Medicine, preparation and composition for treating neuropathic pain and preparation method of medicine, preparation and composition

Abstract

According to the medicine, the preparation and the composition for treating the neuropathic pain and the preparation method of the medicine, the preparation and the composition, the medicine comprises polypeptide CBD3A6K-RhoB and PEG-PLGA, and the polypeptide CBD3A6K-RhoB is wrapped by the PEG-PLGA. In the embodiment of the invention, the nanoparticles formed by PEG-PLGA and the RhB group in the polypeptide CBD3A6K-RhoB have a synergistic effect, and the drug is jointly assisted to penetrate through the cell membrane and enter the cell to play a role, so that the neuropathic pain can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a medicine, preparation and composition for treating neuropathic pain and a preparation method thereof. BACKGROUND

[0002] Neuropathic pain is caused by lesions or diseases involving the somatosensory nervous system, has become a major global health burden, and seriously affects the quality of life of millions of patients, but effective therapies are very limited. The key is that N-type voltage-gated calcium channels (Ca V 2.2) play a central role in its pathogenesis. These channels are densely expressed in dorsal root ganglion (DRG) and spinal neurons, responsible for regulating synaptic neurotransmitter release, and mediating the transmission of pain signals from peripheral nociceptors to the central nervous system. Notably, neuropathic states significantly upregulate Ca V 2.2 activity; nerve injury enhances the transcriptional expression of Ca V 2.2α1b subunit in DRG neurons, which is directly related to mechanical allodynia and thermal hyperalgesia. This disease-specific dysregulation, combined with the low expression level of Ca V 2.2 in non-neural tissues, makes it a target with clear mechanism and therapeutic potential.

[0003] Based on this principle, ziconotide made of synthetic ω-conotoxin MVIIA was approved by FDA for marketing in 2004 as a selective Ca V 2.2 blocker. Despite its targeted design, its clinical application is limited by intrathecal administration only, narrow therapeutic window, and adverse reactions such as dizziness, nausea, and urinary retention due to poor central nervous system selectivity and persistent channel blockade. Therefore, although ziconotide has verified Ca V 2.2 inhibition as an analgesic strategy, its pharmacokinetic and pharmacodynamic limitations require next-generation inhibitors with enhanced specificity and delivery properties. SUMMARY

[0004] Therefore, the present application provides a medicine, preparation and composition for treating neuropathic pain and a preparation method thereof, so as to at least partially solve the defects of the prior art.

[0005] In a first aspect, the embodiments of the present application provide a medicine for treating neuropathic pain, comprising:

[0006] a polypeptide CBD3A6K-RhoB and PEG-PLGA, wherein the PEG-PLGA encapsulates the polypeptide CBD3A6K-RhoB.

[0007] In one possible implementation, the neuropathic pain is selected from one or more of sciatica, trigeminal neuralgia, diabetic neuropathy, postherpetic neuralgia, and spinal cord injury.

[0008] Secondly, embodiments of this application provide an agent for treating neuropathic pain, comprising:

[0009] The drugs and excipients mentioned in the first aspect.

[0010] In one possible implementation, the dosage form of the formulation includes:

[0011] One or more of the following: tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections.

[0012] Thirdly, embodiments of this application provide a pharmaceutical composition for treating neuropathic pain, comprising:

[0013] The medicine described in the first aspect, or the preparation described in the second aspect;

[0014] Other active ingredients, which are any ingredients that can relieve symptoms caused by or associated with neuropathic pain, but are different from CBD3A6K-RhB peptide and its derivatives.

[0015] In one possible implementation, the dosage form of the pharmaceutical composition includes:

[0016] One or more of the following: tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections.

[0017] In one possible implementation, the pharmaceutical composition further includes:

[0018] Coloring agents, preservatives, flavorings, flavoring agents and / or sweeteners.

[0019] Fourthly, embodiments of this application provide a method for preparing the polypeptide CBD3A6K-RhoB, comprising:

[0020] Step 1: Rink Amide resin was deprotected using 20% ​​piperidine to remove the Fmoc protecting groups and expose the amino groups, thus obtaining NH2-Resin;

[0021] Step 2: Add Fmoc-amino acid, HCTU and AEEA to NH2-Resin in a ratio of 1:3:3:6, and react at 30°C for 25-90 minutes to obtain Fmoc-NH-Aa-CONH-Resin;

[0022] Step 3: Add Fmoc-amino acids, HCTU and AEEA to Fmoc-NH-Aa-CONH-Resin in a ratio of 1:3:3:6, react at 30°C for 25-90 minutes, repeat the process multiple times, adding the corresponding amino acids in sequence to obtain a full-chain polypeptide that matches the polypeptide sequence ARSRLKELRGVPRGL.

[0023] Step 4: Add HOAT, HATU, DIEA and AEEA to the full-chain peptide for treatment, then add Rhodamine B, HCTU and DIEA for treatment to obtain crude peptide;

[0024] Step 5: The crude peptide was cleaved at a ratio of TFA:H2O:PhOH:TIPs = 88:5:5:2 and purified using high performance liquid chromatography to obtain peptide CBD3A6K-RhB.

[0025] Fifthly, embodiments of this application provide a method for preparing a medicament for treating neuropathic pain, comprising:

[0026] The inner oil phase, intermediate aqueous phase, and outer oil phase are injected into the microfluidic device through independent inlets, forming dual emulsion droplets which are then collected.

[0027] The dual emulsion was placed in a membrane bag and dialyzed overnight in distilled water to remove organic solvents;

[0028] The dialyzed aqueous solution of nanoparticles was freeze-dried to obtain the pharmaceutical composition of claim 1.

[0029] The inner oil phase is a dichloromethane solution containing 8% PEG-PLGA, the intermediate aqueous phase consists of 1% polyvinyl alcohol and 0.5% polypeptide CBD3A6K-RhoB, and the outer oil phase is a toluene solution containing 10% Span80.

[0030] In one possible implementation, the polypeptide CBD3A6K-RhoB is prepared using the method of claim 8.

[0031] The medicament for treating neuropathic pain prepared according to the embodiments of this application has at least the following advantages:

[0032] 1. Compared to the original CBD3 peptide, the modified CBD3A6K peptide exhibits superior analgesic properties and achieves significant reversal of neuropathic pain in a tibial nerve injury (TNI) model.

[0033] 2. The nanoparticles formed by PEG-PLGA have a synergistic effect with the RhB group in the peptide CBD3A6K-RhoB, which together help the drug penetrate the cell membrane and enter the cell to exert its effect, thus improving neuropathic pain. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 A schematic diagram illustrating the implementation principle of CRPPNs provided in this application embodiment;

[0036] Figure 2 A flowchart of a solid-phase peptide synthesis method (SPPS) based on 9-fluorenylmethoxycarbonyl (Fmoc) is provided for embodiments of this application.

[0037] Figure 3A A model diagram of the polypeptide CBD3A6K-RhoB prepared for the embodiments of this application;

[0038] Figure 3B The structural formula of the polypeptide CBD3A6K-RhoB prepared in the embodiments of this application;

[0039] Figure 4 This is a schematic diagram of a mass spectrometry analysis for peptide characterization provided in an embodiment of this application;

[0040] Figure 5 This is a schematic diagram of a high-performance liquid chromatography (HPLC) analysis and detection of peptide characterization provided in an embodiment of this application;

[0041] Figure 6 A schematic diagram illustrating the preliminary molecular docking analysis of CBD3A6K(A) and CBD3A6K-RhB(B) with CaV2.2 channel protein, provided for embodiments of this application;

[0042] Figure 7 Morphological images of CRPPNs prepared for embodiments of this application under a transmission electron microscope;

[0043] Figure 8 Particle size distribution diagram of CRPPNs prepared in the embodiments of this application;

[0044] Figure 9 A schematic diagram of drug characterization of CRPPNs prepared in the embodiments of this application after EDS energy dispersive spectroscopy analysis;

[0045] Figure 10 The results of stability analysis of CRPPNs prepared in the embodiments of this application under simulated normal and slightly acidic pH environments in vitro;

[0046] Figure 11 The results of in vitro release experiments of CRPPN nanoparticles prepared in the embodiments of this application;

[0047] Figure 12 This is a control diagram of an animal behavioral experiment of a drug provided in an embodiment of this application. Detailed Implementation

[0048] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. It should be noted that the described embodiments are merely some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort should fall within the scope of protection of this application.

[0049] Unless otherwise specified, all test materials used in the following examples are available through conventional commercial channels.

[0050] The interaction between CRMP2 and the pore-forming subunit of calcium channel protein 2.2 (α1b) constitutes a key regulatory mechanism for neural signal transduction. CRMP2 co-localizes with calcium channel protein 2.2 in various neuronal compartments, including immature and mature synapses, growth cones, and dorsal root ganglion neurons, where it enhances pain signal transduction. Recent research has found that the 15-amino acid peptide CBD3 (ARSRLKELRGVPRGL) composed of CRMP2 residues 484-498 can act as a selective calcium channel protein 2.2 receptor. V Inhibitor 2.2 inhibits CRMP2-Ca V2.2 Interactions, thereby weakening calcium ion influx and inhibiting downstream pain signals. Based on this finding, we successfully developed a modified CBD3A6K peptide by replacing alanine (A) at position 6 with lysine (K) for structural optimization. Compared to the original CBD3 peptide, this modified peptide exhibits superior analgesic properties and achieved significant reversal of neuropathic pain in a tibial nerve injury (TNI) model. Furthermore, further optimization with aptamers that bind to fluorescent groups such as rhodamine B not only improves intracellular internalization efficiency but also enables real-time tracking of peptide dynamics. Despite breakthroughs in drug development, peptide therapeutics such as CBD3A6K derivatives still face delivery challenges: susceptibility to protease degradation and oxidative damage, poor membrane permeability leading to low intracellular bioavailability, and systemic off-target effects caused by non-specific distribution. Although engineered conjugates such as TAT-CBD3A6K and R9-CBD3A6K have shown significant progress in improving neuronal uptake, their widespread application still faces challenges in enhancing tissue specificity and optimizing safety. Traditional drug delivery methods, such as intravenous and intramuscular injections, have also revealed problems of insufficient targeting and low drug utilization efficiency.

[0051] To address these limitations, nanotechnology-based delivery systems have emerged as a promising alternative. Biodegradable polymer nanocarriers—especially those using synthetic polyester materials such as polylactic-co-glycolic acid copolymer (PLGA)—offer integrated solutions through a triple mechanism: protecting therapeutic peptides from enzymatic degradation, utilizing nanocarrier-mediated endocytosis to overcome membrane permeability barriers, and achieving tissue targeting through surface modification to reduce off-target effects. Among these, polyethylene glycol-modified PLGA (PEG-PLGA) copolymer nanoparticles exhibit key advantages: PEGylation reduces systemic circulation limitations caused by immune clearance, while retained polymer function precisely regulates drug release kinetics and achieves targeted distribution to lesion sites. In summary, these properties address the core challenges of CBD3A6K peptide drug delivery: ensuring the active ingredient is protected from enzymatic degradation, promoting intracellular delivery through alternative uptake pathways, and achieving neuronal-specific targeting. This comprehensive solution makes the PEG-PLGA nanoplatform an ideal choice for the treatment of neuropathic pain.

[0052] Based on this nanotechnology, we developed CBD3A6K-RhB@PEG-PLGA nanoparticles (CRPPNs) to enhance intraneural delivery to primary sensory neurons. This nanodelivery system achieves enhanced delivery of CRPPNs to CRMP2-Ca through controlled peptide release. V 2.2 Continuous regulation of interactions effectively inhibits neurotransmitter membrane transport and exocytosis.

[0053] See Figure 1This is a schematic diagram illustrating the implementation principle of CRPPNs provided in an embodiment of this application. Figure 1 As shown, the therapeutic peptide CBD3A6K-RhB selectively inhibits the CRMP2-Cav2.2 pathway within nerve cells, thereby inhibiting the release of calcium currents and the calcitonin gene-related peptide (CGRP), a pain-related neurotransmitter, and thus blocking pain signal transmission. The therapeutic peptide CBD3A6K-RhB was prepared using solid-phase peptide synthesis technology and encapsulated in PEG-PLGA nanoparticles using microfluidic technology, constructing a comprehensive therapeutic nanodelivery system integrating enzyme protection of bioactive peptides, sustained-release kinetics to maintain therapeutic concentrations, and rhodamine B fluorescence tracking functionality. Comprehensive in vitro and in vivo evaluations confirmed that the system possesses excellent biocompatibility and functional activity lasting for more than ten days. Experiments with subdermal injection into the sciatic nerve in rats showed that this method can prolong analgesic effects and avoid systemic toxicity caused by traditional drug delivery methods. Our developed nanodelivery system significantly improves the utilization efficiency and biosafety of peptide drugs, laying a solid translational medical foundation for the clinical management of neuropathic pain.

[0054] The CBD3A6K-RhB peptide described herein is an optimized variant of the parent CBD3, namely CBD3A6K(ARSRLKELRGVPRGL), prepared by replacing the A at position 6 of the peptide CBD3 (composed of amino acid positions 484-498 of CRMP-2) with K, and then attaching a new group—rhodamine b—to its amino terminus. It can bind to CaV2.2, thereby inhibiting the interaction between CRMP-2 and CaV2.2.

[0055] Pharmacodynamics: After being encapsulated with PEG-PLGA, CBD3A6K-RhB peptide can maintain its effect for about 2 weeks, after which the detectable drug concentration decreases significantly. In animal pain sensitivity experiments, rat pain sensitivity was significantly increased 2 weeks after administration.

[0056] The term "PLGA" used in this article refers to polylactic-co-glycolic acid, which is a copolymer formed by the polymerization reaction of lactic acid and glycolic acid. It is a biodegradable functional high molecular weight organic compound that is non-toxic and has good biocompatibility, encapsulation and film-forming properties. The PLGA used in this invention is PEG-PLGA formed by the polymerization of PLGA and polyethylene glycol (PEG).

[0057] To improve cellular uptake efficiency, facilitate tracking and localization, and enable imaging monitoring of the therapeutic peptide CBD3A6K, we coupled Rhodamine B (RhB) to the N-terminus of CBD3A6K via a 2-aminoethoxyethoxyacetic acid (AEEA) linker, resulting in RhB-AEEA-CBD3A6K. Rhodamine B, being negatively charged, readily crosses negatively charged cell membranes and, compared to the long CBD3A6K peptide chain (ARSRLKELRGVPRGL), is a large molecule, exhibiting a certain degree of membrane-drag effect, significantly increasing cell penetration and nuclear targeting, and allowing for real-time monitoring of peptide distribution. However, because Rhodamine B (479 Da) is a large-molecule fluorescent dye with high steric hindrance, incomplete N-terminal coupling may occur, potentially affecting the peptide's stable secondary structure and increasing its instability. Therefore, AEEA (8-amino-3,6-dioxanoic acid) was chosen as the linker arm. As a hydrophilic spacer arm, its addition between RhB and the terminal Ala amino acid sequence increases the distance between the linker and the peptide chain, reducing steric hindrance. Simultaneously, AEEA increases water solubility. The peptide was synthesized using a solid-phase peptide synthesis method (SPPS) based on 9-fluorenemethoxycarbonyl (Fmoc) (see attached). Figure 1 It was artificially synthesized in a conventional air bath constant temperature shaker, using Rink amide AM resin as the carrier of Fmoc-SPPS.

[0058] To facilitate understanding, the preparation method of peptide CBD3A6K-RhoB is described in detail below with reference to the accompanying drawings.

[0059] See Figure 2 This document provides a flowchart of a solid-phase peptide synthesis (SPPS) method based on 9-fluorenemethoxycarbonyl (Fmoc) as an embodiment of this application. Figure 2 As shown, it mainly includes the following steps.

[0060] Step 1: Rink Amide resin (Fmoc-NH-Resin) is deprotected using 20% ​​piperidine to remove the Fmoc protecting group and expose the amino group (-NH2) to obtain NH2-Resin.

[0061] Specifically, the Fmoc group is sensitive to bases, and piperidine causes it to undergo a β-elimination reaction, generating a highly conjugated fluorene dibenzofuran derivative which is then removed, exposing the free α-amino group.

[0062] Step 2: Add Fmoc-amino acid (Fmoc-NH-Aa-OH), HCTU and DIEA to NH2-Resin in a ratio of 1:3:3:6, and react at 30°C for 25-90 minutes to obtain Fmoc-NH-Aa-CONH-Resin.

[0063] Specifically, HCTU, as a condensing agent, reacts with the carboxyl group of amino acids via chlorine atoms to generate a highly reactive O-acylurea onium salt intermediate, significantly enhancing the nucleophilicity of the carboxyl group. Simultaneously, compared to traditional condensing agents (such as DIC), the hexafluorophosphate structure of HCTU reduces racemic side reactions. DIEA, as an activating base, neutralizes the HCl released during condensation, driving the reaction towards peptide bond formation while maintaining the pH of the reaction system within a suitable range of 8-9. It also enhances the nucleophilic attack efficiency of the HCTU-activated carboxyl group on the free amino group, accelerating the coupling reaction.

[0064] The complex peptide structure of RhB-AEEA-CBD3A6K contains multiple consecutive basic residues (such as `RSR` and `RGVPR`), which easily form intramolecular / intermolecular hydrogen bonds, making the peptide difficult to synthesize, easy to aggregate, and difficult to dissolve. Therefore, the HCTU / DIEA condensation system is adopted.

[0065] It should be noted that in this step, the added Fmoc-amino acid should be the first amino acid in the polypeptide sequence ARSRLKELRGVPRGL, namely leucine (L).

[0066] Step 3: Add Fmoc-amino acids, HCTU and AEEA to Fmoc-NH-Aa-CONH-Resin in a ratio of 1:3:3:6, react at 30°C for 25-90 minutes, repeat the process multiple times, adding the corresponding amino acids in sequence to obtain a full-chain peptide ([full-chain-peptides]-Resin) that matches the peptide sequence ARSRLKELRGVPRGL.

[0067] It should be noted that in this step, starting from the second amino acid in the polypeptide sequence ARSRLKELRGVPRGL, the corresponding amino acids are added sequentially to the polypeptide chain until all amino acids are added, thus obtaining a full-chain polypeptide that matches the polypeptide sequence ARSRLKELRGVPRGL.

[0068] It is understandable that the second amino acid in the polypeptide sequence ARSRLKELRGVPRGL is glycine (G). Therefore, in this step, glycine (G), arginine (R), proline (P), and so on, are added to the peptide chain in sequence.

[0069] Step 4: Add HOAT, HATU, DIEA and AEEA to the full-chain peptide Resin, then add rhodamine B, HCTU and DIEA to obtain the crude peptide ([Hybrid peptide]).

[0070] Specifically, for sequences of amino acids that are prone to epimerization, such as AEEA, HATU exhibits superior inhibitory effects on racemization. HOAt, as an additive, can capture HOBt (1-hydroxybenzotriazole) released during HATU activation, preventing it from competitively binding to the carboxyl group of amino acids, thus improving coupling efficiency and reducing condensation side reactions caused by HOBt accumulation, making it particularly suitable for the synthesis of long-chain peptides.

[0071] Step 5: The crude peptide ([Hybrid peptide]) was subjected to peptide cleavage at a ratio of TFA:H2O:PhOH:TIPs = 88:5:5:2, and then purified using high-performance liquid chromatography (RP-HPLC) to obtain the final product peptide CBD3A6K-RhB. The peptide sequence is: RhB-AEEA-ARSRLKELRGVPRGL-NH2.

[0072] Specifically, 88% TFA ensures sufficient acid concentration for rapid cleavage (typically 30 minutes to 2 hours) while avoiding excessive acidification that could lead to side-chain modifications of sensitive amino acids. 5% H2O balances cleavage efficiency with side-reaction inhibition; too low a concentration can result in incomplete cleavage, while too high a concentration may induce peptide hydrolysis. 5% PhOH + 2% TIS minimizes carbocation side reactions through a dual scavenging mechanism.

[0073] TFA can remove the protecting groups of all amino acid side chains and cleave the synthesized crude peptide from the resin. In the purification step, the crude peptide is dissolved in a medium consisting of distilled water and a small amount of acetonitrile, followed by high-performance liquid chromatography (HPLC) separation.

[0074] The core principle of the preparation method of the polypeptide CBD3A6K-RhoB provided in this application embodiment is as follows: the C-terminus of the peptide chain is covalently fixed on a solid support, and then, starting from the N-terminus, appropriately protected amino acid monomers are added sequentially. After repeated removal of protecting groups (exposing reaction sites), coupling with excess reagent (extending peptide chains), and washing (purifying intermediates), the side chain deprotection and cleavage from the resin are finally completed in one step under strong acid conditions to obtain the target polypeptide.

[0075] See Figure 3A This is a model diagram of the polypeptide CBD3A6K-RhoB prepared in the embodiments of this application; see also Figure 3B , is the structural formula of the polypeptide CBD3A6K-RhoB prepared in the embodiments of this application.

[0076] Analysis of the rationality of the preparation method of the polypeptide CBD3A6K-RhoB provided in the embodiments of this application:

[0077] (1) Strategy selection

[0078] Fmoc is more suitable for this sequence (the side chain protecting group is cleaved under acidic conditions, avoiding the destruction of strong acid-sensitive groups such as RhB).

[0079] Choose RinkAmide resin: ensure the C-terminus is an amide (-CONH2).

[0080] (2) Step Design

[0081] The first amino acid, Fmoc-Leu-OH, is fixed at the C-terminus and ligated to Rink resin to form Leu-NH2 (meeting the sequence end requirement).

[0082] RhB coupling: After peptide chain synthesis, RhB-AEEA-COOH is directly coupled onto the resin (to avoid purification losses due to solution phase modification).

[0083] The cleavage reagent of TFA:H2O:PhOH:TIPs = 88:5:5:2 is more suitable for this long-chain peptide to remove the side chain protecting groups and cleave the peptide chain.

[0084] Analysis of the difficulties in preparing the polypeptide CBD3A6K-RhoB provided in the embodiments of this application:

[0085] (1) Rhodamine B (479Da) is a macromolecular fluorescent dye with high steric hindrance, which may lead to incomplete N-terminal coupling, easily affecting the stable secondary structure of the peptide itself and increasing the instability of the peptide itself.

[0086] Solution:

[0087] AEEA (8-amino-3,6-dioxanoic acid) is a hydrophilic spacer arm. Adding it between RhB and the terminal Ala amino acid sequence can increase its distance from the peptide chain and reduce steric hindrance interference.

[0088] Pre-activated RhB-AEEA: RhB-AEEA-COOH and HATU / HOAt / DIEA are pre-activated in DMF for 5 min before adding the resin.

[0089] Extend the reaction time: extend the coupling reaction to 2.5 hours (room temperature).

[0090] The Kaiser Test confirms whether the N-terminal amino group has reacted completely.

[0091] (2) The sequence contains multiple consecutive basic residues (such as `RSR`, `RGVPR`), which are prone to forming intramolecular / intermolecular hydrogen bonds, making the polypeptide difficult to synthesize, easy to aggregate, and difficult to dissolve.

[0092] Solution: Select HCTU as the activator (more efficient than HBTU, reduces racemization), and extend the reaction time in the step of adding basic amino acids.

[0093] (3) The peptide chain contains multiple hydrophobic residues (Leu, Val, Pro). After cleavage, the crude peptide may precipitate in ether in a gel-like manner, making it difficult to purify.

[0094] Solution:

[0095] Cutting mixture optimization: Use TFA:H2O:PhOH:TIPs = 88:5:5:2 to protect side chains and improve solubility.

[0096] Precipitation solvent replacement: Use acetonitrile instead of diethyl ether to reduce the aggregation of hydrophobic peptides.

[0097] See Figure 4 This is a schematic diagram of a mass spectrometry analysis for peptide characterization provided in an embodiment of this application. Figure 4 As shown, electrospray ionization mass spectrometry (ESI-MS) analysis revealed that the calculated molecular mass of the peptide, derived from the three main peaks of CBD3A6K-RhB, was (570.000*4-4+759.650*3-3+1139.200*2-2) / 3 = 2276.117. This is within the acceptable error range and not significantly different from the theoretical molecular mass of 2278.762 determined based on the peptide structure, indicating that CBD3A6K-RhB was successfully synthesized.

[0098] See Figure 5 This is a schematic diagram illustrating a high-performance liquid chromatography (HPLC) method for peptide characterization, provided in an embodiment of this application. Figure 5 As shown, by analytical reversed-phase high-performance liquid chromatography (RP-HPLC), the main peak area of ​​CBD3A6K-RhB accounts for about 92% of the area of ​​all peaks, indicating that the purity of the peptide after purification is relatively high and exceeds 90%.

[0099] In summary, the CBD3A6K and its RhB derivatives were successfully synthesized by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS), and the RP-HPLC purity exceeded 90%.

[0100] See Figure 6 This is a schematic diagram illustrating the preliminary molecular docking analysis of CBD3A6K(A) and CBD3A6K-RhB(B) with CaV2.2 channel protein, as provided in an embodiment of this application.

[0101] Preliminary molecular docking analysis of CBD3A6K and CaV2.2 channel protein revealed stable binding, with an optimal docking energy of -5.0 kcal / mol. Figure 6 (A) To assess whether RhB-AEEA modification affects the binding affinity of the peptide to CaV2.2, we performed molecular docking analysis on CBD3A6K-RhB. The results showed that its binding stability was comparable to that of the unmodified peptide, with an optimal binding energy of -4.9 kcal / mol. Figure 6 (B in the original text). This indicates that the coupling strategy did not significantly reduce the predicted binding affinity of the peptide to the CaV2.2 channel protein. In summary, we successfully synthesized high-purity CBD3A6K and its RhB-conjugated derivative CBD3A6K-RhB, and characterized their properties. Furthermore, CBD3A6K and CBD3A6K-RhB were found to have similar secondary structures and comparable CaV2.2 binding affinity, supporting their potential to inhibit CRMP-2 / CaV2.2 interactions.

[0102] Based on the prepared peptide CBD3A6K-RhB, CBD3A6K-RhB@PEG-PLGA nanoparticles (CRPPNs) were further prepared. The CBD3A6K-RhB@PEG-PLGA nanoparticles were prepared using classical microfluidic technology. In the preparation of CRPPNs, the inner oil phase was a dichloromethane solution containing 8% (w / v) PEG-PLGA, the middle aqueous phase consisted of 1% (w / v) polyvinyl alcohol and 0.5% (w / v) peptide, and the outer oil phase was a toluene solution containing 10% (w / w) Span 80. These three phases were injected into the microfluidic device through independent inlets, forming double emulsion droplets which were then collected. The droplets were subsequently dialyzed overnight in distilled water using a membrane bag to remove organic solvents. Finally, the nanoparticles were freeze-dried to obtain the CRPPNs.

[0103] See Figure 7 The image shows the morphological characteristics of CRPPNs prepared according to embodiments of this application under a transmission electron microscope. Figure 7 As shown, CRPPNs include the peptide CBD3A6K-RhoB and PEG-PLGA, wherein the PEG-PLGA encapsulates the peptide CBD3A6K-RhoB.

[0104] It is understood that the CRPPNs prepared in the embodiments of this application are a drug for treating neuropathic pain. The neuropathic pain is selected from one or more of the following: sciatica, trigeminal neuralgia, diabetic neuropathy, postherpetic neuralgia, and spinal cord injury.

[0105] The test and experimental results of the CRPPNs prepared in the embodiments of this application are described below.

[0106] 1. Particle size and zeta potential determination

[0107] A small amount of the prepared nanoparticles was diluted to an appropriate concentration with 1 ml of double-distilled water as the solute. The particle size and zeta potential of the nanoparticles were determined using a dynamic light scattering particle size analyzer (instrument name to be determined). Under stable conditions, the particle size of the CBD3A6K nanoparticles was measured to be approximately 180 nm (e.g., ...). Figure 8 As shown in the figure, the zeta potential was measured to be approximately -13mV.

[0108] 2. Morphological examination

[0109] Similarly, a small amount of the prepared nanoparticles was diluted to an appropriate concentration with a small amount of double-distilled water as a solute. One or two drops of the nanoparticle colloidal solution were placed on a copper grid, stained with 10.0 g·L⁻¹ phosphotungstic acid, air-dried at room temperature, and the morphology of the nanoparticles was observed and photographed under a transmission electron microscope (TEM).

[0110] 3. EDS energy dispersive spectroscopy analysis

[0111] A small amount of the prepared nanoparticles were placed on a conductive carbon adhesive for scanning electron microscopy (SEM), and gold was sputtered onto it to give our material a certain degree of conductivity. EDS (Energy Dispersive Spectroscopy) analysis was then performed under an SEM. Because the outer shell of our nanoparticles is made of the polymer PEG-PLGA, which consists only of C, N, and O elements, the results for empty PEG-PLGA nanoparticle shells, within the allowable error range, only show the presence of these three elements. However, after encapsulating peptides containing N and rhodamine b containing Cl, the results showed that nanoparticles encapsulated with CBD3A6K and CBD3A6K-rhodamine b could additionally show a certain amount of N and N / Cl elements, respectively.

[0112] See Figure 9 This is a schematic diagram illustrating the drug characterization of CRPPNs prepared in the embodiments of this application after EDS energy dispersive spectroscopy analysis. Figure 9 As shown, EDS energy dispersive spectroscopy analysis of CRPPN nanoparticles revealed that the particles contain a certain proportion of N and Cl atoms. These elements are only present in the polypeptide CBD3A6K-RhB, indicating that the polypeptide drug has been encapsulated within the nanoparticles, demonstrating successful drug loading.

[0113] 4. Determination of drug loading and encapsulation efficiency

[0114] By measuring the absorbance of CBD3A6K-Rhodamine B at 562 nm at different concentrations, an absorbance-concentration standard curve can be plotted. By measuring the absorbance of the supernatant after centrifuging CBD3A6K-Rhodamine B, and substituting it into the standard curve formula, the concentration of peptides not loaded into the nanoparticles can be determined, and the mass of peptides encapsulated in the nanoparticles can then be calculated.

[0115] The formulas are as follows: Encapsulation efficiency (EE) = m_packet / m_peptide × 100% to calculate the encapsulation efficiency (EE) of the drug-loaded nanoparticles; Drug loading (DL) = m_packet / m_total × 100% to calculate the drug loading (DL%) of the drug-loaded nanoparticles. Where m_packet is the mass of the peptide encapsulated in the nanoparticle (mg), m_peptide is the total mass of the added peptide (mg), and m_total is the total mass of the drug-loaded nanoparticles (mg). The measured drug loading and encapsulation efficiency were 12.6% ± 0.74% and 81.3% ± 3.24%, respectively.

[0116] 5. In vitro stability test of CRPPNs

[0117] A small amount of the prepared nanoparticles were dissolved in double-distilled water. The prepared colloidal solution of nanoparticles was placed in a refrigerator at 4°C, and the particle size changes of the nanoparticles were measured at different time points: immediately, 1 hour, 6 hours, 12 hours, 24 hours, and 3 days. The measured particle size changes of the nanoparticles at different time points were not significant, and the PDI value was small, indicating that our drug has good stability.

[0118] See Figure 10 The results are the stability analysis results of the CRPPNs prepared in the embodiments of this application under simulated normal and slightly acidic pH environments in vitro.

[0119] like Figure 10 As shown, the particle size and distribution coefficient (PDI) of CRPPN nanoparticles remained consistent over a long period of time under different pH environments (pH 7.4 and 6.5), indicating that the peptides have strong stability and are basically unaffected by pH.

[0120] 6. In vitro release assay of CRPPNs

[0121] Phosphate-buffered saline (PBS) was selected as the release medium. PBS solutions of CBD3A6K-RhB at different concentrations (0-1000 μg / mL) were prepared, and absorbance was measured at 214 nm to plot a standard curve. A certain amount of nanoparticle drug was dissolved in PBS solutions at different pH levels, and absorbance was measured at different time points (2 hours, 5 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, and 20 days) using a UV-Vis spectrophotometer. The absorbance was then substituted into the standard curve to determine the peptide content in the samples, and the cumulative release amount and cumulative release percentage of the drug were calculated.

[0122] See Figure 11 The results are from the in vitro release experiment of the CRPPNs nanoparticles prepared in the embodiments of this application.

[0123] like Figure 11As shown, the release rate of CRPPN nanoparticles reaches its maximum within 4-6 days, and the maximum release is basically completed around the 10th day, with a maximum release ratio of about 82%. The release rate is slightly faster in a slightly acidic environment (pH=6.5).

[0124] 7. Experiment on the reduction of pain sensitivity in rats by CRPPNs

[0125] We established a CCI model to simulate clinical traumatic painful peripheral neuropathy. Experimental animals were anesthetized with isoflurane at a 4% induction concentration and a 2% maintenance concentration. Under anesthesia, the right sciatic nerve was exposed under aseptic conditions by blunt dissection of the biceps femoris muscle. Four loose ligatures were created using 4-0 silk sutures at approximately 1 mm intervals, forming a 4-5 mm long damaged nerve segment. Mild compression of the superficial artery was applied under a surgical microscope to ensure tight ligation while maintaining blood flow. The model was considered successfully established when rats exhibited typical neuropathic pain symptoms (hindlimb weight-bearing, paw curling, licking, and lameness). Postoperatively, the covered muscles and skin were sutured. Rats in the sham-operated group underwent only all the above procedures without nerve ligation and transection.

[0126] Rats that underwent CCI surgery exhibited typical spontaneously triggered pain symptoms, consistent with previous findings, including curling up, raising their bodies, or placing the affected paw sideways in a standing or sitting position to avoid contact with the ground, thereby alleviating pain. One month post-surgery, behavioral test data from the normal group, sham-operated group, and CCI group were used not only to assess the development of CCI-induced pain behaviors but also as a baseline for comparing the efficacy of drug administration.

[0127] One week after modeling, nanoparticle colloidal solution or simple CBD3A6K peptide suspension was microinjected into the proximal sciatic nerve on the ipsilateral side of the CCI. During injection, the microsyringe was held as close as possible to the tangential direction of the injection surface (horizontal angle 25-30°), and the needle tip was slowly advanced until it retracted to the epineurium. After successful penetration, the syringe was gently withdrawn until the tissue pressure disappeared to minimize pressure on the pipette nozzle. After injection, the pipette tip was left to stand for 3 minutes to promote sealing of the surrounding tissue. Then, 20 μL of solution was slowly injected at a rate of 20 nL / s. The pipette was immobilized for 5 minutes after injection to allow for tissue fluid pressure equalization. Finally, the animals were returned to the animal facility and continued to be housed according to the experimental design requirements.

[0128] See Figure 12 This is a control diagram of an animal behavioral experiment for a drug provided in an embodiment of this application. Figure 12As shown, the paw retraction phenomenon in rats was observed and recorded before CCI administration and at 3, 14 and 20 days after CCI administration in the CBD3A6K group and the CRPPNs group. It was found that rats exhibited long-term pain sensitivity in the affected limb, i.e., paw retraction behavior, after CCI. CBD3A6K administration did not reverse this phenomenon, while CRPPNs administration showed a long-term reversal of pain sensitivity. At 20 days, a weakening of the therapeutic effect was observed, indicating that the drug can be continuously released to rats for no more than 20 days.

[0129] Corresponding to the above embodiments, this application also provides a formulation for treating neuropathic pain, the formulation comprising: CRPPNs and excipients.

[0130] In one possible implementation, the dosage form of the formulation includes one or more of the following: tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections.

[0131] Corresponding to the above embodiments, this application also provides a pharmaceutical composition for treating neuropathic pain, comprising: the above-mentioned CRPPNs drug or preparation; and other active ingredients, wherein the other active ingredients are any of those that can relieve symptoms caused or accompanied by neuropathic pain, but are different from CBD3A6K-RhB peptide and its derivatives.

[0132] In one possible implementation, the dosage form of the pharmaceutical composition includes one or more of the following: tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections.

[0133] In one possible implementation, the pharmaceutical composition further includes: a colorant, a preservative, a flavoring agent, a taster, and / or a sweetener.

[0134] The above description is merely a specific embodiment of this application. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the protection scope of this application. The protection scope of this application should be determined by the protection scope of the claims.

Claims

1. A drug for treating neuropathic pain, characterized in that, include: The peptide CBD3A6K-RhoB and PEG-PLGA, wherein the PEG-PLGA encapsulates the peptide CBD3A6K-RhoB.

2. The pharmaceutical composition according to claim 1, characterized in that, The neuropathic pain is selected from one or more of the following: sciatica, trigeminal neuralgia, diabetic neuropathy, postherpetic neuralgia, and spinal cord injury.

3. A preparation for treating neuropathic pain, characterized in that, include: The drug and excipients as described in claim 1 or 2.

4. The formulation according to claim 3, characterized in that, The dosage forms of the formulation include: One or more of the following: tablets, capsules, droplets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections.

5. A pharmaceutical composition for treating neuropathic pain, characterized in that, include: The drug according to claim 1 or 2, or the formulation according to claim 3 or 4; Other active ingredients, which are any ingredients that can relieve symptoms caused by or associated with neuropathic pain, but are different from CBD3A6K-RhB peptide and its derivatives.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage forms of the pharmaceutical composition include: One or more of the following: tablets, capsules, droplets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections.

7. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further includes: Coloring agents, preservatives, flavorings, flavoring agents and / or sweeteners.

8. A method for preparing the polypeptide CBD3A6K-RhoB, characterized in that, include: Step 1: Rink Amide resin was deprotected using 20% ​​piperidine to remove the Fmoc protecting groups and expose the amino groups, thus obtaining NH2-Resin; Step 2: Add Fmoc-amino acids, HCTU and AEEA to NH2-Resin in a ratio of 1:3:3:6, and react at 30°C for 25-90 minutes to obtain Fmoc-NH-Aa-CONH-Resin; Step 3: Add Fmoc-amino acids, HCTU and AEEA to Fmoc-NH-Aa-CONH-Resin in a ratio of 1:3:3:6, react at 30°C for 25-90 minutes, repeat the process multiple times, adding the corresponding amino acids in sequence to obtain a full-chain polypeptide that matches the polypeptide sequence ARSRLKELRGVPRGL. Step 4: Add HOAT, HATU, DIEA and AEEA to the full-chain peptide for treatment, then add Rhodamine B, HCTU and DIEA for treatment to obtain crude peptide; Step 5: The crude peptide was cleaved at a ratio of TFA:H2O:PhOH:TIPs = 88:5:5:2 and purified by high performance liquid chromatography to obtain peptide CBD3A6K-RhB.

9. A method for preparing a medicament for treating neuropathic pain, characterized in that, include: The inner oil phase, intermediate aqueous phase, and outer oil phase are injected into the microfluidic device through independent inlets, forming dual emulsion droplets which are then collected. The dual emulsion was placed in a membrane bag and dialyzed overnight in distilled water to remove organic solvents; The dialyzed aqueous solution of nanoparticles was freeze-dried to obtain the pharmaceutical composition of claim 1. The inner oil phase is a dichloromethane solution containing 8% PEG-PLGA, the intermediate aqueous phase consists of 1% polyvinyl alcohol and 0.5% polypeptide CBD3A6K-RhoB, and the outer oil phase is a toluene solution containing 10% Span80.

10. The method according to claim 9, characterized in that, The polypeptide CBD3A6K-RhoB was prepared using the method described in claim 8.

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