CN115151271A - Application of glatiramer acetate in preparation of Abeta 42 toxicity inhibitor and scavenger - Google Patents

Abstract

Use of glatiramer acetate having a high affinity for a β 42 for scavenging a β 42 and inhibiting toxicity thereof.

Description

Application of glatiramer acetate in preparation of Abeta 42 toxicity inhibitor and scavenger Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to glatiramer acetate Lei Zaizhi for preparing a beta-amyloid A beta protein _1-42 Use of (Abeta 42) toxicity inhibitor and scavenger for treating Alzheimer's diseaseAlzheimer's disease.

Background

Alzheimer's Disease (AD) imposes a huge social and economic burden on society, affecting more than 2600 million people worldwide. There is no cure available and existing treatments can only ameliorate mild to moderate symptoms for a limited period of time. To date, even advanced treatments have not been proposed.

Innate immunity serves as a basic biological function, maintaining homeostasis in the body. Natural phagocytosis is one of the important components of innate immunity and does not require antibodies or complements to recognize and phagocytose apoptotic cells, cell debris, protein aggregates and invading bacteria. Several genome-wide association studies (GWASs) and other genetic studies in the field of discrete AD have identified a group of AD risk genomes that belong to the core innate immune pathway, including CD33, CR1, MS4A6A, MS A4E, ABCA and TREM2. Variations of these genes, especially TREM2 and CD33, are associated with impaired restriction of phagocytic function of monocytes/macrophages in the brain of AD patients and altered accumulation of a β. Notably, complement receptor 1 (CR 1, also known as CD 35) is expressed predominantly in peripheral blood leukocytes and erythrocytes, but not in the brain under normal physiological conditions. Thus, in addition to excessive neuroinflammation, systemic defects involving phagocytic function of the Central Nervous System (CNS) and peripheral tissues may be the primary cause of a β deposition and failure to clear.

The deposition and aggregation of a β forms amyloid plaques, and the established special features of AD are widely thought to be associated with disease etiology. Thus, current drug discovery for AD has focused primarily on the use of antibodies to clear existing amyloid deposits. However, since 1998, more than 100 clinical trials for a β failed or showed only "first line hope". Until recently, one of these antibody-based approaches, "aducanmab", has shown some potential for treating AD, probably due to its high Blood Brain Barrier (BBB) penetration capacity, but this capacity also carries significant side effects, such as brain hematomas and the like, manifested by a high incidence of amyloid-related imaging abnormalities (ARIA, 13-47%). Therefore, effective treatment strategies for AD are urgently needed.

Several studies from three different groups have reported the safety and beneficial effects of glatiramer acetate in AD mouse models at doses ranging from 5 to 10 mg/kg/week. However, in these studies glatiramer acetate was considered as an immunological vaccine and the mechanism of action was not clear.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of glatiramer acetate in preparation of an A beta 42 inhibitor.

Natural phagocytosis is the most important component of the innate immune system of the body and is critical to the development and homeostasis of the body. Natural phagocytosis does not require opsonization to recognize and eliminate apoptotic cells, cell debris, and invading microorganisms. Professional scavenger cells (phagocytes) that rapidly clear senescent apoptotic or already dead neuronal cells are vital in our body to avoid inflammation and allow the neurogenic central nervous system: rapid clearance of misfolded protein complexes is critical to avoid neurodegeneration; the rapid elimination of invading microorganisms is the first line of defense of our bodies, is particularly important for the central nervous system lacking acquired immunity, and is an important measure for avoiding subsequent neuroinflammation. However, this important biological function has long been overlooked and the concept of "natural phagocytosis" has never even been proposed.

Doctor Gu Baijun (Baijun Gu or Ben j. Gu) and its team, who originally presented the concept of "native Phagocytosis" (also known as "Innate Phagocytosis"), have been working on this study for the past decade. The inventor takes the purinergic receptor P2X7 as a model, firstly invents a method for quantifying the natural phagocytic capacity in vitro: real-time multicolor flow cytometry (FIG. 1), and on this basis it was demonstrated through years of effort, a series of theoretical bases of how this receptor functions as a scavenger receptor in conjunction with the relevant cytoskeletal proteins, and how it recognizes and eliminates apoptotic fine particles by cysteine disulfide bond formation before necrotic death and the resulting inflammation occursCan reduce inflammatory reaction. The inventors have also found three important features of natural phagocytosis: (1) Closely related to the temperature, the natural phagocytic capacity can be obviously improved when the environmental temperature is increased from 37 ℃ to 39 ℃; (2) The effect is strongest in a serum-free environment, and the natural phagocytic function can be greatly reduced by 1-5% of serum; (3) The natural phagocytic function gradually decreases with age. Therefore, the natural phagocytosis plays an important role in serum-free environments such as the central nervous system, eyes, bone joints and the like, and can be used as a target for treating severe infections such as septicemia and viral pneumonia, and senile degenerative diseases such as senile macular degeneration (AMD) and Alzheimer's Disease (AD). Large-scale genetic sequencing analysis found several mutations and groups of mutations that could alter P2X 7-mediated native phagocytic function: the Arg307Gln mutation is more effective in maintaining the natural phagocytic function of P2X7, and was found to reduce the risk of Multiple Sclerosis (MS) by half; the common mutation of the P2X7 receptor His150Arg and the P2X4 receptor Tyr315Cys thoroughly destroys the natural phagocytic function mediated by the P2X7 receptor, so that the incidence rate of the age-related macular degeneration is improved by about 4 times. In the long-term studies of alzheimer patients, with the AIBL platform (the australian organization of research on medical imaging, biomarkers and lifestyle of alzheimer patients, also the world's largest long-term follow-up cohort for elderly), the inventors found that Mild Cognitive Impairment (MCI) and alzheimer patients (AD) are disordered in their natural phagocytic function of peripheral blood mononuclear cells and are associated with the size of ap plaques in their brains, whereas P2X7 receptor-mediated natural phagocytic function is also closely associated with ap plaque area. The inventors have further found that glatiramer acetate (GA, a drug for clinical treatment of multiple sclerosis, registered drug brand name

) Can improve the natural phagocytic function of the mononuclear cells of the patient, and the degree of the improvement is closely related to the clinical diagnosis and the A beta plaque area.

The present invention focuses on a more effective approach to prevent accumulation of a β or other proteins and to inhibit or even partially reverse the progression of senile dementia by promoting natural phagocytosis to clear these fragments.

Our in vitro and in vivo experimental data indicate that glatiramer acetate is able to tightly bind to a β and antagonize the toxic effects that a β 42 exhibits in the long-range potentiation (LTP). The combined A beta is embedded into a mononuclear cell membrane before A beta 42, membrane fluidity is enhanced, and the memory and learning capacity of neurons are promoted. In vivo experiments, the APP/PS1 transgenic aged mice (an AD animal model with the life expectancy of 26-27 months) with 24 months old age mice injected directly with glatiramer acetate Lei Cheng were implanted using osmotic minipumps, and their behavior was significantly improved within three weeks, with an increase in LTP after 5 weeks, as compared to the blank group. Soluble a β levels found in the brain were reduced to half and immunohistochemical staining of a β showed predictable lysis occurring in the center of the deposited a β plaques.

These results suggest a new therapeutic approach to AD by direct infusion of glatiramer acetate into the Central Nervous System (CNS). Nasal sprays, spinal punctures, or implantation devices by intrathecal injection, such as o Ma Yenang (Ommaya Reservoir, a ventricular catheter system implanted under the scalp, infuses drugs into the cerebrospinal fluid) may be chosen. Because glatiramer acetate exhibits high affinity for a β and a strong effect in increasing brain neuron LTP levels in aged AD mice, this therapeutic approach can be used to treat AD, not just another disease-modifying therapy. Glatiramer acetate can be used alone or in combination with other compounds such as P2X7 antagonists, and has synergistic therapeutic effect.

Drawings

FIG. 1: phagocytosis of YO microbeads by monocyte subpopulations. Fresh human PBMCs were labeled with APC-CD14 and FITC-CD16 monoclonal antibodies prior to addition of 1 μm yellow orange fluorescent YO microbeads, and the fluorescence intensity of the YO microbeads in selected cells was then analyzed by real-time flow cytometry. a typical example of a CD14 versus CD16 density plot. Monocytes were first selected using forward and side scatter. b. Typical real-time flow cytometry assays for YO microsphere uptake curves, CD14dimCD16+ (green), CD14+ CD16+ (red), CD14+ CD16- (brown) monocytes. The CD 14-lymphocytes (black) have the lowest phagocytic capacity.

FIG. 2: study of glatiramer acetate

Influence on phagocytic function of yellow orange fluorescent (YO) microbead cells of human monocyte subpopulation. Fresh human peripheral blood cells were collected from normal healthy Humans (HC) (n = 59). Cells were labeled with APC-CD14 and FITC-CD16 monoclonal antibodies and incubated with Copaxone 100. Mu.g/ml for 10 min before addition of 1 μm YO microbeads. The phagocytic capacity was quantified as the area unit under the YO bead uptake curve in the 1 st to 6 th minutes after addition of the YO beads. The monocyte subpopulations are differentiated by the degree of cell surface CD14 and CD16 expression. Subjects were grouped according to their cerebral amyloid burden (Gu et al, acta neuropathology, 2016)

FIG. 3: glatiramer acetate (

CPX) enhances the phagocytic capacity of monocytes to phagocytose fluorescent latex beads. (a) Fresh human monocytes (2X 106/mL) were labeled with APC-bound anti-CD 14 and FITC-bound anti-CD 16 antibodies, respectively, and analyzed for fluorescence intensity of typical CD14+ CD 16-monocytes by real-time flow cytometry. Cells were pretreated for 10 min at 37 ℃ with or without 100. Mu.g/mL CPX and then placed on ice or held for an additional 5min at 37 ℃. As shown, the yellow orange latex bead absorption test was performed at 37 ℃ or 5 ℃ for 6 minutes. The results are representative data from three individuals. (b) Concentration-dependent promotion of phagocytosis of yellow-green latex beads by CPX. Human monocytes were pretreated with glatiramer acetate at various concentrations for 10 minutes at 37 ℃. The increase in phagocytosis stimulated by glatiramer acetate was normalized to the percentage of basal phagocytosis levels (n = 3) (Gu et al, acta neuropathology, 2016, suppl fig. s 4).

FIG. 4 is a schematic view of: glatiramer Acetate (GA) promotes the innate phagocytosis of yellow-green YG fluorescent microbeads in vivo. YG microspheres were injected intravenously into the ear rim of a New Zealand white rabbit at 1.5ml, and 2ml/kg GA or 4% mannitol as a control. Blood was collected before injection and 5, 10, 15, 20, 30, 60, and 120 minutes after injection, respectively. After the blood was treated with the erythrocyte lysis buffer, it was labeled with Alexa647-CD14 monoclonal antibody, and the YG bead fluorescence intensity in the selected cell population was measured by flow cytometry. The upper panel shows the Mean Fluorescence Intensity (MFI) of monocytes and neutrophils after phagocytosis of YG beads 30 minutes after injection. The lower two panels show the time course of the uptake of YG beads into monocytes (left) and neutrophils (right).

FIG. 5: glatiramer acetate has high affinity for direct binding to a β 1-42. HiLyte Fluor 488-labeled A β 1-42 (80 nM, in PBS), 1:1 were mixed in volume to serially dilute the pro-phagocytosis peptide. Measurements were performed in standard process capillaries using a 95% LED and a 40% IR-laser power NT.115 system.

FIG. 6: interaction of a β 42 with Glatiramer Acetate (GA). A. Circular dichroism spectroscopy (CD) was used to determine the secondary structure of A.beta. (0.2 mg/ml, 44. Mu.M). B. Secondary structure of glatiramer acetate GA (44 μ M). C. When A.beta.and GA are combined, the structure is determined. In the implementation of Sreerama et al, the secondary structure was analyzed using the computer program CDSSTR and the reference data set 3.

FIG. 7: glatiramer acetate binds tightly to a β 42. A β 42 (10 μ g 2.2 nmol) was mixed with varying amounts of glatiramer acetate 0,0.22,1.1,2.2,6.6, 15.4nmol (lanes 1-6) or seroset 2.2nmol (lane 7). The mixture (30. Mu.l each) was diluted with 50. Mu.M DTT and heated at 90 ℃ for 5 minutes, followed by SDS-PAGE (4-12% NuPage gel, MES buffer, 100V 50 minutes) electrophoresis. anti-A.beta.monoclonal antibody (W0-2 antibody) transferred and probed proteins. A. Western blot electrophoretic pictures showed abeta staining. The semi-quantitative results for 4.5kD A β 42 monomer per lane show the bar at the bottom. Ponceau S staining, proteins were transferred to a nitrocellulose membrane fraction. A large amount of glatiramer acetate and Bovine Serum Albumin (BSA) were stained red. Protein size was estimated using the seeplus prestained protein standard.

FIG. 8: glatiramer Acetate (GA) antagonizes the long-range potentiating effect (LTP) of the toxic effects of A β 1-42. LTP changes were recorded from mouse brain sections using a multi-electrode array (MEA) electrophysiological method. Fresh mouse hippocampal slices were mounted on a 3D-MEA chip and treated with 60 spikes and a 30 μm high electrode spacing of 200 μm. Sections were perfused continuously with artificial cerebrospinal fluid (aCSF, 3mL/min,32 ℃). Data were collected using a multichannel system (MCS GmbH, luo Yite Lin Gen, germany). The Schaffer-collator injection biphasic current waveforms (100 μ s) were stimulated by selecting one electrode at 0.033 Hz. The peak-to-peak values of the CA1 proximal radiolayer excitatory postsynaptic potentials (fEPSPs) were analyzed by LTP-analyzer.

FIG. 9: human monocyte a β dimer was stained with W0-2 monoclonal antibody. A. A β staining of different subsets of human monocytes of an AD patient. Staining cells with mouse anti-a β monoclonal antibody (clone W0-2 antibody) and FITC-conjugated secondary antibody, followed by labeling of the monocyte subpopulation with fluorescent CD16 and CD14 monoclonal antibodies; w0-2 monoclonal antibody stained the surface A β of CD14+ CD16+ monocytes (the highest phagocytic subset). The figure shows a population with very high W0-2 binding levels; C. comparison of CCR2 staining and W0-2 monoclonal antibody Abeta staining for channel monocytes in one AD patient. Cells with high levels of W0-2 binding also exhibit high binding to CCR2, CCR2 being a marker of high migration potential; D. human Peripheral Blood Mononuclear Cells (PBMCs) were classified by CCR2/CD14 expression and side scatter using flow cytometry. Cells were lysed and the microchip was coated with W0-2 monoclonal antibody to capture A.beta.and then analyzed for binding proteins using SELDI-TOF.

FIG. 10: glatiramer Acetate (GA) and the P2X7 antagonist AZ10606120 inhibit insertion of a β 1-42 into the monocyte membrane. Abeta 1-42 (10. Mu.g/ml) was added to whole blood with or without 100. Mu.g/ml GA or 1. Mu.M AZ10606120 (AZ) for 15 minutes. Cells were counterstained with CD14 and CD16 after staining with anti-A β monoclonal antibody (clone W0-2 antibody). A. The three types of leukocytes were stained with W0-2; B. the cell surfaces of the three monocyte subpopulations bind to W0-2; w0-2 stained CD14+ monocytes.

FIG. 11: the increase in membrane fluidity induced by A.beta.1-42 and ATP can be inhibited by Glatiramer Acetate (GA) and the P2X7 antagonist AZ10606120 (AZ). a. Cells were incubated with different concentrations of freshly solubilized A β 1-40 and A β 1-42 polypeptides for 15 minutes prior to addition of TMA-DPH. b. Cells were cultured with 50. Mu.g/mL A.beta.1-42, 100. Mu.g/mL GA,1.0mM ATP or 10. Mu.M of AZ10606120 (AZ), respectively, and then viable monocytes (CD 14+ and 7-AAD-) were selected for flow cytometry analysis.

FIG. 12: the therapeutic effect of Glatiramer Acetate (GA) directly perfused with a micropump brain on AD animal models was evaluated. The ALZET mini osmotic pump (model 1004, 100 μ L reservoir volume, for four weeks) was filled with GA (20 mg/ml) or PBS plus 4% mannitol (control), implanted in the subcutaneous space in APP/PS1 transgenic mice (23-24 months old), and brain-attached ALZET infusion device 3 penetrated 2.5mm below the surface of the skull, lateral ventricles.

FIG. 13: open field behavior tests were performed 3 weeks after implantation. A. There was no significant difference in the number of times two groups of mice entered the open field center; ga treated mice had a longer percentage of residence time in the open field center than control mice; C. there was no significant difference in the distance traveled by the two groups of mice in the open field.

FIG. 14 is a schematic view of: the Elevated Plus Maze (EPM) test was used 3 weeks after implantation. Ga-treated mice showed preference for closed arms (Post hoc plus Sidak multiple comparison test); control mice showed a deeper preference than GA treated mice (Post hoc plus Sidak multiple comparison test). (P =0.0001 two-way ANOVA analysis of variance); B. there was no significant difference in the distance traveled in the elevated + maze by the two groups of mice.

FIG. 15: the Y maze behavior test was performed 3 weeks after implantation. A. Mice treated with GA showed a clear preference for the novel arm, in contrast to control mice (P =0.0067, two-way ANOVA analysis of variance); B. there was no significant difference in the distance traveled in the Y maze by the two groups of mice.

FIG. 16: social interaction tests were performed 3-4 weeks after implantation. Ga-treated mice showed a preference for stimulation chambers (P = 0.0114) (two-way ANOVA analysis of variance); B. mice treated with GA showed greater preference for regions of interest in the stimulation chamber compared to control mice (P = 0.0016); ga treated mice and control mice both showed a preference for a new stimulation chamber (P = 0.0032); D. mice treated with GA showed a preference for new stimulation chamber regions of interest, in contrast to control mice (P = 0.0011).

FIG. 17: long-range potentiation potential (LTP) was measured 5 weeks post-operatively in freshly prepared mouse hippocampal slices.

FIG. 18 is a schematic view of: after 5 weeks of implantation, mice were tested for brain soluble and insoluble a β by ELISA.

FIG. 19: glatiramer Acetate (GA) treated mice had a reduced number/size of a β plaques. Mouse brain sections were stained with anti-a β monoclonal antibody (clone 1E 8). Images were analyzed using ImageJ to calculate the number and area of Α β plaques in the hippocampus (a & B) and cortex (C & D) regions. Typical channels are shown in the a & C regions, respectively. Magnification is 40 times. (n =4 per group).

FIG. 20: mouse brain a β immunohistochemical staining and anti-a β monoclonal antibody (clone 1E 8). Glatiramer Acetate (GA) treated mice showed erosive ring-shaped Α β plaques whereas control mice did not. Magnification is 200 times.

FIG. 21: australian sheep subjected to ventricular puncture surgery.

FIG. 22: typical sheep experimental records. Sheep received a ventriculoperitopuncture procedure on day one, followed by 8 Glatiramer Acetate (GA) administrations directly via the ventricles (indicated by arrows) in the following 3 months, 0.5mL (10 mg) each time drinking water and urination (up), forage consumption and defecation (middle), anal body temperature (down) and respiration, heart beat, etc. were recorded daily.

FIG. 23: preliminary pharmacokinetic experiments with Glatiramer Acetate (GA). Alexa488 (A & B) or Alexa647 (C & D) -labeled glatiramer acetate (GA, 10 mg) was injected directly into the ventricles, and 3-4 days thereafter cerebrospinal fluid (A & C), anticoagulated blood and urine (B & D) were collected and fluorescence intensity was measured, and comparison with the respective standard curves gave the GA content.

Detailed Description

Through intensive research on the activation of purinergic ion channels by Adenosine Triphosphate (ATP), named "P2X7", a series of findings have been made in the field of natural phagocytosis. This channel mediates pro-inflammatory responses in the presence of extracellular ATP. In microglia and macrophages, transient exposure to extracellular ATP, which activates the P2X7 receptor, opens cation selective channels, and prolonged exposure to high concentrations of ATP (> 30 seconds) results in the formation of cell membrane pores and massive potassium efflux, stimulating the assembly of "inflammasome" leading to the maturation and secretion of IL-18 and IL-1 β from monocytes. Activation of the caspase cascade also leads to apoptotic changes in cell morphology that become irreversible after a few hours. Since 2007, another "hidden" function of P2X7 was discovered by professor Ben j. Gu and James s s.wiley: a scavenger receptor. In the absence of extracellular ATP, the P2X7 receptor can directly clear the particle, and this function does not require opsonization. We have demonstrated that the P2X7 receptor has a tight molecular association with non-myosin heavy chain IIA (NMMHC-IIA) in monocytes, this complex molecular association mediating phagocytosis of non-opsonic latex beads, live and dead bacteria and apoptotic cells. As a physiological activator of P2X 7-mediated pore formation and proinflammatory responses, ATP is actually an antagonist of P2X 7-mediated phagocytosis because it breaks down the P2X7-NMMHC-IIA complex, which is required for capture particle internalization. The reduced part of phagocytic function by exogenous ATP therefore represents the part of P2X7 mediated native phagocytosis. In addition, P2X 7-mediated phagocytosis becomes active in cerebrospinal fluid, while only 1-5% of serum completely inhibits the scavenger function of P2X7, suggesting that this function of P2X7 may be of particular importance in the central nervous system in the absence of serum. Not only can remove aged dead nerve cells, but also can promote the reconstruction of a neural network of neural stem cells.

The invention adopts a method for measuring the function of the leukocyte subset by real-time multicolor flow cytometry to report the evidence of the non-function of a P2X7 receptor for the first time, and then further develops a method for measuring the function of a P2X7 channel/hole and the interaction between phagocytosis and protein (figure 1). This trichromatic real-time flow cytometry was recently used to quantify the phagocytic capacity of the monocyte subpopulations of healthy controls and patients with Mild Cognitive Impairment (MCI) and Alzheimer's Disease (AD). By using this approach, we found the first human evidence demonstrating that the natural phagocytic function of discrete AD is disturbed. We tested from MCI, ADIsolating mononuclear cells from peripheral blood of the patient or the age-matched healthy control group of the patient, and treating the isolated mononuclear cells with glatiramer acetate (glatiramer acetate)

The clinically approved drug, which we found altered native phagocytosis, both altered native phagocytosis and altered values correlated with clinical diagnosis and brain a β burden as measured by a β positron Scan (a β -PET Scan) (fig. 2). This finding presents a new insight into the development of AD involving natural phagocytosis and is expected to be useful in the diagnosis, prognosis and treatment of this disease.

Using this method, we have found that Glatiramer Acetate (GA) strongly promotes monocytes, in particular CD14 ⁺ CD16 ^- Typical of monocyte phagocytic function, EC ₅₀ About 20. Mu.g/mL. This effect was completely abolished at low temperature (5 ℃), indicating that its route of action was by affecting phagocytic function rather than non-specific adhesion, since low temperature inhibited phagocytic function but did not affect non-specific adhesion (FIG. 3).

We first tested whether glatiramer acetate also promotes natural phagocytosis in vivo. Female New Zealand white rabbits (2.5 Kg) were selected for this experiment. Healthy rabbits were divided into two groups (3 per group). Both groups were injected with 1 μm fluorescent latex beads (YG beads) intravenously via the ear rim. One group was intravenously injected with 2mL/kg of glatiramer acetate through the marginal ear vein, and the other group was intravenously injected with 2mL/kg of 4% mannitol through the marginal ear vein. To ensure that the detected bead fluorescence came from the circulation, arterial blood samples were taken from the other ear 5, 10, 15, 20, 30, 60 and 120min after the injection of the beads, respectively. No fluorescence was detected in the pre-injection leukocytes. YG microsphere injection 5min later, CD14 ⁺ Microsphere fluorescence was detected in monocytes. In this particular experiment, the highest level of fluorescence was detected at a time point of 30min, and then the detected fluorescence decreased with time. Experimental rabbits showed a higher level of phagocytosis after receiving glatiramer acetate (fig. 4). During the period, the experimental rabbits are also found in the 15 th, 20 th groups,30. Fluorescence was detected at the 60 and 120min time nodes, whereas more YG fluorescence was found in glatiramer acetate treated experimental rabbits (fig. 4). In addition, the neutrophils in both groups of the experimental rabbits treated with glatiramer acetate and mannitol phagocytosed a small amount of YG beads, however, no significant difference was seen between the experimental group and the control group (fig. 4), which is consistent with the in vitro results, i.e., glatiramer acetate did not alter the phagocytosis of neutrophils.

The ability to target binding well is also required for bridge-linked molecules to be phagocytosed. We then examined whether Glatiramer Acetate (GA) interacted with a β. The method was to use micro-scale thermal electrophoresis (MST) to determine the binding affinity between GA and a β 42. A.beta.42 (80 nM, in PBS) was fluorescently labeled with HiLyte Fluor488 and serially diluted at a ratio (volume) of 1:1 to have an average molecular weight of 6.5K _D The GA of (1). Measurements were then made in standard process capillaries of the Monolith NT.115 system using 95% LEDs and 40% IR-laser power. The results show that GA and A beta 42 show high affinity, K _D =6.6nM, the monomer ratio of the two combined is 3:1. k even after A.beta.42 had been left to stand at 4 ℃ for one week to produce more oligomers _D The value still reached 25.4nM (fig. 5), similar to the affinity of the a β monoclonal antibody.

Circular dichroism spectroscopy (CD) is an effective method to study peptide-peptide interactions in solution. The CD in the extreme ultraviolet (178-260 nm) is derived from an amide of the protein backbone, which is sensitive to the conformation of the protein. Thus, when they interact, detection of CD can determine whether the conformation of the peptide has changed. CD spectra can show a β 42 binding to GA (fig. 6). CD is a quantitative technique, and the amount of CD spectrum that varies is directly proportional to the amount of peptide-peptide complex formed after the interaction of A.beta.42 and GA. The results indicate that beta-sheet is the preferred structure for A.beta.42, and that the structure of GA is less pronounced. The complex shows a distinct structure, indicating an interaction between a β 42 and GA (fig. 6).

We further investigated the reaction between a β 42 and GA using western blot immunoelectrophoresis. A.beta.42 was mixed with different contents of GA in different ratios, and then the mixture was subjected to SDS-PAGE electrophoresis in an unnatural state. Even in the unnatural state (by breaking protein disulfide bonds, adding detergent to boil to break all possible binding) GA still binds tightly to a β 42 in a dose-related manner (fig. 7).

After the interaction between GA and a β 42 was determined, we continued to examine whether GA had inhibitory or toxic effects on a β 42. Long-range potentiation (LTP) is an important in vitro measure for assessing neuronal memory and learning. It is well known that a β 42 has a toxic effect on neurons and as shown in figure 8, can significantly reduce LTP levels. The presence of GA alone did not alter the LTP basal levels, however, when a β 42 was present, 50 μ g/mL GA was sufficient to completely prevent the toxic effects of a β 42 (fig. 8). Furthermore, the GA/A β 42 complex increased even the basal level of LTP when the GA concentration was increased to 100. Mu.g/mL, and very few drugs are known to achieve this effect. Our results indicate that GA has the potential to reverse the toxic effects of a β 42 in the brain, thereby restoring memory and learning to the patient.

There is a great deal of evidence that peripheral blood mononuclear cells/macrophages (CD 45) ^high Is a marker for all peripheral blood leukocytes) may migrate to the brain and we found that in APP/PS1 mouse models these cells surround congo red positive amyloid plaques. Recently, we used different anti-a β antibodies or Amyloid Precursor Protein (APP) to detect APP expression on monocytes. Clone 1E8 (anti A.beta.. Beta.) _1-2 ) 4G8 (anti-Abeta) _17-24 ) 6E10 (anti-Abeta) _1-17 ) 22C11 (anti-APP) _66-81 ) And clone W0-2 (anti A.beta.. Beta.) _5-8 Identification of Abeta _1-40 ，Aβ _1-42 And can dissolve APP). Monocytes showed high affinity for W0-2, reduced binding to 6E10 and 4G8, but little binding to 22C11, indicating the presence of A β/APP pools on the surface of monocytes. Few CD14 ⁺ CD16 ⁺ The intermediate monocytes (a subset with the highest phagocytic function) bound W0-2 to a very high degree (FIG. 9A, B). Chemotaxis of these cells to the cell migration marker C-CThe daughter receptor type 2 (CCR 2 or CD192, expressed on monocytes with high migration potential) was also highly positive (fig. 9C). We sorted the cells by flow cytometry based on CCR2 and CD14 expression, lysed, and captured the cell membrane lysed proteins using a microchip coated with W0-2 monoclonal antibody, followed by analysis of their immunoprecipitation by SELDI-TOF. The results show CCR2 ^high CD14 ⁺ Presence of Abeta 42 dimer, CCR2, on monocytes ^low CD14 ⁺ The dimers were reduced on monocytes compared to the dimers, and a β 42 dimers were absent on lymphocytes (fig. 9D).

After recognizing cell surface a β 42 dimers, we examined whether a β 42 could adhere directly to the cell membrane without being phagocytosed by phagocytosis or endocytosis, as this could suggest how a β 42 enters our body. After culturing whole blood containing a β 42 for 15min, the binding capacity of monocytes to W0-2 antibody was significantly increased, but lymphocytes and neutrophils were absent (fig. 10A). Further studies showed that the intermediate monocyte CD14 ⁺ CD16 ⁺ Highest ability to bind A β 42, followed by CD14 ^dim CD16 ⁺ Atypical monocytes, typical monocytes CD14 ⁺ CD16 ^– A β 42 rarely adhered (fig. 10B). We also found that binding of a β to cell membranes was reduced by the presence of GA or the P2X7 antagonist AZ10606120, which together synergistically prevented a β 42 adhesion (fig. 10C), suggesting that the combined use of GA and P2X7 antagonists may be a more effective treatment for AD.

We have also found that another pathway can explain the toxic effects of a β 42. Cell membrane stability is a fundamental factor in many pathological changes in senile diseases. The fluidity of cell membranes plays an important role not only in the processing of Amyloid Precursor Protein (APP), but also as a basis for phagocytosis by macrophages. Since we found that a β can intercalate into the cell membrane, further investigation of its effect on plasma membranes can understand how a β causes cytotoxicity. We used a fluorescence polarization probe TMA-DPH following the method in Cold spring harbor laboratory Manual to measure membrane fluidity. We added APC fluorescently labeled anti-CD 14 monoclonal antibody, FITC fluorescently labeled anti-CD 16 monoclonal antibody and cytoactive dye 7-amino actinomycin D (7-AAD) to Peripheral Blood Mononuclear Cells (PBMCs), then treated with 10 μ M TMA-DPH at 37 ℃ for 5min, lysed erythrocytes with BD FACS lysate, and then washed the cells once with PBS, and 7-AAD negative viable cells were analyzed with a three laser (405nm, 488nm, 633nm) flow cytometer. We found that a β 42 promotes monocyte membrane fluidity, whereas a β 40 does not (fig. 11A), and this promotion was inhibited by P2X7 antagonists (AZ 10606120) or GA (fig. 11B).

We further investigated the therapeutic effect of glatiramer acetate on AD mouse model (APP/PS 1) by implantation of mini-osmotic pump direct brain perfusion (fig. 12). APP/PS1 transgenic mice are widely used in AD animal models. These mice had a β accumulation already at four months, but mice showed cognitive impairment typically after 10 months. The life expectancy of this mouse model is between 25 and 27 months. To better mimic human disease, we performed this study using 23-34 month old female aged APP/PS1 mice, which is equivalent to elderly people 80-90 years old and who have had a history of 40 years old dementia.

All animals were surgically implanted with a micro-osmotic pump (ALZET, model 1004, 100 μ L volume, sustained release of drug for 28 days) and ALZET brain infusion package. GA (20 mg/mL) (n = 10) or blank control (PBS with 4% mannitol) (n = 9) (100 μ L each) was injected in a micro osmotic pump. Animals were anesthetized with isoflurane inhalation and a micropump was implanted in the dorsal subcutaneous space, penetrating the skull 2.5mm subcutaneously, which is suitable for lateral ventricle positioning in adult mice (fig. 12). The whole process is basically carried out according to the method reported in the literature. The animal ethics Committee of the Flori institute, university of Melbourne approved this study (16-076 and 17-032).

Three weeks after implantation, we performed behavioral testing on these mice for two consecutive weeks (twist balance, open field behavior, Y maze, elevated maze, and social interaction). After completion of these behavioral tests, mice were sacrificed and half brain sections were collected for LTP measurement, and Α β was stained using immunohistochemistry, and after homogenization of the other half brain, soluble and insoluble Α β in brain was quantified by ELISA test.

In the behavior test, the GA group was improved in the first day of the swivel balance test in the balancing time and the completion speed as compared with the control group, but in the following days, the difference disappeared. The center of the GA group was longer in the open field experiments in mice, indicating that these mice were more interested in new environmental exploration (fig. 13). In the elevated maze test, both groups showed better performance on seal arms, however, the GA group's preference for seal arms and open arms was less pronounced than the control group compared to the control experimental mice (fig. 14). In the Y maze test, the GA group of mice used the new arm significantly longer than the familiar old arm, indicating that these mice had more motivation to explore (fig. 15). In the social interaction test, although no difference was found between the GA group and the control group in the first stage, the two groups did differ in the second stage and the third stage (fig. 16). In the second phase, GA mice showed a preference for the stimulation chamber, whereas control mice did not. Consistent results were obtained later in the third phase, and these GA mice showed a preference for new stimulation chambers compared to control mice (fig. 16). These behavioral experiments indicate that GA-treated mice are more motivated to explore new environments.

After 5 weeks of implantation, mice were sacrificed. When fresh brain sections of mice were used to determine LTP, basal LTP levels were significantly increased in GA-treated mice compared to control mice (fig. 17), indicating that GA treatment improved memory and learning in vivo in aged AD mice. This is consistent with our observations in behavioral testing. Furthermore, the brain soluble a β number decreased from 39 ± 23 to 26 ± 12 pg/' mg proteins after GA treatment, while the ratio of soluble and insoluble a β decreased from 0.0040 to 0.0029 (P = 0.023) (fig. 18).

The brains of some mice also received immunohistochemical staining for IBA-1 (microglia marker) and a β. Circular microglia were found in both groups (data not shown). Using image analysis tools (image J), Α β staining showed a reduction in the number of plaques in the hippocampal region and a reduction in the area of plaques in the cortical region of near GA treated mice (fig. 19). Furthermore, etched annular plaques were found in all GA treated mice, predominantly in the cortical region. In contrast, only a small number of etched annular plaques were found in the control mice (fig. 20). This unique plaque shape may represent the lysis of a β plaques in GA treated mice.

In conclusion, our in vitro experimental data indicate that GA can act as a bridging molecule, promoting natural phagocytosis in vitro. GA binds tightly to A.beta.and antagonizes the toxic effects of A.beta.42 during LTP depletion, and A.beta.inserts into the cell membrane of monocytes and increases membrane fluidity. We found that GA in the presence of A.beta.42 can restore or even promote neuronal memory and learning. These findings are consistent with our in vivo experimental data of direct GA injection into AD mouse brains. GA treated mice showed improved behavioral testing, increased basal levels of LTP, decreased brain soluble a β and evidence of decreased number/area of a β plaques and dissolved plaques. These results indicate a great potential for GA in the treatment of alzheimer's disease.

We have preliminarily demonstrated the safety and efficacy of Glatiramer Acetate (GA) administration by intravenous injection in vivo and direct intracerebroventricular administration using animal experiments in new zealand white rabbits and mice. To further verify its safety and to obtain pharmacokinetic data, three adult Australian sheep were selected for preliminary experiments (# 1, ewe, 36.5 kg; #2, ram, 35.8 kg; #3, ram, 48.6 kg). The experiment was approved by the animal Committee on the Lung of the research institute of Flori, university of Melbourne (18-010).

We first performed Ventricular puncture Surgery (ICV) on sheep following the standard procedure of the Flori institute (SOP 026): sheep were induced by intravenous injection of 5% sodium phthalate (0.4 mg/kg), intubated and then hung on an anaesthesia machine with a 2% isoflurane/air/oxygen mixture and monitored for respiration, heart rate and blood oxygen levels throughout the procedure. Sheep were placed in a stereotactic frame and held in place by mouth, eyes and ear bars. The mouth is located below the front jaw. The eye stick is placed on top of the orbital bone and the ear stick is placed in the ear canal. A 30-40mm diameter skin hole is cut at the top of the head to expose the anterior halogen spot (the point on the top of the skull where all 3 skull plates meet). The skull was scraped to clean its periosteum and cleaned with hydrogen peroxide. Five small screws were screwed into the skull at the portion around the incision. These help anchor the acrylic to the skull. Then two small holes are drilled on the skull, and the two small holes are respectively positioned 10mm on the left side and the right side of the front 10mm in front of the front halogen point. A catheter (# 8 needle size) is then inserted through the hole into the brain. When the catheter entered the lateral ventricle, the pressure sensor was connected to the saline infusion tube and the catheter showed a pressure drop, leaving the catheter in place. It is now located in the center of the lateral ventricle. The process is then repeated on the other ventricle. The wells were filled with Surgicel to prevent leakage of liquid and acrylic acid into the brain. Dental acrylic is then molded onto the skull to form a hard protective surface to hold the catheter in place. Stopping bleeding with an electric coagulation hemostatic pen, and suturing the wound. The sheep were then removed from the isoflurane anesthetic and placed on an air/oxygen mixture. When the swallowing reflex was evident, the cannula was removed and the sheep was returned to the operating table. Each animal received a 1mL (1 mg/kg) intramuscular injection of Flunixin meglamine to relieve pain. The sheep were then returned to the cage and monitored for several hours. The next three days were injected with penicillin once daily (fig. 21).

We first examined the safety of GA. On day 1 post ICV surgery, we injected 1mL of the carrier solution (4% mannitol in artificial cerebrospinal fluid, sterile filtered) from the ICV catheter at a rate of 0.5mL/min, all at the same rate. 0.5mL of cerebrospinal fluid, 5mL of blood and 5mL of urine were collected. Used to measure the established baseline. GA was then injected once a week at a standard dose of 10mg/0.5mL, typically all at Monday 8 times during which blood was collected for one to two deliveries. The heartbeat respiration, the anus temperature, the forage consumption, the drinking water, the defecation, the urination and the like are observed and recorded every day. We found that there was typically a 0.5 ℃ rise in body temperature within 24 hours after each GA injection, but body temperature remained between 37.8-39.5 ℃ throughout the experiment without any body surface abnormalities. The most obvious change since the beginning of GA injection is a large increase in food consumption by the animals, with forage consumption rising from the initial 400-800 grams to 800-2400 grams, and sometimes even over 3000 grams. While drinking water rises from the first 4-5 liters to 8-16 liters. At the same time, there was a large increase in faecal excretion, on average 1-2 fold, and several fold increase in urination, reaching a surprising 12 liter maximum volume of the urine bucket (fig. 22). This continued until one month after the end of the entire injection procedure, indicating that ventricular GA injection greatly enhanced the metabolic capacity of the animals. It should be noted that the animals were examined for blood routine, urine routine, thyroid function, liver function, kidney function, etc., without any abnormality or significant change. The weight of the animals did not increase significantly. It is well known that the brain is the organ consuming the most energy in the human body, and our results suggest that the increased metabolism caused by GA is mainly to supplement the energy required by the brain, indicating that some brain functions are activated.

We next investigated the pharmacokinetics of GA. We first labeled GA with the fluorescent dyes Alexa488 and Alexa647 and measured the concentration with Direct Detect (Merck) and developed a standard curve with a spectrofluorometer. After 4-6 weeks of the last GA injection, we performed an external jugular vein cannulation of the animals to collect blood samples. Briefly, sheep will first receive local anesthesia (2% lidocaine) by subcutaneous injection, place a single lumen 18 gauge central venous catheter in the external jugular vein aseptically, and tape the fabric

The site is bandaged to protect it from animal damage. The sleeve will be secured by the cloth tape to the sleeve sutured to the skin. The catheter was flushed with 1-2mL heparin anticoagulant saline (100 IU/mL) before and after sampling. The cannula will stay in the sheep for no more than four days. After the first collection of cerebrospinal fluid, blood and urine, we injected 10mg of fluorescently labeled GA through the ventricles, followed by collection of the sample and determination of the fluorescence intensity in the sample. Cerebrospinal fluid (0.5 mL each) collection was collected via intracerebroventricular catheter at 0.5, 1,2, 4, 8, 24, 48, and 72 hours post-injection, blood (5 mL each) was collected from the external jugular vein cannula, urine (5 mL each) was collected from urine samplesCollecting in a container. The results show that the GA content in cerebrospinal fluid decreases rapidly after 24 hours and almost completely disappears at 48 hours; the GA content in the blood increased after 24 hours, at least for 72 hours; the urine cannot be accurately measured due to the deep fluorescence background.

The total amount of cerebrospinal fluid in sheep was approximately 14mL, and we injected 10mg of GA directly into the ventricles of sheep during the experiment, at an initial concentration of approximately 0.7mg/mL, well beyond the working concentration of GA (0.1 mg/mL). Even at such high concentrations, sheep did not show any discomfort. The total cerebrospinal fluid of human is about 120mL, and only 12mg of GA is needed to reach the working concentration each time, so that the safety is guaranteed. GA is rapidly metabolized in the brain into peripheral blood, suggesting that it is blood-brain barrier permeable.

Claims (6)

1. Application of glatiramer acetate in preparing A beta 42 toxicity inhibitor and scavenger.
2. Application of glatiramer acetate serving as an Abeta 42 toxicity inhibitor and scavenger in preparation of medicaments for treating Alzheimer's disease.
3. A method of treating alzheimer's disease by direct infusion of glatiramer acetate into the central nervous system.
4. The method of claim 3, wherein the direct infusion into the central nervous system is by intravenous drip, nasal spray, spinal cord puncture, or by intrathecal injection of an implant device.
5. The method of claim 3 or4, wherein the glatiramer acetate is used alone or in combination with other compounds.
6. The method of claim 5, wherein the additional compound is a P2X7 antagonist.

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