Cohen JA,et al.Dose comparison trial ofsustained-release fampridine in multiple sclerosis.Neurology.2008；71:1134-1141.

●Goodman AD,Brown TR,Krupp LB,et al.Sustained-release oralfampridine in multiple sclerosis:a randomized,double-blind,controlledtrial.Lancet.2009；373:732-38.

●Goodman AD,Brown TR,Edwards KR,et al.A Phase2trial of extendedrelease oral dalfmapridine in multiple sclerosis.Ann Neurol.2010；373:494-50.

●Menon B,Shorvon S.Ischaemic stroke in adults and epilepsy.EpilepsyRes.2009；87:1-11.

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●Szaflarski J,Rackley A,Kleindorfer D,et al.Incidence of seizures inthe acute phase of stroke:A population-based study.Epilepsia.2008；49(6):974-981.

●Krakow K,Sitzer M,Rosenow F,et al.Predictors of acute post-strokeseizures.Cerebrovasc Dis.2010；30:584-589.

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6.17Example 17: effect of oral administration of 4-AP: function was restored in rats after MCA occlusion (MCAO). Blind method and carrier Dose escalation studies of body controls.

In rats with stable motor deficits, the ability of 4-AP to promote functional sensorimotor improvement following ischemic stroke was evaluated at a time distant from their ischemic event. The animal model for ischemic stroke, i.e. the MCAO model, used in this example was the same as the animal model described in example 2.

In the MCAO model, recovery began to reach plateau by 4 weeks after MCAO, at which time there was still a measurable defect in sensorimotor function. However, endogenous recovery may continue slowly 4 weeks after MCAO. For these reasons, treatment in this example was initiated 56 days post MCAO, a time point further from the initial ischemic event, allowing the animal to reach a more stable level of sensorimotor deficiency following endogenous recovery.

Design of experiments

In this experiment, Sprague Dawley rats were operated to cause Middle Cerebral Artery Occlusion (MCAO) using either vehicle (water) or 4-aminopyridine treatment as described below and behavioral assessments were performed as described below.

Animals: 30 male Sprague Dawley rats, 300-275 g (obtained from Charles river laboratories, coming to the laboratory 7-10 days before surgery, weighing 250-275g) were used. Animals were randomly assigned to treatment groups.

The terms: the terminology used for study days is as follows: day 0 is the day of MCAO and the subsequent days are numbered sequentially (day 1, day 2, day 3, etc.); day-1 represents the day before MCAO.

Grouping details: the amount of time required for some procedures in this study required the division of 2 treatment groups (see table 26 below) into 4 working groups. The 6 animals received stroke surgery daily. If the animal died within the 8 day surgical period of the study, it was replaced with a backup. If not, the animal is not replaced. Most animal deaths (< 5% of total) occurred within a period of time from immediately to 7 days post op.

Anesthesia: anesthesia was performed as described above in example 2.

Temperature: 37.0 +/-1 ℃.

And (3) surgical procedures: the procedure was performed as described above in example 2.

And (3) monitoring after operation: post-operative monitoring was performed as described in example 2 above.

Feeding, surgery and injection schedules: the feeding, surgery and injection schedules were the same as described in example 2 above.

Treatment and dosage: rats were treated according to the treatment schedule shown in table 26. Table 27 shows the doses. 4-aminopyridine was dissolved in water for injection (WFI, Cellgro) and sterile filtered. Solutions of 0.25mg/mL, 0.5mg/mL, and 1.0mg/mL 4-aminopyridine were delivered by gavage at 2mL/kg, resulting in final doses of 0.5mg/kg, 1mg/kg, or 2.0mg/kg, respectively. The vehicle control treatment was WFI delivered at 2mL/kg by gavage. Animals received gavage of solution (2mL/kg) at approximately 12 hour intervals beginning on day 56 post MCAO. Vehicle controls were treated with water for all doses on days 56-65. For the treatment group, 6 doses of 4-aminopyridine were delivered at 0.5mg/kg on days 56-59, followed by 6 doses at 1.0mg/kg on days 59-62 and 6 doses at 2.0mg/kg on days 62-65. Animals in all groups were untreated during days 66-70. p.o. ═ oral.

Table 26:

table 27:

treatment ID	Treatment of
		V	Carrier (Water)
Low dose	0.5mg/kg4-AP，b.i.d,p.o.
		Middle dose	1mg/kg4-AP，b.i.d.,p.o.
High dose	2.0mg/kg4-AP，b.i.d,p.o.

Treatment group: animals were subjected to MCAO surgery and allowed to recover for 56 days. Animals were then randomly assigned to groups 2 based on their baseline behavior. Oral dose administration was started 2 times 1 day on the evening 56 days after MCAO. 1 hour after dose administration, behavioral testing of the dose administration period was initiated. Blood was collected via the saphenous vein just prior to or during the treatment period after the behavioral testing (90 minutes after dose administration). All dose administrations were performed by gavage, with a volume not exceeding 2 mL/kg.

Blood sampling: a 300 microliter blood sample was taken from the saphenous vein of each animal prior to the 1 st dose on day 56, and then at 90 minutes after the 6 th dose at each dose level. Blood was collected, centrifuged, stored and analyzed as described in example 2 above.

Behavior test details: behavioral assessments were performed by evaluators whose treatment assignment was unknown. Using limb placement and body swing behavior testing, blind evaluations of sensorimotor function were performed immediately prior to MCAO surgery, 24 hours after MCAO surgery, and weekly thereafter, up to phase 1 of dose administration. As described above, the behavior assessment time is accurately made as a function of the dose administration time. Animals were given a first dose and behavioral assessment was initiated 60 minutes later. Animals were tested 1 hour after the 6 th dose (days 59, 62 and 65) and at the end of the 5 day washout period on day 70 at each dose level.

Placing the limbs: evaluation was performed on day-1 (before operation), day 1, day 7, day 14, day 21, day 28, day 35, day 42, day 49, day 56, day 59, day 62, day 65, and day 70. The limb placement test is divided into forelimb and hindlimb tests. The forelimb and hindlimb placement tests and the scoring of these tests are described in example 2 above.

Body swinging: evaluation was performed on day-1 (before operation), day 1, day 7, day 14, day 21, day 28, day 35, day 42, day 49, day 56, day 59, day 62, day 65, and day 70. The body swing test and scoring of the test is described in example 2 above.

Euthanasia and post-mortem treatments: on day 70 post MCAO, rats were anesthetized as described in example 2 above.

Infarct measurement: infarct measurements were performed as described in example 2 above.

The statistical method comprises the following steps: at each time point evaluated after dose administration, changes from pretreatment baseline values (day 56) were calculated for each behavioral score. The mean behavior parameter data were subjected to analysis of variance (ANOVA). Infarct volume data was analyzed by ANOVA. All data are expressed as mean ± SEM.

Regulatory compliance: the regulatory compliance of this study was the same as described in example 2 above.

Quality Assurance (QA): QA for this study was the same as described in example 2 above.

Results

Two groups of animals (vehicle and 4-aminopyridine treatment) showed a typical recovery response to MCAO-induced ischemia with a normal score of 0 just before surgery (day-1) and complete loss of function within 24 hours after occlusion (day 1) (forelimb, score 12; hindlimb, 6). During the subsequent 8-week untreated period, the forelimb and hindlimb scores improved to about 4.5 and 2.5, respectively, and a restored plateau level was reached (see fig. 11 and 12). Specifically, after complete loss of function, the animal partially recovered and reached a plateau at about 30 days. The animals were maintained at this functional level until day 56 of treatment initiation.

Sensorimotor performance was evaluated using forelimb and hindlimb placement and body swing testing. The forelimb placement test shows the effect of treatment on forelimb function (see figure 11). Hindlimb placement tests showed the effect of treatment on hindlimb function (see figure 12). The body swing test shows the effect of treatment on systemic control (see figure 13).

The vehicle group showed small and statistically insignificant behavioral changes compared to the last evaluation before the start of the dose. In contrast, animals receiving 0.5mg/kg 4-aminopyridine (low dose) significantly improved forelimb placement (p <0.001) compared to vehicle (see figure 11, day 59). Hind limb placement scores were improved with low doses but not significant (see figure 12, day 59). Increasing the dose of 4-aminopyridine to 1mg/kg resulted in measurable improvement in forelimb and hindlimb tests compared to vehicle (p <0.001 and p <0.05, fig. 11 and 12, day 62, respectively). The final dose escalation to 2mg/kg 4-aminopyridine was associated with significant improvement in forelimb and hindlimb function compared to vehicle treated animals (p <0.0001 and p <0.001, fig. 11 and 12, day 65, respectively). When treatment was stopped for 5 days, the improved raw score was partially reduced, although hind limb scores were still greater than the vehicle treated group (p <0.05, fig. 12, day 70). It is likely that the extended and stable dose period required additional time to clear completely compared to the vehicle treated group. However, given the short serum half-life of 4-aminopyridine, it seems more likely that the training effect of repeated tests occurs over a relatively short period of time. Since only slight improvement was observed in vehicle treated animals throughout the treatment, the deficiencies of vehicle treated animals remained stable.

Thus, figure 11 shows that treatment with low, medium, or high dose 4-aminopyridine was effective in improving forelimb function in rats 8 weeks after ischemic brain injury. Figure 11 also shows that this effect is dose-responsive. It is also reversible as the effect diminishes after withdrawal. Figure 12 shows that treatment with low-dose 4-aminopyridine may be effective to improve hindlimb function in rats 8 weeks after ischemic injury, and that treatment with medium-dose or high-dose 4-aminopyridine is effective to improve hindlimb function in rats 8 weeks after ischemic brain injury. Furthermore, figure 12 shows that this effect is dose responsive due to improved performance scores resulting from treatment with high doses relative to treatment with low doses or vehicle controls. Figure 12 also shows that the effect is at least partially reversible.

At the evaluation time point, the body swing performance was not extensively characterized. Although there appeared to be a treatment effect in body swing performance at the time of initial drug evaluation compared to the pretreatment score at day 56, no conclusions could be drawn from the data as a whole based on the divergence in body swing asymmetry observed between vehicle and 4-aminopyridine groups before treatment initiation (figure 13). It should be noted that the age and size of the animals used in this example were significantly larger than the study provided in example 2, which may play a role in the general aggressiveness and performance of the animals in this particular test.

4-aminopyridine plasma levels: when the animal is treated with the vehicle, the level of 4-aminopyridine in the drawn blood sample is below the lower limit of quantitation for this method. When the animals received 4-aminopyridine, samples were drawn to confirm that exposure at the time of the behavioral test correlated appropriately with dose level. The 4-aminopyridine plasma levels are shown in table 28.

Table 28: plasma levels of 4-aminopyridine.

SE, standard deviation; lower limit of quantitation (<1.0ng/mL)

Thus, the data show a significant reversible and dose-dependent improvement in forelimb and hindlimb sensorimotor function when 4-AP is at detectable plasma levels in animals.

Table 29 shows that no difference in infarct volume was observed between the vehicle (water)) and 4-aminopyridine. Standard deviation of SE ═

Table 29:

group of	Mean (SE) infarct volume (%)
		Carrier (Water)	38.5(2.4)
4-AP	40.0(2.3)

In this study, infarct volume analysis of brain tissue was included as a typical outcome measure in preclinical stroke studies. No difference in infarct volume was observed between any of the groups within the study, and the infarct volume was similar between the studies provided in this example and example 2.

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Various references, such as patents, patent applications, and patent publications, are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims (50)

1. Use of a 4-aminopyridine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating stroke-associated sensorimotor impairment in a human patient suffering from a stroke.
2. 2. The use of claim 1, wherein the medicament is an oral formulation.
3. 3. The use of claim 2, wherein the stroke-related sensorimotor impairment is impairment of limb function, impairment of balance or coordination or impairment of systemic control.
4. 4. The use of claim 2, wherein the stroke-related sensorimotor impairment is impaired lower limb function.
5. 5. The use of claim 2, wherein the stroke-related sensorimotor impairment is upper limb functional impairment.
6. 6. The use of claim 2, wherein the stroke-related sensorimotor impairment is impairment of hand function.
7. 7. The use of claim 2, wherein the stroke-related sensorimotor impairment is fine hand coordination impairment or grip strength impairment.
8. 8. The use of claim 2, wherein the stroke-related sensorimotor impairment is impairment of jaw function.
9. 9. The use of claim 2, wherein the stroke-related sensorimotor impairment is mastication impairment, jaw bite impairment, or dysphonia.
10. 10. The use of claim 2, wherein the stroke-related sensorimotor impairment is walking impairment.
11. 11. The use of claim 10, wherein the walking impairment is a decrease in walking speed.
12. 12. Use according to claim 11, wherein the walking speed decline is shown by using a timed 25 foot walk (T25 FW).
13. 13. The use of claim 2, wherein the treatment comprises administration of the medicament during a stable chronic period following stroke.
14. 14. The use of claim 10, wherein the treatment comprises administration of the medicament during a stable chronic period following stroke.
15. 15. The use of claim 2, wherein the treatment comprises administering the medicament at or after 6 months from the patient having a stroke.
16. 16. The use of claim 10, wherein the treatment comprises administering the medicament at or after 6 months from when the patient had a stroke.
17. 17. The use of claim 2, wherein the treatment comprises administration of the medicament beginning at least 6 months from when the patient has stroke.
18. 18. The use of claim 10, wherein the treatment comprises administration of the medicament beginning at least 6 months from when the patient has stroke.
19. 19. The use of any one of claims 1-12, wherein the treatment comprises administration of the medicament beginning at least 8 months from the time the patient suffered from stroke.
20. 20. The use of any one of claims 1-12, wherein the treatment comprises administration of the medicament beginning at least 4 months from the time the patient suffered from stroke.
21. 21. The use of any one of claims 1-12, wherein the treatment comprises administration of the medicament beginning at least 8 weeks from the time the patient suffers from stroke.
22. 22. The use of any one of claims 1-12, wherein the treatment comprises administration of the medicament beginning at least 4 weeks from the time the patient suffers from stroke.
23. 23. The use of any one of claims 1-12, wherein the treatment comprises administration of the medicament beginning at least 1 week from the time the patient suffers from stroke.
24. 24. The use of any one of claims 1-12, wherein the treatment comprises administration of the medicament beginning 2-7 days from when the patient has a stroke.
25. 25. The use of any one of claims 1-12, wherein the treatment comprises starting administration of the medicament within 6 hours or within 2 days from when the patient suffers from stroke.
26. 26. Use according to any one of claims 1 to 18 which is a 4-aminopyridine other than a pharmaceutically acceptable salt thereof, for the manufacture of a medicament.
27. 27. The use of claim 26, wherein the medicament comprises 4-aminopyridine in a sustained release composition for oral administration, and wherein the stroke is ischemic stroke.
28. 28. The use according to any one of claims 1-18, wherein the medicament comprises the 4-aminopyridine or a pharmaceutically acceptable salt thereof in a sustained release composition.
29. 29. The use of claim 28, wherein the sustained release composition provides a time to maximum measured plasma concentration (T) in a human after 2 hours to 6 hours of dose administration_max)。
30. 30. The use of any one of claims 1-18, wherein the medicament comprises the 4-aminopyridine or a pharmaceutically acceptable salt thereof in an immediate release composition.
31. 31. The use of any one of claims 1-18, wherein the stroke is an ischemic stroke.
32. 32. The use of any one of claims 1-18, wherein the medicament is formulated in tablet form.
33. 33. The use of any one of claims 1-18, wherein the medicament is administered to the patient twice daily.
34. 34. The use according to any one of claims 1-18, wherein the amount of the 4-aminopyridine in the medicament is in the range of 5 to 15mg for oral administration twice daily in the form of a slow release composition.
35. 35. The use of claim 34, wherein the amount of 4-aminopyridine in the medicament is 5 mg.
36. 36. The use of claim 34, wherein the amount of 4-aminopyridine in the medicament is 7.5 mg.
37. 37. The use of claim 34, wherein the amount of 4-aminopyridine in the medicament is 10 mg.
38. 38. The use of claim 34, wherein the amount of 4-aminopyridine in the medicament is 12.5 mg.
39. 39. The use of any one of claims 1-18, wherein the medicament is administered to the patient once a day.
40. 40. The use according to any one of claims 1-18, wherein the amount of the 4-aminopyridine in the medicament is in the range of 8 to 30mg for once daily oral administration in the form of a slow release composition.
41. 41. The use of claim 40, wherein the amount of 4-aminopyridine in the medicament is 10 mg.
42. 42. The use of claim 40, wherein the amount of 4-aminopyridine in the medicament is 15 mg.
43. 43. The use according to any one of claims 1 to 18, wherein the 4-aminopyridine or a pharmaceutically acceptable salt thereof in the medicament achieves a minimum measured plasma concentration (Cmin) at steady state of at least 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20ng/ml when administered to a human_minss) Or average C_minss。
44. 44. The use according to any one of claims 1 to 18, wherein the 4-aminopyridine or a pharmaceutically acceptable salt thereof in the medicament, when administered to a human, achieves a minimum measured plasma concentration (Cmin) at steady state in the range of 12ng/ml to 20ng/ml_minss) Or average C_minss。
45. 45. The use of any one of claims 1-18, wherein the medicament does not comprise choline or a source of choline.
46. Use of 4-aminopyridine in the manufacture of a medicament for improving walking in a human patient suffering from stroke and having walking impairment, wherein the medicament comprises the 4-aminopyridine in a sustained release composition for oral administration.
47. 47. The use of claim 46, wherein the amount of 4-aminopyridine in the medicament is 10mg, administered twice daily.
48. 48. The use of claim 46 or 47, wherein the improving walking is increasing walking speed.
49. 49. The use of claim 46, wherein the medicament is for administration (i) during a stable chronic period following a stroke, or (ii) at or after 6 months from when the patient had a stroke.
50. 50. The use of any one of claims 46, 47, or 49, wherein the stroke is an ischemic stroke.