WO2018028117A1 - 一种蝎毒耐热合成肽及其用途 - Google Patents

一种蝎毒耐热合成肽及其用途 Download PDF

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WO2018028117A1
WO2018028117A1 PCT/CN2016/112078 CN2016112078W WO2018028117A1 WO 2018028117 A1 WO2018028117 A1 WO 2018028117A1 CN 2016112078 W CN2016112078 W CN 2016112078W WO 2018028117 A1 WO2018028117 A1 WO 2018028117A1
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scorpion
resistant
peptide
heat
synthetic peptide
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French (fr)
Chinese (zh)
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赵杰
李韶
张万琴
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Dalian Medical University
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Dalian Medical University
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Priority to ES16905684T priority Critical patent/ES2945407T3/es
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Priority to CA2984569A priority patent/CA2984569C/en
Priority to JP2017554073A priority patent/JP6483858B2/ja
Priority to US15/800,802 priority patent/US10442837B2/en
Publication of WO2018028117A1 publication Critical patent/WO2018028117A1/zh
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Definitions

  • the invention belongs to the field of polypeptide drug research and development, and particularly relates to an amino acid sequence of a scorpion venom heat-resistant peptide (SVHRP) obtained from a traditional Chinese medicine scorpion venom and its synthetic product scorpion venom heat-resistant synthetic peptide (SVHRSP) in the treatment of epilepsy, Alz Application of Haimer's disease and Parkinson's disease.
  • SVHRP scorpion venom heat-resistant peptide
  • SVHRSP synthetic product scorpion venom heat-resistant synthetic peptide
  • PD Parkinson's Disease
  • DA dopamine
  • NCs pars compacta
  • BmK Buthus martensii Karsch
  • SmK Scorpion Venom
  • toxin action mechanism it can be divided into neurotoxin and cytotoxin.
  • neurotoxin is classified into long-chain scorpion toxin (containing 60-70 amino acid residues) and short-chain scorpion toxin (containing 30-40 amino acid residues).
  • the target of long-chain scorpion toxin is mainly voltage-dependent Na + channels on the neuro-excitable membrane, and short-chain scorpion toxin can act on Ca 2+ channel, K + channel or Cl - aisle.
  • Scorpion toxin has been widely used in the development of tools and antitoxins for membrane ion channels. It is a traditional Chinese medicine in China. So far, China has isolated and purified the scorpion toxin from the BmK scorpion venom with anti-tumor, anti-pain, anti-epileptic, anti-thromb, anti-inflammatory, anti-rheumatic and antibacterial functions. The toxicity of scorpion venom is second only to snake venom.
  • the national invention patent CN1324621 confirmed the effectiveness of the venom-scorpion venom-removed epilepsy (RE) on the medicinal part of the natural drug, and the safety of the Refractory Epilepsy (RE).
  • the National Invention Patent Application discloses the removal of toxic components which are not heat-resistant and heat-resistant from the SV of BmK, and obtains a safer extract of scorpion-resistant heat-resistant peptide.
  • the scorpion venous peptide extract has a low peptide yield, so obtaining chemically synthesized scorpion phytol peptide is a key link in determining whether this original research and development can be transformed and developed and industrialized.
  • the content of the application mainly includes the determination of the amino acid sequence of the scorpion phytolophilic peptide, the solid phase chemical synthesis according to the sequence, and the pharmacodynamic activity and safety detection of the scorpion venom heat-resistant synthetic peptide (SVHRSP).
  • the amino acid sequence of the scorpion venom resistant synthetic peptide (SVHRSP) of the present invention is as follows:
  • SEQ ID No. 1 (N-terminal) Lys-Val-Leu-Asn-Gly-Pro-Glu-Glu-Glu-Ala-Ala-Ala-Pro-Ala-Glu (C-terminal) scorpion venom heat-resistant synthetic peptide
  • a synthetic active polypeptide comprising 15 amino acid residues having a molecular weight of 1524 Da.
  • the scorpion venom heat-resistant synthetic peptide (SVHRSP) of the present invention first obtains the amino acid sequence of scorpion phytohormone from the traditional Chinese medicine BmK scorpion venom: mainly includes an experimental sample confirmed by an animal experiment (xiedu 20160112-peptide summary) for refractory epilepsy, Parkinson's disease and senile dementia have obvious preventive and therapeutic effects; after the sample is further subjected to LaGM composite material and repeated rapid magnetic separation, the mass spectrometry parallel experiment of nanoliter-electrospray ionization mass spectrometry (nanoLC-ESI-MS) is used for detection.
  • nanoLC-ESI-MS nanoliter-electrospray ionization mass spectrometry
  • scorpion phytohormone obtained by solid phase chemical synthesis, chromatographic purification and mass spectrometry to obtain a scorpion venom resistant synthetic peptide (SVHRSP) whose amino acid sequence is shown in SEQ ID No. 1, this sequence is maintained.
  • SVHRSP scorpion venom resistant synthetic peptide
  • the method for extracting the venom heat-resistant peptide of the present invention is as follows:
  • the sample was coarsely divided by Superdex Peptide 10/300GL molecular sieve column (Optimum Separation range (peptides) M, 100-7000 Da) (see Figure 1), and the sample was subdivided by HPLC (see Figure 2).
  • the chromatographic conditions were: :Zorbax SB-C18 4.6*250 5 ⁇ m (AgilentUSA), mobile phase: liquid A: acetonitrile/water: 2:98 (containing 0.1% trifluoroacetic acid); liquid B: acetonitrile/water: 98:2 (containing trifluoroacetic acid 0.08%); 0–40% B approximately 3-6 column volumes, 40–100% B 0.5-1 column volumes, 100% B 1-3 column volumes (CV); flow rate: 0.8 ml/min; UV The detector has a detection wavelength of 280 nm / 258 nm / 214 nm.
  • the polypeptide synthesis technique is to obtain a target molecule by orienting an amide bond method according to the amino acid sequence of the scorpion phytolophilic peptide.
  • Solid phase synthesis is to connect the carboxyl group of one amino acid of the drug substance to the solid phase carrier (Fmoc resin) in the form of a covalent bond, and then use the amino group of this amino acid as the starting point of synthesis to make the carboxyl group with the adjacent amino acid (amino group).
  • An acylation reaction occurs to form a peptide bond.
  • the amino group of the resin peptide containing these two amino acids is then deprotected and reacted with the carboxyl group of the next amino acid, and the process is repeated until the target peptide is formed.
  • the scorpion venom heat-resistant synthetic peptide of the present invention uses the Morris water maze test method to detect the effect of scorpion venom heat-resistant synthetic peptide (SVHRSP) on learning and memory of AD mice, and the results indicate that scorpion venom heat-resistant synthetic peptide (SVHRSP) has space for AD mice.
  • SVHRSP scorpion venom heat-resistant synthetic peptide
  • scorpion venom heat-resistant synthetic peptide SVHRSP
  • the toxic heat-resistant synthetic peptide SVHRSP
  • the effects of scorpion venom-resistant synthetic peptide (SVHRSP) on type II astrocytes were observed.
  • the results showed that scorpion venom-resistant synthetic peptide (SVHRSP) can promote type II astrocyte reprogramming (reverse differentiation) into brain. Endogenous neural stem cells.
  • SVHRSP scorpion venom-resistant synthetic peptide
  • Figure 1 is a gel filtration chromatogram of a sample of a venom heat-resistant peptide extract
  • Fig. 3 Morris water maze test to detect the effect of scorpion venom heat-resistant synthetic peptide on spatial learning and memory ability in senile dementia (AD) mice;
  • Figure 4 venom heat-resistant synthetic peptide scavenging 6-OHDA PD cell model ROS production
  • Figure 5 venom-resistant synthetic peptide inhibits epileptic discharge of hippocampal slices
  • Figure 7 shows the results of MS identification of venom-resistant synthetic peptide
  • Figure 10 Inhibition of sodium currents in primary cultured neurons by venom-resistant synthetic peptides
  • Figure 11 venom heat-resistant synthetic peptide has protective effect on NMDA-induced SH-SY5Y cell injury
  • Figure 12 venom heat-resistant synthetic peptide promotes type II astrocyte reprogramming (reverse differentiation) endogenous stem cells in the brain;
  • Figure 13 venom heat-resistant synthetic peptide promotes dedifferentiation of OPC cells into endogenous stem cells
  • Figure 14 The venom heat-resistant synthetic peptide up-regulates Nestin protein expression.
  • the centrifugal ultrafiltration tube with molecular weight cutoff of 50kDa and 30kDa filter is used to centrifuge the ultrafiltration of scorpion toxic heat-resistant component extract. Liquid, to obtain a venom heat-resistant peptide extract.
  • the muscarinic peptide extract was subjected to coarse fractionation using Superdex Peptide 10/300GL molecular sieve column (Optimum Separation range (peptides) M, 100-7000 Da) (see Figure 1), and the sample was subdivided by HPLC (see Figure 2).
  • the chromatographic conditions were: column: Zorbax SB-C18 4.6*250 5um (Agilent.USA), mobile phase: liquid A: acetonitrile/water: 2:98 (containing 0.1% trifluoroacetic acid); liquid B: acetonitrile/water : 98:2 (containing 0.08% trifluoroacetic acid); 0–40% B approximately 3-6 column volumes, 40–100% B 0.5-1 column volumes, 100% B 1-3 column volumes (CV) ; Flow rate: 0.8 ml/min; detection wavelength: 280 nm / 258 nm / 214 nm for subdivision. A purified extract of the venom heat-resistant polypeptide is obtained.
  • the analytical column is a C18 reversed phase column (75 ⁇ m x 15cm C18-3 ⁇ m) ChromXPEksigent), the gradient used in the experiment was increased from 5% to 80% in 70 min.
  • the scanning time of the _MS single image is 250ms. Under the IDA cycle, a maximum of 35 secondary spectra with 2+ to 8+ charge and a single second count greater than 100 are collected. The cumulative time of each secondary spectrum is 80ms. Each cycle time was fixed at 2.5 seconds and the collision cell energy setting was applied to all precursor ion collision induced dissociation (CID) with a dynamic exclusion setting of 11 seconds.
  • CID precursor ion collision induced dissociation
  • the scorpion venous heat-resistant peptide (SVHRP) extract was freeze-dried by LaGM composite and repeated rapid magnetic separation.
  • the lyophilized polypeptide sample was redissolved in Nano-RPLC Buffer A, the online Nano-RPLC liquid chromatography in EksigentnanoLC-Ultra TM 2D system (AB SCIEX), maintaining a flow rate of flushing desalted 10min.
  • Mass spectrometry was performed using a TripleTOF 5600 system (AB SCIEX) in combination with a nanoliter spray III ion source (AB SCIEX, USA), the original wiff map file acquired by mass spectrometry, and data processing using Protein Pilot Software v.4.5 (AB SCIEX, USA) software. And search analysis, the amino acid sequence of the scorpion venom polypeptide was detected.
  • Solution B containing 0.1% trifluoroacetic acid water
  • the water maze detects behavioral changes, and the navigational experiment lasts for 5 days, training 4 times a day, recording the time when the mouse finds the platform (the platform is placed in the first quadrant), that is, escaping the incubation period, and letting the mouse stay on the platform for 20s; if small No platform was found in the mouse for 60s, and the escape latency was recorded as 60s.
  • the space search experiment removed the platform after 4 trainings on the 5th day of the navigation test, and then placed the mice in the pool at the third quadrant water inlet to start the test.
  • the swimming distance and swimming time percentage of the mouse in the target quadrant (1st quadrant) were calculated for a total duration of 60 s. The number of mice crossing the hidden platform was measured.
  • the hidden platform of water maze obtained the escape latency of mice in the AD model group on the experimental test day (Day 5) (5d, 12 ⁇ 2) Time) was significantly shorter than the escape latency of the AD model group (day 5, 22 ⁇ 2 times) **p ⁇ 0.01, and the swimming distance was reduced from 500 cm on the 5th day of the model group to 300 cm in the g administration group.
  • the 5th increase in the model group was 8 times in the drug-administered group.
  • Synchronized transgenic nematode CL2355 and its control strain CL2122 were given different concentrations of SVHRSP (2, 20, 40 ⁇ g/ml) and the same amount of three distilled water, and were administered from the eggs, and the nematodes were transferred to new drugs every day.
  • the NGM dish After all the nematodes were cultured at 16 ° C for 36 hours, the incubator temperature was raised to 23 ° C, and further culture was carried out for 36 hours. The above nematodes were collected and washed 3 times with M9 buffer.
  • the petri dish used for the chemotaxis experiment was a 100 mm dish.
  • the solid medium was mixed with 1.9% agar, 1 mM CaCl 2 , 1 mM MgSO 4 , and 25 mM phosphate buffer, pH 6.0, and then poured into a dish and cooled. At the bottom of the Petri dish, two vertical lines are drawn with a marker on the center of the dish, and the whole dish is divided into four quadrants. About 60 nematodes in each group were placed in the center of the chemotaxis, and 1 ⁇ l of benzaldehyde (concentrated at 0.1%, diluted with absolute ethanol) and 1 ⁇ 0.25 M sodium azide were dropped to the center of the 1 and 3 quadrants.
  • Benzaldehyde is a chemical substance with a high concentration of odor. The nematode is attracted to the odor of benzaldehyde and moves to its area. When exposed to sodium azide, it will be paralyzed in situ.
  • the chemotaxis index is calculated by counting the number of nematodes in the 1 and 3 quadrants (the quadrant of the attraction) - the number of nematodes in the 2 and 4 quadrants divided by the total number of nematodes.
  • the scorpion venom-resistant synthetic peptide can increase the CI of CL2355 in a dose-dependent manner.
  • the protective effect of 40 ⁇ g/ml SVHRSP on neurons is most obvious, and the chemotaxis behavior damage caused by A ⁇ expression is almost completely reversed.
  • p ⁇ 0.01 compared with the two indicates that SVHRSP can protect neurons, counteract the toxic effects caused by A ⁇ , and improve the expression of A ⁇ caused by neurons.
  • the chemotaxis behavior is abnormal, see Table 2.
  • Fluorescence probe DCFH-DA was used to detect the effect of SVHRSP on 6-OHDA-induced SH-SY5Y oxidative stress.
  • the experiment was divided into normal control group, ROSup positive control group, 6-OHDA model group and different concentrations (2 ⁇ g/ml, 5 ⁇ g). /ml, 10 ⁇ g/ml, 20 ⁇ g/ml) SVHRSP drug group.
  • the SH-SY5H cells in the exponential growth phase were taken, the culture solution was discarded, washed once with PBS after sterilization, and then added with 0.25% trypsin for digestion and blown into individual cells.
  • the cells were counted by a cell counting plate, seeded in a 96-well plate at 1.5 ⁇ 10 4 /ml, and the cells were allowed to settle at the bottom of the plate, placed in a 5% CO 2 , and cultured in a 37 ° C incubator for 24 hours. After adding the concentration of 20 ⁇ g/ml SVHRSP drug group for 1 h, 6-OHDA was added, and 5% CO 2 was further added, and cultured in a 37 ° C incubator for 24 hours.
  • the ROSup was diluted with the medium at a dilution ratio of 1:500, 100 ⁇ l was added to each well, cultured for 27 minutes, and cultured for 27 minutes, diluted with DCFH-DA in a serum-free medium at a dilution ratio of 1:2000, and discarded at a final concentration of 5 ⁇ mol/L.
  • the medium was added with 50 ⁇ l of diluted DCFH-DA solution per well, gently shaken, placed in 5% CO 2 , and cultured in a 37 ° C incubator for 20 min. The supernatant was discarded, washed 3 times with PBS, and detected by fluorescence detection at 488 nm excitation wavelength and 525 nm emission wavelength in a fluorescent plate reader. Measure the OD value and compare.
  • SVHRSP for 6-OHDA-induced SH-SY5Y intracellular production
  • 6-OHDA 100 ⁇ M was given for 24h.
  • the expression of ROS in the cells was detected by fluorescence microplate reader at excitation wavelength of 488nm and emission wavelength of 525nm.
  • 6-OHDA can significantly increase intracellular ROS and is statistically significant (control 1 ⁇ 0.2vs6-OHDA 3.4 ⁇ 0.4p ⁇ 0.01), but when added to SVHRSP, it can clear intracellular ROS, and also Statistically significant (control 3.4 ⁇ 0.4 vs 1.5 ⁇ 0.4 p ⁇ 0.05).
  • ip pilocarpine epilepsy model + NS
  • lithium-pilocarpine epilepsy model + sodium valproate Observe and record the level of epilepsy, according to Racine , s classification as the evaluation criteria:
  • SVHRSP has a significant control effect on seizures in chronic model of rats with lithium-pilocarpine epilepsy, which can significantly reduce the number of seizures;
  • SVHRSP is stronger than the positive control valproic acid in reducing the level of seizure and reducing the number of seizures in the chronic model of rats with lithium-pilocarpine epilepsy.
  • the ml ml-coated plates were placed in an incubator at 37 ° C and 10% CO 2 for cultivation.
  • all the planting liquids were replaced with the culture solution, and on the 4th day, cytarabine (3 ⁇ g/ml) was added to the culture solution to inhibit excessive proliferation of non-neuronal cells.
  • 50% of the fresh culture medium was replaced every 3 days, and the hippocampal neurons cultured in vitro were cultured until 9-12 days, and the neurons were cultured for 10 days.
  • the drugs used were all dosed through a BPS-8 perfusion device.
  • the diameter of each tube was 0.2 mm, and the tube was about 100 ⁇ m from the recorded cells.
  • the sodium channel current was recorded by patch clamp in the primary cultured neuron system, and the currents were compared before and after the same cells were added with scorpion refractory synthetic peptide (SVHRSP).
  • SVHRSP scorpion refractory synthetic peptide
  • SH-SY5Y cells are derived from human neuroblastoma cell lines.
  • NMDA model NMDA 20 mM + Gly 10 ⁇ M.
  • Experimental procedure Before the experiment, grouped according to the purpose of the experiment. SH-SY5Y was seeded in 96-well plates (0.8 ⁇ 10 4 /well). After 24 h, SVHRSP and peptide were added for pre-incubation for 24 h. Then, according to the group, NMDA 20 mM+Gly10 ⁇ M was added for 24 h, and the cell viability was detected by MTT kit. See Figure 11. The results showed that SVHRSP had protective effects on NMDA-induced SH-SY5Y cell injury.
  • Type II astrocytes form pluripotent neural stem cells with proliferation and differentiation ability by reverse differentiation
  • type II astrocyte inducing solution (20% DMEM). After 4-5 days, continue to culture. Relatively purified type II astrocytes can be obtained. After 5 days, type II astrocytes were digested and planted in ultra-low adhesion 6-well plates. After changing the culture medium for stem cell culture for 12 days, stem cell spheres were observed. After 12 days, the cells in the ultra-low adhesion 6-well plate were aspirated and placed in a 24-well plate covered with polylysine-treated cells. After 24 hours, the slides were taken out and fixed with 4% paraformaldehyde.
  • SVHRSP scorpion venom thermosuppressant peptide
  • MTD 1Maximal tolerancedose
  • MLD 2 Minimal lethal dose
  • the maximum non-toxic reaction dose (the highest non-toxic): the No observed adverse effect level (NOAEL), that is, the highest dose of no damage found in the animal within a certain period of time;
  • NOAEL No observed adverse effect level
  • the minimum dose of toxic reaction in an animal the dose that just caused the toxic reaction is the lowest dose at which the animal develops a toxic reaction.
  • Test animals Kunming mice, female and male (half healthy adult, weight 19-20g;
  • Test substance SVHRSP: a.
  • Source Henan Yichang Xinxin Yangshuo Plant (2012-05-28) purchased BmK scorpion venom extract;
  • the optimal administration capacity of the species of animals was selected according to the route of administration (different doses, but the administration volume was the same), and the ip administration route of this experiment was 0.2 ml/20 g BW.
  • MTD maximum tolerance dose
  • the range between the dose and the minimum lethal dose is a range of approximate lethal doses; if the animal dies at the dose, the dose is The range between the maximum tolerated doses is approximately the lethal dose range.
  • the results showed that the medicinal safety and pharmacodynamic activity ratio of the scorpion venom heat-resistant synthetic peptide (SVHRSP) of the present invention was greater than 2000/0.05 40,000 times.
  • the maximum non-toxic reaction dose (highest non-toxic) has not been reached: the maximum dose without toxicity.

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ES16905684T ES2945407T3 (es) 2016-08-08 2016-12-26 Péptido sintético termorresistente del veneno de escorpión y aplicaciones del mismo
EP16905684.3A EP3505530B1 (en) 2016-08-08 2016-12-26 Scorpion venom heat-resistant synthesized peptide and uses thereof
CA2984569A CA2984569C (en) 2016-08-08 2016-12-26 A scorpion venom heat-resistant synthetic peptide and applications thereof
JP2017554073A JP6483858B2 (ja) 2016-08-08 2016-12-26 サソリ毒耐熱合成ペプチド
US15/800,802 US10442837B2 (en) 2016-08-08 2017-11-01 Scorpion venom heat-resistant synthetic peptide and applications thereof
US16/573,495 US10870680B2 (en) 2016-08-08 2019-09-17 Methods of reducing neuronal injury or toxicity in epilepsy, Alzheimer's disease or Parkinson's disease using a scorpion venom heat-resistant synthetic peptide (SVHRSP)

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CN106220713B (zh) * 2016-08-08 2017-09-01 大连医科大学 一种蝎毒耐热合成肽及其用途
CN109232718B (zh) * 2018-11-09 2020-04-14 泰安市启航生物科技有限公司 一种合成肽sp2及其应用
CN111647051B (zh) * 2019-03-04 2022-02-01 中国医科大学 一种基于钠通道结构的短肽及其应用
CN111896651B (zh) * 2020-07-30 2021-04-23 山东大学 一种白眉蝮蛇蛇毒类凝血酶特征多肽及其应用
CN114028537B (zh) * 2021-11-27 2024-03-29 上海万锦医药科技有限公司 一种含有svhrsp蝎毒肽的药物组合物及其制备方法
CN117224655B (zh) * 2023-09-14 2024-05-14 大连医科大学 蝎毒耐热合成肽(svhrsp)在制备治疗肠道炎症的药物中的应用

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