WO2019095639A1 - 一类具有镇痛活性的多肽及其应用 - Google Patents

一类具有镇痛活性的多肽及其应用 Download PDF

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WO2019095639A1
WO2019095639A1 PCT/CN2018/085275 CN2018085275W WO2019095639A1 WO 2019095639 A1 WO2019095639 A1 WO 2019095639A1 CN 2018085275 W CN2018085275 W CN 2018085275W WO 2019095639 A1 WO2019095639 A1 WO 2019095639A1
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polypeptide
pain
ser
group
cys
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PCT/CN2018/085275
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English (en)
French (fr)
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徐寒梅
刘晨
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南京安吉生物科技有限公司
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Priority to EP18879579.3A priority Critical patent/EP3702362A4/en
Priority to US16/762,948 priority patent/US11192921B2/en
Priority to AU2018367163A priority patent/AU2018367163B2/en
Priority to JP2020544084A priority patent/JP7026245B2/ja
Publication of WO2019095639A1 publication Critical patent/WO2019095639A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biopharmaceuticals, and more particularly to a class of polypeptides having analgesic activity and uses thereof.
  • the process of pain production is complicated, involving factors such as pain receptors, conduction nerves, and pain centers.
  • the main causes of pain are as follows: (1) a series of mechanical stimuli existing outside the body and local edema of the tissue in the event of an inflammatory reaction, which will cause pain in the nerve endings; (2) stimuli of physical and chemical properties will pass through the feeling Neurons cause excitement and pain in the pain center; (3) Pathological damage of peripheral nerves causes stimulation of sympathetic nerve fibers, which ultimately stimulates excessive excitation of the cerebral cortex and causes neuropathic pain; (4) Damaged cells after external stimulation by the body Or the painful substances released by nociceptors can stimulate local receptors, causing pain in the central nervous system.
  • analgesics can be broadly classified into non-steroidal (aspirin), opioid receptors (conotoxin), and other analgesics (anticonvulsants). They have a good effect on chronic pain, neuropathic pain and inflammation, but limitations and side effects have always been a bottleneck problem that is not well improved. For example, aspirin has a great damage to the human circulatory system, especially the digestive and hematopoietic system; conotoxin has a series of adverse reactions such as severe addiction, tolerance, respiratory distress, etc. Allergic and side effects of anticonvulsant drugs have been Not getting a good improvement. Finding new targets for analgesics or creating optimized new structural compounds has always been an urgent problem for pharmacy workers.
  • ion channel polypeptides Compared with traditional analgesics, ion channel polypeptides have brought a new dawn to researchers due to their wide range of sources, small side effects, long half-life and obvious efficacy.
  • the ion channels of biomembrane are a kind of membrane proteins that penetrate the lipid bilayer of the cell membrane and have hydrophilic channels in the center. They are the basis of bioelectricity production of nerves, skeletal muscles and cardiomyocytes. This channel is closely related to various life processes such as receptor potential generation, nerve excitation conduction and central nervous system regulation, and is highly selective.
  • a specific biofilm ion channel only one or several specific ions are allowed to hypertonic, and can be classified into a sodium ion channel, a potassium ion channel, a calcium ion channel, and an acid sensitive ion channel.
  • Ca 2+ ions and K + ions are balanced inside and outside the cell membrane.
  • the activity of Na + ion channels determines the formation and transmission of pain sensation.
  • Many analgesics block the pain-sensing conduction pathway by inhibiting voltage-gated sodium channels.
  • conotoxin polypeptides The study of conotoxin polypeptides has been a hot topic in the field.
  • the Chinese patent application number is 201480082283.7, and the patent application filed on May 31, 2017 discloses a conotoxin polypeptide ⁇ -CPTx-bt105. Preparation methods and applications.
  • the conotoxin polypeptide consists of 16 amino acids with a molecular weight of 1626.62 Daltons and the entire sequence is GICCVDDTCTTHSGCL.
  • the conotoxin polypeptide has an inhibitory effect on potassium ion channel current and analgesia.
  • the method for extracting the conotoxin polypeptide from the snail is relatively complicated, and the conotoxin polypeptide has certain side effects, which may have serious dependence on the physiological and psychological.
  • the present invention provides a kind of polypeptide having analgesic activity and application thereof, and has significant improvement in structural stability and analgesic activity compared with conotoxin, in body damage, etc. Both side effects were weak and conotoxin.
  • a class of analgesic polypeptides having the amino acid sequence X a -X b -Cys-Ser-Thr-Pro-Pro-X c -X d -Val-Leu-Tyr-X e or a pharmaceutically acceptable salt.
  • the X a is Gly or a deletion
  • the X b is one of Ser or Lys
  • the X c is one of d-Cys or Ser or Asp
  • the X is d is Ala or a deletion
  • the X e is Cys or Ser.
  • amino acid sequence of the polypeptide is:
  • Peptide ⁇ Gly-Ser-Cys-Ser-Thr-Pro-Pro-d ⁇ Cys ⁇ -Val-Leu-Tyr-Ser;
  • Peptide ⁇ Gly-Ser-Cys-Ser-Thr-Pro-Pro-Ser-Ala-Val-Leu-Tyr-Cys; or
  • Peptide ⁇ Lys-Cys-Ser-Thr-Pro-Pro-Asp-Ala-Val-Leu-Tyr-Cys.
  • polypeptide sequence is a polypeptide having an analgesic effect after deletion, substitution or addition of one or more amino acids on the basis of the above sequence, or a pharmaceutically acceptable salt of the polypeptide.
  • a product for preventing and/or treating pain wherein the active ingredient is the above polypeptide.
  • the above products may specifically be drugs.
  • one or more pharmaceutically acceptable excipients may be added to the above-mentioned drugs, including excipients, excipients, fillers, binders, humectants, absorption promotion conventional in the pharmaceutical field. Agents, surfactants, lubricants and stabilizers.
  • the medicament of the present invention can be prepared into various forms such as an injection solution, a dry powder injection, a tablet or a granule.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.
  • the pain includes physicochemical irritant, pathological or neuropathic pain.
  • anti-inflammatory analgesics and opioid analgesics are the main clinical factors.
  • Anti-inflammatory analgesics are mainly used for chronic dull pain such as headache, toothache, muscle and joint pain, dysmenorrhea, etc. These drugs are not easily addictive and are widely used in clinical practice.
  • Opioid analgesics are drugs that eliminate or reduce pain and alter the emotional response to pain, but have side effects such as constipation, lethargy, dullness, nausea, vomiting, and breathing. Inhibition and dependence, etc., have serious dependence on physiology and psychology, so it is severely limited in its clinical application.
  • narcotic analgesics many drugs that act on the central nervous system are used to control pain, such as antidepressants, anticonvulsants, neuroleptics, etc., to a certain extent, have direct analgesic effects or potential towns. Pain activity, but the side effects become more intense as the dose increases. For example, aspirin has a great damage to the human circulatory system, especially the digestive and hematopoietic system; conotoxin has a series of adverse reactions such as severe addiction, tolerance, respiratory distress, etc. Allergic and side effects of anticonvulsant drugs have been Not getting a good improvement.
  • the invention adopts computer three-dimensional simulation technology, based on the analysis of a large number of traditional structures and pharmacological experiments, the self-designed polypeptide structure has stronger analgesic activity and less toxic and side effects.
  • the invention independently designs an analgesic polypeptide having an entirely new structure acting on an ion channel by analyzing the structure of various analgesic polypeptides. It has been proved by various experimental models that it has the same analgesic effect as toxin and has low adverse reactions such as tolerance, which is very promising.
  • the polypeptide of the present invention has a simple structure, is easy to synthesize, isolate and purify, and effectively retains and enhances the analgesic activity of conotoxin;
  • polypeptide of the present invention has weak side effects and toxic side effects
  • the analgesic effect of the polypeptide involved in the present invention is good in each experimental model, and the specific effect is to significantly increase the pain threshold of the mouse, prolong the heat tolerance time of the mouse, and the adverse reactions such as spontaneous movement and excitability of the mouse are lower than that. Normal value.
  • the present invention relates to an analgesic activity polypeptide having a parent core structure having an amino acid sequence X a -X b -Cys-Ser-Thr-Pro-Pro-X c -X d -Val-Leu-Tyr-X e Or the pharmaceutically acceptable salt of the polypeptide, wherein X a may be Gly or a deletion, X b may be one of Ser or Lys; X c may be one of d-Cys or Ser or Asp, X d may be Ala or a deletion; X e may be Cys or Ser. Specifically, the amino acid sequence of such a polypeptide is
  • Peptide ⁇ Gly-Ser-Cys-Ser-Thr-Pro-Pro-d ⁇ Cys ⁇ -Val-Leu-Tyr-Ser;
  • Peptide ⁇ Gly-Ser-Cys-Ser-Thr-Pro-Pro-Ser-Ala-Val-Leu-Tyr-Cys; or
  • Peptide ⁇ Lys-Cys-Ser-Thr-Pro-Pro-Asp-Ala-Val-Leu-Tyr-Cys.
  • a polypeptide having an analgesic effect after one or more amino acids on the polypeptide sequence is deleted, substituted or added, and a pair of disulfide bonds are formed between the two cysteines in the sequence, and the polypeptide is physicochemically stimulated.
  • sexual, pathological, and neuropathic pain have a good inhibitory effect.
  • polypeptide ⁇ , polypeptide ⁇ and polypeptide ⁇ were mainly studied for their activity of inhibiting pain.
  • the peptide was commissioned by Nanjing Jinsrui Biotechnology Co., Ltd. and the purity was over 95%.
  • Kunming mice female, weighing 20 ⁇ 2g. The room temperature is maintained at 22 to 25 °C. The mice were placed on a constant temperature metal plate at a temperature of 55 ° C, and the temperature was changed (within ⁇ 0.5 ° C). The mouse was exposed to the metal plate and the time to the hind paw or jump reaction was the pain threshold. .
  • mice were housed in the experimental cage cage with ventilation system; the feeding temperature was about 23 °C; normal day and night replacement; all experimental animals were free to drink water; the purchased experimental animals were kept in the laboratory for 3 to 5 days to adapt to the experimental environment; all animal experiments All were carried out from 8:00 to 18:00; experimental animals were kept in single cages after surgery.
  • Experimental reagent conotoxin, 0.9% NaCl solution, polypeptide I, polypeptide II, polypeptide ⁇ .
  • the conotoxin polypeptide (typed is BuIA) structure used in the present invention is Gly-Cys-Cys-Ser-Thr-Pro-Pro-Cys-Ala-Val-Leu-Tyr-Cys, wherein C1-C3 is looped. , C2-C4 is looped.
  • mice The basic pain threshold was measured before the experiment, and the average value was calculated twice, with an interval of 5 minutes. Mice with a response latency of less than 5 s or greater than 30 s were excluded. To prevent burns on the feet of mice, set 60s as the cut-off time. The screened mice were divided into a negative control group, a positive control group and a conotoxin group according to a random number table, with 10 rats in each group.
  • the saline group was 0.9% NaCl solution as the negative control group; the conotoxin group was 1 ⁇ g/kg as the positive control group; the polypeptide group was administered as the experimental group, and the dosage was 2 nmol/kg.
  • Route of administration intraperitoneal injection.
  • the pain threshold of the mice was recorded again after 15, 30, 45, 60, 75, and 90 min after administration, and the experiment was repeated 3 times independently.
  • Representation and statistical T test compared with saline negative group, *P ⁇ 0.05 is a significant difference, **P ⁇ 0.01 is a very significant difference; compared with conotoxin positive group, ⁇ P ⁇ 0.05 is significant
  • the difference, ⁇ P ⁇ 0.01 is a very significant difference.
  • the tolerance time of the hot plates in the mice before and after the administration was compared in each group, and the pain threshold of each group was compared between the groups. The maximum possible analgesic rate after administration was calculated, and the analgesic effect of the drug was evaluated.
  • the peptide group can exert analgesic effect in the hot plate analgesic animal model.
  • the results are shown in Table 1: Compared with the saline negative group, the peptides I, II and III can effectively increase the thermal threshold of mice. The increase in the pain threshold of peptide II was significantly higher than that in the saline group and the conotoxin group. The experimental results were statistically significant.
  • the PAMP was calculated to be about 47.57%, and the analgesic effect was not significantly different from that of the conotoxin group, and it had a good analgesic effect.
  • Kunming mice male and female, weighing 20 ⁇ 2g. The room temperature is maintained at 22-25 °C. A small spotlight is used to generate a beam of intensity that is focused by the lens to illuminate the tail of the mouse.
  • the rat tail was placed on the tail groove of the appendix pain tester, so that the light spot fell on the lower third of the tail of the rat (marked with a pen before the experiment, so that each test point is in the same position) Top)
  • the latencies (TFL, Tail Flick Latency) from the onset of radiant heat to the appearance of the appendix reaction are indicators of pain response.
  • mice with a response latency of less than 1 s or greater than 3 s were excluded.
  • 10 s was set as the cut-off time.
  • the mice with the basic pain threshold met the criteria were divided into the negative control group, the positive control group and the conotoxin group according to the random number table, with 10 rats in each group.
  • the saline group was used as the negative control group; the conotoxin group was given 1 ⁇ g/kg as the positive control group; the polypeptide group was administered at 2 nmol/kg.
  • the administration route is administered to the lateral ventricle.
  • the pain thresholds at 5, 15, 30, 45, 60, 90, and 120 minutes after administration were recorded separately.
  • the changes in the tolerance time of the mice to heat radiation before and after the administration were compared in the group, and the pain threshold values of the mice in each group were compared between the groups.
  • the percentage increase in pain threshold after administration was calculated, and the analgesic effect of the drug was evaluated.
  • the peptide group can exert analgesic effect in the mouse thermal radiation appendix model.
  • the results are shown in Table 2.
  • the polypeptide group can effectively increase the thermal threshold of mice.
  • the peptide I was significantly different from the conotoxin group at 2nmol/kg ( ⁇ P ⁇ 0.01).
  • the peptide III was significantly different from the normal saline group at the dose of 2nmol/kg. **P ⁇ 0.01).
  • the experimental results were statistically significant.
  • PAMP was calculated to be about 38.32%, and the analgesic effect was not significantly different from that of the conotoxin group, and it had a good analgesic effect.
  • the computer real-time monitoring and analysis system SMART VIDEO-TRACKING (US, SMARTv 3.0.02) was used.
  • the instrument is mainly composed of four 40cm ⁇ 40cm ⁇ 40cm experimental open boxes.
  • the camera is connected with the recording system in the center of the top of the box.
  • the mouse real-time image is analyzed systematically to obtain the behavior data of the mouse in the open field.
  • mice were subjected to body mass statistical test, and the unqualified mice were excluded and classified into a negative control group, a positive control group, a polypeptide I, a polypeptide II, and a polypeptide III group according to a random number table.
  • the saline group was injected as a negative control group with 0.9% NaCl solution; the conotoxin group was given 1 ⁇ g/kg as a positive control group; the polypeptide group was administered at a dose of 2 nmol/kg.
  • the mode of administration is intraperitoneal injection.
  • the market equipment is clean and tasteless. Record the number, date and status of the mouse in the operating software.
  • the animal experiment must be sent to the temporary cage for the behavioral laboratory in advance, adapt to the environment for about 3 hours, reduce the animal's tension; remove the mouse from the cage (small The mouse is turned back to the experimenter.
  • the device cover is quickly and gently closed, and the video recording system is opened to record the activity of the mouse in the market. The total time is 5min.
  • the inner wall and the bottom surface of the square box were cleaned with 75% ethanol in time to avoid the information left over from the last animal (such as the animal's large, urinal, smell) affecting the next test result.
  • mice were again tested for relevant indicators according to the above steps at intervals of 1 h and 24 h after observation.
  • the mouse center residence time, exercise time, number of wall climbs, and number of crossing grids were counted as the final data using Origin 8.0 software.
  • the experimental devices were all made by the laboratory; beakers, constant temperature water baths, stopwatches, counters, O'Neill ARC Core HD D1080P HD camera.
  • mice Twenty-five mice were divided into a negative control group, a positive control group, a polypeptide I, a polypeptide II, and a polypeptide III group according to a random number table before the experiment.
  • the saline group was injected as a negative control group with 0.9% NaCl solution; the conotoxin group was given 1 ⁇ g/kg as a positive control group; the polypeptide group was administered at a dose of 2 nmol/kg.
  • the mode of administration is intraperitoneal injection.
  • the operator placed the mouse in a round beaker with warm water, 10 cm in diameter, 10 cm in water depth, and 23 to 25 ° C in water temperature.
  • the state of motion of the animal within 5 minutes was recorded by a camera system.
  • the state of motion means that the animal is actively struggling and the body is in a floating and twisting state.
  • the mice were again tested for movement time in water at intervals of 1 h and 24 h after observation.
  • the improvement of exercise endurance is the most direct manifestation of the anti-fatigue ability.
  • the length of swimming can reflect the degree of animal fatigue. The lower the exercise time, the greater the side effects.
  • the experimental devices are all made by the laboratory; including the hanging tail box (20cm ⁇ 20cm ⁇ 30cm), side standing, the perimeter wall and the bottom are black, stopwatch, counter, O'Neill ARC Core HD1080P HD camera.
  • mice were divided into a negative control group, a positive control group, and peptides I, II, and III according to a random number table before the experiment.
  • the saline group was injected as a negative control group with 0.9% NaCl solution; the conotoxin group was given 1 ⁇ g/kg as a positive control group; the polypeptide group was administered at a dose of 2 nmol/kg.
  • the mode of administration is intraperitoneal injection.
  • the operator suspends 1/3 of the tail tip of the mouse in the hanging tail box with non-adhesive glue, so that the head is facing the lens about 10 cm away from the bottom of the box, and the camera system is used to record the immobile period of the animal within 6 min and within 4 min.
  • the state of the immobile state means that the animal gives up the active struggle, and the body is in a state of being overhanging and not twisting.
  • Kunming mice male and female, weighing 20 ⁇ 2g. The room temperature is maintained at 23 to 24 °C. The mice were grouped according to a random number table, and a negative control group, a positive control group, and a polypeptide group were set, respectively, with 10 rats in each group. The 0.9% NaCl solution group was used as the negative control group; the conotoxin group was 1 ⁇ g/kg as the positive control group; the analgesic polypeptide group I, II and III were administered at a dose of 0.2 nmol/head.
  • mice After 5 minutes of administration in each group of rats, 20 ⁇ L of 5% formalin (5% 5%) solution was injected subcutaneously into the right hind paw of the mice using a 1 mL syringe. Immediately after the injection, the mice were placed in a large glass beaker. The pain response within 1 h was based on the right foot time as an indicator of behavioral response. A mirror with an angle of about 30° to the platform was placed behind the beaker. At the same time, the cumulative time of the sputum, bite, and shaking of the injected foot was recorded every 5 s from the front and the mirror.
  • the continuous observation time was 60 min, which was phase I ( 0-10 min) and phase II (10-60 min), using a formalin-induced mouse inflammatory pain model to examine the analgesic effect of the test drug after acute administration.
  • the inhibition rates of the positive control group and the conotoxin to the phase I and phase II pain responses were calculated.
  • mice Male BALB/c mice have a body weight of 25-30 g.
  • the latent period of heat radiation stimulation was significantly shortened by 8 seconds, and on the 14th day after inoculation, it was significantly increased by 15 seconds, especially in the peptide group I.
  • mice On the 10th day after inoculation, part of the mice were observed to have foot drop and the cumulative duration of the right hind foot increased slightly. On the 14th day, the left foot behavior was observed in the peptides I and II.
  • mice 7 weeks old, weighing 20-25 g.
  • Filament mechanical pain tester fine fibrils (equivalent to 0.0688g strength); coarse fibrils (equivalent to 1.4798g strength); paclitaxel; peptides I, II, III; cloxaprofen; physiological saline.
  • mice were used for testing after 1 week in an animal room at a temperature of (22 ⁇ 1) ° C and a relative humidity of 55% ⁇ 10%. Thirty-six mice were divided into a model group (only paclitaxel), a blank control group (no paclitaxel), a cloxaprofen group, and a polypeptide group I, II, and III were administered for 3 days before paclitaxel administration for 7 days. 6 mice per group
  • mice were injected intraperitoneally with paclitaxel 10 mg/kg.
  • mice were placed in a metal mesh divided into 3 compartments for 15 minutes to measure the mechanical stimulation of the hind limbs and ankles.
  • the abnormal fibrils were first evaluated for fine pain and then coarse. Filaments assess the extent of hyperalgesia.
  • the response was stimulated 5 times for a total of 10 points. Judging criteria (0 points: rapid lifting action; 2 points: severe squat or lame movement.
  • the measurement time was before paclitaxel administration and 24, 48, 72, 96 hours after administration).
  • Measurement data were expressed as M ⁇ SD, and differences between groups were analyzed by t test.
  • mice When the mice were intraperitoneally administered with paclitaxel 10 mg/kg, allergic pain and hyperalgesia were peaked at 24 hours after administration of paclitaxel, and then gradually decreased, and returned to the pre-dose level after 96 hours.
  • the pain-related fractional allodynia at 24 hours before and after administration was 0.5 ⁇ 0.2, 1.8 ⁇ 0.6, and the hyperalgesia was 2.3 ⁇ 0.5, 5.5 ⁇ 0.6.
  • the pain-related scores of peptides I, II, and III were significantly lower than those of the model group (P ⁇ 0.05).
  • the pain-related scores of the peptide group showed a downward trend compared with the model group, and there was no significant difference.
  • Regarding the effects of hyperalgesia there were no significant differences in pain-related scores between the cloxaprofen, peptides I, II, and III groups compared with the single group.
  • Adaptation training was given before the experiment.
  • the model was induced by trigeminal neuralgia in a quiet and room temperature condition about 2 weeks after surgery.
  • the model was established when any one or more of the following reactions occurred in the rats: 1 After the stimulation, it showed a rapid retreat. Turning and other dodging actions, the rats contracted the body to avoid the irritant and moved closer to the cage wall, or hide the head and face under the body to protect the face from being touched by the irritant; 2 licking the face, showing at least three consecutive times The action of the facial stimulation area; 3 rats quickly grasp the stimuli and make an attack action.
  • the animals were injected with 50ug/kg, 100ug/kg, 300ug/kg, and 0.1mL/kg of normal saline in each abdominal cavity.
  • the pain threshold was measured every 1 hour for 5 hours. , record pain threshold changes.
  • the experimental data were expressed by M ⁇ SD.
  • the non-parametric Kruskal-Wallis test was used for one-way ANOVA and Mann ⁇ Whitney U test.
  • the area under the aging curve was calculated by the trapezoidal method, and the area under the aging curve between the two groups was analyzed by t test. P ⁇ 0.05 was considered statistically significant.
  • the highest pain threshold on the surgical side was (7.4 ⁇ 0.9) g
  • the contralateral side was (3.2 ⁇ 1.3) g
  • the effect on the surgical side lasted about 8 hours.
  • the pain threshold was not significantly increased in the 300 ug/kg group of subcutaneously injected peptides I, II, and III.

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Abstract

公开了一类具有镇痛活性的多肽及其应用，所述多肽具有氨基酸序列X a-X b-Cys-Ser-Thr-Pro-Pro-X c-X d-Val-Leu-Tyr-X e，或其药学上可以接受的盐，所述的X a为Gly或缺失，所述的X b为Ser或Lys中的一种；所述的X c为d-Cys或Ser或Asp中的一种，所述的X d为Ala或缺失；所述的X e为Cys或Ser。序列中两个半胱氨酸之间形成一对二硫键。

Description

一类具有镇痛活性的多肽及其应用 技术领域
本发明属于生物药物领域，更具体地说，涉及一类具有镇痛活性的多肽及其应用。
背景技术
疼痛的产生过程比较复杂，涉及到疼痛感受器、传导神经、疼痛中枢等因素。引发疼痛的原因主要有以下几点：(1)外界存在的一系列机械性刺激以及发生炎症反应时组织的局部水肿，会压迫神经末梢引起疼痛；(2)物理及化学性质的刺激会通过感觉神经元使痛觉中枢产生兴奋，引起疼痛；(3)周围神经的病理性损伤使交感神经纤维受到刺激，最终刺激大脑皮质过度兴奋，引起神经病理性疼痛；(4)机体受到外界刺激后由损伤细胞或者伤害性感受器释放的致痛物质会刺激局部感受器，使中枢神经系统兴奋引起疼痛。
早期的镇痛药大致可以分为非甾体类(阿司匹林)，阿片受体类(芋螺毒素)以及其他辅助性镇痛药(抗惊厥)。它们对于慢性疼痛、神经病理性疼痛以及炎症都具有较好的作用效果，但局限性和副反应一直是一个瓶颈问题得不到很好的改善。例如阿司匹林对人体循环系统损伤较大，尤其是消化与造血系统；芋螺毒素具有严重的成瘾性、耐受性、呼吸窘迫等一系列不良反应；抗惊厥药物的过敏性及副反应也一直得不到很好的改善。寻找新靶点的镇痛药或创造优化新结构化合物一直是药学工作者需要迫切解决的问题。相对于传统镇痛药而言，作用于离子通道类镇痛多肽由于来源广泛、毒副作用小、半衰期长、药效明显等特点为研究者带来了新的曙光。生物膜离子通道(ion channels of biomembrane)是一类贯穿细胞膜脂质双分子层且中央带有亲水性孔道的膜蛋白，是神经、骨骼肌、心肌细胞等生物电产生的基础。此通道与感受器电位发生、神经兴奋传导和中枢神经系统调控等多种生命活动过程密切相关，并伴有高度选择性。根据某一种特定的生物膜离子通道只允许一种或几种特定的离子发生高渗，可分为钠离子通道、钾离子通道、钙离子通道和酸性敏感离子通道等。一般Ca 2+离子和K +离子在细胞膜内外保持平衡，通过调节中枢内钾离子和钠离子的浓度，可以抑制神经元的兴奋性，这也是中枢神经系统发挥中枢镇痛作用的一种机制。而Na +离子通道的活动性决定了痛觉冲动的形成和传递，许多镇痛药都是通过抑制电压门控钠离子通道，阻断痛觉兴奋传导通路。近年，随着关于疼痛分子机理研究的深入，研究者发现离子通道的构象变化与机体是否产生疼痛息息相关，其中钠离子通道(Na V)起到了最主要作用。现代分子生物学研究表明该亚型Na V1.7是一种治疗疼痛的新靶点，在发挥正常生理功能时可使兴奋细胞去极化，并将信号分子通过突触从灰质后角传输到伤害性感受器，最终使机体出现痛觉反应。在动物毒素中存在大量作用于离子通道的多肽，极具开发空间。现研究较为透彻的几种多肽结构有 从少棘蜈蚣中提取的μ-SLPTXSsm6a；从东亚钳蝎中提取的BmKAGAP；从虎纹捕鸟蛛中提取的HWTX-I；从黑曼巴蛇中提取的Mambalgins等。
芋螺毒素多肽的研究一直是领域内的热点，例如，中国专利申请号为201480082283.7，申请公布日为2017年5月31日的专利文件公开了一种芋螺毒素多肽κ-CPTx-bt105，其制备方法以及应用。所述芋螺毒素多肽由16个氨基酸组成，分子量为1626.62道尔顿，全序列为GICCVDDTCTTHSGCL。所述芋螺毒素多肽具有抑制钾离子通道电流作用和镇痛作用。但是目前从芋螺中提取芋螺毒素多肽的方法比较复杂，而且芋螺毒素多肽存在一定的副作用，对生理与心理会产生严重的依赖性。
发明内容
1.要解决的问题
针对现有的芋螺毒素多肽存在副作用等问题，本发明提供一类具有镇痛活性的多肽及其应用，在结构稳定性、镇痛活性方面较芋螺毒素具有显著性提升，在机体损伤等不良反应方面均弱与芋螺毒素。
2.技术方案
为了解决上述问题，本发明所采用的技术方案如下：
一类具有镇痛活性的多肽，具有氨基酸序列X a-X b-Cys-Ser-Thr-Pro-Pro-X c-X d-Val-Leu-Tyr-X e，或其药学上可以接受的盐。
更进一步地，所述的X a为Gly或缺失，所述的X b为Ser或Lys中的一种；所述的X c为d-Cys或Ser或Asp中的一种，所述的X d为Ala或缺失；所述的X e为Cys或Ser。
更进一步地，所述的多肽氨基酸序列为：
多肽Ι：Gly-Ser-Cys-Ser-Thr-Pro-Pro-d{Cys}-Val-Leu-Tyr-Ser；
多肽ΙΙ：Gly-Ser-Cys-Ser-Thr-Pro-Pro-Ser-Ala-Val-Leu-Tyr-Cys；或
多肽ΙΙΙ：Lys-Cys-Ser-Thr-Pro-Pro-Asp-Ala-Val-Leu-Tyr-Cys。
更进一步地，所述的每条多肽序列中两个半胱氨酸之间形成一对二硫键。
更进一步地，所述的多肽序列在上述的序列基础上，一个或多个氨基酸被删除、置换或添加后仍具有镇痛作用的多肽，或该多肽药学上可以接受的盐。
上述的多肽在制备预防和/或治疗疼痛的产品中的应用。
一种预防和/或治疗疼痛的产品，其活性成分为上述的多肽。
上述产品具体可为药物。
需要的时候，在上述药物中还可以加入一种或多种药学上可接受的辅料，所述辅料包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、吸收促进剂、表面活性剂、润 滑剂和稳定剂等。
本发明的药物可以制成注射液、干粉针剂、片剂或粒剂等多种形式。上述各种剂型的药物均可以按照药学领域的常规方法制备。
更进一步地，所述的疼痛包括理化刺激性、病理性或神经性疼痛。
现阶段临床中以抗炎性镇痛药及阿片类镇痛药为主。抗炎性镇痛药临床主要用于慢性钝痛如头痛、牙痛、肌肉关节痛、痛经等，这类药不易成瘾，在临床上运用十分广泛。阿片类镇痛药(麻醉性镇痛药)，是一类能消除或减轻疼痛并改变对疼痛情绪反应的药物，但是会存在一定的副作用，如便秘、嗜睡、感觉迟钝、恶心、呕吐、呼吸抑制和依赖性等，对生理与心理会产生严重的依赖性，所以严重限制了它在临床上的广泛应用。除麻醉性镇痛药，很多作用于中枢神经系统的药物都被应用于控制疼痛，如抗抑郁药、抗惊厥药、神经安定药等，在一定程度上具有直接的镇痛作用或潜在的镇痛活性，但副反应随着剂量的增大越发剧烈。例如阿司匹林对人体循环系统损伤较大，尤其是消化与造血系统；芋螺毒素具有严重的成瘾性、耐受性、呼吸窘迫等一系列不良反应；抗惊厥药物的过敏性及副反应也一直得不到很好的改善。因此，本发明通过计算机三维模拟技术，在对大量传统结构进行解析以及药理实验基础上，自主设计的多肽结构取具有更强镇痛活性和更低毒副性。本发明通过对现有各种镇痛多肽结构的解析，利用三维结构自主设计出具有全新结构的作用于离子通道的镇痛多肽。经各种实验模型验证，与芋螺毒素具有相同镇痛效果且耐受性等不良反应较低，极具开发前景。
3.有益效果
相比于现有技术，本发明的有益效果为：
(1)本发明的多肽结构简单，容易合成、分离和纯化，有效保留并提高芋螺毒素的镇痛活性；
(2)本发明的多肽不良反应和毒副性反应弱；
(3)本发明涉及的多肽镇痛效果在各实验模型中表现良好，具体效果为显著提高小鼠痛阈值，延长小鼠热耐受时间，小鼠自主运动、兴奋性等不良反应均低于正常值。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。以下所述，仅是本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员均可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修饰或等同变化，均落在本发明的保护范围内。
实施例1
本发明涉及的一种具有镇痛活性的多肽，其母核结构具有氨基酸序列X a-X b-Cys-Ser-Thr-Pro-Pro-X c-X d-Val-Leu-Tyr-X e，或者该多肽在药学上可接受的盐，其中X a可以为Gly或缺失，X b可以为Ser或Lys中的一种；X c可以为d-Cys或Ser或Asp中的一种，X d可以为Ala或缺失；X e可以为Cys或Ser。具体地，这类多肽的氨基酸序列为
多肽Ι：Gly-Ser-Cys-Ser-Thr-Pro-Pro-d{Cys}-Val-Leu-Tyr-Ser；
多肽ΙΙ：Gly-Ser-Cys-Ser-Thr-Pro-Pro-Ser-Ala-Val-Leu-Tyr-Cys；或
多肽ΙΙΙ：Lys-Cys-Ser-Thr-Pro-Pro-Asp-Ala-Val-Leu-Tyr-Cys。
或该类多肽序列上的一个或多个氨基酸被删除、置换或添加后仍具有镇痛作用的多肽，序列中两个半胱氨酸之间形成一对二硫键，这类多肽对理化刺激性、病理性、神经性疼痛具有良好抑制作用。
实施例中主要以该多肽Ι、多肽ΙΙ和多肽ΙΙΙ作为对象，研究其抑制疼痛的活性。该多肽委托南京金斯瑞生物科技有限公司合成，纯度为95％以上。
镇痛多肽在镇痛热板实验中对疼痛的抑制作用
昆明种小鼠，雌性，体重20±2g。室内温度保持在22～25℃。将小鼠放在温度为55℃的恒温金属板上，恒温(变化在±0.5℃内)，以小鼠接触金属板开始计时，至其出现舔后足或跳跃反应的时间为其痛阈指标。
1.实验动物：昆明种雌性小白鼠
小鼠饲养于实验鼠笼具通风系统中；饲养温度23℃左右；正常昼夜更替；所有实验动物自由饮水摄食；所购实验动物，在本实验室饲养3～5天适应实验环境；所有动物实验均在8:00～18:00进行；实验动物手术后均为单笼饲养。
2.实验设备：电子天平、热板测试仪、笼具通风系统、1mL注射器、Ultra超纯水系统
3.实验试剂：芋螺毒素、0.9％的NaCl溶液、多肽I、多肽II、多肽ΙΙΙ。
本发明中使用的芋螺毒素多肽(分型是BuIA)结构为Gly-Cys-Cys-Ser-Thr-Pro-Pro-Cys-Ala-Val-Leu-Tyr-Cys，其中，C1-C3成环，C2-C4成环。
4.实验方法：实验前先测基础痛阈，测两次取平均值计算，两次中间间隔5分钟。剔除反应潜伏期小于5s或大于30s的小鼠。为防止小鼠足部烫伤，设60s为截止时间。将筛选合格的小鼠按随机数字表分为阴性对照组、阳性对照组和芋螺毒素组，每组10只。
生理盐水、芋螺毒素、多肽I、多肽II和多肽III共5组，每组10只。其中生理盐水组为0.9％的NaCl溶液，作为阴性对照组；芋螺毒素组剂量为1μg/kg，作为阳性对照组；多肽组作为实验组给药，给药剂量均为2nmol/kg。给药途径：腹腔注射。
分别在给药后15、30、45、60、75、90min后再次记录小鼠痛阈值，实验独立重复3 次，结果用
Figure PCTCN2018085275-appb-000001
表示，并进行统计T检验，与生理盐水阴性组对比，*P<0.05为显著性差异，**P<0.01为极显著性差异；与芋螺毒素阳性组对比，^P<0.05为显著性差异，^^P<0.01为极显著性差异。各组内比较给药前后小鼠对热板的耐受时间变化，组间比较各组小鼠痛阈值大小。计算给药后可能最大镇痛百分率，评价药物的镇痛效果。
Figure PCTCN2018085275-appb-000002
表1 小鼠热板实验中各时间段痛阈值的检测结果
Figure PCTCN2018085275-appb-000003
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
5.实验结果：
5.1 小鼠热阈值检测结果
多肽组在热板镇痛动物模型中都能发挥镇痛作用，结果见表1：与生理盐水阴性组相比，多肽I、II、III组均可有效提高小鼠热阈值。多肽II痛阈值提高水平显著高于生理盐水组及芋螺毒素组。实验结果具有统计学意义。
5.2 PMAP评价结果
PAMP经计算得出约为47.57％，镇痛效果相比较芋螺毒素组无明显差异性，具有很好的镇痛效果。
实施例2
镇痛多肽在热辐射甩尾实验中对疼痛的抑制作用
昆明种小鼠，雌雄各半，体重20±2g。室内温度保持在22-25℃。用小型聚光灯产生一定强度的光束，通过透镜聚焦照射小鼠的尾巴来致痛。实验时，将鼠尾置于甩尾测痛仪的尾槽上，使光点落在鼠尾中下1/3处(实验前先用笔涂色作标记，使每次测痛点在同一位置上)从辐射热照射开始到出现甩尾反应的潜伏期(TFL，Tail Flick Latency)为痛反应的指标。
给药前，首先测定所有小鼠的基础痛阈，剔除反应潜伏期小于1s或大于3s的小鼠，为 防止小鼠尾部烫伤，设10s为截止时间。基础痛阈值符合标准的小鼠按随机数字表分为阴性对照组、阳性对照组、芋螺毒素组，每组10只。
生理盐水、芋螺毒素、多肽I、多肽II和多肽III组，每组10只。其中生理盐水组作为阴性对照组；芋螺毒素组剂量为1μg/kg，作为阳性对照组；多肽组给药剂量均为2nmol/kg。给药途径侧脑室给药。
分别记录给药后5、15、30、45、60、90、120min时的痛阈值，结果用
Figure PCTCN2018085275-appb-000004
表示，组内比较给药前后小鼠对热辐射的耐受时间变化，组间比较各组小鼠痛阈值大小。计算给药后痛阈提高百分率，评价药物的镇痛效果。
Figure PCTCN2018085275-appb-000005
表2 CTX改造肽对小鼠甩尾法镇痛作用的影响(n＝10)(i.p)
Figure PCTCN2018085275-appb-000006
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
实验结果：
1.小鼠热阈值检测结果
多肽组在小鼠热辐射甩尾动物模型中都能发挥镇痛作用，结果见表2：与生理盐水阴性组相比，多肽组均可有效提高小鼠热阈值。某时间段多肽I在2nmol/kg剂量下与芋螺毒素组比较极显著性差异(^^P＜0.01)；某时间段多肽III在2nmol/kg剂量下与生理盐水组比较极显著性差异(**P＜0.01)。实验结果具有统计学意义。
2.PMAP评价结果
PAMP经计算得出约为38.32％，镇痛效果相比较芋螺毒素组无显著性差异，具有很好的镇痛效果。
实施例3
镇痛多肽在旷场实验中对小鼠自发性活动与探索行为的影响作用
1.材料与方法
1.1 实验动物
昆明雌性小鼠60只。
饲养条件：室温(23±1)℃；湿度(50±5)％；日光灯采光，光暗周期12/12(光照时间为7:00～19:00)；动物自由获取水和食物。
1.2实验仪器
采用计算机实时监测分析系统SMART VIDEO-TRACKING(美国、SMARTv3.0.02)。该仪器主要由4个40cm×40cm×40cm实验敞箱组成，箱子顶部中央安置有与记录系统相连接的摄像机，通过系统分析小鼠实时图像以获得小鼠在旷场中的行为数据。
2.实验方法：
实验开始前对每只小鼠进行体质量统计学检测，剔除检测不合格的小鼠，并按照随机数字表分为阴性对照组、阳性对照组、多肽I、多肽II和多肽III组。其中生理盐水组作为阴性对照组注射0.9％NaCl溶液；芋螺毒素组剂量为1μg/kg，作为阳性对照组；多肽组给药剂量均为2nmol/kg。给药方式为腹腔注射。
首先确认旷场装置是清洁、无味道的。在操作软件中记录小鼠的编号、日期、状态；其次，动物实验必须提前送到行为学实验室专用临时笼架，适应环境3小时左右，减少动物紧张；将小鼠从笼内取出(小鼠背向实验者)，将小鼠放置在装置中央后迅速轻柔关闭装置上盖，打开录像记录系统，记录小鼠在旷场内的活动。总时间5min。观察完后用75％乙醇及时清洗方箱内壁及底面，以免上次动物余留的信息(如动物的大、小便、气味)影响下次测试结果。最后将本轮小鼠放回笼内，开始进行下一批小鼠测试。所有小鼠观察完后间隔1h和24h分别按以上步骤再次检测相关指标。用Origin 8.0软件统计小鼠中心停留时间、运动时间、爬壁次数以及穿越格子数作为最终数据。
3.实验结果：
表3 小鼠旷场实验运动距离(m)
Figure PCTCN2018085275-appb-000007
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
表4 小鼠旷场实验中心区域进入次数
Figure PCTCN2018085275-appb-000008
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
表5 小鼠旷场实验外周区域进入次数
Figure PCTCN2018085275-appb-000009
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
表6 小鼠旷场实验中心区域停留时间(s)
Figure PCTCN2018085275-appb-000010
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
表7 小鼠旷场实验外周区域停留时间/s
Figure PCTCN2018085275-appb-000011
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
表8 小鼠旷场实验中心区域运动距离(m)
Figure PCTCN2018085275-appb-000012
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
表9 小鼠旷场实验外周区域运动距离/m
Figure PCTCN2018085275-appb-000013
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
多肽I、多肽II和多肽III在旷场实验中各项指标与生理盐水组对比均无显著性差异，与芋螺毒素组对比多数具有显著性差异，说明多肽I、多肽II和多肽III相比较芋螺毒素具有更低的副反应效应，能明显改善小鼠自发性行为等不良状况。详见表3～9。实验结果具有统计学意义。
实施例4
镇痛多肽在强迫游泳实验中对小鼠抗运动疲劳的影响作用
1.材料与方法
1.1 实验动物
昆明雌性小鼠40只
室温18～22℃，光照12h(6:00～18:00)和黑暗12h(18:00～6:00)环境，自由进水饮食饲养；预适应环境1周后，选取20只体质量18～22g小鼠进入正式实验(所有实验均于19:00～24:00进行)。
1.2.实验装置
实验装置均由本实验室自行制作；烧杯，恒温水浴锅，秒表，计数器，奥尼ANC酷睿H D1080P高清摄像头。
1.3.方法
实验前将25只小鼠按照随机数字表分为阴性对照组、阳性对照组、多肽I、多肽II和多肽III组。其中生理盐水组作为阴性对照组注射0.9％NaCl溶液；芋螺毒素组剂量为1μg/kg，作为阳性对照组；多肽组给药剂量均为2nmol/kg。给药方式为腹腔注射。
操作者将小鼠放入装有温水的圆形烧杯，直径10cm，水深10cm，水温23～25℃，用摄像系统记录动物5min内的运动状态时间。运动状态是指动物主动挣扎，躯体处于漂浮扭动状态。所有小鼠观察完后间隔1h和24h分别按以上步骤再次检测小鼠在水中的运动时间。
2.实验结果
表10 小鼠强迫游泳实验运动时间(s)
Figure PCTCN2018085275-appb-000014
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
运动耐力的提高是抗疲劳能力加强最直接的表现，游泳时间长短可以反应动物运动疲劳的程度，运动时间越低，毒副作用越大。多肽组在强迫游泳实验中的数据与生理盐水组对比均无显著性差异，与芋螺毒素阳性组对比多数具有显著性差异，少部分具有极显著性差异。说明多肽I、II、III相比较芋螺毒素具有更低的毒副反应效应。详见表10。实验结果具有统计学意义。
实施例5
镇痛多肽在悬尾实验中对小鼠抗抑郁性的影响作用
1.材料与方法
1.1 实验动物
昆明雌性小鼠50只
室温18～22℃，光照12h(6:00～18:00)和黑暗12h(18:00～6:00)环境，自由进水饮食饲养；预适应环境1周后，选取20只体质量18～22g小鼠进入正式实验(所有实验均于19:00～24:00进行)。
1.2.实验装置
实验装置均由本实验室自行制作；包括悬尾箱(20cm×20cm×30cm)，侧立，周壁及底面均为黑色，秒表，计数器，奥尼ANC酷睿HD1080P高清摄像头。
1.3.方法
实验前将20只小鼠按照随机数字表分为阴性对照组、阳性对照组、多肽I、II、III组。其中生理盐水组作为阴性对照组注射0.9％NaCl溶液；芋螺毒素组剂量为1μg/kg，作为阳性对照组；多肽组给药剂量均为2nmol/kg。给药方式为腹腔注射。
操作者用不粘胶将小鼠尾尖部1/3处悬于悬尾箱内，使其头部正对镜头离箱底约10cm，用摄像系统记录动物6min内不动状态潜伏期及及4min内不动状态持续时间。不动状态是指动物放弃主动挣扎，躯体处于悬垂不扭动状态。
2.实验结果
表11 小鼠悬尾实验不动状态维持时间/s
Figure PCTCN2018085275-appb-000015
注：与生理盐水组比，*P<0.05，**P＜0.01；与芋螺毒素阳性对照组比，^P<0.05，^^P＜0.01
利用小鼠悬尾后企图逃脱但又无法逃脱，从而放弃挣扎，进入特有的抑郁不动状态，实验过程中记录动物不动时间来反映抑郁状态，悬尾运动时间越小，毒副作用越大。多肽I、II和III组在悬尾实验中的数据与生理盐水组对比均无显著性差异，与芋螺毒素阳性组对比多数具有显著性差异。说明镇痛多肽I、II、III相比较芋螺毒素具有更低的毒副反应效应。详见表11。实验结果具有统计学意义。
实施例6
镇痛多肽在福尔马林致痛实验中对疼痛抑制率的影响
模拟急性组织损伤所致的持续性疼痛。在动物一肢足背皮下注射稀释的福尔马林溶液，导致动物的行为改变如安静时屈腿、运动时跛行以及舔足等。这些行为的程度(如舔足时间)与福尔马林浓度成正比，一般认为它是疼痛的象征。此外，其它行为如理毛、探索和运动活动等也受福尔马林注射的影响。
昆明种小鼠，雌雄各半，体重20±2g。室内温度保持在23～24℃。依据随机数字表将小鼠分组，分别设置阴性对照组、阳性对照组和多肽组，每组10只。其中0.9％的NaCl溶液组作为阴性对照组；芋螺毒素组剂量为1μg/kg，作为阳性对照组；镇痛多肽组I、II和III给药剂量为0.2nmol/只。各组实验鼠给药5min后，使用1mL注射器向小鼠右后足底皮下注射5％福尔马林(甲酵含量5％)溶液20μL，注射后立即将小鼠放置于大玻璃烧杯中观察其1h内的痛反应，以舔右足时间作为行为学反应指标。在烧杯后面置一面与平台约成30°角的镜子，同时从正面和镜面用秒表记录每5s出现舔、咬、抖被注射足的累积时间，持续观察记录时间为60min，分别为Ⅰ相(0-10min)和Ⅱ相(10-60min)，利用福尔马林致小鼠炎性疼痛模型考察受试药急性给药后的镇痛作用。计算阳性对照组和芋螺毒素分别对Ⅰ相和Ⅱ相疼痛反应的抑制率。
Figure PCTCN2018085275-appb-000016
实验结果：
表12 各组小鼠Ⅰ相和Ⅱ相舔足累计时间比较
Figure PCTCN2018085275-appb-000017
注：与阴性组比，*P<0.05，**P＜0.01；与阳性对照组比，^P<0.05，^^P＜0.01
自主设计的镇痛多肽在福尔马林致痛实验中的数据与生理盐水组对比均具有显著性差异，与芋螺毒素阳性组对比多数具有显著性差异。说明多肽I、II和III均具有良好的镇痛效果。详见表11。实验结果具有统计学意义。
实施例7
镇痛多肽在癌症侵袭痛模型CIP中对神经痛的影响作用
1.材料
1.1.动物
雄性BALB/c小鼠若干，体重25～30g。
1.2.实验装置及试剂
vonFery单纤丝；微量注射器、5mg/mL多肽I、II、III溶液；生理盐水。
1.3方法
BALB/c小鼠，暴露右侧坐骨神经，将含5×10 4Meth A肉瘤细胞的腹水注射至远离后二头肌腱分支处坐骨神经转子附近的近端神经，封闭伤口，左侧实施假手术注入相同体积的生理盐水。
1.3.1 热敏测试
接种后的第4、7、10、14天观察动物热辐射刺激出现抬足的潜伏期，热辐射强度调整至以正常小鼠抬足潜伏期为(10±2)秒为基准。
表13 各组小鼠热敏测试结果对比
Figure PCTCN2018085275-appb-000018
Figure PCTCN2018085275-appb-000019
1.3.2 机械敏感度测试
接种后的第4、7、10、14天使用von Fery单纤丝测试双足的机械敏感度，采用翻转测试法测定出现50％缩足反应的阈值，测试从0.4g开始。
1.3.3 Gross行为
接种后的第4、7、10、14天，在出现热敏及机械痛敏等行为之前，首先出现自发性疼痛以及足下垂等，计算10分钟内抬右后足的累积时间。
2.结果
所有数据均以M±SD表示，采用单因素方差分析。
2.1 热增敏发生
在接种后的第10天热辐射刺激出现抬足的潜伏期明显缩短了8秒，接种后的第14天明显增长了15秒，其中多肽I组尤为明显。
2.2 机械敏感度改变
对会出现痛超敏感及热敏过敏的症状，同时还可以观察到自发痛。第10天可以检测到von Fery单纤丝机械痛超敏，但到第14天，小鼠出现机械痛低敏。各组别无差异性。
2.3 自发性疼痛神经综合征
到接种后第10天，部分小鼠步法观察出现足下垂，抬右后足累计持续时间略有增加，第14天，多肽I、II组明显观察到抬左足行为。
实施例8
镇痛多肽对紫杉醇诱发疼痛小鼠模型中对疼痛的抑制作用
1.材料
1.1.动物
ddY系雄性小鼠，7周龄、体重20～25g。
1.2.实验装置及试剂
纤丝机械测痛仪；细纤丝(相当于0.0688g强度)；粗纤丝(相当于1.4798g强度)；紫杉醇；多肽I、II、III；氯索洛芬；生理盐水。
1.3方法
1.3.1 动物分组
小鼠在温度(22±1)℃，相对湿度55％±10％的动物室内饲养1周后用于试验。将36只 小鼠分为模型组(只给予紫杉醇)、空白对照组(不给予紫杉醇)、氯索洛芬组和多肽I、II、III组于给予紫杉醇前3天给予，共7天。每组6只小鼠
1.3.2 实验过程
紫杉醇诱发疼痛模型的制备：小鼠腹腔注射紫杉醇10mg/kg造模。
小鼠疼痛行为学测试：将小鼠放在分为3个分隔间的金属网上静置15分钟，测定后肢足跖部机械性刺激的反应，先用细纤丝评价异常性疼痛，再用粗纤丝评价痛觉过敏的程度。刺激反应5次，共为10分。评判标准(0分：迅速抬足动作；2分：剧烈甩足或舔足动作。测定时间为紫杉醇给药前以及给药后24、48、72、96小时)。
2.实验结果
计量资料以M±SD表示，组间差异采用t检验。
表14 多肽对紫杉醇诱发疼痛小鼠模型中对疼痛的抑制作用结果
Figure PCTCN2018085275-appb-000020
2.1 小鼠给予紫杉醇后的疼痛反应
小鼠腹腔内给予紫杉醇10mg/kg时，异常性疼痛与痛觉过敏均在给予紫杉醇24小时出现高峰，此后慢慢降低，96小时后恢复到给药前水平。给药前后24小时的疼痛相关分数异常性疼痛为0.5±0.2、1.8±0.6，痛觉过敏为2.3±0.5、5.5±0.6。
2.2氯索洛芬与多肽注射液的疗效
对异常性疼痛的影响，多肽I、II、III组与模型组相比疼痛相关分数均明显降低(P＜0.05)；多肽组与模型组相比疼痛相关分数呈下降趋势，无明显差异。对痛觉过敏的影响，氯索洛芬、多肽I、II、III组与单独组相比疼痛相关分数均无显著性差异。
实施例9
镇痛多肽对三叉神经疼痛阈值的影响
1.材料
1.1.动物
SD大鼠，雌雄不限，体重170～200g。
1.2.实验装置及试剂
机械阈值测试器(美国Stoelting，Wood Dale公司)；显微镜；多肽I、II、III；生理盐水。
1.3方法
1.3.1 动物模型制备(所有操作均在无菌条件下完成)。
1.3.2 三叉神经痛诱发及模型动物阳性标准
实验前给予适应性训练。术后2周左右在安静、室温条件下进行本模型三叉神经痛诱发实验，当大鼠出现以下反应中任意一项或一项以上即可判断模型建立：①一经刺激即表现为快速的后退、转身等闪避动作，大鼠为躲避刺激物将身体蜷缩并向笼壁靠拢，或将头面部藏在身下，以保护面部避免被刺激物触及；②搔抓面部，表现为至少连续三次搔抓面部刺激区域的动作；③大鼠快速抓咬刺激物并作出攻击动作。
1.3.3 实验方法
选用体重175～200g SD大鼠60只，雌雄不限，随机分为6组：生理盐水对照组(6只)；50ug/kg多肽I、II、III组各6只；100ug/kg多肽I、II、III组各6只；300ug/kg多肽I、I I、III组各6只。在术后2周出现痛觉超敏后，各组动物腹腔分别注射多肽注射液50ug/kg，100ug/kg，300ug/kg，生理盐水0.1mL/kg后，每1小时测痛阈值，连续5小时，记录痛阈变化。
表15 镇痛多肽对三叉神经疼痛阈值的影响结果
Figure PCTCN2018085275-appb-000021
Figure PCTCN2018085275-appb-000022
2.实验结果
实验数据用M±SD表示，使用非参数Kruskal-Wallis检验单因素方差分析和Mann±Whit ney u检验，时效曲线下面积用梯形法计算，两组间时效曲线下面积用t检验。P<0.05为具有统计学意义。
术后14天大鼠出现痛觉超敏[手术侧痛阈降为(0.38±0.04)g，手术对侧为(0.43±0.04)g，假手术组为12.5g时，皮下注射多肽I、II、III注射溶液50ug/kg，100ug/kg后，动物反应阈值明显增高。多肽I、II、III50ug/kg组注射3小时后，手术侧最高疼痛阈值为(2.3±0.5)g，对侧为(1.2±0.2)g，且持续1小时。多肽I、II、III的100ug/kg组注射3小时后，手术侧最高疼痛阈值为(7.4±0.9)g，对侧为(3.2±1.3)g，并且在手术侧作用持续8小时左右。而皮下注射多肽I、II、III的300ug/kg组则痛阈值未见明显增高。

Claims (8)

  1. 一类具有镇痛活性的多肽，其特征在于：具有氨基酸序列X a-X b-Cys-Ser-Thr-Pro-Pro-X c-X d-Val-Leu-Tyr-X e，或其药学上可以接受的盐。
  2. 根据权利要求1所述的一类具有镇痛活性的多肽，其特征在于：所述的X a为Gly或缺失，所述的X b为Ser或Lys中的一种；所述的X c为d-Cys或Ser或Asp中的一种，所述的X d为Ala或缺失；所述的X e为Cys或Ser。
  3. 根据权利要求1或2所述的一类具有镇痛活性的多肽，其特征在于：所述的多肽氨基酸序列为：
    多肽Ι：Gly-Ser-Cys-Ser-Thr-Pro-Pro-d{Cys}-Val-Leu-Tyr-Ser；
    多肽ΙΙ：Gly-Ser-Cys-Ser-Thr-Pro-Pro-Ser-Ala-Val-Leu-Tyr-Cys；或
    多肽ΙΙΙ：Lys-Cys-Ser-Thr-Pro-Pro-Asp-Ala-Val-Leu-Tyr-Cys。
  4. 根据权利要求3所述的一类具有镇痛活性的多肽，其特征在于：所述的每条多肽序列中两个半胱氨酸之间形成一对二硫键。
  5. 根据权利要求3所述的一类具有镇痛活性的多肽，其特征在于：所述的多肽序列在权利要求3的序列基础上，一个或多个氨基酸被删除、置换或添加后仍具有镇痛作用的多肽，或该多肽药学上可以接受的盐。
  6. 权利要求1或2或3中所述的多肽在制备预防和/或治疗疼痛的产品中的应用。
  7. 一种预防和/或治疗疼痛的产品，其活性成分为权利要求1或2或3中所述的多肽。
  8. 根据权利要求7所述的一种预防和/或治疗疼痛的产品，其特征在于：所述的疼痛包括理化刺激性、病理性或神经性疼痛。
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