Compositions containing apoaequorin and methods for treating neuronal inflammation using the same

Abstract

This disclosure relates to compositions containing apomylated jellyfish luminescent protein and methods for treating neuronal inflammation. The invention relates to a method of premodulating neurons to reduce neuronal inflammation in a subject. The method includes the step of administering apomylated jellyfish luminescent protein to the subject, wherein the subject's neurons are premodulated to reduce subsequent neuronal inflammation in the subject.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201580061431.1.

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Application 62 / 078,099, filed November 11, 2014, which is incorporated herein by reference in its entirety for all purposes. Invention Field

[0004] This invention generally relates to compositions that can be used to treat neuronal inflammation. More specifically, this invention relates to compositions containing apomeric jellyfish luminescent proteins and methods of using those compositions to treat neuronal inflammation. Background of the Invention

[0005] In 2009, stroke accounted for approximately one in 19 deaths in the United States, making it the third leading cause of death after heart disease and cancer. Therefore, it is imperative to seek ways to mitigate post-stroke damage. Significant attention has been paid to the role of calcium in ischemia and the potential neuroprotection offered by blocking its toxic effects after ischemia.

[0006] Calcium (Ca 2+ Neurotransmitters play a crucial role in various neuronal processes, including neurotransmitter release and synaptic plasticity. Due to their sustained activity, neurons continuously undergo intracellular Ca2+ processes. 2+ Fluctuations in intracellular Ca 2+ Excessive or sustained increases in Ca2+ can be toxic to neurons. Therefore, intracellular Ca2+ in neurons... 2+ It is subject to extremely strict regulation and possesses the ability to allow neurons to limit or control cytoplasmic calcium. 2+ Several mechanisms at the level. Specifically, calcium-binding proteins (CaBPs; such as calcium-binding proteins, parvalbumin, and calreticulin) are involved in the binding of calcium to cytoplasmic calcium. 2+ It is important for the combination and buffering of [something].

[0007] Studies on the hippocampus have shown that the presence of CaBP provides some protection against excitotoxic damage, which typically leads to cell death. Interestingly, decreased CaBP levels have been observed with age and in neurodegenerative diseases, including Alzheimer's and Parkinson's. The aim is to reduce the toxicity of CaBP during ischemic events by administering CaBP prior to ischemic injury. 2+Minimized treatment has also shown positive results. For example, Yenari et al. treated animals with calcium-binding protein before inducing local ischemia and found that overexpression of calcium-binding protein was neuroprotective. Furthermore, Fan et al. treated rats with calcium-binding protein before local ischemia and showed that the treated animals had smaller infarct volumes, better behavioral recovery, and reduced apoptosis. In fact, a large body of research has focused on understanding the detrimental effects of stroke. Interestingly, a major risk factor for stroke is aging, and a prominent hypothesis of brain aging is the neuroprotective effect of calcium-binding protein on stroke. 2+ The hypothesis argues that age-related changes in the ability to regulate calcium and calcium-dependent processes are key contributing factors to increased susceptibility to cognitive decline and neurodegenerative diseases. Given these age-related changes in calcium... 2+ Changes, and Ca 2+ Ca2+ plays a crucial role in ischemic cell death, and a large body of research has focused on its role in neurons and glial cells. 2+ Abnormal regulation.

[0008] It is known that excessive intracellular calcium occurs after local ischemia. 2+ Accumulation can exacerbate cell death through excitotoxicity. Following ischemic injury, Ca... 2+ Ca through voltage gating 2+ VGCCs (vegetative-cell channels) accumulate in cells via NMDA receptors and through release from intracellular organelles. Numerous studies have shown that blocking Ca2+ pathways via NMDA receptors, VGCCs, or both can reduce the risk of cancer cell death. 2+ Entry may possess neuroprotective properties against localized ischemia. Interestingly, when NMDA receptor blockers entered clinical trials, they failed to provide neuroprotection and produced undesirable side effects such as hallucinations and coma. While it is not yet clear why NMDA receptor blockers failed in clinical trials, it is clear that continued research is needed to mitigate their devastating effects in ischemic stroke.

[0009] Despite progress, new alternative therapies for treating neuronal inflammation are still needed. Specifically, there is a need for pharmaceutical or nutritional compositions with fewer side effects compared to previous formulations, which, if developed, would meet a long-standing need in the medical and nutritional health communities. Summary of the Invention

[0010] This invention is partly based on the inventors' recent research on apoaequorin, a calcium-binding protein, in which the inventors unexpectedly discovered novel neuroprotective capabilities. Specifically, apoaequorin has been found to be used to precondition neurons in subjects to reduce subsequent neuronal inflammation. Therefore, this invention provides compositions containing apoaequorin and methods of using them, which offer substantial benefits in neuroprotective applications.

[0011] In a first aspect, the present invention relates to a method for pre-regulating neurons to reduce neuronal inflammation in a subject. The method includes the step of administering an apomylated jellyfish luminescent protein to the subject, wherein the neurons of the subject are pre-regulated to reduce neuronal inflammation.

[0012] In one embodiment, the substance is administered to the subject by injection. In another embodiment, the substance is administered orally, for example, using a unit dosage form of apomyelin luminescent protein, the unit dosage form being selected from tablets or capsules. In some embodiments, the apomyelin luminescent protein is administered to the subject in the form of a nutritional composition.

[0013] It is understood that the present invention covers apomyelin for premodulating neurons to reduce neuronal inflammation in a subject, and the use of apomyelin in the manufacture of compositions for premodulating neurons to reduce neuronal inflammation in a subject.

[0014] On the other hand, the present invention relates to a method for reducing the level of tumor necrosis factor α (TNFα) protein in a subject. The method includes the step of administering apopolymerized jellyfish luminescent protein to the subject, wherein the TNFα protein level in the subject is reduced.

[0015] In some embodiments, the substance is administered to the subject by injection. In other embodiments, the substance is administered orally, for example, using a unit dosage form of apomyelin luminescent protein selected from tablets or capsules. In some embodiments, the apomyelin luminescent protein is administered to the subject in the form of a nutritional composition.

[0016] It is understood that the present invention covers apomyelin luminescent protein for reducing TNFα protein levels in a subject, and the use of apomyelin luminescent protein in the preparation of compositions for reducing TNFα protein levels in a subject.

[0017] The present invention provides several advantages over prior art compositions and methods, including providing an overall improvement in the physical and mental health of the subject through its neuroprotective function.

[0018] Other objectives, features, and advantages of the invention will become apparent after reading the specification and claims. Attached Figure Description

[0019] Figure 1 AC describes the effect of oxygen-glucose deprivation on cell death in short-term hippocampal slices. A The experimental design is illustrated in the diagram. Coronal hippocampal sections were incubated in artificial cerebrospinal fluid (aCSF) for 1 hour. Half of the sections were transferred to ischemic conditions, subjected to oxygen-glucose deprivation (OGD) for 5 minutes, while the other half remained oxygenated (without OGD). All sections were then transferred to aCSF for 30 minutes of reperfusion and trypan blue staining. The sections were then fixed in 10% neutral buffered formalin. B Representative images of trypan blue staining in the CA1 region of the hippocampus in sections with normal oxygenation (no OGD) and sections that underwent 5 minutes of OGD. Note that less staining is present in the sections with normal oxygenation compared to the OGD sections. C Compared to sections with normal oxygen levels, sections that underwent 5 minutes of OGD showed a significant increase in the number of trypan blue-stained neurons in the CA1 region of the hippocampus. , p < .01).

[0020] Figure 2 AC describes the dose-dependent effect of apomylated jellyfish luminescent protein on ischemic cell death. A Experimental design diagram. In rats with bilateral cannulation in the dorsal hippocampus, 0, 0.4, 1, or 4% apopolymerized luminescent protein (AQ) was infused into one hemisphere, while the other hemisphere received a carrier (0% AQ). One day after infusion, sections of the coronal hippocampus were excised and incubated in artificial cerebrospinal fluid (aCSF) for 1 hour. All sections were transferred to ischemic conditions for 5 minutes of oxygen-glucose deprivation (OGD). The sections were then transferred to aCSF for 30 minutes of reperfusion and trypan blue staining. Finally, the sections were fixed in 10% neutral buffered formalin. B Typical image of trypan blue staining in CA1 region of the hippocampus after local ischemia in sections treated with the carrier or 4% AQ. Note that less staining is present in AQ-treated sections compared to those treated with the carrier. C) Graphs show the relationship between neuroprotection (percentage of rescued cells) and the dosage of apomyelin. Significant neuroprotection was observed in rats treated with 1% or 4% AQ (but not 0.4% AQ) compared to 0% AQ (carrier). , p < .01).

[0021] Figure 3 AC describes the time-dependent effect of apopolymerized jellyfish luminescent protein on ischemic cell death. A Experimental design diagram. In rats with bilateral cannulation in the dorsal hippocampus, 4% apo-coated luminescent protein (AQ) was infused into one hemisphere, while the other hemisphere received a carrier (0% AQ). Coronal hippocampal sections were excised at 1 hour, 1 day, 2 days, 3 days, or 5 days post-infusion and incubated in artificial cerebrospinal fluid (aCSF) for 1 hour. All sections were transferred to ischemic conditions for 5 minutes of oxygen-glucose deprivation (OGD). The sections were then transferred to aCSF for 30 minutes of reperfusion and trypan blue staining. The sections were then fixed in 10% neutral-buffered formalin. In a second group of rats, 4% AQ was bilaterally infused, and the brain was excised at 1 hour, 1 day, 2 days, or 3 days post-infusion for Western blot analysis. B Infusion of 4% AQ 1 or 2 days prior to local ischemia provided significant neuroprotection, but the neuroprotective effect was no longer evident 3 or 5 days after infusion. It was noted that AQ also lacked neuroprotective properties when infused only 1 hour prior to local ischemia. p = 0.78). C Western blot analysis of 22 kD AQ protein. AQ was present in the dorsal hippocampus (AQ-dhpc) at 1 hour and 1 day post-infusion, but was absent at 3 days post-infusion. At 2 days post-infusion, a band was present in only 29% of rats. Notably, no band was ever observed in the ventral hippocampus (AQ-vhpc), regardless of infusion time. Analysis of β-actin (45 kD) showed that protein loading at any time point had no effect in either the dorsal (actin-dhpc) or ventral (actin-vhpc) hippocampus. , p < .01.

[0022] Figure 4 AB describes the effect of apopolymerized jellyfish luminescent protein on interleukin-10 mRNA expression. A One hour after 4% AQ infusion into the dorsal hippocampus, interleukin-10 (IL-10) mRNA expression significantly increased. This statistically significant increase was transient, as IL-10 mRNA expression returned to near baseline levels within 1–2 days, although a biologically relevant 2–3 fold increase was still observed. B There was no significant difference in β-actin mRNA expression between the 4% AQ and load-treated hemispheres. p = 0.52). For both graphs, the data are presented as fold changes compared to the control hemisphere treated with the carrier.

[0023] Figure 5 The experimental methods used in Example 2 are described below.

[0024] Figure 6 The data described by AB show that intrahippocampal infusion of AQ is neuroprotective.

[0025] Figure 7 AD describes the expression of cytokines after AQ infusion.

[0026] Figure 8 The data from the AC study indicate that oral administration of AQ is neuroprotective.

[0027] Figure 9 Data described by AC showed that AQ infusion altered the expression of IL-10 and TNF-α proteins.

[0028] Figure 10 AC indicates AQ infusion and trace fear conditioning in aging rats.

[0029] Figure 11 AC describes oral administration of AQ as time- and dose-dependent.

[0030] Figure 12 The experimental methods used in Example 4 are described.

[0031] Figure 13 AC describes oral administration of AQ as neuroprotective.

[0032] Figure 14 Data from AD showed that oral administration of AQ altered the expression of cytokine proteins.

[0033] Figure 15 The data presented in the AB description indicate that intrahippocampal infusion of AQ alters cytokine protein expression.

[0034] Figure 16 The data showed that IL-10 nAb reversed the neuroprotective effect of AQ. Invention Details

[0035] I. Overview

[0036] Before describing the materials and methods of this invention, it should be understood that the invention is not limited to the specific methods and materials described, and variations are possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and should not be used to limit the scope of the invention, which is limited only by the appended claims.

[0037] It should be noted that the singular forms “a,” “an,” and “this” used herein and in the appended claims include the plural meaning unless otherwise expressly stated. Therefore, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] animal. Ninety-two male adult F344 rats were used. The rats were housed on a 14 / 10 hour day / night cycle, with freely available food and water. The weight of each animal was recorded twice weekly to calculate significant weight gain and / or loss.

[0040] drug. Apopolymerized jellyfish luminescent protein (AQ; Quincy Bioscience) was prepared in doubly deionized water at a concentration of 7.4%. In a dose-dependent experiment (n = 18), the experimental groups received AQ mixed into their daily PB at doses of 0 (n = 4), 3.6 (n = 5), 48 (n = 4), 240 (n = 3), or 480 mg / kg. For the remainder of the study, rats ( n = 73) Rats received an AQ of 48 mg / kg mixed into their daily PB. Animals were assigned to one of five groups: no AQ (n = 12), 1-hour AQ (n = 17), 1-day AQ (n = 15), 2-day AQ (n = 15), and 7-day AQ (n = 14). Each day, rats received ¼ teaspoon of PB placed in a Piper dish in their cage at the designated time. The dish was removed only after all PB had been consumed. Animals were weighed twice a week to maintain an appropriate AQ dose.

[0041] The AQ used for the infusion study was prepared as previously described (Detert et al., 2013). IL-10 neutralizing antibody (nAb) and its IgG control were prepared in sterile PBS. 0.5 μg was infused at a rate of 1 μL / min using a 1 μL Hamilton syringe.

[0042] Oxygen-glucose deprivation. On the last day of administration, after PB consumption, rats were allowed to digest for 1 hour, then deeply anesthetized with isoflurane, and the dorsal hippocampus (dhpc; anterior fontanelle) was prepared using a standard procedure (Moyer and Brown, 2007). - 3.14 - -4.16 (Paxinos and Watson, 1998) Coronal sections (400 μm). After recovery in aCSF for 1 hour, one hemisphere of each brain (balanced) underwent in vitro local ischemia for 5 minutes by transferring the sections to an oxygen-glucose deprivation chamber (glucose replaced with fructose and bubbling with 95% N2–5% CO2 instead of 95% O2–5% CO2), while the other hemisphere remained in recovery. All sections were then placed in oxidized aCSF containing 0.2% trypan blue for a 30-minute reperfusion phase. Trypan blue stains dead cells but not live cells (DeRenzis and Schechtman, 1973). The sections were rinsed twice in oxidized room temperature aCSF and then fixed overnight in 10% neutral buffered formalin in a refrigerator. Then, the sections were frozen in 30% sucrose for protection, sectioned (40 μm) on a cryostat, and mounted on embedding slides for cell counting.

[0043] Cell count. Sections were visually examined at 10X magnification under an Olympus microscope (equipped with a digital camera) and photographed (CellSens). Trypan blue-stained neurons in CA1 (approximately 800 μm) were counted by researchers unaware of the experimental conditions. Statistical analysis was performed using SPSS (v 21.0.0; IBM; Armonk, NY). ANOVA was used to evaluate drug effects, and Fisher's LSD causality assessment method was used to evaluate group interactions. (Astro) )instruct p < .05.

[0044] Western Imprint. Animals were deeply anesthetized with isoflurane, their brains were rapidly removed, frozen, and stored at -80°C. C. After the dissection time, from dhpc (anterior fontanelle) - 3.14 - -Samples were cut at 4.16 mm. After homogenization, the samples were centrifuged at 4000 RPM for 20 minutes, and the supernatant was removed. Protein analysis was performed using the Bradford Protein Assay Kit (Bio-Rad). Protein samples were standardized and loaded for SDS-PAGE (12%). Protein (30 µg) was transferred to a PVDF membrane using the Turbo Transfer System (Bio-Rad). The membranes were incubated in the following conditions: blocking buffer (2 h), primary antibody (4°C overnight; 1:1000 mouse anti-jellyfish luminescent protein [Chemicon] or 1:1000 rabbit anti-β-actin [Cell Signaling Technology]), and secondary antibody (90 min; 1:20,000 goat anti-mouse [Santa Cruz Biotechnology] or 1:40,000 goat anti-rabbit [Millipore]). The membranes were then rinsed, placed in chemiluminescent solution (Thermo Scientific), and imaged using a Syngene GBox. Images were acquired using Geneys software (v1.2.4.0; Synoptics camera 4.2MP), and fluorescence of each band was evaluated using GeneTools software (v 4.02; Cambridge, UK). Values ​​are expressed as a percentage compared to control animals. Statistical analysis was performed using SPSS (v. 21).

[0045] While the invention may be practiced or tested using any methods and materials similar to or equivalent to those described herein, preferred methods and materials are described hereafter. All publications and patents specifically mentioned herein are incorporated herein by reference in their entirety for all purposes, including describing and disclosing chemical substances, devices, statistical analyses, and methods reported in those publications that may be used in connection with the invention. All references cited in this specification should be considered as indications of the level of expertise in the art. Nothing herein should be construed as an admission that the invention did not precede these disclosures by virtue of prior invention.

[0046] II. This invention

[0047] In the United States, approximately 795,000 people are affected by ischemic stroke each year, with an estimated annual cost of $73.7 billion. Calcium is crucial in various neuronal signaling cascades; however, during local ischemia, excessive calcium influx can trigger excitotoxic cell death. Calcium-binding proteins assist neurons in regulating / buffering intracellular calcium levels during local ischemia. The jellyfish bioluminescent protein is isolated from the jellyfish *Echeveria virens* (also known as the bioluminescent jellyfish). Aequorea victoriaApo-Jewel luminescent protein (the calcium-binding component of luminescent protein) has been used as a calcium indicator for many years, but its neuroprotective properties are largely unknown. This study used in vitro rat brain slices to test the hypothesis that intrahippocampal infusion of apo-Jewel luminescent protein (the calcium-binding component of luminescent protein) protects neurons from ischemic cell death. One hemisphere of bilaterally cannulated rats received apo-Jewel luminescent protein infusion, while the other hemisphere received a carrier control. Hippocampal slices were then prepared and subjected to 5 minutes of oxygen-glucose deprivation (OGD), with cell death measured by trypan blue exclusion. Apo-Jewel luminescent protein dose-dependently protected neurons from OGD – doses of 1% and 4% (but not 0.4%) significantly reduced the number of trypan blue-labeled neurons. This effect was also time-dependent, lasting up to 48 hours. This time-dependent effect paralleled changes in cytokine and chemokine expression, indicating that apo-Jewel luminescent protein protects neurons through neuroimmunomodulatory mechanisms. These data support the hypothesis that pretreatment with apopolymerized jellyfish luminescent protein protects neurons from ischemic cell death and may have effective neurotherapeutic properties.

[0048] Spectrophotoprotein is a photoprotein that is initially isolated from spectral jellyfish and other marine organisms. The spectral protein complex contains a 22,285-Dalton apopolymerized spectral protein, molecular oxygen, and luciferin from the luminescent coelenterate. When three Ca... 2+ When ions bind to this complex, coelenterate luciferin is oxidized to coelentermide, accompanied by the release of carbon dioxide and blue light. Jellyfish luciferin is neither exported nor secreted by cells, nor is it segmented or isolated within cells. Therefore, jellyfish luciferin measurements have been used to detect the presence of Ca2+ over relatively long periods. 2+ Changes. In several experimental systems, the luminescence of the jellyfish bioluminescent protein was detectable several hours to several days after cell loading. It is also known that the jellyfish bioluminescent protein does not disrupt cell function or embryonic development.

[0049] Because its Ca 2+ Dependent luminescence, the jellyfish luminescent protein complex has been widely used as an intracellular Ca2+. 2+ Indicator. Specifically, the Victoria multituberculate jellyfish ( Aequorea victoria ) Jellyfish luminescent proteins have been used to: (1) analyze the secretory response of a single adrenal chromaffin cell to nicotinic cholinergic agonists; (2) elucidate Ca 2+ The role of release in myocardial injury; (3) showing Ca 2+ The massive release during fertilization; (4) Research on sarcoplasmic reticulum Ca 2+ (5) Regulation of pump expression in developing chick myoblasts; and (6) Calibration of micropipettes with injection volumes as low as three picoliters.

[0050] The molecular weight of apomyelin is approximately 22 kDa. By reducing the disulfide bonds in apomyelin, it can be used to regenerate jellyfish luminescent proteins. Calcium-loaded apomyelin retains the same compact scaffold and overall folding pattern as unreacted luminescent proteins containing bound substrates.

[0051] jellyfish bioluminescent proteins from the jellyfish Victoria multituberculatus ( Aequorea victoria Conventional purification of jellyfish luminescent protein requires laborious extraction operations and sometimes yields substantially heterogeneous or toxic preparations to the organism under investigation. Generally, two tons of jellyfish yield approximately 125 mg of purified luminescent protein. In contrast, recombinant jellyfish luminescent protein is preferably produced by purifying apopolymerized jellyfish luminescent protein from genetically engineered *E. coli*, followed by in vitro reconstruction of the jellyfish luminescent protein complex using pure coelenterate luciferin. The apopolymerized jellyfish luminescent protein used in this invention has been described and is commercially available via purification methods and / or synthetic methods known to those skilled in the art. S. Inouye, S. Zenno, Y. Sakaki, and F. Tsuji. High-level expression and purification of apopolymerized jellyfish luminescent protein (… High level expression and purification of apoaequorin ) . (1991) Protein Expression and Purification 2,122-126.

[0052] The jellyfish bioluminescent protein was isolated from the cnidarian jellyfish *Eichhornia victoria*. Aequorea victoria The luminescent protein belongs to the EF-hand family of CaBP, in which the EF-hand is closely associated with CaBP in mammals. Furthermore, the luminescent protein has been used as a CaBP... 2+ The indicator has been used for many years and has been shown to be cellularly safe and well-tolerated. However, its therapeutic potential has not yet been investigated. The jellyfish luminescent protein consists of two components—a calcium-binding component, apomeric luminescent protein (AQ), and a chemiluminescent molecule, coelenterate luciferin. Because the AQ portion of the protein contains a calcium-binding domain, AQ is used in the current study.

[0053] For our current experiment, we employed an in vitro model of global-local ischemia using short-term hippocampal slices. In these short-term slices, OGD-induced damage was most pronounced in the CA1 region of the hippocampus, similar to what was observed in vivo. Short-term hippocampal slices offer several advantages over cell cultures and in vivo models, including relatively unchanged tissue morphology compared to intact animals, extracellular ion concentration changes and neurotransmitter release similar to those reported in vivo, and the absence of uncontrollable vascular or other systemic responses in vivo. Neuronal damage following OGD was observed in short-term slices within the first 30 minutes of reperfusion; however, due to the short lifespan of the slices, only early changes in local ischemia could be analyzed. Because hippocampal neurons are prone to cell death after local ischemia, we tested the hypothesis that direct infusion of AQ into the hippocampus prior to ischemic injury would have neuroprotective effects.

[0054] This invention relates to administering to a subject compositions containing apomeric jellyfish luminescent proteins to generally correct or maintain calcium balance in the subject. It is understood that maintaining ionic calcium concentrations in plasma and body fluids is crucial for many bodily functions, including but not limited to neuronal excitability, muscle contraction, membrane permeability, cell differentiation, hormone secretion, bone mineralization, or prevention of cell death following local ischemia. It is known that disruption of calcium homeostasis, i.e., calcium imbalance, causes and / or is associated with a variety of diseases, syndromes, and conditions. Exemplary diseases, syndromes, and conditions include those related to sleep quality, energy quality, mood quality, memory quality, and pain perception. Research on CaBP has led to their identification as protective factors playing a role in maintaining appropriate ionic calcium levels.

[0055] In some embodiments, the method of the present invention includes administering apomylated jellyfish luminescent protein as the sole active ingredient to provide neuroprotection, to delay the progression of neuronal inflammation, to prevent the onset of neuronal inflammation, and to prevent and / or treat the recurrence of neuronal inflammation. In some embodiments, the present invention provides a method comprising administering apomylated jellyfish luminescent protein and one or more other agents having known therapeutic or nutritional value.

[0056] The term "treatment" as used herein includes both preventative and dysregulation-relieving treatments. The terms "reduction," "reduction," "relief," "stop," and "inhibition" as used herein have the meaning of reduction or lessening as commonly understood. The term "development" as used herein refers to an increase, progression, growth, or worsening of the extent or severity of the disease. The term "relapse" as used herein refers to the recurrence of the disease after a period of remission.

[0057] As used herein, the term “give” means to expose a patient, tissue, organ, or cell to the apomyelin luminescent protein. Giving as used herein can be performed in vitro, i.e., in a test tube, or in vivo, i.e., in the cells or tissues of a living organism (e.g., a human). In a preferred embodiment, the invention comprises giving a patient or subject a composition useful in the invention. “Patient” or “subject” herein has the same meaning as “mammalian,” preferably human, who: (1) has neuronal inflammation that can be cured or treated by giving the apomyelin luminescent protein; or (2) is susceptible to neuronal inflammation that can be prevented by giving the apomyelin luminescent protein.

[0058] As used herein, the terms “effective amount” and “therapeutic effective amount” refer to the amount of an active agent sufficient to produce the desired therapeutic response without excessive adverse side effects, such as toxicity, irritation, or anaphylactic response. The specific “effective amount” will obviously vary with a variety of factors, such as the specific disease being treated, the patient’s physical condition, the type of animal being treated, the duration of treatment, the nature of any concurrent treatments, and the structure of the specific formulation and compound or its derivatives used. In this case, an amount is considered therapeutically effective if it results in one or more of the following: (1) prevention of neuronal inflammation; and (2) reversal or stabilization of neuronal inflammation. The optimal effective amount can be readily determined by those skilled in the art using routine experiments.

[0059] In certain preferred compositions for oral administration, apopolymerized jellyfish luminescent protein is formulated with at least one acceptable carrier at a dose of about 10 mg / dose, preferably in capsule form, having a recommended dose of about 10 mg / day (i.e., one capsule / day) for the subject.

[0060] The compositions of the present invention comprise liquid or lyophilized or otherwise dried formulations, and include diluents of various buffering substances (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent surface absorption, detergents (e.g., Tween 20, Tween 80, Prönkel F68, bile salts), solubilizers (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thiomercurate, benzyl alcohol, parabens), swelling agents or stress modifiers (e.g., lactose, mannitol), covalent linkage of polymers such as polyethylene glycol to proteins, coordination with metal ions, or introduction of materials into or onto polymer compounds such as polylactic acid, polyglycolic acid, or hydrogel microparticle formulations, or onto liposomes, microemulsions, micelles, sheets or multilayer carriers, erythrocyte shadows or protoplasts. The compositions will affect physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. Controlled or sustained-release compositions include formulations in the form of lipophilic libraries (e.g., fatty acids, waxes, oils).

[0061] This invention also covers methods of administering particulate compositions coated with polymers (e.g., poloxamer or poloxamine). Other embodiments of the compositions incorporate protective coatings in particulate form, protease inhibitors, or permeability enhancers for use via a variety of routes of administration, including parenteral, pulmonary, nasal, and oral. In some embodiments, the compositions are administered via: parenteral, paracancerally, through mucosa, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intravenous, intracranial, or intratumoral administration.

[0062] Furthermore, the term "pharmaceutically acceptable carrier" as used herein is well known to those skilled in the art and includes, but is not limited to, 0.01-0.1M, and preferably 0.05M phosphate buffer or 0.9% saline. Additionally, the pharmaceutically acceptable carrier can be an aqueous or non-aqueous solution, suspension, or emulsion. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic solutions / aqueous solutions, emulsions, or suspensions, including saline and buffer media.

[0063] Parenteral carriers include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, and fixed oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextran. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, collating agents, and inert gases.

[0064] The compositions of the present invention containing apo-coated jellyfish luminescent protein are particularly useful for formulation into injectable drug doses, including combinations of apo-coated jellyfish luminescent protein with injectable delivery systems. As used herein, injectable and infusion formulations (i.e., parenteral formulations) include, but are not limited to, liposome injections or lipid bilayer carriers having phospholipids encapsulating the active drug. Injection includes sterile preparations for parenteral use.

[0065] UPS defines five different categories of injectable preparations: emulsions, lipids, powders, solutions, and suspensions. Emulsion injections comprise an emulsion containing a sterile, pyrogen-free preparation for parenteral administration. Lipid complexes and powders for solution injections are aseptically prepared and designed to be reconstituted to form a solution for parenteral use. Powders for suspension injections are aseptically prepared and designed to be reconstituted to form a suspension for parenteral use. Lyophilized powders for liposome suspension injections are sterile, lyophilized formulations designed to be reconstituted for parenteral use, formulated in a manner that allows the inclusion of liposomes (e.g., lipid bilayer carriers with phospholipids used to encapsulate the active drug within a lipid bilayer or an aqueous space), thereby enabling the formation of the formulation after reconstitution. Lyophilized powders for solution injections are dosage forms for solutions prepared by lyophilization (“lyophilization”), a process involving the removal of water from a frozen product under extremely low pressure, followed by the addition of liquid to produce a solution that meets all injectability requirements. Powders prepared for suspension injection by lyophilization are liquid preparations intended for parenteral use, containing solids suspended in a suitable liquid medium and meeting the requirements of a sterile suspension in all respects, thus preparing pharmaceutical formulations for suspension by lyophilization. Solution injections relate to liquid preparations containing one or more pharmaceutical substances dissolved in a suitable solvent or a mixture of mutually miscible solvents suitable for injection. Concentrated solution injections relate to sterile preparations for parenteral use, producing a solution that meets the requirements for injection in all respects upon the addition of a suitable solvent. Suspension injections relate to (injectable) liquid preparations containing solid particles dispersed in a liquid phase, said particles being insoluble, with an oil phase dispersed in an aqueous phase or vice versa. Liposome suspension injections are (injectable) liquid formulations having an oil phase dispersed throughout the aqueous phase in a manner that forms liposomes (a lipid bilayer carrier, typically containing phospholipids for encapsulating the active drug within a lipid bilayer or an aqueous space). An ultrasonically injected suspension is a liquid preparation (suitable for injection) containing solid particles dispersed throughout the aqueous phase, making the particles insoluble. Furthermore, the product can be ultrasonically treated as gas bubbles emerge from the suspension, causing the solid particles to form microspheres.

[0066] Gastrointestinal delivery carrier systems include one or more pharmaceutically suitable excipients, such as solvents or cosolvents, solubilizers, wetting agents, suspending agents, thickeners, emulsifiers, chelating agents, buffers, pH adjusters, antioxidants, reducing agents, antimicrobial preservatives, swelling agents, protective agents, tension modifiers, and special additives.

[0067] The controlled or sustained-release compositions available according to the invention comprise formulations in the form of lipophilic libraries (e.g., fatty acids, waxes, oils). The invention also covers particulate compositions coated with a polymer (e.g., poloxamer or poloxamine) and a compound conjugated to an antibody, said antibody being directed against a tissue-specific receptor, ligand, or antigen, or a ligand conjugated to a tissue-specific receptor.

[0068] Other embodiments of the compositions available according to the invention incorporate a protective coating in particulate form, a protease inhibitor, or a permeability enhancer for use in a variety of routes of administration, including parenteral, pulmonary, nasal, ocular, and oral.

[0069] Chemical entities modified by covalently linking water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextrose, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline are known to exhibit substantially longer blood half-lives after intravenous injection than their unmodified counterparts. These modifications can also increase the solubility of the chemical entity in aqueous solutions, eliminate aggregation, enhance the physical and chemical stability of the compound, and significantly reduce its immunogenicity and reactivity. Therefore, the desired in vivo bioactivity can be achieved by administering the polymer-entity adduct at a lower frequency or lower dose compared to the unmodified entity.

[0070] According to another method of the invention, the composition can be delivered in the form of a controlled-release system. For example, the substance can be delivered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other delivery modalities. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be employed. In yet another embodiment, the controlled-release system can be placed near the therapeutic target (i.e., the brain), thus requiring only a portion of the systemic dose.

[0071] The composition may contain only apomylated jellyfish luminescent protein, or may also include a pharmaceutically acceptable carrier, and may be in solid or liquid form, such as tablets, powders, capsules, clumps, solutions, suspensions, elixirs, syrups, beverages, emulsions, gels, creams, ophthalmic preparations, or suppositories, including rectal and urethral suppositories. Pharmaceutically acceptable carriers also include gums, starches, sugars, cellulose materials, and mixtures thereof. The composition containing apomylated jellyfish luminescent protein may be administered to a patient, for example, by subcutaneous implantation of a clump. In another embodiment, the clump provides controlled release of apomylated jellyfish luminescent protein over a period of time. The composition may also be administered intravenously, intra-arterially, intramuscularly, or orally in liquid or solid form, or by topical application. Administration may also be performed using rectal or urethral suppositories.

[0072] The compositions of this invention can be prepared by known dissolution, mixing, granulation, or tableting processes. For oral administration, the apopolymerized jellyfish luminescent protein or its physiologically tolerable derivatives, such as salts, esters, nitrides, etc., are mixed with additives conventionally used for this purpose, such as carriers, stabilizers, or inert diluents, and converted by conventional methods into a form suitable for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic, or oily solutions.

[0073] Examples of suitable inert carriers are conventional tablet matrices, such as lactose, sucrose, or corn starch, with binders such as gum arabic, corn starch, or gelatin, accompanied by disintegrants such as corn starch, potato starch, or alginate, or with lubricants such as stearic acid or magnesium stearate.

[0074] Examples of suitable oily carriers or solvents are vegetable or animal oils, such as sunflower seed oil or cod liver oil. The composition can function in dry or wet granule form. For parenteral administration (subcutaneous, intravenous, intra-arterial, or intramuscular injection), the chemical entity or its physiologically tolerable derivatives, such as salts, esters, nitrogen oxides, etc., are converted into a solution, suspension, or expulsion, if necessary for and suitable for the intended use of the substance, for example, with solubilizers or other adjuvants.

[0075] Examples include sterile liquids such as water and oils, with or without surfactants and other pharmaceutically acceptable adjuvants. Examples of oils are petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, or mineral oil. Generally, water, saline, dextrose solutions and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are preferred liquid carriers, especially for injectable solutions.

[0076] The preparation of compositions containing active ingredients is well known in the art. These compositions can be prepared in an aerosol form for delivery to the nasopharynx, or in an injectable form as a liquid solution or suspension; however, they can also be prepared in a solid form suitable for forming a solution or suspension in a liquid prior to injection. The composition can also be emulsified. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextran, glycerol, ethanol, etc., or any combination thereof. Furthermore, the composition may contain small amounts of adjuvants such as wetting agents or emulsifiers, pH buffers, which enhance the effectiveness of the active ingredient.

[0077] The active ingredient can be formulated into a composition in a pharmaceutically acceptable salt form. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as hydroxides of sodium, potassium, ammonium, calcium, or iron, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0078] For topical administration, such as in creams, gels, drops, etc., apo-coated jellyfish luminescent protein or its physiologically tolerable derivatives are prepared or applied in the form of a solution, suspension, or emulsion in a physiologically acceptable diluent, with or without a drug carrier.

[0079] In another method of the invention, the active component may be delivered in a carrier, specifically, liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss Press, New York, pp. 353-365 (1989).

[0080] The salt of the apomylated jellyfish luminescent protein is preferably a pharmaceutically acceptable salt. However, other salts may be used in the preparation of the compositions of the present invention or their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include acid addition salts, which can be formed, for example, by mixing a solution of the apomylated jellyfish luminescent protein with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid.

[0081] Furthermore, the compositions containing apomylated jellyfish luminescent protein described herein may be provided in the form of nutritional compositions, wherein the apomylated jellyfish luminescent protein prevents the onset of various harmful effects of neuronal inflammation, or reduces or stabilizes various harmful effects of neuronal inflammation. For the purposes of this specification, the term "nutrition" or "nutritional composition" refers to food, or a portion of food, which provides medical and health benefits, including the prevention and / or treatment of disease. The nutritional compositions of this invention may contain only apomylated jellyfish luminescent protein as the active ingredient, or may also include a mixture of food supplements (including vitamins, coenzymes, minerals, herbs, amino acids, etc.) that supplement the diet by increasing the total intake of such substances.

[0082] Therefore, the present invention provides a method for providing nutritional benefits to a patient, the method comprising the step of administering a nutritional composition containing apomylated jellyfish luminescent protein to the patient. The composition generally comprises a "nutritionally acceptable carrier," which, as described herein, is any carrier suitable for oral delivery, including the aforementioned pharmaceutically acceptable carriers suitable for oral administration. In some embodiments, the nutritional composition of the present invention comprises a food supplement defined based on its function as including immune enhancers, anti-inflammatory agents, antioxidants, antiviral agents, or mixtures thereof.

[0083] Immunostimulants and / or antiviral agents can be used to accelerate wound healing and improve immune function; and these include those derived from the genera *Cinnamomum* or *Echinacea*. Echinacea Extracts from herbs of the genus *Sambuca* ( Sambuca Extracts of herbs, including North American Coptis chinensis extract. (Astragalus genus) Astragalus Herbal remedies, whether natural or processed, are also effective immune boosters. Astragalus stimulates the development of stem cells and active immune cells in the bone marrow and lymphatic tissue. Zinc and its bioactive salts, such as zinc gluconate and zinc acetate, can also be used as immune boosters to treat the common cold.

[0084] Antioxidants include the naturally occurring sulfur-containing amino acid allicin, which works by increasing the level of antioxidant enzymes in the blood. Herbs or herbal extracts, such as garlic, which contains allicin, are also effective antioxidants. Catechins, and herbal extracts such as green tea containing catechins, are also effective antioxidants. (Asarum spp.) Astragalus Extracts of [the substance] also exhibit antioxidant activity. Bioflavonoids, such as quercetin, hesperidin, rutin, and mixtures thereof, are also effective antioxidants. A major beneficial effect of bioflavonoids may be protecting vitamin C in the body from oxidation. This allows more vitamin C, or ascorbic acid, to be utilized by the body.

[0085] Bioflavonoids, such as quercetin, are also effective anti-inflammatory agents and can therefore be used in the compositions of the present invention. Anti-inflammatory herbal supplements and compounds derived from plants or herbs can also be used as anti-inflammatory agents in the compositions of the present invention. These include bromelain (a protease found in pineapple); extracts of tea and nettle; turmeric, turmeric extract, or curcumin, a yellow pigment isolated from turmeric.

[0086] Other supplements that can be used in this invention may be ginger, which is derived from the genus Zingiber (Zingiber). Zingiber Ginger is an herb. It has been found to have cardiotonic activity, attributed to compounds such as gingerol and related compound shogaol, and provides benefits in the treatment of dizziness and vestibular disorders. Ginger is also effective in treating nausea and other stomach upsets.

[0087] Supplements that help rebuild soft tissue structure, especially cartilage, can be used in compositions to treat pain associated with arthritis and other joint conditions. Glucosamine, glucosamine sulfate, and chondroitin are available from a variety of sources, such as elk antler. Marine liquid complexes, omega-3 fatty acid complexes, and fish oil are also known to be beneficial in treating arthritis-related pain.

[0088] Supplements that may help treat migraines include wild chamomile and ginkgo. Gingko biloba The main active ingredient in wild chamomile is the sesquiterpene lactone chamomile, which inhibits the secretion of prostaglandins, which in turn cause pain due to vasospasm in blood vessels. Wild chamomile also exhibits anti-inflammatory properties. Fish oil, due to its platelet-stabilizing and antivasospasmodic effects, can also be used to treat migraines. Herbal ginkgo (Ginkgo biloba) Gingko biloba It also helps treat migraines by stabilizing arteries and promoting blood circulation.

[0089] The invention can be more fully understood by considering the following non-limiting embodiments.

[0090] Example

[0091] Example 1. Pretreatment with apomylated jellyfish luminescent protein protects hippocampal CA1 neurons from oxygen-glucose deprivation.

[0092] Materials and methods

[0093] object

[0094] The subjects were 142 adult male F344 rats (mean age: 4.0 ± 0.1 months; Harlan). They were housed in an AAALAC-level facility with a 14-hour light–10-hour dark cycle, and were provided with free access to food and water, and were kept separately.

[0095] Operation

[0096] Rats were administered ibuprofen solution (15 mg / kg / day) at least one day before and two days after surgery. On the day of surgery, rats were anesthetized with isoflurane and fixed to a stereotactic apparatus. Under aseptic conditions, bilateral 26-gauge stainless steel guiding cannulas were inserted into the dorsal hippocampus (relative to the anterior fontanelle: AP -3.5 mm, L±2.6 mm, V -3.0 mm). The cannulas were secured to the skull with stainless steel screws and acrylic bone cement. Stainless steel caps were placed in the guiding cannulas to prevent occlusion, and rats were allowed at least 7 days to recover before infusion.

[0097] Hippocampal infusion

[0098] Jellyfish luminescent protein consists of two components: apomyelin and coelenterate luciferin. The apomyelin component (AQ) contains Ca2+-bound protein. 2+ The EF hand

[51] is therefore the component used in the current study. Zero Ca infusion was administered to rats. 2+ AQ was administered in artificial cerebrospinal fluid (aCSF; mM: 124.00 NaCl, 2.80 KCl, 2.00 MgSO4, 1.25 NaH2PO4, 26.00 NaHCO3, 10.00 D-glucose, and 0.40 sodium ascorbate), which also contained 6% DMSO to promote neuronal uptake of AQ. Rats received bilateral infusions (0.5 µl / hemispheric) for more than 60 seconds, then the infusion cannula was held in place for an additional 2 minutes to ensure diffusion from the tip. The 33-gauge infusion cannula was cut to protrude 0.5 mm from the guide cannula. To determine the dose-dependent neuroprotective effects of AQ, one hemisphere of the animal was infused with 0.4, 1, or 4% AQ (w / v; Quincy Bioscience, Madison, Wisconsin) in a balanced manner, while the other hemisphere was infused with the carrier. In addition, two hemispheres of a rat subgroup were infused with the carrier (0% AQ) as a control (n = 11 in each group).

[0099] Slice preparation

[0100] To determine the neuroprotective effect of AQ in a short-term brain slice model of localized ischemia, 94 male F344 rats (mean age 4.4 ± 0.2 months) were used. Brain slices were prepared as previously described from control rats (0% AQ, n = 10) or from rats that received AQ at one of the following time points after infusion: 1 hour (n = 10), 1 day (n = 10), 2 days (n = 10), 3 days (n = 10), or 5 days (n = 5). In short, rats were deeply anesthetized with isoflurane and perfused via the ascending aorta with cold, oxidized (95% O2 / 5% CO2) sucrose-CSF (mM: 206.00 sucrose, 2.80 KCl, 2.00 MgSO4, 1.25 NaH2PO4, 1.00 CaCl2, 1.00 MgCl2, 26.00 NaHCO3, 10.00 D-glucose, and 0.40 sodium ascorbate), and the brain was rapidly excised and placed in the cold, oxidized sucrose-CSF. The brain was sealed near the cannulation site, and 400 µm thick coronal sections were cut on a temperature-controlled vibratory microtome, as previously described. Only the first five sections immediately after cannulation (without any visible cannulation marks) were collected and used in the experiments described below. Sections were incubated on a mesh submerged in oxidized (95% O2 / 5% CO2) aCSF (composition, in mM: 124.00 NaCl, 2.80 KCl, 2.00 MgSO4, 1.25 NaH2PO4, 2.00 CaCl2, 26.00 NaHCO3, 10.00 D-glucose, and 0.40 sodium ascorbate) at 35°C. After 1 hour of recovery, sections underwent 5 minutes of oxygen-glucose deprivation (OGD) to induce local ischemia. OGD was induced by transferring sections to a 35°C solution of fructose-CSF (in which fructose replaces glucose at an equimolar concentration) bubbled with 95% N2 / 5% CO2 (in which N2 replaces O2). Following OGD, sections were transferred to a 35°C solution containing oxidized aCSF and 0.2% trypan blue (Sigma-Aldrich, St. Louis, Missouri) for 30 minutes before reperfusion. Trypan blue penetrates dead and dying cells, staining them blue while leaving live cells unstained. Sections were then briefly rinsed at room temperature, treated with oxidized aCSF, and immediately fixed overnight in 10% neutral-buffered formalin. Sections were cryoprotected with 30% sucrose for at least one day, after which they were fractionated in 40µm sections using a cryostat, fixed onto gelatin-coated slides, dehydrated with incremental ethanol, and covered with Permount mounting medium.

[0101] Cell Count

[0102] Sections were visually examined under an upright microscope (Olympus BX51) equipped with a digital camera (Olympus DP70) and a 10X objective. Photographs were taken of the CA1 cell body layer (the apex of the upper lobe of the dentate gyrus) in each 40-µm fraction. To avoid overstaining due to neuronal damage attributable to the initial hippocampal section preparation, only internal fractions were photographed for analysis. Then, neurons stained with trypan blue throughout the images were counted by a person unaware of the processing conditions. Data from only one fraction were counted. The percentage of neuroprotection in each animal was assessed by normalizing data from the AQ-treated hemisphere to the drug-treated hemisphere.

[0103] Western blot analysis

[0104] To determine the duration of AQ retention in the dorsal hippocampus following infusion, 4% AQ was infused into both hemispheres of 24 adult male F344 rats (mean age 4.2 ± 0.1 months). At 1 hour (n = 4), 1 day (n = 7), 2 days (n = 7), or 3 days (n = 6) post-infusion, rats were anesthetized with an overdose of isoflurane, and their brains were excised, rapidly frozen on dry ice, and stored at -80°C. Two bilateral brain regions (dorsolateral and ventral hippocampus; dhpc and vhpc) were excised from each rat and homogenized separately. Samples were centrifuged at 4000 rpm, and the supernatant was removed and analyzed using a Bradford protein assay kit (Bio-Rad, Hercules, CA). Protein samples were normalized (50 or 150 µg / lane) and loaded for SDS-PAGE (10%). Proteins were transferred to PVDF membranes using a semi-dry transfer device (Bio-Rad, Hercules, CA). The membranes were then incubated in blocking buffer (3% skim milk) for 2 hours, followed by incubation with primary antibody (4°C overnight; 1:5000 mouse anti-jellyfish luminescent protein [Millipore, Billerica, MA] or 1:1000 rabbit anti-... Incubate with actin [Cell Signaling Technology, Boston, MA], then incubate with secondary antibody (90 min; 1:5000 goat anti-mouse [Santa Cruz Biotechnology, Santa Cruz, CA] or 1:5000 goat anti-rabbit [Millipore]). The membrane is then rinsed (0.05% Tween 20 in Tris-buffered saline), placed in chemiluminescent solution (Santa Cruz Biotechnology), and exposed to Hyperfilm MP. Images are acquired and analyzed using NIH ImageJ software via densitometric analysis. A band is considered positive if its densitometric value (minus the background in each lane) is more than 2 standard deviations above the mean of the ventral hippocampal bands. From this quantification, 100% of the 1-hour bands, 83% of the 1-day bands, 29% of the 2-day bands, 0% of the 3-day bands, and 0% of the VHPC lanes were observed to be positive. The ventral hippocampus was compared to this near-brain structure, which should not contain AQ, and therefore served as a negative control.

[0105] Quantitative RT-PCR

[0106] Twelve male rats (each 3.8 months old) received a unilateral infusion of 4% AQ (as described above), and tissues were collected 1 hour, 1 day, or 2 days post-infusion (n = 4 per group). The hippocampus was excised and immediately placed in TRIzol reagent (Life Technologies Corp., Carlsbad, CA). Tissues were homogenized using a 25-gauge needle and syringe, and samples were stored at -80°C. C. RNA isolation was performed simultaneously using the TRIzol method (Life Technologies Corp., Carlsbad, CA) according to the manufacturer's instructions. The isolated RNA was dissolved in 50 µl of RNase-free H₂O, and RNA purity was calculated based on the absorbance ratio at 260 nm and 280 nm. Absorbance readings between 1.8 and 2.1 were considered sufficiently pure for reverse transcription. Samples with an absorbance ratio less than 1.8 were further purified using the Qiager RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions, and the purified RNA was resuspended in 50 µl of RNase-free H₂O. Total RNA from all samples was reverse transcribed into cDNA using the Qiager RT2 HT First Strand Kit-96 (Qiagen). Samples were purified in triplicate in 96-well plates using primers specific to rat IL-10 and β-actin (RT2 qPRC primer assay; Qiagen) and RT²SYBR Green qPCR master mix (Qiagen), on a StepOne real-time PCR system and software (Life Technologies Corp., Carlsbad, CA). Changes in IL-10 gene expression relative to load treatment with AQ were calculated using the Pfaffl equation. β-actin expression in corresponding samples at each time point was normalized and compared to hippocampal isolates from load-treated rats. Primer efficiency was calculated based on dilution profiles of two randomly selected samples targeting IL-10 and β-actin. AQ infusion did not alter β-actin expression compared to tissues infused with aCSF, indicating that AQ infusion generally does not or specifically affect gene transcription.

[0107] Gene expression array

[0108] cDNA was obtained from rats and used for RT-PCR (see Methods). PCR analysis targeting overall genetic markers of inflammatory cytokines and receptors was performed using a Qiagen RT2 Profiler array according to the manufacturer's protocol. Briefly, 2X RT2 SYBR Green master mix, cDNA (see above), and RNase-free water were combined, and 25 µl of this mixture was added to each well of a 96-well PCR Profiler array plate. Samples were processed using the StepOne real-time PCR system and software; those with multiple melting curves were excluded from the analysis (n = 2). A total of one animal was excluded from this study because its gene expression deviated from the mean by more than two standard deviations. Gene expression changes were calculated using Qiagen's web-based RT2 Profiler PCR array analysis software v3.5.

[0109] Data analysis and statistics

[0110] Statistical analyses were performed using Statview (v 5.0; SAS Institute, Inc., Culley, NAT). ANOVA was used to evaluate therapeutic effects. Fisher's PLSD was used for causal relationship comparisons. Data are reported as mean ± standard error of mean.

[0111] result

[0112] Oxygen-glucose deprivation leads to significant cell death.

[0113] Prepare short-duration hippocampal sections, expose them to oxygen-glucose deprivation (OGD) for 5 minutes, and stain them by transferring them to oxidized aCSF containing trypan blue (see Methods). Figure 1 As shown, OGD induced significantly more cell death than the control slides that did not undergo OGD. ANOVA analysis of the mean number of cells stained with trypan blue in both ischemic and non-ischemic conditions showed a statistically significant effect of local ischemia. F (1, 12) = 9.65, p < 0.01. These results are consistent with previous studies indicating that OGD causes significant cell death in the CA1 region of the hippocampus

[52] .

[0114] Reduced cell death after treatment with apomylated jellyfish luminescent protein

[0115] To test the potential neuroprotective effects of intrahippocampal infusion of apomylated luminescent protein (AQ) prior to OGD, rats were infused with 0, 0.4, 1, or 4% AQ 24 hours before OGD (see [link to OGD test]). Figure 2A). AQ exerts its neuroprotective effect in a dose-dependent manner; therefore, intracavitary infusion of 1% or 4% AQ before local ischemia results in a significant increase in neuroprotection compared to carrier (0% AQ) infusion. F (3, 40) = 3.61, p < 0.05 ( Figure 2 (B and C). Causal relationship analysis revealed that, compared with the 0% AQ group, infusion of 1 or 4% AQ significantly enhanced neuroprotection. p < 0.01, and the infusion of 0.4% AQ was not statistically different from any other group. It is also noteworthy that the amount of neuroprotection was not different between the 1% and 4% AQ treatment groups.

[0116] To evaluate the duration of neuroprotective effects of AQ, rats were infused with 4% AQ at multiple time points (1 hour, 1 day, 2 days, 3 days, or 5 days) before OGD. Figure 3 A). Unidirectional ANOVA indicates that time significantly affects the ability of hippocampal AQ infusion to protect neurons from subsequent OGD. F (5, 49) = 3.35, p < 0.05. Causal relationship tests revealed that the neuroprotective effects of AQ required at least one day to appear and lasted for at least two days (at various time points). p < 0.05). No statistically significant neuroprotection was observed when the slices underwent OGD 3 or 5 days after AQ infusion (with respective values ​​of < 0.05). p = .10 and p = .47).

[0117] Western blot analysis of apomyelin luminescent protein

[0118] To determine how long AQ remains in the dorsal hippocampus after intra-hippocampal infusion, Western blot analysis was used to test AQ protein levels at different time points (1 hour, 1 day, 2 days, or 3 days) after bilateral infusion of 4% AQ into the dorsal hippocampus. Figure 3 C indicates that AQ was present in the dorsal hippocampus at 1 hour and 1 day, almost invisible at 2 days, and no longer present by 3 days post-infusion. Therefore, 100% positive bands were observed at 1 hour, 83% at 1 day, 29% at 2 days, and 0% in the 3-day lane. As expected, AQ was not detected in the ventral hippocampus (vhpc), which was used as a negative control structure because it is far from the injection site (see [link to relevant documentation]). Figure 3C). To ensure sufficient protein loading into the gel to allow for the observation of extremely weak bands, Western blots were repeated on the animal subgroups, but the gels were loaded with 150 µg of protein per lane (instead of the normal 50 µg per lane). In these blots, additional bands appeared in lanes at days 2 and 3, resulting in 57% of lanes at day 2 and 25% at day 3 showing positive bands, indicating that AQ remains detectable in the dorsal hippocampus up to 3 days after infusion. Importantly, in the sample targeting - No time-dependent changes were observed during actin staining, suggesting that these differences reflect time-dependent changes in the presence of AQ, rather than general changes in protein content (see [link to article]). Figure 3 C).

[0119] Cytokine and chemokine expression after AQ infusion

[0120] The significant neuroprotective effect of AQ infusion into the hippocampus at time points with minimal protein presence suggests that AQ may trigger a cascade of events that ultimately protect neurons from ischemic injury. One possibility is that AQ induces a premodulation-like effect, leading to reduced cell death at a later time point. Ischemic premodulation is a phenomenon by which short-term ischemic events mitigate the damage caused by subsequent, more severe ischemic injury. Recent evidence has shown that multiple cytokines and chemokines are associated with ischemic premodulation. Given the link between ischemic premodulation and changes in cytokine production, we tested the hypothesis that AQ infusion may lead to increased expression of cytokines or chemokines, which could ultimately affect the neuronal tolerance to subsequent ischemic injury. RT-PCR was used to investigate changes in the mRNA of the anti-inflammatory cytokine interleukin-10 (IL-10), and PCR arrays were used to observe changes in the expression of multiple genes following AQ infusion. Adult rats received an infusion of 4% AQ in one hemisphere and a carrier in the other hemisphere, as described in this paper (see Methods). Hippocampus was excised and quantitative RT-PCR was performed at different time points following AQ infusion (1 hour, 1 day, or 2 days later) to evaluate time- and treatment-dependent changes. One-way ANOVA indicated significant differences between the four treatment groups. F (3, 19) = 9.55, p < 0.0005. Causal relationship analysis revealed that IL-10 mRNA significantly increased 1 hour after AQ infusion, relative to the load-treated hemispheres ( p < .001; see also Figure 4 A). Furthermore, the AQ-induced increase in IL-10 expression at 1 hour was significantly greater than that at 1 day (A). p < .001) or 2 days ( pThe enhancement was observed at < 0.001). Although IL-10 expression increased 2-3 times at later time points, these were not statistically significant compared to the hemispheres treated with the load, suggesting that the significant increase in IL-10 observed at 1 hour could be attributed to a short-term response to AQ infusion.

[0121] To investigate whether AQ-related cytokine expression changes were limited to IL-10 rather than a more holistic change in mRNA expression patterns, PCR arrays were performed. A total of 82 genes associated with cytokine and chemokine responses were investigated. Of these genes, 80 showed the degree of change in the control hemisphere, and two genes (CCR8, chemokine receptor 8; and CRP, C-reactive protein) were undetectable. Of the 80 detectable genes, only 16 differed significantly between the AQ- and load-treated hemispheres (see Table 1, data organized by response time). Most genes increased 1 hour after AQ infusion, then decreased to or near baseline levels by day 1. Of the eight genes significantly upregulated at 1 hour, only one remained elevated by day 2 post-infusion: chemokine ligand 10 (CXCL10). Six genes were not significantly upregulated at 1 hour but were upregulated 1 day after AQ infusion. Of these six genes, only two did not remain elevated at day 2 – chemokine ligand 11 (CXCL11) and interleukin-1 receptor type II (IL-1rII). Only two genes were significantly upregulated two days after AQ infusion – chemokine receptor 1 (XCR1) and complement component 3 (C3). These results indicate that AQ infusion into the dorsal hippocampus has a significant impact on cytokine and chemokine mRNA expression at both short-term and long-term time points.

[0122] discuss

[0123] Current research shows that the calcium-binding protein apomeric luminescent protein (AQ) has a time- and dose-dependent neuroprotective effect when administered prior to ischemic injury. Infusion of 1% or 4% AQ into the hippocampus resulted in significantly fewer dead or dying neurons compared to the control group (see [link to study]). Figure 2 This neuroprotection is time-dependent, meaning it takes at most 1 or 2 days to develop and weakens by 3 to 5 days. The neuroprotection may involve a pre-regulation-like effect, whereby AQ infusion modulates cytokine and chemokine expression, subsequently protecting neurons from oxygen-glucose deprivation (OGD).

[0124] Previous studies have demonstrated the neuroprotective effects of CaBP. For example, neurons containing CaBP calcium-binding protein are more resistant to excitotoxicity and ischemia-related injury than neurons lacking calcium-binding protein. Furthermore, some studies have noted increased calcium-binding protein expression following traumatic brain injury and ischemia, suggesting that calcium-binding protein expression may increase to maintain calcium levels.2+ Homeostasis and protection against excitotoxicity. Similarly, overexpression of CaBP prior to ischemia has been found to be neuroprotective using gene therapy or protein transduction. However, the presence of calcium-binding proteins in the dentate gyrus (a region resistant to ischemic cell death) and CA1 (a region prone to cell death) has been used to refute the role of calcium-binding proteins in neuroprotection. Finally, others have reported enhanced recovery from ischemia in mice with calcium-binding protein knockout. Given that these are not inducible knockouts, other compensatory mechanisms may play a role in the observed neuroprotection.

[0125] Studies testing the effects of artificial calcium chelators (e.g., BAPTA-AM, EGTA, etc.) on excitotoxicity have yielded mixed results, with some finding neuroprotection while others found increased vulnerability to cell death. Nikonenko et al. showed neuroprotection in rat organoid hippocampal culture sections treated with EGTA, BAPTA, mibeprazole, Kurtoxin, nickel, zinc, and piracetam after OGD. Conversely, Abdel-Hamid and Baimbridge loaded hippocampal neurons with the calcium chelator BAPTA-AM and found enhanced glutamate excitotoxicity in those neurons. These authors demonstrate the existence of artificial calcium chelators that affect normal calcium excitotoxicity. 2+ Interference with dependency mechanisms, which prevent Ca 2+ Inflow into cells. These contradictory results can be attributed to a variety of factors, including the pattern of inducing excitotoxicity, the Ca used, and so on. 2+ The type of chelating agent, or its application compared to short-time brain slice culture of neurons.

[0126] Interestingly, when AQ protein was most readily detectable in the dorsal hippocampus, no neuroprotection was observed 1 hour post-infusion (see [link to article]). Figure 3 Although it is unknown how or whether AQ enters cells, the current study used DMSO accompanied by AQ for infusion, which is used to transport drugs across membranes. Therefore, it is possible that AQ has the opportunity to enter cells. Furthermore, the centrifugation process used for the Western blot samples was designed to separate intracellular components of cells (by low-speed centrifugation), and the presence of AQ in these samples strongly suggests its presence in cells. Although significant neuroprotection was evident 1 and 2 days post-infusion, much less AQ was observed in the dorsal hippocampus. Figure 3 C), this indicates that neuroprotection is not only mediated by AQ binding to Ca. 2+This is due to the immediate effect of AQ. Furthermore, data suggest that an event cascade induced by AQ infusion results in neuroprotection. Given that neuroprotective effects were observed 1 and 2 days post-infusion when the protein was almost absent or undetectable (but not at 1 hour when AQ expression is at its peak), this cascade may be attributed to other AQ-triggered mechanisms, including post-infusion pre-regulation-like effects. This type of effect may take time to develop, which may explain why neuroprotection was not observed immediately (e.g., 1 hour post-infusion). Pre-regulation may also explain why robust neuroprotection was observed 1 or 2 days post-infusion, regardless of the detection of lower protein levels at these time points. Although the exact mechanism is not yet known, studies have involved cytokines and chemokines in pre-regulation.

[0127] To investigate whether the observed neuroprotective effect following AQ infusion was attributable to a premodulation-like effect, we examined changes in IL-10 mRNA, an anti-inflammatory cytokine known to be involved in premodulation. A statistically significant increase in IL-10 mRNA was observed 1 hour after infusion. Although not statistically significant, a biologically significant (>2-fold) increase in IL-10 mRNA was observed for up to 2 days after AQ infusion (see [link to relevant documentation]). Figure 4 A) Anti-inflammatory cytokines act by recruiting protective cell populations that secrete cytokines, thereby preventing or downregulating the induction of destructive pro-inflammatory immune responses and actively protecting against future damage. The increased IL-10 expression 1 hour after AQ infusion plays a protective role against future OGD damage, thus preparing the brain for ischemic injury 1-2 days later. This effect is short-lived, showing a near-absent to non-existent neuroprotective effect up to 3-5 days after AQ infusion.

[0128] Given the increase in IL-10 mRNA 1 hour after AQ infusion, indicating a pre-regulation-like effect, a multi-gene PCR array was used to evaluate the effects of AQ on the expression of a wide range of cytokines and chemokines (see Table 1). These studies revealed that, compared to the carrier-treated hemispheres, AQ infusion differentially regulated the expression of multiple cytokine and cytokine receptor genes in a time-dependent manner. Of the 82 genes detected in the array, 16 were significantly upregulated after AQ infusion. Among these 16 genes, a time-dependent effect was observed, with 8 rapidly upregulated immediately after AQ infusion, while the remaining 8 were upregulated only after a delay of 1 or 2 days.

[0129] Table 1. Fold change of gene corpuscle after 4% AQ infusion, grouped by response time.

[0130] The numbers represent a multiple change compared to the carrier-infusion hemisphere. p< 0.05;† p < .01).

[0131] Of the cytokines upregulated after AQ infusion, preregulation was detected in only four: (1) interleukin-1β (IL-1β), (2) IL-10, (3) tumor necrosis factor-α (TNF-α), and (4) complement component 3 (C3). All four of these cytokines showed an increase after preregulation. IL-1β, a pro-inflammatory cytokine, showed an increase within 6 hours after preregulation, after which it returned to baseline within 3–4 days. This is consistent with existing studies showing a rapid increase in IL-1β mRNA, followed by a return to baseline levels by 2 days after AQ (Table 1). Although IL-1β is a pro-inflammatory cytokine, a moderate increase can also have neuroprotective effects. Similarly, IL-10 also showed a rapid increase after preregulation, similarly returning to baseline quickly. We now use quantitative RT-PCR (… Figure 4 PCR arrays (Table 1) showed that IL-10 was significantly upregulated 1 hour after AQ infusion. IL-10 was shown to reduce TNF-α release and decrease brain injury following focal ischemia in rats. TNF-α was rapidly upregulated after pre-regulation, lasting up to 2 days, and was no longer detectable after 3-4 days. The current experiment showed an increase in TNF-α gene expression 1 hour after AQ infusion, but not 1 or 2 days after AQ infusion. C3 was significantly upregulated 24 hours after lipopolysaccharide (LPS) pre-regulation. We observed a significant increase in C3 gene expression 2 days after AQ infusion. Activation of the complement host defense system (including C3) showed both disruptive and protective effects. In conclusion, these data suggest that the increases in IL-1β, IL-10, TNF-α, and C3 observed in the current experiment may be one of the reasons for the neuroprotective effect of AQ infusion.

[0132] Although only four upregulated cytokines were detected during pre-regulation, almost all 16 were detected after cerebral ischemia. Only chemokine ligand-9 (CXCL9), chemokine ligand-11 (CXCL11), and chemokine receptor-1 (XCR1) were not detected, although their relationship with cerebral ischemia has been previously investigated to our knowledge. Of the other cytokines, all showed an increase after ischemia, except for interleukin-2 receptor β (IL-2rβ). Under normal conditions, IL-2rβ is present in the cell membrane of CA1 pyramidal neurons in the hippocampus. After ischemia, IL-2rβ is not only reduced in CA1, but it also translocates from the cell membrane to the cytoplasm and nucleus. The role of some cytokines after ischemia may depend on their expression profile, which can affect when or whether they exert neuroprotective effects. For example, CD40 ligands play a role in inflammation and tissue damage, and they are upregulated after lesion ischemia. However, CD40 ligands also protect neurons from neuronal stress, and CD40 ligand deficiency leads to neuronal dysfunction, indicating that CD40 ligands are important for general neuronal function. Existing data indicate that CD40 ligand levels significantly increase 1 and 2 days after AQ infusion. This sustained increase in CD40 ligand levels may affect the duration of the neuroprotective effect we observed. Although beyond the scope of current research, it would be important (and the data suggest it warrants further investigation) for further evaluation of the neuroprotective effect of AQ over longer durations using an in vivo model of local ischemia.

[0133] In summary, the current experiments support the hypothesis that AQ protects neurons from local ischemia when administered directly to the brain prior to ischemic injury. These effects are dose- and time-dependent, with single intrahippocampal AQ infusion protecting neurons from OGD for up to 2 days. Furthermore, AQ infusion activates cytokine and chemokine gene expression in a similar manner to those seen in ischemic premodulation. Therefore, pretreatment with AQ may be an effective way to protect neurons from ischemic stroke by acting as a chemical premodulator.

[0134] Example 2. Effect of intrahippocampal infusion of apo-co-mediated jellyfish luminescent protein on cytokine protein expression.

[0135] In previous experiments, our lab has shown that a single intrahippocampal infusion of AQ 24 and 48 hours prior to ischemic injury in vitro significantly reduced cell death (Detert et al., 2013). Simultaneous changes in cytokine mRNAs, including interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α; Detert et al., 2013), have also been observed after AQ infusion. These data suggest that the neuroprotective mechanism of AQ may involve the regulation of certain anti-inflammatory and pro-inflammatory molecules, potentially involving pre-regulatory-like effects. This current study was designed to further investigate whether cytokine protein expression changes in a time-dependent manner following intrahippocampal AQ infusion. By focusing on possible changes at the protein level, we hope to gain a better understanding of the extent to which AQ regulates multiple cytokines and ultimately, the mechanisms by which AQ protects neurons from oxygen-glucose deprivation.

[0136] Our lab has previously shown that infusion of apo-co-electroluminescent protein (AQ) into the CA1 region of the hippocampus can exert neuroprotective effects in a time- and dose-dependent manner (Detert et al., 2013).

[0137] Significant neuroprotection was observed 1 and 2 days after AQ infusion, but not 1 hour. This occurred in parallel with altered cytokine mRNA expression, suggesting that this ischemic neuroprotection may involve a neuroimmune regulatory response (Detert et al., 2013).

[0138] Mild stress stimulation can trigger ischemic preconditioning through the regulation of inflammatory cytokine expression (Gidday, 2006).

[0139] IL-10 protects neurons from ischemic damage, both in vitro and in vivo (Grilli et al., 2000).

[0140] IL-10 inhibits the upregulation of TNF-α (a pro-inflammatory cytokine), which is involved in the pathological mechanism of hemorrhagic stroke (Ewen et al., 2013).

[0141] The current embodiment shows that intrahippocampal infusion of AQ initiates a neuroimmune regulatory response, which triggers IL-10 and TNF-α proteins. Changes in white expression. Data supporting this conclusion are shown in... Figure 5 , 6 AB, 7A-D, and 8A-C.

[0142] Example 3. Neurotherapeutic effects of calcium-binding protein apopolymerized jellyfish luminescent protein.

[0143] Calcium-binding protein (CaBP) reduces ischemic cell death.

[0144] Data from our laboratory show that CaBP apopolymer luminescent protein (AQ) has neuroprotective effects when infused into the dorsal hippocampus before local ischemia in vitro, and leads to time-specific increases in IL-10 and TNF-α mRNA, suggesting a role in preregulation (Detert et al., 2013).

[0145] The current embodiment shows , A single AQ hippocampal infusion differentially modulates the expression of IL-10 and TNF-α proteins.

[0146] Calcium toxicity is evident in normal aging. According to the calcium hypothesis of aging, disruption of calcium homeostasis leads to cognitive decline in normal aging (Khachaturian, 1987).

[0147] Age-related reductions in CaBP are observed (DeJong et al., 1996; Bu et al., 2003; Moyer et al., 2011), and our laboratory results show reduced CaBP expression in the hippocampus (a key structure for tracer fear learning (McEchron et al., 1998)). Tracer fear conditioning is impaired in normal aging (Villarreal et al., 2004; McEchron et al., 2004; Moyer et al., 2006). Reduction of excess calcium leads to improved cognitive function in aging animals (Deyo et al., 1989; Veng et al., 2003).

[0148] This embodiment also shows A single AQ hippocampal infusion will reduce age-related deficits in the acquisition of trace fear conditioning.

[0149] Oral administration is used as a delivery method. The compound can be delivered orally to rats using hazelnut butter Nutella® or peanut butter as a carrier (Isaksson et al., 2011; Cundell et al., 2003).

[0150] Recent data from our laboratory show that AQ is neuroprotective when administered orally at a single dose prior to local ischemia in vitro (Adams et al., SfN 2013). This example also shows that the neuroprotective effect of oral AQ is dose- and time-dependent.

[0151] Figure 9 AC, 10A-C, and 11A-C support the following conclusion: Direct infusion of AQ resulted in altered IL-10 and TNF-α protein expression relative to the load. Both IL-10 and TNF-α showed differential expression profiles after AQ infusion, suggesting that the neuroprotective effect of AQ may be mediated through an immune-regulating response.

[0152] AQ infusion did not resolve trace fear learning impairment in aged animals, nor did it interfere with learning of the task in adult animals. Aged rats showed reduced rigidity 24 hours after conditioning compared to adult animals, but as expected, AQ administration did not cause increased rigidity in aged rats.

[0153] Oral administration of AQ resulted in time- and dose-dependent neuroprotection. A 7-day oral administration of 48 mg / kg AQ significantly reduced cell death following local ischemia.

[0154] Example 4. Oral administration of AQ showed neuroprotective effects in a short-time slice model.

[0155] Our lab recently demonstrated that apomeric luminescent protein (AQ) has neuroprotective effects in a short-term brain slice model of ischemic stroke known as oxygen-glucose deprivation (OGD). Rats receiving 4% AQ infusion showed reduced cell death following OGD (Detert et al., 2013).

[0156] This example demonstrates that the reduction in cell death is attributed to an immune regulatory mechanism involving time-dependent changes in cytokine mRNA.

[0157] The compound was orally administered to rats via hazelnut spread Nutella. ® (Isaksson et al., 2011) or peanut butter (Cundell et al., 2003) were used as carriers. Recently, AQ has been shown to be non-toxic when administered to rats via tube feeding (Moran et al., 2013). Oral administration of AQ delivered in a carrier (e.g., peanut butter) is less invasive than other methods (e.g., viral delivery, direct infusion, or tube feeding), and the oral delivery system is applicable to human studies.

[0158] This embodiment demonstrates that oral administration of AQ protects against neuronal immune oxygen glucose deprivation-induced cell death.

[0159] method

[0160] animal. Ninety-two male adult F344 rats were used. The rats were housed on a 14 / 10 hour day / night cycle, with freely available food and water. The weight of each animal was recorded twice weekly to calculate significant weight gain and / or loss.

[0161] drug.Apopolymerized jellyfish luminescent protein (AQ; Quincy Bioscience) was prepared in doubly deionized water at a concentration of 7.4%. In a dose-dependent experiment (n = 18), experimental groups received AQ mixed into their daily PB at doses of 0 (n = 4), 3.6 (n = 5), 48 (n = 4), 240 (n = 3), or 480 mg / kg. For the remainder of the study, rats ( n = 73) Rats received an AQ of 48 mg / kg mixed into their daily PB. Animals were assigned to one of five groups: no AQ (n = 12), 1-hour AQ (n = 17), 1-day AQ (n = 15), 2-day AQ (n = 15), and 7-day AQ (n = 14). Each day, rats received ¼ teaspoon of PB placed in a Piper dish in their cage at the designated time. The dish was removed only after all PB had been consumed. Animals were weighed twice a week to maintain an appropriate AQ dose.

[0162] The AQ used for the infusion study was prepared as previously described (Detert et al., 2013). IL-10 neutralizing antibody (nAb) and its IgG control were prepared in sterile PBS. 0.5 μg was infused at a rate of 1 μL / min using a 1 μL Hamilton syringe.

[0163] Oxygen-glucose deprivation. On the last day of administration, after PB consumption, rats were allowed to digest for 1 hour, then deeply anesthetized with isoflurane, and the dorsal hippocampus (dhpc; anterior fontanelle) was prepared using a standard procedure (Moyer and Brown, 2007). - 3.14 - - 4.16 (Paxinos and Watson, 1998) Coronal sections (400 μm). After recovery in aCSF for 1 hour, one hemisphere of each brain (balanced) underwent in vitro local ischemia for 5 minutes by transferring the sections to an oxygen-glucose deprivation chamber (glucose replaced with fructose and bubbling with 95% N2–5% CO2 instead of 95% O2–5% CO2), while the other hemisphere remained in recovery. All sections were then placed in oxidized aCSF containing 0.2% trypan blue for a 30-minute reperfusion phase. Trypan blue stains dead cells but not live cells (DeRenzis and Schechtman, 1973). The sections were rinsed twice in oxidized room temperature aCSF and then fixed overnight in 10% neutral buffered formalin in a refrigerator. Then, the sections were frozen in 30% sucrose for protection, sectioned (40 μm) on a cryostat, and mounted on embedding slides for cell counting.

[0164] Cell count.Sections were visually examined at 10X magnification under an Olympus microscope (equipped with a digital camera) and photographed (CellSens). Trypan blue-stained neurons in CA1 (approximately 800 μm) were counted by researchers unaware of the experimental conditions. Statistical analysis was performed using SPSS (v 21.0.0; IBM; Armonk, NY). ANOVA was used to evaluate drug effects, and Fisher's LSD causality assessment method was used to evaluate group interactions. (Astro) )instruct p < .05.

[0165] Western Imprint. Animals were deeply anesthetized with isoflurane, their brains were rapidly removed, frozen, and stored at -80°C. C. After the dissection time, from dhpc (anterior fontanelle) - 3.14 - - Samples were cut at 4.16 mm. After homogenization, the samples were centrifuged at 4000 RPM for 20 minutes, and the supernatant was removed. Protein analysis was performed using the Bradford Protein Assay Kit (Bio-Rad). Protein samples were standardized and loaded for SDS-PAGE (12%). Protein (30 µg) was transferred to a PVDF membrane using the Turbo Transfer System (Bio-Rad). The membranes were incubated with the following conditions: blocking buffer (2 h), primary antibody (4°C overnight; 1:1000 mouse anti-jellyfish luminescent protein [Chemicon] or 1:1000 rabbit anti-β-actin [Cell Signaling Technology]), and secondary antibody (90 min; 1:20,000 goat anti-mouse [Santa Cruz Biotechnology] or 1:40,000 goat anti-rabbit [Millipore]). The membranes were then rinsed, placed in chemiluminescent solution (Thermo Scientific), and imaged using Syngene GBox. Images were acquired using Geneys software (v 1.2.4.0; Synoptics camera 4.2MP), and fluorescence of each band was evaluated using GeneTools software (v 4.02; Cambridge, UK). Values ​​are expressed as a percentage compared to control animals. Statistical analysis was performed using SPSS (v. 21).

[0166] Overview

[0167] Figure 12 , 13 AC, 14A-D, 15A-B, and 16 support the conclusion that the neuroprotective effect of apomyelin is dose-dependent. Oral administration of AQ at a dose of 48 mg / kg protected against OGD-induced cell death.

[0168] Apopolymerized jellyfish luminescent protein exhibits durable neuroprotective effects. Brain slices from rats that received oral administration of AQ for 1 hour, 1 day, 2 days, or 7 days showed neuroprotective effects.

[0169] Apopolymerized jellyfish luminescent protein alters the expression of cytokine proteins.

[0170] TNF-α protein expression increased after oral administration of AQ for 2 days, while IL-10 protein expression remained unchanged.

[0171] During infusion, IL-10 protein expression increased at 1 hour compared to the hemisphere infused with the carrier. Furthermore, TNF-α increased at 1 day, and then protein levels returned to baseline. 4. The neuroprotective effect of apomyelin was reversed by IL-10 neutralizing antibodies.

[0172] When AQ is infused one day before OGD in vitro, its neuroprotective effect is eliminated if it is paired with IL-10 nAb.

[0173] Existing data suggest that the neuroprotective effect of AQ involves IL-10; it works through the neutralization or downstream cascade of IL-10.

[0174] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art will understand that various modifications or changes can be made accordingly, and these are included within the scope of the spirit and benefits of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

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Claims

1. Use of apo-jellyfish luminescent protein in the preparation of a composition for premodulating neurons to reduce neuronal cell death after ischemia in a subject susceptible to ischemic cell death, wherein the apo-jellyfish luminescent protein is provided at a therapeutically effective dose to premodulate neurons in a subject to reduce neuronal cell death, and wherein the apo-jellyfish luminescent protein is formulated for oral administration to the subject.

2. Use of apo-jellyfish luminescent protein in the preparation of a composition for premodulating neurons to reduce ischemic cell death in a subject susceptible to ischemic cell death, wherein the apo-jellyfish luminescent protein is provided at a therapeutically effective dose to premodulate neurons in the subject to reduce ischemic cell death, and wherein the apo-jellyfish luminescent protein is formulated for oral administration to the subject.

3. Use of apo-jellyfish luminescent protein in the preparation of a composition for premodulating neurons to reduce neuronal inflammation in a subject, wherein the apo-jellyfish luminescent protein is provided at a therapeutically effective dose to reduce neuronal inflammation in the subject, and wherein the apo-jellyfish luminescent protein is formulated for oral administration to the subject.

4. Use of apo-coated jellyfish luminescent protein in the preparation of a composition for reducing tumor necrosis factor α (TNFα) protein levels in a subject, wherein the apo-coated jellyfish luminescent protein is provided at a therapeutically effective dose to reduce TNFα protein levels in the subject, and wherein the apo-coated jellyfish luminescent protein is formulated for oral administration to the subject.

5. The use according to any one of claims 1-4, wherein the composition is in unit dosage form, the unit dosage form being selected from tablets or capsules.

6. The use as described in any one of claims 1-4, wherein the apopolymerized jellyfish luminescent protein is in the form of a nutrient composition.

7. An orally administered composition for premodulating neurons to reduce post-ischemic neuronal cell death in subjects susceptible to ischemic cell death, for premodulating neurons to reduce ischemic cell death in subjects susceptible to ischemic cell death, or for premodulating neurons to reduce neuronal inflammation in subjects, wherein the composition comprises a therapeutically effective amount of apo-co-phosphorylated jellyfish luminescent protein.

8. An orally administered composition for reducing tumor necrosis factor α (TNFα) protein levels in a subject, wherein the composition comprises a therapeutically effective amount of apo-co-luminescent jellyfish protein.

9. The composition of claim 7 or 8, wherein the composition is in unit dosage form selected from tablets or capsules.

10. The composition of claim 7 or 8, wherein the decozyme luminescent protein is in the form of a nutrient composition.

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