CN106632603A - Peptides as well as pharmaceutical composition and application thereof - Google Patents

Abstract

The invention provides a group of peptides as well as a pharmaceutical composition and application thereof. The peptides are as shown in sequence tables; the peptides have amino acid sequences with an analgesic effect, or are pharmacologically acceptable salts of the peptides. A ring is formed by two cysteines at the head end and the tail end of each peptide. A pharmacophore (a structural domain) which has abirritation in the GsMTx-4 peptide is determined by comparing multiple inhibitor cystein knot (ICK)-containing peptide sequences such as GsMTx-4 and GsMTX-2 and carrying out behavior test on a rat pain model.

Description

One group of peptide and its pharmaceutical composition and application

Technical field

The invention belongs to biomedicine technical field, is related to one group of new type of peptides, that what is more particularly to applied in medical science has The peptide of analgesic activity or their pharmaceutically acceptable salt.Moreover, it relates to this group of peptide is used to prepare drug regimen Thing.

Background technology

2000, the U.S. about state university Frederick doctors Sachs were from a kind of Chilean tarantula (Chilean Tarantula polypeptide GsMTx-4 (Sachs F, et a., J Gen Physiol.115, Po583-) are successfully separated out in venom 598,2000), it is reported that, this is a kind of polypeptide to mechanosensitive ion channels with Specific Inhibitory Effect. GsMTx-4 is found the congestive heart failure that can be used for preventing to be produced by cellular swelling, especially at positions such as lung, liver, legs (Sachs F.et al.,JGP 2000).Also report points out that GsMTx-4 can suppress rabbit auricular fibrillation and not to heart Other functions produce impact, thus it can be used for treating heart disease (Sachs, F.et al., Nature 409, pp 35～ 36,2001；Hamill, 0.P., Martinac, B., Physiol.Rev.81, pp 685～740,2001.).Additionally, GsMTx- 4 may can treat brain tumor (Sachs, F.et al., J.Gen.Physiol.115, pp 583～598,2000).

United States Patent (USP) 12153942 (16.07.2009), 10550102 (12.10.2006), 04723309 (28.12.2005) low molecular weight polypeptide (the Low-molecular weight peptides of ion channel activity can, be suppressed inhibiting ion channel activity).B1 (the Pub.No. of European patent EP 1 609 861：WO/2004/ 085647) low molecular weight polypeptide (English of ion channel activity is suppressed：Low-molecular weight peptides inhibiting ion channel activity；Japanese：イ オ Application チ ャ ネ Le activity The encumbers The Ru low molecule ペ プ チ De) above content Japan also applied for patent.It is that same group of researcher and Japanese firm jointly apply.The invention is carried A series of short chain polypeptides from derived from total length GsMTx-4 are out supplied, the short chain polypeptides of these low-molecular-weights are found can be with Specifically suppress the activity of the big conductance potassium-channel SAKca of the dirty middle mechano-sensitive of heart.

Polypeptide GsMTx-4 mentioned above, containing 34 amino acid, its molecular weight is 1,094Da, wherein there is 6 half Guangs Histidine residue, forms three couples of cysteine knot (ICK：Inhibitor cysteine Knot motif) structure characteristic (Robert.et al.,J.Biol.Chem.277(37)：34443-34450,2002).This structuring limits its chemical synthesis With yield during biosynthesis, reason is to be difficult to fold into correct conformation.Although have recently patent for instruct in yeast Middle synthesis total length GsMTx-4 polypeptide, but cost and yield remain the bottleneck of drug development.

The content of the invention

In view of developing the problem for existing to analgesic medicine in above-mentioned and/or existing biomedicine technical field, propose The present invention.

Therefore, one of purpose of the invention is to provide one group of small peptide with analgesic activity.

To solve above-mentioned technical problem, according to an aspect of the present invention, the invention provides following technical scheme：One group Peptide, it has the arbitrary amino acid sequence as shown in (1)～(8)：(1) the amino acid sequence as shown in sequence table SEQ ID No.1 Row；(2) amino acid sequence as shown in sequence table SEQ ID No.2；(3) amino acid as shown in sequence table SEQ ID No.3 Sequence；(4) amino acid sequence as shown in sequence table SEQ ID No.4；(5) amino as shown in sequence table SEQ ID No.5 Acid sequence；(6) amino acid sequence as shown in sequence table SEQ ID No.6；(7) ammonia as shown in sequence table SEQ ID No.7 Base acid sequence；(8) amino acid sequence as shown in sequence table SEQ ID No.8；Or, their pharmaceutically acceptable salt.

As a kind of preferred version of peptide of the present invention, wherein：On the basis of head end and terminal cysteine are present, In the arbitrary peptide in (1) described in claim 1～(8), one or more amino acid still have analgesia after being deleted, replace or adding The polypeptide of effect is or, their pharmaceutically acceptable salt.

As a kind of preferred version of peptide of the present invention, wherein：It is arbitrary with analgesia work shown in (1)～(8) Amino acid sequence or their pharmaceutically acceptable salt, wherein, two cysteines of head end and end form ring.

As a kind of preferred version of peptide of the present invention, wherein：It is passed through chemical synthesis or is obtained by recombinant technique.

As a kind of preferred version of peptide of the present invention, wherein：Itself and protein fusion.

As a kind of preferred version of peptide of the present invention, wherein：It is coupled with polymer.

As a kind of preferred version of peptide of the present invention, wherein：The peptide is connected with carrier.

Another purpose of the invention is to provide a kind of pharmaceutical composition with analgesic activity.

To solve above-mentioned technical problem, according to another aspect of the present invention, the invention provides following technical scheme： A kind of pharmaceutical composition, it is characterised in that include, (1) effective dose as the peptide described in active component；(2) it is selective Pharmaceutically acceptable carrier.

As a kind of preferred version of pharmaceutical composition of the present invention, wherein：The pharmaceutically acceptable carrier choosing From：Solvent, diluent, suspending agent, emulsifying agent, antioxidant, pharmacy preservative, colouring agent, flavouring agent, medium, oiliness substrate, One or more in excipient.

It is a still further object of the present invention to provide one group of peptide or they pharmaceutically acceptable salt or pharmaceutical composition exist The application of ease pain.

The present invention is passed through by comparing various peptide sequences containing cysteine knot (ICK) such as GsMTx-4, GsMTX-2 The behaviouristics of rat pain model is detected, the pharmacophore (domain) that analgesic activity is played in GsMTx-4 polypeptides is determined.This Invention finds that the part of polypeptide fragment of GsMTx-4 plays the role of the inhibition of pain no less than total length.Its principle is polypeptide GsMTx-4 Only have 11% identical (28% is similar) with GsMTx-2 amino acid sequences, but belong to the special suppression of mechanosensitive ion channels together Agent, although other several toxin amino acid sequences are higher with GsMTx-4 homologys (such as：The ammonia of GsMTx-4 and Hanatoxin Base acid sequence has nearly 28% identical, and 37% is similar), but the blocking agent of other different type ion channels.It is so of the invention Sequence of the people based on polypeptide GsMTx-4 key structures domains, a series of short chain polypeptides by business-like chemical synthesis.By many Testing result of the peptide to rat nociceptive pain model, inventor confirms to have obtained the series of the polypeptide short chain with analgesic activity, Complete the present invention.

Description of the drawings

In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below will be to use needed for embodiment description Accompanying drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for this For the those of ordinary skill of field, without having to pay creative labor, can be obtaining other according to these accompanying drawings Accompanying drawing.Wherein：

Fig. 1 is using vola local injection administering mode, SEQ ID No.1, GsMTx-4 and morphine in rats hyperalgia Analgesic effect schematic diagram.To make data reliability high, we are with Normal Saline (Saline) as Negative controls, morphine (Morphine) as positive control.The present invention relates to all test and take double-blind method.

Fig. 2 is using intraperitoneal injection mode, SEQ ID No.1, GsMTx-4 and the hyperalgesic town of morphine in rats Pain effect schematic diagram.

Fig. 3 is that short chain polypeptides SEQ ID No.1 are eased pain with total length GsMTx-4 by vola (A), abdominal cavity (B) injection system Effect is contrasted, and SEQ ID No.1 and GsMTx-4 analgesic activity dose dependent schematic diagram (C).

Fig. 4 is that SEQ ID No.2 pass through to ease pain rat when vola (A, B) or abdominal cavity (C, D) drug administration by injection mode respectively Effect schematic diagram, and respectively with the contrast schematic diagram (B, D) of full-length polypeptide GsMTx-4 analgesic effects.

Fig. 5 is to the rat pain sensation when SEQ ID No.3 are administered respectively by vola (A, B) or abdominal cavity (C, D) injection system The schematic diagram of the analgesia benefit of allergy, and respectively with the contrast schematic diagram (B, D) of full-length polypeptide GsMTx-4 analgesic effects.

Fig. 6 is SEQ ID No.4 respectively by vola (A, B) or during abdominal cavity (C, D) drug administration by injection to rat hyperalgia Analgesia benefit schematic diagram, and respectively with the contrast schematic diagram (B, D) of full-length polypeptide GsMTx-4 analgesic effects.

Fig. 7 is that SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 pass through respectively Rats With Unilateral Analgesic activity schematic diagram when hind leg vola local injection mode is administered.

Fig. 8 be SEQ ID No.6 (A, B) and SEQ ID No.8 (C, D) respectively by during lumbar injection mode to rat pain Feel allergy inhibitory action schematic diagram.

Fig. 9 is that SEQ ID No.1, SEQ ID No.3 and full-length polypeptide GsMTx-4 (are not set up pain to normal rat During pain model) pain threshold do not have influential schematic diagram, and morphine is right as the positive influential on normal rat pain threshold tool According to.

Specific embodiment

It is understandable to enable the above objects, features and advantages of the present invention to become apparent from, with reference to Figure of description pair The specific embodiment of the present invention is described in detail.

Many details are elaborated in the following description in order to fully understand the present invention, but the present invention can be with Implemented using alternate manner described here is different from, those skilled in the art can be in the situation without prejudice to intension of the present invention Under do similar popularization, therefore the present invention is not limited by following public specific embodiment.

Secondly, " one embodiment " or " embodiment " referred to herein is referred to and may be included at least one realization side of the invention Special characteristic, structure or characteristic in formula." in one embodiment " that in this manual different places occur not refers both to Same embodiment, nor single or selectively mutually exclusive with other embodiment embodiment.

According to all of peptide sequence that general agreement writing book text is referred to, wherein N-terminal amino acid is on the left side, and C-terminal amino Acid is on the right.Short-term between two amino acid residues indicates peptide bond.

The compound of the present invention can be provided in the form of pharmaceutical salts.The example of preferred salt be with pharmaceutically acceptable organic acid and Polymeric acid formed those and with inorganic acid formed salt, the organic acid such as acetic acid, lactic acid, maleic acid, citric acid, apple Acid, ascorbic acid, butanedioic acid, benzoic acid, salicylic acid, methanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid or pamoic acid, the polymerization Acid such as tannic acid or carboxymethylcellulose calcium, the inorganic acid such as halogen acids (for example, hydrochloric acid, sulfuric acid or phosphoric acid etc.).Can be using this The known any method for obtaining pharmaceutical salts of art personnel.

In order to describe the convenience of the present invention, the routine and unconventional abbreviation using various amino acid residues.These abbreviations are Familiar to the person skilled in the art, but in order to clearly be listed below：

Asp=D=aspartic acids；Ala=A=alanine；Arg=R=arginine；

Asn=N=asparagines；Gly=G=glycine；Glu=E=glutamic acid；

Gln=Q=glutamine；His=H=histidines；Ile=I=isoleucines；

Leu=L=leucines；Lys=K=lysines；Met=M=methionines；

Phe=F=phenylalanines；Pro=P=proline；Ser=S=serines；

Thr=T=threonines；Trp=W=tryptophans；Tyr=Y=tyrosine；

Val=V=valines；Cys=C=cysteines.

Overview

Present disclosure is characterized in that for treating pain (such as all pain such as Acute Pain, chronic ache, cancer pain Type) new type of peptides.Peptide can reduce pain sensitivity, therefore with analgesic activity.The peptide can produce long-acting analgesic and/or anti-wound Evil sensitization.There is also described herein the concrete application method (such as treat pain) and the medicine containing this kind of peptide of this kind of peptide Or composition.

Pain and nociception

International pain EASD (International Association for the Study of Pain, IASP) pain be defined as " a kind of offending sensibility, emotionality impression, we mainly contact it with histologic lesion Together, or in terms of histologic lesion it is described, or not only with histologic lesion had been associated but also had been described in terms of infringement ". Pain can be the symptom of potential disease or illness, or pain itself is a kind of disease.Pain may be generally divided into two classes Type：It is acute and chronic.Primary difference is that Acute Pain shields after infringement, and chronic ache does not play this effect. Acute Pain is pain symptom；And chronic ache is pain disease.

Most of Acute Pain is caused by disease, inflammation or tissue damage.The types of pain is typically in such as wound or outer Occur suddenly after section's operation, and can be with anxiety or depressed.The reason for generally may diagnose that Acute Pain and to it Treated, and pain is often self limiting, that is to say, that it is limited to special time period and serious.In some cases, Acute Pain can be transformed into chronic ache.

Generally believe chronic ache inherently disease.Environment and psychological factor can be allowed to deteriorate.Chronic ache is than acute Durante dolors is longer, and can have resistance to many drug therapies.

According to Clinical symptoms, the main Types of deducibility constant pain mechanism.Presumptive physiopathologic classification, Broadly pain syndrome is divided into nociception, neuropathic, spirituality, mixed type or special hair style.

Analgesia peptide

Present disclosure is provided to be had analgesic properties and can be used to treat pain (such as Acute Pain, chronic ache or cancer Pain etc.) peptide.The peptide can have Antinociceptive effect.

One of purpose of the present invention is to provide one group to have the peptide of analgesic activity (such as SEQ ID No.1～SEQ ID No.8).And the preparation method of this group of peptide is by synthesis, purifying, wash-out so that the sample purity of final synthetic peptide> 99%, and its molecular weight is determined using electrospray ionization mass spectrum (ESI-MS).

Certainly, those skilled in the art will be understood that, on the basis of head end and terminal cysteine are present, SEQ ID In the arbitrary peptides of No.1～SEQ ID No.8, one or more amino acid still have analgesic activity after being deleted, replace or adding Polypeptide or, their pharmaceutically acceptable salt, still fall within protection scope of the present invention.

It is passed through chemical synthesis or is obtained by recombinant technique peptide shown in SEQ ID No.1～SEQ ID No.8, and it is not But can also can be coupled with polymer with protein fusion, can be being connected with carrier.Further study show that, this group of peptide has Special structure, the arbitrary amino acid sequence with analgesic activity or their medicines shown in SEQ ID No.1～SEQ ID No.8 Acceptable salt on, wherein, two cysteines of head end and end form ring.Such as following structures：

Embodiment

1. animal used as test, instrument and reagent

Adult Sprague-Dawley male rats (more than 8 weeks), body weight 180g-220g buys happy in Jinan, Shandong Province friend Animal used as test breeds Co., Ltd.

Carrageenan (Carrageenan is abbreviated as Carr) is U.S.'s sigma Products, and 1% Irish moss is prepared respectively Glue (0.1g carrageenans are dissolved in the normal saline solutions of 10ml 0.9%, are dispensed to 10 EP pipes) and 2% carrageenan (0.2g Carrageenan is dissolved in the normal saline solutions of 10ml 0.9%, is dispensed to 10 EP pipes), in -20 DEG C of preservations, using front half an hour Take out and place on ice.

, from U.S.'s alomone labs purchases, SEQ ID No.1～all polypeptides of SEQ ID No.8 are with height for GsMTx-4 Deionized water (DD water) after temperature sterilizing is configured to the mother liquor of 5mM or 10mM, and is divided in EP pipes after -80 DEG C of preservations, Concentration when using with normal saline dilution to needed for testing.

38500-PAM pressure application measuring systems (Μ go Basile Biological Research Apparat μ s, Comerio-Varese, Italy) it is made up of two parts, a part is handheld unit, and another part is integrated electronicses unit.Hand It is force snesor to hold unit, and the force snesor is designed according to Randall-Selitto pain threshold detectors, and integrated electronicses unit can be certainly Dynamic record is applied to the maximum mechanical paw withdrawal reflex threshold in rats with left metapedes vola.

2. the preparation of inflammatory pain model：We take two kinds of different inflammatory pain model preparation methods, are respectively used to vola Detected with the analgesic effect of abdominal cavity drug injection：

2-1：The carrageenan inflammatory pain models of 6 μ l 1%：According to document (Alessandri-Haber N, et al.Neurosci.Vol 29(19),pp 6217-6288,2009)；The method of introduction prepares carrageenan inflammatory pain model.Should Method is used for the detection of subplantar injection administering mode medicine analgesic effect.Briefly：From inserting needle between the left back foot 2 of rat, 3 toes, 1% carrageenan is injected, injection volume is 6 μ l, and the injection concentration is inducible to produce hyperalgia, i.e.,：After injection in half an hour There is swelling and the reduction of pain threshold in In The Rat Sole.Mechanical paw withdrawal reflex threshold is scorching as carrageenan in below 50gf persons Property pain modeling success rat.Inflammatory threshold value gives in the inflammatory model rat of more than 50gf and rejects.

2-2：The carrageenan inflammatory pain models of 50 μ l 2%：According to document (Seung Pyo Park, et al.2008.Pain.137:208-217) method introduced prepares 2% carrageenan inflammatory pain model.The method is used for lumbar injection The detection of administering mode medicine analgesic effect.Briefly：From inserting needle between the left back foot 2 of rat, 3 toes, 2% carrageenan is injected 50 μ l, the injection concentration is inducible to produce hyperalgia, i.e.,：After injection there is more serious swelling in In The Rat Sole in half an hour The reduction of swollen and pain threshold.It is qualified as carrageenan inflammatory pain modeling in below 50gf that mechanical paw withdrawal reflex threshold is reduced Rat.Inflammatory threshold value gives in the inflammatory model rat of more than 50gf and rejects.

3. experiment packet and animal are processed

The 6 week old SD rats that all experimental rats are bought from Jinan, Shandong Province Peng Yue Company of Animals Ltd..Experimental rat is adapted to Property raise 2～4 weeks, feeding environment temperature is 22～26 DEG C, humidity 30%～50%, and adaptability selects every time body weight after raising The rat of 180～220g 10～12, measurement rats with left metapedes machinery paw withdrawal threshold value, takes the average that every rat measures 5 times As basic value (Baseline is abbreviated as BL) before the rat inflammatory modeling, reject basic value difference it is big or to mechanical stimulus without The rat of reaction；Then according to the method that document is introduced (is used in the carrageenans of 6 μ l of rats with left metapedes vola intracutaneous injection 1% In subplantar injection dosing method) or the carrageenans of 50 μ l 2% (being used for lumbar injection dosing method), detect that rat is left after 1 hour Side metapedes mechanicalness paw withdrawal threshold value, rejects the rat of modeling failure (inflammatory threshold value is in more than 50gf), by the successful rat of modeling It is equally divided into two groups of medicine group and physiological saline group.More than operate in every occur that modeling is unsuccessful, the feelings that rat is disallowable Condition, then plus test to guarantee that per group of rat for statistics is at least 5～8.

4.GsMTx-4 and its small peptide induce effect --- the measure of mechanical paw withdrawal reflex threshold in hyperalgia in inflammatory

4.1 subplantar injection morphines, GsMTx-4 and small peptide suppress the measure of Inflammatory hyperalgesia

GsMTx- is evaluated with the left back sufficient mechanicalness paw withdrawal threshold value of 38500-PAM pressure applications measuring system measure rat 4 and its effect of the small peptide in rat mechanical hyperalgesia.Need to carry out paw withdrawal to rat before all rat behavior tests Training, until every rat all can start recording rat basis paw withdrawal threshold value after paw withdrawal.10～12 rats that the same day need to be surveyed In being put into same mouse cage, it is numbered from 1～10 (or 12) on the tail of each rat, and relief rat is fitted in measurement room 30min is answered, after rat peace and quiet test is started.It is arranged on according to the force snesor of Randall-Selitto pain threshold detectors design In one hand-held device unit of 38500-PAM pressure application measuring systems.It is 0.45gf that measuring system arranges minimum force value, Maximum force value is 450gf.Successively by number Digital size is successively with 38500-PAM pressure applications measuring system for operating personnel Every rats with left metapedes machinery paw withdrawal threshold value of measurement.Concrete grammar is first the cone point in handheld unit to be placed on into a rat left side Side metapedes vola middle part, then applies a power with constant power rate (30gf/s) to rats with left metapedes, exerted forces Maximum time is that in 15s, 15s times when tested rat hindleg bounces back, operator stops force, and now electronic unit will be automatic Record is applied to the maximum mechanicalness paw withdrawal threshold value in rats with left metapedes vola, if left side metapedes does not go out yet after tested rat 15s Existing paw withdrawal reflection, system meeting automatic alarm, operator stops measurement, duplicate measurements after 10min.If the multiple 15s of certain rat Afterwards there is not paw withdrawal reflection in left side metapedes, rejects this rat.Measure successively according to rat tail numerical digit size successively, repeat Measurement 5 times (each stimulus intervals time is 10min), takes the mean value of 5 experiments,.Measurement finishes the μ l of metapedes bottom intracutaneous injection 6 1% carrageenan, sets up rat inflammatory pain model.Injection carrageenan detects that rats with left metapedes mechanicalness contracts after 1 hour Sufficient threshold value, rejects the abnormal rat (modeling failure) of threshold size.The rat that inflammatory threshold value is higher and threshold value is relatively low is mixed into flat It is divided into 2 groups：Medicine group and physiological saline group, 5～6 per group so that two groups of rat inflammatory threshold value distributions are similar.Medicine group exists μ g/5 μ l GsMTx-4 (with suitable concn is front diluted to) of rats with left metapedes vola intracutaneous injection 1.2 or one of which small peptide, Or 5mg/kg morphines, or the isopyknic physiological saline of rats with left metapedes vola 5 μ l of subcutaneous same position injection, after injection respectively In 1h, 3h, 5h, 7h and 24h detection each group rats with left metapedes machinery paw withdrawal reflex threshold, the equal retest of every rat 5 times (each test interval is 10min), the mean value for taking 5 paw withdrawal reflex thresholds is the actual paw withdrawal reflex threshold of the rat (unit is：g).

Note：It is every in operating above the situation that modeling is unsuccessful, rat is disallowable occur, then plus test to guarantee per group Rat for Exemplary statistical data is at least 5～8.

4.2 lumbar injection GsMTx-4 and its small peptide suppress the measure of Inflammatory hyperalgesia

Lumbar injection GsMTx-4 and its small peptide suppress the method and step of Inflammatory hyperalgesia similar to subplantar injection, but have Some is different：

(1) it is 0.75gf that the measuring system arranges minimum force value, and maximum force value is 750gf

(2) in view of lumbar injection morphine mechanicalness paw withdrawal threshold value may be caused significantly to rise, by exerted forces most The big time is adjusted to 25s

(3) the μ g/kg GsMTx-4 of medicine group lumbar injection 270 or one of which small peptide or 10mg/kg morphines, physiological saline The lumbar injection amount of group such as is at the SPSS of quality.

Based on above-mentioned experiment, Fig. 1 is GsMTx-4, morphine and SEQ ID No.1 by vola subcutaneous administrations mode The schematic diagram of (volume injected is 6 μ l) to rat hyperalgia inhibition.Wherein, A figures show left back in rat in advance After carrageenan (Carr) the induced rat inflammatory models of 6 μ l of whole bottom intracutaneous injection 1%, in In The Rat Sole swelling injection location 5 Physiological saline 1h, the 3h of μ l, after 5h, rat machinery paw withdrawal threshold value and inflammatory threshold value no significant difference；And with modeling before machinery contracting Foot reflex threshold value is compared, and the morphine (B) of equal volume amounts (5 μ l), GsMTx-4 (C) or SEQ ID No.1 (D) are significantly reduced Rat machinery paw withdrawal reflex threshold.The such as isopyknic GsMTx-4 of In The Rat Sole swelling position hypodermic injection or small peptide SEQ ID No.1 (injection volume be 1.2 μ g/5 μ l) 1h, 3h, after 5h, rat machinery paw withdrawal threshold value has and increases, and 3h reaches most after injection It is high.Notice that morphine drug effect after administration 1h reaches maximum, but increasing for rat machinery paw withdrawal threshold value greatly exceed rat baseline Mechanical paw withdrawal threshold value when (Base Line, be abbreviated as BL).It is considered that more than baseline may with morphine it is additive, Rely related to its toxicity by property.E figures are with big in the left back carrageenans (Carr) of whole 6 μ l of bottom intracutaneous injection 1% induction of rat Compare GsMTx-4, small peptide SEQ ID No.1 and physiological saline and morphine in rats mechanical hyperalgesia after mouse inflammatory model In inhibitory action.

Fig. 2 is GsMTx-4, morphine and SEQ ID No.1 right by intraperitoneal injection mode (volume injected is 50 μ l) The contrast schematic diagram of rat hyperalgia inhibitory action.We obtained and above Fig. 1 (subplantar injection) it is similar to pain sensation mistake Quick inhibitory action, but GsMTx-4 and SEQ ID No.1 higher suppression works when there is the mode than be administered from vola intracutaneous injection With：They reach maximum suppression effect after 3h, and can be totally turned over by carrageenan (Carr：Injection volume is 50 μ l 2%) the inflammatory pain threshold value (B, C) of induction, and physiological saline is not acted on (A).Additionally, morphine (injection volume is 10mg/Kg) Highest is reached to rat hyperalgia inhibitory action after injection 1h, although than GsMTx-4 (injection volume is 270 μ g/Kg, 50 μ l) One times (see E) is higher by with the analgesic activity of SEQ ID No.1 (injection volume is 270 μ g/Kg), but with subplantar injection administering mode As a result it is similar to：Although morphine analgesic activity is higher, the raising to rat machinery paw withdrawal threshold value greatly exceed rat Baseline threshold (Base Line, be abbreviated as BL), it is believed that this part is related to the toxicity of morphine.Additionally, after 3h morphine town Pain acts on rapid decline, reaches the effect (E) for having similar analgesic activity to GsMTx-4 and SEQ ID No.1.Due to the present invention Even if the short chain polypeptides with most strong analgesic activity for proposing do not affect (see Fig. 9) on the normal rat pain sensation, therefore this The polypeptide of bright discovery is possible to develop into the hyperalgesic best analgesic for the treatment of or intermixture.

Fig. 3 is the contrast schematic diagram of SEQ ID No.1 and total length GsMTx-4 analgesic activity.SEQ ID No.1 and GsMTx- 4 by subplantar injection mode when being administered, and analgesic activity does not have notable difference (A, C in figure), and wherein C is SEQ ID No.1 (right side) The dose-dependant figure for passing through analgesic activity during subplantar injection mode with total length GsMTx-4 (left side).But when both pass through respectively abdominal cavity When injection system is administered, SEQ ID No.1 may have higher analgesic effect (B in figure).

Fig. 4 is that SEQ ID No.2 pass through respectively vola (A), the analgesic activity schematic diagram of abdominal cavity (C) administering mode.In order to More preferably compare the analgesic effect of short chain polypeptides, the analgesic effect of equivalent total length GsMTx-4 is compared (B, D) by light grey expression. Wherein A is illustrated after the left back carrageenans (Carr) of whole 6 μ l of bottom intracutaneous injection 1% of rat, with modeling before mechanical paw withdrawal it is anti- Penetrate threshold value to compare, hence it is evident that reduce rat machinery paw withdrawal reflex threshold, (injection volume is 1.2 μ to vola hypodermic injection SEQ ID No.2 G/5 μ l) 1h, 3h, 5h, after 7h, rat machinery paw withdrawal threshold value increases, and 1-3h reaches highest after injection.C diagrams are intended to rat (injection volume is 270 μ for the left back carrageenans (Carr) of whole 50 μ l of bottom intracutaneous injection 2% 1h afterwards, lumbar injection SEQ ID No.2 G/Kg) 1h, 3h, 5h, rat machinery paw withdrawal threshold value has and increases after 7h, and inhibitory action is most strong during injection 3h, it is evident that note in abdominal cavity The analgesic activity for penetrating administration is better than vola topical modes.In addition lumbar injection SEQ ID No.2 have and total length GsMTx-4 Similar analgesic activity (D).

Fig. 5 is that SEQ ID No.3 are shown hyperalgesic inhibitory action respectively by vola (A), abdominal cavity (C) administering mode It is intended to.Wherein, A figure shown in after the left back carrageenans (Carr) of whole 6 μ l of bottom intracutaneous injection 1% of rat, with modeling before machinery Paw withdrawal reflex threshold is compared, vola hypodermic injection SEQ ID No.3 (injection volume is 1.2 μ g/5 μ l) 1h, 3h, 5h, after 7h substantially Rat machinery paw withdrawal reflex threshold is reduced, and 3h reaches highest after injection.SEQ ID No.3 shown in B figures are subcutaneous with vola Quality, equal-volume GsMTx-4 (1.2 μ g/5 μ l) or physiological saline 1h, 3h, the 5h such as injection, rat machinery paw withdrawal reflection threshold after 7h The comparison of value, SEQ ID No.3 do not have notable difference (B) to hyperalgesic inhibitory action and full-length polypeptide GsMTx-4 small peptides, Saline rats machinery paw withdrawal threshold value does not change.C figures are shown in left back 2% jiao of the 50 μ l of whole bottom intracutaneous injection of rat After fork dish glue (Carr), by lumbar injection SEQ ID No.2 (injection volume is 270 μ g/Kg) 1h, 3h, 5h, after 7h with modeling before Mechanical paw withdrawal reflex threshold is compared, and rat machinery paw withdrawal threshold value substantially increases, and 3h is totally turned over the machine of rat after injection Tool paw withdrawal threshold value, with the rat baseline (baseline before modeling:BL) without notable difference.Therefore given by lumbar injection mode SEQ ID No.3 may have than waiting quality full-length polypeptide GsMTx-4 higher analgesic effect (D) during medicine.

Fig. 6 is that SEQ ID No.4 are shown hyperalgesic inhibitory action respectively by vola (A), abdominal cavity (C) administering mode It is intended to.Wherein, A figure shown in after the left back carrageenans (Carr) of whole 6 μ l of bottom intracutaneous injection 1% of rat, with modeling before machinery Paw withdrawal reflex threshold is compared, vola hypodermic injection SEQ ID No.4 (injection volume is 1.2 μ g/5 μ l) 1h, 3h, 5h, after 7h substantially Rat machinery paw withdrawal reflex threshold is reduced, and 3h reaches highest after injection.B figures show SEQ ID No.4 and in vola skin Physiological saline 1h, 3h, the 5h of the quality/equal-volume GsMTx-4 (1.2 μ g/5 μ l) such as lower injection or equal-volume (5 μ l), rat after 7h The comparison of mechanical paw withdrawal reflex threshold, SEQ ID No.4 to hyperalgesic inhibitory action than full-length polypeptide GsMTx-4 small peptides slightly It is weak.C is shown in after the inflammation inducing swelling of the left back carrageenans (Carr) of whole 50 μ l of bottom intracutaneous injection 2% of rat, by abdominal cavity Injection SEQ ID No.4 (injection volume is 270 μ g/Kg) are compared with mechanical paw withdrawal reflex threshold before modeling, and 1h, 3h, 5h are big after 7h Mouse machinery paw withdrawal threshold value substantially increases, and rat machinery paw withdrawal threshold value is no with rat baseline (BL) obvious after 3h after injection Difference.When being therefore administered by lumbar injection mode, analgesic activity and the grade quality full-length polypeptide GsMTx-4 of SEQ ID No.4 Analgesic activity there is no notable difference (D).

Fig. 7 shows SEQ ID No.5, SEQ ID No.6, tri- kinds of small peptides of SEQ ID No.7 by lumbar injection mode To the hyperalgesic inhibitory action of rat during administration.Wherein, A illustrates vola hypodermic injection SEQ ID No.5 (1.2 μ g/5 μ l) After 1h, 3h, 5h, 7h, compared with Carr inflammatory models rat machinery paw withdrawal threshold value, mechanical paw withdrawal threshold value is all significantly improved, And reach highest in 3h.B illustrates vola hypodermic injection SEQ ID No.6 (1.2 μ g/5 μ l) 1h, after 3h, with Carr inflammatories Threshold value is compared, and mechanical paw withdrawal threshold value is all significantly improved, and reaches highest in 3h.And C illustrates vola hypodermic injection SEQ ID No.7 (1.2 μ g/5 μ l) 1h, 3h, 5h, after 7h, compared with Carr inflammatory threshold values, mechanical paw withdrawal threshold value is all significantly improved, and Highest is reached in 3h.D illustrates vola hypodermic injection SEQ ID No.8 (1.2 μ g/5 μ l) 1h, 3h, 5h, scorching with Carr after 7h Property threshold value is compared, and mechanical paw withdrawal threshold value is all significantly improved, and reaches highest in 3h.

To rat inflammatory pain when Fig. 8 shows that SEQ ID No.5 and SEQ ID No.8 are administered by lumbar injection mode Analgesic activity.Wherein A and C figures are illustrated respectively in the left back carrageenans (Carr) of whole 50 μ l of bottom intracutaneous injection 2% of rat and lure After leading inflammatory pain, then respectively by lumbar injection SEQ ID No.5 (A figures) or SEQ ID No.8 (C figures), (injection volume is 270μg/Kg).Compared with rat inflammatory model machinery paw withdrawal reflex threshold (Carr), 1h, 3h, 5h, rat machinery paw withdrawal after 7h Threshold value substantially increases, and 3h reaches maximum after injection.Therefore, lumbar injection SEQ ID No.5 (B figures) and SEQ ID No.8 (D figures) has similar analgesic activity to total length GsMTx-4, and wherein SEQ ID No.8 effects are slightly higher, hold time more It is long.There is no with the analgesic activity of full-length polypeptide notable difference (D figures) after 5h.

Fig. 9 diagram GsMTx-4, SEQ ID No.1 and SEQ ID No.3 do not have shadow to the mechanical paw withdrawal threshold value of normal rat Ring.A is shown in the subcutaneous direct injection morphine in the left back whole bottom of normal rat, and (injection volume is 5mg/kg, and the dosage is usually morphine The minimum dose of dosage) after 1h, more than one times (threshold of mechanical paw withdrawal reflex threshold of the further strong inhibition normal rat of morphine Value rises to 550g from the average～250g of normal rat), it is believed that while this plays analgesic activity with morphine, have It is additive related to toxicity.B, C, D figure be shown in respectively subcutaneous direct injection GsMTx-4 (A figures) in the left back whole bottom of normal rat, (injection volume is 1.2 μ g/5 μ l, and the dosage is GsMTx-4 and SEQ for SEQ ID No.1 (B figures) or SEQ ID No.3 (C figures) Suppress rat hyperalgesic maximum dose when ID No.1 are administered by subplantar injection mode) 1h, 3h, 5h, after 7h, equal not shadows Ring the mechanical paw withdrawal reflex threshold of normal rat.Because GsMTx-4, SEQ ID No.1 and SEQ ID No.3 are involved by the present invention And the polypeptide short chain with most strong analgesic activity in peptide, therefore the group experiment demonstrates again that from the negative polypeptide pair according to the present invention Normal rat is without pair (toxicity) effect.

Pharmaceutical composition

The peptide of present disclosure can be configured to for example give subject the pharmaceutical composition to treat pain.Peptide can be with Individually give, or can be given in combination with other Pain treatments in same composition or as single composition.

Pharmaceutical composition generally includes pharmaceutically acceptable carrier." pharmaceutically acceptable carrier " used herein is wrapped Include solvent, decentralized medium, coating material, antibacterial agent and antifungal agent, isotonic agent and the suction of any and all physical compatibility Receive delayer etc..Composition may include pharmaceutically acceptable salt, such as acid-addition salts or base addition salts.

Drug preparation technique is generally accepted technology, and more details see, for example, Gennaro, The Science and Practice of Pharmacy, the 20th edition, Lippincott, Williams＆Wilkins (2000) (ISBN： 0683306472)；Ansel etc., Pharmaceutical Dosage Forms and Drug Delivery Systems, the 7th Version, Lippincott Williams＆Wilkins Publishers (1999) (ISBN：0683305727)；And Kibbe, Handbook of Pharmaceutical Excipients American Pharmaceutical Association, the 3rd Version (2000) (ISBN：091733096X).

In one embodiment, (for example salt solution, sodium chloride, disodium hydrogen phosphate seven are hydrated the peptide with excipient materials Thing, sodium dihydrogen phosphate) and stabilizer prepare together.Can also provide by suitable concentration in such as cushioning liquid.

Pharmaceutical composition can take various forms.These include such as liquid, semi-solid and solid dosage forms, such as liquid solution Agent (for example injecting and be transfused solution), dispersant or supensoid agent, tablet, pill, powder, liposome and suppository.Preferred shape Formula may depend on set administering mode and treatment use.

Composition can be given by parenteral (such as intravenous, subcutaneous, intraperitoneal or intramuscular injection).It is used herein Term " parenteral " and " parenterally giving " refer to enteral and the administering mode beyond the administration of local, and usual Jing is administered to Medicine, include, without being limited in intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, socket of the eye, in heart, intradermal, intraperitoneal, transtracheal, Under subcutaneous, epidermis, in joint, under capsule, under arachnoid, in backbone, Epidural cavity and breastbone inner injection and infusion.

Can enteral interior approach give composition, such as by alimentary canal, such as oral administration.For example, can be with tablet, glue Wafer, caplet agent, pill, powder, drops, supensoid agent, solution, paste, gel or other peroral dosage forms give composition.

Enteral route includes Jing stomach feeding tubes, duodenum feeding tube or gastrostomy or rectal administration, such as with suppository or bowel lavage The composition of dosage form formula.

Composition can locally be administered, such as in painful area.Local administration includes such as epidermis, intranasal, suction and vagina Administration.Skin (such as burn, blister or cut), lip, gum, tooth, oral cavity, eye, ear, nail matrix or throat can be given by composition Deng such as painful area.The composition of local administration can be cream, gel, lotion or ointment etc..

Pharmaceutical composition may include the peptide as herein described of " therapeutically effective amount ".The effective dose can be according to institute's drug (example Such as peptide) effect determine, or if during using more than one medicines, then determined according to combination effect.The medicine of therapeutically effective amount Thing can also change according to following factor：The types of pain of such as subject, disease condition, age, sex and body weight and Medicine causes the ability of required reaction (for example improving pain) in subject's body.Therapeutically effective amount is also wherein composition Treatment beneficial effect exceedes the amount of any toxicity or illeffects.

Based on the present invention, be possible to obtain in the future it is a kind of it is new, for analgesic polypeptide drugs, or containing the polypeptide Effectively ease pain mixture.The present invention has the advantage that：

1. primary spider toxin GsMTx-4 polypeptides form four circuluses in water, require that polypeptide must be rolled over during synthesis Build up and keep the space structure of the native polypeptide that just there is corresponding drug effect, therefore the loaded down with trivial details high cost of synthesis procedure.The present invention finds Play the pharmacophore of analgesic activity in native polypeptide, therefore reduce the synthesis cost of polypeptide, make industrially to produce in enormous quantities Become efficient and cheap；

2. because the present invention reduces polypeptide length, immunogenicity is reduced, it is easier to absorption of human body.In clinical practice Drug effect can more be given full play to.Because the polypeptide does not affect on the pain threshold of normal big category, compare with the medicine such as morphine without into Addiction, without drug dependence and nontoxicity.Therefore the present invention may be developed to clinically wide variety of analgesic.

It should be noted that above example is only unrestricted to illustrate technical scheme, although with reference to preferably Embodiment has been described in detail to the present invention, it will be understood by those within the art that, can be to the technology of the present invention Scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it all should cover at this In the middle of bright right.

SEQUENCE LISTING

<110>Xuzhou medical university

<120>One group of peptide and its pharmaceutical composition and application

<130> 2016

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 17

<212> PRT

<213>Artificial sequence

<400> 1

Trp Lys Cys Asn Pro Asn Asp Asp Lys Cys Cys Arg Pro Lys Leu Lys

1 5 10 15

Cys

<210> 2

<211> 11

<212> PRT

<213>Artificial sequence

<400> 2

Trp Trp Lys Cys Asn Pro Asn Asp Asp Lys Cys

1 5 10

<210> 3

<211> 10

<212> PRT

<213>Artificial sequence

<400> 3

Trp Lys Cys Ala Pro Ala Asp Asp Lys Cys

1 5 10

<210> 4

<211> 11

<212> PRT

<213>Artificial sequence

<400> 4

Trp Lys Cys Ala Pro Ala Asp Asp Lys Cys Arg

1 5 10

<210> 5

<211> 10

<212> PRT

<213>Artificial sequence

<400> 5

Trp Cys Asn Pro Asn Asp Asp Lys Cys Ala

1 5 10

<210> 6

<211> 9

<212> PRT

<213>Artificial sequence

<400> 6

Trp Cys Lys Pro Asn Asp Asp Lys Cys

1 5

<210> 7

<211> 7

<212> PRT

<213>Artificial sequence

<400> 7

Trp Cys Asn Asp Asp Lys Cys

1 5

<210> 8

<211> 7

<212> PRT

<213>Artificial sequence

<400> 8

Trp Cys Lys Asp Asp Lys Cys

1 5

<210> 9

<211> 7

<212> PRT

<213>Artificial sequence

<400> 9

Trp Cys Lys Asp Asp Lys Trp

1 5

Claims (10)

1. one group of peptide, it is characterised in that：With the arbitrary amino acid sequence as shown in (1)～(8)：

(1) amino acid sequence as shown in sequence table SEQ ID No.1；

(2) amino acid sequence as shown in sequence table SEQ ID No.2；

(3) amino acid sequence as shown in sequence table SEQ ID No.3；

(4) amino acid sequence as shown in sequence table SEQ ID No.4；

(5) amino acid sequence as shown in sequence table SEQ ID No.5；

(6) amino acid sequence as shown in sequence table SEQ ID No.6；

(7) amino acid sequence as shown in sequence table SEQ ID No.7；

(8) amino acid sequence as shown in sequence table SEQ ID No.8；Or,

Their pharmaceutically acceptable salt.

2. according to the peptide described in claim 1, it is characterised in that：On the basis of head end and terminal cysteine are present, power Profit requires in the arbitrary peptide in (1) described in 1～(8) that one or more amino acid after being deleted, replace or adding still there is analgesia to make Polypeptide is or, their pharmaceutically acceptable salt.

3. peptide according to claim 1 and 2, it is characterised in that：It is arbitrary with analgesic activity shown in (1)～(8) Amino acid sequence or their pharmaceutically acceptable salt, wherein, two cysteines of head end and end form ring.

4. peptide according to claim 3, it is characterised in that：It is passed through chemical synthesis or is obtained by recombinant technique.

5. according to the arbitrary described peptide in claim 1,2 or 4, it is characterised in that：Itself and protein fusion.

6. according to the arbitrary described peptide in claim 1,2 or 4, it is characterised in that：It is coupled with polymer.

7. according to the arbitrary described peptide in claim 1,2 or 4, it is characterised in that：The peptide is connected with carrier.

8. a kind of pharmaceutical composition, it is characterised in that include,

(1) peptide as described in claim 1～7 is arbitrary as active component of effective dose；

(2) selective pharmaceutically acceptable carrier.

9. pharmaceutical composition according to claim 7, it is characterised in that the pharmaceutically acceptable carrier is selected from：It is solvent, dilute In releasing agent, suspending agent, emulsifying agent, antioxidant, pharmacy preservative, colouring agent, flavouring agent, medium, oiliness substrate, excipient One or more.

10. one group of peptide as described in claim 1,2 or 4 are arbitrary or their pharmaceutically acceptable salt or such as claim 8 Or the pharmaceutical composition described in 9 is in the application of ease pain.