CN114601912A - Itch-caused polypeptide caused by tick and mosquito bites and itch-resisting application thereof - Google Patents

Itch-caused polypeptide caused by tick and mosquito bites and itch-resisting application thereof Download PDF

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CN114601912A
CN114601912A CN202011441138.7A CN202011441138A CN114601912A CN 114601912 A CN114601912 A CN 114601912A CN 202011441138 A CN202011441138 A CN 202011441138A CN 114601912 A CN114601912 A CN 114601912A
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itch
polypeptide
mrgprc11
mrgprx1
tick
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曹志贱
吴英亮
李文鑫
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Wuhan University WHU
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Abstract

The invention discloses an itch-causing polypeptide caused by tick and mosquito bites and an anti-itch application thereof, and belongs to the technical field of biology. The polypeptide IP and SW secreted by tick and mosquito bites with the amino acid sequences shown as SEQ ID No.1 and 2 respectively can be used as an itch-causing agent to cause itch by activating a MrgprC11/MrgprX1-TRPV1 signal channel, and an MrgprC11/MrgprX1 antagonist or a TRPV1 inhibitor can be used for preparing a medicament for treating or preventing itch diseases caused by arthropod bites. The MrgprC11/MrgprX1 antagonist or TRPV1 inhibitor is prepared into an external gel preparation, and the gel preparation has obvious effect and obvious treatment effect on ticks and mosquito bites.

Description

Itch-caused polypeptide caused by tick and mosquito bites and itch-resisting application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to application of polypeptide secreted by tick and mosquito bites in itching, an external gel for treating the tick and mosquito bites and a preparation method thereof.
Background
Itching caused by stinging or biting by arthropods is a relatively common clinical phenomenon. Many arthropod bites or stings represent one of the most important itch-inducing factors, and venom from ticks, mosquitoes, centipedes, hornets, bees, etc., that sting the stinger can cause a localized or systemic itch response. The material basis and associated molecular mechanisms underlying itch induction by these arthropod bites or stings have not been known for a long time. This situation leads to clinically limited treatment of pruritus caused by arthropod bites or stings.
Ticks belong to the arachnids. Ticks cause itching through biting stimuli. It is well known that tick bites can transmit many fatal pathogens, such as fever with thrombocytopenia syndrome virus (SFTSV), tick-borne encephalitis virus (TBEV), and borrelia burgdorferi (LYME pathogen), which pose a significant threat to human and animal health. Itching is also a typical symptom of diseases associated with tick bites, but it is not known at all whether the itching is caused by the pathogen transmitted by the tick bite or by its salivary components, and therefore the composition of the substance causing the itching by the tick bite and its associated molecular, cellular and neural mechanisms have never been explored.
Mosquitoes belong to the family of the order diptera of the phylum arthropoda, also known as "mosquitoes", the most important group of medical insects. Mosquitoes are widely distributed and various, and mosquitoes of 3 subfamilies (macromuscidae, anopheles and culex subfamily), 35 genus, 3600 variety and subspecies have been recorded all over the world so far. More than 370 species have been found in china, of which more than half of the 3 mosquito species of anopheles, culex, aedes. Except Antarctic, there are mosquitoes in all continents. Typically, females feed on blood, while males suck plant sap. Female mosquitoes that take blood are intermediate hosts to other pathogens such as dengue fever, malaria, yellow fever, filariasis, Japanese encephalitis, and the like. When bitten by a mosquito, the toxin is released to the skin surface, which causes degranulation of mast cells and induces vasodilatation. A distinct red oedema wheal is seen and the allergenic substance stimulates the peripheral nerves and produces itching symptoms, all of which are caused by toxins.
Itching caused by stinging or biting of arthropods such as ticks and mosquitoes is a skin allergy phenomenon, and toxins are combined with toxin antibodies to stimulate degranulation of mast cells to generate allergic substances such as histamine, and the allergic substances are combined with receptors (such as histamine receptors 1 and 4) on the cell membrane of peripheral nerves to stimulate peripheral nerves and generate itching symptoms. However, in fact, the itch-causing sources associated with bites or stings of arthropods such as ticks and mosquitoes and their molecular, cellular and nerve conduction mechanisms are unclear. For example, some conventional antihistamine allergy medicines have a poor curative effect in clinically treating itching caused by biting or stinging of arthropods such as ticks and mosquitoes.
Disclosure of Invention
The invention discloses an itching-causing source and an itching-causing molecular signal channel for tick and mosquito bites. Based on the situation, the invention mainly aims to provide application of polypeptide secreted by ticks and mosquitoes and a medicament for inhibiting or treating pruritus bites of ticks and mosquitoes.
The invention firstly virtually screens and prepares the polypeptide from the secreted polypeptide from the tick and mosquito bites with high flux, and concretely comprises analyzing the secreted polypeptide from the tick and mosquito bites, selecting potential polypeptide causing itch, and then preparing the secreted polypeptide IP and SW from the tick and mosquito bites by using the technologies and methods such as polypeptide chemical synthesis, mass spectrum and the like. Mouse animal experiments are adopted to find that the function of secreting the polypeptides IP and SW by tick and mosquito bites can generate pruritus. Further screening and identifying MrgprC11/MrgprX1-TRPV1 signal channels and inhibitors thereof on target peripheral neurons resisting ticks and mosquito biting polypeptide IP and SW itch. An exogenous gel containing anti-tick and mosquito-biting polypeptide IP and SW itch-causing inhibitor components is developed, and the gel is proved to have the treatment and protection effects on tick and mosquito-biting itch.
The purpose of the invention is realized by the following technical scheme:
the polypeptide IP secreted by tick bites and the polypeptide SW secreted by mosquito bites can cause pruritus as an itching-causing agent, and the amino acid sequences of the IP and the SW are shown as follows:
IP：GFGCPFNQGACHRHCRSIGRRGGYCAGLFKQTCTCYSR(SEQ ID NO.1)；
SW：ATCDLASGFGVGSSLCAAHCIARRYRGGYCNSQQVCVCRN(SEQ ID NO.2)。
the MrgprC11/MrgprX1-TRPV1 signal channel can be used as a drug target to screen drugs for resisting itch diseases caused by arthropod bite.
The MrgprC11/MrgprX1 antagonist or the TRPV1 inhibitor can be used for preparing medicaments for treating or preventing itch diseases caused by the bite of arthropods. The medicine comprises injection, paste, spray, external gel preparation and the like.
A gel preparation for external use for treating or preventing itch caused by arthropod bite comprises MrgprC11/MrgprX1 antagonist or TRPV1 inhibitor, preferably 0.2-0.5% (by mass). The gel matrix used by the external gel preparation is hydroxymethyl cellulose.
Further, the external gel preparation for treating or preventing the itch disease caused by the bite of arthropod comprises the following components by mass percent: 5-15% of propylene glycol, 5-15% of ethanol, 0.5-1.5% of triethanolamine, 0.5-1.5% of hydroxymethyl cellulose, 0.2-0.5% of MrgprC11/MrgprX1 antagonist or TRPV1 inhibitor, 0.05-0.3% of ethylparaben and the balance of sterile water.
The arthropods include ticks, mosquitoes, and the like.
The MrgprC11/MrgprX1 antagonists include but are not limited to 2, 4-diaminopyrimidine derivatives, azabicyclooctane; such TRPV1 inhibitors include, but are not limited to, BCTC, AMG9810, SB-366791, A889425.
The 2, 4-diaminopyrimidine derivatives include, but are not limited to, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl) -N- (3-morpholinopropyl) acetamide.
The azabicyclooctane includes but is not limited to 1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) prop-2-en-1-one.
The preparation method of the external gel preparation for treating or preventing the itching disease caused by the bite of arthropod comprises the following steps: spraying hydroxymethyl cellulose on sterile water surface, standing to obtain gel matrix, standing, vacuum defoaming, mixing the rest components, gradually adding into gel matrix, and mixing to avoid excessive bubbles.
The invention discovers the polypeptide causing the ticks from the tick bites and the effective inhibitor thereof, and a new medicament prepared by taking the inhibitor for preventing the ticks from the tick bites and the mosquito bites as the effective component has obvious effect and obvious treatment effect when being used for treating the ticks from the tick bites and the mosquito bites. The invention has good curative effect, quick effect, no toxic or side effect and no sequelae. The medicine can really achieve the effects of quick response, no side effect and relapse prevention after healing.
Drawings
FIG. 1 is a graph of the results of reverse phase high performance liquid chromatography (RP-HPLC), mass spectrometry and circular dichroism spectroscopy analyses of chemically synthesized linear (IP-R) and cyclized (IP-O) IP polypeptides. In the figure: a, RP-HPLC profiles of linear (IP-R) and cyclic (IP-O) IP polypeptides; b, mass spectra of linear (IP-R) and cyclic (IP-O) IP polypeptides; circular dichroism spectra of C, linear (IP-R) and cyclic (IP-O) IP polypeptides.
FIG. 2 is a graph showing the results of the itch-causing effect of the cyclized IP polypeptide (IP-O) on mice. In the figure: negative control, solvent control; positive control, histamine positive drug control; IP-O (Low), IP-O Low dose; IP-O (middle): IP-O medium dosage; IP-O (high), high dosage of IP-O.
FIG. 3 is a graph showing the results of the itch-causing effect of the cyclized SW polypeptide (SW-O) on mice. In the figure: negative control, solvent control; positive control, histamine positive drug control; SW-O (Low), SW-O Low dose; SW-O (middle), SW-O middle dose; SW-O (high), SW-O high dose.
FIG. 4 is a graph of the effect of cyclized IP polypeptides (IP-O) and SW polypeptides (SW-O) on the activation of peripheral neuronal itch receptors MrgprC11 and MrgprX 1. In the figure: activation effect of a, IP-O on MrgprX 1; b, activation effect of IP-O on MrgprC 11; the activating effect of C, SW-O on MrgprX 1; activation effects of D, SW-O on MrgprC 11; BAM, BAM22-8 positive control.
FIG. 5 is a graph showing the results of the molecular signaling mechanism of TRPV1 signaling pathway mediating the itch-causing activity of IP-O polypeptide. In the figure: a, activation effect of TRPV1 agonist capsaicin (Cap) on IP-O polypeptide itch neurons; b and C, insensitivity of IP-O to Trpv1 knockout neurons; d, differential pruritus response of intradermal injection of IP-O polypeptides in wild-type mice (white) and TRPV1 knock-out mice (black); AMG9810, TRPV1 antagonists; AITC, TRPA1 agonists; HIS, histamine; CQ, chloroquine.
FIG. 6 is a graph showing the results of identifying the MPrgprC 11/X1 and TRPV1 inhibitors which are antipruritic to the secretion of the polypeptide IP from tick bites. In the figure: p, IP-O polypeptides; i1, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide; i2, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide; i3, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl) -N- (3-morpholinopropyl) acetamide; i4, 1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) prop-2-en-1-one; i5, BCTC; i6, AMG 9810; i7, SB-366791; i8, a 889425.
FIG. 7 is a graph of the results of identifying an inhibitor of the secreted polypeptide SW against mosquito bites, MrgprC11/X1 and TRPV1, which are itchy. In the figure: a P, SW-O polypeptide; i1, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide; i2, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide; i3, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl) -N- (3-morpholinopropyl) acetamide; i4, 1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) prop-2-en-1-one; i5, BCTC; i6, AMG 9810; i7, SB-366791; i8, a 889425.
Detailed Description
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
Example 1: preparation and identification of secreted polypeptides IP and SW from tick and mosquito bites
According to the amino acid sequence (GFGCPFNQGACHRHCRSIGRRGGYCAGLFKQTCTCYSR) of IP and the amino acid sequence (ATCDLASGFGVGSSLCAAHCIARRYRGGYCNSQQVCVCRN) of SW, reduced linear peptide with the purity of more than 95% is obtained by chemical synthesis, and then a disulfide bond is formed by applying the cysteine directional pairing technology to prepare oxidized cyclized peptide. Oxidized cyclized peptides were used in the following examples.
I preparation and identification of tick bite secretion polypeptide IP
A: chemical synthesis of reduced linear IP polypeptides
Tick and mosquito bite secreted polypeptide IP is consigned to Shanghai Qiangyao biotech Limited (China Peptides Co., Ltd.), and the purity is more than 95 percent and the molecular weight of the synthesized polypeptide is correct by the map identification of High Performance Liquid Chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) (figure 1).
B: renaturation of oxidized cyclic IP polypeptides
The specific operation steps are as follows:
(1) the pH value of the 0.2M Tris-HCl buffer solution is adjusted to 8.0 according to the isoelectric point of the polypeptide IP.
(2) 5mg of chemically synthesized reduced linear polypeptide was placed in 2.0mL sterilized cell cryopreservation tubes.
(3) 1mL of 1.0M Tris-HCl buffer at the corresponding pH was added.
(4) The solution was gently aspirated with a pipette to speed up the dissolution of the polypeptide.
(5) And putting the cell freezing tube containing the polypeptide solution into a constant-temperature shaking table, and incubating for about 48 hours at the temperature of 26-28 ℃ and at the speed of 150 rpm.
(6) High speed centrifugation at 12000rmp for 3min at room temperature.
(7) The supernatant was transferred to another clean, sterilized 5mL EP tube.
(8) Stored at-80 ℃ or used for subsequent experiments.
C: separation and purification of oxidized cyclic IP polypeptide (IP-O)
Separating and purifying the prepared oxidized cyclized IP polypeptide by using RP-HPLC, which comprises the following steps:
(1) and (3) setting related parameters after the self-checking of the instrument is passed after the instrument is started: flow rate is 4 mL/min; mobile phase: 95% solution B (95mL sterilized ddH)2O, 5mL of chromatographic acetonitrile and 100. mu.L of chromatographic TFA mixed well) and 5% solution D (95mL of chromatographic acetonitrile, 5mL of sterilized ddH)2O and 100 μ L chromatographic TFA mixed well); and the ultraviolet measurement wavelength is 230 nm.
(2) And after the baseline is stable, setting the absorbance to zero, sucking the sample to be separated into a sample injection needle, and injecting the sample into a manual sample injector (the sample loading volume of the semi-preparation strain is not more than 5mL, and the sample loading volume of the analytical column is not more than 20 muL). Adopting a gradient elution mode, wherein the elution conditions are as follows: firstly, 95% → 5% of solution B and 5% → 95% of solution D; ② the total elution time is 60 min; and the ultraviolet measurement wavelength is 230 nm.
(3) During the HPLC separation, the effluent corresponding to each absorption peak was collected in a clean centrifuge tube and marked with a marker for Retention Time (RT) and chromatographic peak flow number. Liquid A for mobile phase exchange (80mL of chromatographic methanol and 20mL of sterilized ddH2O is fully mixed), the flow rate is 4mL/min, and the chromatographic column is flushed until the base line is stable (about 30min is needed); the collected effluent was pre-frozen in a freezer at-80 ℃ for 4 h.
(4) The freeze vacuum drier was opened to pre-cool for about 1 h. And (4) when the temperature is reduced to be close to-100 ℃, putting the pre-frozen sample, closing the air inlet valve, covering the gland, and opening the oil pump. After about 12h of freeze vacuum drying, the oil pump and the freeze dryer are closed, the air inlet valve is opened slowly, and the centrifuge tube is taken out.
(5) A small amount of the lyophilized solid polypeptide was picked up with a sterilized pipette tip, transferred to a 200. mu.L PCR thin-wall tube and labeled (for mass spectrometry to determine the molecular weight of the material corresponding to each chromatographic peak).
(6) All solid oxidized cyclized IP polypeptides obtained after freeze vacuum drying are stored in a refrigerator at the temperature of-80 ℃ or used for subsequent experiments.
D: molecular weight characterization of oxidized cyclized IP polypeptides (IP-O)
The method comprises the following specific steps:
(1) about 15. mu.g of the desalted, purified and lyophilized oxidized cyclized IP polypeptide was removed and sterilized with 20. mu.L of ddH2And dissolving the O.
(2) The IP-O polypeptide solution and 5. mu.L of MALDI-matrix solution were mixed uniformly in a ratio of 1: 1.
(3) mu.L of the mixture of IP-O polypeptide peptide samples was spotted on a MALDI target plate and air-dried at room temperature.
(4) Mass spectrometry was performed by Flex Control software (version 3.0, Bruker Daltonics) with a molecular mass range m/z of 1000-.
(5) The molecular weight of the IP-O polypeptide is determined using a positive ion linear mode at an accelerating voltage of 25 kV.
II following the procedures of A, B, C and D for the IP polypeptide, chemical synthesis, renaturation, isolation and purification and molecular weight identification of oxidized cyclized SW polypeptide (SW-O) were carried out.
Example 2: itching-causing effects of tick and mosquito bites secreting polypeptides IP and SW
The oxidized polypeptide obtained in example 1 was injected into the face of a mouse by subcutaneous injection, and the number of times the mouse grasped the face was monitored.
(1) The positive drug control was histamine, 20 μ g/mouse, with the number n ═ 6 per group of mice. Negative drug control was 0.9% saline, n-6. Low, medium and high dose groups of cyclized polypeptide drug: 10 ug, 20 ug and 40 ug per mouse, with the number n of mice per group being 6. The injection volume should be controlled at 10-20. mu.L/tube.
(2) C57/BL mice, 6-8 weeks old, Hubei province disease control center, purchased before the experiment. C57/BL mice acclimate for 2 days, anaesthetize and remove a small part of facial hair, continue acclimating for 1 day, place into an observation container to acclimate for 30min and observe scratching times, and inject medicine intradermally and continue to observe scratching times for 30 min.
The results of the experiments (FIGS. 2 and 3) show that the concentration dependence of the IP-O polypeptide and SW-O polypeptide can cause the mice to generate pruritus. The IP-O polypeptide and the SW-O polypeptide are respectively new substances causing itching caused by tick and mosquito bites.
Example 3: molecule signal path for ticking caused by polypeptide IP and SW secreted by tick and mosquito bites
(1) Known itch receptors such as H1R, H4R, PAR2, PAR4 and MRGPRs or other unknown itch receptor genes such as Kvs, SKCas, TRPs and GPCRs are constructed on pEGFP-N1 vector (Clontech), then transfected into HEK293 cells, the known itch receptors or the unknown itch receptors are over-expressed on HEK293 cell membranes, and whether the IP-O polypeptide and the SW-O polypeptide can activate the receptors is researched by adopting a calcium imaging technology.
(2) In a HEK293 cell system, a calcium imaging technology is adopted to research whether an inhibitor of TRPs influences the activation effect of an itch-causing tick venom polypeptide IP-O and an itch-causing mosquito venom polypeptide SW-O on cells; on the animal level, a mouse itch model is constructed by adopting an itch receptor downstream coupled ion channel gene TrpV1 knockout mouse based on a back and cheek subcutaneous injection mode, and whether the TRPV1 ion channel knockout influences the itch causing effect of tick venom polypeptide IP-O is tested.
The experimental results (figures 4 and 5) show that the IP-O polypeptide and the SW-O polypeptide can effectively activate human itch receptor MrgprX1 and mouse itch receptor MrgprC11 as well as positive polypeptide medicament bovine adrenal medulla polypeptide 8-22(BAM8-22, BAM). Furthermore, the downstream ion channel of the IP-O polypeptide activated itch receptor MrgprX1/MrgprC11 was also demonstrated to be TRPV 1.
Example 4: screening and identification of anti-tick and mosquito-bite secreted polypeptide IP and SW itch-causing inhibitor
(1) C57/BL mice, 6-8 weeks old, Hubei province disease control center, purchased before the experiment. C57/BL mice acclimate for 2 days, anaesthetize and remove a small part of facial hair, continue acclimating for 1 day, place into an observation container to acclimate for 30min and observe scratching times, and inject medicine intradermally and continue to observe scratching times for 30 min. The number of animals tested per group was 6.
(2) Grouping of subcutaneous injection drugs:
2, 4-diaminopyrimidine derivatives [2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl) -N- (3-morpholinopropyl) acetamide (available from shanghai hong science and technology limited with purity of more than 95%), azabicyclooctane [1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) propan-2-en-1-one ] (available from wuhan kabda chemical limited with purity of more than 95%), BCTC, AMG9810, SB-366791, a889425 (available from eimeria science and technology limited with purity of more than 95%);
an IP-O or SW-O polypeptide;
2, 4-diaminopyrimidine derivatives [2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl) -N- (3-morpholinopropyl) acetamide, azabicyclooctane [1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) prop-2-en-1-one ], BCTC, AMG9810, SB-366791, A889425+ IP-O or SW-O polypeptide.
The experimental results (fig. 6 and 7) show that the IP-O polypeptide and the SW-O polypeptide can remarkably cause the pruritus behavior of the mice, and the mice are treated by the MrgprX1/MrgprC11 antagonist or the TRPV1 inhibitor alone without influencing the pruritus behavior of the mice. However, when mice were treated with IP-O polypeptide, SW-O polypeptide in combination with either MrgprX1/MrgprC11 antagonist or TRPV1 inhibitor, the number of scratching in mice was also very low, indicating that treatment with either MrgprX1/MrgprC11 antagonist or TRPV1 inhibitor was effective in preventing pruritic behavior induced by IP-O polypeptide, SW-O polypeptide.
Example 5: preparation of gel preparation for resisting ticks and mosquitoes to bite and secrete polypeptide IP and SW to cause itching
(1) Prescription: 2, 4-diaminopyrimidine derivatives [2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl) -N- (3-morpholinopropyl) acetamide, azabicyclooctane [1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) prop-2-en-1-one ], BCTC, AMG9810, SB-366791 or A8894250.3 g, propylene glycol 10 g, ethanol 10 g, hydroxymethylcellulose 1.0 g, triethanolamine 1.0 g, ethylparaben 0.1 g, sterile water up to 100 g.
(2) Preparation: spraying hydroxymethyl cellulose on sterile water surface, standing overnight to obtain gel matrix, vacuum defoaming, mixing triethanolamine with other components, gradually adding into slurry, and mixing to avoid excessive bubbles. Packaging to obtain gel preparation.
(3) Blank control gel: the preparation is prepared by the same method except that the preparation does not contain 2, 4-diaminopyrimidine derivative, azabicyclooctane, BCTC, AMG9810, SB-366791 or A889425.
Example 6: therapeutic effect of gel preparation of MrgprC11/X1 antagonist or TRPV1 inhibitor on ticks and mosquito bite pruritus
(1) Case selection: the selected patients are from the dermatology clinic of the people hospital of Wuhan university and are clinically diagnosed with tick and mosquito bite allergy patients. Excluding patients who have used other drugs, serious liver and kidney insufficiency and pregnant women in lactation period.
(2) Grouping experiments: the method for observing 60 patients by adopting multi-center open parallel control is divided into an experimental group and a control group. Experimental groups: 30, 16 men, 14 women, 18 to 65 years of age. Control group: 30, 17 men, 13 women, age 19 to 64 years. Compared with index values such as age, sex, disease stage and skin damage degree among 2 groups, the difference has no obvious meaning and is comparable.
(3) The experimental method comprises the following steps: experimental groups the compounds prepared in example 5 were administered to a patient containing the 2, 4-diaminopyrimidine derivatives [2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chloroethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl-N- (3-morpholinopropyl) acetamide ], azabicyclooctane [1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) prop-2-en-1-one ], BCTC, AMG9810, SB-366791 or A889425 gel formulations are applied topically to patients with ticks and mosquito bites. The medication method comprises the following steps: washing face with warm water and soap solution or sulfur medicated soap, cleaning face putty, dipping with cotton swab, and repeatedly applying to affected part, wherein the application is carried out once every morning and evening, and 1 week is a treatment course. The control group was treated with a gel formulation without MrgprC11/X1 antagonist or TRPV1 inhibitor as placebo.
(4) The curative effect results are as follows: the group treated with gel formulations containing either MrgprC11/X1 antagonist or TRPV1 inhibitor showed significantly less scrapie than placebo-treated patients for 2 hours of administration. The effective rates are 95% and 100% respectively at 4 and 6 hours of medication. Compared with a control group, the effective rate difference between the two groups is obvious.
(5) Adverse reactions: no adverse reaction occurred in 1 of 30 patients in the experimental group.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Wuhan university
<120> polypeptide causing itching due to tick and mosquito bites and anti-itching application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> PRT
<213> tick (Ixodoidea)
<400> 1
Gly Phe Gly Cys Pro Phe Asn Gln Gly Ala Cys His Arg His Cys Arg
1 5 10 15
Ser Ile Gly Arg Arg Gly Gly Tyr Cys Ala Gly Leu Phe Lys Gln Thr
20 25 30
Cys Thr Cys Tyr Ser Arg
35
<210> 2
<211> 40
<212> PRT
<213> mosquito (Curicidae)
<400> 2
Ala Thr Cys Asp Leu Ala Ser Gly Phe Gly Val Gly Ser Ser Leu Cys
1 5 10 15
Ala Ala His Cys Ile Ala Arg Arg Tyr Arg Gly Gly Tyr Cys Asn Ser
20 25 30
Gln Gln Val Cys Val Cys Arg Asn
35 40

Claims (10)

  1. The application of the IP polypeptide as an itch causing source for tick bites is characterized in that: the amino acid sequence of the IP polypeptide is GFGCPFNQGACHRHCRSIGRRGGYCAGLFKQTCTCYSR.
  2. The application of SW polypeptide as the itch-causing source for mosquito bites is characterized in that: the amino acid sequence of the SW polypeptide is ATCDLASGFGVGSSLCAAHCIARRYRGGYCNSQQVCVCRN.
  3. The application of the MrgprC11/MrgprX1-TRPV1 signal channel as a drug target in screening drugs for resisting pruritus diseases caused by arthropod bite.
  4. Use of an MrgprC11/MrgprX1 antagonist or a TRPV1 inhibitor for the preparation of a medicament for the treatment or prevention of itch caused by arthropod bites.
  5. 5. Use according to claim 3 or 4, characterized in that: the medicine comprises injection, paste, spray and external gel preparation.
  6. 6. A gel preparation for external use for treating or preventing itch disease caused by arthropod bite, characterized in that: comprising an MrgprC11/MrgprX1 antagonist or a TRPV1 inhibitor.
  7. 7. The topical gel formulation according to claim 6, wherein: the gel matrix is hydroxymethyl cellulose.
  8. 8. The topical gel formulation according to claim 6, wherein: comprises the following components in percentage by mass: 5-15% of propylene glycol, 5-15% of ethanol, 0.5-1.5% of triethanolamine, 0.5-1.5% of hydroxymethyl cellulose, 0.2-0.5% of MrgprC11/MrgprX1 antagonist or TRPV1 inhibitor, 0.05-0.3% of ethylparaben and the balance of sterile water.
  9. 9. The use according to claim 4 or 5 or the topical gel formulation according to any one of claims 6 to 8, wherein: the MrgprC11/MrgprX1 antagonist comprises 2, 4-diaminopyrimidine derivatives, azabicyclooctane; the TRPV1 inhibitor comprises BCTC, AMG9810, SB-366791 and A889425.
  10. 10. The use or topical gel formulation according to claim 9, characterized in that: the 2, 4-diaminopyrimidine derivatives include 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (furan-2-ylmethyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- (3-methoxypropyl) -N- (pyridin-4-ylmethyl) acetamide, 2- (4- (2- ((4-chlorophenethyl) amino) pyrimidin-4-yl) piperazin-1-yl) -N- ((3, 5-dichloropyridin-4-yl) methyl) -N- (3-morpholinopropyl) acetamide; the azabicyclooctane comprises 1- (2- ((2-benzhydrylquinuclidin-3-yl) oxy) phenyl) prop-2-en-1-one.
CN202011441138.7A 2020-12-08 2020-12-08 Itch-caused polypeptide caused by tick and mosquito bites and itch-resisting application thereof Pending CN114601912A (en)

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CN105233286A (en) * 2015-09-10 2016-01-13 上海交通大学医学院 Preparation containing acid-sensing ion channel regulating agent and usage of preparation containing acid-sensing ion channel regulating agent for treatment of pruritus
CN106990200A (en) * 2017-05-16 2017-07-28 暨南大学 A kind of cell membrane solid phase chromatography for screening Chinese medicine psychoactive ingredient and its application
CN109640981A (en) * 2016-06-29 2019-04-16 曼隆治疗公司 Neurokinine-1 antagonist is used to treat the purposes of a variety of pruritic conditions

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CN105233286A (en) * 2015-09-10 2016-01-13 上海交通大学医学院 Preparation containing acid-sensing ion channel regulating agent and usage of preparation containing acid-sensing ion channel regulating agent for treatment of pruritus
CN109640981A (en) * 2016-06-29 2019-04-16 曼隆治疗公司 Neurokinine-1 antagonist is used to treat the purposes of a variety of pruritic conditions
CN106990200A (en) * 2017-05-16 2017-07-28 暨南大学 A kind of cell membrane solid phase chromatography for screening Chinese medicine psychoactive ingredient and its application

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115583988A (en) * 2022-10-14 2023-01-10 深圳市维琪医药研发有限公司 Synthetic peptides, cosmetic or pharmaceutical compositions and uses thereof
CN115583988B (en) * 2022-10-14 2023-08-18 深圳市维琪科技股份有限公司 Synthetic peptides, compositions and uses thereof

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