WO2018108185A1 - 一组具有镇痛作用的肽及其药物组合物和应用 - Google Patents

一组具有镇痛作用的肽及其药物组合物和应用 Download PDF

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WO2018108185A1
WO2018108185A1 PCT/CN2017/116992 CN2017116992W WO2018108185A1 WO 2018108185 A1 WO2018108185 A1 WO 2018108185A1 CN 2017116992 W CN2017116992 W CN 2017116992W WO 2018108185 A1 WO2018108185 A1 WO 2018108185A1
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seq
pharmaceutically acceptable
pain
peptide
rats
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PCT/CN2017/116992
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • 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 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 6 cysteine residues form a three-pair cysteine knot (ICK: Inhibitor cysteine Knot motif) structure. Characteristics (Robert. et al., J. Biol. Chem. 277 (37): 34443-34450, 2002). This structure limits its yield during chemical synthesis and biosynthesis because it is difficult to fold into the correct conformation. Although recent patents have been used to direct the synthesis of full-length GsMTx-4 polypeptides in yeast, cost and yield remain a bottleneck in drug development.
  • the inventors applied the patents (Application No.: 201611128087.6) on December 9, 2016 by comparing various polypeptide sequences containing cysteine knots (ICK) such as GsMTx-4 and GsMTX-2, and by pain in rats.
  • ICK cysteine knots
  • the behavioral test of the model identified a pharmacophore that acts as an analgesic effect in the GsMTx-4 polypeptide (located in the domain of loop 2) and found that part of the polypeptide fragment of GsMTx-4 is not inferior to the full-length inhibition of pain. .
  • 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. Higher (eg, 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. However, it is limited to analgesic effects after one or more amino acids in Loop 2 are deleted, replaced, or added.
  • the polypeptide is shown in the following structural formula:
  • Loop 3 It is not known whether one or more amino acids in Loop 3 are still analgesic after being deleted, replaced, or added.
  • 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 solution: a group of an analgesic peptide having an amino acid sequence as shown in (1) or (2): (1) The amino acid sequence shown in SEQ ID No. 1 of the Sequence Listing; (2) the amino acid sequence shown in SEQ ID No. 2 of the Sequence Listing or a pharmaceutically acceptable salt thereof.
  • a preferred embodiment of the peptide of the present invention wherein: based on the presence of the head end and the terminal cysteine, the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 of the Sequence Listing, A polypeptide having an analgesic effect after deletion, substitution, or addition of a plurality of amino acids, or a pharmaceutically acceptable salt thereof.
  • a preferred embodiment of the peptide of the present invention wherein: an analgesic amino acid sequence represented by SEQ ID No. 1 or SEQ ID No. 2 of the Sequence Listing or a pharmaceutically acceptable salt thereof, wherein The two cysteines at the ends and ends form a loop.
  • a preferred embodiment of the peptide of the present invention wherein: it is synthesized by chemical synthesis or by recombination Obtained.
  • 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 present invention is to provide an analgesic effect of a group of analgesic peptides or their pharmaceutically acceptable salts or pharmaceutical compositions.
  • the present inventors have found that a part of the polypeptide fragment of GsMTx-4 has an effect of suppressing pain which is not inferior to the full length, and determines a pharmacophore which plays an analgesic role in the GsMTx-4 polypeptide by behavioral detection of a rat pain model ( Domain).
  • the present invention thus forms a polypeptide by deleting, replacing, or adding one or more amino acids in Loop 3 of GsMTx-4, and then screening the polypeptide for an analgesic polypeptide.
  • 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 and morphine on hyperalgesia in rats using local injection of the plantar.
  • Saline normal saline
  • Morphine a positive control.
  • Figure 2 is a graphical representation of the analgesic effect of SEQ ID No. 1 and morphine on hyperalgesia in rats by intraperitoneal administration.
  • Figure 3 is a short-chain polypeptide SEQ ID No. 1 and full-length GsMTx-4 through the sole (A), abdominal cavity (B) Comparison of analgesic effects of injection methods.
  • Figure 4 is a schematic view showing the analgesic effect of SEQ ID No. 2 on the rats by intraplantal (A) or intraperitoneal (B) injection, respectively, in which the same dose is passed through the sole with normal saline (Saline).
  • A or intraperitoneal (B) injection mode of administration as a negative control of the analgesic effect of the polypeptide.
  • Figure 5 is a graphical representation of SEQ ID No. 1 (A) and SEQ ID No. 2 (B) having no effect on the pain threshold of normal rats when no pain model is established, wherein morphine has an effect on the pain threshold of normal rats. Positive control.
  • Fig. 6 is a schematic view showing the analgesic effect of the comparative example, in which the polypeptide P13573 of the comparative example showed no significant analgesic effect.
  • 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.
  • 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.). These peptides have reduced pain sensitivity and therefore have an analgesic effect. The peptide produces long-lasting analgesic and/or antinociceptive effects.
  • Pain is a phenomenon of integrated sensation, emotion, and cognition. For patients suffering from pain, there is no physical pathological change. Conceptually, pain can be thought of as consisting of three levels: sensory-resolved composition (eg, location, intensity, nature), motivation-emotional composition (eg, depression, anxiety), and cognitive-evaluation components (eg, regarding pain causes and Cognition of meaning).
  • sensory-resolved composition eg, location, intensity, nature
  • motivation-emotional composition eg, depression, anxiety
  • cognitive-evaluation components eg, regarding pain causes and Cognition of meaning.
  • the basis for the perception of pain is the sensory neuron (nociceptor) system and the specific afferent pathways that specifically respond to potentially harmful tissue-damaging stimuli.
  • the activity of the afferent pathway is affected by non-nociceptive pathophysiology (eg, abnormal processing of the nervous system) or psychological factors.
  • Nociceptive Pain Mechanism In clinical practice, pain can be referred to as "nociception” if it can be inferred that pain is associated with nociceptor stimulation and subsequent sensory nerve fiber activation as a result of tissue damage initiation. Nociceptive pain involves the normal activation of the nociceptive system by unwanted stimuli. Nociception consists of four processes: transduction, transmission, perception, and regulation.
  • the nociception process begins with transduction (depolarization) on peripheral nociceptors in response to noxious stimuli. Delivery is the process by which these stimuli travel along the primary afferent nociceptive axons to the spinal cord and then to the advanced center. Only when the impulse reaches the brain can it be recognized as pain through brain power. This is perception.
  • the ultimate pain perception depends on the activity of the afferent system and its regulation at multiple levels of the nervous system. Pain regulation depends on the activity of the endogenous opiidergic system and other pain regulation systems.
  • opioid energy system analgesia is mediated by binding of endogenous opioid compounds to opioid receptors, primarily mu, delta and kappa opioid receptors.
  • Endogenous opioids are widely distributed and are closely related to systems known to regulate homeostasis, respond to stress and pain.
  • other neurotransmitters such as serotonin and norepinephrine also play a role in the endogenous pain regulation system, but the present invention relates to the possibility that the polypeptide may directly act by inhibiting certain specific force-sensitive ion channels.
  • the action potential is prevented from being produced or further transmitted to the analgesia in the nerve, which is why our peptide does not need to pass the blood-brain barrier to exert a pharmacodynamic mechanism.
  • 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.
  • the peptides described herein can be prepared in a biological system or chemically synthesized.
  • peptides can be prepared by recombinant DNA techniques.
  • an expression vector encoding a nucleic acid sequence comprising a peptide described herein can be introduced into a biological system (e.g., a bacterial, yeast, plant, insect or mammalian expression system) and expressed using standard techniques.
  • the peptide is then purified from biological systems (e.g., cells or culture media) using standard purification techniques (e.g., using peptide-based physical or chemical separation techniques or affinity purification techniques).
  • standard purification techniques e.g., using peptide-based physical or chemical separation techniques or affinity purification techniques.
  • Such techniques are known in the art. See, for example, Current Protocols in Molecular Biology, 3rd edition, John Wiley and Sons, Inc, New York, NY.
  • Peptides can be synthesized chemically, for example by liquid phase synthesis or solid phase synthesis. Such techniques are standard techniques in the art, see, for example, Atherton, E., Sheppard, RC Solid Phap synthesis: Practical approach. IRL Press, Oxford, England, 1989; Stewart JM, Young, JDSolid phase peptide synthesis, 2nd edition , Pierce Chemical Company, Rockford, 1984; Carpino, LA 19921-Hydroxy-7-azabenzotriazole. An efficient Peptide Coupling Additive (1-hydroxy-7-azabenzotriazole: an effective peptide coupling additive). J .Am.Chem.Soc. 115, 4397-4398. The peptide is synthesized by coupling the carboxy terminus or C terminus of one amino acid to the amino terminus or the N terminus of the other.
  • Liquid phase synthesis is a classic peptide synthesis method. It can be used in industrial large scale production of peptides.
  • Solid phase synthesis Solid phase peptide synthesis (SPPS) is a currently accepted method for the synthesis of peptides and proteins in a laboratory. SPPS allows, for example, the synthesis of natural peptides that are difficult to express in bacteria, the incorporation of non-natural amino acids, peptide/protein backbone modifications, and the synthesis of D-proteins and D-peptides containing D-amino acids. The synthesis can be carried out by hand, for example, as described herein, or by an automated synthesizer.
  • One of the objects of the present invention is to provide a group of peptides having an analgesic action (such as SEQ ID No. 1 or SEQ ID No. 2).
  • 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 and SEQ ID No. 2 are obtained by chemical synthesis or by recombinant techniques, which may be fused not only to proteins but also to polymers, and may also be linked to a carrier. Further studies have found that the group of peptides has a special structure, any of the analgesic amino acid sequences shown in SEQ ID No. 1 or SEQ ID No. 2 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 polypeptides of SEQ ID No. 1 and SEQ ID No. 2 were formulated into 5 mM or 10 mM mother liquor with high temperature sterilized deionized water (DD water) and dispensed into EP tubes. 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 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 SEQ ID No. 1 and morphine on hyperalgesia in rats by subcutaneous injection of 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.
  • Carr carrageenan
  • the rat's plantar swelling site was injected subcutaneously with an equal volume of short peptide SEQ ID No. 1 (injection volume of 1.2 ⁇ g/5 ⁇ l) for 1 h, 3 h, 5 h, 7 h, and the mechanical shrinkage threshold of the rats increased. The highest was reached 3 hours after the drug.
  • Fig. 2 is a comparative diagram showing the inhibitory effect of SEQ ID No. 1 and morphine on hyperalgesia by intraperitoneal administration (injection volume of 50 ⁇ l).
  • SEQ ID No. 1 has a stronger inhibitory effect than the mode of intradermal injection from the plantar, ie: The cross-peptide SEQ ID No. 1 (injection volume 50 ⁇ l, 270 ⁇ g/Kg) was injected for 1 h, 3 h, 5 h, 7 h after the inflammatory pain threshold induced by carrageenan (Carr: injection volume 50 ⁇ l 2%).
  • the analgesic effect of morphine at 3 h had rapidly decreased to baseline (BL). Since the short-chain polypeptide of the present invention which has an analgesic effect has no effect on the pain sensation of normal rats (see Fig. 5), the polypeptides discovered by the present invention may develop into a better analgesic drug or a mixture for treating hyperalgesia. Agent.
  • 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 When GsMTx-4 was administered by plantar injection, the analgesic effect was slightly weaker after 3 h ( Figure A), but when both were administered by intraperitoneal injection, SEQ ID No. 1 and full-length GsMTx were 3 h later. There was no significant difference in the analgesic effect of -4 (B in the figure).
  • Figure 4 is a schematic illustration of the analgesic effect of SEQ ID No. 2 by means of the sole (A) and the abdominal cavity (B), respectively.
  • the top two panels in Figure 4 are negative controls when an equal volume of saline is injected.
  • the A map indicates that the foot is injected subcutaneously with SEQ ID No. 2 (injection amount is compared with the rat mechanical contraction threshold after intradermal injection of 6 ⁇ l of 1% carrageenan (Carr) in the left hind foot of the rat. After 1.2 ⁇ g/5 ⁇ l) 1h, 3h, 5h, 7h, the threshold of mechanical contraction of rats increased, and reached the highest 3h after injection.
  • Carr carrageenan
  • Panel B 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 was obvious that the analgesic effect of intraperitoneal injection was stronger than that of the sole.
  • Figure 5 shows that SEQ ID No. 1 and SEQ ID No. 2 have no effect on the mechanical withdrawal threshold of normal rats.
  • a and B images respectively show direct injection of SEQ ID No 1 (A) or SEQ ID No. 2 (B) subcutaneously in the left hind foot of normal rats (the injection volume is 1.2 ⁇ g/5 ⁇ l, the dose is the full length
  • the maximum dose of GsMTx-4 and SEQ ID No.1 inhibiting hyperalgesia in rats when administered by plantar injection did not affect the mechanical paw withdrawal reflex threshold of normal rats after 1 h, 3 h, and 5 h.
  • C 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).
  • morphine further strongly inhibits mechanical contraction reflex in normal rats.
  • the threshold is more than doubled.
  • This part of the threshold (BL) is related to the analgesic effect of morphine and the addiction and dependence.
  • the analgesic peptide required for the present invention needs to be screened. See Fig. 6, which is a schematic diagram of the analgesic effect of the comparative example. In the figure, the comparative peptide P13573 (WA C NPNAAA C A) is not obvious. Analgesic effect.
  • 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.
  • 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, epidermis, intranasal, and suction Into 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 inexpensive.
  • 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.

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Abstract

一组具有镇痛作用的肽及其药物组合物和应用，其具有如序列表SEQ ID No.1或序列表SEQ ID No.2所示的氨基酸序列或，它们药学上可以接受的盐。其在首端和末端半胱氨酸存在的基础上，如序列表SEQ ID No.1或SEQ ID No.2所示的氨基酸序列，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。对GsMTx-4的Loop 3中一个或多个氨基酸的删除、置换、或添加后形成多肽，然后对所述多肽筛选出具有镇痛作用的多肽。通过多肽对大鼠伤害性疼痛模型的检测结果，证实得到了具有镇痛作用的多肽短链系列。

Description

一组具有镇痛作用的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)。
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。
发明人于2016年12月9日申请的专利(申请号：201611128087.6)中通过比较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(位于环2的结构域)，发现了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。但其限于对Loop 2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，如下结构式所示：
Figure PCTCN2017116992-appb-000001
对于环3(Loop 3)中一个或多个氨基酸被删除、置换、或添加后是否仍具有镇痛作用，目前尚未可知。
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：一组具有镇痛作用的肽，其具有如(1)或(2)所示的氨基酸序列：(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，如序列表SEQ ID No.1或SEQ ID No.2所示的氨基酸序列，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：如序列表SEQ ID No.1或SEQ ID No.2所示的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具有镇痛作用的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，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)。本发明由此对GsMTx-4的Loop 3中一个或多个氨基酸的删除、置换、或添加后形成多肽，然后对所述多肽筛选出具有镇痛作用的多肽。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。
图2是利用腹腔注射给药方式，SEQ ID No.1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。
图4是SEQ ID No.2分别通过足底(A)或腹腔(B)注射给药方式时对大鼠镇痛作用示意图，图中分别是用普通生理盐水(Saline)以相同剂量通过足底(A)或腹腔(B)注射给药方式时作为多肽镇痛效果的负性对照。
图5是在没有建立疼痛模型时，SEQ ID No.1(A)和SEQ ID No.2(B)对正常大鼠疼痛阈值没有影响的示意图，其中，吗啡作为对正常大鼠疼痛阈值具有影响的阳性对照。
图6是对比实施例的镇痛作用示意图，图中，对比例的多肽P13573并没有明显的镇痛作用。
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。
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。
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。这些肽可以降低疼痛敏感性，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。
疼痛和伤害感受
疼痛是一种综合感觉、情绪和认知的现象，对于遭受疼痛的患者，不一定存在身体上的病理变化。可从概念上将疼痛视为由三个层次水平构成：感觉-分辨组成(例如部位、强度、性质)、动机-情绪组成(例如抑郁、焦虑)和认知-评价组成(例如关于疼痛起因和意义的认知)。疼痛的感知的支持基础在于感觉神经元(伤害性感受器)系统和具体响应潜在有害的组织损害性刺激的神经传入途径。所述传入途径的活动受非伤害感受病理生理(例如神经系统处理异常)或心理因素的影响。
伤害感受性疼痛机制：在临床上，如果可以推知疼痛与随组织损伤引发过程而来的伤害性感受器刺激和随后的感觉神经纤维活化有关，则疼痛可称为“伤害感受”。伤害感受性疼痛包括受有害刺激而正常激活伤害感受系统。伤害感受由四个过程组成：转导、传递、感知和调节。
伤害感受过程始于响应有害刺激时在外周伤害性感受器上的转导(去极化)。传递是由此将这些刺激沿初级传入伤害感受轴突行进到脊髓然后再行进到高级中心的过程。只有当冲动到达脑部时，才能通过脑力识别为疼痛。这就是感知。
最终的疼痛感知取决于所述传入系统的活动及其在多个神经系统水平上的调节。疼痛调节取决于内源性阿片样物质能系统(opioidergic system)和其它疼痛调节系统的活动。在阿片样物质能系统中，通过内源性阿片样化合物与阿片样物质受体(主要为μ、δ和κ阿片样物质受体)结合介导痛觉缺失。内源性阿片样物质分布广，并与已知调节稳态、响应应激和疼痛的系统密切相关。在这个复杂的系统中，5-羟色胺和去甲肾上腺素等其它神经递质也在内源疼痛调节系统中发挥作用，但本发明涉及多肽可能是直接通过抑制某些特殊力敏感离子通道的活性，从而阻止动作电位在神经中的产生或进一步传递到达镇痛的作用，这也是为什么我们的肽不需要通过血脑屏障即可发挥药效的作用机制。
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。
本文所述的肽可在生物系统中制备或经化学合成。
为了在生物系统中产生肽，可通过重组DNA技术制备肽。例如，可将含有本文所述肽的编码核酸序列的表达载体导入生物系统(例如细菌、酵母、植物、昆虫或哺乳动物表达系统)中，并应用标准技术进行表达。然后应用标准纯化技术(例如应用基于肽的物理或化学性质的分离技术或者亲和纯化技术)，从生物系统(例如细胞或培养基)中将肽纯化出来。这类技术为本领域所知。参见例如Current Protocols inMolecular Biology第3版，John Wiley和Sons，Inc，New York，NY。
肽可用化学方法合成，例如采用液相合成或固相合成。这类技术是本领域的标准技术，参见例如Atherton，E.，Sheppard，R.C.SolidPhase peptide synthesis：Apractical approach.IRL Press，Oxford，England，1989；Stewart J.M.，Young，J.D.Solid phase peptide synthesis，第2版，Pierce Chemical Company，Rockford，1984；Carpino，L.A.19921-Hydroxy-7-azabenzotriazole.An efficient Peptide Coupling Additive(1-羟基-7-氮杂苯并三唑：一种有效的肽偶联添加剂).J.Am.Chem.Soc.115，4397-4398。通过将一个氨基酸的羧基端或C端与另一个的氨基端或N端偶联来合成肽。
液相合成法：液相肽合成法是经典的肽合成方法。它可用于工业大规模生产肽。
固相合成法：固相肽合成法(SPPS)是目前普遍公认的在实验室以合成方式制备肽和蛋白质的方法。SPPS允许例如难以在细菌中表达的天然肽的合成、掺入非天然氨基酸、肽/蛋白质主链修饰和含有D-氨基酸的D-蛋白质和D-肽的合成。可按照例如本文所述方法以手工进行合成，或者采用自动合成仪进行合成。
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或SEQ ID No.2)。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或SEQ ID No.2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和SEQ ID No.2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或SEQ ID No.2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 PCTCN2017116992-appb-000002
实施例(本发明涉及所有实验均并采取双盲测试方法)
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和SEQ ID No.2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SEQ ID No.1和吗啡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，7h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SEQ ID No.1(B)或吗啡(C)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短肽SEQ ID No.1(注射量为1.2μg/5μl)1h，3h，5h，7h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、赖受性和其毒性均相关。
图2是SEQ ID No.1和吗啡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，即：与由角叉菜胶(Carr：注射量为50μl 2％)诱导的炎性疼痛阈值相比，短肽SEQ ID No.1(注射量为50μl，270μg/Kg)注射1h，3h，5h，7h后，大鼠机械缩足阈值均有显著增高，且在3h后达到最大抑制效果(B)，，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SEQ ID No.1(注射量为270μg/Kg)的镇痛强(见C)，但是同足底注射给药方式结果类似：吗啡的镇痛作用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时吗啡的镇痛作用已经迅速下降到基线指(BL)。由于本发明提出的即使具有镇痛作用的短链多肽对正常大鼠痛觉均没有影响(见图5)，因此本发明发现的多肽有可能发展成为治疗痛觉过敏的更好的镇痛药物或混合剂。
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，镇痛作用在3h后稍弱(图中A)，但当两者通过腹腔注射方式给药时，3h后SEQ ID No.1与全长GsMTx-4的镇痛效果没有明显差异(图中B)。
图4是SEQ ID No.2分别通过足底(A)、腹腔(B)给药方式的镇痛作用示意图。图4中最上端两图是注射等体积的生理盐水时的负性对照。其中，A图表示与大鼠左后足足底皮内注射6μl 1％角叉菜胶(Carr)后大鼠机械缩足反射阈值相比，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3h达到最高。B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。
图5图示SEQ ID No.1和SEQ ID No.2对正常大鼠的机械缩足阈值没有影响。A，B图分别图示在正常大鼠左后足足底皮下直接注射SEQ ID No 1(A)或SEQ ID No.2(B)(注射量均为1.2μg/5μl，该剂量为全长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后，均不影响正常大鼠的机械缩足反射阈值。C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，这个超出基础阈值(BL)的部分为吗啡发挥镇痛作用的同时，具有的成瘾性和赖受性有关。该组实验从反面再次证明本发明涉及的多肽对正常大鼠疼痛阈值没有影响，因而没有副(毒性)作用的特性。
本发明所需的具有镇痛作用的肽需要进行筛选，参见图6，图6是对比实施例的镇痛作用示意图，图中，对比例的多肽P13573(WACNPNAAACA)并没有明显的镇痛作用。
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。
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.目前已知的多肽均是通过血脑屏障才能发挥其药效功能，但由于多肽本身的特点它们又不能直接通过血脑屏障，因此这已是目前多肽开发为药物的瓶颈。由于本发明的多肽可以直接通过局部注射或腹腔注射方式发挥阵痛作用，因此，我们的发明使药物的转化进一步成为可能。
3.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. 一组具有镇痛作用的肽，其特征在于：具有如(1)或(2)所示的氨基酸序列：
    (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；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，如序列表SEQ ID No.1或SEQ ID No.2所示的氨基酸序列，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：如序列表SEQ ID No.1或SEQ ID No.2所示的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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