Organophosphorus poisoning rescue composite enzyme and application thereof

Abstract

The application provides an organic phosphorus poisoning relief composite enzyme and application thereof, and belongs to the technical field of biological medicine. The organic phosphorus poisoning relief composite enzyme provided by the application comprises polyethylene glycol modified organic phosphorus hydrolase and butyrylcholine esterase, and the mass ratio of the polyethylene glycol modified organic phosphorus hydrolase to the butyrylcholine esterase is (4-8):(2500-10000). The rat acute organic phosphorus poisoning treatment result shows that the organic phosphorus poisoning relief composite enzyme provided by the application fully plays the efficient catalytic hydrolysis capacity of the enzyme as a catalyst, efficiently removes organic phosphorus and acetylcholine by using the organic phosphorus hydrolase and the butyrylcholine esterase respectively, provides a drug combination for treating the cause for the organic phosphorus poisoning injury first aid, and the treatment effect is significantly better than that of the single drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a complex enzyme for the treatment of organophosphate poisoning and its application. Background Technology

[0002] Since their introduction in the mid-20th century, organophosphate compounds have been widely used as agricultural pesticides and chemical warfare agents. While they have played a role in increasing agricultural yields and providing military deterrence, they have also brought serious poisoning problems. Organophosphate compounds irreversibly inhibit cholinesterase, leading to the accumulation of acetylcholine in the synaptic cleft, thereby blocking cholinergic neurotransmission. Inhibition of cholinesterase in the peripheral nervous system (such as ganglia and neuromuscular junctions) and the central nervous system is the main cause of acute toxicity, often triggering severe cholinergic syndrome. Most fatal organophosphate poisoning cases stem from acute hypoxia caused by the combined effects of peripheral acute cholinergic effects and central respiratory arrest. Therefore, rapidly clearing free organophosphates from the body in the early stages of acute poisoning, reducing blood drug concentrations, preventing redistribution, and clearing accumulated acetylcholine are crucial factors in preventing death.

[0003] Currently, clinical treatment for acute organophosphate poisoning mainly relies on traditional atropine and oxime reactivators, which have been used for over 60 years. However, the mortality rate of this treatment regimen remains as high as 10%–40%, and its efficacy is highly controversial. This is primarily due to the limitation of oxime drugs exhibiting a "aging" phenomenon: organophosphates undergo dealkylation after binding with cholinesterase, forming aged enzymes that cannot be reactivated by any oxime drug. Atropine, as the only drug with clear evidence of efficacy, rapidly alleviates poisoning symptoms by competitively antagonizing muscarinic receptors and blocking the effects of accumulated acetylcholine; however, its dosage lacks objective indicators, it has significant side effects, and an ideal dosage regimen has yet to be established.

[0004] Organophosphorus hydrolases (OPHs) use organophosphorus compounds as substrates to rapidly catalyze their hydrolysis into non-toxic or low-toxic products. Previous studies have shown that OPHs modified with polyethylene glycol at the N-terminus, administered via tail vein injection, can effectively prevent damage from lethal doses of ethylparaben in animals (CN 118516331 A). However, when used to treat organophosphorus poisoning, they only clear the organophosphorus compounds and cannot address the accumulated acetylcholine in the body, thus failing to prevent the development of cholinergic symptoms. Butyrylcholinesterase (BuChE) has been reported as a drug for the prevention and treatment of organophosphorus poisoning. It can both clear organophosphorus compounds through stoichiometric reactions and efficiently catalyze the hydrolysis of accumulated acetylcholine. However, butyrylcholinesterase requires large doses, is difficult to obtain, and, importantly, its stoichiometric rate of clearing organophosphorus compounds is much lower than that of organophosphorus hydrolases (kJ / L). cat / Km =1.9×10 6 M -1. s -1 ) (M. Ma, Y. Zhai, Q.Jin, et al., N-terminal PEGylation enhances organophosphorus hydrolasecatalysis for a fast promising and long-acting prophylactic candidate, JHazard Mater, 488 (2025) 137336. https: / / doi.org / 10.1016 / j.jhazmat.2025.137336.; T. ade , D. Koli G. Inko, et al., Assessment of four organophosphorus pesticides as inhibitors of human acetylcholinesterase and butyrylcholinesterase, Scientific Reports, 11 21486. ​​https: / / doi.org / 10.1038 / s41598-021-00953-9.), but the hydrolysis of acetylcholine is a catalytic reaction with extremely high efficiency, and the reaction rate constant can reach 2 × 10⁻⁶. 6 M -1. s -1 (AR Mukhametgalieva, AR Aglyamova, SV Lushchekina, et al., Time-course of human cholinesterases-catalyzed competing substrate kinetics, Chemico-biological interactions, 310 108702. https: / / doi.org / 10.1016 / j.cbi.2019.06.015.). Based on the highly efficient catalytic hydrolysis capabilities of organophosphate hydrolases and butyrylcholinesterases for organophosphates and acetylcholine, respectively, combining these two enzymes represents a feasible and innovative strategy for organophosphate remediation. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a complex enzyme for the treatment of organophosphate poisoning and its application, wherein the complex enzyme comprises a polyethylene glycol-modified organophosphate hydrolase and a butyrylcholinesterase; the complex enzyme of the present invention can effectively clear free organophosphate compounds in the body and regulate the metabolism of acetylcholine, providing an efficient detoxification strategy for organophosphate poisoning.

[0006] This invention, recognizing the heterogeneity of organophosphate hydrolases, prepared and characterized polyethylene glycol-modified organophosphate hydrolases; established an animal model of ethyl paraoxon poisoning; and evaluated the potential therapeutic effect of a composite enzyme consisting of polyethylene glycol-modified organophosphate hydrolases and butyrylcholinesterase on poisoned animals. This study fully utilizes the highly efficient catalytic hydrolysis capabilities of enzymes as catalysts, employing organophosphate hydrolases and butyrylcholinesterase to efficiently remove organophosphates and acetylcholine, respectively, providing a "causal treatment" drug combination for the emergency treatment of organophosphate poisoning injuries.

[0007] The technical problem to be solved by this invention is how to simultaneously achieve effective clearance of free organophosphate compounds in the body and metabolic regulation of acetylcholine, so as to provide a new treatment strategy for organophosphate poisoning.

[0008] This invention provides a complex enzyme for treating organophosphate poisoning, comprising a polyethylene glycol-modified organophosphate hydrolase and a butyrylcholinesterase, wherein the mass ratio of the polyethylene glycol-modified organophosphate hydrolase to the butyrylcholinesterase is (4~8):(2500~10000).

[0009] Preferably, the mass ratio of the polyethylene glycol-modified organophosphorus hydrolase to butyrylcholinesterase is (5~6):(5000~10000).

[0010] Preferably, the dosage of the polyethylene glycol-modified organophosphorus hydrolase is 40-80 μg / kg, and the dosage of the butyrylcholinesterase is 25-100 mg / kg.

[0011] Preferably, the polyethylene glycol-modified organophosphorus hydrolase includes wild-type organophosphorus hydrolase or mutant organophosphorus hydrolase; the amino acid sequence of the wild-type organophosphorus hydrolase is shown in SEQ ID NO.1, and the amino acid sequence of the mutant organophosphorus hydrolase is shown in SEQ ID NO.2.

[0012] Preferably, the nucleotide sequence encoding the wild-type organophosphorus hydrolase gene is shown in SEQ ID NO.3, and the nucleotide sequence encoding the mutant organophosphorus hydrolase gene is shown in SEQ ID NO.4.

[0013] Preferably, the polyethylene glycol has a molecular weight of 8-12 kDa, and the polyethylene glycol modification includes one or more of the following: N-terminal, C-terminal, lysine side chain, and cysteine ​​side chain site-directed modification.

[0014] This invention provides the application of the aforementioned organophosphate poisoning rescue complex enzyme in the preparation of drugs for treating organophosphate poisoning.

[0015] This invention provides the application of the aforementioned organophosphorus poisoning rescue complex enzyme in the preparation of reagents for the detection or removal of organophosphorus compounds.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The organophosphate poisoning rescue complex enzyme provided by the present invention comprises polyethylene glycol-modified organophosphate hydrolase and butyrylcholinesterase, wherein the mass ratio of the polyethylene glycol-modified organophosphate hydrolase to butyrylcholinesterase is (4~8):(2500~10000). According to the examples, the treatment results for acute organophosphate poisoning in rats show that using 60 μg / kg PEG... M-ALD-10KD -OPHDS5 and 100 mg / kg BuChE, 60 μg / kgPEG M-ALD-10KD -All patients in the OPHDS5 and 50 mg / kg BuChE groups survived, with PEG M-ALD-10KD Increased concentration of OPHDS5 leads to faster distribution in the body, quicker clearance of organophosphates, and reduced accumulation of acetylcholine at the neuromuscular junction and central nervous system. Meanwhile, BuChE can hydrolyze excess acetylcholine, alleviating cholinergic crisis. Attached Figure Description

[0017] Figure 1 The vector map of the recombinant expression vector pET28a-OPHDS5; Figure 2 The UV absorption spectrum for hydrophobic chromatography purification; Figure 3 For hydrophobic chromatography purification of SDS-PAGE; Figure 4 The UV absorption spectrum is for cation exchange chromatography purification. Figure 5 Purification of SDS-PAGE by cation exchange chromatography Figure 6 The UV absorption spectrum is for cation exchange chromatography purification. Figure 7 Cation exchange for removal of free PEG SDS-PAGE; Figure 8 Image showing iodine staining for cation exchange removal of free PEG; Figure 9The images show the UV absorption spectra of gel filtration chromatography, where (A) represents OPHDS5 and (B) represents PEG. M-ALD-10kDa -OPHDS5; Figure 10 The non-denaturing gel electrophoresis SDS-PAGE consisted of (1) Marker, (2) OPHDS5, and (3) PEG. M-ALD-10kDa -OPHDS5; Figure 11 This is the standard curve for p-nitrophenol; Figure 12 The enzyme activity of polyethylene glycols of different molecular weights modified with ethyl para-phosphorus as substrate was determined at pH 6.0 (n=3). Figure 13 The molecular weight of OPDHS5 was characterized by matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry. Figure 14 Characterization of PEG by matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry M-ALD-10KDa -OPHDS5 molecular weight; Figure 15 To analyze the characterization of PEG using ultracentrifugation technology M-ALD-10KDa -OPHDS5 and OPHDS5; Figure 16 Extracted ion chromatogram and first-order mass spectrum (peptide AMITNSGDR) at m / z 482.729. Figure 17 Extracted ion chromatogram and first-order mass spectrum (peptide ALIDQGYMK) at m / z 519.768. Figure 18 For OPHDS5 and PEG M-ALD-10kDa -OPHDS5 enzyme kinetic curve; Figure 19 The inhibition kinetics curve of BuChE enzyme; Figure 20 The enzyme kinetics curve for the hydrolysis of thioacetylcholine by BuChE; Figure 21 To investigate the effect of single-drug administration on the survival rate of rats after acute organophosphate poisoning; Figure 22 To investigate the effect of combined drug administration on the survival rate of rats after acute organophosphate poisoning. Detailed Implementation

[0018] This invention provides a complex enzyme for treating organophosphate poisoning, comprising a polyethylene glycol-modified organophosphate hydrolase and a butyrylcholinesterase, wherein the mass ratio of the polyethylene glycol-modified organophosphate hydrolase to the butyrylcholinesterase is (4-8):(2500-10000). In this invention, the preferred mass ratio of the polyethylene glycol-modified organophosphate hydrolase to the butyrylcholinesterase is (5-6):(5000-10000), more preferably 6:(5000-10000). In this invention, the preferred dosage of the polyethylene glycol-modified organophosphate hydrolase is 40-80 μg / kg, specifically 40 μg / kg, 50 μg / kg, 60 μg / kg, or 70 μg / kg; the preferred dosage of the butyrylcholinesterase is 25-100 mg / kg, specifically 25 mg / kg, 50 mg / kg, or 100 mg / kg.

[0019] In this invention, the polyethylene glycol-modified organophosphorus hydrolase includes wild-type organophosphorus hydrolase or mutant organophosphorus hydrolase; the amino acid sequence of the wild-type organophosphorus hydrolase (named OPHDS5) is shown in SEQ ID NO.1, and is as follows: MAMITNSGDRINTVRGPITISEAAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRRARAAGVRTIVDVSTFDIGRDVSLLAEVSRAADVHIVAATGLWFDPPLSMRLRSVEELTQFFLREIQYGIEDTGIRAGIIKVATTGKATPFQELVLRAAARASLATGV PVTTHTAASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTALAARGYLIGLDHIPHSAIGLEDNASASALLGIRSWQTRALLIKALIDQGYMKQILVSNDWLFGFSSYVTNIMDVMDSVNPDGMAFIPLRVIPFLREKGVPQETLAGITVTNPARFLSPPTLRAS.

[0020] The amino acid sequence of the mutant organophosphorus hydrolase is shown in SEQ ID NO.2, and is as follows: MITNSGDRINTVRGPITISEAAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRRARAAGVRTIVDVSTFDIGRDVSLLAEVSRAADVHIVAATGLWFDPPLSMRLRSVEELTQFFLREIQYGIEDTGIRAGIIKVATTGKATPFQELVLRAAARASLATGVP VTTHTAASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTALAARGYLIGLDHIPHSAIGLEDNASASALLGIRSWQTRALLIKALIDQGYMKQILVSNDWLFGFSSYVTNIMDVMDSVNPDGMAFIPLRVIPFLREKGVPQETLAGITVTNPARFLSPPTLRAS.

[0021] In this invention, the gene encoding the wild-type organophosphorus hydrolase (named...) ophds5 The nucleotides of ) are shown in SEQ ID NO.3, as follows: The nucleotide sequence of the gene encoding the mutant organophosphorus hydrolase is shown in SEQ ID NO.4, and is as follows:

[0022] In this invention, the wild-type or mutant organophosphorus hydrolase can be obtained by first synthesizing its encoding gene and then expressing it biologically. In one specific embodiment of this invention, it is obtained by expressing the encoding gene in a host microorganism. Specifically, the encoding gene is synthesized and ligated into an expression vector to construct a recombinant expression vector; the constructed recombinant expression vector is introduced into host cells, positive clones are screened, and protein expression and purification are performed to obtain the wild-type or mutant organophosphorus hydrolase.

[0023] Furthermore, the recombinant expression vector of the organophosphorus hydrolase OPHDS5 is named pET28a-OPHDS5. It is a recombinant expression vector obtained by replacing the fragment between the Nco I and EcoRI restriction sites of pET28a(+) with the nucleotide sequence shown in SEQ ID NO:3, while keeping the other parts of the sequence unchanged. This recombinant expression vector expresses the protein with the amino acid sequence of SEQ ID NO:1.

[0024] Furthermore, the recombinant expression host of the fusion protein can be a prokaryotic or eukaryotic organism, and in this invention, it is Escherichia coli.

[0025] In this invention, the polyethylene glycol-modified organophosphorus hydrolase is the aforementioned organophosphorus hydrolase and its mutants, prepared by different polyethylene glycol modification strategies. The modification sites include, but are not limited to, one or more of the N-terminus, C-terminus, lysine side chain, and cysteine ​​side chain of the protein. In this invention, the molecular weight of the polyethylene glycol is preferably 8-12 kDa, more preferably 10 kDa; the polyethylene glycol type includes, but is not limited to, linear PEG, branched PEG, and multi-arm PEG.

[0026] In this invention, the butyrylcholinesterase includes, but is not limited to, butyrylcholinesterase and its mutants that have the function of hydrolyzing acetylcholine and are obtained from animal sources and through artificial recombinant technology.

[0027] This invention provides a process for preparing a polyethylene glycol-modified organophosphorus hydrolase, including the preparation of the organophosphorus hydrolase, a method for site-directed N-terminal polyethylene glycol modification, product characterization, and process optimization. Further, the organophosphorus hydrolase OHDS5 can be obtained by purification via ammonium sulfate precipitation, hydrophobic chromatography, ion exchange chromatography, and gel filtration chromatography. Further, this invention utilizes a PEG-aldehyde reduction alkylation reaction to perform N-terminal site-directed modification of the purified organophosphorus hydrolase OHDS5, using 10 kDa PEG. Further, this invention provides a method for the preparation of OHDS5 and its polyethylene glycol-modified product (PEG). M-ALD-10KDCharacterization methods for OPHDS5: This invention employs matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) to determine the molecular weight of unmodified OHDS5 and the average molecular weight increment of the PEGylated product to confirm successful coupling. This invention uses analytical ultracentrifugation to determine the sedimentation coefficient, hydrodynamic radius, and aggregation state of the product in solution to confirm its existence as a dimer. This invention uses liquid chromatography-tandem mass spectrometry (LC-MS / MS), using unmodified OHDS5 as a control, to precisely locate specific amino acid residues involved in PEGylation and calculate the modification ratio.

[0028] This invention provides a systematic kinetic evaluation of the catalytic function of the enzymes involved, specifically including OPDHS5 and PEG. M-ALD-10KD Determination of kinetic parameters for OPHDS5 and BuChE: Furthermore, this invention analyzes OPDHS5 and PEG. M-ALD-10KD The reaction kinetics of OPHDS5 with ethyl paraoxon was studied, and its enzyme kinetic parameters were determined. K m , k cat , V max、 k cat / K m This allows for a quantitative assessment of the effects of PEG modification on enzyme catalytic efficiency and substrate affinity.

[0029] Furthermore, this invention evaluates BuChE's ability to neutralize toxins as a "biological scavenger" and its inherent cholinesterase activity by determining the reaction rate or kinetic parameters of BuChE with ethyl paraoxonium and the acetylcholine analog thioacetylcholine using the Ellman method.

[0030] Furthermore, this invention clarifies the effective treatment window for acute organophosphate poisoning through animal experiments, allowing intervention to be administered after poisoning but before the condition enters the second stage (i.e., the depolarization blockade phase). The effects of the following treatment regimens were compared: atropine group, atropine combined with pralidoxime group, and PEG group. M-ALD-10KD -OPHDS5 monotherapy, BuChE monotherapy, PEG M-ALD-10KD -OPHDS5 and BuChE joint group: Furthermore, the present invention validates the superiority of combined drug therapy through survival rate.

[0031] The wild-type or mutant organophosphorus hydrolase provided by this invention possesses organophosphorus hydrolase activity and exhibits broad substrate specificity for a variety of organophosphorus compounds, effectively decomposing compounds containing PO, P-CN, PF, and PS bonds. Specifically, the organophosphorus hydrolase can hydrolyze organophosphorus compounds containing PO bonds and linked to 4-nitrophenyl groups (e.g., paraoxon and methyl parathion) to p-nitrophenol; for organophosphorus compounds containing PS bonds (e.g., malathion, VX nerve agent (O-ethyl-S-[2-(diisopropylamino)ethyl]methyl thiophosphate), dimethoate, phorate, ethion, and terbufos), it can hydrolyze to yield some sulfides; for compounds containing P-CN bonds (e.g., tabun), it can hydrolyze to yield hydrocyanic acid; and for Class G agents containing PF bonds (e.g., sarin, soman, and cyclosarrin), it can hydrolyze to yield fluorides.

[0032] Furthermore, the organophosphorus compound described in this invention may specifically be at least one of parathion, methyl parathion, dimethoate, malathion, phorate, ethion, and terbufos, or may be a nerve agent, including at least one of Class G agents (sarin, soman, tabun, cyclosarin) and VX nerve agents.

[0033] This invention provides the application of the aforementioned organophosphate poisoning rescue complex enzyme in the preparation of drugs for treating organophosphate poisoning.

[0034] The organophosphate poisoning rescue complex enzyme provided by this invention not only possesses organophosphate hydrolase activity in vitro, but also counteracts organophosphate compounds in vivo, and can serve as a potential drug for the prevention and / or treatment of organophosphate poisoning. The dosage form of the drug can be selected from one of the following: tablets, capsules, granules, oral liquids, suspensions, sprays, inhalers, ointments, lotions, gels, eye drops, suppositories, transdermal patches, injections, or lyophilized powder injections.

[0035] In practical applications, the drug of this invention can be administered directly to the subject or mixed with a pharmaceutically acceptable carrier or excipient before administration. The carriers here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to injections, tablets, capsules, oral liquids, granules, ointments, creams, creams, eye drops, suspensions, and other external or topical dosage forms. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms into injectable formulations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. To formulate unit-dose dosage forms into tablets, a wide variety of carriers known in the art can be widely used. Examples of carriers include, for instance, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills.Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. These dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections. Injection is the preferred route of administration. When patients experience local poisoning symptoms due to organophosphate contamination of the skin, mucous membranes, or eyes, topical or local application formulations can be used.

[0036] The drug described in this invention can be administered orally, intravenously, intra-arterially, intraperitoneally, intrathecally, transdermally, via the respiratory tract, rectally, or locally. The subject can be a mammal. Mammals can be selected from bovine, equine, feline, canine, lagomorph, suidae, camel, rodent, and primate animals, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, and orangutans), and humans, preferably cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys, and humans.

[0037] The dosage of the drug described in this invention can be appropriately adjusted based on the method of administration, route of administration, age and / or weight of the subject, and individual circumstances of the subject.

[0038] This invention also provides the application of the aforementioned organophosphorus poisoning rescue complex enzyme in the preparation of reagents for the detection or removal of organophosphorus compounds.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Establishment of an expression and purification system for organophosphorus hydrolases

[0042] 1.1 Microbial fermentation

[0043] Take 50 µL of BL21-opd-1 (containing plasmid pET28a-OPHDS5) (prepared by the method described in Example 8 of Chinese Patent 202410608657.X) and inoculate it into an Erlenmeyer flask containing 100 mL of LB medium (containing 50 µg / mL Kan). Incubate at 37°C and 180 r / min for 16 h in a shaker. In a clean bench, add the revived bacterial culture (16 h) to TB medium, adding 100 mL of the revived bacterial culture per 1 L of TB medium. Incubate at 37°C and 180 r / min for 5 h in a shaker. Take out the bacterial culture that has been incubated at 37°C for 5 h, add 250 μL of 1M IPTG (concentration in the medium is 0.25 mM), and incubate at 25°C and 180 r / min for 24 h in a shaker. Take out the bacterial culture that has been fermented and cultured at 25℃ for 24 h, centrifuge at 4℃ and 9000 r / min for 3 min, discard the supernatant, weigh the bacterial cells, and use them directly for subsequent protein purification or store them temporarily at -80℃.

[0044] 1.2 Cell disruption

[0045] After centrifugation, the bacterial cells were resuspended in 20 mM HEPES at pH 8.0. After complete dissolution, the bacterial cells were homogenized at 4°C under the following conditions: pressure 900-1000 bar, flow rate 40 mL / min, for 4 cycles. After homogenization, the cells were centrifuged at 4°C and 9000 r / min for 30 min. The supernatant was collected and placed on ice for later use.

[0046] 1.3 Precipitation of protamine sulfate

[0047] Take the supernatant after centrifugation, add 0.2 g of protamine sulfate per 100 mL, and react in an ice bath for 30 min. After the reaction is complete, centrifuge at 9000 r / min for 30 min, and take the supernatant and place it on ice for later use.

[0048] 1.4 Ammonium sulfate precipitation

[0049] After centrifugation, take the supernatant and add 0.326 g of ammonium sulfate to each 1 mL. Let it react for 30 min. After the reaction, centrifuge at 9000 r / min for 30 min. Take the precipitate and resuspend it in 20 mM Tris-HCl (containing 0.8 M ammonium sulfate) at pH 7.0. After it is fully dissolved, centrifuge at 10000 r / min for 10 min. Take the supernatant and filter it through a 0.45 μm filter membrane for hydrophobic chromatography.

[0050] 1.5 Hydrophobic Chromatography Purification

[0051] Using HiTrap TMPre-packed Capto Phenyl Impres hydrophobic interaction chromatography column, flow rate 2 mL / min, column pressure alarm 0.3 MPa. First, rinse with deionized water for 5 column volumes, then equilibrate with 20 mM, pH 7.0 Tris-HCl (containing 0.8 M ammonium sulfate), i.e., solution A, for 5 column volumes. Begin sample loading. Continuous loading is used; avoid exceeding the column's maximum loading capacity to prevent protein loss. Elution buffer is 20 mM, pH 7.0 Tris-HCl (containing 2.5% isopropanol), i.e., solution B. Linear binding isocratic elution is used: 0-25% B linear elution for 5 column volumes, 25% B isocratic elution for 5 column volumes, 25-100% B linear elution for 5 column volumes, and 100% B isocratic elution for 5 column volumes. Collect fractions based on the UV absorption spectrum (…). Figure 2 SDS-PAGE verification of separation effect ( Figure 3 At the end of the experiment, the hydrophobic chromatography column was stored in 20% ethanol.

[0052] 1.6 Cation exchange chromatography purification

[0053] SDS-PAGE showed that hydrophobic chromatography effectively separated the target protein from other proteins, but the purity was not yet satisfactory. Therefore, based on this, the target protein was collected and fully replaced with 20 mM CH3COOH-CH3COONa (solution A) at pH 5.5 for concentration. HiTrap was then used... TM SP HP strong cation exchange chromatography pre-packed column for further purification, flow rate 2 mL / min, alarm column pressure 0.3 MPa. First, wash with deionized water for 5 column volumes, then equilibrate with solution A for 5 column volumes before loading the sample using a loading loop. Elution buffer was 20 mM, pH 5.5 CH3COOH-CH3COONa (containing 1M sodium chloride), i.e., solution B. The process used was linear-isocratic elution: 0-5% B linear elution for 5 column volumes, 5% B isocratic elution for 5 column volumes, 5-50% B linear elution for 5 column volumes, and 50% B isocratic elution for 5 column volumes. The fractional elution was performed based on the UV absorption spectrum (…). Figure 4 SDS-PAGE verification of separation effect ( Figure 5 At the end of the experiment, the cation exchange column was stored in 20% ethanol (containing 0.2M sodium acetate).

[0054] Example 2

[0055] Preparation and purification of PEGylated OPHDS5

[0056] 2.1 Preparation of PEGylated OPDHS5

[0057] The concentrated OPHDS5 obtained after cation exchange chromatography was stored separately in a 20 mM, pH 6.0 CH3COOH-CH3COONa reaction system. Protein concentration was determined according to the BCA kit instructions (Bierce™ BCAProtein Assay Kit, Thermo Fisher Scientific, catalog number: 23227). 20 mL of OPHDS5 solution was taken, and M-ALD-10K, M-ALD-20K, Y-PALD-20K, and Y-AALD-40K, dissolved in the same medium, were added at an OPHDS5:PEG molar ratio of 1:2. 20 mM sodium cyanoborohydride was added, bringing the total reaction volume to 30 mL. After the sodium cyanoborohydride and PEG were completely dissolved in the 20 mM, pH 6.0 CH3COOH-CH3COONa solution, they were added to the OPHDS5 solution, and the mixture was incubated at 4°C with shaking for 16 h.

[0058] 2.2 Purification of PEGylated OPDHS5 (using PEG) M-ALD-10KDa (Taking OPHDS5 as an example)

[0059] After the reaction, the sample was concentrated by centrifugation at 5000 r / min at 4℃ using a 10 kDa ultrafiltration tube. It was then completely replaced and concentrated using 20 mM, pH 5.5 CH3COOH-CH3COONa solution (solution A). HiTrap was then used for further concentration. TM SP HP was further purified at a flow rate of 2 mL / min and an alarm column pressure of 0.3 MPa. Five column volumes were first rinsed with deionized water, followed by equilibration with solution A for another five column volumes. Sample loading was then initiated using a loading loop. The elution buffer was 20 mM, pH 5.5 CH3COOH-CH3COONa (containing 1 M sodium chloride), i.e., solution B. Linear and isocratic elution was employed, specifically: 0-5% B linear elution for five column volumes, 5% B isocratic elution for five column volumes, 5-50% B linear elution for five column volumes, and 50% B isocratic elution for five column volumes. The elution was then determined based on the UV absorption spectrum (…). Figure 6 The samples were collected in segments, and the cation exchange column was stored in 20% ethanol (containing 0.2M sodium acetate) after the experiment.

[0060] Staining with Coomassie brilliant blue ( Figure 7 ), iodine staining ( Figure 8 ), gel filtration chromatography ( Figure 9 ) and non-denaturing gel electrophoresis ( Figure 10 It was found that cation exchange chromatography can effectively separate unreacted PEG, OPDHS5 and modified products.

[0061] 2.3 In vitro enzyme activity measurement

[0062] OPHDS5 catalyzes the hydrolysis of its substrate, ethyl para-oxophosphate, to p-nitrophenol, with a molar ratio of ethyl para-oxophosphate to p-nitrophenol of 1:1. Therefore, the enzyme activity of a sample can be evaluated by measuring the amount of p-nitrophenol produced under the same conditions. Different volumes of 6 mM p-nitrophenol solution (20.86 mg of p-nitrophenol dissolved in 5 mL of deionized water and diluted to 25 mL in a volumetric flask) were prepared, and 1 mL of 50 mM HEPES buffer (pH=8.0) was added to bring the volume to 1 mL. Then, 1 mL of 10% (w / v) trichloroacetic acid and 10% (w / v) sodium carbonate solution were added sequentially, resulting in a final p-nitrophenol concentration range of 0 to 192 μmol / L (in 8 μmol / L increments), with a total of 13 concentration points. The absorbance at 405 nm was measured. The experiment was performed in triplicate, and the average value was taken. A standard curve was plotted by linearly fitting the absorbance values ​​to the p-nitrophenol concentration. Figure 11 ).

[0063] The concentration of each protein sample was determined by the BCA method, and then diluted to 500 ng / mL using 50 mM Hepes at pH 8.0.

[0064] Twenty-one 5 mL centrifuge tubes were used, with tubes 1, 2, and 3 designated as blank controls and numbered sequentially as the experimental groups. 5 μL of 10 mg / mL ethyl paraoxonate solution and 900 μL of 50 mM pH 8.0 HEPES buffer were added to each tube. 1 mL of 10% trichloroacetic acid was added to tube 1, followed by 100 μL of the protein solution to be tested (10% trichloroacetic acid inactivates the enzyme and terminates the reaction), and the tube was placed in a 37°C water bath. Then, 100 μL of the protein solution was added every 15 seconds. After 10 minutes, tube 1 was removed, and then every 30 seconds, tube 1 was removed and immediately treated with 10% trichloroacetic acid. 1 mL of 10% sodium carbonate solution was added to each of the remaining 15 centrifuge tubes for color development. After mixing, 200 μL of each sample was taken and the absorbance at 405 nm was measured using a microplate reader. Each sample was measured in triplicate. Enzyme activity = amount of p-nitrophenol (μmol) × (1 mg / (0.1 mL)) Sample concentration) / 10 min, and a control group was set up for each sample during the measurement process.

[0065] The activity of OPDHS5 enzyme modified with PEG aldehydes of different molecular weights was measured, and no significant changes were found. Furthermore, the modification rate of M-type PEG aldehyde (M-PEG-ALD, 10 kDa) was found to be higher at 5.4%, and the recovery rate of unreacted enzyme was 29.04%. The enzyme activity remained unchanged and could be used for further modification. Therefore, PEG... M-ALD-10KDa -OPHDS5 was used for subsequent experiments.

[0066] Example 3

[0067] Characterization of N-terminal site-directed modification of polyethylene glycol OPHDS5

[0068] 3.1 Matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry characterization of molecular weight before and after modification

[0069] OPDHS5 and PEG M-ALD-10KD -OPHDS5 was placed in a 10 kDa ultrafiltration tube for buffer replacement, centrifuged at 5000 r / min for 10 min at 4 °C, and the buffer was replaced with deionized water to concentrate to a concentration of 1 mg / mL.

[0070] The sinapic acid matrix solution and the sample solution were mixed at a volume ratio of 1:1. The mixed sample was spotted onto a stainless steel target plate and left at room temperature until the solvent evaporated and co-crystallization was formed. Mass spectrometry was performed using a 337 nm laser in linear positive ion mode.

[0071] Based on the amino acid sequence, the theoretical molecular weight of OPDHS5 is calculated to be 73.28 kDa, and its theoretical molecular weight after N-terminal attachment of a 10 kDa PEG should be 83.28 kDa. However, the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS) results ( Figure 13 , Figure 14 The results showed that OPHDS5 exhibited a main absorption peak at 36.8 kDa, while the PEGylated sample (PEG...) showed a different peak. M-ALD-10KDa Apart from this position, -OPHDS5 shows only a weak absorption peak at 47.4 kDa, far below the expected quality of the complete PEGylated dimer. This phenomenon can be attributed to the following factors: under the action of laser and matrix, some dimers dissociate, releasing monomer signals; at the same time, the polydispersity of PEG itself disperses the already weak complete modification peak signal into multiple m / z intervals, thereby further weakening the intensity of the target peak.

[0072] 3.2 Ultracentrifugation (AUC) was used to characterize the molecular weight before and after modification under natural conditions.

[0073] OPHDS5 with an OD280 of 0.8 and its modified product PEG were used. M-ALD-10KDa400 μL of each OPHDS5 sample was collected and sedimentation rate experiments were performed using a Beckman Coulter Optima AUC at 20 °C and 40,000 r / min, following the method described in the literature (H. Zhao, CA Brautigam, R. Ghirlando, et al., Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation, CP Protein Science, 71. https: / / doi.org / 10.1002 / 0471140864.ps2012s71.). All samples were in deionized water. Absorbance scans were performed at 280 nm with 180 s intervals. Data analysis was performed using SEDFIT(16p36) and the continuous c(s) distribution model described by Schuck et al.

[0074] Experimental results are as follows Figure 15 As shown, although no complete molecular weight peak was observed in the mass spectrometry, AUC analysis still indicated that PEG modification did not disrupt the dimer structure of OPDHS5. The AUC results, as shown in Table 1, indicate that the Stokes radius of the PEGylated protein increased, the sedimentation coefficient decreased, and the friction ratio increased. These changes all point to the "dynamic hydration layer" formed by the PEG chain on the protein surface: its random coil conformation and strong hydration together increase the hydrodynamic volume of the molecule and introduce significant asymmetry, thereby slowing down the sedimentation rate and enhancing the friction effect overall.

[0075] Furthermore, AUC measurements revealed an apparent molecular weight of OPHDS5 of 65.4 kDa and a PEGylated form of 73.7 kDa, both lower than theoretical values. This discrepancy may be related to the partial specific capacitance (PSC) used in the calculations. (≈0.73 ml / g, which is a typical reference value for globular proteins). Given... Errors are transmitted to the final molecular weight result by approximately three times, and there is a deviation between the actual value in solution and the theoretical model, resulting in a reasonable gap between the calculated value and the theoretical expectation. Combining AUC and mass spectrometry data, it can be concluded that PEG modification did not disrupt the dimer configuration of OPDHS5, with only one PEG molecule bound to the OPDHS5 dimer. Its main impact is reflected in the change of hydrodynamic properties.

[0076] Table 1. Analysis of changes in relevant parameters before and after PEG modification using ultracentrifugation technology.

[0077] 3.3 Identification of PEGylated OPDHS5 modification sites

[0078] Sample pretreatment: Take 500 μL of the sample before and after modification, add 50 mL of 1 mol / L Tris-HCl (pH 8.0) solution, add 20 μg of trypsin, react at 37℃ for 18 h, remove, add 50 μL of 10% formic acid to stop the enzymatic reaction, centrifuge (10000 r / min, 10 min), and put the supernatant into a sample bottle for analysis; Chromatographic conditions: Mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was an acetonitrile solution containing 0.1% formic acid, and the chromatographic column used was a Peptide CSH C18 (1.0) column. The column diameter was 150 mm (1.7 μm), the flow rate was 0.1 mL / min, the column temperature was 60℃, and the injection volume was 5 μL.

[0079] Mass spectrometry conditions: Ion source spray voltage 3.5 kV, capillary temperature 320℃, sheath flow rate 19.8 mL / min, auxiliary flow rate 5 psi, positive ion scanning; scanevent1 was full-scan, scan range: m / z 400-2000, resolution: 60000, RF lens: 45%, Normalized AGC Target: 300%, maximum ion implantation time: 100 ms; scanevent2 was data-dependent MS / MS, resolution: 15000, isolation window: m / z 1.6, maximum ion implantation time: 200 ms, Normalized AGC Target: 100%, collision energy: 30%, cycle time: 2 s.

[0080] Experimental results are as follows Figure 16 , Figure 17As shown, two specific peptides containing potential modification sites—AMITNSGDR and ALIDQGYMK—were identified from the primary mass spectrometry. By extracting their ion current chromatograms at mass-to-charge ratios of 482.729 and 519.768, and integrating the corresponding peak areas, the PEG modification percentage on AMITNSGDR was calculated to be 82.36%, while on ALIDQGYMK it was only 19.73%. This result indicates that PEG modification exhibits a significant site preference at pH 6.0, primarily concentrated in the N-terminal region of the protein. The lower degree of lysine modification in the intermediate peptide ALIDQGYMK, based on literature reports, may be related to the local microenvironment of this residue: the lysine residue may be located in an exposed hydrophobic region, resulting in a lower pKa value. Under the reaction system conditions of pH 6.0, partial deprotonation occurs, thereby enhancing its reactivity with the PEGylation reagent.

[0081] Example 4

[0082] In vitro enzyme kinetics study of complex enzymes

[0083] 4.1 Determination of kinetic parameters of polyethylene glycolated OPDHS5 enzyme

[0084] Pipettes of 150 μL, 148 μL, 146 μL, 144 μL, 142 μL, 140 μL, 138 μL, 136 μL, 134 μL, 132 μL, 130 μL, 120 μL, 110 μL, 100 μL, 80 μL, 60 μL, and 50 μL of 50 mM HEPEs buffer (pH=8.0) into 1.5 mL centrifuge tubes, and then adds 150 μL of sample (PEG) to each tube. M-ALD-10kDa -OPHDS5 concentration is 100 ng / mL).

[0085] Take 10 mg / mL (36.3 mM) of ethyl paraoxonium and dilute it to 1 mL with 800 μL of deionized water to prepare a 2 mg / mL ethyl paraoxonium solution. Add 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, 20 μL, 30 μL, 40 μL, 50 μL, 70 μL, 90 μL, and 100 μL of the 2 mg / mL ethyl paraoxonium solution sequentially every 30 seconds, and immediately place the tubes in a 37°C water bath. Start timing for 10 min, and continue this process until the last centrifuge tube. After 10 min, add 300 μL of 10% trichloroacetic acid solution sequentially to terminate the reaction, with a 30-second interval between additions. Finally, add 300 μL of 10% sodium carbonate solution for color development. Tube 1 was set as a blank control group. 200 μL of sample was added to each well of the 96-well plate. Each sample was measured in 3 replicates. The absorbance value was measured at OD405.

[0086] Plotting the concentration of ethyl paraoxonium on the x-axis, the values ​​of free enzyme OPHDS5 and its polyethylene glycol-modified product PEG were respectively used to represent the total concentration of OPHDS5. M-ALD-10kDa Plot an enzyme kinetic curve with the rate of reaction between -OPHDS5 and the substrate to produce p-nitrophenol as the ordinate. Figure 18 By combining the double reciprocal plotting method with Michaelis-Menten equation fitting, the kinetic parameters of the two enzymes were obtained: those of OPHDS5. K m =6.5×10 -5 M, V max =4.0×10 -7 M s -1 , k cat =575.3 s -1 , k cat / K m =8.9×10 5 M -1 s -1 PEG M-ALD-10kDa -OPHDS5 K m =1.3×10 -5 M, V max =1.4×10 -7 M s -1 , k cat =230.1 s -1 , kcat / K m =1.7×10 6 M -1 s -1 Kinetic parameter comparisons showed that the substrate affinity of the enzyme was enhanced after N-terminal polyethylene glycol modification. K m However, the maximum reaction rate decreased. V max The number of substrate molecules converted by a single enzyme molecule per unit time also decreases accordingly. k cat This may be due to the steric hindrance effect at the binding sites caused by polyethylene glycolation. However, due to K m The significant decrease in overall catalytic efficiency ( k cat / K m The results still showed a significant improvement. This phenomenon suggests that PEG modification may unexpectedly enhance the catalytic activity by increasing the structural flexibility of the enzyme protein and promoting hydrogen bonding interactions between the enzyme and the substrate.

[0087] 4.2 Determination of BuChE inhibition kinetic parameters

[0088] Take 16 μL of a 10 mg / mL (36.3 mM) ethyl paraoxonium solution and dilute it to 4 mL with 3984 μL of deionized water to prepare a 40 μg / mL ethyl paraoxonium solution. Take 15 μL, 22.5 μL, 37.5 μL, 45 μL, 75 μL, 100 μL, 125 μL, 137.5 μL, and 150 μL of the 40 μg / mL ethyl paraoxonium solution, respectively, and dilute to 1 mL with deionized water.

[0089] Take 10 μL of 40 μg / mL human plasma BuChE, add 25 μL of 20 mM 5,5'-dithiobis(2-nitrobenzoic acid) and 5 μL of 0.1 M thiobutyrylcholine, and record the change in absorbance at 412 nm within 1 min at 37℃. Measure 3 replicates for each sample.

[0090] Take 10 μL of 40 μg / mL BuChE, add 0.6 μg / mL, 0.9 μg / mL, 1.5 μg / mL, and 1.8 μg / mL ethyl paraoxon solution and incubate for 1 min, 2 min, 3 min, 4 min, and 5 min, respectively. After incubation, add 25 μL of 20 mM 5,5'-dithiobis(2-nitrobenzoic acid) and 5 μL of 0.1 M thiobutyrylcholine. Record the absorbance change at 412 nm within 1 min at 37 °C. Measure 3 replicates for each sample.

[0091] Take 10 μL of 40 μg / mL BuChE and add 3 μg / mL, 4 μg / mL, 5 μg / mL, 5.5 μg / mL, and 6 μg / mL ethyl paraoxonate solutions, respectively, and incubate for 1 min, 90 s, 120 s, 150 s, and 180 s. After incubation, add 25 μL of 20 mM 5,5'-dithiobis(2-nitrobenzoic acid) and 5 μL of 0.1 M thiobutyrylcholine. Record the change in absorbance at 412 nm within 1 min at 37℃. Measure 3 replicates for each sample.

[0092] like Figure 19 As shown, at a fixed substrate concentration (ethyl para-oxygen phosphorus), the pseudo-first-order rate constants corresponding to different substrate concentrations (I0) were measured. k’ ), to obtain I0 pairs k’ The isometric hyperbola was fitted using nonlinear regression to obtain the inhibition kinetic parameters of BuChE: k inact =0.04 s -1 K I =1.8×10 -7 M,k inact / K I =2.2×10 5 M -1 s -1 .

[0093] Given that BChE plays a crucial compensatory role in high substrate concentrations and when AChE is inhibited or absent, the enzyme kinetics using thioacetylcholine (ATC) as a substrate were determined by Ellman spectrophotometry to provide a reliable basis for combination therapy. BChE was diluted to 1 μg / mL with 0.1 M PBS buffer (pH 7.4). For assay, 15 μL of the diluted enzyme sample was added to a 96-well plate, followed by 15 μL of 2 mM DTNB, and different volumes (from 2 to 150 μL) of substrate (0.9 mM). The plate was then brought to a final volume of 200 μL with PBS buffer (pH 7.4, 0.1 M). After mixing, the plate was incubated at 37°C for 10 minutes, and the absorbance at 412 nm was monitored using a microplate reader. Enzyme kinetic curves were plotted, as shown below. Figure 20 As shown. The enzyme kinetic parameters were obtained through Prism fitting. Enzyme kinetic parameters: K m =2×10 -4 M, V max =8.7×10 -8 M s -1 , k cat =395.5 s -1 , k cat / K m =2×10 6 M -1 s-1.

[0094] Contains two enzymes (BuChE and PEG) M-ALD-10kDa In complex systems containing OPHDS5 and two substrates (ethyl paraoxon and acetylcholine), substrate allocation follows competitive kinetics: when the substrate concentration is much lower than 0.5%, substrate allocation follows competitive kinetics. K m ([S]<< K m When ), catalytic efficiency ( k cat / K m A higher enzyme-substrate combination dominates the reaction; while at substrate concentrations much higher than... K m ([S]>> K m At high turnover rate ( k cat The combination of high concentrations of enzymes and high concentrations of LD50 plays a dominant role. This was used to analyze the effects of subcutaneous injection of 2-fold LD50 in SD rats. 50 Ethyl parathion (OP, in vivo concentration approximately 3.5 × 10⁻⁶)-5 Dynamic detoxification process after M): Initial exposure period (high substrate concentration): At this time, the OP concentration exceeds that of both enzymes. K m BuChE, with its high affinity ( K I =180 nM) rapidly binds with OP, exerting a stoichiometric neutralization effect. Meanwhile, PEG... M-ALD-10kDa -OPHDS5 also initiates catalytic degradation, although its K m It is relatively high, but thanks to its turnover rate ( k cat =230 s - ¹) This allows it to continuously remove OP. Concentration decline phase (transition to low substrate concentration): as PEG... M-ALD-10kDa -OPHDS5 continued to catalyze, reducing the OP concentration to sub-micromolar levels. At this point, [S] << K m , k cat / K m It becomes a decisive parameter. PEG M-ALD-10kDa -OPHDS5, with its catalytic efficiency 7.7 times higher than BuChE, completely dominates the scavenging process at this stage, efficiently reducing OP concentration. Physiological recovery and synergistic protection: While the OP concentration is effectively reduced, the remaining BChE is prevented from continuous consumption and instead efficiently hydrolyzes acetylcholine (ACh) accumulated due to AChE inhibition. Even if the synaptic ACh concentration may rise to near that of BuChE. K m BuChE's high turnover rate ( k cat =395.5 s - ¹) It can also achieve maximum hydrolysis efficiency. This synergistic mechanism—that is, PEG M-ALD-10kDa -OPHDS5 primarily scavenges ethyl para-oxophosphate to protect BuChE, which in turn efficiently hydrolyzes ACh to restore physiological balance—together forming an effective in vivo detoxification pathway.

[0095] Example 5

[0096] Pharmacodynamic studies of complex enzymes

[0097] Several healthy male SD rats, weighing 200±20 g, were randomly divided into 15 groups of 7 rats each. All rats were subcutaneously injected with twice the absolute lethal dose (0.658 mg / kg) of ethyl paraoxon to establish an acute severe organophosphate poisoning model. Eight minutes after exposure, each group was administered the following antidotes: Control group: physiological saline, 0.9 mg / kg atropine, 0.19 mg / kg atropine + 54 mg / kg pralidoxime; Single-drug group: PEG. M-ALD-10KD -OPHDS5 (30, 60, 120 μg / kg) or BuChE (150, 300 mg / kg); combination therapy group: 0.9 mg / kg atropine + 60 μg / kg PEG M-ALD-10KD -OPHDS5; and different doses of PEG M-ALD-10KD The treatment was administered using a combination of OPHDS5 (30, 60 μg / kg) and BuChE (100, 50, 25 mg / kg). The efficacy of the treatment was evaluated by rat survival rate, erythrocyte membrane acetylcholinesterase activity, improvement in ventilation, and recovery of motor function.

[0098] 5.1 Effects of different drug administration combinations on the survival rate of rats after acute organophosphate poisoning

[0099] The results of the rat survival curve experiment are as follows: Figure 21 As shown, all mice in the saline group died within 15 minutes after injection of ethylparaben into the back. Six mice died in the group given 0.9 mg / kg atropine alone, and the mice given PEG alone also died. M-ALD-10KD One animal died in the OPHDS5 group, three died in the 150 mg / kg BuChE group, and all survived in the 300 mg / kg BuChE group. Overdose of atropine rapidly relieved muscarinic symptoms such as salivation, vomiting, and urinary and fecal incontinence. However, it could not eliminate the persistent damage caused by free organophosphates in the body, such as central respiratory depression. Excessive acetylcholine triggered acute peripheral cholinergic effects, which could only be recovered by the body's own cholinesterase, thus inevitably leading to mass mortality. However, rapidly reducing blood organophosphate levels and preventing redistribution avoids persistent damage. This "first line of defense" mechanism prevents the accumulation of acetylcholine at the neuromuscular junction and central nervous system, preventing progression to acute respiratory failure, seizures, and cardiac arrest, and significantly improves survival rates, but it cannot reverse existing cholinergic system dysfunction. Furthermore, PEG... M-ALD-10KD -OPHDS5 requires a smaller treatment volume and has higher hydrolysis efficiency.

[0100] like Figure 22 The indicated combination therapy consisted of 0.9 mg / kg atropine and 60 μg / kg PEG. M-ALD-10KDThree mice died in the OPHDS5 group, indicating that although competitive antagonism of M-cholinergic receptors can prevent sustained excessive stimulation of cholinergic receptors by acetylcholine while clearing free organophosphates from the body, atropine generally takes effect 1-3 minutes after administration and has no effect on existing cholinergic system disorders. Six mice died in the 0.19 mg / kg atropine and 54 mg / kg pralidoxime groups; those given 30 μg / kg PEG... M-ALD-10KD One animal died in the OPHDS5 and 100 mg / kg BuChE group, and one animal died in the 30 μg / kg PEG group. M-ALD-10KD One animal died from OPHDS5 and 50 mg / kg BuChE, and 30 μg / kg PEG. M-ALD-10KD -OPHDS5 and 25 mg / kg BuChE caused 3 deaths, 60 μg / kg PEG M-ALD-10KD -OPHDS5 and 100 mg / kg BuChE, 60 μg / kg PEG M-ALD-10KD -All patients in the OPHDS5 and 50 mg / kg BuChE groups survived, possibly due to the presence of PEG. M-ALD-10KD Increased concentration of OPHDS5 leads to faster distribution and clearance of organophosphates in the body, reducing the accumulation of acetylcholine at the neuromuscular junction and central nervous system. Simultaneously, BuChE hydrolyzes excess acetylcholine, alleviating cholinergic crisis. 30 μg / kg PEG M-10KD The group treated with OPHDS5 died successively within 24 hours. Combined with in vitro enzyme kinetic parameters, this indicated that the dosage was insufficient to clear free ethylparaben immediately, thus requiring BuChE binding. However, its binding rate was significantly slower than that of PEG. M-ALD-10KD The hydrolysis efficiency of -OPHDS5 is insufficient to prevent the persistent damage to the central nervous system caused by free ethylparaben. Animal studies show that rats exhibit epileptic-like symptoms upon death. Traditional drugs such as atropine and pralidoxime have extremely strict therapeutic windows and are often ineffective in severe cholinergic crises.

[0101] As can be seen from the above embodiments, the organophosphate poisoning rescue complex enzyme provided by the present invention can effectively clear free organophosphate compounds in the body and regulate the metabolism of acetylcholine, providing an efficient detoxification strategy for organophosphate poisoning.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex enzyme for treating organophosphate poisoning, characterized in that, The enzyme includes polyethylene glycol-modified organophosphorus hydrolase and butyrylcholinesterase, wherein the mass ratio of the polyethylene glycol-modified organophosphorus hydrolase to butyrylcholinesterase is (4~8):(2500~10000).

2. The organophosphate poisoning rescue complex enzyme according to claim 1, characterized in that, The mass ratio of the polyethylene glycol-modified organophosphorus hydrolase to butyrylcholinesterase is (5~6):(5000~10000).

3. The organophosphate poisoning rescue complex enzyme according to claim 1 or 2, characterized in that, The dosage of the polyethylene glycol-modified organophosphorus hydrolase is 40-80 μg / kg, and the dosage of the butyrylcholinesterase is 25-100 mg / kg.

4. The organophosphate poisoning rescue complex enzyme according to claim 1, characterized in that, The polyethylene glycol-modified organophosphorus hydrolase includes wild-type organophosphorus hydrolase or mutant organophosphorus hydrolase; the amino acid sequence of the wild-type organophosphorus hydrolase is shown in SEQ ID NO.1, and the amino acid sequence of the mutant organophosphorus hydrolase is shown in SEQ ID NO.

2.

5. The organophosphate poisoning rescue complex enzyme according to claim 4, characterized in that, The nucleotide sequence encoding the wild-type organophosphorus hydrolase is shown in SEQ ID NO.3, and the nucleotide sequence encoding the mutant organophosphorus hydrolase is shown in SEQ ID NO.

4.

6. The organophosphate poisoning rescue complex enzyme according to claim 1, characterized in that, The polyethylene glycol has a molecular weight of 8-12 kDa, and the polyethylene glycol modification includes one or more of the following: N-terminal, C-terminal, lysine side chain, and cysteine ​​side chain site-directed modification.

7. The use of the organophosphate poisoning rescue complex enzyme according to any one of claims 1 to 6 in the preparation of drugs for treating organophosphate poisoning.

8. The use of the organophosphorus poisoning rescue complex enzyme according to any one of claims 1 to 6 in the preparation of organophosphorus compound detection or organophosphorus compound removal reagents.

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