Methods and compositions for the treatment of stroke

Abstract

To provide compositions and methods for treating stroke in mammals. [Solution] The composition and method generally involve administering an effective amount of growth and differentiation factor 11 (GDF11) molecules to a subject initiated within a short time frame after a stroke event, administering GDF11 molecules at a high dose relative to the subject's body weight, and performing such an administration regimen over a short period ranging from 1 to approximately 14 days.

Description

[Technical Field]

[0001] Related Art This patent application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 026,809, filed May 19, 2020, entitled "Methods and Compositions for Treating Stroke", which is incorporated herein by reference.

[0002] Technical Field The present disclosure relates generally to methods and compositions for treating stroke in a subject, characterized by administering growth differentiation factor 11 (GDF11) to the subject. [Background Art]

[0003] Several studies have shown that certain blood-derived factors play a major role in regulating tissue homeostasis and regeneration (see, for example, Conboy et al. (2005) Nature 433(7027):760-764; Ruckh et al. (2012) Cell Stem Cell 10(1):96-103; Loffredo et al. (2013) Cell 153(4):828-839; Katsimpardi et al. (2014) Science 344(6184):630-634; and Sinha et al. (2014) Science 344(6184):649-652). One such circulating blood factor is growth and differentiation factor (GDF11), which has been demonstrated to broadly and consistently stimulate regenerative capacity in several different tissue systems, including the skin and muscle, cardiovascular system, and nervous system (see, for example, International Publication No. 2013 / 142114; International Publication No. 2014 / 168973; International Publication No. 2014 / 201143; and International Publication No. 2015 / 070076).Following the above publications, the following widespread therapeutic and regenerative effects of GDF11 have been recognized and reported: improved tubular regeneration after renal ischemia-reperfusion injury (Zhang et al (2016) Scientific Reports 6(1):34624); protection against endothelial injury (Mei et al. (2016) Molecular Therapy: the Journal of the American Society of Gene Therapy 24(11):1926-1938); enhanced myocardial regeneration after myocardial ischemia-reperfusion injury (Du et al. (2017) Basic Research in Cardiology 112(1):7); improved insulin secretion and glucose tolerance in type 2 diabetes (Li et al. (2017) Diabetes 66(7):db170086-1927); improved lung function in emphysema (COPD) (Onodera et al. (2017) Thorax April); tumor suppression in trinegative breast cancer (Bajikar et al. al. (2017) Developmental Cell 43(4):418-435); Recovery of cardiac hypertrophy (Harper et al. (2018) Circulation Research 7 Sep 2018); Promotion of diabetic limb ischemic angiogenesis and blood flow (Zhang et al. (2018) Diabetes); Improved neurobehavioral recovery and angiogenesis in cerebral ischemia-reperfusion injury (Ma et al. (2018) Brain Research Bulletin 139 (February):38-47); Rescued cognitive function and improved cerebrovascular function in Alzheimer's disease (Zhang et al. (2018) Journal of Alzheimer's Disease 62(2)); Promotion of cerebral angiogenesis, neurogenesis and functional recovery after stroke (Lu et al. (2018) Front Cell Neurosci 12:205); Prevention of intestinal inflammasome activation in ulcerative colitis (Wang et al. al. (2018) Am J Physiology Gastrointest Liver Physiology); and reduced skin inflammation in psoriasis (Wang et al. (2018) Inflammation). [Overview of the project]

[0004] In 2013, approximately 6.9 million people experienced ischemic stroke and 3.4 million experienced hemorrhagic stroke (Global Burden of Disease Study 2013 Collaborators (August 2015) The Lancet 386(9995):743-800). In 2015, stroke was the second leading cause of death after coronary artery disease, accounting for 11% of all deaths (GBD 2015 Mortality and Causes of Death Collaborators (October 2016) The Lancet 388(10053):1459-1544). It is generally accepted that the vast majority of strokes are ischemic, as it is difficult to determine whether hemorrhagic stroke originated from an ischemic event. Current medical practices and treatments for stroke are highly time-dependent. In acute ischemic stroke, existing thrombolytic methods, such as those using recombinant tissue plasminogen activator (rtPA), when administered within 3 hours of onset, provide an overall benefit of approximately 10% in terms of disability-free survival, but do not improve the chances of survival (see, for example, Wardlaw et al. (July 2014) The Cochrane Database of Systemic Reviews 7(7):CD000213 and Emberson et al. (2014) The Lancet 384(9958):1929-1935). The desirable timing for administering thrombolytic agents 3-4 hours and 30 minutes-5 hours after onset is controversial in terms of whether it yields therapeutic effects or causes the possibility of further disability. Intra-arterial fibrinolysis, which involves inserting a catheter through an artery into the brain and injecting drugs at the site of thrombolysis, has shown an effect in improving the prognosis of acute ischemic stroke (see, for example, Lee et al. (2010) Stroke 41(5):932-937).Mechanical removal of blood clots that cause ischemic stroke (mechanical thrombectomy) is another possible treatment for occlusion of the aorta, such as the middle cerebral artery. Published reviews have reported the safety and effectiveness of such procedures in reducing disability when performed within 24 hours of onset, but still not in improving the chance of survival (see, for example, Sardar et al. (2015) European Heart Journal 36(35):2373-2380; Saver et al. (2016) JAMA 316(12):1279-1288; Goyal et al. (2016) The Lancet 387(10029):1723-1731; Mistry et al. (2017) Stroke 48(9):2450-2456; and Powers et al. (2018) Stroke 49(3):e46-e110).

[0005] Given the low therapeutic efficacy of medical interventions administered immediately after a stroke event, stroke treatment has evolved into a traditional three-pillar approach, with the first pillar (prong) consisting of prevention. For example, in populations at high risk of developing myocardial infarction or other cardiovascular diseases, daily aspirin may have some preventive effect. For populations that have previously had a stroke, treatment with aspirin, clopidogrel, and dipyridamole may be effective. Modifiable risk factors such as hypertension, atrial fibrillation, high cholesterol, diabetes, and similar conditions can be treated similarly using methods well known in the medical field. The second pillar of treatment requires the medical and mechanical approaches detailed above, applied to the time frame immediately following a stroke event. The third pillar requires rehabilitation, such as physiotherapy, occupational therapy, and speech-language pathology. In this regard, stroke survivors usually show some functional recovery within the first few months, but often remain with significant neurological impairments, including motor, sensory, and cognitive impairments. Despite the generally accepted belief that nerve cells are rarely replaced and that some circuits are permanently lost due to stroke, several approaches have been outlined that seek to influence a new fourth pillar of stroke treatment, namely the potential medical repair or regeneration of nerve tissue and nervous system damaged by stroke (see, e.g., Iaci et al. (2013) Stroke 44:1942-1950 (dalfampridine) and Iaci et al. (2016) Journal of Neuroscience Research 94:253-265 (Neuregulin 1β3, Glial Growth Factor)). However, to date, there are no approved medicines for the repair or regeneration of nerve tissue and / or nervous system damaged by a stroke event.

[0006] Therefore, there is an urgent and long-standing pressing need in the art to develop and use novel methods and compositions suitable for the repair or regeneration of nerve tissue and / or the nervous system damaged by a stroke event. In response to this urgent and long-standing pressing need, the inventors have surprisingly found that administering high doses of growth and differentiation factor 11 (GDF11) molecules to subjects within approximately 12 hours to 3 days after a stroke event over a limited administration period results in a durable and sustained treatment (repair or regeneration of nerve tissue and / or the nervous system damaged by a stroke event) without concurrent side effects.

[0007] Multiple studies have reported endothelial cell angiogenesis and proliferation with daily administration of rGDF11 in rodent stroke, diabetes-related peripheral artery disease, and AD models (see, e.g., Ma et al. (2018) Brain Research Bulletin 139 (February):38-47; Lu et al. (2018) Front Cell Neurosci 12:205; and Zhang et al. (2018) Journal of Alzheimer's Disease 62(2)). In particular, in an Alzheimer's disease (AD) mouse model, daily intravenous administration of rGDF11 at 0.1 mg / kg for 28 days was found to improve cerebral vascular structure, function, and blood flow, and further rescue cognitive function (Zhang et al. (2018), ibid.). A young adult rat cerebral ischemia / reperfusion (I / R) model demonstrated that daily intravenous administration of 0.1 mg / kg of rGDF11 for 7 or 14 days after IR-improved neurological function recovery increased the number of functional microvessels in the ventricular cortex and promoted endothelial cell proliferation and neurogenesis (Ma et al. (2018), ibid.). Similarly, intraperitoneal administration of 0.1 mg / kg of rGDF11 daily for 7 days in young adult mice after I / R resulted in enhanced neurological function recovery, neurogenesis, and neurogenesis from day 14 onwards (Lu et al. 2018, ibid.). Furthermore, in a stroke model using aged mice, daily injection of 0.1 mg / kg of rGDF11 for 5 days starting 5 days after ischemic stroke resulted in significantly improved mortality (Chauhan et al. (2018), see Stroke 49). Other effects of rGDF11 treatment in this aging mouse stroke model include reduced brain tissue loss and pathological lateral ventricular dilation, increased NeuN+ neuron count, and improved motor function at 14 and 30 days post-stroke, as well as reduced gliosis, increased angiogenesis, restored white matter integrity, and synaptic plasticity (Chauhan et al. (2018), ibid.).

[0008] More recently, daily injections of rGDF11 for 28 days prior to experimental intracerebral hemorrhage (ICH) have been reported to reduce neuropathy and ICH-induced edema, as well as inflammation, apoptosis, oxidative stress, and mitochondrial damage to perivascular tissue in 24-month-old rats (see Anqi et al. (2019) J Clin Neurosci 63:182-188). Pre-treatment with a lentivirus carrying the GDF11 gene has been shown to protect against cerebral infarcts / retrograde thromboembolism (ICH) in rat models (see Zhao et al. (2020) Brain Res. 1737:146802). GDF11 lentiviral pre-treatment reduced cerebral infarct volume and apoptotic cells, promoted behavioral recovery, and facilitated neurogenesis and angiogenesis in the subventricular zone (SVZ). A single injection of 1.25 ng of rGDF11 into the lateral ventricle 24 hours after reperfusion has been reported to reduce cerebral infarct volume, decrease the number of apoptotic cells in the cerebral cortex within the ventricle, and promote behavioral recovery 5 days after transient ischemic stroke (Zhao et al. (2020), ibid.).

[0009] It should be noted that all of the above studies targeted low-dose or so-called "moderate" doses of GDF11 (either a single dose of 1.25 ng of GDF11 or daily administration of 0.1 mg / kg of GDF11 in rodents). The inventors believe this is for two reasons. Firstly, baseline GDF11 levels in mammals are approximately 3–5 ng / mL (unpublished), and therefore, past dosing paradigms have been structured to increase plasma GDF11 levels substantially rather than substantially. Secondly, and perhaps more significantly, several studies have reported that hyperphysiological or so-called "excessive" doses of GDF11 produce significant side effects, including cachexia, muscular atrophy, anorexia, and nephropathy (see, e.g., Hammers et al. (2017) EMBO Molecular Medicine 9(4):531-544; Pons et al. (2018) Surgery (May); and Jones et al. (2018) Cell Reports 22(6):1522-1530). In addition, elevated GDF11 levels have been observed in colorectal cancer (Gu et al. (2018) Cell Mol Biology Noisy-le-grand France 64:80-84). A single study in aging hippocampi reported daily administration of GDF11 at doses of ≤1 mg / kg without observing significant side effects (Ozek et al. (2018) Sci Rep 8:17293), but there is certainly a strong bias in the art regarding the targets of high-dose GDF11 use in any planned therapeutic setting.

[0010] Accordingly, a first aspect of this disclosure provides a method for treating stroke in subjects. The method requires initiating an administration regimen by administering a therapeutically effective dose of the GDF11 molecule to the subject within a time frame of approximately 12 to 72 hours after a stroke event in the subject. The GDF11 molecule is administered in an amount of at least approximately the minimum high dose of GDF11 relative to the subject's body weight over a period of 2 to approximately 14 days. In one aspect of this disclosure, the GDF11 molecule is administered over a period of 2 to approximately 7 days. In one particular aspect of this disclosure, the method requires initiating an administration regimen within approximately 12 to 24 hours after a stroke event. In another particular aspect, the administration of the GDF11 molecule is carried out over a period of 2 to 4 days. In yet another aspect of this disclosure, the GDF11 molecule is administered to the subject at a dose of approximately 1 to 2 mg / kg per day in rodent subjects or at a corresponding dose in large mammal subjects. The practice of the method described herein, namely rapid initiation of the administration regimen (within approximately 12 hours to 3 days of the stroke event), high-dose administration of the GDF11 molecule, and a limited duration of treatment (2 to 14 days), is effective in providing durable and sustained treatment in stroke patients without concurrent side effects.

[0011] The GDF11 molecule used in practice in this method is any therapeutically active form of the GDF11 molecule that may be the same or different in the composition used throughout the administration regimen. In this regard, the native GDF11 protein in humans is encoded by the GDF11 gene and has the same molecular structure in humans, mice, and rats. Therefore, the GDF11 sequence is highly conserved across several mammalian species, and GDF11 is known to be expressed in many tissues, including skeletal muscle, pancreas, kidney, nervous system, and retina. With respect to human GDF11, the propeptide + signal sequence (e.g., precursor polypeptide) is 407 amino acids long. Cleavage of the 24-amino acid signal peptide produces a 383-amino acid propeptide, and cleavage of the propeptide results in a 109-amino acid mature GDF11 polypeptide corresponding to the C-terminal 109 amino acids of the propeptide. The mature form of the GDF11 polypeptide molecule produces a disulfide-bonded homodimer. Therefore, any derivative, variant, or modified form of the "natural" GDF11 molecule can be determined to be a "therapeutic" GDF11 molecule by comparing the pharmacological activity of the target molecule with that of the mature form (in its fully active form as a homodimer) of the natural human GDF11 polypeptide using methods and techniques well known to those skilled in the art.

[0012] Accordingly, in certain preferred embodiments of this disclosure, the GDF11 molecule selected for use in the method may be a mature form of the GDF11 polypeptide, which can be provided in the form of a homodimer. Alternatively, the selected GDF11 molecule may be a polypeptide having at least 91% sequence homology to the natural sequence of human GDF11. Preferably, the GDF11 molecule is a mature recombinant human GDF11 (rhGDF11). In other embodiments, the GDF11 molecule may be a therapeutically active variant or derivative of the human GDF11 molecule. Such variants or derivatives may include one or more amino acid substitutions or deletions from the natural sequence of the human GDF11 molecule and may include one or more amino acid analogs. In yet another embodiment of this disclosure, the selected GDF11 molecule may be a modified GDF11 polypeptide, for example, in which the molecule is phosphorylated, glycated, glycosylated, pegylated, HES-modified, ELP-modified, lipid-modified, acetylated, amidated, end-capped, contains a cyano group or albumin, or is cyclized. Alternatively, the modified GDF11 molecule may be a chimeric polypeptide having at least two parts, a first GDF11 molecule part and a second part, for example, the second part being derived from transferrin, growth hormone, or an Fc fragment. In a preferred embodiment, the modified GDF11 molecule would have an increased half-life compared to the mature form of the natural GDF11 polypeptide. In any case, the selected GDF11 molecule is formulated into a suitable pharmaceutical composition for administration to a subject, which may further contain a pharmaceutically acceptable carrier, excipient, or vehicle.

[0013] Since GDF11 is not thought to cross the blood-brain barrier (BBB) ​​and is most effective when it can be utilized systemically, the GDF11 molecule is administered parenterally. The GDF11 molecule can be administered twice daily (BID), three times daily (TID), four times daily (QID), every hour ("q_h", where "h" indicates the number of hours between doses), or similarly, based on once daily (QD), and each day of treatment may be the same or different throughout the course of treatment. In certain embodiments of this disclosure, administration is performed once daily (QD). For example, a composition containing the GDF11 molecule can be administered intravenously to a subject using a catheter such as a central venous catheter line or a similar intravenous catheter. Alternatively, the GDF11 composition can be administered intravenously, intramuscularly, intraperitoneally or subcutaneously using a standard needle and syringe. Thus, in certain embodiments, the composition can be simply formulated to include a suitable injection medium such as sterile water for injection. In yet another embodiment, the composition may be administered using an external drug pump, such as an infusion pump. The composition may further comprise controlled, sustained, or delayed-release excipients. In practice of such a method, the composition may be provided in the form of nanoparticles, such as liposomes. The composition may further comprise biodegradable polymers or non-polymer controlled-release excipients and may be provided in the form of an injectable liquid implant or microparticles. In practice of the method of this disclosure, the GDF11 molecule may be present in the composition in the form of a liquid, suspension, or emulsion.

[0014] In certain aspects of this disclosure, the precise dosage and duration of treatment used in the practice of the method are a function of the type of stroke and the resulting stroke injury being addressed, and can be empirically determined using known test protocols or by estimation from in vivo or in vitro test data or subsequent clinical trials. It should also be noted that the concentration and dosage values ​​may also vary depending on the severity of the stroke being addressed. For any particular subject, a specific dosing regimen may be adjusted over time according to the individual needs and the professional judgment of the person administering or directing the administration of the GDF11 composition, and the concentration ranges described herein are merely examples and are not intended to limit the scope or practice of the claimed method. In other aspects of this disclosure, a single administration of the composition may be required, or several treatment regimens may be needed that are particularly appropriate for the expected treatment. For example, an appropriate treatment regimen may include a first dose of the GDF11 molecule at a first dose (day 1 of treatment), followed by a second or higher or lower dose of the GDF11 molecule, or subsequent doses (e.g., from day 2 to day 7 of treatment). In certain preferred embodiments, the administration regimen requires a traditional dose setting of the GDF11 molecule, either with escalating or tapering doses, for example, a first dose administered on day 1 of the treatment period at least approximately the minimum high dose of GDF11 relative to the subject's body weight, and a second dose ending at a higher dose. Alternatively, the dose setting of the GDF11 molecule may require a high initial (day 1) dose of the GDF11 molecule and end at a final dose of at least approximately the minimum high dose of GDF11 in the subject. In either dose setting strategy, it may be preferable to administer the GDF11 molecule at a first high dose approaching the median toxic dose (MTD) for this molecule, or at least approaching the maximum dose within the therapeutic range for the GDF11 molecule being administered, and then at lower levels in subsequent doses (or doses). In other aspects of this disclosure, for example, if subsequent treatment is administered 2 to 7 days after the completion of the initial treatment, the GDF11 treatment regimen can be administered multiple times (e.g., repeatedly) according to so-called “drug-free periods,” i.e., planned treatment interruptions, tolerance breakdowns, or treatment interruptions.Again, for any particular subject, a specific dosing regimen may be adjusted over time according to the individual needs and the professional judgment of the person administering or directing the administration of the GDF11 composition. The dosing strategies described herein are merely examples and are not intended to limit the scope or practice of the claimed methods.

[0015] The smooth implementation of this method can be evaluated using diagnostic and clinical testing techniques well known in the art. For example, such techniques are typically based on physical and neurological examinations (such as NIHSS) to assess improved physical and / or cognitive function in the subject, and are often supported by medical imaging techniques such as CT scans, MRI scans, Doppler ultrasound, and angiography, and by auxiliary tests such as electrocardiograms (ECG) and blood tests. Medical imaging techniques can be used to evaluate successful stroke treatment in a subject by visualizing angiogenesis, neurogenesis, improved cerebrovascular structure, and / or function or blood flow in the stroke site or vicinity of the stroke site in the subject, for example, in the area around the stroke where blood flow is locally reduced near the original injury (ischemia). For clarity, successful stroke treatment using the method of this disclosure can be established by evaluating one or more of the above criteria (and any combination thereof) and / or by using one or more of the above diagnostic and imaging techniques.

[0016] These aspects of the present disclosure, as well as others, are described in detail in the following sections of the Application and expressly in the attached claims. [Brief explanation of the drawing]

[0017] [Figure 1]Figure 1 shows the results from one pillar of the middle cerebral artery occlusion (MCAO) test described in Example 1, where the therapeutic efficacy of rGDF11 administration was evaluated by body swing (behavioral) testing. Animals receiving 1 mg / kg once daily (QD) of GDF11 showed superior stroke recovery compared to placebo (vehicle only) animals on days 7 (p<0.001), 14 (p<0.001), 21 (p<0.001), and 28 (p<0.001). n=24 rats. [Figure 2] Figure 2 shows the results from the second pillar of the MCAO study described in Example 1, and the therapeutic efficacy of rGDF11 administration was evaluated by hind limb repositioning (behavioral) testing. Animals receiving 1 mg / kg once daily (QD) of GDF11 showed superior stroke recovery compared to placebo (vehicle only) animals on days 7 (p<0.001), 14 (p<0.001), 21 (p<0.001), and 28 (p<0.001). n=24 rats. [Figure 3] Figure 3 shows the results from the third pillar of the MCAO study described in Example 1. The therapeutic efficacy of rGDF11 administration was evaluated by the forelimb repositioning (behavioral) test. Animals receiving GDF11 at 1 mg / kg once daily (QD) showed superior stroke recovery at day 14 (p<0.05) compared to placebo (vehicle only) and at day 21 (p=0.60). n=24 rats. [Figure 4] Figure 4 shows the results from the fourth pillar of the MCAO study described in Example 1, evaluating the adverse event profile of rGDF11 administration. Animals receiving rGDF11 at 1 mg / kg showed a significant decrease in body weight compared to vehicle-treated animals (p<0.001). The body weight of rGDF11-treated animals decreased by 7.03% on postoperative day 3 compared to the body weight of animals immediately after surgery. Subsequently, rGDF11-treated animals gained body weight at a rate comparable to vehicle-treated animals. n=24 rats. [Figure 5]Figure 5 shows the results from one pillar of the MCAO study described in Example 2, where the therapeutic efficacy of rGDF11 administration was evaluated by forelimb repositioning (behavioral) testing. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 1 to day 7 showed superior recovery compared to vehicle-treated animals on day 3 (p<0.0001), day 7 (p<0.001), day 14 (p<0.001), day 21 (p<0.0001), and day 30 (p<0.001). [Figure 6] Figure 6 shows the results from one pillar of the MCAO study described in Example 2, where the therapeutic efficacy of rGDF11 administration was evaluated by a hindlimb repositioning (behavioral) test. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 1 to day 7 showed superior recovery compared to vehicle-treated animals on day 3 (p<0.001), day 5 (p<0.01), day 7 (p<0.0001), day 14 (p<0.001), day 21 (p<0.0001), and day 30 (p<0.0001). [Figure 7] Figure 7 shows the results from one pillar of the MCAO study described in Example 2, where the therapeutic efficacy of rGDF11 administration was evaluated by body swing (behavioral) testing. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 3 to day 16 showed superior recovery compared to vehicle-treated animals on day 7 (p<0.05), day 14 (p<0.001), day 21 (p<0.001), and day 30 (p<0.01). [Figure 8] Figure 8 shows the results from one pillar of the MCAO study described in Example 3. The therapeutic efficacy of rGDF11 administration was evaluated by forelimb repositioning (behavioral) testing. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on days 3, 5, and 7 showed statistically improved recovery compared to vehicle-treated animals 14 days post-stroke (p<0.05 on day 3 of treatment, p<0.0001 on day 5 of treatment, and p<0.001 on day 7 post-treatment). [Figure 9]Figure 9 shows the results from one pillar of the MCAO study described in Example 3, where the therapeutic efficacy of rGDF11 administration was evaluated by hind limb repositioning (behavioral) testing. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on days 1, 3, 5, and 7 showed statistically improved recovery compared to vehicle-treated animals 14 days post-stroke (p<0.05 on day 1 of treatment; p<0.001 on day 3 of treatment; p<0.0001 on day 5 of treatment; p<0.0001 on day 7 post-treatment). [Figure 10] Figure 10 shows the results from one pillar of the MCAO study described in Example 3, where the therapeutic efficacy of rGDF11 administration was evaluated by the body swing (behavioral) test. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on days 5 and 7 showed statistically improved recovery compared to vehicle-treated animals 14 days post-stroke (p<0.001 on day 5 of treatment, p<0.0001 on day 7 post-treatment). [Figure 11] Figure 11 shows the results from one pillar of the MCAO study described in Example 4, where the therapeutic efficacy of rGDF11 administration was evaluated by the forelimb repositioning (behavioral) test. Animals that received intraperitoneal administration of 1 mg / kg, 2 mg / kg, and 4 mg / kg of rGDF11 showed statistically improved recovery compared to vehicle-treated animals at 28 days post-stroke (p<0.05 for 1 mg / kg, p<0.001 for 2 mg / kg, and p<0.001 for 4 mg / kg). [Figure 12] Figure 12 shows the results from one pillar of the MCAO study described in Example 4, where the therapeutic efficacy of rGDF11 administration was evaluated by a hind limb repositioning (behavioral) test. Animals that received intraperitoneal administration of 1 mg / kg, 2 mg / kg, and 4 mg / kg of rGDF11 showed statistically improved recovery compared to vehicle-treated animals at 28 days post-stroke (p<0.001 for 1 mg / kg, p<0.0001 for 2 mg / kg, and p<0.0001 for 4 mg / kg). [Figure 13]Figure 13 shows the results from one arm of the MCAO assay described in Example 4, wherein the therapeutic efficacy of rGDF11 administration was evaluated by a body swing (behavior) test. Animals that received intraperitoneal administration of 0.5 mg / kg, 1 mg / kg, 2 mg / kg, and 4 mg / kg of rGDF11 showed statistically improved recovery compared with vehicle-treated animals on day 28 after stroke (p<0.001 for 0.5 mg / kg, p<0.001 for 1 mg / kg, p<0.0001 for 2 mg / kg, p<0.0001 for 4 mg / kg). [Figure 14] Figure 14 shows the percentage survival of Group A (rGDF11 treatment group; 1 mg / kg, administered once daily for 7 days) and Group B (vehicle group) in C57Bl6 / j mice as a function of days after ICH. The vehicle group showed a trend toward increased mortality compared with the rGDF11 treatment group. [Figure 15A] Figure 15A shows post-ICH neurological severity scores versus days after injection for Group A and Group B, including dead animals. The vehicle group showed increased neurological severity compared with the rGDF11 treatment group on day 28 (p<0.05). [Figure 15B] Figure 15B shows post-ICH neurological severity scores versus days after injection for Group A and Group B, excluding dead animals. The vehicle group showed increased neurological severity compared with the rGDF11 treatment group on day 28 (p<0.01). [Figure 16A] Figure 16A shows a plot of rotarod latency as a function of days after ICH injury for Group A and Group B, including dead animals. Group A showed improved rotarod latency compared with Group B on day 28 (p<0.05). [Figure 16B] Figure 16B shows a plot of rotarod latency as a function of days after ICH injury for Group A and Group B, excluding dead animals. Group A showed improved rotarod latency compared with Group B on day 28 (p<0.0001). [Figure 17A] Figure 17A shows the average velocity (centimeters per second) 7 days after the first treatment (p<0.05). [Figure 17B]Figure 17B shows the forelimb support base 7 days after the first treatment in the rGDF11 administration group (group A) compared to the vehicle group (group B) (p<0.05). [Figure 18A] Figures 18A-18D show progenitor cell replenishment in the subventricular zone ipsilateral to the injury site on day 29: Figure 18A (vehicle), Figure 18B (1 mg / kg), Figure 18C (2 mg / kg), and Figure 18D (4 mg / kg). [Figure 18B] Figures 18A-18D show progenitor cell replenishment in the subventricular zone ipsilateral to the injury site on day 29: Figure 18A (vehicle), Figure 18B (1 mg / kg), Figure 18C (2 mg / kg), and Figure 18D (4 mg / kg). [Figure 18C] Figures 18A-18D show progenitor cell replenishment in the subventricular zone ipsilateral to the injury site on day 29: Figure 18A (vehicle), Figure 18B (1 mg / kg), Figure 18C (2 mg / kg), and Figure 18D (4 mg / kg). [Figure 18D] Figures 18A-18D show progenitor cell replenishment in the subventricular zone ipsilateral to the injury site on day 29: Figure 18A (vehicle), Figure 18B (1 mg / kg), Figure 18C (2 mg / kg), and Figure 18D (4 mg / kg). [Figure 19] Figure 19 shows the increased neurogenesis in the subventricular zone ipsilateral to the injury site at doses of 1 mg / kg, 2 mg / kg, and 4 mg / kg in GDF11 on day 29, compared to vehicles treated with GDF11. [Figure 20A] Figures 20A–20D show progenitor cell replenishment in the subventricular zone opposite the injury site on day 29: Figure 20A (vehicle), Figure 20B (1 mg / kg), Figure 20C (2 mg / kg), and Figure 20D (4 mg / kg). [Figure 20B] Figures 20A–20D show progenitor cell replenishment in the subventricular zone opposite the injury site on day 29: Figure 20A (vehicle), Figure 20B (1 mg / kg), Figure 20C (2 mg / kg), and Figure 20D (4 mg / kg). [Figure 20C]Figures 20A–20D show progenitor cell replenishment in the subventricular zone opposite the injury site on day 29: Figure 20A (vehicle), Figure 20B (1 mg / kg), Figure 20C (2 mg / kg), and Figure 20D (4 mg / kg). [Figure 20D] Figures 20A–20D show progenitor cell replenishment in the subventricular zone opposite the injury site on day 29: Figure 20A (vehicle), Figure 20B (1 mg / kg), Figure 20C (2 mg / kg), and Figure 20D (4 mg / kg). [Figure 21] Figure 21 shows the increased neurogenesis in GDF11 at doses of 1 mg / kg, 2 mg / kg, and 4 mg / kg in the subventricular zone opposite the injury site. [Figure 22] Figure 22 shows an analysis comparing the ipsilateral and contralateral hemispheres of the injury site. [Modes for carrying out the invention]

[0018] For convenience, all specific terms used throughout this application (including the specification, drawings, and appended claims) are explicitly defined. Unless otherwise expressly defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this disclosure pertains.

[0019] definition When used throughout this specification and the accompanying claims, the singular forms "a," "an," and "the" include plural subjects unless otherwise expressly indicated. Thus, for example, a reference to "pharmaceutically acceptable carrier, excipient or vehicle" includes mixtures of two or more such entities, and similar mixtures.

[0020] As used herein, the term “comprising” means that other elements may be present in addition to the defined present elements. Therefore, the use of “comprising” indicates comprehensiveness, not limitation. The term “consisting of” refers to the compositions, methods, and their respective components described herein, excluding any elements not described in the description of the embodiment. As used herein, the term “consisting essentially of” refers to these elements necessary for a given embodiment. This term permits the presence of elements that do not substantially affect the basic, novel, or functional features of this embodiment of the disclosure.

[0021] In general, the nomenclature used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization as described herein, as well as the nomenclature used in relation to these techniques, are well known and commonly used in the art. The methods and techniques of this disclosure are generally carried out in accordance with the prior art methods described in the various general and more specific references cited and discussed throughout this specification unless otherwise indicated.For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lan, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY(1990);Principles of Neural Science,4th ed.,Eric R.Kandel,James H.Schwart,Thomas M.Jessell eds.McGraw-Hill / Appleton & Lange:New York,NY(2000);The Merck Manual of Diagnosis and Therapy,19th Edition,published by Merck Research Laboratories(2006)(ISBN 0-911910-19-0),Robert See S. Porter et al. eds., The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. (1994) (ISBN 0-632-02182-9); and Current Protocols in Protein Sciences (2009) Wiley Intersciences, Coligan et al., eds.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0023] Except in the operational examples, or unless otherwise indicated, all figures representing amounts of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term “about,” whether expressly indicated or not. When used in relation to percentages, days, doses, or other amounts, the term “about” may mean ±10%.

[0024] As used herein, the terms “administer,” “administering,” and “administered” refer to providing a therapeutic activator to a subject being treated. Administration of the GDF11 molecule may be carried out according to any appropriate criteria, such as once daily (QD), twice daily (BID), three times daily (TID), four times daily (QID), every hour ("q_h," where "h" indicates the number of hours between administrations), or similarly, and each day of treatment may be the same or different throughout the course of treatment. In certain embodiments, the GDF11 molecule is administered in the form of a liquid solution or suspension introduced into the subject by conventional intraperitoneal, subcutaneous, or intravenous delivery techniques, but may include microinjection, stereotactic injection, and / or direct application to specific sites of the subject.

[0025] The terms "biodegradable" and "bioerodible" are used interchangeably herein and refer to substances such as polymers that decompose or corrode in vivo over time to produce lower molecular weight chemical species, which may occur, for example, through enzymatic, chemical, and physical processes. Typically, such substances decompose or corrode by hydrolysis, producing degradation products that do not exhibit significant, harmful, or undesirable effects on the target body. Examples of biodegradable polymers suitable for use in the compositions and methods of this disclosure include, but are not limited to, polylactides; polyglycolides; poly(lactide-co-glycolides); polylactic acid; polyglycolic acid; and poly(lactic acid-co-glycolic acid); polycaprolactone; polymalic acid; polyamides; polyacid anhydrides; polyamino acids; polyorthoesters; polyether esters; polycyanoacrylates; polyphosphazines; polyphosphate esters; polyesteramides; polydioxanones; polyacetals; polyketals; polycarbonates; polyorthocarbonates; biodegradable polyurethanes; polyhydroxybutyrates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; chitin; chitosan; oxidized cellulose; and copolymers, terpolymers, formulations, combinations or mixtures of any of the above substances.

[0026] The term "controlled release" refers to a dosage form or composition of a pharmaceutical product that provides a delayed, slow, continuous, intermittent, or sustained release of a therapeutically active molecule over a period of time.

[0027] In this specification, the terms “decrease,” “reduce,” “reduction,” “decrease,” and “inhibit” are all used interchangeably and generally mean a statistically significant reduction relative to a reference. However, to avoid misunderstanding, such terms usually mean a reduction of at least 10% compared to a reference level, and may include, for example, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, for example, the complete absence of a given entity or parameter and including such absence, or any reduction of 10–99% compared to the absence of a given treatment.

[0028] When used interchangeably in this specification, the phrase “dose of GDF11” or “dose of GDF11 molecule” refers to the amount of GDF11 administered to a subject over a specific period, and such amount is measured relative to the body weight of the subject being treated. To date, all published reports of actual in vivo administration of GDF11 have been in rodent (mouse and rat) animal model systems. From these published studies, two different administration paradigms have emerged. The first paradigm, the therapeutic GDF11 strategy, is clearly defined and is referred to herein collectively as targeting “moderate doses of GDF11.” This moderate dose strategy includes the vast majority of published in vivo GDF11 administration studies that target an approximately 1-4-fold increase in circulating GDF11 in the treated subject, treating mouse or rat subjects with a typical daily dose of GDF11 of approximately 0.1 mg / kg body weight.For example, Zhang et al(2016)Scientific Reports 6(1):34624;Mei et al.(2016)Molecular Therapy:the Journal of the American Society of Gene Therapy 24(11):1926-1938;Du et al.(2017)Basic Research in Cardiology 112(1):7;Li et al.(2017)Diabetes 66(7): db170086-1927;Onodera et al.(2017)Thorax (April);Bajikar et al. (2017) Developmental Cell 43(4):418-435;Harper et al.(2018)Circulation Research 7 Sep 2018;Zhang et al.(2018)Diabetes;Ma et al. al.(2018)Brain Research Bulletin See 139(February):38-47; Zhang et al.(2018) Journal of Alzheimer's Disease 62(2); Lu et al.(2018) Front Cell Neurosci 12:205; Wang et al.(2018) Am J Physiology Gastrointest Liver Physiology; and Wang et al.(2018) Inflammation. Such moderate doses of GDF11 are characterized by a range of 0.001 mg / kg body weight to 0.5 mg / kg body weight (see, for example, U.S. Patent No. 9,434,779; and U.S. Patent Application Publication No. 2016 / 074477 and U.S. Patent Application Publication No. 2016 / 220640). Therefore, as used herein, “moderate dose of GDF11” means a moderate application of a broad range of GDF11 molecules to a subject over a specific period of time, typically ranging from about 0.001 mg / kg body weight to about 0.5 mg / kg body weight daily, and preferably about 0.1 mg / kg body weight daily, as is typically extrapolated to rodent subjects and human subjects, and other large mammalian species including human subjects.As mentioned above, published reports on moderate doses of GDF11 administered in vivo have avoided the upper limit (0.5 mg / kg) and instead focused on a specific moderate dose of 0.1 mg / kg as the objective standard. These publications have demonstrated therapeutic efficacy of such "moderate doses of GDF11" across a broad spectrum of diseases and conditions.

[0029] The second GDF11 administration paradigm, referred to herein as “GDF11 overdose,” is a dose of GDF11 that may have some therapeutic effect or no therapeutic effect at all, but may result in the development of adverse or other unacceptable adverse side effects or adverse side effect profiles in the subject being treated with GDF11. As used herein, “adverse effect” is any pharmacological or physiological effect of GDF11 administration that occurs secondary to the intended subject, and “adverse side effect” is any such secondary effect that is undesirable and / or harmful, and may be reversible or irreversible, and which manifests in the treated subject in terms of prognosis such as morbidity, mortality, dysfunction, or other pathological changes. Several published in vivo GDF11 administration studies targeting GDF11 overdoses, in which mice or rats were treated with GDF11 administered daily at typical doses of approximately 5 mg / kg to 10 mg / kg or more, have reported that such hyperphysiological doses of GDF11 may produce significant adverse effects, including cachexia, muscular atrophy, anorexia, fibrosis, or even death (see, for example, Hammers et al. (2017) EMBO Molecular Medicine 9(4):531-544; Pons et al. (2018) Surgery (May); and Jones et al. (2018) Cell Reports 22(6):1522-1530).

[0030] As used herein, “GDF11” or “GDF11 molecule” refers to “Growth and Differentiation Factor 11” (NCBI gene ID number 10220), a member of the transformed growth factor β ("TGF-β") superfamily of growth factors. GDF11 is known to bind to TGF-β superfamily type I receptors, including ALK4, ALK5, and ALK7. The term “rGDF11” refers to a GDF11 molecule produced using recombinant methods, and the term “rhGDF11” refers to such a molecule derived from the native human GDF11 molecule. rhGDF11 has the amino acid sequence defined in Sequence ID No. 1. For signaling during mammalian development, GDF11 primarily uses ALK4 and ALK5. In some embodiments, GDF11 signaling can also occur by the ACVR2B receptor. GDF11 is closely associated with Growth and Differentiation Factor 8 (GDF8, also known as myostatin). GDF11 can also be called bone morphogenetic protein 11, or BMP11. Therefore, as used herein, “GDF11” or “GDF11 molecule” includes human precursor polypeptide (NCBI Ref Seq: NP_005802); human propeptide; human N-terminal polypeptide; and human mature form of GDF11, as well as homologs from other species, including but not limited to cattle, dogs, cats, chickens, mice, rats, pigs, sheep, turkeys, horses, fish, baboons, and other primates. The term also refers to fragments, derivatives, or variants of GDF11 that, for example, maintain at least 50% of the physiological (therapeutic) effects of mature GDF11 when measured in appropriate animal models. Conservative substitution variants that maintain the appropriate activity of wild-type GDF11 would include the conservative substitutions as defined herein. The identification of amino acids most likely to be tolerant of conservative substitutions while maintaining at least 50% of the wild type is guided, for example, by sequence alignment with GDF11 homologs or paralogs from other species. Amino acids that are identical between GDF11 homologs are less likely to tolerate change, while those showing conservative differences are clearly much more likely to tolerate conservative change in relation to the artificial variant.Similarly, positions with non-conservative differences are less likely to be essential to function and are more likely to tolerate conservative substitutions in artificial variants. For example, variants can be tested for activity by administering them to subjects in appropriate animal models described herein to evaluate therapeutic efficacy. With respect to human GDF11, the propeptide + signal sequence (e.g., precursor polypeptide) is 407 amino acids long. Cleavage of the 24-amino acid signal peptide produces a 383-amino acid propeptide, and cleavage of the propeptide results in a 109-amino acid mature GDF11 polypeptide corresponding to the C-terminal 109 amino acids of the propeptide. The mature form of the polypeptide produces a disulfide-bonded homodimer.

[0031] As used herein, the term “high dose of GDF11” defines a dose of the GDF11 molecule administered to subjects in which the dose lies between the moderate dose and the overdose of GDF11, and which has surprisingly demonstrated an unexpectedly beneficial effect in the treatment of stroke as described herein. In this specification, a high dose of the GDF11 molecule is defined as generally encompassing doses starting above the upper limit of the reported moderate dose of GDF11 and below the lower limit of the reported overdose of GDF11, including both the moderate and overdose ranges of GDF11 reported routinely in subjects in which the subject is in question. More specifically, in this specification, a standard high dose of GDF11 is defined as equivalent to approximately 1 mg / kg body weight in rodent subjects (mice or rats) routinely, and therefore approximately an order of magnitude higher than the normal moderate dose of GDF11 in such species (i.e., approximately 0.1 mg / kg). Therefore, the “minimum high dose of GDF11” is at least about 0.8 mg / kg (body weight) in rodent species, and the same dose in large mammal species is normalized to the molecular weight of rhGDF11 as defined by Sequence ID No. 1. Similarly, the “maximum high dose of GDF11” is about 4 mg / kg (body weight) in rodent species, and the same dose in large mammal species is normalized to the molecular weight of rhGDF11 as defined by Sequence ID No. 1. In this regard, the maximum high dose of GDF11 is the dose that avoids adverse side effects in the treated subject. It should also be noted that the value of a particular dose may vary depending on the severity of the stroke being addressed. For any particular subject, a particular dosing regimen may be adjusted over time according to the individual needs and the professional judgment of the person administering or directing the administration of the GDF11 composition, and the concentration ranges described herein are merely examples and are not intended to limit the scope or practice of the claimed method. The precise dosage can be experimentally predicted by testing in vitro and in vivo systems well known to those skilled in the art, and then estimating from the tests for use in subjects including human subjects. The human dose is then typically fine-tuned in clinical trials until a response is achieved.For the purposes of this disclosure, the high dose of GDF11 as defined for rodents is at least about 0.8 mg / kg to about 4 mg / kg, or at least about 0.9 mg / kg to about 4 mg / kg, and in some cases at least about 1 mg / kg to about 3 mg / kg, and in certain aspects of this disclosure at least about 1 to 2 mg / kg, encompassing the corresponding dose in any large mammalian species.

[0032] As used herein, the term “hydrogel” refers to a polymer that, as it is commonly used in the art, swells in the presence of water or other aqueous systems, shrinks in the absence or reduction of water, can retain a significant proportion of water within its structure, and is not normally soluble in water. Those skilled in the art will understand that there are several standard tests that can be used to determine whether a polymer or polymer system will perform the function of a hydrogel when immersed in an aqueous system, such as when implanted in a mammalian subject or delivered in vivo.

[0033] As used herein, “implant” or “implantable composition or device” refers to any implantable system for use in the delivery of a therapeutically active substance to a subject. Common implantable devices enable local (site-specific) and / or systemic administration of a drug of interest and include, for example, solid structures such as stents or wafers that can be left on or within tissue at a surgical site, rods or microparticles that can be administered by subcutaneous or intramuscular injection using needles and syringes or trocars, and implantable drug pump devices. Solid implantable compositions or devices are typically produced using biodegradable polymers that can provide controlled release of a drug of interest. Injectable implantable compositions or devices can be provided in the form of viscous liquid carriers, hydrogel compositions, nanoparticle compositions, microspheres or microparticles, or plastic polymer carriers.

[0034] The terms “increased,” “increased,” or “enhanced” are all used interchangeably herein and generally mean an increase of a statistically significant amount; to avoid any misunderstanding, these terms indicate an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or an increase of less than or equal to 100%, or an increase of 10% to 100%, or an increase of at least about 2 times, or at least about 3 times, or at least 4 times, or at least 5 times, or at least about 10 times, or any increase of 2 times to 10 times or more compared to a reference level.

[0035] As used herein, the term “kit” means any product (e.g., package or container) containing at least one therapeutic active agent (GDF11 molecule). In a particular kit, the product may be promoted, distributed, or sold as a unit for performing the methods of the present disclosure.

[0036] As used herein, the term “liposome” refers to a spherical vehicle having at least one lipid bilayer formed of a specific lipid (such as phosphatidylcholine or its phospholipids) filled with an aqueous core which may contain other components including the therapeutic active agent of the present disclosure. Liposomes may be about 10 microns or less in size, but preferably, liposomes used in the practice of the compositions and methods of the present disclosure are submicron in size, i.e., in the form of nanoparticles.

[0037] As used herein, in relation to the delivery or administration of a therapeutic active agent to a subject, the terms “topical” or “locally” mean that such agent is delivered to a localized site of the subject, but may not be detectable at a biologically significant level in the subject’s plasma.

[0038] "Nanoparticles" generally refer to particulate matter or a collection of such particles having a size in the range of 1 to 100 nm, and include nanospheres, lipid systems such as liposomes and micelles, nanocrystals, and nanoparticles. Nanospheres may contain drugs (molecules or compounds) and other substances such as inorganic nanoparticles or magnetic particles such as gold, or nanoparticles may contain polymers such as biodegradable polymers, or may be formed from polymers. In practice of the compositions and methods of this disclosure, synthetic polymers such as polyvinyl alcohol, poly-L-lactic acid, polyethylene glycol, and poly(lactic acid-co-glycolic acid), as well as natural polymers such as alginates and chitosan, can be used in nanofabrication of nanoparticles to obtain nanospheres or nanocapsules. Nanoparticles generally remain in the blood circulation system for extended periods, thus enabling sustained drug release and a sustained drug lifecycle. Due to their nanosize, nanoparticle structures readily penetrate tissue systems and facilitate easy uptake by cells to achieve effective delivery to the target site.

[0039] The term “pharmaceutically acceptable” refers to a substance that is approved or eligible for approval for medicinal use by the relevant federal or state regulatory agency and / or listed in the United States Pharmacopeia or another generally accepted pharmacopoeia for use in animals and, more particularly, in humans. “pharmaceutically acceptable carrier, excipient or vehicle” refers together to any medium, diluent, excipient or carrier used to administer a therapeutically active compound.

[0040] "Pharmacokinetically acceptable salt" refers to a salt of a therapeutically active molecule or compound that is pharmacokinetically acceptable and has the desired pharmacological effect of the parent molecule or compound. Examples of pharmacokinetically acceptable salts of therapeutic agents described herein include salts derived from pharmacokinetically acceptable inorganic and organic acids and bases. Suitable salts include acetate, adipine, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, and odorate. Examples include hydrochlorides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, malons, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, palmoates, pectins, persulfates, 3-phenylpropionates, phosphates, picric acid, pivalates, propions, salicylates, succinates, sulfates, tartrates, thiocyans, tosylates, and undecanoates. Although not pharmacodynamically acceptable on their own, other acids such as oxalic acid may be used to prepare salts that are useful as intermediates in obtaining pharmacodynamically acceptable acid addition salts. Examples of salts derived from suitable bases include alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., magnesium), and ammonium salts.

[0041] As used herein, “polymer” refers to any polymer, copolymer, and formulation unless otherwise explicitly defined. Polymers for use in connection with the compositions and methods of this disclosure may be produced using standard polymerization and copolymerization techniques such as graft copolymerization, polycondensation, and polyaddition, with appropriate catalysts as needed. These techniques may be carried out in conventional methods well known in polymer technology with respect to time, temperature, and other parameters. Alternatively, polymers used herein may be produced using standard polymer formulation techniques or copolymer formulations, which may be carried out in conventional methods well known in polymer technology, as before.

[0042] As used herein, the terms “protein” and “polypeptide” are used interchangeably and refer to a series of amino acid residues linked together by peptide bonds between the α-amino and carboxyl groups of adjacent residues. Therefore, the term represents polymers of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of size or function. While “protein” and “polypeptide” are often used to refer to relatively large polypeptides, the term “peptide” is often used to refer to smaller polypeptides; however, the use of these terms in the art is overlapping. The terms “protein” and “polypeptide” are used interchangeably herein when referring to gene expression products and their fragments. Therefore, good examples of polypeptides or proteins include gene expression products, native proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, derivatives, fragments, and the aforementioned analogs.

[0043] As used herein, “stroke event” refers to a medical condition in which insufficient blood flow to the brain leads to neuronal cell death. Stroke events are classified into two main types: ischemic (resulting from an interruption of blood supply to the brain) and hemorrhagic (resulting from a ruptured blood vessel or abnormality of a vascular structure). Both types of stroke events result in a part of the brain that does not function properly. Ischemic stroke events are usually caused by occlusion of a blood vessel, reducing blood supply to a part of the brain and leading to dysfunction of brain tissue in that area. There are four causes of such ischemic strokes: thrombolysis, embolism, decreased systemic blood flow, and cerebral venous sinus thrombosis. Hemorrhagic stroke events are caused by either direct bleeding in the brain or intermeningeal spaces, and such bleeding can be caused by a ruptured cerebral aneurysm. There are two main types of hemorrhagic stroke: intracerebral hemorrhage (bleeding within the brain itself) and subarachnoid hemorrhage (bleeding outside the brain tissue but within the cerebral cavity). The signs and symptoms of stroke events occurring in the subject are well known in the medical field and include sudden onset of facial weakness, upper limb elevation, speech abnormalities, unilateral loss of movement or touch, difficulty understanding, dizziness or vertigo, unilateral vision loss, and severe headache. Ischemic stroke events can be classified into total anterior cerebral infarction (TACI), partial anterior cerebral infarction (PACI), lacunar infarction (LACI), or posterior cerebral infarction (POCI), which predict the severity of the stroke event, the affected brain region, the underlying cause, and the prognosis. The clinical diagnosis of stroke events is also well known in the medical field and is usually based on physical and neurological examinations (such as NIHSS), often supported by medical imaging techniques such as CT scans, MRI scans, Doppler ultrasound, and angiography, and often supported by auxiliary tests such as electrocardiograms (ECG) and blood tests.

[0044] As used herein, the term “subject” means any animal that is to be the recipient of a particular method of treatment (e.g., mammals including, but not limited to, humans, primates, dogs, cattle, cows, horses, kangaroos, pigs, sheep, goats, cats, rabbits, and rodents), transgenic non-human animals, amphibians, reptiles, other vertebrates and invertebrates and similar animals, but not limited to fish, frogs, and salamanders.

[0045] As used herein, the terms “systemic” or “systemic” mean, with respect to the delivery or administration of a therapeutic active agent to a subject, that agent is detectable at a biosignificant level in the subject’s plasma. This term includes oral or parenteral administration of the therapeutic active agent to the subject.

[0046] As used herein, the term “therapeutic activity” may refer to the activity of a GDF11 molecule or compound whose effect is consistent with the desired therapeutic outcome in the target subject. The terms “therapeutic agent,” “therapeutic GDF11 molecule,” or “therapeutic derivative, variant, or modified GDF11” are used interchangeably herein and refer to molecules whose effect is consistent with the desired outcome in the target subject, and in the case of variants, derivatives, and / or modified molecules, have therapeutic activity consistent with the pharmaceutically active parent molecule. Therapeutic activity may be measured using in vitro or in vivo methods well known to those skilled in the art, for example, the desired therapeutic effect can be evaluated in cell culture.

[0047] The “therapeutic dose” represents the amount of therapeutic active agent (molecule or compound) that, when administered to a subject, is sufficient to provide the desired treatment for a disease, condition, complication, or disorder present in the subject. The therapeutic dose of a therapeutic active agent for use in any particular method herein will vary depending on the molecule or compound, the disease, condition, complication, or disorder, and its severity, as well as the age and weight of the subject. Complete therapeutic effect may not necessarily occur with a single dose of the therapeutic active agent (molecule or compound), but may occur only after a series of doses have been administered. The therapeutic dose may also vary depending on the identity of the active agent, the disease, condition, injury, or complication (and its severity) to be addressed, and the age, weight, absorption, distribution, metabolism, and excretion of the relevant active agent in the subject. Therefore, the therapeutic dose may need to be administered in one or more doses to the subject. An appropriate therapeutic dose of a therapeutic active molecule or compound can be determined according to one of several established protocols known to those skilled in the art in the relevant field. For example, animal studies, such as those using mice, rats, or larger mammals, can be used to determine the appropriate dosage of a pharmaceutical compound. Then, the results from such animal studies can be extrapolated to determine the dosage for use in other species, such as humans.

[0048] The term “to treat” or “treatment” means any remission, rehabilitation, rejuvenation, improvement, reduction or mitigation of any one or more effects, complications, a decline in normal or pre-existing function or ability, disability or impairment resulting from a stroke event in the subject and / or progression or exacerbation of such effects, complications, a decline in normal or pre-existing function or ability, or at least one clinical symptom (e.g., stabilization of an identifiable symptom), physiologically (e.g., stabilization of a physical parameter), or both, and / or suppression of at least one physical parameter that may not be identifiable in the subject. When used herein, “to treat” or “treatment” also means the possibility of preventing future or further stroke events in the subject. In practice of the method disclosed herein, treatment of a stroke event involves administering a therapeutically effective dose of the GDF11 molecule (both as defined herein) to the subject using a dosing regimen targeting early initiation (within sometime between approximately 12 hours and 3 days of the stroke event), high-dose administration of the GDF11 molecule (at least 1 mg / kg), and a limited period of daily treatment (once daily over a period of 2 to 7 days), which can be evaluated using diagnostic and clinical laboratory techniques well known in the art. As described herein above, such techniques are typically based on physical and neurological (behavioral) tests (such as NIHSS) to assess the subject's improved physical motor function and / or cognitive function, and may be supported by medical imaging techniques such as CT scans, MRI scans (e.g., spin-echo MRI or cine magnetic resonance imaging), Doppler ultrasound and angiography, and often supported by auxiliary tests such as electrocardiograms (ECG) and blood tests. Using such techniques, a person skilled in the art can evaluate successful stroke treatment in a subject by visualization of angiogenesis, neurogenesis, improved cerebrovascular structure, and / or the stroke site or vicinity of the site in the subject. To clarify again, successful stroke treatment using the methods of the present disclosure can be established by evaluating one or more of the above criteria (and any combination thereof) and / or by using one or more of the above diagnostic and imaging techniques.

[0049] It should be understood that this disclosure is not limited to the specific methods, protocols, reagents, and other matters described herein. The terms used herein are for illustrative purposes only and are not intended to limit the scope of this disclosure, which is defined solely by the claims.

[0050] (Treatment methods, pharmaceutical compositions): The present invention provides a method and composition for the treatment of stroke in mammalian subjects. The method involves administering a therapeutically effective dose of the GDF11 molecule to the subject. The GDF11 molecule is administered to the subject within a limited time frame of approximately 12 hours to 3 days after a stroke event, under a dosing regimen characterized by high-dose administration of the GDF11 molecule, and such administration is continued for a limited period of 2 to approximately 14 days.

[0051] Surprisingly, we have found that in subjects requiring treatment after a stroke event—when administered at selected high doses and over a limited period within a specific time frame after the stroke event—administering a therapeutically effective dose of the GDF11 molecule or a pharmaceutically acceptable salt thereof can effectively treat the stroke as described herein. This disclosure indicates that the therapeutically effective dose of the GDF11 molecule for such daily administration and such structured administration regimens preferably includes at least about 0.8 mg / kg body weight to about 4 mg / kg body weight or less in rodent subjects, or an equivalent (corresponding) dose in large subjects of this sole therapeutic agent.

[0052] Accordingly, a first aspect of this disclosure provides a method for treating stroke in subjects. The method requires initiating an administration regimen by administering a therapeutically effective dose of the GDF11 molecule to the subject within a time frame of approximately 12 to 72 hours after a stroke event in the subject. The GDF11 molecule is administered at a dose of at least approximately 0.8 mg (0.8 mg / kg) per kg of body weight in rodents or a corresponding dose in large mammal subjects, over a period of 2 to approximately 14 days. In one aspect of this disclosure, the GDF11 molecule is administered at a dose of at least approximately 1 mg (1 mg / kg) per kg of body weight in rodents or a corresponding dose in large subjects. In another aspect of this disclosure, the GDF11 molecule is administered over a shorter period of approximately 7 days, and in yet another aspect, the GDF11 molecule is administered over an even shorter period of 2 to 4 days. In one particular aspect of this disclosure, the method requires the administration of a pharmaceutical composition containing the GDF11 molecule. The composition may further comprise a pharmaceutically acceptable carrier, excipient, or vehicle.

[0053] The GDF11 molecule may be administered systemically and / or topically by any suitable parenteral administration technique. For example, the composition may be administered to a subject by standard intravenous, intramuscular, intraperitoneal, or subcutaneous injection. Thus, in certain embodiments, a composition containing the GDF11 molecule may contain a suitable injection medium such as water for injection. Such a composition may further contain a controlled-release excipient. In practice of such a method, the composition may be provided in the form of nanoparticles such as liposomes. These compositions may further contain biodegradable polymers or may be conveniently provided in the form of a solid or injectable implant. In practice of the compositions and methods of this disclosure, the GDF11 molecule may be present in the composition in the form of a liquid, suspension, or emulsion.

[0054] In certain aspects of this disclosure, the methods are suitable for resulting in any remission, rehabilitation, rejuvenation, improvement, reduction or mitigation of any one or more effects, complications, a decline in normal or pre-existing function or ability, impairment or disability resulting from a stroke event in a subject and / or progression or exacerbation of such effects, complications, a decline in normal or pre-existing function or ability, or suppression of at least one clinical symptom (e.g., stabilization of an identifiable symptom), physiologically (e.g., stabilization of a physical parameter), or both, and / or suppression of at least one physical parameter.

[0055] The successful execution of the methods disclosed herein can be evaluated using techniques well known in the art. As described herein, successful treatment of a stroke event in a subject can be evaluated using diagnostic and clinical testing techniques well known in the art, which rely on physical and neurological (behavioral) examinations to assess improved physical and / or cognitive function in the subject and can be supported by medical imaging techniques such as CT scans, MRI scans (e.g., spin-echo MRI), Doppler ultrasound, and angiography, which are captured by auxiliary examinations. Using such techniques, a person skilled in the art can evaluate successful stroke treatment in a subject by visualization of angiogenesis, neurogenesis, improved cerebrovascular structure, and / or the stroke site or vicinity in the subject. As described herein, successful stroke treatment using the methods disclosed herein can be established by evaluating one or more of the above criteria, using one or more of the above diagnostic and imaging techniques, or by one or more of the above criteria (and any combination thereof).

[0056] In practice of the methods of this disclosure, the selected GDF11 molecule is any therapeutically active form of the GDF11 molecule that may be the same or different in the composition used throughout the course of the administration regimen disclosed herein. For example, the mature human form of the GDF11 polypeptide is particularly preferred. Alternatively, any derivative, variant, or modification of the “natural” GDF11 molecule may be used, insofar as such molecule is therapeutically active as defined herein. In some embodiments, the GDF11 molecule administered to the subject may include a GDF11 polypeptide homodimer. The GD11 molecule may also include the GDF11 polypeptide or a fragment thereof. In some specific embodiments of this disclosure, a variant, derivative, or fragment of the GDF11 polypeptide is administered to the subject. In this regard, the variant, derivative, or fragment of GDF11 may be a conserved modified variant, derivative, or fragment of the natural sequence of the mature form of human GDF11. In certain aspects of this disclosure, the subjects include Collectin kidney 1 (e.g., NCBI gene ID No: 78989) (SEQ ID NO: 4), cathepsin D (e.g., NCBI gene ID No: 1509), Dickkopf-related protein 4 (e.g., NCBI gene ID No: 27121), erythrocyte membrane protein 4.1 (e.g., NCBI gene ID No: 2035), esterase D (e.g., NCBI gene ID No: 2098), hemoglobin (e.g., NCBI gene ID No: 3043 or 3047), interleukin-1 receptor accessory protein (e.g., NCBI gene ID No: 3556), natural killer group 2 member D (e.g., NCBI gene ID No: 22914), Ras-related C3 botulinum toxin substrate 1 (e.g., NCBI gene ID No: 5879), GTP-binding nucleoprotein Ran (e.g., NCBI gene ID Modified GDF11 molecules containing a secondary polypeptide moiety selected from a tissue inhibitor of metalloproteinase 3 (e.g., NCBI gene ID No: 7078) or thymidylate synthase (e.g., NCBI gene ID No: 7298) can be administered.

[0057] More specifically, the GDF11 molecule may be a polypeptide obtained by mutations in the native nucleotide sequence. As herein, a “variant” or “derivative” is a polypeptide substantially homologous to the native or reference polypeptide, but having an amino acid sequence that differs from the native or reference polypeptide's amino acid sequence due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the polypeptide molecule, when compared to the native or reference DNA sequence, includes the addition, deletion, or substitution of one or more nucleotides, but encompasses the sequence encoding the variant or derivative protein or polypeptide (or fragment thereof) that retains the relevant biological activity relative to the reference protein. Those skilled in the art will know that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids in the encoded sequence (e.g., less than 5%, or less than 4%, or less than 3%, or less than 1%) are “conserved modified variants” or conserved modified derivatives, where the alteration results in the substitution of an amino acid by a chemically similar amino acid. Some modifications may improve the associated activity, and as a result, the variant or derivative is intended to have at least about 90%, more preferably about 95% or 100% of the activity of wild-type GDF11, and even more preferably about 110% or more of the wild (natural) activity of the associated GDF11 molecule.

[0058] One method for identifying substituted amino acid residues is, for example, aligning human GDF11 with human GDF11 homologs from one or more non-human species. Alignment can provide guidance not only on residues that may be functionally necessary, but also on residues that may tolerate changes. For example, if the alignment shows two identical or similar amino acids at corresponding positions, those sites are likely to be functionally important. Conversely, if the alignment shows residues at corresponding positions that differ significantly in size, charge, hydrophobicity, etc., those sites are likely to tolerate changes in the functional polypeptide. Similarly, alignment with related polypeptides from the same species, such as GDF8 which does not exhibit the same activity, can also provide guidance on regions or structures necessary for GDF11 activity. Variant or derivative amino acid sequences may have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity with the native or reference sequence. The degree of homology (identity percentage) between natural and mutant sequences can be determined, for example, by comparing the two sequences using a freely available computer program commonly used for this purpose on the World Wide Web. Variant amino acids may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more similar to the derived sequence (referred to herein as the “original” sequence). The degree of similarity (similarity percentage) between the original and mutant sequences can be determined, for example, by a similarity matrix. Similarity matrices are well known in the art, and several tools for comparing two sequences using similarity matrices are freely available online, such as BLAST (available on the World Wide Web at http: / / blast.ncbi.nlm.nih.gov) with a default parameter set.

[0059] Furthermore, the mature GDF11 polypeptide likely contains intrachain disulfide bonds between amino acids 313 and 372; 341 and 404; and 345 and 406 (numbering for the full-length polypeptide including the signal sequence), and amino acid 371 is likely involved in interchain disulfide bonds.

[0060] A given amino acid can be substituted with a residue having similar physicochemical properties, for example, by substituting another residue with an aliphatic residue (such as Ile, Val, Leu, or Ala), or by substituting another residue with a polar residue (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, such as substitutions of entire regions having similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested with any of the assays described herein to confirm that they retain the desired apoptotic activity of the natural or reference polypeptide. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. Such conservative modification variants are in addition to, and not excluded from, polymorphic variants, interspecific homologs, and alleles consistent with this disclosure. Typical conservative substitutions include: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); and 5) isoleucine (I), leucine (L), methionine (M).

[0061] By substituting any cysteine ​​residue that does not participate in maintaining the proper conformation of the polypeptide with serine, the oxidative stability of the molecule can be improved and abnormal crosslinking can be prevented. Conversely, by adding cysteine ​​bonds to the polypeptide, its stability can be improved or oligonucleotide formation can be promoted.

[0062] The GDF11 polypeptide molecule administered to a subject may also include one or more amino acid substitutions, modifications, or additions. For example, substitutions and / or modifications or additions can be used to prevent or reduce proteolysis and / or extend the half-life of the GDF11 molecule in the subject. In non-limiting examples, the GDF11 polypeptide may also be modified by conjugating or fusing it to other polypeptides or polypeptide domains, such as transferrin, albumin, growth hormone; cellulose and / or Fc fragments (see, for example, U.S. Patent No. 9,434,779). The GDF11 polypeptide may also be modified by conjugating or fusing it to a growth and differentiation factor 8 (GDF8) precursor moiety, or any fragment or derivative thereof. Alternatively, the GDF11 polypeptides described herein may include at least one peptide bond substitution. One peptide bond or more peptide bonds, for example, two, three, four, five, or six or more, or all peptide bonds may be substituted. In other embodiments, the GDF11 polypeptide molecules described herein may include one type of peptide bond substitution or multiple types of peptide bond substitution, for example, two, three, four, five, or more types of peptide bond substitution. Non-limiting examples of peptide bond substitutions include ureas, thioureas, carbamates, sulfonylureas, trifluoroethylamines, ortho-(aminoalkyl)phenylacetic acid, para-(aminoalkyl)phenylacetic acid, meta-(aminoalkyl)phenylacetic acid, thioamides, tetrazoles, boronic acid esters, olefin groups, and derivatives thereof.

[0063] In yet another aspect of this disclosure, the GDF11 polypeptide molecule for use in the present method may contain natural amino acids commonly found in polypeptides and / or proteins produced by living organisms, such as Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M), Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(O), Asp(D), Glu(E), Lys(K), Arg(R), and His(H). In other aspects, the GDF11 polypeptide molecule may contain alternative amino acids. Non-limiting examples of alternative amino acids include D-amino acids; β-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, γ-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statins, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citrulline, α-methylalanine, para-benzoylphenylalanine, para-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine, diaminobutyric acid, 7-hydroxytetrahydroisoquinolinecarboxylic acid, naphthylalanine, bif Examples include phenylalanine, cyclohexylalanine, aminoisobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecoric acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, aminobenzoic acid, aminonaphthoic acid, γ-aminobutyric acid, difluorophenylalanine, nipecoic acid, α-aminobutyric acid, thienylalanine, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0064] In further embodiments of this disclosure, modified GDF11 polypeptide molecules, such as molecules modified by adding a moiety to one or more amino acids comprising the polypeptide, can be selected. For example, the GDF11 polypeptides described herein may include one or more partial molecules, such as one or more partial molecules per peptide, two or more partial molecules per peptide, five or more partial molecules per peptide, ten or more partial molecules per peptide, or more partial molecules per peptide. A suitable GDF11 modified polypeptide may include another type of modification and / or moiety, such as one type of modification, two types of modification, three types of modification, or more types of modification. Non-limiting examples of modifications and / or moieties include pegylation; glycosylation; HESation; ELPation; lipidation; acetylation; amidation; end-cap modification; cyano group; phosphorylation; albumination, and cyclization. Alternatively, end-cap modification may include N-terminal acetylation, N-terminal acylation, and N-terminal formylation, or end-cap modification may include C-terminal amidation, introduction of a C-terminal alcohol, aldehyde, ester, and thioester moieties. Therefore, the half-life of the modified GDF11 polypeptide can be extended by the addition of a selected moiety, such as PEG or albumin. In any case, the GDF11 molecule can be modified by known medical chemistry techniques to improve at least one of its distribution in the body, ease of administration, metabolic stability, and at least a combination of these.

[0065] In other aspects of this disclosure, modified GDF11 polypeptide molecules can be provided as pharmaceutically acceptable prodrugs. As used herein, “prodrug” refers to a compound that can be converted into a therapeutic agent by several chemical or physiological processes (e.g., enzymatic processes and metabolic hydrolysis). Thus, the term also refers to a precursor of a pharmaceutically acceptable therapeutic agent. A prodrug may be inactive when administered to a subject, but for example, an ester can be converted in vivo to an active compound by hydrolysis to a free carboxylic acid or free hydroxyl. Prodrug molecules often offer advantages such as solubility, tissue compatibility in the subject, or delayed release. A prodrug may also include any covalent carrier that releases the active compound in vivo when such a prodrug is administered to a subject. Prodrugs of active compounds can be prepared by modifying functional groups present in the active molecule so that the modified portion is cleaved back to the parent active molecule, either in a routine operation or in vivo. When administered to a target, a prodrug binds to a group that cleaves a hydroxyl group, amino group, or mercapto group, generating a free hydroxyl group, free amino group, or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, alcohol acetate, formate, and benzoate derivatives, or acetamide, formamide, and benzamide derivatives of amine functional groups in active compounds, and similar compounds. For example, see Harper, Drug Latentiation in Jucker, ed. Progress in Drug Research 4:221-294 (1962); Morozowich et al, Application of Physical Organic Principles to Prodrug Design in EB Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APHA Acad. Pharm. Sci.40(1977) Bioreversible Carriers in Drug in Drug Design,Theory and Application,E.B.Roche,ed.,APHA Acad.Pharm.Sci.(1987);Design of Prodrugs,H.Bundgaard,Elsevier(1985);Wang et al.(1999)Curr.Pharm.Design.5(4):265-287;Pauletti et al.(1997)Adv.Drug.Delivery Rev. 27:235-256;Mizen et al.(1998) Pharm. Biotech. 11: 345-365; Gaignault et al. (1996) Pract. Med. Chem. 671-696; Asgharnejad, Improving Oral Drug Transport, in Transport Processes in Pharmaceutical Systems,G.L.Amidon,P.I.Lee and E.M.Topp,Eds.,Marcell Dekker,p.185-218(2000);Balant et al.(1990)Eur. J. Drug Metab. Pharmacokinet.,15(2):143-53; Balimane et al. (1999) Adv. Drug Delivery Rev.39(1-3):183-209;Browne(1997)Clin.Neuropharmacol.20(1):1-12;Bundgaard H.(1979)Arch. Pharm. Chem 86(1):1-39;Bundgaard H.(1987)Controlled Drug Delivery 17:179-96;Bundgaard H.(1992) Arfv. Drug Delivery Rev.8(1):1-38;Fleisher et al.(1996) Arfv. Drug Delivery Rev.19(2):115-130;Fleisher et al.(1985)Methods Enzymol.112:360-81;Farquhar D,et al.(1983)Pharm.Sci.72(3):324-325; Freeman et al. (1991) Chem. Soc., Chem. Commun. 875-877; Friis et al. (1996) Eur. J. Pharm. Sci. 4:49-59; Gangwar et al. (1977) Des. Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976:409-21; Nathwani et al. (1993) Drugs 45(6):866-94; Sinhababu et al. (1996) Adv. Drug Delivery Rev. 19(2):241-273; Stella et al. (1985) Drugs 29(5):455-73; Tan et al. (1999) Adv. Drug Delivery Rev. 39(1-3):117-151; Taylor (1996) Adv. Drug Delivery Rev. 19(2):131-148; Valentino et al. (1997) Drug Discovery Today 2(4):148-155; Wiebe et al. (1999) Adv. Drug Delivery Rev. 39(1-3):63-80 (1999); and see Waller et al. (1989) Br. J. Clin. Pharmac. 28:497-507..

[0066] Suitable GDF11 polypeptide molecules for use in this specification can be synthesized by known methods, including recombinant methods and chemical synthesis. Recombination methods for peptide production by introducing a vector containing a peptide-encoding nucleic acid into a suitable host cell are well known in the art; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed, Vols 1 to 8, Cold Spring Harbor, NY (1989); MWPennington and BMDunn, Methods in Molecular Biology Peptide Synthesis Protocols, Vol 35, Humana Press, Totawa, NJ (1994).Appropriate polypeptides can also be chemically synthesized using methods well known in the art (e.g., Merrifield et al. (1964) J.Am.Chem.Soc.85: 2149; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, New York, NY; Kimmerlin et al. (2005) Pept.Res.65:229-260; Nilsson et al. (2005) Annu.Rev.Biophys.Biomol.Struct.34:91-118; WCChan and PDWhite (Eds.) Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, Cary, NC (2000); NLBenoiton, Chemistry of Peptide Synthesis, CRC Press, Boca Raton, Fla. (2005); J. Jones, Amino See Acid and Peptide Synthesis, 2nd Ed., Oxford University Press, Cary, NC (2002); and P. Lloyd-Williams, F. Albericio, and E. Giralt, Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, Boca Raton, Fla. (1997). Peptide derivatives can also be prepared as described in U.S. Patent Nos. 4,612,302; 4,853,371 and 4,684,620; and U.S. Patent Application Publication No. 2009 / 0263843.

[0067] Modification of the original amino acid sequence of the GDF11 polypeptide molecule can be carried out by any of several techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing an oligonucleotide containing a mutant sequence adjacent to a restriction site that allows ligation to a fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis procedure can be used to obtain a modified nucleotide sequence having the desired substitution, deletion, or insertion and the modified specific codon. Techniques for making such modifications are disclosed in U.S. Patent No. 9,434,779, which is incorporated herein by reference in its entirety. In some aspects of this disclosure, modified, variant, or derivative GDF11 polypeptide molecules can be chemically synthesized, and mutations can be incorporated as part of the chemical synthesis method.

[0068] The selected GDF11 molecules are then formulated as pharmaceutical compositions for use in administration to a target. For example, the GDF11 molecules can be provided as pharmaceutically acceptable solvates. The term “solvate” refers to the GDF11 molecules described herein in a solid state in which molecules of a suitable solvent are incorporated into the crystal lattice. A solvent suitable for therapeutic administration is physiologically acceptable at the dose to be administered. Examples of solvents suitable for therapeutic administration are ethanol and water. When water is the solvent, the solvate is called a hydrate. In general, solvates are produced by dissolving molecules in a suitable solvent and isolating the solvate by cooling or using a reverse solvent. Solvates are usually dried and azeotropically prepared under environmental conditions.

[0069] Suitable formulations of pharmaceutical compositions containing an active ingredient (GDF11 molecule) dissolved or dispersed in the pharmaceutical composition are well understood in the art and, in general, do not need to be limited by formulation. Typically, such compositions are prepared as injectable preparations, either as a solution or a suspension, but solid forms suitable for liquid or suspension preparations can also be prepared in liquid form before use. Formulations can also be provided as emulsified or liposomal compositions. The GDF11 molecule can be mixed with an excipient that is pharmaceutically acceptable, miscible with the GDF11 molecule, and in an amount suitable for use in the methods described herein. Suitable excipients include, for example, water, saline solution, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, the pharmaceutical composition may optionally contain small amounts of humectants or emulsifiers, pH buffers, and similar adjuvants that enhance the efficacy of the active ingredient. The compositions of this disclosure may contain pharmaceutically acceptable salts of the components in the composition. Examples of pharmaceutically acceptable salts include acid addition salts with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, tartaric acid, or mandelic acid (formed with the free amino group of the polypeptide), and similar products. The salts formed with the free carboxyl group can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and similar products. Pharmaceutically acceptable carriers, excipients, and vehicles are well known in the art. A good example of a liquid carrier is a sterile aqueous solution containing no substances in addition to the active ingredient and water, and / or may contain a buffer such as sodium phosphate, physiological saline, or both, at a physiological pH, such as phosphate-buffered saline. In further embodiments of this disclosure, the aqueous carrier may contain two or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. The liquid composition may also contain a liquid phase in addition to and excluding water. Good examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.The amount of GDF11 molecule used in this method that would be effective in treating stroke disorders or conditions in the subjects depends on the nature of such disorders or conditions and can be determined by standard clinical techniques.

[0070] In certain aspects of this disclosure, the GDF11 molecule may be administered using a controlled-release dosage form or composition. Controlled-release drugs have the common objective of improving upon the drug therapy achieved by their non-controlled-release counterparts. Ideally, the use of an optimally designed controlled-release dosage form or composition in a medical procedure is characterized by the minimum amount of drug substance used to address the disorder or condition in the shortest possible time. Advantages of the controlled-release approach include: 1) extended activity of the GDF11 molecule; 2) reduced dosing frequency; 3) increased compliance; 4) the possibility of using GDF11 in dosage forms with smaller total amounts; 5) reduced local or systemic side effects; 6) minimized drug (GDF11) accumulation; 7) reduced fluctuations in blood levels; 8) improved therapeutic efficacy; 9) reduced enhancement or loss of drug (GDF11) activity; and / or 10) faster control of the disease or condition.

[0071] Conventional dosage forms and compositions generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, the use of conventional dosage forms can result in a wide range of drug concentrations in the target blood and other tissues. These variations can affect several parameters, including administration frequency, onset of action, duration of effect, maintenance of therapeutic blood levels, adverse effects, side effects, and similar factors. Advantageously, controlled-release dosage forms or compositions can be used to control the onset of action, duration of action, plasma levels within the therapeutic frame, and peak blood levels of the GDFD11 molecule. In particular, controlled release can be used to achieve maximum efficacy of the GDF11 molecule while minimizing the potential adverse effects and safety concerns that may arise from both drug overdose (i.e., below the minimum therapeutic level) and exceeding the drug's toxicity threshold.

[0072] Most controlled-release dosage forms or compositions are designed to initially release an amount of drug (active ingredient) to rapidly achieve the desired therapeutic effect, and then gradually and continuously release additional amounts of the drug to maintain this level of pharmacological effect over a longer period. To maintain a constant drug level in the body, the drug must be released from the dosage form at a rate that restores the amount of drug metabolized and excreted from the body. The controlled release of GDF11 molecules from a selected dosage form or composition can be stimulated by a variety of conditions, including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions, molecules, or compounds. Various known controlled-release dosage forms and compositions can be adapted for use in the methods of this disclosure. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185. These dosage forms provide controlled release using excipients such as hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, or osmotic systems, or combinations thereof that provide a desired release profile.

[0073] The GDF11 molecule is included in the composition in an amount sufficient to exert a therapeutically useful effect with no or minimal undesirable side effects in the subject. Such therapeutically effective concentrations may be predicted by testing the GDF11 molecule in in vitro and in vivo systems well known to those skilled in the art, and then extrapolating the human dose from the tests. The human dose is then typically fine-tuned in clinical trials to achieve the desired therapeutic response. To formulate a composition, a weight fraction of the compound is dissolved, suspended, dispersed, or mixed with a selected carrier, excipient, or vehicle at an effective concentration. The formulated pharmaceutical composition containing the GDF11 molecule can then be conventionally administered, for example, in the form of a unit dose. The term "unit dose," as used in relation to pharmaceutical compositions, represents a physically separate unit suitable as a unit dose for the subject, each unit containing a predetermined amount of GDF11 molecules calculated to obtain the desired pharmacological effect together with a pharmaceutically acceptable carrier, excipient, or vehicle. Examples of unit dosage forms include ampoules and syringes. Accordingly, in one preferred embodiment of the present disclosure, the GDF11 molecule is provided in the form of a pharmaceutical composition comprising water for injection. In a related embodiment, a syringe formulation is provided containing a therapeutically effective amount of the GDF11 molecule in the pharmaceutical composition. The unit dose may be administered in fractional or multiple units. A multi-dose formulation is multiple identical unit doses packaged in a single container, administered in separate unit doses. Examples of multi-dose formulations include vials, bottles of tablets or capsules, or pint or gallon bottles. Thus, a multi-dose formulation is multiple unit doses that are not separated within a package. Alternatively, the GDF11 composition is provided in a kit (package or container) containing at least one therapeutically active agent (GDF11 molecule). In a particular kit, the product may be labeled, promoted, distributed, or marketed as a unit for carrying out the methods of the present disclosure.

[0074] As described herein, preferred routes of administration for the composition are parenteral, for example, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Liquids or suspensions used for such parenteral administration may include: sterile diluents such as water for injection, saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and isotonic modifiers such as sodium chloride or dextrose. The pH of the composition may be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be sealed in ampoules, disposable syringes, or glass or plastic multi-dose vials. Suitable pharmaceutical compositions for injection include sterile aqueous solutions (water-soluble) or dispersants, emulsions, or suspensions, and sterile powders for immediate formulations of injectable sterile solutions or dispersants. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremofor EL.™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In any case, the composition must be sterile and fluid enough to pass easily through a needle. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier or medium may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and similar), and suitable mixtures thereof. The appropriate fluidity of the composition can be maintained, for example, by the use of a coating such as lecithin, maintaining a selected particle size in the case of a dispersant, and using a surfactant. Prevention of microbial activity can be carried out with various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and similar. In some cases, isotonic agents, such as sugars, polyalcohols like mannitol or sorbitol, or sodium chloride, may be included in the composition.Delayed absorption of an injectable composition can be achieved by including an absorption-delaying excipient, such as aluminum monostearate or gelatin, in the composition.

[0075] Injectable sterile solutions can be formulated by combining GDF11 molecules in a specific amount in a suitable solvent with one or a combination of the components listed above, and then, if necessary, by sterile filtration. Generally, formulations are made by incorporating GDF11 molecules into a sterile medium containing a basic dispersion medium and other components selected from those listed above or other components known in the art. In the case of sterile powders for the formulation of sterile injectable solutions, formulation methods include vacuum drying and freeze-drying, to obtain a powder of GDF11 molecules + any desired component from a sterile powder solution that has been previously sterile filtered.

[0076] In certain embodiments of this disclosure, the composition is suitable for implantation in a subject. For example, the implantable device or system may be constructed as a molded article such as a sphere, rod, plate, thin film, fiber, cylinder, sheet, tube, or other suitable shape and size including fine particles, microspheres, and / or microcapsules. The implant can be provided in any size and shape suitable for a specific location, such as a catheter, shunt, subarachnoid continuous infusion device, feeding tube, solid implant to prevent surgical adhesions, uterine implant, artificial sphincter, periurethral implant, splint, ophthalmic implant, contact lens, plastic surgical implant, esophageal stent, gastrointestinal stent, vascular stent, biliary stent, colon stent, pancreatic stent, ureteral stent, urethral stent, lacrimal gland stent, Eustachian tube stent, Fallopian tube stent, nasal stent, sinus stent, tracheal stent, or bronchial stent (containing or coated with an active agent), or a venous access device, implantation port, epidural catheter, or central venous catheter (PICC) port. The implant can be implanted surgically or using minimally invasive techniques such as trocars, catheters, or the like to the desired site. Alternatively, the implant can be implanted into any suitable tissue using standard techniques, such as intradermally, subdermally, subcutaneously, intraperitoneally, intramuscularly, or intraluminally (e.g., intraarterially, intraveinally, intravaginally, intrarectally, or periodontal ligament space). Alternatively, the implant can be fabricated as part of a matrix, graft, prosthesis, or coating. If the implantable device is manufactured in granular form, for example as microparticles, microspheres, or microcapsules, the device can be implanted into the suitable tissue using a cannula, needle and syringe, or similar device for injecting a suspension of particles.

[0077] The GDF11 molecule is typically administered intravenously using a catheter, such as a central venous catheter line or a similar intravenous catheter. Alternatively, the GDF11 composition can be administered intravenously, intramuscularly, intraperitoneally, or subcutaneously using a standard needle and syringe. Thus, in certain embodiments, the composition can be simply formulated to include a suitable injection medium, such as sterile water for injection. In yet another embodiment, the composition can be administered using an external drug pump, such as an infusion pump. In practice of the methods of this disclosure, the GDF11 molecule may be present in the composition in the form of a liquid, suspension, or emulsion.

[0078] In certain aspects of this disclosure, the precise dosage and duration of treatment used in the practice of the method are a function of the type of stroke and the resulting stroke injury addressed, and can be determined empirically using known test protocols or by estimation from in vivo or in vitro test data or subsequent clinical trials. It should also be noted that the concentration and dosage values ​​may vary depending on the severity of the stroke to be addressed. For any particular subject, a specific dosing regimen may be adjusted over time according to the individual needs and the professional judgment of the person administering or directing the administration of the GDF11 composition, and the concentration ranges described herein are merely examples and are not intended to limit the scope or practice of the claimed method.

[0079] In other embodiments of the Disclosure, the composition may be administered on any appropriate basis, such as once daily (QD), twice daily (BID), three times daily (TID), four times daily (QID), every hour ("q_h", where h is the number of hours between doses), or similarly, and each day of treatment may be the same or different throughout the course of treatment. In other embodiments of the Disclosure, treatment may be performed once, or any number of treatment regimens appropriate to the expected particular treatment may be required. For example, an appropriate treatment regimen may include a first dose of the GDF11 molecule at a first dose (day 1 of treatment), followed by a second or higher or lower dose of the GDF11 molecule or subsequent doses (e.g., from day 2 to day 14 of treatment). In some variations, the GDF11 treatment regimen may be performed over a period of one day. In some variations, the GDF11 treatment regimen may be performed over a period of two days. In some variations, the GDF11 treatment regimen may be performed over a period of three days. In some variations, the GDF11 treatment regimen can be administered over a 4-day period. In some variations, the GDF11 treatment regimen can be administered over a 5-day period. In some variations, the GDF11 treatment regimen can be administered over a 6-day period. In some variations, the GDF11 treatment regimen can be administered over a 7-day period. In some variations, the GDF11 treatment regimen can be administered on intermittent days. In such administration, any combination of one, two, or three days can be skipped. For example, days 1, 3, and 6; days 1, 3, and 7; days 1, 2, 4, and 6; days 1, 1, 2, 3, 5, and 7; and so on.

[0080] In certain embodiments, the administration regimen requires a traditional dose setting of the GDF11 molecule, either with escalating or tapering doses, for example, a first dose of at least the smallest high dose of GDF11 on day 1 of the treatment period, ending with a second, higher dose at any intermediate dose between such first and second doses. Alternatively, the dose setting of the GDF11 molecule may require an initial (day 1) high dose of the GDF11 molecule, ending with a final dose of at least the smallest high dose of GDF11, and again with any intermediate doses between such first and final doses. In either dose setting strategy, it may be preferable to administer a first high dose approaching the median toxic dose (MTD) for this molecule, or at least approaching the maximum dose within the therapeutic framework for the GDF11 molecule being administered, and then administering the GDF11 molecule at lower levels in subsequent doses (or doses).

[0081] In other aspects of this disclosure, for example, if subsequent treatment is administered 2 to 7 days after the completion of the initial treatment, the GDF11 treatment regimen can be administered multiple times (e.g., repeatedly) according to so-called “drug-free periods,” i.e., planned treatment interruptions, tolerance breakdowns, or treatment interruptions. Again, for any particular subject, a specific dosing regimen can be adjusted over time according to the individual needs and the professional judgment of the person administering or directing the administration of the GDF11 composition, and the dosing strategies described herein are merely examples and are not intended to limit the scope or practice of the claimed methods. In one particular regimen, the initial or first few doses of the GDF11 molecule are administered in an intensive care setting where the subject has a catheter, such as a central venous catheter line or a similar intravenous catheter. During transfer to a stabilization and step-down unit, stroke recovery unit, or other appropriate setting, subsequent administrations of the GDF11 molecule can be performed using needles and syringes. Subsequent treatment regimens (e.g., after drug-free periods) can be performed using implants or external drug pumps. Subsequent treatment regimens can target the same high dose and short duration as the initial treatment, or they can target lower doses of the GDF11 molecule, whether the treatment is long-term or not.

[0082] In yet another embodiment, the above method is performed, in which a therapeutically effective dose of the GDF11 molecule is administered in combination with at least one additional activator to achieve an additional or synergistic effect. In certain preferred embodiments of the present disclosure, the GDF11 molecule and the second therapeutic activator may be administered together, as a mixture, separately and in parallel, separately and simultaneously, or separately and sequentially. For example, the GDF11 molecule can be administered together with the additional activator according to one of the following methods of administration: i) simultaneously but separately administering at least one dose of the additional activator and at least one dose of the GDF11 molecule; ii) together as a mixture of at least one dose of the additional activator and at least one dose of the GDF11 molecule; iii) sequentially administering at least one dose of the additional activator and at least one dose of the GDF11 molecule, with at least one dose of the additional activator administered before at least one dose of the GDF11 molecule; iv) sequentially administering at least one dose of the additional activator and at least one dose of the GDF11 molecule, administering at least one dose of the additional activator, and then administering at least one dose of the GDF11 molecule; and v) sequentially and together administering at least one dose of the additional activator and at least one dose of the GDF11 molecule in a mixture. Such dosing strategies can be experimentally predicted by testing various combinations and sequences in in vitro and in vivo systems well known to those skilled in the art, and then extrapolating the test results for use in human subjects. The human dose is then typically fine-tuned in clinical trials until a response is achieved.

[0083] Accordingly, a summary of the claimed aspects of the present invention supported by the above specification and the following examples is as follows: (1) A method for treating a stroke in a subject, the method comprising administering a therapeutically effective amount of growth and differentiation factor 11 (GDF11) molecules to the subject within 12 to 72 hours after a stroke event in the subject, wherein the GDF11 molecules are administered at a minimum high dose of GDF11 per day relative to the subject's body weight for a treatment period of 1 to approximately 14 days;

[0084] (2) The method according to (1), wherein administration of the GDF11 molecule is initiated within 12 to 24 hours after the stroke event;

[0085] (3) The method according to (1) or (2), wherein the GDF11 molecule is administered over a treatment period of 2 to 4 days;

[0086] (4) The method according to any one of (1) to (3), wherein the GDF11 molecule is administered to the subject once daily (QD) or once daily on intermittent days;

[0087] (5) The GDF11 molecule is the mature form of the GDF11 polypeptide, as described in any one of (1) to (4);

[0088] (6) The mature form of the GDF11 polypeptide forms a homodimer, as described in (5);

[0089] (7) The method according to any one of (1) to (6), wherein the GDF11 molecule is a polypeptide having at least 91% sequence homology to the natural sequence of the human GDF11 molecule;

[0090] (8) The method according to (7), wherein the GDF11 molecule is recombinant human GDF11 (rhGDF11);

[0091] (9) The method according to (7), wherein the GDF11 molecule is a therapeutically active variant of the human GDF11 molecule;

[0092] (10) The method according to (7), wherein the GDF11 molecule is a therapeutically active derivative of the human GDF11 molecule;

[0093] (11) The variant or derivative GDF11 molecule comprising one or more amino acid substitutions or deletions from the natural sequence of the human GDF11 molecule, as described in (9) or (10);

[0094] (12) The method according to (11), wherein the GDF11 molecule comprises an amino acid analog;

[0095] (13) The method according to (7), wherein the GDF11 molecule is a modified GDF11 polypeptide;

[0096] (14) The modified GDF11 polypeptide is phosphorylated, glycated, glycosylated, pegylated, HES-modified, ELP-modified, lipid-modified, acetylated, amidated, end-capped, or contains a cyano group, albumin, or is cyclized, as described in (13);

[0097] (15) The modified GDF11 polypeptide is a chimeric polypeptide comprising a first GDF11 molecular part and a second part, according to the method of (13);

[0098] (16) The second part is derived from transferrin, growth hormone, or an Fc fragment, as described in (15);

[0099] (17) The modified GDF11 polypeptide having an increased half-life compared to the natural GDF11 polypeptide, according to any one of (13) to (16);

[0100] (18) The method according to any one of (1) to (17), wherein the GDF11 molecule is administered to the subject in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, or vehicle;

[0101] (19) The pharmaceutical composition comprising water for injection, as described in (18);

[0102] (20) The method according to (19), wherein the pharmaceutical composition is administered to the subject by intravenous injection;

[0103] (21) The method according to any one of (1) to (20), wherein the dose of the GDF11 molecule is set upward from the first minimum high dose on the first day of the administration period to a higher dose on the last day of the administration period;

[0104] (22) The method according to any one of (1) to (20), wherein the dose of the GDF11 molecule is set downward from an initial high dose to a final minimum high dose on the last day of the administration period;

[0105] (23) The method according to any one of (1) to (22), further comprising a second dose of a therapeutically effective amount of GDF11 molecules to the subject, wherein the second dose is administered 2 to 7 days after the completion of the first dose;

[0106] (24) Treatment of the subject is characterized by the improved physical motor function or cognitive function of the subject, as described in any one of (1) to (23);

[0107] (25) The stroke event is ischemic, as described in any one of (1) to (24);

[0108] (26) The stroke event is hemorrhagic, as described in any one of (1) to (24);

[0109] (27) Treatment of the subject is characterized by neovascularization, improved cerebral vascular structure, function, or blood flow in or near the site of stroke in the subject, according to any one of (1) to (26);

[0110] (28) A composition comprising a therapeutically effective amount of growth and differentiation factor 11 (GDF11) molecules for use in a method of treating a stroke in the subject, the method comprising initiating administration of the composition to the subject within 12 to 72 hours after a stroke event in the subject, wherein the composition comprises at least a minimum high dose of GDF11 molecules per day relative to the subject's body weight, and the composition is administered to the subject over a treatment period of 1 to about 14 days;

[0111] (29) The composition according to (28), wherein the GDF11 molecule is a mature form of the GDF11 molecule.

[0112] (30) The mature form of the GDF11 polypeptide forms a homodimer, the composition according to (29);

[0113] (31) The composition according to any one of (28) to (30), wherein the GDF11 molecule is a polypeptide having at least 91% sequence homology to the natural sequence of the human GDF11 molecule;

[0114] (32) The composition according to (31), wherein the GDF11 molecule is recombinant human GDF11 (rhGDF11);

[0115] (33) The composition according to (31), wherein the GDF11 molecule is a therapeutically active variant of the human GDF11 molecule;

[0116] (34) The composition according to (31), wherein the GDF11 molecule is a therapeutically active derivative of the human GDF11 molecule;

[0117] (35) The variant or derivative GDF11 molecule comprises one or more amino acid substitutions or deletions from the natural sequence of the human GDF11 molecule, as described in (33) or (34);

[0118] (36) The GDF11 molecule comprises an amino acid analog, as described in (31);

[0119] (37) The composition according to (31), wherein the GDF11 molecule is a modified GDF11 polypeptide;

[0120] (38) The composition according to (37), wherein the modified GDF11 polypeptide is phosphorylated, glycated, glycosylated, pegylated, HES-modified, ELP-modified, lipid-modified, acetylated, amidated, end-capped, or contains a cyano group, albumin, or is cyclized;

[0121] (39) The composition according to (37), wherein the modified GDF11 polypeptide is a chimeric polypeptide comprising a first GDF11 molecular part and a second part;

[0122] (40) The second part is the composition according to (39), derived from transferrin, growth hormone, or Fc fragment;

[0123] (41) The modified GDF11 polypeptide has an increased half-life compared to the natural GDF11 polypeptide, as described in any one of (37) to (40);

[0124] (42) The composition according to any one of (28) to (41), further comprising a pharmaceutically acceptable carrier, excipient, or vehicle;

[0125] (43) The pharmaceutical composition is the composition according to (42), comprising water for injection;

[0126] (44) The composition according to (42) or (43), which is prepared for administration to the subject by intravenous injection;

[0127] (45) A composition according to any one of (28) to (44) for use in a method for treating the ischemic stroke of the subject; and

[0128] (46) A composition according to any one of (28) to (44) for use in a method for treating hemorrhagic stroke.

[0129] It should be understood that the above specification is intended to illustrate and not limit. Many other embodiments will be obvious to those skilled in the art upon reading the above specification. It should be noted that further embodiments of this disclosure can be formed by combining specific embodiments described in different parts of this specification and / or shown in the drawings. Accordingly, the scope of this disclosure should be determined by reference to the appended claims, along with the entire scope of equivalents to which such claims are right. All publications, patents and patent documents are incorporated herein by reference as if they were described herein as a whole and separately. [Examples]

[0130] This disclosure is illustrated herein by the experiments described in the following examples, but should not be construed as limiting. Those skilled in the art will understand that this disclosure may be embodied in many different forms and should not be construed as being limited to the forms described herein. Rather, these examples are provided so that this disclosure fully conveys the disclosure to those skilled in the art. Many modifications and other forms of this disclosure will be apparent to those skilled in the art that this disclosure has the benefit of teaching as set forth in the preceding specification. Certain terms are used, but they are used as in the art unless otherwise specified.

[0131] Example 1: The objective of this example was to evaluate the therapeutic effect of a GDF11 molecular administration regimen consisting of a single dose of rGDF11 administered once daily (QD) for 14 days post-occlusion in a rat permanent middle cerebral artery occlusion (pMCAO) model study, in which physical motor function was measured up to 28 days post-occlusion. The pMCAO method and model are validated rodent models of stroke recovery used to support the initiation of human clinical trials (see, e.g., Iaci et al. (2013) Stroke 44:1942-1950 (dalfampridine) and Iaci et al. (2016) Journal of Neuroscience Research 94:253-265 (Neuregulin 1β3, Glial Growth Factor)).

[0132] pMCAO model. Focal cerebral infarction was created by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modified method of Tamura et al. (1986) No To Shinkei 38:747-751; Iaci et al. (2013) Stroke 44(7):1942-1950; and Iaci et al. (2016) J Neurosci Res 94(3):253-265). Male Sprague Dolly rats (300-400g at the time of surgery) were anesthetized with 3% isoflurane in a mixture of N2O:O2 (2:1) and maintained with 2-2.5% isoflurane in a mixture of N2O:O2 (2:1). The temporalis muscle was split into two sections and reflected in an incision midway between the eye and the tympanic canal. The proximal MCA was exposed by subtemporal bone resection craniotomy without removing the zygomatic arch or transversely incising the facial nerve. The artery was then occluded by microbipolar coagulation from just proximal to the inferior cerebral vein. Using a rectal temperature probe and a connected heating pad, the target body temperature was set to 37.0 ± 1°C throughout the procedure. Cefazolin (40 mg / kg; Hospira, lot: 319002.1, expiration date: February 28, 2022) was administered intraperitoneally (ip) preoperatively to prevent infection. Buprenorphine (approximately 0.1 mg / kg, Simbadol, lot: B195093, expiration date: October 31, 2020) administered subcutaneously (sc) was also given as an analgesic before the MCAO procedure.

[0133] Animals and animal products. This study included two experimental groups of 12 animals each, for a total of 24 rats. For acclimatization purposes, all rats were housed and handled for 5 days prior to surgery for behavioral evaluation. At the end of handling, the rats were randomly assigned and each cage was placed in a different group. The above 24 adult male Sprague Dolly rats were used for the study. The rats were randomly assigned and each cage was placed in a different group. Each rat was given a unique identification number by marking its tail.

[0134] Administer. Daily administration was continued until day 16 after MCAO, and then the drug was started in the animals on day 3 after surgical occlusion of the MCA. The animals received 1 ml / kg of rGDF11 once daily via vehicle or 1 mg / kg (1 ml / kg) intraperitoneally.

[0135] body weight. The animals' weight was measured daily from the day of surgery until the final day of administration (day 16), and then again on days 21 and 28 after MCAO.

[0136] Blood sample taken. Blood samples (approximately 300 microliters (μL) of whole blood) were collected one hour after the final dose on day 16 and processed for serum. Serum samples were evaluated for mechanistic biomarkers of rGDF11 activity in vivo.

[0137] Functional behavior testing. Functional activity was evaluated using behavioral tests involving repositioning limbs and twisting the body. Both behavioral tests were performed before drug administration on the days in which the tests were conducted.

[0138] Repositioning the limbs. The limb repositioning test was divided into forelimb and hindlimb tests. The forelimb repositioning test scored the rat's ability to place its forelimb on the table in response to whisker, visual, tactile, or proprioceptive stimuli. The hindlimb repositioning test scored the rat's ability to place its hindlimb on the table in response to tactile and proprioceptive stimuli. In summary, these tests reflect the function and recovery of the sensorimotor system (see, e.g., De Ryck et al. (1992) Brain Res 573:44-60). For the forelimb repositioning test, the examiner brought the rat closer to the table and scored the rat's ability to place its forelimb on the table in response to whisker, visual, tactile, or proprioceptive stimuli. Similarly, for the hindlimb repositioning test, the examiner assessed the rat's ability to place its hindlimb on the table in response to tactile and proprioceptive stimuli. Separate subscores were obtained for each mode of sensory input (half-points could be specified) and added together to obtain a total score (for the forelimb repositioning test, 0 = normal, 12 = worst; for the hindlimb repositioning test, 0 = normal, 6 = worst). Tests were performed on day 1 before surgery (-1 day or the day before), day 1 after surgery (1 day), 3 days (3 days), 7 days (7 days), 14 days (14 days), 21 days (21 days), and 28 days (28 days) after MCAO. (Day 0 = day of MCAO).

[0139] Body twisting test. The rat was held approximately 1 inch from the base of its tail. Then, the rat was lifted 1 inch above the table surface. The rat was held on the vertical axis at a deviation of no more than 10° to either the left or right. A twist was recorded whenever the rat's head moved off the vertical axis to either side. The rat had to return to the vertical position for the next twist to be counted. A total of 30 twists were counted. The body twist test reflects the symmetry of striatal function (see, e.g., Borlongan et al. (1995) J Neurosci 15:5372-5378). Normal rats typically have an equal number of twists to either side. After local ischemia, rats tend to twist to the opposite (left) side. This test was performed concurrently with the limb repositioning test. This test reflects the symmetry of striatal function (see reference), and normal rats typically have an equal number of twists to either side. After local ischemia, rats tend to twist to the opposite (left) side.

[0140] Brain perfusion and harvesting. On day 28 after MCAO, rats were deeply anesthetized with ketamine / xylazine (91 mg / kg ketamine and 9 mg / kg xylazine, respectively). After the rats were in anesthetized state, transcardiac perfusion was performed with physiological saline (containing 2 units / mL of heparin) followed by 4% paraformaldehyde. The brains were removed and stored overnight in 4% paraformaldehyde, then placed in 1×PBS and stored at 0–4°C. Brain samples were used for histological examination and immunofluorescence staining to evaluate cerebral infarct size, neurogenesis, angiogenesis, and various other markers of GDF11.

[0141] result:

[0142] Clinical observations and survival rates. No deaths were observed in this experiment. All animals appeared normal.

[0143] Behavioral test 1 (forelimb repositioning test). The results of the forelimb repositioning test are shown in Figure 1. There was no difference between the two groups before the start of treatment. As can be seen in Figure 1, animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 3 to day 16 showed superior recovery compared to vehicle-treated animals at day 14 (p<0.05); the trend continued at day 21 (p=0.060). No statistically significant differences were observed at day 7 or day 28.

[0144] Behavioral test 2 (hind limb repositioning test). The results of the hind limb repositioning test are shown in Figure 2. There was no difference between the two groups before the start of treatment. As can be seen in Figure 2, animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 3 to day 16 showed superior recovery compared to vehicles-treated animals on day 7 (p<0.001), day 14 (p<0.001), day 21 (p<0.001), and day 28 (p<0.01). No statistically significant difference was observed on day 3.

[0145] Behavioral test 3 (body twisting test). The results of the body twist test are shown in Figure 3. There was no difference between the two groups before the start of treatment. As can be seen in Figure 3, animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 3 to day 16 showed superior recovery compared to vehicle-treated animals on day 7 (p<0.001), day 14 (p<0.001), day 21 (p<0.001), and day 28 (p<0.001). No statistically significant differences were observed on day 3 or day 5.

[0146] Weight change test. The results of the weight change test are shown in Figure 4. There was no difference between the two groups before the start of treatment. As can be seen in Figure 4, animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 3 to day 16 showed a significant weight loss compared to vehicles treated (p<0.001). The body weight of rGDF11-treated animals decreased by 7.03% on day 3 post-surgery compared to the body weight of animals immediately after surgery. Subsequently, rGDF11-treated animals gained weight at a rate comparable to that of vehicles treated.

[0147] Conclusion. Middle cerebral artery occlusion (MCAO) occurred in adult male Sprague-Dolly rats, resulting in unilateral focal cerebral infarction. Vehicle (1 ml / kg) or rGDF11 (1 mg / kg, 1 ml / kg) was administered intraperitoneally from day 3 to day 16 after MCAO. Behavioral assessment of sensorimotor function: Limb repositioning tests were performed before MCAO and on days 1, 3, 7, 14, 21, and 28 after MCAO. Body twisting tests were performed on the same schedule as the limb repositioning tests. As can be seen from the results of this experiment, the study demonstrated a significant enhancement of post-stroke sensorimotor performance, particularly in body twisting, hind limb repositioning, and forelimb repositioning, in the rGDF11 treatment group. The magnitude of these improvements and endurances was greatest in the body twisting and limb repositioning tests. In addition, an initial weight loss was observed in rGDF11-treated animals, resulting in a 7.03% decrease in weight on day 3 postoperatively compared to the weight immediately after surgery. However, while weight loss in rGDF11-treated animals was maintained throughout the study period compared to vehicle-treated controls, after the study period, rGDF11-treated animals gained weight at a rate comparable to that of vehicle-treated animals.

[0148] Example 2: The purpose of this example was to evaluate the therapeutic effect of a GDF11 molecular administration regimen consisting of a single dose of rGDF11 administered once daily (QD) for 7 days, starting on day 1 post-occlusion in a rat permanent middle cerebral artery occlusion (pMCAO) model study. Physical motor function was measured up to day 28 post-occlusion.

[0149] The pMCAO method model, animals, and animal formulations were the same as those described in Example 1.

[0150] Administer. Daily administration was continued until day 7 after MCAO, and then the drug was administered to the animals starting day 1 after surgical occlusion of the MCA. The animals received 1 ml / kg of rGDF11 once daily via vehicle or 1 mg / kg (1 ml / kg) intraperitoneally.

[0151] Weight, blood sampling, functional behavior tests, limb repositioning tests, body twisting tests, and cerebral perfusion and sampling were measured or performed as described in Example 1.

[0152] result:

[0153] Behavioral test 1 (forelimb repositioning test). The results of the forelimb repositioning test are shown in Figure 5. There was no difference between the two groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 1 to day 7 showed superior recovery compared to vehicle-treated animals on day 3 (p<0.0001), day 7 (p<0.001), day 14 (p<0.001), day 21 (p<0.0001), and day 30 (p<0.001). No statistically significant difference was observed on day 5.

[0154] Behavioral test 2 (hind limb repositioning test). The results of the hind limb repositioning test are shown in Figure 6. There was no difference between the two groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 1 to day 7 showed superior recovery compared to vehicle-treated animals on day 3 (p<0.001), day 5 (p<0.01), day 7 (p<0.0001), day 14 (p<0.001), day 21 (p<0.0001), and day 30 (p<0.0001).

[0155] Behavioral test 3 (body twisting test). The results of the body twist test are shown in Figure 7. There was no difference between the two groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg from day 3 to day 7 showed superior recovery compared to vehicle-treated animals on day 7 (p<0.05), day 14 (p<0.001), day 21 (p<0.001), and day 30 (p<0.01). No significant difference was observed on day 3 or day 5.

[0156] Conclusion.

[0157] Middle cerebral artery occlusion (MCAO) occurred in adult male Sprague-Dolly rats, resulting in unilateral focal cerebral infarction. Vehicle (1 ml / kg) or rGDF11 (1 mg / kg, 1 ml / kg) was administered intraperitoneally from day 1 to day 7 after MCAO. Behavioral assessment of sensorimotor function: Limb repositioning tests were performed before MCAO and on days 1, 3, 7, 14, 21, and 30 after MCAO. Body twisting tests were performed on the same schedule as the limb repositioning tests. As can be seen from the results of this experiment, the study demonstrated durable sensorimotor function improvement after stroke in the rGDF11 treatment group, particularly in hind limb repositioning, forelimb repositioning, and body twisting tests. Significant improvement in the limb repositioning test was observed after two doses. Sensorimotor function improvements were maintained for at least 30 days after occlusion.

[0158] Example 3: The purpose of this example was to evaluate the therapeutic effect of a GDF11 molecular administration regimen consisting of a single daily dose of rGDF11 administered during each period from day 1 to day 7 post-occlusion (i.e., treatment on day 1, day 3, day 5, or day 7) in a rat permanent middle cerebral artery occlusion (pMCAO) model study. Physical motor function was measured up to 14 days post-occlusion.

[0159] The pMCAO method model, animals, and animal formulations were the same as those described in Example 1.

[0160] Administer. The animals were administered rGDF11 once daily, starting on day 1 after surgical occlusion of the MCA, with daily administration continuing for 1, 3, 5, or 7 days after MCAO. The animals received 1 ml / kg of rGDF11 per vehicle or 1 mg / kg (1 ml / kg) intraperitoneally once daily.

[0161] Weight, blood sampling, functional behavior tests, limb repositioning tests, body twisting tests, and cerebral perfusion and sampling were measured or performed as described in Example 1.

[0162] result:

[0163] Behavioral test 1 (forelimb repositioning test). The results of the forelimb repositioning test are shown in Figure 8 for animals treated with rGDF11 for 1 day, 3 days, 5 days, and 7 days, as well as for animals treated with a vehicle. There were no differences among the five groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 1 showed superior recovery time compared to vehicles-treated animals on day 3 (p<0.05), but no statistically significant difference was observed on day 5, day 7, or day 14. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 3 showed superior recovery time compared to vehicles-treated animals on day 3 (p<0.001), day 5 (p<0.001), day 7 (p<0.01), and day 14 (p<0.05). Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.01), 5 (p<0.0001), 7 (p<0.0001), and 14 (p<0.0001). Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 7 also showed superior recovery times compared to vehicles-treated animals on days 5 (p<0.01), 7 (p<0.01), and 14 (p<0.001), with no statistically significant difference observed on day 3.

[0164] Behavioral test 2 (hind limb repositioning test). The results of the hind limb repositioning test are shown in Figure 9. There were no differences among the five groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 1 showed superior recovery times compared to vehicles-treated animals on day 3 (p<0.05) and day 14 (p<0.05), and no statistically significant differences were observed on day 5 or day 7. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 3 showed superior recovery times compared to vehicles-treated animals on day 3 (p<0.05), day 5 (p<0.0001), day 7 (p<0.0001), and day 14 (p<0.001). Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 5 showed superior recovery times compared to vehicles-treated animals on days 5 (p<0.0001), 7 (p<0.0001), and 14 (p<0.0001), with no statistically significant difference observed on day 3. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 7 also showed superior recovery times compared to vehicles-treated animals on days 5 (p<0.0001), 7 (p<0.0001), and 14 (p<0.0001), with no statistically significant difference observed on day 3.

[0165] Behavioral test 3 (body twist test). The results of the body twist test are shown in Figure 10. There were no differences among the five groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 1 showed superior recovery times compared to vehicles-treated animals on day 3 (p<0.001) and day 7 (p<0.001), and no statistically significant difference was observed on day 5 or day 14. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 3 showed superior recovery times compared to vehicles-treated animals on day 3 (p<0.001), day 5 (p<0.0001), and day 7 (p<0.001), and no statistically significant difference was observed on day 14. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 5 showed superior recovery times compared to vehicles-treated animals on days 5 (p<0.001), 7 (p<0.0001), and 14 (p<0.001), with no statistically significant difference observed on day 3. Animals that received rGDF11 intraperitoneally at a dose of 1 mg / kg on day 7 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.001), 5 (p<0.0001), 7 (p<0.0001), and 14 (p<0.0001).

[0166] Conclusion.

[0167] Shorter durations of rGDF11 treatment resulted in transient sensorimotor function improvements of 1 or 3 days, but significant improvements in sensorimotor function were observed. Specifically, in the group that received only a single dose of rGDF11 on day 1 post-occlusion, significant improvements were observed in all three assessments on day 3, significant improvements in torsion outcomes on day 7, and significant improvements in hind limb repositioning on day 14. The 5-day treatment regimen was not inferior to the 7-day treatment regimen. Surprisingly, both 5-day and 7-day treatment periods showed durable therapeutic effects.

[0168] Example 4: The objective of this example was to evaluate the therapeutic effect of a GDF11 molecular administration regimen for dose ranges (0.1, 0.5, 1.0, 2.0, and 4.0 mg / kg) in a rat permanent middle cerebral artery occlusion (pMCAO) model study, in which rGDF11 was administered to be started on day 1 post-occlusion. Physical motor function was measured up to day 28 post-occlusion.

[0169] The pMCAO method model, animals, and animal formulations were the same as those described in Example 1.

[0170] Administer. Animals were administered rGDF11 once daily, starting on day 1 after surgical occlusion of the MCA, at five different doses: 0.1, 0.5, 1, 2, and 4 mg / kg, daily for 5 days. Animals received 1 ml / kg of rGDF11 once daily via vehicle or 1 mg / kg (1 ml / kg) intraperitoneally.

[0171] Weight, blood sampling, functional behavior tests, limb repositioning tests, body twisting tests, and cerebral perfusion and sampling were measured or performed as described in Example 1.

[0172] result:

[0173] Behavioral test 1 (forelimb repositioning test). The results of the forelimb repositioning test for animals treated with doses of 0.1, 0.5, 1, and 4 mg / kg, as well as animals treated with a vehicle, are shown in Figure 11. There were no differences among the six groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 0.1 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.01), 5 (p<0.001), 7 (p<0.001), 14 (p<0.05), and 21 (p<0.05), and no statistically significant difference was observed on day 28. Animals that received rGDF11 intraperitoneally at a dose of 0.5 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.0001), 5 (p<0.0001), 7 (p<0.01), and 14 (p<0.05), with no statistically significant differences observed on days 21 or 28. Animals that received rGDF11 intraperitoneally at a dose of 1.0 mg / kg up to day 5 also showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.001), 5 (p<0.0001), 7 (p<0.001), 14 (p<0.05), 21 (p<0.01), and 28 (p<0.05). Animals that received rGDF11 intraperitoneally at a dose of 2.0 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.0001), 5 (p<0.0001), 7 (p<0.001), 14 (p<0.01), 21 (p<0.001), and 28 (p<0.001). Animals that received rGDF11 intraperitoneally at a dose of 4.0 mg / kg up to day 5 also showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.001), 5 (p<0.001), 7 (p<0.001), 14 (p<0.01), 21 (p<0.001), and 28 (p<0.001).

[0174] Behavioral test 2 (hind limb repositioning test). The results of the hind limb repositioning test are shown in Figure 12 for animals treated with doses of 0.1, 0.5, 1, 2, and 4 mg / kg, as well as animals treated with a vehicle. There were no differences among the six groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 0.1 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.01), 5 (p<0.0001), and 7 (p<0.05), and no statistically significant differences were observed on days 14, 21, or 28. Animals that received rGDF11 intraperitoneally at a dose of 0.5 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.0001), 5 (p<0.0001), and 7 (p<0.001), and no statistically significant differences were observed on days 14, 21, or 28. Animals that received rGDF11 intraperitoneally at a dose of 1.0 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.0001), 5 (p<0.0001), 7 (p<0.0001), 14 (p<0.01), 21 (p<0.01), and 28 (p<0.01). Animals that received rGDF11 intraperitoneally at a dose of 2.0 mg / kg up to day 5 also showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.0001), 5 (p<0.0001), 7 (p<0.0001), 14 (p<0.001), 21 (p<0.0001), and 28 (p<0.0001). Animals that received rGDF11 intraperitoneally at a dose of 4.0 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.0001), 5 (p<0.0001), 7 (p<0.0001), 21 (p<0.001), and 28 (p<0.0001), with no statistically significant difference observed on day 14.

[0175] Behavioral test 3 (body twisting test). The results of the body twist test are shown in Figure 13. There were no differences among the six groups before the start of treatment. Animals that received rGDF11 intraperitoneally at a dose of 0.1 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on day 3 (p<0.05), day 5 (p<0.05), and day 14 (p<0.05), and no statistically significant differences were observed on day 7, day 21, or day 28. Animals that received rGDF11 intraperitoneally at a dose of 0.5 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on day 14 (p<0.01), day 21 (p<0.05), and day 28 (p<0.001), and no statistically significant differences were observed on day 3, day 5, or day 7. Animals that received rGDF11 intraperitoneally at a dose of 1.0 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.01), 5 (p<0.001), 14 (p<0.05), 21 (p<0.01), and 28 (p<0.001), with no statistically significant difference observed on day 7. Animals that received rGDF11 intraperitoneally at a dose of 2.0 mg / kg up to day 5 showed superior recovery times compared to vehicles-treated animals on days 3 (p<0.05), 5 (p<0.001), 7 (p<0.01), 14 (p<0.0001), 21 (p<0.0001), and 28 (p<0.0001). Animals that received rGDF11 intraperitoneally at a dose of 4.0 mg / kg up to day 5 showed superior recovery times compared to vehicle-treated animals on days 14 (p<0.01), 21 (p<0.001), and 28 (p<0.0001), and no statistically significant differences were observed on days 3, 5, or 7.

[0176] Conclusion.

[0177] rGDF11 treatment improved sensorimotor function recovery across a wide dose range. Efficacy across all treatment groups at various time points was observed in the 0.1–4.0 mg / kg dose range of rGDF11 administered daily for 5 days. In particular, animals administered 0.1 mg / kg and 0.5 mg / kg showed little to no improvement in forelimb and hindlimb function at 7 days. No statistically significant differences were observed in either the forelimb or hindlimb repositioning test at the final day of the experiment (day 28). However, these animals administered 1.0 mg / kg, 2.0 mg / kg, and 4.0 mg / kg of rGDF11 surprisingly showed statistically significant improvement in both the forelimb and hindlimb repositioning tests up to day 28. Animals receiving rGDF11 doses of 1.0 mg / kg, 2.0 mg / kg, and 4.0 mg / kg showed remarkable and significant long-term therapeutic effects compared to lower doses of 0.1 mg / kg and 0.5 mg / kg.

[0178] Example 5: The purpose of this example was to evaluate the therapeutic effect of a GDF11 molecular administration regimen consisting of a single dose of rGDF11 administered 30 minutes after ICH, and then once daily for 7 days (q24x7), in the C57Bl6 / j mouse model.

[0179] ICH mouse model.

[0180] Eleven-week-old male C57BL / 6J mice (Jackson Laboratory, Bar Harbor, Maine) were housed in standard acrylic cages with free access to food and water, in a 12-hour light-dark cycle. In each experiment, mice were randomly assigned to either a treatment group or a vehicle group before injury.

[0181] The animals were treated in a blinded manner. All procedures and evaluations were performed in a blinded manner.

[0182] In mice, intrastriatal collagenase injection was used to induce ICH. After induction of anesthesia with 4.6% isoflurane, the trachea was intubated and the lungs were mechanically ventilated with 1.5% isoflurane in a 30% / 70% O2 / N2 mixture. Rectal temperature was maintained at 37°C ± 0.2°C by circulating warm water in a lower abdominal waterbed. The animals' heads were fixed to a stereotactic frame. A midline scalp incision was made. After skull exposure, a cranial perforation was made 2.2 mm to the left of the cruciate suture, and a 0.5 μL injection needle (Hamilton, Reno, Nevada, USA) was advanced to a depth of 3 mm from the cortex. Clostridium collagenase type IV-S (Sigma, St. Louis, Missouri, USA) was injected over 2 minutes (0.075 U in 0.4 μL saline). After closing the incision, the animals were allowed to recover to the point of spontaneous ventilation, and then given free access to food and water.

[0183] Animals received intraperitoneal vehicle or rGDF11 starting 30 minutes post-ICH, and continued daily for 7 days. All test substances were labeled with codes. Both surgeons and researchers performing behavioral assessments were blinded to treatment assignments.

[0184] 100 μl of either solution A (1.0 mg / kg rGDF11) or solution B (vehicle) was administered by intraperitoneal (IP) injection 30 minutes after ICH daily, followed by once daily (q24x7) for 7 days.

[0185] Neurobehavioral evaluation. Animals were randomly assigned to either the vehicle group (n=22) or the rGDF11 group (n=22), and mortality, neurological severity score (NSS), rotarod latency (RR), mean speed (catwalk) 7 days post-treatment, and forelimb support base (catwalk) 7 days post-treatment were evaluated.

[0186] Neurological severity score. Animals were assessed based on their behavior in seven categories, including spontaneous activity, symmetry, climbing, balance and coordination, proprioception, tactile sensation, and tactile response. Detailed scoring criteria for the NSS are provided in Appendix A, where 21 = normal and 3 = fatal. These tests were performed before ICH surgery (day 0) and on days 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 after ICH. (Day 1 = day of ICH).

[0187] Rotarod test. The effect of therapeutic intervention on vestibular motor function was evaluated using an automated rotarod (Ugo Basile, Comerio, Italy). On the day before injury, mice underwent two consecutive conditioning tests at a set rotation speed of 16 revolutions / minute for 60 seconds, followed by three additional tests at accelerated (4-40) rotation speeds. The mean time difference of falling from the rotating cylinder in the second set of tests was recorded as the baseline latency. To evaluate motor outcomes, mice underwent the rotarod test on days 1-7, 14, 21, and 28 post-injury. On each day, mice underwent three tests with a 15-minute interval between tests. The mean latency of falling from the rod was recorded.

[0188] Throughout all rotor rod tests, the test is manually stopped under the following conditions, and the latency to stopping is recorded for each test: 1) The mouse is unable to run on the rotating rod and remains still, holding onto the rod for two consecutive rotations; 2) After 20 seconds of rotation, the speed reaches a maximum of 40 rpm.

[0189] Catwalk analysis. Precise features of spontaneous ground walking movements were collected and analyzed using the Catwalk XT system (Noldus Information Technology; Leesburg, Virginia). Illuminated footprint detection of all four limbs was achieved by recording mice as they crossed a glass plate illuminated from within. In this setup, contact between the feet and the floor appeared bright, while the mouse's body appeared dark. All procedures were performed by an experimenter blinded to the treatment group. Mice were acclimatized to the room and system environment and then trained to continuously cross the passage before baseline data collection. Successful running required continuous walking movements crossing the passage without stopping or sniffing. Automated footprint labeling was performed using Catwalk XT software (v10.6). Mislabeled footprints were visually inspected and manually corrected. Automated gait analysis was performed using Catwalk XT software (v10.6), and the parameters of the subject were averaged and analyzed over three successful runs: average speed, support base (forelimbs, hindlimbs), gait, regularity index, single-limb support, and variability. Outlier scores exceeding two standard deviations from the mean were excluded from the analysis (n=1 per group). Intergroup comparisons were performed 7 days after injury using independent sample t-tests.

[0190] For brain / tissue sampling for the ICH test, animals were slaughtered 28 days after injury.

[0191] Statistical analysis: Multiple Kolmogorov-Smirnov tests or two-way analysis of variance (ANOVA) with repeated measures were used to compare neurological severity scores, rotarod latency, and catwalk analysis against time as repeated variables. Bonferroni correction was used for repeated measures techniques in ANOVA. A p-value < 0.05 was considered statistically significant. All values ​​are expressed as mean ± standard error. Statistical analysis was performed using SPSS.

[0192] mortality rate.

[0193] ICH injection resulted in 3 deaths within 24 hours of injury (#9, 11, 14). The vehicle group resulted in 2 deaths within 24 hours (#10, 35), 4 deaths on day 6 (#26, 28, 29, 36), and 1 death on day 10 (#34). [Table 1]

[0194] Figure 14 shows the survival percentages for C57Bl6 / j mice as a function of day 0 and days after ICH for group A (rGDF11 treatment group) and group B (vehicle group). Survival analysis was performed using log-rank (Prism 7.0) with p=0.1893. Group A had 3 deaths within 24 hours of injury and no deaths for the remainder of the following 28 days. Group B had 2 deaths within 24 hours of injury, followed by 4 deaths on day 6 and 1 death on day 10. Group B showed a trend toward decreasing survival.

[0195] Figure 15A shows the post-ICH neurological severity scores for Group A and Group B as days after injection, including dead animals. Neurological severity scores were assessed daily on days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28. A two-way repeated measures analysis of variance (ANOVA) was performed using SPSS, p=0.121. Group A showed measurable improvement in neurological severity scores compared to Group B on days 7, 14, 21, and 28. Daily independent t-tests showed statistically independent results on day 21 (p=0.048) and day 28 (0.019).

[0196] Figure 15B shows the neurological severity scores as a function of days after ICH in groups A and B, excluding dead animals (n=19 in group A and n=15 in group B). A two-way repeated measures ANOVA with Bonferroni correction was performed using Graphpad Prism software. Neurological severity scores in group A showed measurable improvement compared to group B on days 5, 7, 14, 21, and 28. The two-way ANOVA showed statistically independent results on day 21 (p=0.006) and day 28 (p=0.008).

[0197] Figure 16A shows plots of rotorod latency as a function of days after ICH injury for groups A and B, including dead animals. Survival analysis was performed using log-rank (Prism 7.0), p=0.1893. Daily rotorod testing was performed on days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28. Two-way ANOVA with repeated measures was performed using SPSS, p=0.024. Starting on day 4, group A showed improvement in rotorod latency, which continued until day 28. On days 14 and 28, group B showed a statistically significant (p<0.05) decrease in rotorod latency compared to group A (two-sided ANOVA 0.024). On days 6, 7, 14, 21, and 28, the rotorod latency of group A was within the statistical error of the pre-injury rotorod latency. Group B did not improve to the pre-injury rotorod latency level over 14 days. On days 21 and 28, the rotor rod latency of group B was within the statistical error of the pre-injury rotor rod latency.

[0198] Figure 16B shows plots of rotorod latency as a function of days after ICH injury for groups A and B, excluding dead animals. Daily rotorod tests were performed on days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28. Statistical analysis was performed using corrected multiple Kolmogorov-Smirnov tests or two-way ANOVA. Starting on day 4, group A showed improved rotorod latency, which continued until day 28. On days 7, 14, and 28, group B (vehicle) showed a statistically significant (p<0.05) decrease in rotorod latency compared to group A (GDF11). On days 6, 7, 14, 21, and 28, the rotorod latency of group A was within or greater than the statistical error of the pre-injury rotorod latency. After day 14, group B improved to within the statistical error of the pre-injury rotorod latency.

[0199] Catwalk evaluations were performed on mice in groups A and B on days 0, 2, and 7. The catwalk test measured the average speed and forelimb support base for all groups.

[0200] Figures 17A and 17B show the walking ability of group A compared to group B. Figure 17A shows the average speed (cm / second) 7 days after the first treatment. Figure 17B shows the forelimb support base of the rGDF11-administered group (group A) compared to the vehicle group (group B).

[0201] Systematic differences between groups were described as a function of treatment allocation. Group B showed a trend toward non-survival. On days 21 and 28, there was a significant (p<0.05) decrease in NSS in Group B (two-sided ANOVA 0.121). On days 7, 14, 21, and 28, there was a significant (p<0.05) decrease in rotarod in Group B (two-sided ANOVA 0.024). Using catwalk analysis, there was a significant decrease in mean walking speed in Group B (p=0.043). Group B showed a statistically significant decrease in mean walking speed (p=0.043). Group B showed a statistically significant increase in forelimb support base (p=0.015).

[0202] Example 6: The objective of this example was to evaluate neurogenesis in 5-day GDF11 treatment at different doses for stroke recovery during the 24-day post-treatment period. Neurogenesis was measured using the Sox2 pluripotent neural stem cell marker.

[0203] The pMCAO method model, animals, and animal formulations were the same as those described in Example 1.

[0204] Administer. Animals were administered rGDF11 once daily, starting on day 1 after surgical occlusion of the MCA, at five different doses: 1, 2, and 4 mg / kg, for 5 days. Animals received 1 ml / kg of rGDF11 per vehicle or 1 mg / kg (1 ml / kg) intraperitoneally once daily.

[0205] Imaging studies: Rats were perfused with PBS, followed by 4% PFA. Brains were extracted, washed with PBS, and then cryopreserved in 20% sucrose. After cryopreservation, the brains were embedded in an optimal cutting temperature (OCT) compound and stored at -20°C.

[0206] Free-suspension 50 μm sections were collected using a Leica cryostat. Sections from 6 rats were spatially matched and prepared for immunofluorescence staining. Antigen recovery was performed at 90°C for 10 minutes using 1× citrate buffer (pH 6.0). Then, brain sections were washed with PBS and blocked at room temperature in blocking buffer (1× PBS, 0.5% Triton X-100, and 10% normal donkey serum) for 1 hour. After washing the samples in wash buffer (1× PBS, 0.5% Triton X-100), they were incubated with primary antibody. Primary antibody (rabbit polyclonal for SOX2, Abcam; ab97959) was diluted 1:200 in antibody dilution buffer (1× PBS, 1% BSA, and 0.5% Triton X-100) and incubated overnight at 4°C with gentle agitation. After primary antibody incubation, the sections were washed three times with wash buffer and incubated with secondary antibody (donkey anti-rabbit IgG Alexa Fluor 488, Invitrogen; A-21206) diluted 1:2000 in antibody dilution buffer at room temperature for 1 hour. Then, the secondary antibody was removed, and the sections were incubated with 300 nM DAPI in PBS and nuclear staining reagent (Invitrogen; D3571) for 10 minutes, followed by three washes with wash buffer. The slide sections were then placed on microscope slides, dried, and then mounted with mounting medium, coverslips, and nail polish.

[0207] Images were acquired at 20x magnification using an Olympus VS120 and analyzed with ImageJ software. Image analysis was performed anonymously, and the target area of ​​the infarcted hemisphere was cropped to isolate the ventricular zone (VZ) closely adjacent to the stroke. Images of the contralateral hemisphere were acquired from a similar area of ​​the VZ in the non-infarcted hemisphere. Sections were excluded from analysis if the complete VZ could not be preserved due to the stroke. For each image, the number of Sox2-positive cells was quantified using the particle analysis tool in ImageJ, and independent t-tests (designated comparisons) were performed using GraphPad Prism software for statistical analysis.

[0208] result:

[0209] Figures 18A–18D show a series of images illustrating progenitor cell replenishment in the ipsilateral subventricular zone at the site of injury in animals sacrificed 29 days post-injury. Different doses were measured: Figure 18A (vehicle), Figure 18B (1 mg / kg), Figure 18C (2 mg / kg), and Figure 18D (4 mg / kg). Just from observation, scans show substantially increased Sox2-positive cells with rGDF11 administrations of 1 mg / kg, 2 mg / kg, and 4 mg / kg.

[0210] Figure 19 shows the analysis of the data from Figures 18A-D. The data are presented as mean ± SEM using an independent t-test with a p-value < 0.05. Each of the 1 mg / kg, 2 mg / kg, and 4 mg / kg doses of GDF11 shows a statistically significant increase in the number of Sox2-positive cells. The statistically significant increase in neurogenesis corresponds to improved behavioral tests in mice administered rGDF11. In summary, the data indicate progenitor cell replenishment in the subventricular zone ipsilateral to the injury site.

[0211] Figures 20A–20D show progenitor cell replenishment in the subventricular zone opposite the injury site in animals sacrificed 29 days post-injury. Different doses were measured: Figure 20A (vehicle), Figure 20B (1 mg / kg), Figure 20C (2 mg / kg), and Figure 20D (4 mg / kg). Just from observation, scans show substantially increased Sox2-positive cells with rGDF11 administrations of 1 mg / kg, 2 mg / kg, and 4 mg / kg.

[0212] Figure 21 shows the analysis of the data from Figures 20A-D. The data are presented as mean ± SEM using statistically independent t-tests with p-value < 0.05 and p-value < 0.1. Both the 1 mg / kg dose and the 2 mg / kg dose showed statistical significance (p < 0.05). The 4 mg / kg dose of GDF11 had a p < 0.10 in Sox2-positive cell count for each administration group. The statistically significant increase corresponds to improved behavioral tests in mice administered rGDF11. In summary, the data indicate progenitor cell replenishment in the subventricular zone opposite to the injury site.

[0213] Figure 22 shows an analysis comparing the ipsilateral and contralateral hemispheres of the injury site. The data are presented as mean ± SEM values ​​using statistically independent t-tests with p-values ​​< 0.05 and p-values ​​< 0.1. The data indicate that GDF11 has a greater effect on progenitor cell regeneration in the SVZ of the ipsilateral hemisphere of infarction than in the ipsilateral hemisphere. Scalar 1 109 PRT Homo sapiens 1 Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser Glu Ser Arg Cys Cys 1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu 35 40 45 Tyr Met Phe Met Gln Lys Tyr Pro His Thr His Leu Val Gln Gln Ala 50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser 65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser 100 105

Claims

1. A composition comprising a therapeutically effective amount of growth and differentiation factor 11 (GDF11) molecules for use in a method of treating a stroke, wherein the method comprises initiating administration of the composition to the subject within 12 to 72 hours after a stroke event, the composition comprising GDF11 molecules in an amount of at least a minimum high dose of GDF11 relative to the subject's body weight per day, and the composition being administered to the subject for a treatment period of 1 to about 14 days.

2. The composition according to claim 1, wherein the GDF11 molecule is a mature form of the GDF11 molecule.

3. The composition according to claim 2, wherein the mature form of the GDF11 polypeptide forms a homodimer.

4. The composition according to any one of claims 1 to 3, wherein the GDF11 molecule is a polypeptide having at least 91% sequence homology to the natural sequence of a human GDF11 molecule.

5. The composition according to claim 4, wherein the GDF11 molecule is recombinant human GDF11 (rhGDF11).

6. The composition according to claim 4, wherein the GDF11 molecule is a therapeutically active variant of the human GDF11 molecule.

7. The composition according to claim 4, wherein the GDF11 molecule is a therapeutically active derivative of the human GDF11 molecule.

8. The composition according to claim 6 or 7, wherein the variant or derivative GDF11 molecule comprises one or more amino acid substitutions or deletions from the natural sequence of the human GDF11 molecule.

9. The composition according to claim 4, wherein the GDF11 molecule comprises an amino acid analog.

10. The composition according to claim 4, wherein the GDF11 molecule is a modified GDF11 polypeptide.

11. The composition according to claim 10, wherein the modified GDF11 polypeptide is phosphorylated, glycated, glycosylated, pegylated, HES-modified, ELP-modified, lipid-modified, acetylated, amidated, end-capped, or contains a cyano group, albumin, or is cyclized.

12. The composition according to claim 10, wherein the modified GDF11 polypeptide is a chimeric polypeptide comprising a first GDF11 molecular portion and a second portion.

13. The composition according to claim 12, wherein the second portion is derived from transferrin, growth hormone, or an Fc fragment.

14. The composition according to any one of claims 10 to 13, wherein the modified GDF11 polypeptide has an increased half-life compared to the natural GDF11 polypeptide.

15. The composition according to any one of claims 1 to 14, further comprising a pharmaceutically acceptable carrier, excipient, or vehicle.

16. The pharmaceutical composition according to claim 15, comprising water for injection.

17. The composition according to claim 15 or 16, wherein the composition is prepared for administration to the subject by intravenous injection.

18. A composition according to any one of claims 1 to 17 for use in a method for treating the aforementioned ischemic stroke.

19. A composition according to any one of claims 1 to 17 for use in a method for treating the aforementioned hemorrhagic stroke.

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