Application of polypeptide for promoting natural phagocytosis in preparation of medicine for treating Alzheimer's disease

Abstract

The natural phagocytosis promoting polypeptide is applied to preparation of a medicine for treating Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application 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 huge social and economic burden on society, affecting more than 26 million people worldwide. There are currently about 9.83 million AD patients in China, and the disease burden is serious, which brings heavy medical, care and economic burdens to families and society. And one in 10 Australians over the age of 65 and three in those over the age of 85 are affected by Alzheimer's disease. Without medical breakthroughs, by 2050, the number of Australians with Alzheimer's disease is expected to be close to 1 million. There is currently no cure, and existing treatments can only improve mild to moderate symptoms for a limited time. To date, no treatment has even been proposed for the late stage.

[0003] As a basic biological function, innate immunity maintains the body's homeostasis. Innate phagocytosis is one of the important components of the body's immunity, which can recognize and phagocytose apoptotic cells, cell debris, protein aggregates, and invading bacteria without antibodies or complement. Several genome-wide association studies (GWASs) and other scattered genetic studies in the field of AD have identified a set of AD risk gene groups belonging to the core innate immune pathway, including CD33, CR1, MS4A6A, MS4A4E, ABCA7, and TREM2. Variations in these genes, especially TREM2 and CD33, are associated with impaired phagocytic function of monocytes / macrophages in the brain of AD patients and altered Aβ accumulation. 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 defective phagocytosis, involving the central nervous system (CNS) and peripheral tissues, may be the main cause of Aβ deposition and clearance failure.

[0004] The inventors first proposed the concept of "innate phagocytosis" and have been working on this topic in recent years. Using the purinergic P2X7 receptor as a model, the inventors have elucidated how this receptor works together with related cytoskeletal proteins to function as a scavenger receptor to clear apoptotic cells in the central nervous system (CNS) before necrotic death and the resulting inflammation. In addition, although 1-5% serum can completely inhibit scavenger function, innate phagocytosis is most active in the presence of cerebrospinal fluid, indicating that this innate phagocytic function may be important in the brain.

[0005] The deposition and aggregation of Aβ to form amyloid plaques, and the established special features of AD are widely believed to be related to the disease etiology. Therefore, current drug discovery for AD focuses on the use of antibodies to remove existing amyloid deposits. However, since 1998, more than 100 clinical trials for Aβ have failed or only shown a "glimmer of hope". Until recently, these antibody-based approaches, Aducanumab, Lecanemab and Donanemab have shown some potential for the treatment of AD, but these therapies may cause amyloid-related imaging abnormalities (ARIA) due to increased blood-brain barrier (BBB) ​​penetration. In this regard, the inventors are in urgent need of alternative treatment strategies for AD. 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 Aβ or other proteins from accumulating by promoting natural phagocytosis to clear these debris and inhibit chronic neuroinflammation, thereby improving the healthy environment of the brain to completely treat 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] The present invention has identified 12 PPPs, including peptides that mimic the natural sequences of certain proteins, and randomly synthesized peptides, such as glatiramer acetate, which contains both the D-terminal and L-terminal forms of glutamic acid, lysine, alanine, and tyrosine. The common features of PPPs are: (1) isoelectric point>9.6; (2) no cysteine, and (3) rich in arginine and / or lysine.

[0010] In the present invention, in vitro and in vivo experimental data show that these PPPs can promote natural phagocytosis both in vivo and in vitro. Some PPPs can bind Aβ tightly like glatiramer acetate, and can eliminate the toxic effects of Aβ42 that inhibit LTP. In the in vivo experiment, BG01 was injected into 22-month-old APP / PS1 transgenic old mice (an AD animal model with a life expectancy of 26-27 months) using an osmotic micropump, and the LTP level was increased compared with the blank control group after 5 weeks. These results are similar to the results of glatiramer acetate obtained by the inventors before, and have also been verified on transgenic mice. It is suggested that these PPPs with common characteristics can become a new AD therapeutic drug. That is, the common characteristics include ① greatly improving the natural phagocytic ability of cells, ② isoelectric point>9.6, ③ no cysteine, ④ rich in arginine and / or lysine, and PPPs with a length of 10 to 20 amino acids can be used to prepare drugs for the treatment of Alzheimer's disease. Specifically, PPP is a polypeptide including 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 that meet such characteristics. The PPP described in the present invention can be implanted into the cerebrospinal fluid capsule to treat AD by intravenous injection, nasal spray method and direct intrathecal injection. PPP shows a high affinity for Aβ and a strong effect in improving the level of LTP in the brain neurons of old 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, etc., and has a synergistic therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 PPP peptides 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 min, and CD14 + The average fluorescence intensity of YG microspheres in monocytes and time graphs are plotted. A. Typical YG microsphere phagocytosis timeline; B. The percentage of phagocytosis enhanced by each PPP peptide compared to the baseline.

[0012] Figure 2:Microscale thermophoresis (MST) analysis of the binding force between BG01 and Aβ. A. Separation curves of freshly prepared BG01 at different concentrations and freshly prepared 80nM HiLyte Fluor488 fluorescently stained Aβ40 (green dots) and Aβ42 (red dots); B. Separation curves of freshly prepared BG01 at different concentrations and Aβ40 (green dots) and Aβ42 (red dots) after 16 days of storage. The measurements were performed in the standard processing capillaries of the Monolith NT.115 system, using 95% LED and 40% IR-laser power.

[0013] Figure 3 : The phagocytic polypeptide (PPP) antagonizes the toxic effect of Aβ42 on long-term potentiation (LTP). We used multi-electrode array (MEA) electrophysiological recordings of LTP traces in mouse brain slices. Fresh mouse hippocampal slices were mounted on a 3D-MEA chip with 60 electrodes with a tip shape and 30 μm height, with a spacing of 200 μm. Slices were continuously perfused with artificial cerebrospinal fluid (aCSF, 3 mL / min, 32°C), either 5 μM Aβ42, or a mixture of peptide (5 μM) and Aβ42 (5 μM). Data were collected using a multichannel system (MCS GmbH, Reutlingen, Germany). A biphasic current waveform (100 μs) was injected at 0.033 Hz through a selected electrode to stimulate the Schaffer collateral circulation, and the peak-to-peak amplitude of the field excitatory postsynaptic potential (fEPSP) in the proximal stratum radiatum of CA1 was analyzed using the 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 showed different phagocytic abilities after being treated with different peptides. BGX7FD is the all-D-amino acid form of BG01 and does not have the ability to stimulate phagocytosis; E. LTP recording shows that BGX7FD peptide cannot antagonize the effect of Aβ42 on LTP; F. Statistical bar graph.

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

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

[0016] Figure 6 : Preliminary data on the long-term potentiation effect of BG01 administered intracerebroventricularly to 22-month-old APP / PS1 mice. Using PBS containing 4% mannitol as a vehicle control, a mini-osmotic pump containing 100 μL of the agent (28-day release) was implanted in 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 measured on 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 show antagonism to the toxic effects of Aβ42 on long-term potentiation (LTP). Transgenic mice P2X7-307Q were generated using CRISP-CAS9 technology in the C57BL / 6 mouse background, and the arginine at position 307 on the P2X7 receptor was replaced with glutamine (R307Q), corresponding to the sequence of BG01. We used multi-electrode array (MEA) electrophysiology to record LTP traces in brain slices of C57BL / 6 mice and transgenic mice, as well as the effects of Aβ42 on these two types of mice. DETAILED DESCRIPTION

[0018] Natural phagocytosis is the most important component of the human innate immune system and is essential for the development and homeostasis of the organism. Natural phagocytosis does not require conditioning to recognize and remove apoptotic cells, cell debris, and invading microorganisms. Rapid clearance of dead or dying neuronal cells by specialized scavenger cells (phagocytes) is critical to avoid inflammation and allow neurogenesis in the central nervous system; rapid clearance of misfolded protein complexes / fibulae is key to avoid neurodegeneration; and rapid clearance of invading microorganisms is the inventor's body's first line of defense. However, this important biological function has long been overlooked, and the concept of "natural 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, a brief exposure to extracellular ATP that activates the P2X7 receptor opens the cation-selective channel, and more prolonged exposure of P2X7 and ATP leads to the formation of a large number of pores and a huge amount of K + Efflux, which is a stimulus for the assembly of “inflammasomes”, 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 a few hours. Since 2007, the inventors have discovered another “hidden” function of P2X7: a scavenger receptor that clears natural phagocytosis of non-opsonized particles in the absence of extracellular ATP. The inventors have shown that the P2X7 receptor has a tight molecular association with non-myosin heavy chain IIA (NMMHC-IIA) in monocytes and that this complex molecular association mediates the phagocytosis of non-opsonized beads, live and dead bacteria, and apoptotic cells. ATP, a physiological agonist of P2X7-mediated pore formation and pro-inflammatory responses, is actually an antagonist of P2X7-mediated phagocytosis, because it disassembles 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 determining the phagocytic ability of phagocytes. The method of measuring the function of leukocyte subsets using real-time multicolor flow cytometry reported for the first time evidence of the non-functional P2X7 receptor, and later further developed methods for measuring P2X7 channel / pore function, as well as phagocytic function assays and 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 PPP from a peptide library. Using this method, the inventors discovered a group of peptides that promote the phagocytosis of human natural monocytes from the peptide library and their own designed peptides, including certain 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; (4) length of 10 to 20 amino acids.

[0021] Table 1: PPP peptides [Table 1_sm_0001]

[0023] To serve as a bridging molecule for phagocytosis, it also needs the ability to target well. We then tested whether PPP could interact with Aβ. Microscale thermophoresis (MST) was used to determine the binding affinity between PPP and Aβ42. Aβ42 (80nM, dissolved in PBS) was labeled with HiLyte Fluor488 fluorescent stain, and the phagocytic peptide PPP was serially diluted in a 1:1 ratio (volume). Measurements were performed in the 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 the present application, the inventors found that other PPPs also have similar functions. The binding constant K of BG01 and freshly prepared Aβ42 and Aβ40 (both mainly monomers) is D They are 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 days D 3.50×10 -8 M and 1.63×10 - 8 M( Figure 2 ), showing that BG01 prefers to bind to Aβ polymers. Aβ polymers (oligomers) and protofibrils (80-1000KD) are the most toxic forms of Aβ and the most effective targets for anti-amyloid protein treatment.

[0024] After determining the interaction between PPP and Aβ42, the inventors continued to test whether the phagocytic peptide PPP could inhibit the toxic effects of Aβ42. Long-term potentiation (LTP) is an important in vitro measurement of neuronal memory and learning ability. The inventors have previously disclosed that glatiramer acetate has the ability to antagonize the toxic effects of Aβ42 in long-term potentiation. The inventors further tested the remaining PPP and found that BG01, BG02, MBP13, MBP15, MBP28 and MBP30 all had similar effects. BG01 and MBP13 can even be like glatiramer acetate, not only completely restoring the toxic damage caused by Aβ42 but also increasing the baseline level of LTP. When 5μM Aβ42 is present, the same concentration of PPP is sufficient to completely block the toxic effects of molecular Aβ42 ( Figure 3 and Figure 4 As a control, we replaced all L-amino acids in BG01 with D-amino acids (BGX7FD) and found that BGX7FD had no phagocytic stimulating effect ( Figure 3 D), and also lost its antagonistic effect on Aβ42 ( Figure 3 E&F), indicating that there is a close relationship between increasing phagocytic 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 of mice to become obvious, and 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 22-34 month old female aged AD mice for this study.

[0026] All animals were surgically implanted with micro-osmotic pumps (ALZET, model 1004, 100 μL reservoir volume, four-week duration) and ALZET brain perfusion pack 3. BG01 (20 mg / ml) (n=9) or blank control (PBS containing 4% mannitol) (n=8) was injected into the micro-osmotic pump (100 μL each). The animals were anesthetized by inhalation of isoflurane, and the micro-pump was implanted in the dorsal subcutaneous space, penetrating 2.5 mm below the skull, which is suitable for positioning in the lateral ventricle of adult mice. The entire procedure was performed according to the instructions described previously. The agents are expected to be slowly released over a four-week period. The Florey Animal Ethics Committee approved this study (19-089).

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

[0028] In the behavioral tests, although there were differences between the drug-treated group and the control group, no other tests showed statistically significant differences except for the Y-maze ( Figure 5 Five weeks after implantation, the mice were killed. Fresh brain slices were used to measure LTP. Compared with control mice, the basal LTP level of BG01-treated APP / PS1 aged mice was significantly increased ( Figure 6), indicating that BG01 treatment improved the memory and learning ability of elderly AD mice in vivo. This is consistent with the effect of glatiramer acetate previously observed by the inventors.

[0029] The BG01 sequence is derived from the P2X7 receptor (amino acid position 306-320) but contains a mutation site 307 arginine 3 is replaced by glutamine (R307Q), which enhances the natural phagocytic ability of the P2X7 receptor. We then used CRISP-Cas9 technology to establish a transgenic mouse model to mimic this mutation site (R307Q). After successfully obtaining this transgenic mouse, we compared the heterozygous P2X7-307R / Q with the background mouse line C57BL / 6. The baseline level of LTP of P2X7-307R / Q was slightly higher than that of wild-type C57BL / 6, and it was able to resist the toxic effects of Aβ42 ( Figure 7 ). This is consistent with the results of in vitro application of BG01 polypeptide.

[0030] In summary, the in vitro experimental data of the inventors show that, like glatiramer acetate, BG01 and other PPPs can act as bridging molecules to promote natural phagocytosis in vitro. PPP binds tightly to Aβ and can antagonize the toxic effects of Aβ42 during the reduction of LTP. The inventors found that PPP can restore or even promote the memory and learning ability of neurons in the presence of Aβ42. These results suggest a new method for treating AD, namely using PPP to treat AD, especially using multiple PPPs with synergistic enhancement 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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