WO2021160938A1 - Systemic administration of a pharmaceutical composition comprising cdnf or manf polypeptide for use in the treatment of reperfusion injury - Google Patents

Abstract

The present invention provides a pharmaceutical composition comprising a CDNF or MANF polypeptide for use in the treatment of reperfusion injury, said composition being administered systemically to a subject, wherein said CDNF polypeptide comprises the amino acid sequence of a mature CDNF polypeptide, a variant or a functional fragment thereof, and wherein said MANF polypeptide comprises the amino acid sequence of a mature MANF polypeptide, a variant or a functional fragment thereof. The present invention also provides a kit for use in the treatment of reperfusion injury such as ischemic stroke or hemorrhagic brain injury comprising an effective amount of a CDNF or MANF polypeptide and a thrombolytic drug.

Description

Systemic administration of a pharmaceutical composition comprising CDNF or MANF polypeptide for use in the treatment of reperfusion injury

FIELD OF THE INVENTION

The present invention relates to the field of bioactive proteins and also to the field of neurotrophic factors, and more particularly to the field of treating and preventing reperlusion injury after ischemia.

BACKGROUND OF THE INVENTION

Stroke is the fifth leading cause of death and a leading cause of disability. The cause for the latter is slow and incomplete recovery from stroke resulting in a great reduction in the quality of life. Acute care relies on thrombolytic treatment or by mechanical recanalization, but this can be applied only to a small fraction of patients, and its efficacy is not uniform. In the thrombolytic treatment, tissue plasminogen activator (tPA) or analogue thereof is administered to a patient. tPA is a "clot buster" that does not target stroke recovery, but targets the blood vessel that is obstructed in stroke. tPA must be given within 4.5 hours, preferably within 3 hours after stroke because if given later it will cause bleeding into the brain. Currently, there is no stroke therapy that can be given at a time point later than 4.5 hours. Furthermore, recanalization therapy causes a reperfusion injury by reintroducing blood with high oxygen concentrations, and this is detrimental. It was reported that, in 2013, globally, there were nearly 25.7 million stroke survivors, 6.5 million deaths due to stroke, 113 million disability-adjusted life-years (DALYs) lost because of stroke, and 10.3 million new cases of strokes. A majority of the stroke burden was observed in developing countries, accounting for 75.2% of all stroke -related deaths and 81.0% of the associated DALYs lost. Thus, there is a great need for developing treatments that would promote recovery and increase the number of years with a good quality of life after ischemic brain injury.

Notably, spontaneous intracerebral hemorrhage (ICH) accounts for approximately 15% of stroke incidents in Western populations and even higher up to 20 to 30% in Asian populations. Being one of the most lethal forms and destructive type of stroke, mortality of ICH is high at 30% to 50%. The prognosis of patients with ICH is poor. Despite some promising trials, no medical or surgical therapy has been convincingly shown to benefit ICH patients. Early surgical removals of the blood clot have shown no overall benefits when compared with more conservative therapy.

Pathological changes of stroke can be divided into primary and secondary brain injuries. Primary injury occurs rapidly as a result of decreasing blood supply or mass effect of the hematoma leading to rapid oxygen, energy depletion and resulting in cell death through calcium pump dysfunction, excitotoxicity, and oxidative stress. Secondary injury commonly occurs when surrounding area reacts to the reperfusion episode and the invasion of the blood clot breakdown components, initiating the activation of inflammatory cells, brain edema, blood- brain barrier (BBB) disruption and apoptosis. Secondary injury often takes hours to days after stroke insult, making it a practical therapeutic target.

Cerebral dopamine neurotrophic factor (CDNF) is a member of novel family of proteins. CDNF was demonstrated neuroprotective effects in animal models of Parkinson's disease. Increasing evidence indicates that CDNF applied as extracellular proteins into the brain parenchyma or delivered by viral vectors can protect and repair midbrain dopamine neurons in vivo. Importantly, cytoprotective effects of CDNF is not restricted to neurons but also function as a regulator of inflammation in cultured rat primary microglia challenged with LPS, possibly attenuated pro- inflammatory cytokines release by suppressing the activation of the JNK pathway in the microglia. Additionally, injection of CDNF directly into the brain as plasmid and transfected into substantial nigral using nanoparticle carrier neurotensin-polyplex was shown to reduce glial markers and interleukin-6 levels in 6-OHD A- induced parkinsonian rats. Thus, when applied to the brain, CDNF may function as neuroprotective factors by dampening the inflammatory response in the CNS.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) was identified as a secreted neurotrophic factor (18 kDa) for dopaminergic neurons in vitro. Previous studies have shown MANF to be neuroprotective on midbrain dopamine neurons and restore dopamine neurons in PD models. Furthermore, we have found that MANF is neuroprotective in cortical stroke models and that the CXXC motif is critical for MANF's neuroprotective effects and decrease apoptosis. Deletion of MANF in mice leads to loss of pancreatic beta-cells and type 1 diabetes mellitus, and also to the defects in the CNS. Although MANF possesses some properties of classical neurotrophic factors, it has a drastically different mode of action. Similar to other neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF), MANF can reduce apoptosis and promote neuronal survival in vitro and in vivo. However, our findings suggest that MANF is not a classical growth factor and for example, it does not increase the proliferation of neural stem cells, as does GDNF. Furthermore, MANF is localized in the lumen of endoplasmic reticulum (ER) and is mainly ER-resident protein and the majority of the protein resides inside the cells. MANF expression is increased upon ER stress, and it is readily secreted by thapsigargin that causes ER Ca2+ depletion. Recently, it was shown that MANF is important for proper folding of large proteins in the ER lumen. In the brain, MANF transcript levels are upregulated after ischemia and by the accumulation of mis folded proteins.

RU2676701 discloses a method of cell-based and indirect therapy for ischemic stroke comprising a step of administering a genetic material consisting of the gene of the vascular VEGF growth factor, gene of the neurotrophic factor GDNF and gene of the NCAM neural cell adhesion molecule by directly injecting to a patient within 4 hours of the ischemic brain stroke.

WO2018202957 discloses C-terminal CDNF and MANF fragments and an effect thereof in animal models of central nervous system diseases. In the experimental section of WO2018202957, a cortical stroke was induced in a rodent model by occluding the distal middle cerebral artery (dMCA) and a C-terminal CDNF was administered subcutaneously. However, the CDNF fragment was given twice, i.e. before the dMCA occlusion and immediately after reperfusion. Therefore, this neuroprotective experiment only shows that combined administrations of the C-terminal CDNF before and immediately after the insult decrease infarction volume. In WO2017085362, similar results with combined intranasal administrations of recombinant human MANF protein before middle cerebral artery occlusion and immediately after reperfusion are disclosed. Further, CN107149673 discloses a rodent model experiment where animals were preconditioned by intracerebral microinjection of CDNF before ischemia- reperfusion MCAO modelling.

In the present invention, we are the first to show that when MANF is given intravenous route after the ischemic-perfusion injury, it has neuroprotective effects to decrease the lesion size. The intravenous (i.v.) delivered rhMANF at a dose of 3^X 1 5pg is neuroprotective. We extended the protective effect of MANF to be found even when administered 4 hours after stroke. I.v. administration of MANF was found to decrease pro -inflammatory cytokines in the brain and increase anti-inflammatory cytokines. Moreover, we demonstrated that when CDNF is given a subcutaneous (s.c.) route at a dose of 130 mg twice during ischemia-perfusion injury; it also has neuroprotective effects to decrease the infarct size. We extended these findings by giving CDNF as a dose of 30 pg s.c. 15 min after reperfusion and it was found to be protective and also by giving a C-terminal fragment of CDNF i.v. 15 min after reperfusion. Importantly, CDNF can further be used in the treatment of intracerebral hemorrhage (ICH) via suppression of inflammatory cytokines production, brain edema, and blood-brain barrier damage to decrease the hemorrhage-induced brain injury.

SUMMARY OF THE INVENTION Previously, there have been various trials where neuroprotective molecules have been tested in reperfusion injury models, however without success as the mechanisms of detrimental effects seem to be different in acute stroke and the subsequent reperfusion injury. In the present invention, we have found that either MANF or CDNF protein, when systemically delivered, is protective against reperfusion injury following cortical ischemic stroke and hemorrhagic stroke models. Unexpectedly, the present invention shows that neuroprotective MANF and CDNF proteins have positive effects also when administered solely after the time point the reperfusion injury has already occurred.

Another surprising element is the small dose needed for protective effect. To our further surprise, the protective mechanism after peripheral application is related to the modulation of cytokine levels in the brain. These results provide therapies for blood reperfusion and perfusion therapies in ischemic stroke after recanalisation and in hemorrhagic stroke after perfusion injury.

Accordingly, in several embodiments, the present invention provides a pharmaceutical composition comprising a CDNF or MANF polypeptide for use in the treatment of reperfusion injury, said composition being administered systemically to a subject, wherein said CDNF polypeptide comprises the amino acid sequence of SEQ ID NO:l, a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof, and wherein said MANF polypeptide comprises the amino acid sequence of SEQ ID NO:2, a sequence having at least 80% sequence identity with SEQ ID NO:2 or a functional fragment thereof. In other embodiments, the present invention provides a kit for use in the treatment of reperfusion injury comprising an effective amount of a CDNF or MANF polypeptide and a thrombolytic drug.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effect of intravenous administration of rhMANF on infarct volume after distal middle cerebral artery occlusion, dMCAo. a: Experiment design for recombinant human MANF, rhMANF, intravenous, i.v., administration after reperfusion (after 60 min after MCA occlusion); b: Infarction volume in the vehicle and different doses of rhMANF (15 ng, 150 ng and 1.5 pg, i.v.) on day 2 post-stroke (*P < 0.05 vehicle vs. 1.5 pg rhMANF; # p<0.05 150 ng rhMANF vs. 1.5 pg rhMANF, Student’s t- test, n = 7~8). b: Infarction volume in the vehicle and different doses of rhMANF (15 ng, 150 ng and 1.5 pg, i.v.) on day 2 (*P < 0.05 vehicle vs. 1.5 pg rhMANF; # p<0.05 150 ng rhMANF vs. 1.5 pg rhMANF, one-way ANOVA, Bonferroni’s post hoc test n = 7~8). c: Photographs of representative brain sections ofTTC staining show changes of the infarction areas in the vehicle and rhMANF (15 ng, 150 ng and 1.5 pg, i.v.) on post-stroke day 2. d: Experiment design for rhMANF i.v. administration at 4 hours post reperfusion; e: Photographs of representative brain sections ofTTC staining show changes of the infarction areas in the vehicle and rhMANF (3x 1.5 pg, i.v.) on post-stroke day 2. f: Infarction volume measured on post-stroke day 2 in the vehicle and rhMANF (3x1.5 μg, i.v.) treated at 4h after reperfusion from +60 min dMCAo (*P < 0.01, n = 5).

Figure 2. Cortical tissue levels of TNF-a (a), IL-Ib (b), IL-6 (c) and IL-10 (d) in rhMANF treated dMCAo rats. Values are mean + SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle group. Cytokine concentrations were measured from the ipsilateral cortex in the dMCAo groups on day 1 post-stroke and compared to sham + saline group. MANF was administered similarly as in Figure la.

Figure 3. Pre and post-treatment with rhCDNF subcutaneously reduces infarction volume after dMCAO. a: Experiment design of rhCDNF subcutaneous, s.c., administration before and after 60-min dMCAo. b: Histograms ofTTC staining of the infarction volume on day 2 in the vehicle and rhCDNF groups (130 pg, two-times s.c. injections.) (*P < 0.05, n = 7- 8). c: Experiment design ofrhCDNF subcutaneous administration 15 minutes after 60-min dMCAo. d: Photographs of representative brain sections ofTTC staining show changes of the infarction areas in the vehicle and on day 2. e: Histograms ofTTC staining of the infarction volume on day 2 in the vehicle and rhCDNF groups (30pg s.c., *P < 0.05, n = 5-7).

Figure 4. Dose-dependent effect of intracerebroventricular administration of rhCDNF on injured area on day 1, 3 and 7 after ICH. a: Experiment design of rhCDNF administration at one hour after the ICH. b: Photographs of representative brain sections show changes of the injured areas in the ICH + Saline and the ICH + rhCDNF (5 pg, intracerebroventicular, i.c.v.) on days 1, 3 and 7. c: Histograms of morphometry measurement on lesion volume at day 1, 3, and 7 post ICH injury d: Photographs of representative brain sections of injures areas in rhCDNF (1, 5, 10 pg, i.c.v.) on days 3 post-ICH. e: Histograms of morphometry measurement on lesion volume at day 3 post ICH injury with in rhCDNF (1, 5, 10 pg, i.c.v.). Values are mean ± SEM. *P < 0.05, **P < 0.01 , ***P < 0.001 vs. vehicle group.

Figure 5. Therapeutic effects of intracerebroventricular/intravenous administration of rhCDNF on neurobehavior functional assay (mNSS and cylinder test) of ICH rats, a: The mNSS on pre-operated, and day 1 and 3 in rhCDNF (1, 5, 10 pg, i.c.v.) treated ICH rats. b.

The mNSS of rhCDNF (5pg, i.c.v.) on pre-operated, and day 1, 3, and 7 post-ICH. c-e: The limb placement test (Cylinder test) on pre-operated, and day 1 and 3 in rhCDNF (5pg, i.c.v.) treated ICH rats f: The mNSS on pre-operated, and day 1 and 3 in rhCDNF (30 pg, i.v.)- treated rats with ICH. Values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle group.

Figure 6. Effects of intracerebroventricular administration of rhCDNF on brain edema and BBB permeability on day 3 after collagenase-induced ICH. a and b: Images of representative brain sections by DWI show changes of the endemic areas in the vehicle and rhCDNF (5 pg, i.c.v.) treatment on days 1 (a) and 3 (b) post-ICH. c: Histograms of DWI measurement on lesion volume on day 1 and 3 post-ICH injury d: Representative brain coronal sections (2 mm thickness) show Evans blue extravasation on day 3 post-ICH in vehicle and rhCDNF treatment group e: Histograms of dye concentration measurement on day 3 within ipsilateral and contralateral striatum of vehicle and rhCDNF treated group.

Values are mean ± SEM. *P < 0.05 vs. vehicle group.

Figure 7. Cytokine concentrations of ipsilateral striatal tissue in Sham control group, ICH + Saline (vehicle) group, ICH + rhCDNF (5 pg, i.c.v.) group a: TNF-a, b: IFNy , c:

IL-Ib, d: IL-6 and e: IL-10. Values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

# ## ###

ICH + rhCDNF vs. ICH + Saline group; P < 0.05, P < 0.01 , P < 0.001 ICH + Saline vs. Sham + Saline group.

Figure 8. The effect of intravenous rhMANF on blood gasses, electrolytes, and blood pressure. I.v. therapy of rhMANF does not have influence on blood electrolytes, glucose, lactate, PH, and hemoglobulin, but improve arterial partial pressure of oxygen (Pa02) after ischemic stroke a. Blood pressure was measured from the carotid artery of naive animals after i.v. administration of increasing rhMANF doses b-i. Arterial blood gasses and electrolytes were measured at different time points before and after 60 min distal middle cerebral artery occlusion, vehicle n=7; rhMANF n=8. **p<0.01, two-way ANOVA and Bonferroni’s post hoc test. The values are expressed as mean ± SEM. The data are expressed as mean mean ± SEM.

Figure 9. Comparisons of physiological parameters in ICH + Saline, and ICH + rhCDNF (5 pg, icv.) groups. Intracerebroventricular therapy of rhCDNF does not have influence on blood pressure, heart rate (HR), blood electrolytes, glucose, lactate, PH, and hemoglobulin, arterial partial pressure of oxygen (Pa02), carbon dioxide (PaC02) after hemorrhagic stroke a-b: Blood pressure and heart rate were measured from the carotid artery of rats with/without rhCDNF treatment at different time points before and after collagenase-induced ICH. c-j : Arterial blood gasses, electrolytes and hemoglobin were measured at different time points before and after collagenase-induced ICH, each group n=4-7.

Figure 10. Post-treatment with rhCDNF intravenously decreases ICH-induced lesion volume, (a) Experiment design of rhCDNF administration through i.v. route at one -hour post- ICH. (b) Photographs of representative brain coronal sections (2 mm thickness) show brain hemorrhagic areas of the ICH + Saline and the ICH + rhCDNF (15 pg in 500 pL saline, i.v.) at day 3 post-ICH. (c) Lesion volume on day 3 post ICH was determined by morphometric measurement. Values are means ± SEMs. *P < 0.05 vs. ICH + Saline group.

Figure 11. Distribution of rhMANF after systemic administration measured with hMANF ELISA, (a) Timeline of the experiment (b) Bioavailability of rhMANF in serum 30 min and 60 min after subcutaneous and intravenous administration (c) Concentration of rhMANF in the contralateral (contra) and infarcted (ipsi) cortex 60 min after intravenous rhMANF (75 μg) administration. The rhMANF concentration was normalized to total protein concentration in the sample.

Figure 12. The effects of CDNF on inflammatory cytokines after intracerebral hemorrhage. The ICH+rhCDNF group revealed decreased the level of TNF-a, but increased Galectin-1, Fractalkine, IF-1 receptor antagonist (ra), compared to ICH+saline group. CDNF decreased TNF-a (a) concentrations and increased Galectin -1 (b), Fractalkine (c), and IF-lra (d) concentrations in ipsilateral striatum following ICH. All the analyses were conducted in the R programming language V3.5.1 (R Core Team 2017).

Figure 13. GO term enrichment were conducted in two approaches, a: The first one, “GO biological process over-representation analysis” used hypergeometric distribution in the subset of differentially-expressed biomarkers. b:The second one, “GO biological process Gene Set Enrichment Analysis (GSEA)”, considered fold-changes between groups from all the biomarkers in a pathway/GO term, whether the biomarkers were differentially-expressed or not, and then calculated the enrichment score based on the magnitude of the fold changes. The SAM analysis was implemented with R package siggenes 1.56.0. The GO over- representation and GSEA analyses were implemented with the R package clusterProfiler (Yu et al. 2012).

DETAIFED DESCRIPTION OF EMBODIMENTS The present invention is related to cerebral dopamine neurotrophic factor, CDNF. Among CDNF polypeptides are the full-length human CDNF with a signal peptide having the total length of 187 amino acids and the mature human CDNF without the signal peptide having the total length of 161 amino acids (see Table 1). The present invention is also related to mesencephalic astrocyte-derived neurotrophic factor, MANF. Particularly important MANF polypeptides are the full-length human MANF with a signal peptide having the total length of 179 amino acids and the mature human MANF without the signal peptide having the total length of 158 amino acids (see Table 1).

MANF as well as CDNF are small proteins with a molecular weight of 18 kDa and are highly soluble and monomeric in neutral solution. Their primary sequences contain an amino -terminal (N-terminal) signal peptide that directs them to the ER and when cleaved, results in a mature protein which can be secreted. Although MANF/CDNF has many properties of classical neurotrophic factors, NTFs, they have drastically different properties and mechanisms. MANF/CDNF is an ER-lumen resident protein, which is secreted upon ER calcium-depletion. It is suggested that they both are important for the maintenance of ER homeostasis by aiding folding of large proteins and acting together with GRP78. Neuroprotective effects of MANF/CDNF have been demonstrated in animal models of Parkinson's disease. Increasing evidence indicates that CDNF/MANF applied as extracellular proteins or delivered by viral vectors can protect and repair midbrain dopamine neurons in vivo. Importantly, the cytoprotective effects of MANF/CDNF are not restricted to neurons but also function as a regulator of inflammation in primary microglial cell culture or dMCAo-induced cortical ischemic stroked rats.

In the Experimental Section below, it is demonstrated that systemic administration of recombinant human rhMANF/rhCDNF reduces neuronal cytotoxicity, infarction size, hemorrhagic volume, and sensory-motor behavioral deficits via modulation of microglial activation, decreasing inflammatory cytokines production, brain edema, and blood-brain barrier damage.

The present invention is thus directed to a pharmaceutical composition comprising a CDNF or MANF polypeptide for use in the treatment of reperfusion injury, said composition being administered systemically to a subject, wherein said CDNF polypeptide comprises the amino acid sequence of SEQ ID NO: 1 , a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof, and wherein said MANF polypeptide comprises the amino acid sequence of SEQ ID NO:2, a sequence having at least 80% sequence identity with SEQ ID NO:2 or a functional fragment thereof. As used herein, the term “reperfusion injury” relates to a reoxygenation injury caused when blood supply returns to tissue after suffering prolonged lack of oxygen. Reperfusion injury can be caused by thrombectomy, i.e. an interventional procedure of removing a blood clot (thrombus) from a blood vessel. Reperfusion injury is also induced by a treatment with a thrombolytic drug to cure ischemia, i.e. a restriction in blood supply to tissues, causing a shortage of oxygen.

As used herein, the term “systemic administration” or “systemically administered” refers to a route of administration of medication into the circulatory system so that the entire body is affected. Systemic administration can take place via parenteral administration, i.e. intramuscular, subcutaneous or intravenous administration (generally by injection, infusion, or implantation).

As used herein, a "polypeptide" generally refers to a molecule having an amino acid sequence encoded by a polynucleotide. "Polypeptide" also refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic nonnaturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

As used herein, the term "fragment" as applied to a polypeptide, may ordinarily be at least about seven contiguous amino acids, typically, at least about fifteen contiguous amino acids, more typically, at least about thirty contiguous amino acids, typically at least about forty contiguous amino acids, preferably at least about fifty amino acids, even more preferably at least about sixty amino acids and most preferably the peptide fragment will be greater than about seventy contiguous amino acids in length. The term “functional fragment” refers to a fragment, which still retains biological activity of the intact polypeptide. Functional fragments of the MANF and CDNF polypeptides are available for a skilled person as the production of fragments of whole polypeptides is known in the art. Accordingly, the MANF or CDNF protein may be a full length, naturally occurring form or may be a truncated or otherwise derivatised form. Examples of functional MANF or CDNF fragments, such as C-terminal fragments, are disclosed in WO2018202957.

In addition to naturally occurring allelic variants of MANF/CDNF, changes can be introduced by mutation into MANF/CDNF sequences that incur alterations in the amino acid sequences of the encoded MANF/CDNF polypeptide. Nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of a MANF/CDNF polypeptide. MANF/CDNF polypeptides or functional fragments thereof comprising one or more "non-essential" substitutions can be seen as equivalents to wild-type MANF/CDNF polypeptides disclosed herein. These equivalents have preferably 80 %, more preferably 85%, 90%, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to said wild-type polypeptides. Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity between amino acid sequences are the BLASTp and BLASTn 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389- 3402. A "non-essential" amino acid residue is a residue that can be modified in the wild-type sequences of MANF/CDNF without altering its biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the MANF/CDNF molecules of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well known in the art.

In a preferred embodiment, said composition is administered intramuscularly, intravenously or subcutaneously to a subject, preferably intravenously or subcutaneously.

In another preferred embodiment, said treatment prevents reperfusion injury following ischemic stroke or hemorrhagic brain injury. In another preferred embodiment, said CDNF polypeptide consists of the amino acid sequence of SEQ ID NO:l , a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof.

In another preferred embodiment, said MANF polypeptide consists of the amino acid sequence of SEQ ID NO:2, a sequence having at least 80% sequence identity with SEQ ID NO:2 or a functional fragment thereof.

In another preferred embodiment, the reperfusion injury treated is related to ischemic stroke.

In another preferred embodiment, the reperfusion injury treated is related to hemorrhagic brain injury. In preferred embodiments, the CDNF or MANF polypeptide is administered together with a thrombolytic drug.

In another preferred embodiment, the administration(s) of the CDNF or MANF polypeptide and the thrombolytic drug to a subject is(are) conducted simultaneously or consecutively, in any order.

In another preferred embodiment, the thrombolytic drug is selected from the group consisting of: recombinant tissue plasminogen activator (rtPA), streptokinase, anistreplase, alteplase, reteplase, tenecteplase and urokinase.

Pharmaceutical compositions In one embodiment of the present invention, the MANF/CDNF polypeptides can be incorporated into pharmaceutical compositions. Such compositions of the invention are prepared for storage by mixing the peptide having the desired degree of purity with optional physiologically acceptable carriers (such as nanocarriers), preservatives, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 22nd edition, Allen, Loyd V., Jr, Ed., (2012)), e.g., in the form of lyophilized cake or aqueous solutions. Acceptable carriers, preservatives, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium; and/or non-ionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). The MANF/CDNF polypeptides may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the fragment, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels as described by Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982) or polyvinylalcohol, polylactides, or non- degradable ethylene -vinyl acetate (Langer et al., supra).

In an embodiment, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.

Dosage and administration

The actual dosage amount of the MANF/CDNF polypeptides (e.g., an effective amount) that is administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration can determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In other non-limiting examples, a dose of a pharmaceutical composition or formulation can comprise from about 1 ng/kg/body weight of the MANF/CDNF polypeptide, about 5 ng/kg/body weight, about 10 ng/kg/body weight, about 50 ng/kg/body weight, about 100 ng/kg/body weight, about 200 ng/kg/body weight, about 350 ng/kg/body weight, about 500 ng/kg/body weight, 1 pg/kg/body weight, about 5 pg/kg/body weight, about 10 pg/kg/body weight, about 50 pg/kg/body weight, about 100 pg/kg/body weight, about 200 pg/kg/body weight, about 350 pg/kg/body weight, about 500 pg/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight of the MANF/CDNF polypeptide or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 pg/kg/body weight to about 500 milligram/kg/body weight of the MANF/CDNF polypeptides, etc., can be administered, based on the numbers described above.

The route of administration is in accord with known methods as well as the general routes of injection or infusion by intravenous or peripheral administration, intraperitoneal, subcutaneous, intrathecal, intracerebroventricular, intranasal, transdermal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional means, or sustained release systems as noted below. The MANF/CDNF polypeptide or a pharmaceutical composition comprising said MANF/CDNF polypeptide can be administered continuously by infusion or by bolus injection. Generally, where the disorder permits, one should formulate and dose the fragment for site- specific delivery. Administration can be continuous or periodic. Administration can be accomplished by a constant- or programmable-flow implantable pump or by periodic injections. In the present invention, systemic administration is preferred.

In preferred embodiments, the initial administration is performed within 1, 2, 3, 4, 5, 6, 7, or 8 hours after the onset of ischemic cerebral stroke. In preferred embodiments, the administration is performed within 4 hours after the onset of ischemic cerebral stroke.

As used herein, the term “treatment” or “treating” refers to administration of the compound of the invention to a subject/patient, preferably a mammal or human subject/patient, for purposes which include not only complete cure but also prophylaxis, amelioration, or alleviation of disorders or symptoms related to a pathological condition. Therapeutic effect may be assessed by monitoring the symptoms of a patient, biomarkers in blood, or for example a size of an injury or lesion or the length of survival of the patient. Kit

Another embodiment of the present invention is a kit for use in the treatment of reperfusion injury preferably after ischemic stroke or hemorrhagic brain injury. The kit includes a first container of an effective amount of the MANF/CDNF polypeptide as defined herein and a second container of a thrombolytic drug. The kit may also contain include instructions and/or means for delivering the MANF/CDNF polypeptide by any suitable route of administration to a human subject including by intravenous and/or subcutaneous injection including bolus injection or for delivery over a short time period such as one minute. In one preferred form of the invention the MANF/CDNF polypeptide will be in a powdered or lyophilized or other suitable form and the kit will include an acceptable diluent for reconstituting the MANF/CDNF polypeptide.

Table 1. CDNF and MANF amino acid sequences.

Mature CDNF amino acid sequence:

QEAGGRPGAD CEVCKEFLNR FYKSLIDRGV NFSLDTIEKE LISFCLDTKG KENRLCYYLG ATKDAATKIL SEVTRPMSVH MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMRVAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYAATHPKTE L (SEQ ID NO:1)

CDNF amino acid sequence with a signal sequence:

MWCASPVAVV AFCAGLLVSH PVLTQGQEAG GRPGADCEVC KEFLNRFYKS LIDRGVNFSL DTIEKELISF CLDTKGKENR LCYYLGATKD AATKILSEVT RPMSVHMPAM KICEKLKKLD SQICELKYEK TLDLASVDLR KMRVAELKQI LHSWGEECRA CAEKTDYVNL IQELAPKYAA THPKTEL (SEQ ID NO:3)

Mature MANF amino acid sequence:

LRPGDCEVCI SYLGRFYQDL KDRDVTFSPA TIENELIKFC REARGKENRL CYYIGATDDA ATKIINEVSK PLAHHIPVEK ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELKKIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AASARTDL (SEQ ID NO:2) MANF amino acid sequence with a signal sequence:

MWATQGLAVA LALSVLPGSR ALRPGDCEVC ISYLGRFYQD LKDRDVTFSP

ATIENELIKF CREARGKENR LCYYIGATDD AATKIINEVS KPLAHHIPVE KICEKLKKKD SQICELKYDK QIDLSTVDLK KLRVKELKKI LDDWGETCKG

CAEKSDYIRK INELMPKYAP KAASARTDL (SEQ ID NO:4)

Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term such as, for example, about or substantially, the exact numerical value is also disclosed. It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims.

EXPERIMENTAL SECTION

Materials and methods An animal model of cortical ischemia: Distal middle cerebral artery occlusion (dMCAo)

Ligation of the right MCA and bilateral common carotid arteries (CCAs) was performed on adult male Sprague-Dawley rats (250-300g, Envigo), using methods previously described [Airavaara et al., 2009; Anttila et al., 2018; Harvey et al., 2011] under chloral hydrate (0.4 g kg- 1 i.p., Sigma- Aldrich, St. Louis, MO, USA) anesthesia. Briefly, the bilateral CCAs were isolated through a ventral midline cervical incision. Rats, in a stereotaxic apparatus, and a craniotomy was made over the right hemisphere. The right MCA was ligated with a 10-0 suture, and bilateral CCAs were ligated with arterial clamps for 60 min. After 60 min of ischemia, the suture around the MCA and arterial clips were removed. After recovery from anesthesia, the rats were returned to their home cage. Recombinant human CDNF and MANF

The human recombinant CDNF and human recombinant MANF proteins were produced and purified as previously described (Lindholm et al, 2007, Lindholm et al, 2008).

Models for the intra-cerebral hemorrhage (ICH): blood infusion model and collagenase induction model.

The ICH refers to hemorrhage in the striatum, a part of the brain. Male Sprague-Dawley rats (250-300 g) anesthetized with pentobarbital (0.4 g kg-1 i.p., Sigma- Aldrich, St. Louis, MO, USA). The blood infusion model of ICH was induced by intrastriatal (0.0 mm posterior, 3.0 mm right, 5.0 mm ventral to bregma skull surface) infusion of autologous blood (0.1 ml within 1 minute) and stereotaxic administration of bacterial collagenase VII-S into the striatum [Liew et al., 2019; Huang et al., 2018] Briefly, bacterial collagenase VII-S (0.23 U in 1.0 mΐ sterile saline, Sigma- Aldrich) was infused into the striatum throughout 10 minutes. The needle was kept in place for another 10 minutes to prevent backflow. The craniotomies were sealed with bone wax. The rats were allowed to recover in separate cages at room temperature. Experimental design.

All experimental protocols were approved by the Animal Care and Use Committee of Finland and the Buddhist Tzu Chi University, Taiwan in accordance with guidelines set by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed under a 12 hours light/dark cycle with free access to food and water. Utmost efforts were made to minimize the suffering and the number of animals used.

I. In dMCAo experiments, 90 rats were randomly divided into the following four groups:

1. Sham dMCAo control group, n = 6. Rats were infused with 0.5 ml saline (1 ml/minute for 30 seconds) into the tail vein

2. dMCAo + Saline group, n =53. One hour after dMCAo, 21 rats were infused with 500 mΐ saline three times with 10-minutes interval via intravenous route. One hour after dMCAo, 16 rats were infused with 500 mΐ saline via subcutaneous route. 3. dMCAo + rhMANF group, n = 49. One hour after the induction of cortical ischemia, recombinant human MANF (0.015, 0.15 and 1.5 μg in 500 mΐ saline) was infused via intravenous route.

4. dMCAo + rhCDNF group, n = 14. One hour after the induction of cortical ischemia, recombinant human CDNF (30 μg in 500 μΙ saline) was infused via the subcutaneous route.

5. The rats used for ELISA experiments (fig. 11)(1) i.v. saline n=6;(2) iv. rhMANF n=6; (3) s.c. saline n=10; (4) s.c. rhMANF n=10.

The dosages of rhMANF/rhCDNF in systemic administration (i.v. or s.c.) were obtained from experimental trials since there was no precious available data on the systemically therapeutic dosage of rhCDNF/rhMANF for cortical ischemic-reperfusion treatment.

II. In ICH CDNF experiments, totally 118 rats were randomly divided into the following five groups:

1. Sham ICH control group, n = 4. Rats were infused with 1 pi saline (0.1 pl/minute for 10 minutes) into the striatum. 2. ICH + Saline group, collagenase model, n = 39. One hour after infusion with the collagenase

(induction of the ICH), rats received an infusion of saline (0.5 pFminute for 4 minutes) into the lateral ventricle (1.0 mm posterior, 1.4 mm right, 3.4 mm ventral to bregma of dura surface) ipsilateral to the ICH striatum.

3. ICH + rhCDNF group, collagenase model, n = 46. One hour after the induction of ICH, rhCDNF (1, 5 and 10 pg in 2 pi saline) was infused (0.5 pl/minute for 4 minutes) into the lateral ventricle same as group 2.

4. ICH + Saline group, collagenase model (n = 9). A total of 0.5 ml of sterile saline was administrated intravenously to each animal at 60 minutes post-ICH induction.

5. ICH + rhCDNF, collagenase model (n = 10) group. The dose (15 pg in 0.5 ml sterile saline, intravenous route) of rhCDNF was administrated to each animal at 60 minutes post-ICH.

The rhCDNF dosage range in intraventricular or intravenous route for dose-dependent therapeutic effect was done according to the previous studies in cerebral ischemia stroke. Assessment of neurological abnormalities.

The stroke-induced neurological deficits or recovery-promoting effects of treatments were assessed with sensorimotor tests on days 2, 14, and 24 post-stroke: a modified Bederson's neurological test, elevated body swing test, cylinder test, and Rotarod test. In these tests, rats display ischemic injury-induced neurological deficits that resolve spontaneously over time. The elevated body swing test was carried out as described previously (Borlongan et al., 1998). In short, rats were examined for lateral movements/turning when their bodies were suspended 20 cm above the testing table by lifting their tails. The most severe of body asymmetry in stroke animals is 20 contralateral tums/20 trials. In normal rats, the average body asymmetry is 10 contralateral tums/20 trials.

Modified Bederson's score was used to evaluate neurological deficits as described previously [Airavaara et al, 2009] In this test, rats were examined for the degree of abnormal posture when suspended 20-30 cm above the testing table. The following scale was applied:

0= rats extend both forelimbs straight; no observable deficit 1= rats keep the one forelimb to the breast and the other forelimb is straight

2= rats show decreased resistance to lateral push in addition to behavior score 1 3= rats twist the upper half of their body in addition to behavior in score 2

In the cylinder test, the animal was placed inside a transparent vertical tube with a diameter of 35 cm and its movements were video recorded for 5 minutes. The number of first front paw touches to the inner wall of the cylinder after rising on its hind limbs were counted.

Rats were also used in the assessment of the neurological abnormalities with a modified Neurological Severity Score (mNSS) method [Chen et al., 2001] by an investigator blinded to the experimental treatment scheme. The mNSS is a composite test of motor, sensory, and balance functions. The assessment before and on 1, 3 and 7 days after the ICH. Neurological functions were graded on a scale of 0-18 (normal score, 0; maximal deficit score, 18).

Evaluation of physiological parameters.

Rats were randomly assigned for evaluation of physio logical parameters including mean arterial blood pressure, blood gases, body weight changes, and body temperature. Under chloral hydrate(l .0 g/kg bodyweight, intraperitoneally, Sigma- Aldrich) anesthesia, a femoral artery was cannulated with a PE-50 polyethylene tube for fluid supplementation and monitoring of arterial blood pressure and blood gas. Arterial blood pressure was recorded through an amplifier (MP35, BIOP AC system, Inc.) and stored in a PC. Body temperature (rectal temperature) at 37.5 ± 0.5 °C by a rectal temperature sensor and a heating pad (CMA-150, Sweden). The physiological parameters were measured before (baseline) and after treatment (CDNF/MANF or Saline) 0.5, 1 and 3 hours.

Assessment of infarction size

Two days after reperfusion rats were decapitated. The brains were removed and sliced into 2.0- mm-thick sections. The brain slices were incubated in a 2% TTC solution (Sigma, St. Fouis) for 15 min at room temperature and then transferred into a 4% paraformaldehyde solution for fixation. The area of infarction in each slice was measured with a digital scanner and Imagetools programs (University of Texas Health Sciences Center). The volume of infarction in each animal was obtained from the product of average slice thickness (2 mm³) and sum of infarction areas in all brain slices examined.

Assessment of hemorrhagic volume.

Rats were randomly used in assessment of hemorrhagic volume at 24 hours (day 1) post-ICH. The accumulated hemorrhagic volume was quantified by a spectropho to metric assay as reported by Park et al. 2005, with minor modification. Briefly, both contralateral and ipsilateral hemispheres were obtained after transcardial perfusion. PBS was added to the individual hemispheres to make-up a total of 3 ml volume for homogenization and centrifugation (15000 g, 30 minutes). The supernatant (40 ml) was reacted with Drabkin's reagent (160 ml, Sigma) for 15 minutes at room temperature. Optical density was measured at 540 nm with a spectrophotometer (Molecular Devices OptiMax, USA). Equivalent hemorrhage volume (ml) of the supernatant was calculated from a standard curve obtained with known amounts of the blood.

Morphometric measurement of lesion volume.

Rats' brain images were collected from the brain edema and cytokine EFISA assay groups on day 3 post-ICH. The brains were cut coronally through the needle entry site (identifiable on the brain surface), and then serially sliced (2-mm thickness) anteriorly and posteriorly to the needle entry site. Digital photographs of the serial slices the and lesion volume was computed using the image analyzer program (Image J, NIH). The total lesion volume (mm³) was computed by summing the residual blood clot and damaged area in each section and multiplying by the distance between sustains [Jung et al., 2004] Assessment of blood-brain barrier (BBB) disruption.

Rats were randomly used in the assessment of the vascular permeability of BBB with a modified Evans blue extravasation method [Esen et al., 2005] Briefly, 70 hours post-ICH, rats were anesthetized with pentobarbital (0.4 g/kg) and infused, via the right femoral vein with 37°C Evans blue dyes (2% in 0.9% normal saline, 4 ml/kg) over 5 minutes. Two hours later, the rats were perfused with 300 mlof saline to wash out any remaining dye in the blood vessels and then brains were removed and sectioned into 2 mm thickness by a rodent brain matrix. Coronal brain sections were taken starting at + 2 mm and ending at - 4 mm from Bregma. BBB permeability was evaluated in the striatum, cortex, and cerebellum. The cerebellum was used as an internal control. Each portion was weighed immediately and placed in 1 ml of 0.9% normal saline for homogenization of the sample. For protein precipitation, 1 ml of 60% trichloroacetic acid solution was added and vortexed for 2 minutes. The mixture was subsequently cooled for 30 minutes and centrifuged (1500 g at 4°C) for another 30 minutes. The absorbance of Evans blue in the supernatant was then measured with a spectrophotometer (Molecular Devices OptiMax, USA) at 610 nm. The dye concentration was expressed as mg/g of tissue weight and calculated from a standard curve obtained from known amounts of the dye.

Assessment of cytokines.

Rats influence the cytokine assay by ELISA. The ipsilateral striatal tissues were collected before and on days 1, 3 - and 7-days post-ICH. After homogenization in the lysis buffer (PRO-PREPTM, iNtRON Biotechnology, Korea) and centrifuged at 12,000 g for 30 minutes, the supernatants were collected and stored frozen at -80 °C. During quantification, the cytokines (TNFa, IL-Ib, and IL-6, IL-10, IFN-g) were normalized to 100 mg of proteins in the supernatant using a commercial ELISA kit from R & D Systems (Minneapolis, MN, USA) according to the manufacturer's instructions. Evaluation of rhCDNF penetration into the brain.

The rhCDNF was labeled with the Alexa FluorO 488 using Microscale Protein Labeling Kit (A30006, Invitrogen, USA), according to the manufacturer's instruction. The Alexa FluorO 488 dye-labeled rhCDNF (10 mg) with the fluorescence excitation and emission maxima of approximately 494 and 519 nm was administered intraperitoneally one hour after ICH. Three hours after the fluorescence-labeled rhCDNF injection, the rats were re-anesthetized with chloral hydrate (0.4 g/kg) intraperitoneally, and their brains were removed immediately without perfusion and sectioned into 20 mm thickness by a cryostat. After nuclei counter-stained with DAPI, the slides were washed and mounted on cover slips with anti-fading mounting medium (VECTASHIELD®, USA). The penetration of the labeled rhCDNF into the striatum was evaluated under post-ICH fluorescence microscope.

Immunohistochemistry.

Rats were used in immunohistochemistry on day 3 post-ICH. Rats were anesthetized as described above and transcardially perfused with cold 0.1 M phosphate buffer saline followed by cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Brains were removed and immersed in 4% paraformaldehyde for 24 hours and 30% sucrose for another 24 hours. Coronal brain sections (20 mm thickness) were cut and collected from 0.0 to -2.0 mm AP to bregma using a cryostat (Leica CM 1900). Three 20 mm coronal sections, representing the core of the hematoma, were taken starting at 0.0 mm from bregma (the needle entry site). Additional adjacent serial coronal sections taken through the center of the hemorrhagic lesion were processed for the counting of marker-specific cells [Jung et al., 2004] Antibodies of OX-42 (1:100; BD,USA), ED-1 (1 :100; BD, USA) were used as microglial markers, andNeuN (1:200, Chemicon, USA) was used as neuron marker. Tissues sections were incubated with the primary antibodies overnight at 4°C. The bound primary antibody was visualized by incubation with an appropriate biotinylated secondary antibody followedby the Vectastain ABC reagents and color development with 3,3'-diaminobenzidine. Negative control slices from each animal were prepared for immunohistochemical staining processed identically except the primary antibodies were added. The numbers of positive cells in the same focal plane were counted in 6 regions of interest around the ipsilateral striatum (perihematomal region, 1 mm2). All cell counting was done by an independent investigator who counted the positive cells. Magnetic resonance imaging

MRI studies were performed with a 4.7 T scanner (PharmaScan, Bruker BioSpin, Ettlingen, Germany) at the Department of medical research, Tzu Chi post-reperfusion. Imaging Unit using a 90-mm shielded gradient capable of producing a maximum gradient amplitude of 300 mT/^'m with an 80-jis rise time. The linear birdcage RF coil used had an inner diameter of 38 mm. The head was fixed in a holder with a tooth bar to minimize motion artifacts during imaging. T2 weighted images were acquired using a rapid acquisition with relaxation enhancement (RARE) sequence (repetition time=3100 ms, traveled echo time=60 ms, matrix size=256><256, the field of view=40x40 mm, 14 slices with slice thiekness=1.0 mm) encompassing the whole brain, Diffused weighted MRI(DWI) images were acquired using a spin-echo pulse sequence with the following acquisition parameters: number of slices, 12; slice thickness, 1.0 mm; interslice distance, 2.1 mm; field of view=4Qx40 mm; matrix size=256x256; repetition time (TR), 1500 msec; echo time (TE), 37.7 msec; b values 18, 320, 1102 sec/mmi2. T2 -weighted scans served for calculation of ischemic lesion volumes. Manually outlined lesion areas (Para Vision 4.0, Bruker BioSpin, Ettlingen, Germany) in each slice were summed up and multiplied by slice thickness (1 mm), yielding the lesion volume. DWI and FLAIR sequences were converted to ANALYZE format using MRIcro, then de-skulled using the Brain Extraction Tool from this programme [Rorden and Brett, 2000], Lesion identification and quantification were performed using a semiautomatic quantification tool developed at UT Southwestern on MATLAB (Math Works, Inc., Natick, MA). The detailed processing steps have been described previously by Marquez de la Plata et ah, 2007. rhMANF ELISA

The rhMANF protein levels from serum and brain homogenates were analyzed with an i-n house-built double antibody sandwich ELISA specific for human MANF as described previously except without heterophilic antibody blocker (Galli et aia 2016). The samples and rhMANF protein standard dilutions were applied on the ELISA plate as duplicates. Before the brains were collected, the animals were perfused transcardially with 200 ml of saline to remove blood. Blood was collected before the perfusion, let to coagulate for 30min-lh and serum was separated by centrifugation with 2,000 g at RT for 10 min. The whole brain was snapfrozen in isopentane on dry ice, and stored in -80 °C until the infarcted cortex and corresponding contralateral cortex were dissected out, and homogenized with lysis buffer (137 mM NaCl; 20 mM Tris-HCl, pH 8.0; 2.5 mM EDTA; 1% NP40; 10% glycerol) containing protease inhibitors (Complete, Mini, EDTA-free Protease Inhibitor Cocktail, Roche, Mannheim, Germany). The rhMANF concentration in brain lysates was normalized to total protein concentration determined by Lowry method.

Statistical analysis.

Data were analyzed statistically using the software Prism for Student’s /-test and are presented as mean ± standard errors of the mean (SEM). The statistical comparisons among multiple groups were made using one-way ANOVA followed by Bonferroni correction. In all instances, n refers to the number of animals in a particular group. P value of < 0.05 is considered statistically significant.

EXAMPLE 1. Post-treatment with intravenously delivered rhMANF decreased infarct volume and promote behavior recovery after stroke.

Dose-response effects of rhMANF via intravenous route and time course for the dMCAo- induced neurological deficits are shown in Figure 1. Via the intravenous route, the rhMANF administrations produced a dose-dependent reduction in the infarction size at 1.5 pg, (p < 0.05 vs. Saline group by one-way ANOVA followed by Bonferronis post hoc test) (Figure lb). These data demonstrated that post-treatment with rhMANF significantly reduced the dMCAo-induced infarction size on 2 day (Figure lb). Importantly, even when rhMANF was given via intravenous route 4 hours after dMCAo, it was still found to decrease infarction volume when compared to saline -post-treated rats [ p < 0.05 by t-test] (Figure ld-f).

The post-stroke administration of rhMANF reduces pro-inflammatory cytokine levels in peri- infarction area Pro-inflammatory cytokine (TNF-a, IL-Ib, and IL-6) levels of the ipsilateral cortex in the dMCAo + Saline group were significantly increased on day 1 post-dMCAo as compared with day 0 pre-dMCAo of the Sham + Saline group. These high levels of cytokines (TNF-a, IL-1 b, and IL-6) were significantly reduced in the dMCAo + rhMANF group on days 1 post-dMCAo but did not return to the control (normal) at the same day (Figure 2a, b, and c). Contrarily, the level of IL-10 (anti-inflammatory cytokine) in the dMCAo + Saline group was decreased on day 1 post-dMCAo as compared with pre-dMCAo (day 0) level in the Sham + Saline group (Figure 2d); while this increase was also reduced in the dMCAo + rhMANF group on day 1 post-dMCAo.

EXAMPLE 2. Subcutaneously administered CDNF is protective against ischemic stroke and reperfusion injury after cortical ischemia Subcutaneous administration of rhCDNF before and right after dMCAo has a neuroprotective effect, shown in figure 3a and b. Then, we further investigated if rhCDNF is protective against reperfusion injury after cortical ischemia, we gave rhCDNF via subcutaneous route 15 minutes after ligation of right MCA, and arterial clips were removed. Surprisingly, subcutaneous administration of rhCDNF significantly decreased infarction volume on day 2 after stroke (figure 3c-e), suggesting that rhCDNF also has neuroprotective effect in reperfusion injury after ischemia stroke.

CDNF reduces lesion volume but not hemorrhagic volume.

The content of hemoglobin determined by spectrophotometric measurement is a good indication of the hemorrhagic volume. Intracerebroventricular experiment: We utilized collagenase injection to induce intracerebral hemorrhage. The hemorrhagic area developed to the maximum at 24 hours (1 day). Compared to the vehicle group (ICH+ saline group), post-treatment with rhCDNF (the ICH + rhCDNF group) did not change the hemorrhagic volume on day 1 (43.1 ± 10.6 mm2 to 43.9 ± 6.1 mm2 (n = 7-8), indicating that rhCDNF did not affect bleeding (hemorrhagic volume). Since the hemorrhage-induced brain injury would become more deterioration in a few days of the ICH insult, a lesion volume by morphometric measurement was used for the 3-day post-ICH instead. Notably, intracerebroventricular administration of rhCDNF significantly reduced injured area from 48.4 ± 10.6 mm2 to 30.7 ± 10.9 mm2 (p < 0.001, n = 7-8) on day 3 after ICH insult (Figure 4a-c). Also, we determined the dose-response effects of rhCDNF via the subcutaneous and intracerebroventricular route for ICH-induced brain injury (Figure 4d, e).

Intravenous experiment: On day 3 post-ICH, the lesion volume refers to the volume of residual blood clot and damaged tissue. It was significantly reduced in ICH ± rhCDNF as compared with ICH ± Saline group (49.57 ± 19.07 mm3 vs. 94.625 ± 33.78 mm3, P < 0.05) (Figure 10), suggesting that intravenous administration of rhCDNF also can reduce lesion size 3 -day after ICH insult.

The rhCDNF ameliorates neurological deficits.

Dose-response effects of rhCDNF (intracerebroventricular & intravenous) and time course for the ICH-induced neurological deficits are shown in Figure 5. In all rats, the mNSS was 0 before the ICH, indicating a normal neurological function.

In the intracerebroventricular routes, the rhCDNF administrations produced a dose-dependent reduction in the mNSS (an improvement from neurological deficits), most effectively at 5 or 10 mg, (p < 0.001 vs. ICH + Saline group by repeated measures ANOVA followed by Bonferroni test) (Fig. 5a). Also, we demonstrated that post-treatment with rhCDNF significantly reduced the ICH-induced neurological deficits (mNSS, Cylinder test) over 7 days. [mNSS: F (1, 65) = 21.21 and p < 0.0001 by two-way ANOVA; Cylinder test: F (1, 65) = 21.21 and p < 0.0001 by two-way ANOVA] .

In the intravenous routes, the ICH + Saline (control) group, the mNSS peaked up to 9.17 ± 0.75 on day 1 and decreased time-dependently to 7 ± 1.4 on days 3, respectively. In the ICH + rhCDNF (30 mg) group, the mNSS was reduced to 7.22 ± 1.48 on day 1. Then, it would decline to 4.55 ± 1.01 on days 3. These results indicated that intravenous administration of rhCDNF significantly ameliorated neurological deficits on days 1, 3 (P < 0.001 vs. ICH + Saline group) (Fig. 5f). Similar to intracerebroventricular route, peripherally intravenous administration with rhCDNF also acquire the promisingly therapeutic effect on ICH-induced brain injury.

CDNF reduces brain edema.

Diffuse weighted imaging ( DWI) is an effective method to examine water molecule diffusion after an ICH insult DWI did not show abnormalities in the cortex, which was essentially not affected among these groups. In the striatum, DWI revealed high signal intensity at the peri- hematoma site on 1 day after ICH, which sustains a similar intensity on day 3. Diffuse weighted imaging (each slice; mm²/s) of injured areas with the largest proportion of high signal intensity were measured using Function tool software (GEMdeil system Advanta dow). The total edema volume (mm³) was computed by summing the calculated area in each section and multiplying by the distance between sections. On day 1, the volume of edema analyzed by DWI was 12.45 ± 0.21mm³. On day 3, it was 10.8 ± 2.7 mm³. In the ICH + rhCDNF group, perifocal edema of the ipsilateral striatum on dayl was similar to ICH+ saline group. However, on day 3, the volume of edema was smaller 5.99 ± 1.84 mm³ (p < 0.001) (figure 6a; n = 6-8 each group). These findings indicated that the intracerebro ventricular administration of rhCDNF post-treatment significantly reduced the cerebral edema in the ipsilateral striatum.

CDNF attenuates BBB disruption.

Since BBB disruption is very likely a contributory cause of brain edema that peaks on day 3 post-ICH, changes in BBB disruption was determined by Evans blue on the same day.

Intracerebroventricular experiment: On day 3 after the ICH, Evans blue concentrations of the hemorrhagic (ipsilateral) striatum in the ICH + Saline and ICH + rhCDNF groups were 3.63 ± 0.99 pg/g and 1.24 ± 0.31 pg/g (p < 0.05, n = 6 for each group), respectively (Fig. 6d, e), The results indicated that post-treatment with rhCDNF significantly reduced the ICH-induced BBB leakage. The dye concentration of the contralateral striatum was not different between the ICH + Saline and ICH + rhCDNF groups, indicating that the unilateral striatal ICH in the present experimental condition appeared not to affect the BBB of the contralateral striatum.

Penetration of rhCDNF through the BBB into neurons and microglial cells in the ipsilateral striatum.

To examine whether rhCDNF can penetrate into striatal parenchyma to exert its function, we administrated intracerebroventricular of Alexa Fluor^® 488 labeled-rhCDNF one hour after ICH. To test whether rhCDNF can penetrate from cerebral ventricle into striatal parenchyma, we demonstrated that one day after injection of the fluorescence labelled-rhCDNF into one side of the lateral ventricle (LV) of the collagenase-ICH rat, both DAPI and fluorescence-labelled rhCDNF were co-localized in the cells of the ipsilateral but not on the contralateral side (data not shown). Then, we had to demonstrate that fluorescent-labeled cells are none other than rhCDNF-positive cells. These results indicate that the rhCDNF can diffuse from the lateral ventricle into the cells of the same side striatum. In addition, intracerebroventricular injection of fluorescence-labeled rhCDNF, the labelled rhCDNF was localized in NeuN-positive neurons and OX-42-positive microglial cells, but not GFAP-positive astrocytes in the ipsilateral striatum. These results indicate that the intracerebroventricular injection of rhCDNF can diffuse into neurons and microglial in the damaged striatum The rhCDNF reduces pro-inflammatory cytokine levels in striatal tissue. Pro-inflammatory cytokine (TNF-a, IL-1 b, and IL-6, IFN-g) levels of the ipsilateral striatum in the ICH + Saline group were significantly increased on day 1 post-ICH as compared with day 0 pre-ICH of the Sham + Saline group; however, levels of IFN-g (figure 7a), IL-Ib (figure 7c), IL-6 (figure 7d) maintained high, albeit getting lower, throughout days 3 and 7. These high levels of cytokines were significantly reduced in the ICH + rhCDNF group throughout days 1, 3, and 7 post-ICH, but did not return to the control (normal) level in these days. The level of TNF-a (figure 7b) in the ICH + Saline group was also increased on day 1 post-ICH as compared with pre-ICH (day 0) level in the Sham + Saline group; while this increase was also reduced in the ICH + rhCDNF group on day 1, 3 post-ICH. The level of IL-6, however, returned to the control level without rhCDNF treatment on days 7 post-ICH. Also, an anti-inflammatory cytokine (IL-10) levels of the ipsilateral striatum in the ICH + Saline group were significantly decreased on day 1, 3, 7 post-ICH as compared with day 0 pre-ICH of the Sham + Saline group. However, the level of IL-10 (figure 7e) in the ICH + rhCDNF group was less decreased on day 1, 3,7 post-ICH as compared with ICH + Saline group.

EXAMPLE 3. Choose of dosage and administering route for rhMANF /rhCDNF.

The dosages of intracerebroventricular injection of rhCDNF were obtained from empirical trials because there was no previous experiments on the therapeutic dosage of rhCDNF for ICH treatment. Even the high dose (10 pg) of rhCDNF by intracerebroventricular route caused similar effectiveness than the low dose (5 pg) of rhCDNF in attenuating neurological deficits. The mechanisms underlying of such a dose-response relationship are not known. The dose of rhCDNF (30 pg)/ rhMANF (4.5 pg) in a systemic administration did not change physiological parameters, like heart rate, blood pressure, body weight, white blood count (WBC), red blood count (RBC), and electrolytes. Therefore, a safety way of rhMANF/rhCDNF administration thus is demonstrated as using three times intravenous injections of rhMANF with a low dosage, or an intravenous/subcutaneous injection of rhCDNF with high dosage.

EXAMPLE 4. Distribution of rhMANF after systemic administration.

Bioavailability of rhMANF after systemic administration was measured with ELISA specific for human MANF (Galli et al. 2016). A dMCAo was performed and one dose of vehicle or rhMANF was administered subcutaneously or intravenously 30 min after dMCAo reperfusion (figure 11a). Blood samples were collected and after perfusion with saline, also brains were collected. The concentration of rhMANF in serum was measured 30 min and 60 min after subcutaneous (100 pg) and intravenous (75 pg) rhMANF administration and proportioned to the total dose injected for comparison (figure 1 lb). Approximately 0.87% of the dose was still left in the blood circulation 60 min after s.c. administration and 0.22% after i.v. administration. RhMANF was detected also in the infarcted cortex 60 min after i.v. administration (figure 11 c), indicating that rhMANF can penetrate to cerebral tissue, possibly due to infarct-induced BBB disruption that has been reported to occur already 25 min after MCAo (Strbian et al. , 2008; Abo- Ramadan et al., 2009). RhMANF was not detected in the contralateral cortex (figure l ie).

EXAMPLE 5. The analysis of biomarkers in ICH-induced lesion area with/without rhCDNF administration

The analysis on 67 biomarkers in 13 samples on day 1 post-ICH. The 13 samples were divided into 3 groups. In this analysis we focused on comparison among sham+saline, ICH+saline and ICH+rhCDNF groups, including heatmap with clustering, PCA and Gene ontology (GO) terms (figure 12,13). Heatmap with hierarchical clustering was plotted as a heatmap in which the different colors represent biomarker expression levels. The biomarkers and samples were then subjected to hierarchical clustering based on Euclidean distance (figure 12). Each biomarker was summarized by its mean and standard deviation, or median with minimum and maximum responses between ICH+saline and ICH+rhCDNF groups. The significant of expression difference was evaluated by Student’s t-test or Wilcoxon Rank-Sum, respectively. Biomarkers with FDR<0.05 were considered as differentially. Notably, the ICH+rhCDNF group revealed decreased the level of TNF-a (figure 12a), but increased Galectin-1 (figure 12b), Fractalkine (figure 12c), IL-1 receptor antagonist (ra)(figure 12d), compared to ICH+saline group.

Fig 13a: significance Analysis of Microarrays (SAM) identifies differentially-expressed biomarkers. Unlike the above-mentioned statistical test that relay on FDR- value from theoretical probability distribution, SAM compares a t-like statistic di observed across groups with an estimate obtained from randomized permutations of the samples. The biomarkers with deviations larger than predefined (default 1.2) were identified as differentially expressed. Fig. 13b: The scaled data were transformed with principal component analysis (PCA). Each principal component (PC) derived from PCA was a weighted summation of all the input measurements/biomarkers, in which the weights form a vector orthogonal to those of the other PCs. Thus, a sample with p biomarker values was transformed to a datapoint defined by PCs. The PCs were ordered by variation contained/explained by themselves, thus providing a good way for dimension reduction and pattern observation. Also, the biomarkers with large weights in a PC might share some common characteristics. PCA plots of 32 differentially-expressed biomarkers between Group 3(ICH) and Group 2(CDNF). Figure 13 c,d: GO term enrichment were conducted in two approaches. Figure 13c: The first one is “GO biological process over- representation analysis” used hypergeometric distribution in the subset of differentially- expressedbiomarkers. Figure 13d:The second one “GO biological process Gene Set Enrichment Analysis (GSEA)”, considered fold-changes between groups from all the biomarkers in a pathway/GO term, whether the biomarkers were differentially-expressed or not, and then calculated the enrichment score based on the magnitude of the fold changes. The SAM analysis was implemented with R package siggenes 1.56.0. The GO over- representation and GSEA analyses were implemented with the R package clusterPro filer (Yu et al. 2012 and Tusher et al., 2001)

Results summary

Intravenous injections of rhMANF administered after reperfusion decreases lesion volume after 60-min distal middle cerebral artery occlusion (dMCAo) in adult rats when administered either 15 min or 4h after reperfusion. (Figure 1). Intravenous injections of rhMANF reduces pro inflammatory cytokines in the brain one day after 60-min distal middle cerebral artery occlusion (dMCAo) in adult rats (Figure 2).

Subcutaneous injections of rhCDNF are protective against reperfusion injury after 60-min dMCAo in adult rats (Figure 3).

The rhCDNF thereof reduces intracerebral hemorrhage (ICH)-induced lesion volume in adult rats (Figures 4 and 10).

Intraventricular or intravenous administration of rhCDNF lh post-ICH improves sensory-motor function in terms of reducing neurological deficits as determined by the Cylinder test and modified neurological severity scores (mNSS) (Figure 5). Intraventricular injection of rhCDNF ameliorated perihematomal edema and reduces the blood- brain barrier disruption as determined by MRI and Evans blue extravasation assay, respectively. (Figure 6).

The rhCDNF thereof reduces proinflammatory cytokines in ICH rats (Figure 7). Systemic administration of rhMANF or rhCDNF does not alter physiological parameters in rats. (Figures 8 and 9).

We have now made revolutionary findings that post-stroke MANF/CDNF treatment, given as recombinant protein on post-stroke day 3-14 or 7, promotes the functional recovery of rats after a stroke indicating that MANF/CDNF facilitates endogenous repair mechanism after stroke. The progress of MANF-induced promotion of recovery in its robustness is unique.

REFERENCES

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Claims

1. A pharmaceutical composition comprising a CDNF or MANF polypeptide for use in the treatment of reperfusion injury, said composition being administered systemically to a subject, wherein said CDNF polypeptide comprises the amino acid sequence of SEQ ID NO:l, a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof, and wherein said MANF polypeptide comprises the amino acid sequence of SEQ ID NO:2, a sequence having at least 80% sequence identity with SEQ ID NO:2 or a functional fragment thereof.

2. The composition for use according to claim 1, wherein said composition is administered intramuscularly, intravenously or subcutaneously to a subject, preferably intravenously or subcutaneously.

3. The composition for use according to claim 1 or 2, wherein said treatment treats or prevents reperfusion injury following ischemic stroke or hemorrhagic brain injury or wherein said reperfusion injury is induced by a treatment with a thrombolytic drug or thrombectomy.

4. The composition for use according to anyone of claims 1 -3 , wherein the polypeptide is CDNF.

5. The composition for use according to claim 4, wherein said CDNF polypeptide consists of the amino acid sequence of SEQ ID NO:l, a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof.

6. The composition for use according to any one of claims 1-3, wherein the polypeptide is MANF.

7. The composition for use according to claim 6, wherein said MANF polypeptide consists of the amino acid sequence of SEQ ID NO:2, a sequence having at least 80% sequence identity with SEQ ID NO:2 or a functional fragment thereof.

8. The composition for use according to any one of claims 1 -7, wherein ischemic stroke is treated.

9. The composition for use according to any one of claims 1 -7, wherein hemorrhagic brain injury is treated.

10. The composition for use according to claim 9, wherein the CDNF or MANF polypeptide is administered together with a thrombolytic drug.

11. The composition for use according to claim 10, wherein the administration(s) of the CDNF or MANF polypeptide and the thrombolytic drug to a subject is(are) conducted simultaneously or consecutively, in any order.

12. The composition for use according to claim 10 or 11, wherein the thrombolytic drug is selected from the group consisting of: recombinant tissue plasminogen activator (rtPA), streptokinase, anistreplase, alteplase, reteplase, tenecteplase and urokinase.

13. The composition for use according to any one of claims 1-12, wherein the first administration is performed within 4 hours after the onset of ischemic cerebral stroke.

14. A kit for use in the treatment of reperfusion injury, the kit comprising an effective amount of a CDNF or MANF polypeptide and a thrombolytic drug.

15. The kit for use according to claim 14, wherein a reperfusion injury following ischemic stroke or hemorrhagic brain injury is treated or wherein said reperfusion injury is induced by a treatment with a thrombolytic drug or thrombectomy.

16. A method of treating reperfusion injury, said method comprising administering systemically an effective amount of a pharmaceutical composition comprising a CDNF or MANF polypeptide to a subject, wherein said CDNF polypeptide comprises the amino acid sequence of SEQ ID NO:l , a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof, and wherein said MANF polypeptide comprises the amino acid sequence of SEQ IDNO:2, a sequence having at least 80% sequence identity with SEQ IDNO:2 or a functional fragment thereof.

17. The method according to claim 16, wherein said composition is administered intramuscularly, intravenously or subcutaneously to a subject, preferably intravenously or subcutaneously.

18. The method according to claim 16 or 17, wherein the treatment treats or prevents reperfusion injury following ischemic stroke or hemorrhagic brain injury, or wherein said reperfusion injury is induced by a treatment with a thrombolytic drug or thrombectomy.

19. The method according to any one of claims 16-18, wherein the polypeptide is CDNF.

20. The method according to claim 19, wherein said CDNF polypeptide consists of the amino acid sequence of SEQ ID NO: 1 , a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof.

21. The method according to any one of claims 16-18, wherein the polypeptide is MANF.

22. The method according claim 21 , wherein said MANF polypeptide consists of the amino acid sequence of SEQ IDNO:2, a sequence having at least 80% sequence identity with SEQ IDNO:2 or a functional fragment thereof.

23. The method according to any one of claims 16-22, wherein ischemic stroke is treated.

24. The method according to any one of claims 16-22, wherein hemorrhagic brain injury is treated.

25. The method according to claim 23, wherein the CDNF or MANF polypeptide is administered together with a thrombolytic drug.

26. The method according to claim 25, wherein the administration(s) of the CDNF or MANF polypeptide and the thrombolytic drug to a subject is(are) conducted simultaneously or consecutively, in any order.

27. The method according to claim 25 or 26, wherein the thrombolytic drug is selected from the group consisting of: recombinant tissue plasminogen activator (rtPA), streptokinase, anistreplase, alteplase, reteplase, tenecteplase and urokinase.

28. The method according to any one of claims 16-27, wherein the first administration is performed within 4 hours after the onset of ischemic cerebral stroke.

29. Use of a CDNF or MANF polypeptide in the manufacture of a pharmaceutical composition for the treatment of reperfusion injury, said composition being administered systemically to a subject, wherein said CDNF polypeptide comprises the amino acid sequence of SEQ ID NO:l, a sequence having at least 80% sequence identity with SEQ ID NO:l or a functional fragment thereof, and wherein said MANF polypeptide comprises the amino acid sequence of SEQ ID NO:2, a sequence having at least 80% sequence identity with SEQ ID N0:2 or a functional fragment thereof.

30. The use according to claim 29, wherein said composition is administered intramuscularly, intravenously or subcutaneously to a subject, preferably intravenously or subcutaneously.

31. The use according to claim 29 or 30, wherein said treatment treats or prevents reperfusion injury following ischemic stroke and hemorrhagic brain injury, or wherein said reperfusion injury is induced by a treatment with a thrombolytic drug or thrombectomy.

32. The use according to any one of claims 29-31 , wherein the polypeptide is CDNF.

33. The use according to claim 32, wherein said CDNF polypeptide consists of the amino acid sequence of SEQ ID NO: 1, a sequence having at least 80% sequence identity with SEQ IDNO:l or a functional fragment thereof.

34. The use according to any one of claims 29-31 , wherein the polypeptide is MANF.

35. The use according to claim 34, wherein said MANF polypeptide consists of the amino acid sequence of SEQ IDNO:2, a sequence having at least 80% sequence identity with SEQ IDNO:2 or a functional fragment thereof.

36. The use according to any one of claims 29-35, wherein ischemic stroke is treated.

37. The use according to any one of claims 29-35, wherein hemorrhagic brain injury is treated.

38. The use according to claim 36, wherein the CDNF or MANF polypeptide is administered together with a thrombolytic drug.

39. The use according to claim 38, wherein the administration(s) of the CDNF or MANF polypeptide and the thrombolytic drug to a subject is(are) conducted simultaneously or consecutively, in any order.

40. The use according to claim 38 or 39, wherein the thrombolytic drug is selected from the group consisting of: recombinant tissue plasminogen activator (rtPA), streptokinase, anistreplase, alteplase, reteplase, tenecteplase and urokinase.

41. The use according to any one of claims 29-40, wherein the first administration is performed within 4 hours after the onset of ischemic cerebral stroke.