Amphiphilic polypeptide carrier and its application

Abstract

The present invention discloses an amphiphilic polypeptide carrier and its application. The present invention forms a drug sustained-release system by performing supramolecular assembly of an amphiphilic polypeptide carrier and an ion channel blocking biotoxin molecule (conotoxin, tetrodotoxin, etc.), which can effectively block nerve conduction to achieve pain blocking, prolong the analgesic time, and minimize systemic toxicity. In vivo experiments have shown that the drug sustained-release system assembled by the polypeptide carrier and conotoxin (ziconotide), saxitoxin and tetrodotoxin can maintain long-term analgesia, providing a powerful tool for non-addictive drug pain control and biotoxin molecule sustained / controlled release.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical engineering technology, in particular to an amphipathic polypeptide carrier and its application, in particular to its application as an anesthetic and analgesic drug delivery system. Background Art

[0002] During the development of malignant tumors, tumors or their metastases invade the nervous system. The information that the nervous system needs to repair or regulate continues to stimulate the central nervous system, which may eventually cause systemic pain sensitivity. Cancer pain is one of the main causes of suffering in patients with advanced cancer. Among patients with pain, 50% to 80% of the pain is not effectively controlled due to various reasons. Cancer pain, as a global problem, affects the lives of countless patients every day.

[0003] For pain management, the World Health Organization (WHO) has proposed a "three-step" analgesic treatment guideline. The first step is non-steroidal anti-inflammatory analgesics (NSAIDs), which are mainly used to treat mild pain, and non-steroidal anti-inflammatory drugs are preferred for analgesia; the second step is weak opioids. When cancer pain changes from mild to moderate, opioid analgesics such as tramadol and codeine are gradually used; the third step is strong opioids, which are mainly used for severe cancer pain, such as morphine, oxycodone, and hydroxymorphine. The pain caused by cancer is severe, usually moderate to severe pain, and opioid analgesics are mainly selected for clinical treatment. However, opioids are addictive and tolerant, and their side effects are also very obvious. At the same time, nearly 40% of patients are not sensitive to opioids, and cancer pain cannot be relieved. Therefore, the development of non-opioid analgesics is a major demand for clinical cancer pain treatment.

[0004] Studies have found that there are many biological toxins in nature that have anesthetic and analgesic effects that are superior to opioids. These biological toxins exert anesthetic or analgesic effects by blocking the permeability of ion channels to ions and interfering with the conduction of electrical signals in nerve cells. For example, toxins in pufferfish and stone house clams can block NaV1.3, NaV1.6 and NaV1.7, respectively. μ-conotoxin and ω-conotoxin from cone snails act on NaV1.6 and CaV1.3, respectively. ω-conotoxin derivatives act on CaV2.2. The spider-derived HpTx3 toxin peptide mainly acts on the NaV1.7 channel. It has been reported that these biological toxins have good pain suppression effects in inflammatory-induced pain models, surgical-induced pain models, and bone cancer pain models, and are non-addictive, and are expected to become new non-opioid potent analgesics.

[0005] Biotoxins block pain signal transmission by efficiently blocking ion channels, and will not cause addiction and tolerance, but their dosage window is narrow, and direct use is prone to systemic toxicity. In order to use biotoxins to develop non-opioid potent analgesics, it is necessary to construct a suitable dosage form to slow / controlled release biotoxin molecules. However, due to the strong hydrophilicity of biotoxin molecules, traditional drug carriers, such as liposomes and polymer carriers (particles, hydrogels), have low loading efficiency for them, and the preparation process has poor parallelism. Therefore, it is necessary to rationally design new drug delivery systems to achieve efficient loading and slow / controlled release of biotoxin drugs, in order to promote the application of biotoxin molecules in the treatment of severe clinical pain. Summary of the invention

[0006] The purpose of the present invention is to provide an amphiphilic polypeptide carrier and its application, especially its application in the sustained release of biotoxin analgesic drugs.

[0007] The present invention is conceived as follows: Biotoxin molecules (analgesics) achieve nerve blockade by efficiently blocking ion channels, and they show the potential to become non-opioid potent analgesics, but how to develop suitable dosage forms to achieve their safe use still faces huge challenges. Biotoxin molecules can interact with various proteins such as ion channel proteins in organisms, and these proteins are called receptor proteins for biotoxin molecules. Drug carriers constructed from peptides and amino acids selected from the domains where biotoxins bind to receptor proteins are very likely to achieve sustained / controlled release of biotoxin molecules, thereby achieving safe and lasting pain blockade.

[0008] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a polypeptide carrier, whose basic unit is an amphiphilic polypeptide molecule, including a hydrophilic part and a hydrophobic part, and the polypeptide carrier can produce supramolecular interaction with biological toxins; optionally, the hydrophilic part and the hydrophobic part are connected by a connecting module composed of one or more glycine.

[0009] Wherein, the hydrophilic part is designed and modified according to the structural domain on the receptor protein that can bind to the biological toxin;

[0010] The hydrophobic part is composed of hydrophobic alkyl chains and / or hydrophobic, aromatic amino acids;

[0011] The hydrophobic part is located at the C-terminus and / or N-terminus of the polypeptide molecule, or located in the middle of the polypeptide molecule.

[0012] In the present invention, the receptor protein may be an ion channel protein, such as a calcium ion channel protein, a sodium ion channel protein, and the like.

[0013] In the present invention, the biological toxin is an ion channel blocking toxin, and has potential anesthetic and analgesic medicinal effects. Preferably, conotoxin and its derivatives, tetrodotoxin and its derivatives, saxitoxin and its derivatives are selected, and more preferably ω-conotoxin (ziconotide), saxitoxin (STX), tetrodotoxin (TTX), etc.

[0014] Furthermore, the structure of the polypeptide carrier is as shown in any one of Formula I) to Formula III), and excluding the amino acids contained in the hydrophobic part and the connecting module, the number of the remaining amino acids in Formula I) and Formula II) does not exceed 20, and the number of the remaining amino acids in Formula III) does not exceed 40:

[0015] Formula I): (X)n1-T-(X)n2-E-(X)n3-W-(X)n4-(G)n5-U;

[0016] Formula II): U-(G)n5-(X)n1-T-(X)n2-E-(X)n3-W-(X)n4;

[0017] Formula III): (X)n1-T-(X)n2-E-(X)n3-W-(X)n4-(G)n5-U-(G)n5-(X)n4-W-(X)n3-E-(X)n2-T-(X)n1.

[0018] In formula I) to formula III), U represents a hydrophobic part;

[0019] U is a hydrophobic polypeptide composed of 3-10 hydrophobic, aromatic amino acids; or,

[0020] In formula I) and formula III), U is K-Cn, wherein K is lysine; in formula II), U is Cn, and Cn is an alkyl chain containing 6-22 carbon atoms, preferably C12, C16, or C18.

[0021] In formula I) to formula III), (G)n5 represents a linking module, wherein G is glycine.

[0022] In formula I) to formula III), X is any amino acid, T is threonine, E is glutamic acid, W is tryptophan, and n1, n2, n3, n4, and n5 are each independently an integer between 0 and 4.

[0023] In the present invention, the hydrophobic amino acid can be selected from L (leucine), M (methionine), W (tryptophan), V (valine), I (isoleucine), F (phenylalanine), A (alanine), Y (tyrosine) and the like.

[0024] Furthermore, the hydrophilic part is (X)n1-T-(X)n2-E-(X)n3-W-(X)n4, wherein X is any amino acid, T is threonine, E is glutamic acid, W is tryptophan, and n1, n2, n3, n4, and n5 are each independently an integer between 0 and 4.

[0025] Among them, the design of (X)n1-T-(X)n2-E-(X)n3-W-(X)n4 was inspired by the structural domain of ion channel protein binding to toxins.

[0026] (X)n1-T-(X)n2-E-(X)n3-W-(X)n4-(G)n5 all need to be capped, and the end groups include but are not limited to Ac, Benz, OMe, and -NH2.

[0027] Preferably, the amino acid sequence of the hydrophilic part is shown in any one of SEQ ID NOs: 1-5.

[0028] Preferably, the hydrophobic polypeptide is selected from X'X'-FFF, X'X'-FYF, wherein X' is any hydrophobic or aromatic amino acid.

[0029] Preferably, the amino acid sequence of the polypeptide carrier basic unit is shown in any one of SEQ ID NOs: 6-10.

[0030] In a second aspect, the present invention provides any of the following applications of the polypeptide vector:

[0031] 1) Used to prepare drug delivery / sustained release systems;

[0032] 2) Used to prepare detoxification materials for toxin drugs.

[0033] In a third aspect, the present invention provides a composition, which is composed of the polypeptide carrier or a mixture thereof and the biological toxin in a molar ratio of 1:1-100:1, preferably a molar ratio of 2:1-30:1.

[0034] Wherein, the biological toxin is an ion channel blocking toxin, and has potential anesthetic and analgesic medicinal effects. Preferably, conotoxin and its derivatives, tetrodotoxin and its derivatives, saxitoxin and its derivatives are selected, and more preferably, ω-conotoxin, saxitoxin, tetrodotoxin, etc.

[0035] In a fourth aspect, the present invention provides a method for preparing the composition, comprising the following steps:

[0036] S1. dissolving the polypeptide carrier and dispersing it into a medical injection solution to obtain a series of concentrations of polypeptide carrier solutions;

[0037] S2. Mix the product obtained in step S1 and the biological toxin in a medical injection solution according to a certain proportion.

[0038] Preferably, the medical injection solution is selected from water, PBS buffer, physiological saline, and 5% glucose injection.

[0039] In a fifth aspect, the present invention provides the use of the composition in the preparation of anesthetic and analgesic (such as cancer pain) drugs.

[0040] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0041] (I) The present invention successfully established a method for retaining toxins and achieving their slow / controlled release by using a supramolecular assembly conjugate of a receptor-derived polypeptide carrier and a toxin, thereby solving the problem of safe use of toxins. The method delivers toxins to sites requiring nerve blockade, prolongs the time of anesthesia and analgesia, and effectively blocks nerve conduction and thereby pain by chemical nerve block therapy, thereby reducing the reliance on opioids in severe pain management strategies.

[0042] (ii) The preparation method of the polypeptide vector-toxin composition of the present invention is simple and rapid, and does not require harsh and complicated synthesis steps.

[0043] (III) The delivery method of the present invention retains the biological activity and drug efficacy of the polypeptide carrier and the toxin, that is, the supramolecular assembly and combination of the polypeptide carrier and the toxin does not affect the local analgesic effect of the toxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the combination of biotoxin and MA polypeptide sequence into nanostructure through self-assembly in a preferred embodiment of the present invention.

[0045] Figure 2 The morphology of the amphipathic polypeptide designed based on the N-type voltage-gated calcium channel polypeptide sequence and the ω-conotoxin derivative (ziconotide, ZP) after self-assembly is shown in the transmission electron microscope (TEM) image in the preferred embodiment of the present invention. Among them, Q1, Q2 and Q3 are MA1, MA2, MA3 respectively, and ZP is the abbreviation of ziconotide.

[0046] Figure 3 It is the in vitro cumulative release amount of the MA (MA1, MA2, MA3) and ZP assembly after continuous release for 48 hours in a preferred embodiment of the present invention, wherein the molar ratio of MA (MA1, MA2, MA3): ZP is 1:1.

[0047] Figure 4 The figure is a comparison of the duration of sensory nerve blockade by free ZP and assembled ZP / Q3 polypeptide sequences in a preferred embodiment of the present invention.

[0048] Figure 5The figure is a comparison of the duration of sensory nerve block after TTX / polypeptide sequence assembly in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0049] The present invention aims to provide a toxin-specific delivery system that can be widely used to block cancer pain. The delivery system uses toxins (conotoxin, tetrodotoxin and saxitoxin) and polypeptide carriers to form a composition, so as to retain the toxins and achieve their controlled release, prolong the action time of pain signal blocking, effectively block nerve conduction and thus block pain, and deliver the toxins to the most needed sites for cancer pain treatment with a specific delivery system, thereby reducing the dependence of pain management on opioids.

[0050] The present invention adopts the following technical solution:

[0051] The present invention provides a polypeptide carrier-toxin composition, wherein the composition is formed by the association of a polypeptide carrier and a toxin through supramolecular assembly, wherein the polypeptide is an ion channel peptide (X)n1-T-(X)n2-E-(X)n3-W-(X)n4

[0052] The sequence of the polypeptide is as follows:

[0053] (1) Calcium channel peptides A1, A2, and A3: derived from N-type high-potential Ca V 2.2 Ion channel protein (NCBI accession number is NP_000709):

[0054] A1: TMEGWTDI (SEQ ID NO: 1);

[0055] A2: TGEDWNA (SEQ ID NO: 2);

[0056] A3: TGEGWPMV (SEQ ID NO: 3).

[0057] (2) Sodium channel peptides A4 and A5: derived from Na V 1.7 Ion channel protein (NCBI accession number is NP_002968):

[0058] A4: TNDYWEN (SEQ ID NO: 4);

[0059] A5: CGEWLET (SEQ ID NO: 5).

[0060] The composition of the present invention is formed by the supramolecular self-assembly of ion channel peptides and toxins, namely, Ca V 2.2 or Na V1.7 The N / C terminal of the ion channel peptide fragment is combined with the toxins (ziconotide, STX and TTX) through supramolecular assembly via glycine-bound hydrophobic domain structure (M).

[0061] The hydrophobic domain sequence of the present invention is selected from dodecanoic acid (C12), octadecanoic acid (C18), and a polypeptide sequence composed of coupled hydrophobic amino acids (phenylalanine (F), tyrosine (Y), isoleucine (I), valine (V) and leucine (L)) (such as FFF, FYF, LLFFF, LLFYF, IIFFF, IIFYF, etc.).

[0062] The polypeptide carrier-toxin composition of the present invention can be prepared according to the following method, comprising the steps of:

[0063] S1. If the polypeptide carrier can be directly dispersed evenly in the medical buffer, the polypeptide carrier solution is directly prepared; if the polypeptide carrier is not easy to be directly dispersed in the medical buffer, the polypeptide carrier can be dissolved in DMSO and further dispersed in PBS buffer, and then dialyzed to remove DMSO to obtain the self-assembly of the peptide, i.e. A (A1, A2, A3, A4, A5), MA (MA1, MA2, MA3, MA4, MA5);

[0064] S2, reacting the product obtained in step S1 with ziconotide, STX and TTX in a specific molar ratio in PBS buffer or 5% glucose solution at room temperature for 30 minutes to 24 hours to obtain the polypeptide carrier-toxin composition;

[0065] Among them, the molar ratio of polypeptide A (A1, A2 and A3) and / or MA (MA1, MA2 or MA3) to ziconotide is 1:1, 2:1, 5:1, 10:1; the molar ratio of polypeptide A4 (MA4) and A5 (MA5) to TTX is 0:0:1, 1:1:1, 5:5:1, 10:1; the molar ratio of polypeptide A4 (MA4) and A5 (MA5) to STX is 1:1:1.

[0066] In the aforementioned method, step S1 also includes removing unassembled molecules from the generated polypeptide assemblies A (A1, A2, A3, A4, A5) and MA (MA1, MA2, MA3, MA4, MA5) through a dialysis device (Float-A-Lyzer G2 Dialysis Devices, Spectrum Laboratories).

[0067] In the aforementioned method, step S2 further includes characterization by negative staining of uranyl acetate (1.0% w / w) using TEM (Tecnai G2 T20; FEI Company). The average width of the nanofibers was calculated after measuring the thickness of ten nanofibers in the TEM image using Image J. DLS (Delsa Nano; Beckman Coulter) was used to determine whether the channel peptide and toxin contained nanoscale materials.

[0068] The present invention uses a specific delivery system that assembles a receptor-derived polypeptide carrier and a toxin supramolecule into a composite, which not only effectively blocks nerve conduction to block pain, but also controls the sustained release of toxins to prolong the local anesthesia time and minimize systemic toxicity. In vitro experiments have shown that the supramolecular nanostructure delivery system assembled by the polypeptide carrier with ziconotide, STX and TTX can retain toxins and maintain long-term local anesthesia, providing a powerful tool for non-addictive pain control.

[0069] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0070] Example 1 Amphiphilic polypeptides MA1, MA2, MA3 and ziconotide composition delivery system

[0071] 1. Preparation of self-assembly solution of amphiphilic peptides MA1, MA2, and MA3

[0072] 1.76 mg of peptide (MA1, MA2, MA3) was dissolved in 5 μL DMSO, sonicated in a water bath for 1 min, separated in 1 mL 5% glucose solution at a series of concentrations, and then sonicated at 100 W for 3 min at 25°C to obtain a homogenous solution. 1 mL of the solution was placed in a dialysis device (Float-A-Lyzer G2 Dialysis Devices, Spectrum Laboratories) with a cutoff power of 1,000 MW and dialyzed against 1 L 5% glucose solution for 48 h to remove DMSO.

[0073] 2. Ziconotide loading

[0074] Different doses of conotoxin derivatives (ziconotide, ZP), such as 2.64 mg, 1.32 mg, 0.53 mg, 0.26 mg, 0.2 mg (i.e., molar ratios of 1:1, 2:1, 5:1, 10:1, 13.2:1, respectively) were added to the solution obtained in step 1 and mixed, and incubated for 12 hours at room temperature to generate calcium channel peptide-ziconotide compositions (MA1-ZP, MA2-ZP, MA3-ZP). The solution was diluted to 30 μM and characterized by TEM by negative staining with uranyl acetate (1.0% w / w). The average width of the nanofibers was calculated by measuring the thickness of ten nanofibers in the TEM image by Image J. DLS (Delsa Nano; Beckman Coulter) was used to determine whether the calcium channel peptide and ziconotide contained nanoscale materials.

[0075] The three peptide sequences used in this example are as follows:

[0076] MA1(Q1):Ac-TMEGWTDIGGLLFFFF-NH2(SEQ ID NO:6)

[0077] MA2(Q2):Ac-TGEDWNAGGLLFFF-NH2(SEQ ID NO:7)

[0078] MA3(Q3):Ac-TGEGWPMVGGLLFFF-NH2(SEQ ID NO:8)

[0079] Wherein, Ac represents CH3CO-.

[0080] Assembly diagram see Figure 1 , the experimental results are shown in Figure 2 As can be seen from the figure, MA1, MA2, and MA3 not only self-assemble in 5% glucose solution, but also after adding ZP, the system is still an ordered nanofiber structure.

[0081] Example 2 In vitro quantitative drug release test of MA1-ZP, MA2-ZP, and MA3-ZP compositions

[0082] 1. ZP was added to the peptide solutions of MA1, MA2, and MA3, respectively, and incubated for 12 h before use. The ZP concentration in each solution was 500 μM. 1 mL of ZP-loaded peptide was placed in a Slide-A-Lyzer MINI dialysis device (Thermo Scientific) with a pore size of 10,000 MW and dialyzed against 4 ml of 5% glucose solution at 37°C on a platform shaker at 100 rpm for drug release studies. At predetermined time intervals, the dialyzed solution was exchanged with fresh, preheated 37°C 5% glucose solution. The concentration of ZP was quantified by ELISA kit or high-performance liquid chromatography (HPLC).

[0083] 2. Quantification by high performance liquid chromatography. Prepare ZP solutions with concentrations of 1 mg / mL, 0.25 mg / mL, 0.0625 mg / mL, 0.015625 mg / mL, and 0.00390625 mg / mL, respectively, detect the corresponding peak areas by high performance liquid chromatography, and count and draw the ZP standard curve. Transfer the combined samples collected by dialysis for a predetermined time to a liquid phase vial and detect them using the ZP method. The specific detection method for ZP is: C18 chromatographic column, 85% mobile phase A (water / 0.1% TFA), 15% mobile phase B (acetonitrile / 0.1% TFA), detection wavelength 200nm, single injection volume 30μL, peak position 8-10min.

[0084] The experimental results are shown in Figure 3 As can be seen from the figure, compared with the ZP solution, the calcium channel chimeric peptide-ziconotide combination (MA1-ZP, MA2-ZP, MA3-ZP) showed obvious sustained release effect during in vitro release.

[0085] Example 3 Detection of the pain-blocking ability of the MA3-ZP composition in vivo

[0086] 1. Establishment of rat bone cancer pain model

[0087] Six-week-old Wistar female rats weighing 250-300 g were used in the experiment. They were housed under a 12h / 12h light / dark cycle for 7 days, with 6 rats in each group. After anesthesia with isoflurane-oxygen, Walker256 cell line was inoculated into the tibia of the left leg of the rat using a 23-G needle. After successful tumor inoculation, the rats were subjected to imaging, behavioral and molecular biological tests to evaluate the construction of the bone cancer pain model and the central sensitization caused by cancer pain.

[0088] 2. Blockade of sensory nerves by injection of MA3-ZP combination

[0089] The MA3-ZP composition was injected intrathecally into the established rat bone cancer pain model, the molar ratio of MA3 to ZP in the composition was controlled in the range of 1:1-100:1, preferably 2:1-30:1, more preferably 5:1, 10:1, 13.2:1, 30 μL per rat, and the rats were subjected to pain behavioral testing (Von Frey mechanical sting test) at a predetermined time after administration, i.e., mechanical stimulation of the plantar surface of the rat's hind paw was performed, and the magnitude of the mechanical force was recorded until the animal lifted the paw. In order to calculate the duration of nerve block, a mechanical pain foot withdrawal threshold of more than 8G was considered to be a successful nerve block. The measurement was repeated 3 times at each time interval.

[0090] The experimental results are shown in Figure 4 . ZP (0.01 mg / mL, 30 μL / mouse) and MA3-ZP assembly (500 μM MA3+37.89 μM ZP, 30 μL / mouse, i.e. 3 μg each). As can be seen from the figure, both ZP and MA3-ZP combination have mechanical pain analgesic effect at 2 hours; the mechanical pain analgesic effect of ZP has been significantly reduced at 24 hours, almost reaching the level of the control group; the mechanical pain analgesic effect of MA3-ZP combination still exists at 120 hours, and is similar to that of ZP at 8 hours; the analgesic effect of MA3-ZP combination on the contralateral side is better than that on the ipsilateral side.

[0091] Example 4 Amphiphilic polypeptides MA4, MA5 and tetrodotoxin and saxitoxin composition delivery system

[0092] 1. Self-assembly of the amphiphilic peptide MA4MA5 (MA4MA5 means that MA4 and MA5 are mixed in equal molar amounts)

[0093] 63 μmol of peptides (MA4, MA5) were dissolved in 10 μL DMSO, separated in 1 mL PBS at a series of concentrations, and then sonicated at 100 W for 1 min at 25°C to obtain a homogenous solution. 1 mL of the solution was placed in a dialysis device with a pore size of 1,000 MW (Float-A-LyzerG2 Dialysis Devices, Spectrum Laboratories) and dialyzed with 1 L PBS for 48 h to remove DMSO to obtain an assembly of MA4MA5.

[0094] 2. Add TTX to the assembly solution obtained in step 1 and stir at room temperature for 30 minutes to generate an amphiphilic polypeptide-tetrodotoxin composition (MA4MA5-TTX). The molar ratio of MA4, MA5 and TTX is controlled in the range of 1:1:1-100:100:1, preferably 2:2:1-30:30:1, more preferably 10:10:1, 20:20:1. Characterize by negative staining method of uranyl acetate (1.0% w / w) by TEM (Tecnai G2T20; FEI Company). The average width of the nanofibers was calculated by measuring the width of ten nanofibers in the TEM image by Image J. DLS (Delsa Nano; Beckman Coulter) was used to determine whether the amphiphilic polypeptide and TTX contained nanoscale materials.

[0095] 3. The STX operation steps are the same as the assembly of TTX and amphiphilic polypeptide composition.

[0096] The two peptide sequences used in this example are as follows:

[0097] MA4: Ac-FFFLL-GG-TNDYWEN (SEQ ID NO:9);

[0098] MA5: Ac-FFFLL-GG-CGEWLET (SEQ ID NO: 10).

[0099] Example 5 Evaluation of the ability of MA4MA5-TTX to inhibit cancer pain

[0100] 1. Establishment of rat bone cancer pain model (same as Example 3)

[0101] Six-week-old Wistar female rats weighing 250-300 g were used in the experiment. They were housed under a 12h / 12h light / dark cycle for 7 days, with 6 rats in each group. After anesthesia with isoflurane-oxygen, Walker256 cell line was inoculated into the tibia of the left leg of the rat using a 23-G needle. After successful tumor inoculation, the rats were subjected to imaging, behavioral and molecular biological tests to evaluate the construction of the bone cancer pain model and the central sensitization caused by cancer pain.

[0102] 2. Blockade of sensory nerves by injection of MA4MA5-TTX combination

[0103] The MA4MA5-TTX composition was injected intrathecally into the established rat bone cancer pain model. The molar ratio of MA4, MA5 and TTX was controlled in the range of 1:1:1-100:100:1. For example, a 630 μM MA4MA5 solution can be added with TTX with a final concentration of 63 μM, 126 μM, etc., 30 μL each. After administration, the rats were subjected to pain behavioral testing (Von Frey mechanical sting test) at a predetermined time, that is, mechanical stimulation of the plantar surface of the rat's hind paw was performed, and the magnitude of the mechanical force was recorded until the animal lifted the paw. In order to calculate the duration of nerve block, a mechanical pain withdrawal threshold of more than 8G was considered a successful nerve block. The measurement was repeated 3 times at each time interval.

[0104] The experimental results are shown in Figure 5 As can be seen from the figure, both 63μM TTX and 630μM MA4MA5+63μM TTX combination (MA4MA5-TTX) can relieve cancer pain, and the effect of MA4MA5-TTX is better than that of the TTX group.

[0105] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0106] References:

[0107] [1] Mercadante S, Vitrano V: Pain in patients with lung cancer: pathophysiology and treatment. Lung Cancer. 2010,68:10-15.

[0108] [2]Fallon M,Giusti R,Aielli F,Hoskin P,Rolke R,Sharma M,Ripamonti CI:Management of cancer pain in adult patients:ESMO Clinical Practice Guidelines.Ann Oncol.2018,29:iv166-iv191.

[0109] [3]Colvin LA,Bull F,Hales TG:Perioperative opioid analgesia-when isenough too much?Areview of opioid-induced tolerance and hyperalgesia.Lancet2019,393:1558-1568.

[0110] [4]Stein C:New concepts in opioid analgesia.Expert Opin InvestigDrugs.2018,27:765-775.

[0111] [5]Zheng XQ,Wu YH,Huang JF,et al.:Neurophysiological mechanisms ofcancer-induced bone pain.J.Adv.Res.2022,35:117-127.

[0112] [6]Gonzalez-Cano R,Ruiz-Cantero MC,Santos-Caballero M,Gomez-Navas C,Tejada MA,Nieto FR:Tetrodotoxin,a Potential Drug for Neuropathic and CancerPain Relief?Toxins.(Basel)2021,13.

[0113] [7]Dib-Hajj SD,Waxman SG:Sodium Channels in Human Pain Disorders:Genetics and Pharmacogenomics.Annu Rev Neurosci.2019,42:87-106.

[0114] [8]Beckley JT,Pajouhesh H,Luu G,Klas S,Delwig A,Monteleone D,Zhou X,Giuvelis D,Meng ID,Yeomans DC,et al:Antinociceptive properties of an isoform-selective inhibitor of Nav1.7 derived from saxitoxin in mouse models ofpain.Pain.2021,162:1250-1261.

[0115] [9]Shankarappa SA,Tsui JH,Kim KN,Reznor G,Dohlman JC,Langer R,KohaneDS:Prolonged nerve blockade delays the onset of neuropathic pain.Proc NatlAcad Sci U S A.2012,109:17555-17560.

[0116]

[10] Ekberg J,Jayamanne A,Vaughan CW,Aslan S,Thomas L,Mould J,Drinkwater R,Baker MD,Abrahamsen B,Wood JN,et al:muO-conotoxin MrVIBselectively blocks Nav1.8 sensory neuron specific sodium channels and chronicpain behavior without motor deficits.Proc Natl Acad Sci U S A.2006,103:17030-17035.

[0117]

[11] Baddack U,Frahm S,Antolin-Fontes B,Grobe J,Lipp M,Müller G, -Tallon I:Suppression of Peripheral Pain by Blockade of Voltage-Gated Calcium2.2Channels in Nociceptors Induces RANKL and Impairs Recovery FromInflammatory Arthritis in a Mouse Model.Arthritis Rheumatol.2015,67:1657-1667.

[0118]

[12] Hasan MM,Starobova H,Mueller A,Vetter I,Lewis RJ:Subcutaneousω-Conotoxins Alleviate Mechanical Pain in Rodent Models of Acute PeripheralNeuropathy.Mar Drugs.2021,19.

[0119]

[13] Wang YX,Pettus M,Gao D,Phillips C,Scott Bowersox S:Effects ofintrathecal administration of ziconotide,a selective neuronal N-type calciumchannel blocker,on mechanical allodynia and heat hyperalgesia in a rat modelof postoperative pain.Pain.2000,84:151-158.

[0120]

[14] Gao S,Yao X,Yan N:Structure of human Cav2.2 channel blocked bythe painkiller ziconotide.Nature.2021,596:143-147.

Claims

1. An amphiphilic polypeptide carrier, characterized in that: The amphiphilic polypeptide carrier is any one of the following: TMEGWTDI-(G)nU; TGEDWNA-(G)nU; TGEGWPMV-(G)nU; Wherein, U represents the hydrophobic part; U is a hydrophobic polypeptide composed of 3-10 hydrophobic amino acids, wherein the hydrophobic amino acids are selected from one or more of F, Y, and L; (G)n represents a linker module, wherein G is glycine and n is an integer between 0-4.

2. Any of the following uses of the amphiphilic polypeptide carrier according to claim 1: 1) Used to prepare drug delivery or sustained-release systems; 2) Used to prepare detoxification materials for toxin drugs.

3. A composition, characterized in that The composition is composed of the amphiphilic polypeptide carrier according to claim 1 or a mixture thereof and a biological toxin in a molar ratio of 1:1-100:1; Wherein, the biological toxin is ziconotide.

4. The composition according to claim 3, characterized in that The composition is composed of the amphiphilic polypeptide carrier or a mixture thereof and the biological toxin in a molar ratio of 2:1-30:

1.

5. The method for preparing the composition according to claim 3 or 4, characterized in that: The steps include: S1, dissolving the amphiphilic polypeptide carrier and dispersing it into a medical injection solution to obtain a series of amphiphilic polypeptide carrier solutions of concentration; S2. Mix the product obtained in step S1 and the biological toxin in a medical injection solution according to a certain proportion.

6. The method according to claim 5, characterized in that The medical injection solution is selected from one or more of water, PBS buffer, physiological saline, and 5% glucose injection.

7. Use of the composition according to claim 3 or 4 in the preparation of anesthetic or analgesic drugs.

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