WO2018103761A1 - 一组肽及其药物组合物和应用 - Google Patents

一组肽及其药物组合物和应用 Download PDF

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WO2018103761A1
WO2018103761A1 PCT/CN2017/118520 CN2017118520W WO2018103761A1 WO 2018103761 A1 WO2018103761 A1 WO 2018103761A1 CN 2017118520 W CN2017118520 W CN 2017118520W WO 2018103761 A1 WO2018103761 A1 WO 2018103761A1
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seq
amino acid
gsmtx
rats
injection
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PCT/CN2017/118520
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French (fr)
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唐琼瑶
张赭
唐明希
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唐琼瑶
徐州医科大学
西南医科大学附属医院
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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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biomedical technology and relates to a group of novel peptides, in particular to an analgesic peptide or a pharmaceutically acceptable salt thereof for use in medicine. Furthermore, the invention also relates to the use of the group of peptides for the preparation of a pharmaceutical composition.
  • GsMTx-4 In 2000, Dr. Frederick Sachs of the State University of the United States successfully isolated the polypeptide GsMTx-4 from a venom of Chilean tarantula (Sachs F, et a., J Gen Physiol. 115, Po583-598, 2000). It has been reported that this is a polypeptide that specifically inhibits mechanically sensitive ion channels. GsMTx-4 has been found to be useful in preventing congestive heart failure caused by swelling of cells, particularly in the lungs, liver, legs, etc. (Sachs F. et al., JGP 2000). It has also been reported that GsMTx-4 can inhibit rabbit atrial fibrillation without affecting other functions of the heart, so it can be used to treat heart disease (Sachs, F.
  • GsMTx-4 may be able to treat brain tumors (Sachs, F. et al., J. Gen. Physiol. 115, pp 583-598, 2000).
  • the above-mentioned polypeptide GsMTx-4 contains 34 amino acids and has a molecular weight of 1,094 Da, of which there are 6 cysteine residues, forming a structure of a three-pair cysteine knot (ICK: Inhibitor cysteine Knot motif).
  • ICK Inhibitor cysteine Knot motif
  • the present invention has been made in view of the problems in the development of analgesic drugs in the above and/or existing biomedical technology fields.
  • the present invention provides the following technical scheme: a group of peptides having any one of amino acid sequences as shown in (1) to (8): (1) as in the sequence listing SEQ An amino acid sequence represented by ID No. 1; (2) an amino acid sequence as shown in SEQ ID No. 2 of the Sequence Listing; (3) an amino acid sequence as shown in SEQ ID No. 3 of the Sequence Listing; (4) The amino acid sequence shown in SEQ ID No. 4; (5) the amino acid sequence shown in SEQ ID No. 5 of the Sequence Listing; (6) the amino acid sequence shown in SEQ ID No. 6 of the Sequence Listing; (7) The amino acid sequence shown by SEQ ID No. 7; (8) the amino acid sequence shown in SEQ ID No. 8 of the Sequence Listing; or a pharmaceutically acceptable salt thereof.
  • a preferred embodiment of the peptide of the present invention wherein one or more amino acids of any one of the peptides (1) to (8) according to claim 1 are present on the basis of the presence of a terminal end and a terminal cysteine.
  • any one of (1) to (8) has an analgesic amino acid sequence or a pharmaceutically acceptable salt thereof, wherein the head end and the end The two cysteines form a loop.
  • a preferred embodiment of the peptide of the present invention wherein it is obtained by chemical synthesis or by recombinant techniques.
  • a preferred embodiment of the peptide of the present invention wherein: it is fused to a protein.
  • the peptide of the present invention is coupled to a polymer.
  • a preferred embodiment of the peptide of the present invention wherein the peptide is linked to a carrier.
  • Another object of the present invention is to provide a pharmaceutical composition having an analgesic effect.
  • the present invention provides the following technical solution: a pharmaceutical composition comprising: (1) an effective amount of the peptide as an active ingredient; 2) A selective pharmaceutically acceptable carrier.
  • composition of the present invention wherein the pharmaceutically acceptable carrier is selected from the group consisting of a solvent, a diluent, a suspending agent, an emulsifier, an antioxidant, a pharmaceutical preservative, a coloring agent, and a flavoring agent.
  • the pharmaceutically acceptable carrier is selected from the group consisting of a solvent, a diluent, a suspending agent, an emulsifier, an antioxidant, a pharmaceutical preservative, a coloring agent, and a flavoring agent.
  • a medium an oily substrate, and an excipient.
  • Still another object of the invention is to provide a group of peptides or their pharmaceutically acceptable salts or pharmaceutical compositions for use in analgesia.
  • the present invention determines analgesia in GsMTx-4 polypeptide by comparing various cysteine-containing (ICK)-containing polypeptide sequences such as GsMTx-4 and GsMTX-2, and by behavioral detection of a rat pain model.
  • the pharmacophore (domain) of action The present inventors have found that a partial polypeptide fragment of GsMTx-4 has an effect of suppressing pain which is not inferior to the full length.
  • the principle is that the amino acid sequences of the polypeptides GsMTx-4 and GsMTx-2 are only 11% identical (28% similar), but they are specific inhibitors of mechanically sensitive ion channels, and the amino acid sequences of several other toxins are homologous to GsMTx-4.
  • GsMTx-4 is nearly 28% identical to Hanatoxin's amino acid sequence, 37% similar), but is a blocker for other different types of ion channels.
  • the inventors synthesized a series of short-chain polypeptides by commercial chemistry based on the sequence of the key domain of the polypeptide GsMTx-4. The inventors confirmed that a polypeptide short-chain series having an analgesic effect was obtained by the detection result of the polypeptide on the nociceptive model of rats, and completed the present invention.
  • Figure 1 is a schematic illustration of the analgesic effect of SEQ ID No. 1, GsMTx-4 and morphine on hyperalgesia in rats using local injection of the plantar.
  • Saline normal saline
  • Morphine a positive control.
  • the present invention relates to all experiments and employs a double-blind test method.
  • Figure 2 is a graphical representation of the analgesic effect of SEQ ID No. 1, GsMTx-4 and morphine on hyperalgesia in rats by intraperitoneal administration.
  • Figure 3 is a comparison of the analgesic effect of short-chain polypeptide SEQ ID No. 1 and full-length GsMTx-4 by injection of the sole (A) and abdominal cavity (B), and dose-dependent analgesic effect of SEQ ID No. 1 and GsMTx-4.
  • Sexual diagram (C) is a comparison of the analgesic effect of short-chain polypeptide SEQ ID No. 1 and full-length GsMTx-4 by injection of the sole (A) and abdominal cavity (B), and dose-dependent analgesic effect of SEQ ID No. 1 and GsMTx-4.
  • Sexual diagram (C) Sexual diagram
  • Figure 4 is a schematic diagram showing the analgesic effect of SEQ ID No. 2 on the rats by intraplantal (A, B) or intraperitoneal (C, D) injection, respectively, and the analgesic effect of the full-length polypeptide GsMTx-4, respectively. Comparison diagram (B, D).
  • Figure 5 is a schematic illustration of the analgesic benefit of sensation of rat hyperalgesia by SEQ ID No. 3 by intraplantal (A, B) or intraperitoneal (C, D) injection, respectively, and with the full length polypeptide GsMTx-4, respectively.
  • Figure 6 is a graph showing the analgesic effect of SEQ ID No. 4 on hyperalgesia in rats by intraplantal (A, B) or intraperitoneal (C, D) injection, respectively, and analgesia with full-length polypeptide GsMTx-4, respectively. A comparison of the effects (B, D).
  • Figure 7 is a schematic illustration of the analgesic effect of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 by local injection of the unilateral hind limb of the rat.
  • Figure 8 is a graph showing the inhibitory effect of SEQ ID No. 6 (A, B) and SEQ ID No. 8 (C, D) on hyperalgesia in rats by intraperitoneal injection, respectively.
  • Figure 9 is a graphical representation of SEQ ID No. 1, SEQ ID No. 3, and the full length polypeptide GsMTx-4 having no effect on pain threshold in normal rats (when no pain model is established), morphine as an effect on pain threshold in normal rats Positive control.
  • an embodiment or “an embodiment” as used herein refers to a particular feature, structure, or characteristic that can be included in at least one implementation of the invention.
  • the compounds of the invention may be provided in the form of a pharmaceutically acceptable salt.
  • preferred salts are those formed with pharmaceutically acceptable organic acids and polymeric acids and salts with inorganic acids such as acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzene.
  • inorganic acids such as acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzene.
  • the polymeric acid such as citric acid or carboxymethylcellulose
  • the inorganic acid such as hydrohalic acid (eg, hydrochloric acid, Sulfuric acid or phosphoric acid, etc.). Any method known to those skilled in the art for obtaining a pharmaceutically acceptable salt can be used.
  • a feature of the present disclosure is a novel peptide for the treatment of pain (eg, all types of pain such as acute pain, chronic pain, cancer pain, etc.).
  • Peptides reduce pain sensitivity and therefore have an analgesic effect.
  • the peptide produces long-lasting analgesic and/or antinociceptive effects.
  • Specific methods of application of such peptides e.g., for the treatment of pain
  • medicaments or compositions containing such peptides are also described herein.
  • Pain The International Association for the Study of Pain (IASP) defines pain as “an unpleasant sensory, emotional sensation. We primarily associate it with tissue damage, or describe it from organizational damage. Or it is associated with both organizational damage and damage.” The pain can be a symptom of a underlying disease or condition, or the pain itself is a disease. Pain can generally be divided into two types: acute and chronic. The main difference is that acute pain plays a protective role after the damage, while chronic pain does not. Acute pain is a symptom of pain; chronic pain is a painful condition.
  • acute pain is caused by disease, inflammation or tissue damage. This type of pain typically occurs suddenly after, for example, trauma or surgery and may be accompanied by anxiety or depression.
  • the cause of acute pain can usually be diagnosed and treated, and the pain is often self-limiting, that is, it is limited to a specific period of time and is severe. In some cases, acute pain can translate into chronic pain.
  • Chronic pain is generally considered to be a disease. Environmental and psychological factors can make it worse. Chronic pain lasts longer than acute pain and is resistant to many medications.
  • the main types of sustained pain mechanisms can be inferred.
  • the pain syndrome is broadly classified into nociceptive, neuropathic, psychiatric, mixed or special.
  • the present disclosure provides peptides that have analgesic properties and are useful for treating pain (eg, acute pain, chronic pain, or cancer pain, etc.).
  • the peptide may have an antinociceptive effect.
  • One of the objects of the present invention is to provide a group of peptides having analgesic action (e.g., SEQ ID No. 1 to SEQ ID No. 8).
  • the peptides were prepared by synthesis, purification and elution so that the final synthetic peptide samples were >99% pure and their molecular weights were determined by electrospray ionization mass spectrometry (ESI-MS).
  • the peptides represented by SEQ ID No. 1 to SEQ ID No. 8 are obtained by chemical synthesis or by recombinant techniques, and can be fused not only to a protein but also to a polymer, and can also be linked to a carrier. Further studies have found that the peptides of this group have a special structure, any of the analgesic amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 8, or their pharmaceutically acceptable salts, wherein the head end and the end The two cysteines form a loop. As the following structure:
  • Carrageenan (Carr) is a product of American sigma company, which is prepared with 1% carrageenan (0.1g carrageenan dissolved in 10ml 0.9% physiological saline solution and dispensed into 10 EP tubes) And 2% carrageenan (0.2 g of carrageenan dissolved in 10 ml of 0.9% physiological saline solution, dispensed into 10 EP tubes), stored at -20 ° C, and taken out on ice for half an hour before use.
  • GsMTx-4 was purchased from American alomone labs, and all the polypeptides of SEQ ID No. 1 to SEQ ID No. 8 were prepared into 5 mM or 10 mM mother liquor by high temperature sterilized deionized water (DD water) and dispensed in EP tube. After storage at -80 ° C, it was diluted with physiological saline to the concentration required for the experiment.
  • DD water high temperature sterilized deionized water
  • the 38500-PAM Pressure Application Measurement System ( ⁇ go Basile Biological Research Apparat ⁇ s, Comerio–Varese, Italy) consists of two parts, one for the handheld unit and the other for the integrated electronics unit.
  • the hand unit is a force sensor designed according to the Randall-Selitto pain meter.
  • the integrated electronics unit automatically records the maximum mechanical foot reflex threshold applied to the left hind foot of the rat.
  • 2% carrageenan inflammatory pain model was prepared according to the method described in the literature (Seung Pyo Park, et al. 2008. Pain. 137: 208-217). . The method is used for detecting the analgesic effect of drugs by intraperitoneal injection. Briefly: from the left hind foot 2, 3 toe between the rats, injection of 2% carrageenan 50 ⁇ l, the injection concentration can induce hyperalgesia, that is, within half an hour after the injection of the rat sole A more severe swelling and a decrease in the pain threshold occurred. The mechanical contraction threshold was lowered to 50 gf or less as a qualified rat for carrageenan inflammatory pain. Inflammatory model rats with an inflammatory threshold above 50 gf were given rejection.
  • All experimental rats were purchased from Shandong Jinan Pengyue Animal Co., Ltd. for 6-week-old SD rats.
  • the experimental rats were conditioned for 2 to 4 weeks, the temperature of the feeding environment was 22-26 ° C, the humidity was 30% to 50%, and 10 to 12 rats weighing 180-220 g were selected after adaptive feeding.
  • the rats were measured left.
  • the threshold of mechanical reduction of the lateral hind paws was taken as the mean value of 5 times per rat as the base value (Baseline, abbreviated as BL) before inflammatory modeling in the rats, and the difference in the base value was large or the response to mechanical stimulation was not large.
  • Baseline abbreviated as BL
  • Rats were then injected intradermally with 6 ⁇ l of 1% carrageenan (for foot injection) or 50 ⁇ l of 2% carrageenan (for intraperitoneal injection) on the left hind foot of the rat. Dosing method), after 1 hour, the threshold of mechanical retraction of the left hind paw of the rat was detected, and the rats with failed modeling (inflammation threshold of 50 gf or more) were excluded, and the rats with successful modeling were equally divided into drug groups and The saline group was divided into two groups. In the above operations, if the model was unsuccessful and the rats were removed, an experiment was performed to ensure that at least 5 to 8 rats were used for statistical data.
  • the role of GsMTx-4 and its short peptides in mechanical hyperalgesia in rats was evaluated by measuring the mechanical threshold of left hind paw in rats using a 38500-PAM pressure application measurement system. Rats were subjected to contraction training prior to all rat behavioral tests until each rat contracted and began recording the baseline paw withdrawal threshold. 10 to 12 rats to be tested on the day were placed in the same cage, numbered from 1 to 10 (or 12) on the tail of each rat, and then the rats were allowed to acclimate in the measurement room for 30 min. Start testing after being quiet. The force sensor designed according to the Randall-Selitto pain tester is mounted in a handheld unit of the 38500-PAM pressure application measurement system.
  • the measurement system has a minimum force value of 0.45 gf and a maximum force value of 450 gf.
  • the operator used the 38500-PAM pressure application measurement system to sequentially measure the mechanical threshold of the left hind foot of each rat in order of number.
  • the specific method is that the tapered tip in the hand-held unit is placed in the middle of the left hind foot of the rat, and then a force is applied to the left hind paw of the rat at a constant force rate (30 gf/s).
  • the maximum time is 15s.
  • the electronic unit will automatically record the maximum mechanical contraction threshold applied to the left hind foot of the rat.
  • the system will automatically alarm, the operator will stop the measurement, and the measurement will be repeated after 10 minutes. If a rat had no reflexive reflexes in the left hind paw after 15 s, the rats were excluded. The rats were sequentially measured according to the number of the rat tail number, and the measurement was repeated 5 times (each stimulation interval was 10 min), and the average value of the 5 experiments was taken. After the measurement, 6 ⁇ l of 1% carrageenan was intradermally injected into the plantar to establish a model of inflammatory pain in rats. One hour after the injection of carrageenan, the threshold of mechanical retraction of the left hind paw was measured, and the rats with abnormal thresholds were rejected (model failure).
  • Rats with higher inflammatory threshold and lower threshold were divided into two groups: drug group and saline group, with 5-6 groups in each group, which made the inflammatory threshold distribution of the two groups similar.
  • drug group 1.2 ⁇ g/5 ⁇ l of GsMTx-4 (diluted to the appropriate concentration before use) or one of the short peptides, or 5 mg/kg morphine, or the left hind foot of the rat was injected intradermally into the left hind foot of the rat. 5 ⁇ l of the same volume of normal saline was injected at the same position under the subcutaneous layer.
  • the threshold of mechanical reflex of the left hind paw of each group was detected at 1h, 3h, 5h, 7h and 24h, and each rat was tested 5 times. (The interval between each test interval was 10 min), and the average value of the 5 paw withdrawal reflex thresholds was the actual paw withdrawal reflex threshold of the rat (unit: g).
  • the measurement system has a minimum force value of 0.75gf and a maximum force value of 750gf.
  • the maximum time for applying the force is adjusted to 25s.
  • the drug group was intraperitoneally injected with 270 ⁇ g/kg of GsMTx-4 or one of short peptides or 10 mg/kg of morphine, and the amount of intraperitoneal injection of the saline group was an equal mass of sterile physiological saline.
  • Fig. 1 is a schematic diagram showing the inhibitory effect of GsMTx-4, morphine and SEQ ID No. 1 on the hyperalgesia of rats by subcutaneous injection into the plantar skin (injection volume of 6 ⁇ l).
  • the A picture shows that the rat model of inflammatory injury was induced by injecting 6 ⁇ l of 1% carrageenan (Carr) into the left hind foot of the rat in advance, and then injecting 5 ⁇ l of physiological saline into the plantar swelling site of the rat for 1 h. After 3h, 5h, there was no significant difference between the mechanical contraction threshold and the inflammatory threshold in rats.
  • Carr carrageenan
  • the rat foot swelling site was injected subcutaneously with an equal volume of GsMTx-4 or short peptide SEQ ID No. 1 (injection volume of 1.2 ⁇ g/5 ⁇ l) for 1h, 3h, 5h, the mechanical shrinkage threshold of rats increased, and It reached the highest 3h after injection. Note that the effect of morphine reached a maximum after 1 h of administration, but the increase in the threshold of mechanical shrinkage in rats greatly exceeded the threshold of mechanical contraction in the baseline (BL) of rats.
  • E is a comparison of GsMTx-4, short peptide SEQ ID No. 1 and saline and morphine pair after intradermal injection of 6 ⁇ l of 1% carrageenan (Carr) into the rat's left hind foot. Inhibition in mechanical hyperalgesia in rats.
  • Fig. 2 is a comparative diagram showing the inhibitory effect of GsMTx-4, morphine and SEQ ID No. 1 on hyperalgesia in rats by intraperitoneal administration (injection volume of 50 ⁇ l).
  • GsMTx-4 and SEQ ID No. 1 have a stronger inhibitory effect than the mode of intradermal injection from the plantar: they The maximum inhibitory effect was achieved after 3 h, and the inflammatory pain threshold (B, C) induced by carrageenan (Carr: injection volume 50 ⁇ l 2%) was completely reversed, while physiological saline had no effect (A).
  • morphine injection amount of 10 mg/Kg
  • GsMTx-4 injection amount of 270 ⁇ g/Kg, 50 ⁇ l
  • SEQ ID No. 1 injection amount was The analgesic effect of 270 ⁇ g/Kg is twice as high (see E), but the results are similar to those of the plantar injection: although the analgesic effect of morphine is stronger, the threshold of mechanical shrinkage in rats has greatly increased.
  • Rat baseline Base Line, abbreviated as BL
  • the analgesic effect of morphine rapidly decreased after 3 hours, achieving an effect similar to that of GsMTx-4 and SEQ ID No. 1 (E). Since the short-chain polypeptide of the present invention having the strongest analgesic effect has no effect on the pain sensation of normal rats (see FIG. 9), the polypeptides discovered by the present invention may develop into the best analgesic drugs for treating hyperalgesia or Mixture.
  • Figure 3 is a graphical representation of the comparison of the analgesic effect of SEQ ID No. 1 and full length GsMTx-4.
  • SEQ ID No. 1 and GsMTx-4 were administered by plantar injection, there was no significant difference in analgesic effect (A, C in the figure), where C is SEQ ID No. 1 (right) and full-length GsMTx-4 ( Left) Dose-dependent map of analgesic effects by plantar injection. However, when both are administered by intraperitoneal injection, SEQ ID No. 1 may have a stronger analgesic effect (B in the figure).
  • Fig. 4 is a schematic view showing the analgesic action of SEQ ID No. 2 by means of the sole (A) and the abdominal cavity (C), respectively.
  • the analgesic effect of the same amount of full-length GsMTx-4 was compared by light gray (B, D).
  • B, D shows that after intradermal injection of 6 ⁇ l of 1% carrageenan (Carr) into the left hind foot of the rat, the threshold of mechanical contraction reflex in rats is significantly reduced compared with the threshold of mechanical reflex foot reflex before modeling.
  • FIG. 2 shows that 1 hour after intradermal injection of 50 ⁇ l of 2% carrageenan (Carr) into the left hind paw of rats, SEQ ID No. 2 (injection amount of 270 ⁇ g/Kg) was intraperitoneally injected for 1 h, 3 h, 5 h, 7 h.
  • the threshold of mechanical contraction was increased in rats, and the inhibition was the strongest when injected for 3 hours. It is obvious that the analgesic effect of intraperitoneal injection is stronger than the local administration of plantar.
  • intraperitoneal injection of SEQ ID No. 2 has an analgesic effect similar to full-length GsMTx-4 (D).
  • Fig. 5 is a graph showing the inhibitory effect of SEQ ID No. 3 on hyperalgesia by the method of administering the sole (A) and the abdominal cavity (C), respectively.
  • Figure A after intradermal injection of 6 ⁇ l of 1% carrageenan (Carr) into the left hind foot of the rat, the foot was injected subcutaneously with SEQ ID No. 3 compared with the threshold of mechanical reflex foot reflex before modeling. (Injection volume was 1.2 ⁇ g/5 ⁇ l) After 1h, 3h, 5h, 7h, the threshold of mechanical contraction reflex was significantly decreased in rats, and reached the highest at 3h after injection.
  • Figure B shows the comparison of SEQ ID No.
  • SEQ ID No. 3 may have a stronger analgesic effect (D) than the equal-mass full length polypeptide GsMTx-4 when administered by intraperitoneal injection.
  • Fig. 6 is a graph showing the inhibitory effect of SEQ ID No. 4 on hyperalgesia by the method of administering the sole (A) and the abdominal cavity (C), respectively.
  • Figure A after intradermal injection of 6 ⁇ l of 1% carrageenan (Carr) into the left hind foot of the rat, the foot was injected subcutaneously with SEQ ID No. 4 compared with the threshold of mechanical contraction before the modeling. (Injection volume was 1.2 ⁇ g/5 ⁇ l) After 1h, 3h, 5h, 7h, the threshold of mechanical contraction reflex was significantly decreased in rats, and reached the highest at 3h after injection.
  • Figure B shows SEQ ID No.
  • the threshold of mechanical contraction was significantly increased after 1h, 3h, 5h and 7h, and there was no significant difference between the threshold of mechanical contraction and the baseline of rats (3) after 3h. Therefore, when administered by intraperitoneal injection, the analgesic effect of SEQ ID No. 4 was not significantly different from the analgesic effect of the equal-mass full-length polypeptide GsMTx-4 (D).
  • Fig. 7 shows the inhibitory effect of three short peptides of SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7 on hyperalgesia in rats when administered by intraperitoneal injection.
  • the A picture shows the subcutaneous injection of SEQ ID No. 5 (1.2 ⁇ g/5 ⁇ l) for 1h, 3h, 5h, 7h, compared with the mechanical contraction threshold of Carr inflammatory model rats, the mechanical contraction threshold is obvious. Increased and reached its highest level in 3h.
  • Panel B shows subcutaneous injection of SEQ ID No. 6 (1.2 ⁇ g/5 ⁇ l) for 1 h. After 3 h, the mechanical contraction threshold was significantly improved compared with the Carr inflammatory threshold and reached the highest at 3 h.
  • the C picture shows that the subcutaneous injection of SEQ ID No. 7 (1.2 ⁇ g/5 ⁇ l) for 1h, 3h, 5h, 7h, compared with the Carr inflammatory threshold, the mechanical contraction threshold is significantly improved, and reached in 3h highest.
  • Figure D shows subcutaneous injection of SEQ ID No. 8 (1.2 ⁇ g/5 ⁇ l) for 1 h, 3 h, 5 h, 7 h, and the mechanical contraction threshold was significantly improved compared with the Carr inflammatory threshold, and reached the highest at 3 h. .
  • Figure 8 shows the analgesic effect of SEQ ID No. 5 and SEQ ID No. 8 on inflammatory pain in rats when administered by intraperitoneal injection.
  • the graphs A and C respectively indicate that inflammatory pain was induced by intradermal injection of 50 ⁇ l of 2% carrageenan (Carr) into the left hind paw of the rat, respectively, and then intraperitoneally injected with SEQ ID No. 5 (Panel A) or SEQ. ID No. 8 (Fig. C) (injection amounts were 270 ⁇ g/Kg).
  • the threshold of mechanical contraction was significantly increased after 1h, 3h, 5h and 7h, and reached the maximum at 3h after injection.
  • Figure 9 shows that GsMTx-4, SEQ ID No. 1 and SEQ ID No. 3 have no effect on the mechanical withdrawal threshold of normal rats.
  • A shows that morphine is directly injected subcutaneously into the left hind paw of normal rats (injection amount is 5 mg/kg, which is usually the minimum dose of morphine). After 1 h, morphine further strongly inhibits mechanical contraction reflex in normal rats. The threshold was more than doubled (the threshold increased from an average of ⁇ 250g to 550g in normal rats), which we believe is associated with morphine's analgesic effect and its addictiveness and toxicity.
  • B, C, and D diagrams show direct injection of GsMTx-4 (Panel A), SEQ ID No. 1 (P) or SEQ ID No.
  • the peptides of the present disclosure may be formulated, for example, as a pharmaceutical composition for administration to a subject to treat pain.
  • the peptides may be administered alone or in combination with other pain therapies in the same composition or as separate compositions.
  • compositions typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all physiologically compatible solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the composition may include a pharmaceutically acceptable salt such as an acid addition salt or a base addition salt.
  • the peptide is formulated with excipient materials such as saline, sodium chloride, disodium hydrogen phosphate heptahydrate, sodium dihydrogen phosphate, and stabilizers. It may also be provided in a suitable concentration, for example, in a buffer solution.
  • the pharmaceutical composition can take a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.
  • liquid solutions e.g., injectable and infusion solutions
  • dispersions or suspensions tablets, pills, powders, liposomes, and suppositories.
  • the preferred form may depend on the intended mode of administration and therapeutic application.
  • composition can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly).
  • parenteral administration and “parenteral administration” as used herein mean administrations other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial. , intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions .
  • compositions can be administered by the enteral route, for example by the digestive tract, for example by oral administration.
  • the compositions can be administered in the form of tablets, capsules, caplets, pills, powders, drops, suspensions, solutions, pastes, gels, or other oral dosage forms.
  • Enteral routes include administration via a gastric feeding tube, a duodenal feeding tube or a gastrostomy or rectal administration, for example in the form of a suppository or enemas.
  • the composition can be administered topically, for example at the site of pain.
  • Topical administration includes, for example, epidermal, intranasal, inhalation, and vaginal administration.
  • the composition can be administered to the skin (e.g., burns, blistering or rupture), lips, gums, teeth, mouth, eyes, ears, nail bed or throat, etc., such as a painful site.
  • the composition for topical administration may be a cream, a gel, a lotion or an ointment or the like.
  • a pharmaceutical composition can include a "therapeutically effective amount" of a peptide described herein.
  • the effective amount can be determined depending on the effect of the administered drug (e.g., peptide), or if more than one drug is used, based on the combined effect.
  • a therapeutically effective amount of a drug can also vary depending on factors such as the type of pain in the subject, the condition, age, sex, and weight, and the ability of the drug to elicit a desired response (e.g., to ameliorate pain) in the subject.
  • a therapeutically effective amount is also one in which the therapeutically beneficial effects of the composition exceed any toxic or detrimental effects.
  • the native spider toxin GsMTx-4 polypeptide forms four cyclic structures in water, and the synthesis requires that the polypeptide must be folded to maintain the spatial structure of the native polypeptide to have the corresponding pharmacological effect, so the synthesis process is cumbersome and costly.
  • the invention finds a pharmacophore which plays an analgesic role in the native polypeptide, thereby reducing the synthesis cost of the polypeptide, and making the industrial mass production efficient and cheap;
  • the present invention reduces the length of the polypeptide, it reduces the immunogenicity and is more easily absorbed by the human body. In clinical applications, the efficacy can be fully utilized. Since the polypeptide has no effect on the pain threshold of normal large genus, it is not addictive, boring, and non-toxic compared with drugs such as morphine. Therefore, the present invention may be developed as an analgesic drug widely used clinically.

Abstract

提供了一组肽及其药物组合物和应用，其中肽如序列表所示，其是具有镇痛作用的氨基酸序列或它们药学上可以接受的盐。提供肽的首端和末端的两个半胱氨酸形成环。通过比较GsMTx-4、GsMTX-2等多种含半胱氨酸结(ICK)的多肽序列，并通过对大鼠疼痛模型的行为学检测，确定了在GsMTx-4多肽中起镇痛作用的药效团(结构域)。

Description

一组肽及其药物组合物和应用 技术领域
本发明属于生物医药技术领域，涉及一组新型肽，特别涉及在医学中应用的具有镇痛作用的肽或它们药学上可以接受的盐。此外，本发明还涉及该组肽用于制备药物组合物。
背景技术
2000年，美国约州立大学Frederick Sachs博士从一种智利狼蛛(Chilean tarantula)毒液中成功分离出多肽GsMTx-4(Sachs F,et a.,J Gen Physiol.115,Po583-598,2000)，有报道指出，这是一种对机械力敏感离子通道具有特异抑制作用的多肽。GsMTx-4被发现可以用于阻止由细胞肿胀产生的充血性心力衰竭，特别在肺、肝、腿等部位(Sachs F.et al.,JGP 2000)。也有报告指出GsMTx-4可以抑制兔子心房颤动而不对心脏的其它功能产生影响,因此它可以用于治疗心脏病(Sachs,F.et al.,Nature 409,pp 35～36,2001；Hamill,0.P.,Martinac,B.,Physiol.Rev.81,pp 685～740,2001.)。此外，GsMTx-4可能可以治疗脑瘤(Sachs,F.et al.,J.Gen.Physiol.115,pp 583～598,2000)。
美国专利12153942(16.07.2009)，10550102(12.10.2006)，04723309(28.12.2005)，能抑制离子通道活性的低分子量多肽(Low-molecular weight peptides inhibiting ion channel activity)。欧洲专利EP 1 609 861B1(Pub.No.：WO/2004/085647)抑制离子通道活性的低分子量多肽(英语：Low-molecular weight peptides inhibiting ion channel activity；日语：イオンチャネルの活性を阻害する低分子ペプチド)以上内容在日本也申请了专利。均系同一组研究人员及日本公司联手申请。该发明提供了一系列从全长GsMTx-4中衍生出来的短链多肽，这些低分子量的短链多肽被发现可以特异性地抑制鸡心脏中机械力敏感大电导钾离子通道SAKca的活性。
上述提到的多肽GsMTx-4，含有34个氨基酸，其分子量为1,094Da，其中有6个半胱氨酸残基，形成三对半胱氨酸结(ICK：Inhibitor cysteine Knot motif)结构的特性(Robert.et al.,J.Biol.Chem.277(37)：34443-34450,2002)。这种结构限制了它化学合成和生物合成时的产量，原因在于难以折叠成正确的构象。虽然最近有专利用于指导在酵母中合成全长GsMTx-4多肽，但成本和产量仍然是药物开发的瓶颈。
发明内容
鉴于上述和/或现有生物医药技术领域中对镇痛作用药物开发存在的问题，提出了本发明。
因此，本发明其中一个目的是提供一组具有镇痛作用的短肽。
为解决上述技术问题，根据本发明的一个方面，本发明提供了如下技术方案：一组肽，其具有如(1)～(8)所示的任一氨基酸序列：(1)如序列表SEQ ID No.1所示的氨基酸序列；(2)如序列表SEQ ID No.2所示的氨基酸序列；(3)如序列表SEQ ID No.3所示的氨基酸序列；(4)如序列表SEQ ID No.4所示的氨基酸序列；(5)如序列表SEQ ID No.5所示的氨基酸序列；(6)如序列表SEQ ID No.6所示的氨基酸序列；(7)如序列表SEQ ID No.7所示的氨基酸序列；(8)如序列表SEQ ID No.8所示的氨基酸序列；或，它们药学上可以接受的盐。
作为本发明所述肽的一种优选方案，其中：在首端和末端半胱氨酸存在的基础上，权利要求1所述(1)～(8)任一肽中，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，它们药学上可以接受的盐。
作为本发明所述肽的一种优选方案，其中：所述(1)～(8)所示的任一具有镇痛作用的氨基酸序列或它们药学上可以接受的盐，其中，首端和末端的两个半胱氨酸形成环。
作为本发明所述肽的一种优选方案，其中：其通过化学合成或通过重组技术获得。
作为本发明所述肽的一种优选方案，其中：其与蛋白融合。
作为本发明所述肽的一种优选方案，其中：其与聚合物偶联。
作为本发明所述肽的一种优选方案，其中：所述肽与载体连接。
本发明另外一个目的是提供一种具有镇痛作用的药物组合物。
为解决上述技术问题，根据本发明的另外一个方面，本发明提供了如下技术方案：一种药物组合物，其特征在于，包括，(1)有效量的作为活性成分的所述的肽；(2)选择性的药学上可接受的载体。
作为本发明所述药物组合物的一种优选方案，其中：所述药学上可接受的载体选自：溶剂、稀释剂、悬浮剂、乳化剂、抗氧化剂、药学防腐剂、着色剂、香味剂、介质、油性基底、赋形剂中的一种或几种。
本发明还有一个目的是提供一组肽或它们药学上可以接受的盐或药物组合物在镇痛方面的应用。
本发明通过比较GsMTx-4、GsMTX-2等多种含半胱氨酸结(ICK)的多肽序列,并通过对大鼠疼痛模型的行为学检测,确定了在GsMTx-4多肽中起镇痛作用的药效团(结构域)。本发明发现GsMTx-4的部分多肽片段有不逊于全长的抑制疼痛的作用。其原理是多肽GsMTx-4和GsMTx-2氨基酸序列仅有11％相同(28％相似)，但同属机械力敏感离子通道的特异抑制剂，其它几种毒素氨基酸序列虽然与GsMTx-4同源性更高(比如：GsMTx-4与Hanatoxin的氨基酸序列有近28％相同，37％相似)，但却是其它不同类型离子通道的阻断剂。这样本发明人基于多肽GsMTx-4关键结构域的序列，通过商业化的化学合成了一系列短链多肽。通过多肽对大鼠伤害性疼痛模型的检测结果，发明人证实得到了具有镇痛作用的多肽短链系列，完成本发明。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：
图1是利用足底局部注射给药方式，SEQ ID No.1、GsMTx-4和吗啡对大鼠痛觉过敏的镇痛效应示意图。为使数据可信度高，我们用普通生理盐水(Saline)作为负性对照、吗啡(Morphine)作为阳性对照。本发明涉及所有实验均并采取双盲测试方法。
图2是利用腹腔注射给药方式，SEQ ID No.1、GsMTx-4和吗啡对大鼠痛觉过敏的镇痛效应示意图。
图3是短链多肽SEQ ID No.1与全长GsMTx-4通过足底(A)、腹腔(B)注射方式镇痛效应对比，以及SEQ ID No.1和GsMTx-4镇痛作用剂量依赖性示意图(C)。
图4是SEQ ID No.2分别通过足底(A，B)或腹腔(C,D)注射给药方式时对大鼠镇痛作用示意图，以及分别与全长多肽GsMTx-4镇痛效果的对比示意图(B,D)。
图5是SEQ ID No.3分别通过足底(A,B)或腹腔(C,D)注射方式给药 时对大鼠痛觉过敏的镇痛效益的示意图，以及分别与全长多肽GsMTx-4镇痛效果的对比示意图(B,D)。
图6是SEQ ID No.4分别通过足底(A,B)或腹腔(C,D)注射给药时对大鼠痛觉过敏的镇痛效益示意图，以及分别与全长多肽GsMTx-4镇痛效果的对比示意图(B,D)。
图7是SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8分别通过大鼠单侧后肢足底局部注射方式给药时的镇痛作用示意图。
图8是SEQ ID No.6(A,B)和SEQ ID No.8(C,D)分别通过腹腔注射方式时对大鼠痛觉过敏抑制作用示意图。
图9是SEQ ID No.1、SEQ ID No.3以及全长多肽GsMTx-4对正常大鼠(没有建立疼痛模型时)疼痛阈值没有影响的示意图，吗啡作为对正常大鼠疼痛阈值具有影响的阳性对照。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合说明书附图对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
根据一般约定书写本文提及的所有的肽序列，其中N端氨基酸在左边，而C端氨基酸在右边。在两个氨基酸残基之间的短线指示肽键。
本发明的化合物可以以药用盐的形式提供。优选的盐的实例是与药用有机酸以及聚合酸形成的那些和与无机酸形成的盐，所述有机酸如乙酸、乳酸、马来酸、柠檬酸、苹果酸、抗坏血酸、琥珀酸、苯甲酸、水杨酸、甲磺酸、甲苯磺酸、三氟乙酸或双羟萘酸，所述聚合酸如鞣酸或羧甲基纤维素，所述无机酸如氢卤酸(例如，盐酸、硫酸或磷酸等)。可以使用本领域技术人员已知的获 得药用盐的任何方法。
为了描述本发明的方便，使用各种氨基酸残基的常规和非常规缩写。这些缩写是本领域技术人员熟悉的，但是为了清楚在下面列出：
Asp＝D＝天冬氨酸；Ala＝A＝丙氨酸；Arg＝R＝精氨酸；
Asn＝N＝天冬酰胺；Gly＝G＝甘氨酸；Glu＝E＝谷氨酸；
Gln＝Q＝谷氨酰胺；His＝H＝组氨酸；Ile＝I＝异亮氨酸；
Leu＝L＝亮氨酸；Lys＝K＝赖氨酸；Met＝M＝甲硫氨酸；
Phe＝F＝苯丙氨酸；Pro＝P＝脯氨酸；Ser＝S＝丝氨酸；
Thr＝T＝苏氨酸；Trp＝W＝色氨酸；Tyr＝Y＝酪氨酸；
Val＝V＝缬氨酸；Cys＝C＝半胱氨酸。
概况
本公开内容的特征是用于治疗疼痛(例如急性疼痛、慢性疼痛、癌症痛等所有疼痛类型)的新型肽。肽可降低疼痛敏感性，因此具有镇痛作用。该肽可产生长效镇痛和/或抗伤害感受作用。本文还描述了这类肽的具体应用方法(例如用于治疗疼痛)及含有这类肽的药或组合物。
疼痛和伤害感受
国际疼痛研究学会(International Association for the Study of Pain，IASP)将疼痛定义为“一种不愉快的感觉性、情绪性感受，我们主要将其与组织损害联系在一起，或者从组织损害方面进行描述，或者既与组织损害相联系又从损害方面进行描述”。疼痛可以是潜在疾病或病症的症状，或者疼痛本身即为一种疾病。疼痛一般可分成两个类型：急性和慢性。主要的区别是急性疼痛在损害之后起保护作用，而慢性疼痛不起此作用。急性疼痛是疼痛症状；而慢性疼痛是疼痛疾病。
大部分急性疼痛由疾病、炎症或组织损伤造成。该疼痛类型一般在例如创伤或外科手术之后突然发生，并可伴有焦虑或情绪低落。通常可以诊断出急性疼痛的原因并对其进行治疗，且疼痛常常是自限性的，也就是说，它限于特定时间段并且严重。在一些情况下，急性疼痛可转变成慢性疼痛。
普遍认为慢性疼痛本身就是疾病。环境和心理因素可使之恶化。慢性疼痛比急性疼痛持续时间更长，并可对许多药物治疗具有抗性。
根据临床特征，可推断持续疼痛机制的主要类型。基于推测的病理生理的 分类，在广义上将疼痛综合征分成伤害感受性、神经病性、精神性、混合型或特发型。
镇痛肽
本公开内容提供具有镇痛性质并可用于治疗疼痛(例如急性疼痛、慢性疼痛或癌痛等)的肽。该肽可具有抗伤害感受作用。
本发明的其中一个目的在于提供一组具有镇痛作用的肽(如SEQ ID No.1～SEQ ID No.8)。而该组肽的制备方法是通过合成、纯化、洗脱，使得最终合成肽的样品纯度>99％，并采用电喷雾质谱(ESI-MS)测定其分子量。
当然，本领域技术人员能够了解到，在首端和末端半胱氨酸存在的基础上，SEQ ID No.1～SEQ ID No.8任一肽中，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，它们药学上可以接受的盐，仍属于本发明的保护范围。
SEQ ID No.1～SEQ ID No.8所示的肽其通过化学合成或通过重组技术获得，其不但可以与蛋白融合也可以与聚合物偶联，还可以与载体连接。进一步研究发现，该组肽具有特殊的结构，SEQ ID No.1～SEQ ID No.8所示的任一具有镇痛作用的氨基酸序列或它们药学上可以接受的盐，其中，首端和末端的两个半胱氨酸形成环。如下述结构：
Figure PCTCN2017118520-appb-000001
实施例
1.实验动物、仪器和试剂
成年Sprague-Dawley雄性大鼠(8周以上)，体重180g-220g，购买于山东济南朋悦实验动物繁育有限公司。
角叉菜胶(Carrageenan,简写为Carr)为美国sigma公司产品，分别配制1％角叉菜胶(0.1g角叉菜胶溶于10ml 0.9％生理盐水溶液中，分装至10个EP管)和2％角叉菜胶(0.2g角叉菜胶溶于10ml 0.9％生理盐水溶液中，分装至10个EP管)，于-20℃保存，使用前半小时取出放置冰上。
GsMTx-4从美国alomone labs购买，SEQ ID No.1～SEQ ID No.8所有多肽均用高温灭菌后的去离子水(DD water)配制成5mM或10mM的母液，并分装在EP管后于-80℃保存，使用时用生理盐水稀释至实验所需的浓度。
38500-PAM压力应用测量系统(Μgo Basile Biological Research Apparatμs,Comerio–Varese,Italy)由两部分组成，一部分为手持单元，另一部分为综合电子单元。手持单元为力传感器，该力传感器是根据Randall-Selitto测痛仪而设计，综合电子单元可自动记录施加在大鼠左侧后足足底最大的机械缩足反射阈值。
2.炎性痛模型的制备：我们采取两种不同的炎性痛模型制备方法，分别用于足底和腹腔药物注射的镇痛效果进行检测：
2-1：6μl 1％角叉菜胶炎性痛模型：按照文献(Alessandri-Haber N,et al.Neurosci.Vol 29(19),pp 6217-6288,2009)；介绍的方法制备角叉菜胶炎性痛模型。该法用于足底注射给药方式药物镇痛效果的检测。简单地说：从大鼠左后足2、3趾之间进针，注射1％角叉菜胶，注射量为6μl，该注射浓度可诱导产生痛觉过敏，即：在注射后半小时内大鼠足底即出现肿胀和痛觉阈值的降低。机械缩足反射阈值在50gf以下者作为角叉菜胶炎性痛造模成功大鼠。炎性阈值在50gf以上的炎性模型大鼠给予剔除。
2-2：50μl 2％角叉菜胶炎性痛模型：按照文献(Seung Pyo Park,et al.2008.Pain.137:208-217)介绍的方法制备2％角叉菜胶炎性痛模型。该法用于腹腔注射给药方式药物镇痛效果的检测。简单地说：从大鼠左后足2、3趾之间进针，注射2％角叉菜胶50μl，该注射浓度可诱导产生痛觉过敏，即：在注射后半小时内大鼠足底即出现较严重的肿胀和痛觉阈值的降低。机械缩足反射阈值降低在50gf以下的作为角叉菜胶炎性痛造模合格大鼠。炎性阈值在50gf以上的炎性模型大鼠给予剔除。
3.实验分组和动物处理
所有实验大鼠从山东济南朋悦动物有限公司购入的6周龄SD大鼠。实验大鼠适应性饲养2～4周，饲养环境温度为22～26℃，湿度30％～50％，适应性饲养后每次选择体重180～220g的大鼠10～12只，测量大鼠左侧后足机械缩足阈值，取每只大鼠测量5次的均值作为该大鼠炎性造模前基础值(Baseline,缩写为BL)，剔除基础值差异大或对机械刺激无反应的大鼠；然后按照文献介绍的方法于大鼠左侧后足足底皮内注射6μl 1％角叉菜胶(用于足底注射加药方式)或50μl 2％角叉菜胶(用于腹腔注射加药方式)，1小时后检测大鼠左侧后足机械性缩足阈值，剔除造模失败(炎性阈值在50gf以上)的大鼠，将造模成功的大鼠平均分为药物组和生理盐水组两组。以上操作中凡是有出现造模不成功、大鼠被剔除的情况，再加做实验以确保每组用于统计数据的大鼠至少为5～8只。
4.GsMTx-4及其短肽在炎性诱发痛觉过敏中的作用——机械缩足反射阈值的测定
4.1足底注射吗啡、GsMTx-4及短肽抑制炎性痛觉过敏的测定
用38500-PAM压力应用测量系统测定大鼠的左后足机械性缩足阈值来评价GsMTx-4及其短肽在大鼠机械性痛觉过敏中的作用。所有大鼠行为学测试之前需对大鼠进行缩足训练，直到每只大鼠都会缩足后开始记录大鼠基础缩足阈值。将当天需测的10～12只大鼠放入同一鼠笼中，在各只大鼠的尾巴上从1～10(或12)进行编号，而后让大鼠在测量房间适应30min，待大鼠安静后开始测试。根据Randall-Selitto测痛仪设计的力传感器安装在38500-PAM压力应用测量系统的一个手持装置单元中。测量系统设置最小的力值为0.45gf，最大的力值为450gf。操作人员运用38500-PAM压力应用测量系统依次按编号数字大小依次测量每只大鼠左侧后足机械缩足阈值。具体方法为先将手持单元中的锥形尖端放在大鼠左侧后足足底中间部位，接着以恒定力率(30gf/s)给大鼠左侧后足施加一个力，所施加力的最大时间为15s，15s时间内当受试大鼠后足回缩时，操作员停止施力，此时电子单元将自动记录施加在大鼠左侧后足足底最大的机械性缩足阈值，若受试大鼠15s后左侧后足仍未出现缩足反射，系统会自动报警，操作者即停止测量，10min后重复测量。若某只大鼠多次15s后左侧后足未出现缩足反射，剔除此大鼠。依次按照大鼠尾巴编号数字大小依次测量，重复测量5次(每次刺激间隔时间为10min)，取5次实验的平均值，。 测量完毕后足底皮内注射6μl 1％角叉菜胶，建立大鼠炎性疼痛模型。注射角叉菜胶1小时后检测大鼠左侧后足机械性缩足阈值，剔除阈值大小异常的大鼠(造模失败)。将炎性阈值较高与阈值较低的大鼠混合平均分为2组：药物组和生理盐水组，每组5～6只，使得两组大鼠炎性阈值分布相似。药物组在大鼠左侧后足足底皮内注射1.2μg/5μl GsMTx-4(用前稀释到合适浓度)或其中一种短肽，或5mg/kg吗啡，或大鼠左侧后足足底皮下相同位置注射5μl等体积的生理盐水，注药后分别于1h、3h、5h、7h及24h检测各组大鼠左侧后足机械缩足反射阈值，每只大鼠均重复测试5次(每次测试间隔时间为10min)，取5次缩足反射阈值的平均值为该大鼠实际缩足反射阈值(单位为：g)。
注：以上操作中凡是出现造模不成功、大鼠被剔除的情况，再加做实验以确保每组用于实验统计数据的大鼠至少为5～8只。
4.2腹腔注射GsMTx-4及其短肽抑制炎性痛觉过敏的测定
腹腔注射GsMTx-4及其短肽抑制炎性痛觉过敏的方法步骤与足底注射相似，但有几点不同：
(1)该测量系统设置最小的力值为0.75gf，最大的力值为750gf
(2)考虑到腹腔注射吗啡可能导致机械性缩足阈值大幅度上升，将所施加力的最大时间调为25s
(3)药物组腹腔注射270μg/kg GsMTx-4或其中一种短肽或10mg/kg吗啡，生理盐水组的腹腔注射量为等质量的无菌生理盐水。
基于上述实验，图1为GsMTx-4、吗啡和SEQ ID No.1通过足底皮下注射给药方式(注射体积均为6μl)对大鼠痛觉过敏抑制效果的示意图。其中，A图所示为事先在大鼠左后足足底皮内注射6μl 1％角叉菜胶(Carr)诱导大鼠炎性模型后，在大鼠足底肿胀部位注射5μl的生理盐水1h，3h，5h后，大鼠机械缩足阈值与炎性阈值无明显差异；而与造模前机械缩足反射阈值相比，等体积量(5μl)的吗啡(B)、GsMTx-4(C)或SEQ ID No.1(D)都明显降低了大鼠机械缩足反射阈值。比如大鼠足底肿胀部位皮下注射等体积的GsMTx-4或短肽SEQ ID No.1(注射量为1.2μg/5μl)1h，3h，5h后，大鼠机械缩足阈值均有增高，且在注药后3h达到最高。注意吗啡在给药1h后药效达最大值，但大鼠机械缩足阈值的增高大大超过了大鼠基线(Base Line,简写为BL)时的机械缩足阈值。我们认为超过基线部分可能与吗啡的成瘾性、赖受性和其毒性相关。 E图是以在大鼠左后足足底皮内注射6μl 1％角叉菜胶(Carr)诱导大鼠炎性模型后比较GsMTx-4、短肽SEQ ID No.1和生理盐水以及吗啡对大鼠机械性痛觉过敏中的抑制作用。
图2是GsMTx-4、吗啡和SEQ ID No.1通过腹腔注射给药方式(注射体积为50μl)对大鼠痛觉过敏抑制作用的对比示意图。我们得到了和前面图1(足底注射)相似的对痛觉过敏抑制作用，但GsMTx-4和SEQ ID No.1具有比从足底皮内注射给药的方式时更强的抑制作用：它们在3h后达到最大抑制效果，并且都可以完全翻转由角叉菜胶(Carr：注射量为50μl 2％)诱导的炎性疼痛阈值(B,C)，而生理盐水没有作用(A)。此外，吗啡(注射量为10mg/Kg)在注射1h后对大鼠痛觉过敏抑制作用达到最高，虽然比GsMTx-4(注射量为270μg/Kg，50μl)和SEQ ID No.1(注射量为270μg/Kg)的镇痛作用高出一倍(见E)，但是同足底注射给药方式结果类似：吗啡虽然镇痛作用更强，但却对大鼠机械缩足阈值的提高已经大大超过了大鼠基线阈值(Base Line,简写为BL)，我们认为这部分与吗啡的毒性相关。此外，3h后吗啡的镇痛作用迅速下降，达到与GsMTx-4和SEQ ID No.1具有相似镇痛作用的效果(E)。由于本发明提出的即使具有最强镇痛作用的短链多肽对正常大鼠痛觉均没有影响(见图9)，因此本发明发现的多肽有可能发展成为治疗痛觉过敏的最好镇痛药物或混合剂。
图3是SEQ ID No.1与全长GsMTx-4镇痛作用的对比示意图。SEQ ID No.1和GsMTx-4通过足底注射方式给药时，镇痛作用没有明显差异(图中A、C)，其中C是SEQ ID No.1(右)和全长GsMTx-4(左)通过足底注射方式时镇痛作用的剂量依赖图。但当两者分别通过腹腔注射方式给药时，SEQ ID No.1可能具有更强的镇痛效果(图中B)。
图4是SEQ ID No.2分别通过足底(A)、腹腔(C)给药方式的镇痛作用示意图。为了更好比较短链多肽的镇痛效果，等量全长GsMTx-4的镇痛效果由浅灰色表示作对比(B,D)。其中A图示出在大鼠左后足足底皮内注射6μl 1％角叉菜胶(Carr)后，与造模前机械缩足反射阈值相比，明显降低大鼠机械缩足反射阈值，足底皮下注射SEQ ID No.2(注射量为1.2μg/5μl)1h，3h，5h，7h后，大鼠机械缩足阈值增高，且在注药后1-3h达到最高。C图示意在大鼠左后足足底皮内注射50μl 2％角叉菜胶(Carr)后1h,腹腔注射SEQ ID No.2(注 射量为270μg/Kg)1h，3h，5h，7h后大鼠机械缩足阈值均有增高，注药3h时抑制作用最强，很明显腹腔注射给药的镇痛作用强于足底局部给药方式。此外腹腔注射SEQ ID No.2具有与全长GsMTx-4相似的镇痛作用(D)。
图5是SEQ ID No.3分别通过足底(A)、腹腔(C)给药方式对痛觉过敏的抑制作用示意图。其中，A图所示在大鼠左后足足底皮内注射6μl 1％角叉菜胶(Carr)后，与造模前机械缩足反射阈值相比，足底皮下注射SEQ ID No.3(注射量为1.2μg/5μl)1h，3h，5h，7h后明显降低大鼠机械缩足反射阈值，且在注药后3h达到最高。B图所示SEQ ID No.3与在足底皮下注射等质量、等体积GsMTx-4(1.2μg/5μl)或生理盐水1h，3h，5h，7h后大鼠机械缩足反射阈值的比较，SEQ ID No.3对痛觉过敏的抑制作用与全长多肽GsMTx-4短肽没有明显差异(B)，生理盐水组大鼠机械缩足阈值并未改变。C图图示在大鼠左后足足底皮内注射50μl 2％角叉菜胶(Carr)后，通过腹腔注射SEQ ID No.2(注射量为270μg/Kg)1h，3h，5h，7h后与造模前机械缩足反射阈值相比，大鼠机械缩足阈值均明显增高，且在注药后3h完全翻转大鼠的机械缩足阈值，与大鼠基线(造模前的baseline:BL)没有明显差异。因此通过腹腔注射方式给药时SEQ ID No.3可能具有比等质量全长多肽GsMTx-4更强的镇痛效果(D)。
图6是SEQ ID No.4分别通过足底(A)、腹腔(C)给药方式对痛觉过敏的抑制作用示意图。其中，A图所示在大鼠左后足足底皮内注射6μl 1％角叉菜胶(Carr)后，与造模前机械缩足反射阈值相比，足底皮下注射SEQ ID No.4(注射量为1.2μg/5μl)1h，3h，5h，7h后明显降低大鼠机械缩足反射阈值，且在注药后3h达到最高。B图所示为SEQ ID No.4与在足底皮下注射等质量/等体积GsMTx-4(1.2μg/5μl)或等体积(5μl)的生理盐水1h，3h，5h，7h后大鼠机械缩足反射阈值的比较，SEQ ID No.4对痛觉过敏的抑制作用比全长多肽GsMTx-4短肽稍弱。C图示在大鼠左后足足底皮内注射50μl 2％角叉菜胶(Carr)诱导炎性肿胀后，通过腹腔注射SEQ ID No.4(注射量为270μg/Kg)与造模前机械缩足反射阈值相比，1h，3h，5h，7h后大鼠机械缩足阈值均明显增高，且在注药后3h后大鼠机械缩足阈值与大鼠基线(BL)没有明显差异。因此通过腹腔注射方式给药时，SEQ ID No.4的镇痛作用与等质量全长多肽GsMTx-4的镇痛作用没有明显差异(D)。
图7示出了SEQ ID No.5、SEQ ID No.6、SEQ ID No.7三种短肽通过腹 腔注射方式给药时对大鼠痛觉过敏的抑制作用。其中，A图示出足底皮下注射SEQ ID No.5(1.2μg/5μl)1h，3h，5h，7h后，与Carr炎性模型大鼠机械缩足阈值相比，机械缩足阈值都有明显的提高，且在3h达到最高。B图示出了足底皮下注射SEQ ID No.6(1.2μg/5μl)1h，3h后，与Carr炎性阈值相比，机械缩足阈值都有明显提高，且在3h达到最高。而C图示出足底皮下注射SEQ ID No.7(1.2μg/5μl)1h，3h，5h，7h后，与Carr炎性阈值相比，机械缩足阈值都有明显的提高，且在3h达到最高。D图示出足底皮下注射SEQ ID No.8(1.2μg/5μl)1h，3h，5h，7h后，与Carr炎性阈值相比，机械缩足阈值都有明显的提高，且在3h达到最高。
图8示出了SEQ ID No.5和SEQ ID No.8通过腹腔注射方式给药时对大鼠炎性疼痛的镇痛作用。其中A和C图分别表示在大鼠左后足足底皮内注射50μl 2％角叉菜胶(Carr)诱导炎性疼痛后，再分别通过腹腔注射SEQ ID No.5(A图)或SEQ ID No.8(C图)(注射量均为270μg/Kg)。与大鼠炎性模型机械缩足反射阈值(Carr)相比，1h，3h，5h，7h后大鼠机械缩足阈值均明显增高，且均在注药后3h达最大值。因此，腹腔注射SEQ ID No.5(B图)和SEQ ID No.8(D图)与全长GsMTx-4具有相似镇痛作用，其中SEQ ID No.8作用略为更强，维持时间更长。5h后与全长多肽的镇痛作用没有明显差异(D图)。
图9图示GsMTx-4、SEQ ID No.1和SEQ ID No.3对正常大鼠的机械缩足阈值没有影响。A图示在正常大鼠左后足足底皮下直接注射吗啡(注射量为5mg/kg,该剂量通常为吗啡用药量的最小剂量)1h后，吗啡进一步强烈抑制正常大鼠的机械缩足反射阈值一倍以上(阈值从正常大鼠的平均～250g增长到550g)，我们认为这与吗啡发挥镇痛作用的同时，具有的成瘾性和毒性相关。B,C,D图分别图示在正常大鼠左后足足底皮下直接注射GsMTx-4(A图)、SEQ ID No.1(B图)或SEQ ID No.3(C图)(注射量均为1.2μg/5μl，该剂量为GsMTx-4和SEQ ID No.1通过足底注射方式给药时抑制大鼠痛觉过敏的最大剂量)1h，3h，5h，7h后，均不影响正常大鼠的机械缩足反射阈值。由于GsMTx-4、SEQ ID No.1和SEQ ID No.3是本发明所涉及肽中具有最强镇痛作用的多肽短链，因此该组实验从反面再次证明本发明涉及的多肽对正常大鼠没有副(毒性)作用。
药物组合物
本公开内容的肽可以配制成例如给予受治疗者以治疗疼痛的药物组合物。肽可以单独给予，或者可在同一组合物中或作为单独的组合物，与其它疼痛疗法联合给予。
药物组合物通常包括药学上可接受的载体。本文所用的“药学上可接受的载体”包括任何及所有生理上相容的溶剂、分散介质、包衣材料、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。组合物可包括药学上可接受的盐，例如酸加成盐或碱加成盐。
药物制剂技术是普遍接受的技术，更多详情参见例如Gennaro，The Science and Practice of Pharmacy，第20版，Lippincott，Williams&Wilkins(2000)(ISBN：0683306472)；Ansel等，Pharmaceutical Dosage Forms and Drug Delivery Systems，第7版，Lippincott Williams&Wilkins Publishers(1999)(ISBN：0683305727)；以及Kibbe，Handbook of Pharmaceutical Excipients American Pharmaceutical Association，第3版(2000)(ISBN：091733096X)。
在一个实施方案中，所述肽与赋形剂材料(例如盐水、氯化钠、磷酸氢二钠七水合物、磷酸二氢钠)和稳定剂一起配制。还可在例如缓冲溶液中按合适的浓度提供。
药物组合物可呈各种形式。这些包括例如液体、半固体和固体剂型，例如液体溶液剂(例如注射和输注溶液剂)、分散剂或混悬剂、片剂、丸剂、散剂、脂质体和栓剂。优选的形式可取决于既定的给药方式和治疗应用。
可通过胃肠外方式(例如静脉内、皮下、腹膜内或肌内注射)给予组合物。本文所用术语“胃肠外给药”和“经胃肠外给予”是指肠内和局部给药以外的给药方式，通常经注射给药，包括而不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心脏内、真皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外及胸骨内注射和输注。
可经肠内途径给予组合物，例如通过消化道，例如经口服给药。例如，可以片剂、胶囊剂、囊片剂、丸剂、散剂、滴剂、混悬剂、溶液剂、糊剂、凝胶剂或其它口服剂型给予组合物。
肠内途径包括经胃饲管、十二指肠饲管或胃造口或直肠给予，例如以栓剂或灌肠剂形式的组合物。
组合物可局部给药，例如在疼痛部位。局部给药包括例如表皮、鼻内、吸入和阴道给药。可将组合物给予皮肤(例如烧伤、疱或破口)、唇、牙龈、牙、口腔、眼、耳、甲床或咽喉等，例如疼痛部位。局部给药的组合物可为乳膏剂、凝胶剂、洗剂或软膏剂等。
药物组合物可包括“治疗有效量”的本文所述的肽。该有效量可根据所给药物(例如肽)的效果确定，或者如果使用一种以上药物时，则根据联用效果确定。治疗有效量的药物还可根据以下因素而变化：例如受治疗者的疼痛类型、疾病状况、年龄、性别和体重以及药物在受治疗者体内引起所需反应(例如改善疼痛)的能力。治疗有效量也是其中组合物的治疗有益作用超过任何毒性或有害作用的量。
基于本发明，将来有可能获得一种新型的、用于镇痛的多肽药物，或含有该多肽的有效镇痛混合物。本发明具有以下优势：
1.原生蜘蛛毒素GsMTx-4多肽在水中形成四个环状结构，合成时要求多肽必须折叠成保持该原生多肽的空间结构才具有相应药效，因此合成工序繁琐成本高。本发明找到了原生多肽中起镇痛作用的药效团，因此降低了多肽的合成成本，使在工业上大批量生产变得高效而廉价；
2.由于本发明缩减了多肽长度，降低了免疫原性，更易于人体吸收。在临床应用中更能充分发挥药效。由于该多肽对正常大属的疼痛阈值没有影响，和吗啡等药物相比无成瘾性、无赖药性和无毒性。因此本发明可能被开发为临床上广泛应用的镇痛药物。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (10)

  1. 一组肽，其特征在于：具有如(1)～(8)所示的任一氨基酸序列：
    (1)如序列表SEQ ID No.1所示的氨基酸序列；
    (2)如序列表SEQ ID No.2所示的氨基酸序列；
    (3)如序列表SEQ ID No.3所示的氨基酸序列；
    (4)如序列表SEQ ID No.4所示的氨基酸序列；
    (5)如序列表SEQ ID No.5所示的氨基酸序列；
    (6)如序列表SEQ ID No.6所示的氨基酸序列；
    (7)如序列表SEQ ID No.7所示的氨基酸序列；
    (8)如序列表SEQ ID No.8所示的氨基酸序列；或，
    它们药学上可以接受的盐。
  2. 根据权利要求1中所述的肽，其特征在于：在首端和末端半胱氨酸存在的基础上，权利要求1所述(1)～(8)任一肽中，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，它们药学上可以接受的盐。
  3. 根据权利要求1或2所述的肽，其特征在于：所述(1)～(8)所示的任一具有镇痛作用的氨基酸序列或它们药学上可以接受的盐，其中，首端和末端的两个半胱氨酸形成环。
  4. 根据权利要求3所述的肽，其特征在于：其通过化学合成或通过重组技术获得。
  5. 根据权利要求1、2或4任一所述的肽，其特征在于：其与蛋白融合。
  6. 根据权利要求1、2或4任一所述的肽，其特征在于：其与聚合物偶联。
  7. 根据权利要求1、2或4任一所述的肽，其特征在于：所述肽与载体连接。
  8. 一种药物组合物，其特征在于，包括，
    (1)有效量的作为活性成分的如权利要求1～7任一所述的肽；
    (2)选择性的药学上可接受的载体。
  9. 根据权利要求7的药物组合物，其特征在于，所述药学上可接受的载体选自：溶剂、稀释剂、悬浮剂、乳化剂、抗氧化剂、药学防腐剂、着色剂、香味剂、介质、油性基底、赋形剂中的一种或几种。
  10. 一组如权利要求1、2或4任一所述的肽或它们药学上可以接受的盐或如权利要求8或9所述的药物组合物在镇痛方面的应用。
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