WO2020056990A1 - Use of improved prialttm in preparation of medicament - Google Patents

Abstract

Use of an improved prialtTM peptide in the preparation of a medicament. The C-terminal of prialtTM and the N-terminal of a cell membrane penetrating peptide are linked by means of two glycines, and the obtained fusion peptide can pass through the blood-brain barrier and is suitable for intravenous, intraperitoneal or nasal administration.

Description

Application of improved zicorinide in preparing medicine Technical field

The invention belongs to the technical field of polypeptide medicine, and particularly relates to the application of a fused polypeptide of zicornol in the preparation of a medicine.

Background technique

Zicoronide (trade name PrialtTM, Elan Pharmaceuticals) is the first conotoxin drug approved by the US Food and Drug Administration (FDA) in 2004. Its targeting site is an N-type voltage-gated calcium ion channel, which is a first-line drug for compound analgesia in the subarachnoid space (intrathecal). Ziconoxide is an artificial synthesis of the hydrophilic peptide ω-MVIIA in the venom peptide of Pacific snails-Snails-Snails. It is the first new type of non-morphine analgesics used in the clinic. Its molecular formula is C ₁₀₂ H ₁₇₂ N ₃₆ O ₃₂ S ₇ , structural formula: H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly- Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH ₂ .

Ziconotide can clinically treat postherpetic neuralgia, phantom limb pain, AIDS-related neuropathic pain, refractory cancer pain, postoperative pain, intolerance or rejection of other treatment methods such as systemic analgesics, Adjuvant treatment, alleviate the pain of intrathecal injection of opioids and so on. Unlike salicylate, NSAIDs and local anesthetics, which mainly pass through peripheral nerves / nociceptors, opioids and general anesthetics work mainly at the brain level to eliminate pain and consciousness. The mechanism of action of zicoronide is through its binding to N Of Calcium Channel Receptors. N-type calcium channel receptors are located on the major nociceptive A-δ and C-slow fiber pain fibers (nociceptors) in the layers of Rexed's I and II on the posterior surface of the spinal cord, which can alleviate the ineffectiveness of other treatments including intrathecal injection of morphine Pain and long-term use of the drug will not produce tolerance and addiction, the application indication is to treat chronic pain related to trauma, tumor and neuralgia, especially in the treatment of refractory insensitive to opioids Patients with sexual pain or intolerance to opioids have unique advantages. However, zicoronide cannot cross the blood-brain barrier. Currently, only intrathecal intubation is used for infusion. The intubation and infusion pump are buried under the skin, which requires surgery to complete, which is not convenient for clinical use. At present, it is only used for long-term and permanent treatment of existing pain-resistant drugs and chronic pain. This mode of administration greatly limits the clinical application of the inherent advantages of the drug.

The blood-brain barrier (BBB) is a complex cell system that exists between the brain tissue and the blood. It can control the transport of substances on both sides of the blood-brain, thereby ensuring the stability of the central nervous system's internal environment. Various receptors interact and are transported into the brain according to the needs of the body to play a role; some toxic and harmful substances are shielded from the brain tissue by this barrier to prevent damage to the brain. The special protective effect of the blood-brain barrier (BBB) has made it difficult for most drugs to enter the brain, and the treatment and administration of this central nervous system disease have caused problems.

Cell penetrating peptides (CPPs) are a class of short peptides (typically less than 35 amino acid residues) that can enter cells through biofilms. The discovery originated in 1988. Some scholars found that the transactivator of HIV-1 Tat can be transduced into the cell across the membrane, and then someone found that the Drosophila transcription protein has similar characteristics. Since then, many other CPPs have been discovered one after another. CPP shows diversity in relative molecular mass, amino acid composition, and amino acid sequence. The number and type of amino acids contained in CPP are different, and the polarity and charged amount are also different. However, they have some common characteristics. Features, such as: under low concentration conditions, it can pass through the cell membrane to enter the cell without causing significant damage and damage to the membrane; it has the ability to penetrate the membrane itself, and can also mediate various substances including small molecules, nucleic acids, Protein peptides and nanoparticles enter the cell; high efficiency and low toxicity. In recent years, studies have found that the use of cell penetrating peptides to connect with drug molecules can achieve the effect of crossing the blood-brain barrier, which has brought new directions to central nervous system drug delivery.

At present, there are technical methods in the prior art that use cell penetrating peptides to assist the conotoxin to cross the blood-brain barrier. For example, zicoronide is packaged in a virus particle and a TAT polypeptide is connected to the surface of the virus particle. It is possible to deliver zicoronide through the blood-brain barrier. However, the preparation process of this method is complicated, the packaging process of the virus cannot be well controlled, and it is difficult to apply it on a large scale in the industry. As another example, a fusion polypeptide is prepared by using two GGs as the linking unit of the TAT peptide and the N-terminus of conotoxin. The fusion polypeptide can pass through the blood-brain barrier and is suitable for intravenous administration. However, the N-terminated fusion polypeptide By intravenous injection, the analgesic effect and time in the body cannot meet the requirements of clinical application, and it cannot be promoted on a large scale. Therefore, to obtain an improved zicoronide that can overcome the shortcomings of intrathecal intubation administration through the blood-brain barrier and can be used on a large scale in clinical practice is an urgent problem to be solved.

Summary of the Invention

It is an object of the present invention to provide an improved zicoronide. The inventors have discovered through long-term research that the improved zicoronide fusion peptide obtained by linking the C-terminus of the zicoronide to the N-terminus of the cell membrane penetrating peptide can overcome the shortcomings of the prior art and is suitable for intravenous, It can be used intraperitoneally or nasally, and has good analgesic effect and long time in the body, and can be used on a large scale clinically.

Specifically, the technical solution to achieve the above-mentioned objective is shown below.

A fusion polypeptide is composed of zicorinide and a cell membrane penetrating peptide. Preferably, the fusion polypeptide is composed of zicorin peptide connected to the cell membrane penetrating peptide through the C-terminus, or the C-terminus of zicoronide peptide is connected to the N-terminus of the cell membrane penetrating peptide through a linker. Preferably, the linkage The daughter is two glycine (GG).

Further, the amino acid of zicoronide is CKGKGAKCSRLMYDCCTGSCRSGKC (as shown in SEQ ID NO.1), or the zicoronide in the fusion polypeptide can also be CKGKGAKCSRLMYDCCTGSCRSGKC (as shown in SEQ ID NO.1). Variants of amino acids with fewer than 8, less than 6, less than 4, 2 or 1 amino acids deleted, mutated or inserted.

Further, the cell membrane penetrating peptide may be Penetratin, TAT peptide, Pep-1 peptide, S4 ₁₃ -PV, Magainin 2 or Buforin 2.

Among them, the TAT peptide is derived from HIV-1 transactivator protein Tat, which can transduce into the cell across the membrane. The amino acid of the TAT peptide is YGRKKRRQRRR (as shown in SEQ ID No. 2), or the TAT peptide in the fusion polypeptide can also be YGRKKRRQRRR (as shown in SEQ ID NO. 2). By less than 10, less than 8, less Variants of amino acids with deletions, mutations or insertions of 6, or less than 4, 2 or 1 amino acids, or peptidomimetics thereof.

Preferably, the amino acid sequence of the aforementioned polypeptide or fusion polypeptide or modified zicorno peptide is CKGKGAKCSRLMYDCCTGSCRSGKCGGYGRKKRRQRRR (as shown in SEQ ID No. 3) or, by less than 10, less than 8, less than 6, and less than A variant of 4, 2, or 1 amino acid with deletions, mutations or insertions, or a peptidomimetic.

The peptidomimetic refers to a synthetic chemical compound having substantially the same structural and / or functional characteristics as a peptide composed of natural amino acids. Peptidomimetics can completely include synthetic unnatural analogs of amino acids, or chimeric molecules of partially natural peptide amino acids and partially unnatural amino acid analogs. Peptidomimetics can also incorporate conservative substitution sites in any number of natural amino acids, so long as such substitutions do not substantially alter the structure and / or inhibitory or binding activity of the mimetic. Polypeptide mimetic components may contain any combination of non-natural structural components, which generally come from 3 structural groups: a) residue linking groups linked by non-natural amide bonds ("peptide bonds"); b) Non-natural residues that replace naturally occurring amino acid residues; or c) Induces secondary structure simulations, ie, residues that induce or stabilize secondary structures such as β-turns, γ-turns, β-sheets, α-helical conformations, and the like.

A second object of the present invention is to provide a pharmaceutical composition or formulation, preferably a pharmaceutical formulation. Further, the pharmaceutical composition or formulation / pharmaceutical formulation comprises a polypeptide of the present invention and / or an acceptable carrier.

The pharmaceutical composition or formulation / pharmaceutical formulation may provide a pharmaceutical composition in unit dosage form (i.e., a dosage for a single administration) comprising any of the dosages shown below. It can be prepared by conventional mixing, dissolving, granulating, preparing lozenge, grinding, emulsifying, encapsulating, embedding or lyophilizing methods. The pharmaceutical composition or formulation / pharmaceutical formulation may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or excipients that facilitate processing of the active agent into a pharmaceutically acceptable formulation. A suitable formulation depends on the route of administration chosen.

The mode of administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Intravenous or intraperitoneal injection is preferred. The pharmaceutical composition or formulation / pharmaceutical formulation for parenteral administration is preferably sterile and substantially isotonic. For injection, the active agent can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulations such as suspending, stabilizing and / or dispersing agents.

A third object of the present invention is to provide the use of an improved zicoronide. The use is: for preparing a drug, preferably for preparing an analgesic drug, and preferably, the analgesic drug acts on a calcium channel.

Further, the drug can be used to treat pain, pain-related diseases, for example, diseases that can cause chronic pain include diabetes, arthritis (eg, osteoarthritis, rheumatoid arthritis, and juvenile chronic arthritis), cancer, or chemotherapy Toxic effects, fibromyalgia, shingles, irritable bowel syndrome, vascular problems or sickle cell disease.

Diseases associated with occasional general pain include rheumatic polymyalgia, speculative disease, depression, diabetes, malignant anemia, sickle cell disease, and syphilis. Diseases related to neuropathic pain include neuralgia (e.g., trigeminal neuralgia, atypical facial pain, and shingles neuralgia caused by shingles or herpes), peripheral neuropathy, Charcot-Marie-Tooth disease, Fury Dreich ataxia, diabetes (e.g., diabetic neuropathy), dietary defects (especially vitamin B-12), excessive alcohol use (alcoholic neuropathy), uremia (from kidney failure), cancer, AIDS, Hepatitis, Colorado tick heat transfer, diphtheria, G-Pak syndrome, HIV infection that has not progressed to AIDS, leprosy, Lyme disease, multiple nodular arteritis, rheumatoid arthritis, sarcoidosis, homeostasis Glenn syndrome, syphilis, systemic lupus erythematosus, and exposure to toxic compounds.

Diseases associated with inflammatory pain include: (A) arthritic diseases such as rheumatoid arthritis; juvenile chronic arthritis; systemic lupus erythematosus (SLE); gouty arthritis; scleroderma; osteoarthritis; psoriasis Disease arthritis; ankylosing spondylitis; Wright's syndrome (reactive arthritis); adult Still's disease; arthritis from viral infections; arthritis from bacterial infections, such as gonorrhea arthritis and non-gonorrhea Bacterial arthritis (septic arthritis); tertiary Lyme disease; tuberculous arthritis; and arthritis from fungal infections, such as yeast disease; (B) autoimmune diseases, such as Gaubach syndrome , Hashimoto's thyroiditis, malignant anemia, Addison's disease, type I diabetes, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Lyttle syndrome and Gray Fuss disease. (C) connective tissue diseases, such as spinal arthritis, dermatomyositis, and fibromyalgia; (D) inflammation caused by injury; (E) infections, such as tuberculosis or interstitial keratitis; and (G) arthritis, such as Bursitis or tendinitis. The types of headaches include muscular / myogenic headaches, vascular headaches, traction or inflammatory headaches, cluster headaches, hormonal headaches, rebound headaches or chronic sinusitis headaches.

Physical pain can be related to excessive muscle contractions, repetitive motion disorders, muscle disorders such as polymyositis, dermatomyositis, lupus, fibromyalgia, rheumatic polymyalgia, and rhabdomyolysis, myalgia, infections such as muscle Abscess, trichinellosis, influenza, Lyme disease, malaria, Rocky Mountain spotted fever, avian influenza, common cold, socially acquired pneumonia, meningitis, monkeypox, severe acute respiratory syndrome, toxic shock synthesis Symptoms, trichinellosis, typhoid fever, and upper respiratory infections. Visceral pain can be associated with diseases such as: irritable bowel syndrome, chronic functional abdominal pain (CFAP), functional constipation, functional dyspepsia, non-cardiac chest pain (NCCP) and chronic abdominal pain, chronic gastroenteritis, such as gastritis Inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, microcolitis, diverticulitis and gastroenteritis; interstitial cystitis; intestinal ischemia; cholecystitis; appendicitis; gastroesophageal reflux; ulcers, Kidney stones, urinary tract infections, pancreatitis, and hernias.

A fourth object of the present invention is to provide a method for preparing an improved zicoronide. Preferably, the improved zicoronide can be prepared by chemical synthesis. It is further preferred that the improved zicornol peptide is prepared by a solid-phase synthesis method or a recombinant expression method, and further, an F-moc full-automatic solid-phase synthesis method is used to prepare the polypeptide of the present invention.

Compared with the prior art, the present invention has the beneficial effects that: by connecting the C-terminus of zicoronopeptide with a cell membrane penetrating peptide, an improved zicornor peptide is obtained, which overcomes the inability of zicornor peptide to pass through the blood brain barrier Cannot be injected intramuscularly, mainly through intracerebroventricular and spinal canal administration, which brings high surgical risk and high infection risk. The improved zicornol peptide of the present invention can pass through the blood-brain barrier, and is suitable for intravenous, intraperitoneal or nasal administration. The operation is convenient and the clinical risk is small. Through intravenous, intraperitoneal or nasal administration, its pharmacological effect in the body is long. , The analgesic effect is excellent, and the improved zicorinide of the present invention has small side effects and is suitable for large-scale clinical applications. In addition, the improved zicornor peptide of the present invention is simple to prepare, and the quality is controllable during the preparation process and the preparation process, which is suitable for large-scale industrial production.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: MVIIA and MVIIA-a, b, c, d one-step oxidation folding HPLC analysis spectra;

Figure 2: Circular chromatograms of MVIIA and MVIIA-a, b, c, d. The final concentration of the peptide is 35 μmol / L, which is dissolved in phosphate buffer solution (10mM, pH = 7.2).

Figure 3: MVIIA and MVIIA-a, b, c, d inhibit CaV2.2 channel current. The dose-response curve of MVIIA is shown in Figure 3A, and the dose-response curve of MVIIA variants is shown in Figures 3B-3E. The data of the semi-inhibitory concentration and the slope value are shown on the graph, and the data are expressed as the mean ± standard error, with 5 mice per group. As shown in Figure F, at 10 μM L-MVIIA (blue) and 2 μM MVIIA (red), the superimposition of the whole-cell calcium channel current traces excited by a voltage step from -80mv to 10mv; G graph is MVIIA and Summary table of semi-inhibitory concentrations of its variants;

Figure 4: MVIIA and MVIIA-c hot plate pain comparison results, in vivo analgesic effect after MVIIA (Figure 4A) in the lateral ventricle, MVIIA (Figure 4B) and MVIIA-c (Figure 4C) in the tail vein. The analgesic effect is expressed in response latency. Data are expressed as mean ± standard error, 6-8 mice per group. * p <0.05, ** p <0.01 and *** p <0.001 represent comparison with the normal saline group (data analysis was performed by repeated multivariate analysis of variance and Duncan's multiple range test);

Figure 5: MVIIA-a, b, d hot plate pain test results, Figures 5A-5C show the analgesic effect in vivo after tail vein administration of MVIIA-a, b, d polypeptide. The analgesic effect is expressed as a percentage of the largest possible effect (% MPE). Data are expressed as mean ± standard error, 8-10 mice per group. * p <0.05, ** p <0.01 and *** p <0.001 are compared with the normal saline group;

Figure 6: The analgesic effect of MVIIA and MVIIA-a, b, c, d in the acetic acid writhing experiment. Within 5 to 20 minutes after intraperitoneal injection of 1% acetic acid, the number of rolls of the mice was recorded; The effect of intraperitoneal injection of 1% acetic acid 30 minutes after lateral ventricular administration; as shown in Figure B, the effect of intraperitoneal injection of 1% acetic acid 30 minutes after tail vein administration; #, compared with the saline group (saline ) Group comparison; *, compared with MVIIA group; &, compared with MVIIA-C group; *, #, &, p <0.05; ***, ###, &&&, p <0.001. Data are expressed as mean ± standard error, 9-11 mice per group;

Figure 7: Effects of MVIIA and MVIIA-a, b, c, d on tremor time in mice, 6 μL of peptide (0.9 nmol / kg) and saline were administered to the lateral ventricle. 30 minutes and 120 minutes after the administration, the cumulative tremor time of the mice was recorded within 5 minutes. Data are expressed as mean ± standard error (n = 12);

Figure 8: Mass spectrum of MVIIA;

Figure 9: Mass spectrum of MVIIA-a;

Figure 10: Mass spectrum of MVIIA-b;

Figure 11: Mass spectrum of MVIIA-c;

Figure 12: Mass spectrum of MVIIA-d;

Figure 13: Analgesic ability of MVIIA and different doses of MVIIA-c after nasal administration;

Figure 14: MVIIA-a, b, d Analgesic ability in hot plate pain experiments when nasally administered.

detailed description

In order to overcome the shortcomings of zicoronotide in the prior art, the inventors have found through long-term research that the improved zicoronide fusion peptide is obtained by connecting the C-terminus of zicoronide to the N-terminus of the cell membrane penetrating peptide. , Suitable for intravenous or intraperitoneal administration. In order to further study the analgesic effect of different types of improved zicoronide, the present invention designs and synthesizes a variety of different types and structures of fusion polypeptides, including, without using a linker, through the C-terminus of zicoronide and the cell membrane A fusion polypeptide directly linked to the N-terminus of the transpeptide; a fusion polypeptide constructed using one or more glycines as linkers. Further, the structural characterization, cell experiments, in vivo experiments, and side-effect verification experiments of the different types of fusion polypeptides described above are used to illustrate the effects of different types of improved zicoronide

In order to better understand the technical solution of the present invention, detailed description is given below with reference to the embodiments.

Example 1 Preparation of Different Types of Improved Zicoronide

Four different types of modified zicorno peptides were prepared and named as protected peptides MVIIA-a, MVIIA-b, MVIIA-c, and MVIIA-d, respectively. At the same time, zicoronide was prepared and named MVIIA as a control. This experiment uses F-moc full-automatic solid-phase synthesis. The specific steps are as follows:

Polypeptide synthesis: A model of a 433A automatic synthesizer (ABI, Foster City, CA) was used to assemble the protected polypeptide and its derivatives on the resin. The peptide resin was incubated in the suspension at room temperature for 2.5 hours to deprotect the group. The suspension system was composed of 10 ml of TFA, 0.75 g of phenol, 0.25 ml of 1,2-ethanedithiol, 0.5 ml of anisole and 0.5 ml of water. (Wmmethoxycarbonyl (Fmoc), a common alkoxycarbonyl amino protecting group). The resin was isolated from the peptide deprotection mixture by filtration. The crude polypeptide was precipitated in 150 ml of a pre-cooled ether solution, and purified by chromatography on a dextran gel G-25 column using 10% glacial acetic acid as an eluent. Subsequently, the polypeptide-containing components were pooled and lyophilized, and the purity of the crude polypeptide was determined to be about 80% using high-performance liquid chromatography.

Polypeptide folding: MVIIA contains six cysteine residues and maintains three disulfide bond structures. Folding under oxidative conditions can produce a variety of isomers. After screening through the redox system, buffer, salt, concentration and temperature, two efficient folding conditions for MVIIA were selected: (a) 0.5M NH4Ac buffer (pH 7.9), which contained 1mM GSH, 0.1mM GSSG, 1 mM EDTA, and 0.2 mg / mL MVIIA; (b) 0.5 M NH4Ac buffer, which contains 1 mM cysteine, 1 mM EDTA, and 0.2 mg / mL MVIIA. At 4 ° C, the linear polypeptide MVIIA was folded under conditions a for 48-72 hours and conditions b for 24-48 h.

Peptide purification and characterization: After MVIIA is oxidized, the reaction mixture is acidified (pH <4.5) with acetic acid, and then filtered. The filtrate was directly applied to a Zorbax 21.2 × 250mm C18 liquid chromatography column, in which a preparative high performance liquid chromatography pump (Waters 2000 series, Milford, MA) was used. The C18 column was pre-washed with buffer A (0.1% TFA in water) and then 10-40% buffer B (0.1% TFA in cyanocyanine) was used to perform a 40-minute linear gradient elution at 8 mL / min. . The obtained fraction was a concentrated solution containing 90% MVIIA, and then we used a semi-prepared reversed-phase high-performance liquid chromatography with a 9.4 × 250mm Zorbax C18 liquid chromatography column for further purification. Finally, we used a 20% acetic acid solution as the eluent in a dextran gel G-25 column to convert the final product from the TFA salt solution to the acetate solution. The purity of the peptide was evaluated by analytical reversed-phase high-performance liquid chromatography. The evaluation was performed at a flow rate of 1 ml per minute using a Zorbax C18 liquid chromatography column (4.6 × 250mm) with 8-40% buffer B (0.1% TFA in acetonitrile) was subjected to a linear gradient elution for 25 minutes. In the end, the purity of the final product-polypeptide we obtained was 98%.

Example 2: Chemical properties and structural characterization of different types of improved zicoronide

1. Chemical properties of MVIIA and its variants

At 4 ° C, the buffer treated the linear peptide for 24-48 hours, and then analyzed by high performance liquid chromatography. It was found that the folding of the linear peptide caused a major peak and several small peaks. The buffer system includes 1 mM glutathione, 0.1 mM oxidized glutathione, 1 mM EDTA, and 0.2 mg / mL linear peptide. The pH of the solution is 7.9. The main product was purified and evaluated by analytical reversed-phase high performance liquid chromatography, and the purity of the peptide was determined to be greater than 98%. Determination was performed with an Ultraflex III TOF / TOF mass spectrometer (Bruker). The prepared polypeptide sequence is shown in Table 1, and its one-step oxidative folding HPLC analysis chart is shown in Figure 1.

Table 1: Prepared peptide sequences

name

sequence

MⅦA (SEQ ID No.1)	CKGKGAKCSRLMYDCCTGSCRSGKC
MⅦA-a (SEQ ID No.4)	CKGKGAKCSRLMYDCCTGSCRSGKCYGRKKRRQRRR
MⅦA-b (SEQ ID No.5)	CKGKGAKCSRLMYDCCTGSCRSGKCGYGRKKRRQRRR
MⅦA-c (SEQ ID No. 3)	CKGKGAKCSRLMYDCCTGSCRSGKCGGYGRKKRRQRRR
MⅦA-d (SEQ ID No. 6)	CKGKGAKCSRLMYDCCTGSCRSGKCGGGYGRKKRRQRRR

2. Circular dichroism

The peptide was dissolved in a PBS (10 mM, pH = 7.2) solution to a final concentration of 35 μM. At room temperature, a circular dichroism spectrum in the wavelength range of 190nm to 260nm was detected using a Chirascan Plus Spectral Polarimeter (Applied Photophysics Ltd., Leatherhead, Surrey, UK). The detection index is set as follows: step resolution 1.0nm; speed 20nm / min, and cell path length 1.0mm.

As shown in Figure 2, MVIIA showed a clear β-sheet structure at 195nm-205nm. We found that the TAT variant has a similar random coil structure, and around 200 nm, a significant attenuation band appears. These results indicate that the secondary structure of the polypeptide did not change when the length of the linker sequence between MVIIA and TAT was increased. When the ligation sequence was amplified, the molar ellipticity of the TAT variant deepened, indicating that the amplification of the ligation sequence between MVIIA and TAT helped to form a random coil structure. Mass spectrometry (using a Voyager MALDI-TOF spectrometer) was used to identify the exact molecular weight of the product peptide, as shown in Table 2. The mass spectra of MVIIA and MVIIA-a, b, c, d are shown in Figures 8-12. The disulfide bridge bridging pattern is assigned due to methods that partially reduce cysteine coupling and amino acid silencing. The synthetic peptides were consistent with the HPLC and circular chromatograms of MVIIA standards.

Table 2. Molecular weights of MVIIA and its variants

sample	Theoretical MW	Measured m / z	Difference between theoretical and measured values
MⅦA	2645.54	2639.0198	6.5202
MⅦA-a	4186.0784	4180.0108	6.0676
MⅦA-b	4243.0978	4237.0300	6.0678
MⅦA-c	4299.1353	4292.0362	7.0991
MⅦA-d	4356.1568	4351.0842	5.0726

Example 3: Electrophysiological experiments of different types of modified zicoronide

In order to further study the electrophysiological effects of different types of improved zicoronotide and the inhibitory effect on calcium ion (CaV2.2) channels, the following experiments were performed:

HEK293T cells (expressing SV40 large T antigen) were cultured in DMEM high-sugar medium (Gibco) containing 10% fetal bovine serum, 1% penicillin and streptomycin. The incubator environment was 37 ° C, 5% CO ₂ . Providing Dr.Diane Lipscombe CaV2.2 channels in rat α _1B splice variants e37a, auxiliary subunit α ₂ δ ₁ and β ₃ plasmids (Addgene plasmid # 26569, # 26575 , # 26574). Then three plasmids (3 μg), 0.4 μg enhanced green fluorescent protein gene and liposomes were transiently transfected into HEK293T cells. 24 hours after transfection, cells were seeded on glass slides, in an incubator _{(37 ℃, 5% CO 2} ) incubated for at least 6 hours, followed by electrophysiological recordings.

This study was recorded according to the method of cell voltage clamp recording in previously published research literature (F. Wang et al., 2016). In simple terms, the recording electrode has a resistance of about 3 MΩ and is filled with an internal solution. The internal solution contained 135 mM CsCl, 10 mM NaCl, 10 mM HEPES, and 5 mM EGTA, and the pH of the solution was adjusted to 7.2 with CsOH. The extracellular recording solution contained 135 mM N-Methyl-D-glucamine, 10 mM BaCl ₂ .2H ₂ O, 2 mM MgCl ₂ .6H ₂ O, and 10 mM HEPES. The final pH of the solution was 7.4. At room temperature (~ 22 ° C), a MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, CA) and a Clampex 10.3 / Digidata1440A data acquisition system and a digital-to-analog converter were used to record the collected current. The membrane current was filtered at 2 kHz and then sampled at 10 kHz. All data were analyzed using the data analysis system clampfit 10.3 (Molecular Devices) and expressed as mean ± standard error. The dose-response curve of the toxin that blocked the N-type Ca ion current was drawn using GraphPad Prism (GraphPad Software, San Diego, CA) software. The current amplitude inhibition curve was used as a function of drug concentration and fitted using Hill's equation.

The main amino acid sequences of MVIIA and its variants MVIIA-a, b, c, d and their electrophysiological activities are shown in Table 3.

Table 3. Main amino acid sequences of MVIIA and its variants and their electrophysiological activities

Inhibition of MVIIA and its variants on calcium ion (CaV2.2) channels

It is well known that MVIIA is a selective CaV2.2 channel blocker. MVIIA at a concentration of 2 μM can block the Ca _V 2.2 pathway by more than 90%. (F.Wang. 2016, and other articles) In this study, we recorded Ca ²⁺ peak currents (ICa) of CaV2.2 channels (α _1B , α ₂ δ _{1 and} β ₃ ) in 293T cells. All currents are excited by voltage steps from -80mv to 10mv in 100ms. 1 μM concentration of MVIIA, MVIIA-a, MVIIA-b, MVIIA-c and MVIIA-d treatment can reduce the Ca ²⁺ peak current, the reduction values are 98.24 ± 0.708%, 89.45 ± 0.752%, 91.70 ± 1.477%, 98.81 ± 0.427% and 84.26 ± 3.127%. We found that MVIIA-c and MVIIA have similar ability to block Ca _V 2.2 channel. The ability of L-MVIIA to block the Ca _V 2.2 channel was significantly reduced, and at a concentration of 10 μM, it only reduced the Ca ²⁺ peak current by 23.28 ± 3.347%. The semi-inhibitory concentration of the MVIIA concentration and the inhibition response of the Ca _V 2.2 channel is 0.0436 μM, which is almost 5-10 times larger than the TAT variant. The half inhibitory concentrations of the TAT variants (MVIIA-a, MVIIA-b, MVIIA-c and MVIIA-d) were 0.413, 0.379, 0.237 and 0.345 μM, respectively, as shown in FIG. 3. These results indicate that MVIIA-a, MVIIA-b, MVIIA-c, and MVIIA-d have a certain inhibitory effect on the Ca _V 2.2 channel, and the length of the linker sequence between the MVIIA and TAT variants can affect the Ca _V 2.2 channel binding ability.

Example 4: Experiments of in vivo analgesic effect of different types of modified zicoronide

Hot plate pain experiment

1.1 Experimental methods

In this experiment, a total of nine groups of mice, 6-8 mice in each group, were administered MVIIA (0.11,0.33 or 1.00 nmol / kg) in the lateral ventricle, and MVIIA and MVIIA-a, MVIIA-b were administered in the tail vein. MVIIA-c and MVIIA-d (0.33, 1.00 or 3.00 μmol / kg). When the two routes were administered, the saline group was used as a blank control group. The animals were placed on an iron with a constant temperature of 55 ± 0.5 ° C. The delay time was recorded from the time the mouse was placed on the surface of the iron to the first time the hind paw licked or jumped for the first time as pain. The threshold of the index (Eddy and Leimbach, 1953). Take the time of 60s as the limit. If the time exceeds 60s, remove the mouse to avoid damage to the mouse tissue. Before administration, the delay time was measured in advance as a baseline value; subsequently, 0.5, 1, 2, 3, 4, 6 after administration of MVIIA, MVIIA-c, and Saline (lateral ventricle or tail vein) were recorded. , 8, 10, and 12h delay times. Mice with a delay time of less than 5 s or more than 20 s compared to the delayed baseline time were considered to be insensitive and hyper-sensitive mice and were subsequently rejected. The analgesic effect is expressed in incubation time.

1.2 Comparison of Analgesic Ability

As shown in Fig. 4, one hour after the administration of MVIIA (0.11,0.33 and 1.00 nmol / kg) to the lateral ventricle, the efficacy of MVIIA reached the highest value; by 4 hours, the efficacy of MVIIA basically disappeared (Fig. 4A). However, at the time of tail vein injection, multiple doses of MVIIA did not produce efficacy (Figure 4B). MVIIA-c is the variant that has the strongest effect of inhibiting CaV2.2 channel current among the TAT variants of MVIIA. As shown in FIG. 4C, MVIIA-c exhibited the strongest drug effect when administered for 3 hours, and its strongest drug effect lasted for about 4 hours, and the drug effect duration was 12 hours.

As shown in Figure 5, one hour after injection of different doses of MVIIA-a, b, and d (0.11umol / kg, 0.33umol / kg, and 1.00μmol / kg) in the tail vein, they all showed analgesic effects and were administered 2-3 hours show the strongest effect, the effect lasts about 4 hours, and gradually decreases with time. After 12 hours of administration, there is still a significant difference between the administration group and the saline group, and the duration of effect is 12 hours.

2.Acetic acid twist test (Koster et al., 1959)

2.1 Experimental methods

Three doses of the MVIIA-a, b, c, and d peptide groups (0.6, 1.8, and 5.4 nmol / kg, low, medium, and high doses in the figure), saline control group (saline), and three doses of the positive reference drug group Animals were treated with MVIIA (0.11,0.33 and 1.00 nmol / kg, low, medium, and high doses in the figure). In writhing experiments, MVIIA (lateral ventricle) or MVIIA-a, b, c, d (lateral ventricle) were administered 30 minutes before intraperitoneal injection of 1% acetic acid, and then MVIIA and MVIIA-MVIIA-a, b, c were measured. , d in vivo analgesic effect. In order to test the ability of MVIIA and MVIIA-a, b, c, d to cross the blood-brain barrier, MVIIA and MVIIA-a, b, c were administered via tail vein administration 3 hours before intraperitoneal injection of 1% acetic acid. d. The normal saline group was used as a blank control group (administrated lateral ventricle or tail vein). The number of tumblings of the mice within 5 to 20 minutes after the acetic acid injection was recorded (Galeotti et al., 2008). The number of torsional movements was recorded as a contraction of the abdominal muscles accompanied by stretching of the hind limbs and elongation of the body. The percent inhibition is calculated using the following equation:

Inhibition (%) = (N ₀ -N ₁ ) × 100 / N ₀

N ₀ refers to the number of twists in the saline group, and N ₁ refers to the number of twists in the administration group.

2.2 Comparison of Analgesic Ability

In the acetic acid writhing experiment, three doses of the MVIIA-a, b, c, and d peptide groups (0.6, 1.8, and 5.4 nmol / kg, low, medium, and high doses in FIG. 6), saline control group (saline), Three doses of the positive reference drug group MVIIA (0.11,0.33 and 1.00 nmol / kg, low, medium, and high doses in Figure 6) treated animals. Comparison was made between the three groups at three different doses, intravenous and lateral ventricular. The number of rolls down. It was found that the MVIIA-a, b, c, d peptide group and the positive reference drug group MVIIA both reduced the number of acetic acid-induced rolls in a dose-dependent manner. Under the condition of lateral ventricular administration, MVIIA, MVIIA-a, b, c, and d respectively reduced the number of rolls of mice (relative to the saline group): MVIIA 8.97%, 53.37%, 76.88%; MVIIA-A, 2.94 %, 13.36%, 48.35%; MVIIA-B, 10.82%, 42.79%, 77.60%; MVIIA-C, 14.75%, 39.53%, 81.777%; MVIIA-D, 12.08%, 23.95%, 56.54%. Under the condition of intravenous administration, the positive reference drug MVIIA did not reduce the number of rolls in mice. MVIIA-a, b, c, and d respectively reduced the number of rolls in mice (relative to the saline group): MVIIA-a, 10.47%, 27.82%, 30.03%; MVIIA-b, 17.08%, 45.94%, 51.79%; MVIIA-c, 19.81%, 49.30%, 62.95%; MVIIA-d, 6.33%, 35.86%, 47.57%, as shown in the figure 6 shown.

Conclusion: From the above experimental results, it can be seen that compared with MVIIA, MVIIA-a, b, c, d polypeptides can be analgesic and dose-dependent in the case of intravenous injection, especially in medium. In the case of high doses, MVIIA-a, b, c, and d polypeptides can achieve good analgesic effects by intravenous injection, and meet the needs of clinical use. Furthermore, compared with MVIIA, MVIIA-a, b, c, and d are administered by intravenous injection for up to 12 hours and have a good sustained-release effect in the body.

The aforementioned analgesic experiment used one-way analysis of variance (one-way ANOVA), repeated measurement of multi-factor analysis of variance (two-way ANOVA with repeated measures), and analysis between groups using Duncan or Newman-Cole test. All data are presented as mean ± SD or standard error or 95% confidence interval. When the difference in p-value is less than 0.05, the data are considered statistically significant.

Example 5: Side Effects of Different Types of Improved Zicoronide

In order to further study the side effects of different types of improved zicoronide in vivo, the following experiments were performed:

Experimental method

Tremor time is considered to be a typical side effect such as zicoronide. Tremor time is the total time that rhythmic vibrations of the limbs, head, and trunk of a mouse are recorded over a period of time. Mice were randomly divided into groups: MVIIA (0.9nmol / kg) group, MVIIA-a, b, c, d (0.9nmol / kg) group and normal control group (6 μL, lateral ventricular administration; n = 12, male and female half) . 30 minutes and 120 minutes after dosing, a dynamic video of the mouse within 5 minutes was recorded with a digital camera, and the cumulative tremor time within 5 minutes of each mouse was counted by a person who did not understand the experiment.

Toxicology experiments were analyzed by one-way ANOVA and Newman-Cole test. All data are presented as mean ± SD or standard error or 95% confidence interval. When the difference in p-value is less than 0.05, the data are considered statistically significant.

2.1 side effects comparison

As shown in Fig. 7, when administered for 30 minutes, MVIIA caused more obvious tremor symptoms and longer tremor time; when administered for 120 minutes, the peptides of each group caused tremor symptoms and longer tremor compared with MVIIA. No significant difference in time. It can be seen from the above results that there is no significant difference in side effects between MVIIA and MVIIA-a, b, c, d polypeptides, and even at the beginning of administration, the side effects of MVIIA-a, b, c, d are even lower In MVIIA, it can be seen that the MVIIA-a, b, c, d polypeptides of the present application have less toxic and side effects.

Example 6: Comparison of MVII-A intraventricular administration with MVIIA-a, b, c, d nasal analgesia test

1.1 Hot plate pain experimental method

The hot plate pain test method is as described above. Mice In this experiment, a total of nine groups of mice, 10 mice in each group, were given MVIIA (1.00nmol / kg, 5ul / 10g) intraventricularly as a positive control group (intranasal administration of MVIIA was found to be ineffective in the experiment), and the nasal cavity They were given normal saline (saline, 2ul / 10g) and MVIIA-C (3.3, 6.6 or 9.9nmol / kg, 2ul / 10g). The saline group was used as a blank control group. Record the delay time of 0.5, 1, 2, 3, 4, 6, 8, and 10 h after intraventricular administration of MVIIA, nasal administration of MVIIA-c, and Saline. Mice with a delay time of less than 5 s or more than 20 s compared to the delayed baseline time were considered insensitive and hypersensitive mice and were subsequently kicked out.

The analgesic effect is expressed as a percentage of the maximum possible effect (% MPE), and finally calculated using the following equation:% MPE = (T ₁ -T ₀ ) × 100 / (T ₂ -T ₀ )

Among them, T ₀ and T ₁ respectively represent the delay time before and after administration, and T ₂ is the limit time of each test.

1.2 Experimental results

The analgesic ability of MVIIA and different doses of MVIIA-c after nasal administration is shown in FIG. 13. Figure 13 shows the analgesic effect of MVIIA ventricle and MVIIA-c nasal administration in a hot plate pain experiment. After intracerebroventricular administration of MVIIA (1.00 nmol / kg), the effect lasted for 4 hours. MVIIA-C (3.3, 6.6, 9.9 nmol / kg) quickly responded after nasal administration, and high-dose MVIIA-C had a long duration of efficacy, which was still significantly different from the normal saline group at 8 hours, and 10 hours after administration After the effect disappeared. * p <0.05, ** p <0.01 and *** p <0.001 are compared with the normal saline group.

1.3MVIIA-a, b, d Nasal analgesia test

As shown in Figure 14, the analgesic effect of MVIIA-a, b, d in the hot plate pain experiment when nasal administration. Similar to MVIIA-C, MVIIA-a, b, d (9.9nmol / kg) is effective immediately after nasal administration, MVII-b is still significantly different from the normal saline group at 8 hours, and the effect is 10 hours after administration disappear. * p <0.05, *** p <0.001 means comparison with the normal saline group.

In the foregoing, the present invention has been described in detail with the general description and specific embodiments, but it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention belong to the scope of protection of the present invention.

Claims (9)

1. The use of a polypeptide in the preparation of a medicament, preferably, the medicament is used for analgesia, the medicament is administered intravenously, intraperitoneally or nasally, wherein the polypeptide comprises zicoronide and a cell membrane penetrating peptide.
2. The use according to claim 1, characterized in that the polypeptide is composed of zicoronopeptide connected to a cell membrane penetrating peptide through a C-terminus; preferably, the C-terminus of the zicoronotide penetrates to a cell membrane through a linker The N-terminus of the peptide is attached.
3. The use according to any one of claims 1-2, wherein the linker is two glycines.
4. The use according to any one of claims 1 to 3, wherein the amino acid of the zicoronide is shown in SEQ ID No. 1, or the amino acid of the zicoronide is shown in SEQ ID No. 1. Variants of fewer than 10 deleted, mutated or inserted amino acids.
5. The use according to any one of claims 1-4, wherein the cell membrane penetrating peptide is selected from the group consisting of: Penetratin, TAT peptide, Pep-1 peptide, S4 ₁₃ -PV, Magainin 2 or Buforin 2; preferably, all The amino acid of the TAT peptide is shown in SEQ ID NO. 2, or the TAT peptide is a variant of a deletion, mutation or insertion of less than 10 amino acids shown in SEQ ID NO. 2, or a peptidomimetic thereof.
6. The use according to any one of claims 1 to 5, characterized in that the amino acid sequence of the polypeptide is shown in SEQ ID NO.3.
7. The use according to any one of claims 1-6, the polypeptide is prepared synthetically.
8. The use according to any one of claims 1-7, wherein the medicament is administered intravenously, intraperitoneally or nasally.
9. The use according to any one of claims 1-8, the polypeptide is prepared into a preparation for intravenous, intraperitoneal or nasal administration, and is administered by means of intravenous, intraperitoneal or nasal administration.

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