Use of innate phagocytosis-promoting polypeptide in preparing drug for treating alzheimer's disease

Abstract

Use of an innate phagocytosis-promoting polypeptide in preparing a drug for treating Alzheimer's disease.

Description

Application of natural phagocytosis-promoting polypeptide in the preparation of drugs for treating Alzheimer's disease Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a natural phagocytosis-promoting polypeptide in the preparation of a drug for treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) imposes a significant social and economic burden on society, affecting over 26 million people worldwide. China currently has approximately 9.83 million AD patients, resulting in a severe disease burden that places heavy medical, caregiving, and economic demands on families and society. One in 10 Australians over the age of 65, and three in 10 over the age of 85, are affected by AD. Without medical breakthroughs, the number of Australians with AD is projected to approach one million by 2050. Currently, there is no cure, and existing treatments can only improve mild to moderate symptoms for a limited time. To date, there are no treatments proposed for even advanced stages of the disease.

[0003] Innate immunity, a fundamental biological function, maintains body homeostasis. Innate phagocytosis is a crucial component of immunity, enabling the recognition and phagocytosis of apoptotic cells, cellular debris, protein aggregates, and invading bacteria without the need for antibodies or complement. Several genome-wide association studies (GWASs) and other sporadic genetic studies in the AD field have identified a cluster of AD risk genes within the core innate immune pathway, including CD33, CR1, MS4A6A, MS4A4E, ABCA7, and TREM2. Variants in these genes, particularly TREM2 and CD33, are associated with impaired phagocytic function of monocytes / macrophages and altered Aβ accumulation in the brains of AD patients. Notably, complement receptor 1 (CR1, also known as CD35) is primarily expressed in peripheral blood leukocytes and erythrocytes but is not expressed in the brain under normal physiological conditions. Therefore, in addition to excessive neuroinflammation, systemic phagocytosis, involving both the central nervous system (CNS) and peripheral tissues, may be the primary cause of Aβ accumulation and clearance failure.

[0004] The inventors first proposed the concept of "innate phagocytosis" and have been dedicated to this research over the past few years. Using the purinergic P2X7 receptor as a model, the inventors have elucidated how this receptor, in conjunction with related cytoskeletal proteins, functions as a scavenger receptor, clearing apoptotic cells from the central nervous system (CNS) before necrotic death and the resulting inflammation. Furthermore, although scavenger function can be completely inhibited by 1-5% serum, innate phagocytosis is most active in the presence of cerebrospinal fluid, suggesting that this innate phagocytic function may be important in the brain.

[0005] The deposition and aggregation of Aβ, forming amyloid plaques, are well-established hallmarks of AD and are widely considered to be related to the disease's etiology. Consequently, current drug discovery for AD focuses primarily on using antibodies to clear existing amyloid deposits. However, since 1998, over 100 clinical trials targeting Aβ have failed or shown only a glimmer of hope. Only recently have the antibody-based approaches—Aducanumab, Lecanemab, and Donanemab—showed some potential for treating AD. However, these therapies can potentially induce amyloid-associated imaging abnormalities (ARIA) due to their enhanced blood-brain barrier (BBB) ​​penetration. Consequently, alternative treatment strategies for AD are urgently needed. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies of the prior art and provide an application of a natural phagocytosis-promoting polypeptide in the preparation of a drug for treating Alzheimer's disease.

[0007] The present invention focuses on a more effective way to prevent the accumulation of Aβ or other proteins by promoting the natural phagocytic clearance of these fragments and inhibiting chronic neuroinflammation, thereby improving the healthy environment of the brain and completely treating the disease.

[0008] Phagocytosis promoting peptides (PPP) are a group of peptides that act as linker molecules between scavenger receptors to phagocytic cells and their targets (> 0.5 µm in size), such as apoptotic cells, cell debris, accumulated proteins such as Aβ, microorganisms, and microbeads, thereby immediately promoting phagocytosis of these targets.

[0009] Twelve PPPs have been identified, including peptides that mimic the natural sequences of certain proteins, as well as randomly synthesized peptides, such as glatiramer acetate, which contains both D-terminal and L-terminal forms of glutamic acid, lysine, alanine, and tyrosine. Common features of PPPs include: (1) an isoelectric point > 9.6; (2) no cysteine; and (3) rich in arginine and / or lysine.

[0010] In vitro and in vivo experimental data from the present invention demonstrate that these PPPs can promote natural phagocytosis both in vitro and in vivo. Some PPPs can bind to Aβ as tightly as glatiramer acetate, potentially eliminating the toxic effects of Aβ42 that inhibit long-term protein production (LTP). In vivo experiments, BG01 was injected into 22-month-old APP / PS1 transgenic mice (an animal model of Alzheimer's disease with a lifespan of 26-27 months) using an osmotic minipump. Five weeks later, LTP levels increased compared to a control group. These results are similar to those previously obtained by the inventors for glatiramer acetate, also validated in transgenic mice. These PPPs, sharing common characteristics, could potentially serve as novel therapeutic agents for Alzheimer's disease. Specifically, these common characteristics include: ① significantly enhancing the natural phagocytic capacity of cells; ② an isoelectric point > 9.6; ③ lacking cysteine; and ④ being rich in arginine and / or lysine. PPPs ranging in length from 10 to 20 amino acids could be used to develop therapeutics for Alzheimer's disease. Specifically, PPP includes polypeptides with amino acid sequences such as those shown in SEQ ID NO.1 to SEQ ID NO.12, as well as derivative polypeptides based on these polypeptides and polypeptides meeting such characteristics. The PPP described in the present invention can be implanted into the cerebrospinal fluid sac to treat AD via intravenous injection, nasal spray, and direct intrathecal injection. PPP has shown a high affinity for Aβ and a strong effect in increasing the level of LTP in the brain neurons of elderly AD mice. It is speculated that this treatment method can be used to treat AD, rather than just another disease-correcting therapy. PPP can be used alone or in combination with other compounds such as P2X7 antagonists to have a synergistic therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1: PPP peptides pass through CD14 + Human monocytes promote phagocytosis of YG microspheres. Before adding YG microspheres, cells were labeled with APC-anti-CD14 monoclonal antibody and incubated with peptides (10 µg / mL) or glatiramer acetate (GA 100 µg / mL) for 10 minutes, and CD14 + The mean fluorescence intensity of YG microspheres in monocytes was plotted with time. A. Typical YG microsphere phagocytosis timeline; B. The percentage of phagocytic ability enhanced by each PPP peptide compared with the baseline.

[0012] Figure 2: Microscale thermophoresis (MST) analysis of BG01 binding to Aβ. A. Separation curves of freshly prepared BG01 at varying concentrations and freshly prepared 80 nM HiLyte Fluor 488-labeled Aβ40 (green dots) and Aβ42 (red dots). B. Separation curves of freshly prepared BG01 at varying concentrations and 16-day-old Aβ40 (green dots) and Aβ42 (red dots). Measurements were performed in standard processing capillaries of the Monolith NT.115 system, using 95% LED and 40% IR laser power.

[0013] Figure 3: Tufts-promoting polypeptide (PPP) antagonizes the toxic effects of Aβ42 on long-term potentiation (LTP). We used a multi-electrode array (MEA) electrophysiological recording of LTP traces in mouse brain slices. Fresh mouse hippocampal slices were mounted on a 3D-MEA chip with 60 electrodes with 30 µm high tips and a spacing of 200 µm. The slices were continuously perfused with artificial cerebrospinal fluid (aCSF, 3 mL / min, 32°C) or 5 µM Aβ42. ， Alternatively, a mixture of peptide (5μM) and Aβ42 (5μM) was used. Data were collected using a multichannel system (MCS GmbH, Reutlingen, Germany). Schaffer collaterals were stimulated by injecting a biphasic current waveform (100 μs) at 0.033 Hz through a selected electrode. Peak-to-peak amplitudes of field excitatory postsynaptic potentials (fEPSPs) in the proximal stratum radiatum of CA1 were then analyzed using an LTP-Analyzer. A. Typical LTP recording curve; B. Statistical results after integration of multiple curves; C. Statistical bar graph; D. Human THP-1 monocyte phagocytosis of YG microspheres. Cells treated with different peptides exhibited varying phagocytic capacities. BGX7FD, the all-D-amino acid form of BG01, does not stimulate phagocytosis; E. LTP recordings show that the BGX7FD peptide cannot antagonize the effects of Aβ42 on LTP; F. Statistical bar graph.

[0014] Figure 4: Tuftsin peptide (PPP) antagonizes the toxic effects of Aβ42 on long-term potentiation (LTP). Different PPPs demonstrated varying degrees of antagonism against the inhibitory effects of Aβ42 on LTP. A. & B.: MBP13; C. & D.: MBP30; E. & F.: MBP15; G. & H.: MBP28. The infusion concentration of both peptides and Aβ42 was 5 μM.

[0015] Figure 5: Direct intracerebroventricular administration of the phagocytic polypeptide (PPP) BG01 alters behavioral traits in aged APP / PS1 mice. Three weeks after administration, mice in the PPP group (n=7) and control group (n=7) underwent a series of behavioral tests, including open field time (LOF), elevated maze (EPM), rota rod balance, buried food search, Y maze, T maze, and social interaction.

[0016] Figure 6: Preliminary data on the long-term potentiation effect of BG01 administered intracerebroventricularly in 22-month-old APP / PS1 mice. PBS containing 4% mannitol served as a vehicle control. A mini-osmotic pump containing 100 µL of the agent (28-day release) was implanted into the back of the mouse and connected to the ventricle via a brain infusion kit. Behavioral testing was performed three weeks later, and LTP was assessed in brain slices 4-5 weeks later. (n (mouse) = 3-5; n (slice) = 7-8; n (electrode) = 21-27) (**P < 0.01, ****P < 0.0001).

[0017] Figure 7: Transgenic mice, P2X7-307Q, antagonize the toxic effects of Aβ42 on long-term potentiation (LTP). The transgenic mice, P2X7-307Q, were generated using CRISP-CAS9 technology in the C57BL / 6 mouse background. Arginine at position 307 on the P2X7 receptor was replaced with glutamine (R307Q), corresponding to the sequence of BG01. Multielectrode array (MEA) electrophysiological recordings were performed in brain slices from C57BL / 6 mice and transgenic mice, as well as the effects of Aβ42 on both mouse models. DETAILED DESCRIPTION

[0018] Innate phagocytosis is the most important component of the human innate immune system and is crucial for the body's development and homeostasis. Innate phagocytosis recognizes and eliminates apoptotic cells, cell debris, and invading microorganisms without the need for conditioning. Rapid clearance of dead or dying neuronal cells by specialized scavenger cells (phagocytes) is crucial to avoid inflammation and allow neurogenesis in the central nervous system; rapid clearance of misfolded protein complexes / fibulae is key to avoiding neurodegeneration; and rapid clearance of invading microorganisms is the body's first line of defense. However, this important biological function has long been overlooked, and the concept of "innate phagocytosis" has never even been proposed.

[0019] Since 2007, the inventors have made a series of discoveries in the field of natural phagocytosis by studying an adenosine triphosphate (ATP)-gated ion channel called "P2X7", which mediates proinflammatory responses in the presence of extracellular ATP. In microglia and macrophages, short-term exposure to extracellular ATP that activates the P2X7 receptor opens the cation-selective channel, and more prolonged exposure to P2X7 and ATP leads to the formation of a large number of pores and a huge amount of K + Efflux, a stimulus for inflammasome assembly, leads 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 several hours. Since 2007, the inventors have discovered another "hidden" function of P2X7: a scavenger receptor that clears the natural phagocytosis of non-opsonized particles in the absence of extracellular ATP. The inventors have demonstrated that the P2X7 receptor has a tight molecular association with non-myosin heavy chain IIA (NMMHC-IIA) in monocytes, and that this complex molecular relationship mediates the phagocytosis of non-opsonized beads, live and dead bacteria, and apoptotic cells. While a physiological agonist of P2X7-mediated pore formation and proinflammatory responses, ATP is actually an antagonist of P2X7-mediated phagocytosis because it disaggregates the P2X7-NMMHC-IIA complex, which is required for the internalization of captured particles. In addition, P2X7-mediated phagocytosis is most active in the presence of cerebrospinal fluid, and only 1-5% serum can completely inhibit the scavenger function of P2X7, suggesting that this function of P2X7 may be of special importance in the central nervous system in the absence of serum.

[0020] The inventors have previously developed a quantitative method for measuring the phagocytic ability of phagocytes. The method of measuring the function of leukocyte subsets using real-time multicolor flow cytometry first reported evidence of P2X7 receptor non-function. Later, methods for measuring P2X7 channel / pore function and phagocytic function assays were further developed with protein interactions based on fluorescence resonance energy transfer (FRET). This three-color real-time flow cytometry method for quantitative cell phagocytic function was used to screen PPPs from a peptide library. Using this method, the inventors discovered a group of peptides that promote phagocytosis of human natural monocytes from the peptide library and their own designed peptides, including some peptides that mimic the natural ordering of specific proteins (Table 1, Figure 1). The common characteristics of these phagocytic peptides (PPPs) include: (1) isoelectric point > 9.6; (2) no cysteine; (3) rich in arginine and / or lysine; and (4) length of 10 to 20 amino acids.

[0021] Table 1: PPP peptides

[0022] Name Amino acid sequence Molecular weight Isoelectric point BG01KQTLIKVFGIRFDIL (SEQ ID NO.1) 17919.99 BG02KRTLIKVFGIRFDIL (SEQ ID NO.2) 181911.53 MP013RGLSLSRFSWGAEGQRPGFG (SEQ ID NO.3) 216612.2 MP023LDVMASQKRPSQRHGSKYLA (SEQ ID NO.4) 227310.88 MP219PVVHFFKNIVT (SEQ ID NO.5) 13019.69 MP146SKYLATASTMDHARHGFLPR (SEQ ID NO.6) 225910.45 MP028RTPPPSQGKGRGLSLSRFSW (SEQ ID NO.7) 221512.81 MP014YGGRASDYKSAHKGFKGVDA (SEQ ID NO.8) 21149.81EB482AEGLRALLARSHVER (SEQ ID NO.9) 167810.31SX121RSKAFDDIATYFSKKEWKKM (SEQ ID NO.10) 248010.37MP015QGTLSKIFKLGGRDSRSGSP (SEQ ID NO.11) 209111.65SX441KSSEKIVYVYMKLNYEVMTK (SEQ ID NO.12) 24549.66

[0023] To serve as a bridging molecule for phagocytosis, it also needs to have good targeting and binding capabilities. We then tested whether PPP could interact with Aβ. Microscale thermophoresis (MST) was used to determine the binding affinity between PPP and Aβ42. Aβ42 (80 nM, dissolved in PBS) was labeled with HiLyte Fluor488 fluorescent dye, and the phagocytic peptide PPP was serially diluted in a 1:1 ratio (volume). Measurements were performed in standard processing capillaries of the Monolith NT.115 system using 95% LED and 40% IR-laser power. The inventors' previous patent applications have disclosed that glatiramer acetate and Aβ4 have a high affinity (K D = 6.6 nM). In this application, the inventors found that other PPPs also have similar functions. The binding constants K of BG01 and freshly prepared Aβ42 and Aβ40 (both mainly monomeric) are D 6.10×10 -8 M and 4.53×10 -8 M, and the binding constant K of Aβ42 and Aβ40 (mainly polymers) after being placed at 4°C for 16 daysD 3.50×10 -8 M and 1.63×10 -8 M (Figure 2), indicating that BG01 prefers to bind to Aβ polymers. Aβ polymers (oligomers) and protofibrils (80-1000 kD) are the most toxic forms of Aβ and the most effective targets for anti-amyloid therapy.

[0024] After confirming the interaction between PPP and Aβ42, the inventors went on to test whether the phagocytic peptide PPP could inhibit the toxic effects of Aβ42. Long-term potentiation (LTP) is an important in vitro measure of neuronal memory and learning. The inventors have previously demonstrated that glatiramer acetate antagonizes the toxic effects of Aβ42 in LTP. The inventors further tested other PPPs and found that BG01, BG02, MBP13, MBP15, MBP28, and MBP30 all exhibited similar effects. BG01 and MBP13 even demonstrated, similar to glatiramer acetate, not only complete restoration of the toxic damage caused by Aβ42 but also increased baseline LTP levels. In the presence of 5µM Aβ42, the same concentration of PPP was sufficient to completely block the toxic effects of molecular Aβ42 (Figures 3 and 4). As a control, we replaced all the L-amino acids in BG01 with D-amino acids (BGX7FD) and found that BGX7FD had no effect on stimulating phagocytosis (Figure 3D) and also had no effect on antagonizing Aβ42 (Figure 3E&F), indicating that there is a close connection between increasing phagocytosis levels and antagonizing the toxic effects of Aβ42.

[0025] The inventor's previous patent application has disclosed that glatiramer acetate showed considerable therapeutic effects in AD animal models. In this patent application, the inventor further studied the therapeutic effects of other PPPs on AD mouse models by implanting mini-osmotic pumps for direct brain perfusion. APP / PS1 transgenic mice are widely used in AD animal models. These mice have been treated with Aβ accumulation for more than four months, but it takes 10 months for cognitive impairment in mice to become obvious. The life expectancy of this type of mouse model is between 25 and 27 months. In order to better simulate human diseases, the inventor used female aged AD mice aged 22-34 months for this study.

[0026] All animals were surgically implanted with osmotic micropumps (ALZET, Model 1004, 100 µL reservoir volume, four-week duration) and ALZET Brain Perfusion Pack 3. BG01 (20 mg / mL) (n = 9) or a control (PBS containing 4% mannitol) (n = 8) were infused into the micropumps (100 µL each). Animals were anesthetized with inhaled isoflurane, and the micropumps were implanted in the dorsal subcutaneous space, 2.5 mm below the skull, which is suitable for intracerebroventricular positioning in adult mice. The procedure was performed as previously described. The agents were expected to be slowly released over four weeks. The Florey Animal Ethics Committee approved this study (19-089).

[0027] Three weeks after implantation, behavioral tests (rotational balance, open field, Y-maze, elevated maze, and social interaction) were performed for two consecutive weeks. After completion of these behavioral tests, the mice were sacrificed and their brains were collected for LTP measurements, Aβ loading was stained using immunohistochemistry, and soluble and insoluble Aβ were quantified using ELISA.

[0028] While behavioral tests showed differences between the treated and control groups, no other tests showed statistically significant differences except for the Y-maze (Figure 5). Five weeks after implantation, the inventors sacrificed the mice. Fresh brain slices were used to measure long-term protein translation (LTP). Compared to control mice, basal LTP levels were significantly elevated in BG01-treated APP / PS1 aged mice (Figure 6), indicating that BG01 treatment improved memory and learning abilities in aged AD mice. This is consistent with the effects previously observed by the inventors with glatiramer acetate.

[0029] The BG01 sequence is derived from the P2X7 receptor (amino acid positions 306-320) but contains a mutation at position 307, replacing arginine 3 with glutamine (R307Q). This mutation enhances the natural phagocytic ability of the P2X7 receptor. We therefore used CRISP-Cas9 technology to generate a transgenic mouse model that mimics this mutation (R307Q). After successfully generating these transgenic mice, we compared heterozygous P2X7-307R / Q mice with the C57BL / 6 background strain. The P2X7-307R / Q mice showed slightly higher baseline LTP levels than wild-type C57BL / 6 mice and were also able to resist the toxic effects of Aβ42 (Figure 7). This is consistent with the in vitro results using the BG01 peptide.

[0030] In summary, the inventors' in vitro experimental data demonstrate that, like glatiramer acetate, BG01 and other PPPs can act as bridging molecules, promoting natural phagocytosis in vitro. PPPs bind tightly to Aβ and can antagonize the toxic effects of Aβ42 during the reduction of LTP. The inventors found that PPPs can restore and even enhance neuronal memory and learning abilities in the presence of Aβ42. These results suggest a new approach to treating AD, namely, utilizing PPPs, and particularly using multiple PPPs with synergistic enhancing effects as drugs to treat AD.

Claims

1. Application of natural phagocytic polypeptide in the preparation of drugs for the treatment of Alzheimer's disease.

2. The use according to claim 1, characterized in that In addition to being able to promote the phagocytic function of cells, the common characteristics of the natural phagocytosis-promoting polypeptides include: ① isoelectric point> 9.6; ② no cysteine; ③ rich in arginine and / or lysine; ④ length of 10 to 20 amino acids.

3. The use according to claim 2, characterized in that The natural phagocytosis-promoting polypeptides include polypeptides with amino acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.12, as well as derivative polypeptides based on these polypeptides and polypeptides meeting such characteristics.

4. A drug for treating Alzheimer's disease, characterized in that: The drug is a natural phagocytosis-promoting polypeptide. In addition to being able to promote the natural phagocytosis function of cells, the common characteristics of the natural phagocytosis-promoting polypeptide also include: ① isoelectric point> 9.6; ② no cysteine; ③ rich in arginine and / or lysine; ④ length of 10 to 20 amino acids.

5. The drug according to claim 4, characterized in that The natural phagocytosis-promoting polypeptides include polypeptides with amino acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.12, as well as derivative polypeptides based on these polypeptides and polypeptides meeting such characteristics.

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