WO2022061812A1 - USE OF GLATIRAMER ACETATE IN PREPARATION OF Aβ42 TOXICITY INHIBITOR AND SCAVENGER - Google Patents

Abstract

The use of glatiramer acetate with a high affinity to Aβ42 in eliminating Aβ42 and inhibiting the toxicity thereof.

Description

Application of glatiramer acetate in the preparation of Aβ42 toxicity inhibitor and scavenger technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of glatiramer acetate in the preparation of a toxicity inhibitor and a scavenger for β-amyloid Aβ _1-42 (abbreviated as Aβ42), and then for the treatment of Alzheimer's disease .

Background technique

Alzheimer's disease (AD) imposes a huge social and economic burden on society, affecting more than 26 million people worldwide. There is currently no cure, and existing treatments only improve mild to moderate symptoms for a limited time. To date, no treatment has even been proposed for advanced stages.

As a basic biological function, innate immunity maintains the body's homeostasis. Innate phagocytosis is one of the important components of innate immunity and does not require antibodies or complement to recognize and phagocytose apoptotic cells, cellular 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 set of AD risk genes that belong to core innate immune pathways, including CD33, CR1, MS4A6A, MS4A4E, ABCA7, and TREM2. Variations in these genes, especially TREM2 and CD33, were associated with impaired restriction of phagocytosis of monocytes/macrophages and altered Aβ accumulation in the brains of AD patients. Notably, complement receptor 1 (CR1, also known as CD35) is mainly expressed in peripheral blood leukocytes and erythrocytes, but not in the brain under normal physiological conditions. Therefore, in addition to excessive neuroinflammation, systemic deficits involving phagocytosis of the central nervous system (CNS) and peripheral tissues may be the main reasons for the failure of Aβ deposition and clearance.

The deposition and aggregation of A[beta] forms amyloid plaques, and the well-established peculiar features of AD are widely believed to be associated with disease etiology. Therefore, current drug discovery in AD focuses on the use of antibodies to clear existing amyloid deposits. However, since 1998, more than 100 clinical trials targeting Aβ have either failed or only showed "a glimmer of hope". Only recently, one of these antibody-based approaches, "Aducanumab," has shown some potential for treating AD, possibly due to its high blood-brain barrier (BBB) penetration ability, but this ability also brings significant Side effects, such as cerebral hematoma, are manifested by a high incidence of amyloid-related imaging abnormalities (ARIA, 13-47%). Therefore, we urgently need effective therapeutic strategies for AD.

Several studies from three different research 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 immune vaccine, and its mechanism of action was unclear.

SUMMARY OF THE INVENTION

The invention aims to overcome the deficiencies of the prior art and provides the application of glatiramer acetate in the preparation of Aβ42 inhibitor.

Natural phagocytosis is the most important component of the human innate immune system and is critical to the development and homeostasis of the body. Natural phagocytosis does not require opsonization to identify and remove apoptotic cells, cellular debris, and invading microorganisms. Professional scavenger cells (phagocytes) that rapidly clear senescent apoptotic or dead neuronal cells are critical in our bodies to avoid inflammation and allow neurogenesis Central nervous system: rapid clearance of misfolded protein complexes It is the key to avoiding neurodegeneration; rapid removal of invading microorganisms is our body's first line of defense, especially important for the central nervous system lacking acquired immunity, and is an important measure to avoid subsequent neuroinflammation. However, this important biological function has been neglected for a long time, and the concept of "natural phagocytosis" has never even been proposed.

Dr. Baijun Gu (or Ben J. Gu), the inventor who first proposed the concept of "Innate Phagocytosis" (also translated as "innate phagocytosis"), and his team have been working on this research for more than a decade. Using the purinergic receptor P2X7 as a model, the inventors first invented a method that can quantify native phagocytic capacity in vitro: real-time multicolor flow cytometry (Figure 1), and on this basis, through years of efforts to clarify this receptor. A set of rationales for how the body and related cytoskeletal proteins function as scavenger receptors, and how it is recognized and cleared by cysteine disulfide bond formation prior to necrotic death and resulting inflammation Apoptotic cells to reduce inflammatory response. The inventors also found three important characteristics of natural phagocytosis: (1) It is closely related to temperature, and the increase of the ambient temperature from 37°C to 39°C can significantly improve the natural phagocytic ability; (2) It has the strongest effect in a serum-free environment, The 1-5% serum can greatly reduce the natural phagocytic function; (3) the natural phagocytic function gradually declines with the increase of age. Therefore, natural phagocytosis plays an important role in serum-free environments such as the central nervous system, eyes, bones and joints, and can be used as a treatment for severe infections such as sepsis and viral pneumonia, as well as senile degenerative diseases such as age-related macular degeneration (AMD) and Alzheimer's A target for Alzheimer's disease (AD). Large-scale genetic sequencing analysis identified some mutation points and groups of mutations that could alter P2X7-mediated innate phagocytosis: Arg307Gln mutation, which more efficiently maintains P2X7's innate phagocytosis, was found to alter multiple sclerosis (MS) The risk of the disease is reduced by half; the co-mutation of the P2X7 receptor His150Arg and the P2X4 receptor Tyr315Cys completely destroys the natural phagocytosis mediated by the P2X7 receptor, resulting in a nearly 4-fold increase in the incidence of age-related macular degeneration. In the long-term study of Alzheimer's patients, with the help of the AIBL platform (Australia's research organization for medical imaging, biomarkers and lifestyle of Alzheimer's patients, and the world's largest long-term follow-up cohort of elderly), The inventors found that the natural phagocytic function of peripheral blood mononuclear cells in patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) is disturbed and correlated with the size of Aβ plaques in their brains, while P2X7 is affected by Body-mediated natural phagocytosis is also closely related to Aβ plaque size. The inventors have further discovered that glatiramer acetate (GA, a clinical drug for the treatment of multiple sclerosis, registered drug trade name)

) can improve the natural phagocytic function of monocytes in patients, and the degree of improvement is closely related to clinical diagnosis and Aβ plaque area.

The present invention focuses on a more effective method to prevent the accumulation of A[beta] or other proteins and to inhibit or even partially reverse the progression of Alzheimer's disease by promoting natural phagocytosis to clear these fragments.

Our in vitro and in vivo experimental data demonstrate that glatiramer acetate is able to bind tightly to Aβ and antagonize the toxic effects exhibited by Aβ42 in long-term potentiation (LTP). The bound Aβ embeds the monocyte membrane before Aβ42 and enhances membrane fluidity, promoting the memory and learning ability of neurons. In vivo experiments, glatiramer acetate was injected directly into 24-month-old APP/PS1 transgenic mice (an AD animal model with a life expectancy of 26-27 months), implanted using an osmotic minipump, and The behavior of these aged mice was significantly improved within three weeks, and LTP was increased after 5 weeks, compared with the control group. The level of soluble Aβ found in the brain was reduced to half and Aβ immunohistochemical staining showed predictable lysis in the center of the deposited Aβ plaques.

These results suggest a new approach to AD treatment, namely the direct infusion of glatiramer acetate into the central nervous system (CNS). Nasal sprays, spinal taps, or intrathecal injection of an implanted device such as the Ommaya Reservoir (a ventricular catheter system implanted under the scalp to inject the drug into the cerebrospinal fluid) are options. Because glatiramer acetate shows high affinity for Aβ and a strong effect in increasing LTP levels in brain neurons of aged AD mice, this therapeutic modality could be used to treat AD rather than just another disease-modifying therapy . Glatiramer acetate can be used alone or in combination with other compounds such as P2X7 antagonists, which have a synergistic therapeutic effect.

Description of drawings

Figure 1: Phagocytosis of YO microbeads by monocyte subsets. Before adding 1 μm yellow-orange fluorescent YO microbeads, fresh human PBMCs were labeled with APC-CD14 and FITC-CD16 mAbs, and then the fluorescence intensity of YO microbeads in the selected cells was analyzed by real-time flow cytometry. a. Typical example of CD14 and CD16 density maps. Monocytes were first selected using forward and side scatter. b. Typical real-time flow cytometry detection of YO microsphere uptake curve, CD14dimCD16+ (green), CD14+CD16+ (red), CD14+CD16- (brown) monocytes. The phagocytic capacity of CD14-lymphocytes (black) was minimal.

Figure 2: Study glatiramer acetate

Effects on phagocytosis of yellow-orange fluorescent (YO) microbead cells in human monocyte subsets. 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 μg/ml for 10 minutes before adding 1 μm YO beads. Phagocytosis was quantified as the unit of area under the YO bead uptake curve within 1 to 6 minutes after the addition of the YO beads. Monocyte subsets were differentiated by the extent of cell surface CD14 and CD16 expression. Subjects were grouped according to their brain beta-amyloid burden (Gu et al, Acta Neuropathologica, 2016)

Figure 3: Glatiramer acetate (

CPX) enhances the ability of monocytes to phagocytose fluorescent latex beads. (a) Fresh human monocytes (2 × 106/mL) were labeled with APC-conjugated anti-CD14 and FITC-conjugated anti-CD16 antibodies, respectively, and analyzed by real-time flow cytometry for typical CD14+CD16− monocytes. The fluorescence intensity. Cells were pretreated with or without 100 μg/mL CPX at 37°C for 10 minutes and then placed on ice or kept at 37°C for an additional 5 minutes. As shown, yellow-orange latex beads absorption tests were performed at 37°C or 5°C for 6 minutes. 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 various concentrations of glatiramer acetate for 10 min at 37°C. The increase in phagocytosis stimulated by glatiramer acetate was normalized as a percentage of basal phagocytosis levels (n=3) (Gu et al, Acta Neuropathologica, 2016, Suppl Fig. S4).

Figure 4: Glatiramer acetate (GA) promotes innate phagocytosis of yellow-green YG fluorescent microbeads in vivo. New Zealand white rabbits were injected with 1.5 ml of YG microspheres and 2 ml/kg GA or 4% mannitol as controls. Blood was collected before injection and at 5, 10, 15, 20, 30, 60, and 120 minutes after injection, respectively. After the blood was treated with erythrocyte lysis buffer, it was labeled with Alexa647-CD14 monoclonal antibody, and the fluorescence intensity of YG beads in the selected cell population was detected by flow cytometry. The upper panel shows the mean fluorescence intensity (MFI) after phagocytosis of YG beads by monocytes and neutrophils 30 minutes after injection. The lower two graphs show the time course of YG beads uptake by monocytes (left) and neutrophils (right).

Figure 5: Glatiramer acetate has high affinity for direct binding to Aβ1-42. HiLyte Fluor488-labeled Aβ1-42 (80 nM in PBS), 1:1 volume mix for serial dilution of pro-phagocytosis peptides. Measurements were performed in standard process capillaries using an NT.115 system with 95% LED and 40% IR-laser power.

Figure 6: Interaction of A[beta]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 μM). C. When Aβ and GA are combined, determine its structure. In the implementation of Sreerama et al., secondary structure was analyzed using the computer program CDSSTR and reference data set 3.

Figure 7: Glatiramer acetate binds tightly to A[beta]42. Aβ42 (10 μg 2.2 nmol) was mixed with varying amounts of glatiramer acetate 0, 0.22, 1.1, 2.2, 6.6, 15.4 nmol (lanes 1-6) or serum pool 2.2 nmol (lane 7). The mixtures (30 μl each) were diluted with 50 μM DTT and heated at 90° C. for 5 min before SDS-PAGE (4-12% NuPage gel, MES buffer, 100 V for 50 min) electrophoresis. Anti-A[beta] monoclonal antibody (WO-2 antibody) transferred and probed the protein. A. Western blot electrophoresis images showing Aβ staining. Semi-quantitative results for the 4.5KD Aβ42 monomer per lane are shown in the bottom bar. After B. Ponceau S staining, proteins were transferred to a nitrocellulose membrane fraction. Large amounts of glatiramer acetate and bovine serum albumin (BSA) were stained red. Protein size was estimated using Seeblue pre-stained protein standards.

Figure 8: Long-term potentiation (LTP) of glatiramer acetate (GA) antagonizing the toxic effects of A[beta]1-42. Multi-electrode array (MEA) electrophysiological methods were used to record LTP changes in mouse brain slices. Fresh mouse hippocampal slices were mounted on a 3D-MEA chip and treated with 60 spikes and 30 μm high electrode spacing of 200 μm. Sections were continuously perfused with artificial cerebrospinal fluid (aCSF, 3 mL/min, 32°C). Data were collected using a multi-channel system (MCS GmbH, Reutlingen, Germany). By choosing an electrode at 0.033 Hz, stimulate the Schaffer-collateral to inject a biphasic current waveform (100 μs). The peak-to-peak excitatory postsynaptic potentials (fEPSPs) of the CA1 proximal radial layer were analyzed by LTP-analyzer.

Figure 9: Staining of human monocyte A[beta] dimer with WO-2 monoclonal antibody. A. A[beta] staining of different subsets of human monocytes from an AD patient. Cells were stained with mouse anti-Aβ monoclonal antibody (clone WO-2 antibody) and FITC-conjugated secondary antibody, followed by labeling of monocyte subsets with fluorescent CD16 and CD14 monoclonal antibodies; B.W0-2 monoclonal antibody to CD14 Surface A[beta] staining of +CD16+ monocytes (the highest phagocytic subpopulation). A population with very high levels of W0-2 binding is shown; C. Comparison of CCR2 staining and W0-2 mAb A[beta] staining on channel monocytes from an AD patient. Cells with high levels of WO-2 binding also showed high binding to CCR2, which is a marker of high migration potential; D. Human peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry according to CCR2/CD14 expression and positive, side scatter for classification. Cells were lysed, and Aβ was captured on a microchip coated with WO-2 monoclonal antibody, and then bound protein was analyzed by SELDI-TOF.

Figure 10: Glatiramer acetate (GA) and the P2X7 antagonist AZ10606120 inhibit the insertion of A[beta]1-42 into the monocyte membrane. Aβ1-42 (10 μg/ml) was added to whole blood with or without 100 μg/ml GA or 1 μM AZ10606120 (AZ) for 15 minutes. Cells were stained with anti-A[beta] monoclonal antibody (clone WO-2 antibody) and counterstained with CD14 and CD16. A. Three types of leukocytes were stained by W0-2; B. The cell surface of three monocyte subsets was bound to W0-2; C. W0-2 stained CD14+ monocytes.

Figure 11: A[beta]1-42 and ATP-induced increase in membrane fluidity can be inhibited by glatiramer acetate (GA) and the P2X7 antagonist AZ10606120 (AZ). a. Cells were incubated with various concentrations of freshly lysed Aβ1-40 and Aβ1-42 polypeptides for 15 minutes prior to addition of TMA-DPH. b. Cells were cultured with 50 μg/ml Aβ1-42, 100 μg/mL GA, 1.0 mM ATP or 10 μM of AZ10606120(AZ), and then viable monocytes (CD14+ and 7-AAD-) were selected for flow cytometry analysis .

Figure 12: Evaluation of the therapeutic effect of direct infusion of glatiramer acetate (GA) into the brain of a mini-pump on AD animal models. ALZET micro-osmotic pumps (model 1004, 100 μL stock volume, for a period of four weeks) were filled with GA (20 mg/ml) or PBS plus 4% mannitol (control control) and implanted in the subcutaneous space in APP/PS1 transgenic mice ( 23-24 months old), connect the brain to the ALZET infusion device 3 penetrating 2.5 mm below the surface of the skull, to the lateral ventricle.

Figure 13: Open field behavioral testing 3 weeks after implantation. A. There was no significant difference in the number of times the two groups of mice entered the center of the open field; B. The percentage of time that the GA-treated mice stayed in the center of the open field was longer than that of the control mice; C. The distance moved by the two groups of mice in the open field No significant difference.

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

Figure 15: Y-maze behavioral test 3 weeks after implantation. A. Mice treated with GA showed a clear preference for the novel arm, in contrast, control mice did not (P=0.0067, two-way ANOVA); B. Distance traveled in the Y-maze by both groups of mice No significant difference.

Figure 16: Social interaction tests performed 3-4 weeks after implantation. A. GA-treated mice showed a preference for stimulation chambers (P=0.0114) (two-way ANOVA analysis of variance); B. GA-treated mice showed an area of interest for stimulation chambers compared to control mice Greater preference (P=0.0016); C. Both GA-treated and control mice showed preference for novel stimulation chambers (P=0.0032); D. GA-treated mice showed preference for novel stimulation chambers (P=0.0032); Preference for AOI in novel stimulation chambers, compared to none in control mice (P=0.0011).

Figure 17: Measurement of long-range potentiation potential (LTP) in freshly prepared mouse hippocampal slices 5 weeks after surgery.

Figure 18: ELISA detection of soluble and insoluble A[beta] in mouse brain 5 weeks after implantation.

Figure 19: Decreased number/size of A[beta] plaques in glatiramer acetate (GA) treated mice. Mouse brain sections were stained with anti-Aβ monoclonal antibody (clone 1E8). Images were analyzed using ImageJ to calculate the number and area of Aβ plaques in the hippocampal (A&B) and cortical (C&D) regions. Typical channels are displayed separately in the A&C area. 40x magnification. (n=4 per group).

Figure 20: Mouse brain A[beta] immunohistochemical staining and anti-A[beta] monoclonal antibody (clone 1E8). Corrosion-ring-shaped Aβ plaques were displayed in glatiramer acetate (GA)-treated mice, but not in control mice. 200x magnification.

Figure 21: Australian sheep after ventriculocentesis.

Figure 22: Typical sheep experimental recording. Sheep underwent ventriculocentesis on day 1 and received 8 direct administrations of glatiramer acetate (GA) via the ventricle (indicated by arrows), 0.5 mL (10 mg) each time over the next 3 months. Drinking water was recorded daily and urination (top), forage consumption and defecation (middle), anal body temperature (bottom), and respiration, heartbeat, etc.

Figure 23: Preliminary pharmacokinetic experiments of glatiramer acetate (GA). Alexa488 (A&B) or Alexa647 (C&D)-labeled glatiramer acetate (GA, 10 mg) was injected directly into the ventricle, and cerebrospinal fluid (A&C), anticoagulation and urine (B&D) were collected for 3-4 days thereafter and assayed The fluorescence intensity was compared with the respective standard curve to obtain the content of GA.

detailed description

A series of discoveries have been made in the field of natural phagocytosis by in-depth study of the activation of purinergic ion channels by adenosine triphosphate (ATP) called "P2X7". This channel mediates pro-inflammatory responses in the presence of extracellular ATP. In microglia and macrophages, brief exposure to extracellular ATP that activates the P2X7 receptor opens cation-selective channels, and prolonged exposure to high concentrations of ATP (>30 s) results in the formation of membrane pores and massive The efflux of potassium ions stimulates the assembly of the "inflammasome" leading to the maturation and secretion of IL-18 and IL-1β from monocytes. Activation of the caspase cascade also leads to apoptotic changes in cell morphology that become irreversible after a few hours. Since 2007, inventors Dr. Ben J. Gu and Prof. James S. Wiley have discovered another "hidden" function of P2X7: a scavenger receptor. In the absence of extracellular ATP, P2X7 receptors can clear particles directly and do not require opsonization for this function. We have shown that the P2X7 receptor has a tight molecular association with non-myosin heavy chain IIA (NMMHC-IIA) in monocytes, and this complex molecular association mediates non-opsonized latex beads, live and dead bacteria, and Phagocytosis of apoptotic cells. As a physiological activator of P2X7-mediated pore formation and pro-inflammatory responses, ATP is actually an antagonist of P2X7-mediated phagocytosis, as it breaks down the P2X7-NMMHC-IIA complex, which in turn is required to capture particle internalization. Thus, the decrease in phagocytosis caused by exogenous ATP represents part of the natural phagocytosis mediated by P2X7. Furthermore, P2X7-mediated phagocytosis becomes active in the cerebrospinal fluid, whereas only 1-5% serum completely inhibits the scavenger function of P2X7, suggesting that this function of P2X7 may have a role in the CNS in the absence of serum special importance. It can not only remove senescent and dead nerve cells, but also promote the reconstruction of neural network of neural stem cells.

The method of measuring the function of leukocyte subsets by real-time multicolor flow cytometry in the present invention first reported the evidence that the P2X7 receptor was non-functioning, and later further developed the measurement method of the P2X7 channel/pore function, as well as the interaction between phagocytosis and protein ( figure 1). This three-color real-time flow cytometry was recently used to quantify the phagocytic capacity of monocyte subsets in healthy controls and patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). Using this approach, we found the first human evidence that the native phagocytic function of discrete AD is disturbed. We isolated monocytes from the peripheral blood of MCI, AD subjects or age-matched healthy controls and treated with glatiramer acetate (

Among clinically approved drugs, we found that they can alter natural phagocytic function, and both the altered natural phagocytic function and the altered value were correlated with clinical diagnosis and brain Aβ burden measured by Aβ-PET Scan (Figure 2). This finding presents a new insight into AD development involving innate phagocytosis and holds promise for the diagnosis, prognosis and treatment of this disease.

Using this method, we found that glatiramer acetate (GA) has a strong effect of promoting the typical monocyte phagocytosis of monocytes, especially CD14 ⁺ CD16 ^- , with an EC ₅₀ of about 20 μg/mL. This effect was completely abolished at low temperature (5°C), suggesting that its pathway of action is by affecting phagocytosis rather than non-specific adhesion, as low temperature inhibits phagocytosis but not non-specific adhesion (Figure 3).

We first tested whether glatiramer acetate also promotes native phagocytosis in vivo. In this experiment, female New Zealand white rabbits (~2.5Kg) were used. Healthy rabbits were divided into two groups (3 in each group). Both groups were injected with 1 μm fluorescent latex beads (YG beads) through the ear vein. One group was intravenously injected with 2 mL/kg of glatiramer acetate via the marginal ear vein, and the other group was injected with 2 mL/kg of 4% mannitol via the marginal ear vein. To ensure that the detected bead fluorescence was from the circulation, arterial blood samples were collected from the other ear 5, 10, 15, 20, 30, 60 and 120 min after injection of the beads. No fluorescence was detected in leukocytes prior to injection. 5 min after YG microsphere injection, microsphere fluorescence was detected in CD14 ⁺ monocytes. In this particular experiment, the highest level of fluorescence was detected at the 30 min time point, and then the detected fluorescence diminished over time. Experimental rabbits showed higher levels of phagocytosis after receiving glatiramer acetate (Figure 4). During the two groups, it was also found that the experimental rabbits detected fluorescence at the 15th, 20th, 30th, 60th, and 120min time points, while more YG fluorescence was found in the experimental rabbits treated with glatiramer acetate (Figure 4). In addition, neutrophils in both groups of experimental rabbits treated with glatiramer acetate and mannitol phagocytosed a small amount of YG beads, however, there was no significant difference between the experimental group and the control group (Fig. 4). ), which is consistent with the in vitro results that glatiramer acetate did not alter neutrophil phagocytosis.

To act as a bridge linking molecule for phagocytosis, the ability to bind well on target is also required. We then tested whether glatiramer acetate (GA) could interact with Aβ. The method was to use microscale thermoelectrophoresis (MST) to determine the binding affinity between GA and Aβ42. Aβ42 (80 nM in PBS) was labeled with HiLyte Fluor488 fluorescent staining, and GA with an average molecular weight of 6.5K _D was serially diluted in a 1:1 ratio (by volume). Measurements were then performed in a standard processing capillary of the Monolith NT.115 system using 95% LED and 40% IR-laser power. The results showed that GA and Aβ42 exhibited high affinity, K _D =6.6nM, and the monomer dosage ratio of the two was 3:1. Even after Aβ42 was kept at 4°C for a week, more oligomers were produced, and the K _D value still reached 25.4 nM (Fig. 5), which is similar to the affinity of Aβ monoclonal antibody.

Circular dichroism (CD) is an effective method to study peptide-peptide interactions in solution. The CD in the far ultraviolet region (178-260 nm) is derived from the amides of the protein backbone, which are sensitive to protein conformation. Therefore, detection of CD can determine whether the conformation of the peptide changes as they interact. CD spectroscopy can show A[beta]42 and GA binding (Figure 6). CD is a quantitative technique, and the amount of change in the CD spectrum is proportional to the amount of peptide-peptide complexes formed after Aβ42 and GA interact with each other. The results show that β-sheet is the preferred structure of Aβ42, and the structure of GA is less obvious. The complex displayed a distinctly different structure, suggesting a reciprocal reaction between A[beta]42 and GA (Fig. 6).

We further studied the reaction between Aβ42 and GA by western blot immunoelectrophoresis. A[beta]42 was mixed with different amounts of GA in different ratios, and then the mixture was run in a non-native state by SDS-PAGE. Even in the unnatural state (by breaking protein disulfide bonds, boiling with detergent to disengage all possible bindings) GA still bound tightly to A[beta]42 in a dose-dependent manner (Fig. 7).

After determining the interaction between GA and Aβ42, we went on to examine whether GA has inhibitory or toxic effects on Aβ42. Long-term potentiation (LTP) is an important in vitro measure to evaluate neuronal memory and learning ability. Aβ42 is known to have toxic effects on neurons, as shown in Figure 8, and can significantly reduce LTP levels. The presence of GA alone did not alter basal LTP levels, however, when Aβ42 was present, 50 μg/mL GA was sufficient to completely block the toxic effects of Aβ42 (Figure 8). Furthermore, when the GA concentration was increased to 100 μg/mL, the GA/Aβ42 complex even increased the basal LTP level, an effect that few known drugs can achieve. Our results suggest that GA has the potential to reverse the toxic effects of Aβ42 in the brain, thereby restoring memory and learning in patients.

There is substantial evidence that peripheral blood monocytes/macrophages ( ^CD45high , a marker of all peripheral blood leukocytes) may migrate to the brain, and we found that in the APP/PS1 mouse model these cells surround Congo red Positive amyloid plaques. Recently, we used different anti-Aβ antibodies or amyloid precursor protein (APP) to detect the expression of APP on monocytes. Clone 1E8 (anti-Aβ _1-2 ), 4G8 (anti-Aβ _17-24 ), 6E10 (anti-Aβ _1-17 ), 22C11 (anti-APP _66-81 ) and clone WO-2 (anti-Aβ _5-8 , recognize Aβ _1-40 , Aβ _1-42 and soluble APP). Monocytes showed high affinity for W0-2, decreased binding to 6E10 and 4G8, but little to 22C11, indicating the presence of Aβ/APP pools on the surface of monocytes. A small number of CD14 ⁺ CD16 ⁺ intermediate monocytes, a subset with the highest phagocytic function, bound to WO-2 to a very high degree (Fig. 9A,B). These cells were also highly positive for the cell migration marker CC chemokine receptor type 2 (CCR2 or CD192, expressed on monocytes with high migration potential) (Figure 9C). Cells were sorted by flow cytometry based on the expression of CCR2 and CD14, lysed, and the lysed proteins were captured by microchips coated with WO-2 monoclonal antibody, and their immunoprecipitation was analyzed by SELDI-TOF. The results showed that Aβ42 dimer was present on CCR2 ^high CD14 ⁺ monocytes, and the content of this dimer was reduced on CCR2 ^low CD14 ⁺ monocytes, and there was no Aβ42 dimer on lymphocytes (Fig. 9D). .

After identifying the Aβ42 dimer on the cell surface, we examined whether Aβ42 could directly adhere to the cell membrane without being phagocytosed by phagocytosis or endocytosis, as this could suggest how Aβ42 enters our body. After culturing whole blood containing A[beta]42 for 15 min, the binding capacity of monocytes to the WO-2 antibody was significantly increased, but not to lymphocytes and neutrophils (Fig. 10A). Further studies showed that intermediate monocytes CD14 ⁺ CD16 ⁺ had the highest ability to bind Aβ42, followed by CD14 ^dim CD16 ⁺ atypical monocytes, ^and typical monocytes CD14 ⁺ CD16– rarely adhered to Aβ42 (Fig. 10B). At the same time, we also found that Aβ binding to the cell membrane was reduced by the presence of GA or the P2X7 antagonist AZ10606120, which together synergistically prevented the adhesion of Aβ42 (Fig. 10C), indicating that the combination of GA and P2X7 antagonist may be a More effective treatments for AD.

We also discovered another pathway that could explain the toxic effects of Aβ42. Cell membrane stability is a fundamental factor for many pathological changes in senile diseases. The fluidity of the cell membrane not only plays an important role in the processing of amyloid precursor protein (APP), but also underlies the phagocytosis of macrophages. Because we found that Aβ is able to intercalate into the cell membrane, further investigation of its effects on the plasma membrane could provide insight into how Aβ contributes to cytotoxicity. We used the fluorescence polarization probe TMA-DPH to measure membrane fluidity following the methods in the Cold Spring Harbor Laboratory Guidelines. We added APC fluorescently labeled anti-CD14 monoclonal antibody, FITC fluorescently labeled anti-CD16 monoclonal antibody and cell viability dye 7-aminoactinomycin D (7-AAD) to peripheral blood mononuclear cells (PBMCs). ), then treated with 10 μM TMA-DPH for 5 min at 37°C, lysed erythrocytes with BD FACS lysate, then washed cells once with PBS, and analyzed 7-AAD-negative cells by flow cytometry with three lasers (405 nm, 488 nm, 633 nm). Living cells. We found that Aβ42 promoted monocyte membrane fluidity, while Aβ40 did not (Fig. 11A), and this promotion could be inhibited by P2X7 antagonist (AZ10606120) or GA (Fig. 11B).

We further investigated the therapeutic effect of glatiramer acetate in a mouse model of AD (APP/PS1) by direct cerebral perfusion with implanted mini-osmotic pumps (Figure 12). APP/PS1 transgenic mice are widely used in AD animal models. These mice already had Aβ accumulation at four months, but the mice generally showed cognitive impairment after 10 months. The life expectancy of this mouse model is between 25 and 27 months. To better model human disease, we performed this study using 23-34 month old female aged APP/PS1 mice, which is equivalent to an 80-90 year old with a 40-year history of Alzheimer's disease.

We surgically implanted a micro-osmotic pump (ALZET, model 1004, 100 μL volume, sustained drug release for 28 days) and an ALZET cerebral perfusion pack to all animals. GA (20 mg/mL) (n=10) or blank control (4% mannitol in PBS) (n=9) (100 μL each) was injected in an osmotic micropump. Animals were anesthetized with inhalation isoflurane, and a minipump was implanted in the dorsal subcutaneous space, penetrating 2.5 mm subcutaneously in the skull, which is suitable for lateral ventricle localization in adult mice (Figure 12). The whole process is basically carried out according to the method reported in the previous literature. The Animal Ethics Committee of the Florey Institute, University of Melbourne approved this study (16-076 and 17-032).

Three weeks after implantation, we performed behavioral tests (turn balance, open-field behavior, Y-maze, elevated maze, and social interaction) on these mice for two consecutive weeks. After completion of these behavioral tests, mice were sacrificed and half of the brain was collected to measure LTP, immunohistochemically stained for Aβ, and the other half of the brain was homogenized to quantify soluble and insoluble Aβ in the brain by ELISA.

On behavioral tests, the GA group experienced an improvement in equilibration time and speed of completion compared to the control group on the first day of the swivel balance test, but this difference disappeared over the next few days. In the mouse open field experiment, the center duration of the GA group was longer, indicating that these mice were more interested in exploring new environments (Figure 13). In the elevated maze test, both groups showed better performance on closed arms, however, the preference for closed and open arms was less pronounced in the GA group than in the control mice (Figure 14). In the Y-maze test, mice in the GA group used the new arm significantly longer than the familiar old arm, indicating that these mice had more desire to explore (Figure 15). In the social interaction test, although no differences were found between the GA group and the control group in the first stage, the two groups did differ in the second and third stages (Figure 16). During the second stage, GA mice showed a preference for the stimulation chamber, while control mice did not. Consistent results were obtained late in the third phase, with these GA mice showing a preference for new stimulation chambers compared to control mice (Figure 16). These behavioral experiments showed that GA-treated mice had a greater desire to explore new environments.

Five weeks after implantation, mice were sacrificed. Fresh mouse brain slices were used to measure LTP, and basal LTP levels were significantly increased in GA-treated mice compared to control mice (Figure 17), indicating that GA treatment improved in vivo memory and learning in aged AD mice. This is consistent with our observations in behavioral testing. In addition, after GA treatment, the amount of brain soluble Aβ decreased from 39 ± 23 to 26 ± 12 pg/㎎ protein, while the ratio of soluble and insoluble Aβ decreased from 0.0040 to 0.0029 (P = 0.023) (Figure 18).

Some mouse brains were also subjected to immunohistochemical staining for IBA-1 (a marker of microglia) and Aβ. Ring-shaped microglia were found in both groups (data not shown). Using the image analysis tool (Image J), Aβ 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 the near-GA-treated mice (Figure 19). In addition, etched annular plaques were found in all GA-treated mice, mainly in cortical areas. In contrast, only a few etched annular plaques were found in control mice (Figure 20). This unique plaque shape may represent the lysis of Aβ plaques in GA-treated mice.

Taken together, our in vitro data suggest that GA can act as a bridging molecule to facilitate in vivo native phagocytosis in vitro. GA binds tightly to Aβ and can antagonize the toxic effect of Aβ42 in the process of LTP reduction, Aβ inserts into the cell membrane of monocytes and increases membrane fluidity. We found that GA could restore or even promote neuronal memory and learning in the presence of Aβ42. These findings are consistent with our in vivo experimental data of direct injection of GA into AD mouse brains. GA-treated mice showed improved behavioral tests, increased basal levels of LTP, decreased brain soluble Aβ and reduced Aβ plaque number/region with evidence of lytic plaques. These results demonstrate the great potential of GA in the treatment of Alzheimer's disease.

We preliminarily demonstrated the safety and efficacy of in vivo intravenous administration and direct intracerebroventricular administration of glatiramer acetate (GA) by animal experiments in New Zealand white rabbits and mice. In order to further verify its safety and obtain pharmacokinetic data, we conducted preliminary experiments with three adult Australian sheep (#1, ewe, 36.5 kg; #2, ram, 35.8 kg; #3, ram , 48.6 kg). This experiment was approved by the Animal Ethics Committee of the Florey Institute, University of Melbourne (18-010).

We first performed intraventricular puncture (Intra Cerebro Ventricular Surgery, ICV) on sheep according to the Florey Institute standard operating procedure (SOP 026): intravenous injection of 5% sodium phthalate (0.4 mg/kg) to induce sheep, insert The tube was then hooked to an anesthesia machine with a 2% isoflurane/air/oxygen mixture and its respiration, heart rate, and blood oxygen levels were monitored throughout the procedure. Sheep were placed in a stereotaxic frame and held in place by mouth, eye, and ear rods. The mouth is located under the front jaw. The eye stick is located on top of the orbital bone, and the ear stick is placed in the ear canal. A 30-40 mm diameter skin hole was cut on the top of the head to expose the anterior junction (the point on the top of the skull where all 3 cranial plates meet). The skull was scraped to clean its periosteum and cleaned with hydrogen peroxide. Five small screws are partially screwed into the skull around the incision. These help anchor the acrylic to the skull. Then drill two small holes in the skull, 10mm to the left and 10mm to the left and 10mm in front of the anterior brine point. A catheter (#8 needle size) was then inserted into the brain through the hole. As the catheter entered the lateral ventricle, a pressure transducer was attached to the saline infusion line, the catheter showed a drop in pressure, and the catheter remained in place. It is now in the center of the lateral ventricle. The process is then repeated in the other ventricle. The holes are filled with Surgicel to prevent fluid leakage and acrylic from entering the brain. Then, dental acrylic is molded over the skull to create a hard, protective surface that holds the catheter in place. The bleeding was stopped with an electrocoagulation hemostatic pen, and the wound was sutured. The sheep were then removed from the isoflurane anesthetic and placed on an air/oxygen mixture. When the swallowing reflex is evident, the cannula is removed and the sheep is returned to the operating table. Each animal received an intramuscular injection of Flunixin meglamine 1 mL (1 mg/kg) for pain relief. The sheep were then returned to their cages and monitored for several hours. Penicillin was injected daily for the next three days (Figure 21).

We first examined the safety of GA. On day 1 after ICV surgery, we injected 1 mL of vehicle solution (4% mannitol in artificial cerebrospinal fluid, sterile filtered) at a rate of 0.5 mL/min from the ICV catheter at the same rate for all injections. Collect 0.5 mL of cerebrospinal fluid, 5 mL of blood, and 5 mL of urine. Used to measure to establish a baseline. Then GA is injected once a week, the standard dose is 10mg/0.5mL, usually on Mondays, a total of 8 times, during which blood is collected once or twice for inspection. Daily observation and recording of heartbeat, respiration, anal temperature, forage consumption, water intake, defecation, urination, etc. We found that within 24 hours after each injection of GA, the body temperature generally increased by 0.5 °C, but the body temperature remained between 37.8-39.5 °C throughout the experiment, and no body surface abnormalities were found. Since the beginning of GA injection, the most obvious change is that the animal's diet has increased greatly, and the forage consumption has increased from the initial 400-800 grams to 800-2400 grams, and sometimes even more than 3000 grams. The drinking water is increased from the initial 4-5 liters to 8-16 liters. At the same time, excretion was also greatly increased, with fecal excretion increasing by an average of 1-2 times, while urination increased several times, reaching an astonishing 12 liters (the maximum volume of a urine bucket) (Figure 22). This continued until one month after the end of the entire injection procedure, showing that intraventricular injection of GA greatly enhanced the metabolic capacity of the animals. It should be pointed out that we checked the blood routine, urine routine, thyroid function, liver function, kidney function, etc. of the animals, and there was no abnormality or significant change. There was no significant increase in body weight of the animals. It is generally known that the brain is the organ that consumes the most energy in the human body. Our results suggest that the increase in metabolism caused by GA is mainly to replenish the energy required by the brain, indicating that some brain functions are activated.

We next investigated the pharmacokinetics of GA. We first labelled GA with the fluorescent dyes Alexa488 and Alexa647, and determined the concentration with Direct Detect (Merck) and developed a standard curve with a spectrofluorometer. 4-6 weeks after the animals' last injection of GA, the animals were cannulated in the external jugular vein for blood sampling. Briefly, sheep will first receive local anesthesia (2% lidocaine) by subcutaneous injection, a single lumen 18-gauge central venous catheter is aseptically placed in the external jugular vein, and tape is tapered with fabric

Bandage the site to protect it from animals. The cannula secures the quilt tape to the cannula that is sutured to the skin. The catheter was flushed with 1-2 mL of heparin-anticoagulated saline (100 IU/mL) before and after sampling. The cannula will remain in the sheep for no more than four days. After the first collection of cerebrospinal fluid, blood, and urine, we injected 10 mg of fluorescently labeled GA via the ventricle, followed by collecting the samples, and measuring the fluorescence intensity in the samples. Cerebrospinal fluid (0.5 mL each) was collected by intraventricular catheter at 0.5, 1, 2, 4, 8, 24, 48 and 72 hours after injection, blood (5 mL each) was collected from external jugular vein cannula, urine (5 mL each) collected from the urine sample collector. The results showed that the GA content in the cerebrospinal fluid decreased rapidly after 24 hours and almost completely disappeared at 48 hours; while the GA content in the blood increased after 24 hours and lasted at least 72 hours; because of the dark fluorescence background in the urine, cannot be measured accurately.

The total amount of sheep cerebrospinal fluid is about 14mL. In the experiment, we directly injected 10mg GA into the sheep ventricle, and the initial concentration was about 0.7mg/mL, which was far beyond the working concentration of GA (0.1mg/mL). Even at this high concentration, the sheep did not show any discomfort. The total amount of human cerebrospinal fluid is about 120mL, and only 12mg GA is needed to reach the working concentration each time, so the safety is guaranteed. GA can be rapidly metabolized into peripheral blood in the brain, indicating that it can penetrate the blood-brain barrier.

Claims (6)

1. Application of glatiramer acetate in the preparation of Aβ42 toxicity inhibitor and scavenger.
2. Application of glatiramer acetate as Aβ42 toxicity inhibitor and scavenger in the preparation of drugs for treating Alzheimer's disease.
3. A method for treating Alzheimer's disease, characterized in that the method is to directly infuse glatiramer acetate into the central nervous system.
4. The method of claim 3, wherein the method of direct infusion into the central nervous system can be by intravenous drip, nasal spray, spinal tap, or infusion through an intrathecal injection implanted device.
5. The method of claim 3 or 4, wherein the glatiramer acetate can be used alone or in combination with other compounds.
6. The method of claim 5, wherein the other compound is a P2X7 antagonist.

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