CN102127151B - Antagonistic peptide of anti-opioid peptide and application thereof - Google Patents
Antagonistic peptide of anti-opioid peptide and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-opioid peptide, as well as an antagonistic peptide and application thereof. The anti-opioid peptide is one of the following amino acid residue sequences: (1) SEQ ID NO:1 in a sequence table and (2) a polypeptide which is obtained by substituting, deleting or adding 1-10 amino acid residues on an amino acid residue sequence of SEQ ID NO:1 in the sequence table and has an antinociceptic effect on antagonistic morphine. The antagonistic peptide is one of the following amino acid residue sequences: (1) SEQ ID NO:5-7 in a sequence table and (2) a polypeptide which is obtained by substituting, deleting or adding 1-10 amino acid residues on an amino acid residue sequence of SEQ ID NO:5-7 in the sequence table and has the effects of enhancing morphine analgesia, reversing morphine tolerance and/or weakening or eliminating abstinence syndromes. The anti-opioid peptide as well as an active segment and the antagonistic peptide thereof play an important role in the field of preparation of medical and medicament abstinence medicaments, and have wide application prospects.
Description
The application is that application number is 200710100384.4, the applying date is to divide an application for " application of anti-opioid and antagonism peptide thereof and this antagonism peptide " on 06 11st, 2007, invention and created name.
Technical field
The present invention relates to peptide section and encoding sox thereof and application; Particularly relate to a kind of anti-opioid and antagonism peptide thereof, strengthen morphine analgesia with this antagonism peptide in preparation, reverse the morphine tolerance and/or weaken or eliminate the application in the medicine of Withrawal symptom effect with antagonism morphine antinociceptic effect.
Background technology
As everyone knows, worldwide rampant drug trafficking and addiction activity has become a malignant tumor of harm society, has seriously disturbed orderly economic construction and social stability, and in addition, IDU has also caused the soaring of AIDS crowd's infection rate.At present; Though all strengthening, national governments unite the dynamics of combatting drug trafficking; But take drugs, narco-trafficking still remains incessant after repeated prohibition, one of them important reasons is exactly that morphine tolerance and the principle that relies on really or are not fully illustrated as yet, does not still have effective drug rehabilitation means in the world.External employing at first " alternative medicine " be that the medicine with one type of methadone replaces opium in withdrawal, this therapy will cause tolerance and the dependence of patient to methadone class medicine.At home, Mr. Yang Guodong of Ningbo narcotic house adopts hyoscine to cooperate the other medicines drug rehabilitation, does not also separate resolution addiction problem fully.Professor Han Jisheng adopts acupuncture and moxibustion therapeutic apparatus treatment Withrawal symptom; That attempts that electric pulse stimulation through CF improves endogenous opiate in the body synthesizes to come mitigation symptoms, but its greatest drawback be the efficient limited of acupuncture analgesia among the crowd and also constantly acupuncture also have the tolerance problem.Recently, Shijiazhuang tcm-expert Wang Yan debates disease executing to the opium Withrawal symptom and controls, and adopts " tortoise beetle ball " treatment withdrawal syndrome, through clinical further check and perfect.Therefore, press for a kind of can and the dependence from thorough elimination morphine tolerance on the source, and the anti-additive medicament that weakens or eliminate Withrawal symptom.
Summary of the invention
The purpose of this invention is to provide a kind of anti-opioid with antagonism morphine antinociceptic effect.
Anti-opioid provided by the present invention, called after 99A is one of following amino acid residue sequences:
1) the SEQ ID NO:1 in the sequence table;
2), has the polypeptide of antagonism morphine antinociceptic effect with replacement, disappearance or the interpolation of the amino acid residue sequence of SEQ ID NO:1 in the sequence table through one to ten amino-acid residue.
One to ten amino-acid residue of said replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.
SEQ ID NO:1 in the sequence table is made up of 86 amino-acid residues.
The gene of code book invention anti-opioid 99A is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID NO:11 in the sequence table;
2) dna sequence dna of SEQ ID NO:1 in the code sequence tabulation;
3) with sequence table in the nucleotide sequence that limits of SEQ ID NO:11 have 90% above homology and have the nucleotide sequence of antagonism morphine antinociceptic effect;
The nucleotide sequence of the dna sequence dna hybridization that 4) under the rigorous condition of height, can limit with the SEQ ID NO:11 in the sequence table.
The rigorous condition of said height is: (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.
SEQ ID NO:11 in the sequence table is by 258 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:1 in the sequence table.
The active fragments of above-mentioned anti-opioid 99A can be one of following amino acid residue sequences:
1) the SEQ ID NO:2 in the sequence table;
2) the SEQ ID NO:3 in the sequence table;
3) the SEQ ID NO:4 in the sequence table;
4) amino acid residue sequence of SEQ ID NO:2-4 in the sequence table had the polypeptide of antagonism morphine antinociceptic effect through replacement, disappearance or the interpolation of one to ten amino-acid residue.
One to ten amino-acid residue of said replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.
SEQ ID NO:2 in the sequence table is made up of 14 amino-acid residues, will have the anti-opioid called after 99A-16 of SEQ ID NO:2 amino acid residue sequence; SEQ ID NO:3 in the sequence table is made up of 19 amino-acid residues, will have the anti-opioid called after 99A-19 of SEQ ID NO:3 amino acid residue sequence; SEQ ID NO:4 in the sequence table is made up of 14 amino-acid residues, will have the anti-opioid called after DBI-16 of SEQID NO:4 amino acid residue sequence.
The encode gene of above-mentioned anti-opioid active fragments is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID NO:12 in the sequence table;
2) dna sequence dna of SEQ ID NO:13 in the sequence table;
3) dna sequence dna of SEQ ID NO:14 in the sequence table;
4) dna sequence dna of SEQ ID NO:2-4 in the code sequence tabulation;
5) with sequence table in the nucleotide sequence that limits of SEQ ID NO:12-14 have 90% above homology and have the nucleotide sequence that plays an important role in the antagonism morphine antinociceptic effect;
The nucleotide sequence of the dna sequence dna hybridization that 6) under the rigorous condition of height, can limit with the SEQ ID NO:12-14 in the sequence table.
The rigorous condition of said height is: (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.
SEQ ID NO:12 in the sequence table is by 48 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:2 in the sequence table; SEQ ID NO:13 in the sequence table is by 57 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:3 in the sequence table; SEQ ID NO:14 in the sequence table is by 48 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:4 in the sequence table.
The antagonism peptide of above-mentioned anti-opioid 99A also is that the present invention will protect, and can be one of following amino acid residue sequences:
1) the SEQ ID NO:5 in the sequence table;
2) the SEQ ID NO:6 in the sequence table;
3) the SEQ ID NO:7 in the sequence table;
4) replacement, disappearance or the interpolation through one to ten amino-acid residue of the amino acid residue sequence of SEQ ID NO:5-7 in the sequence table had the morphine analgesia of enhancing, reverses the morphine tolerance or/and weaken or eliminate the polypeptide of Withrawal symptom effect.
One to ten amino-acid residue of said replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.
SEQ ID NO:5 in the sequence table is made up of 21 amino-acid residues, will have the antagonism peptide called after CP-99A-16+5 of the anti-opioid of SEQ ID NO:5 amino acid residue sequence; SEQ ID NO:6 in the sequence table is made up of 19 amino-acid residues, will have the antagonism peptide called after CP-99A-19 of the anti-opioid of SEQ ID NO:6 amino acid residue sequence; SEQ ID NO:7 in the sequence table is made up of 19 amino-acid residues, will have the antagonism peptide called after CP-DBI-16+5 of the anti-opioid of SEQ ID NO:7 amino acid residue sequence.
The gene of code book invention anti-opioid 99A antagonism peptide is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID NO:8 in the sequence table;
2) dna sequence dna of SEQ ID NO:9 in the sequence table;
3) dna sequence dna of SEQ ID NO:10 in the sequence table;
4) dna sequence dna of SEQ ID NO:5-7 in the code sequence tabulation;
5) with sequence table in the nucleotide sequence that limits of SEQ ID NO:8-10 have 90% above homology and strengthening morphine analgesia, reversing morphine and tolerate or/and weaken or eliminate the nucleotide sequence that plays an important role in the Withrawal symptom;
The nucleotide sequence of the dna sequence dna hybridization that 6) under the rigorous condition of height, can limit with the SEQ ID NO:8-10 in the sequence table.
The rigorous condition of said height is: (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.
SEQ ID NO:8 in the sequence table is by 63 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:5 in the sequence table; SEQ ID NO:9 in the sequence table is by 57 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:6 in the sequence table; SEQ ID NO:10 in the sequence table is by 63 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:7 in the sequence table.
Contain that arbitrary segmental primer also belongs to protection scope of the present invention in antagonism peptide expression carrier, transgenic cell line and the host bacterium of above-mentioned anti-opioid 99A and this gene that increases.
The antagonism peptide of above-mentioned anti-opioid 99A can adopt conventional artificial synthesis directly to obtain; Method synthetic such as solid-phase synthesis like available fmoc protection; Maybe can entrust and beautiful join (Xi'an) bio tech ltd or Shanghai to dodge biotech firm such as the synthetic ltd of brilliant biological polypeptide synthetic; In addition, the also available method of utilizing microbial fermentation to express obtains.
The present invention also provides a kind of method of expressing the antagonism peptide of above-mentioned anti-opioid 99A.
The method of the above-mentioned antagonism peptide of expression provided by the present invention is with the recombinant expression vector importing host cell of the antagonism peptide gene that contains above-mentioned anti-opioid 99A, expresses the antagonism peptide that obtains anti-opioid 99A.
Said host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Said intestinal bacteria can be E.coli BL21 (DE3), E.coli DH5 α or E.coli Top10 etc.
Be used to make up the said carrier that sets out that contains the antagonism peptide expression carrier of anti-opioid 99A can be any one can be at the prokaryotic expression carrier of expression in escherichia coli foreign gene, like pET-22b, pET-11c, pET-30a, pET-28a, pET-28b or pET-28c etc.
Be the carrier that sets out with pET-22b, the antagonism peptide expression carrier that contains anti-opioid 99A of structure is pET-CP-99A-16, pET-CP-99A-19 or pET-CP-DBI-16.
Above-mentioned recombinant expression vector all can make up according to ordinary method.
The method that above-mentioned recombinant expression vector is transformed the host bacterium can be method for transformation commonly used in the bioengineering field, like the protoplast transformation method of heat shock method, electrotransformation, joint conversion method or PEG mediation etc.
Cultivation contains the substratum and the culture condition of host cell of the antagonism peptide gene of anti-opioid 21Kd, all can be substratum and the culture condition of cultivating the host that sets out.Wherein, need add inductor when cultivating said recombination bacillus coli host, like IPTG etc.; Add IPTG concentration can be 0.1-1.0mmol/L, be preferably 0.2mmol/L, inducing temperature can be 14-37 ℃; Be preferably 30 ℃, induction time can be 2-4 hour, is preferably 3 hours.
Another object of the present invention provides and a kind ofly strengthens morphine analgesia, reverses the morphine tolerance or/and weaken or eliminate the medicine of Withrawal symptom.
The activeconstituents of medicine provided by the present invention is the antagonism peptide of above-mentioned anti-opioid 99A; Wherein, the antagonism peptide of said anti-opioid is selected from one of following amino acid residue sequence:
1) the SEQ ID NO:5 in the sequence table;
2) the SEQ ID NO:6 in the sequence table;
3) the SEQ ID NO:7 in the sequence table;
4) replacement, disappearance or the interpolation through one to ten amino-acid residue of the amino acid residue sequence of SEQ ID NO:5-7 in the sequence table had the morphine analgesia of enhancing, reverses the morphine tolerance or/and weaken or eliminate the polypeptide of Withrawal symptom effect.
One to ten amino-acid residue of said replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.
When needing; In said medicine, can also add one or more acceptable accessories, said auxiliary material comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, absorption enhancer, tensio-active agent, lubricant and the stablizer etc. that pharmaceutical field is conventional.
Medicine of the present invention can be processed various ways such as injection liquid, dry powder injection, tablet or granula.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
Adult's consumption of said medicine is generally 0.01-0.5mg/kg body weight/inferior, can use by one or many, is generally the course of treatment 10 to 20 days.
The present invention provides that separation of pure dissolves a kind of anti-opioid 99A and active fragments thereof from the pig brain, and it has antagonism morphine antinociceptic effect, and the increase that the drug abuse process is accompanied by drugs dosage can impel this proteic synthetic increase, the formation that causes the morphine tolerance and rely on.The present invention utilizes to have interactional principle between the protein molecule, according to the sequence of three active fragmentss, has designed their antagonism peptide.Because the molecular weight of these antagonism peptides is easier to through hemato encephalic barrier less than 2000 dalton, play the effect of counteracting endogenous anti-opioid effect in the region of interest of cns.The artificial synthetic antagonism peptide of experiment proof can be used as the suppressor factor of endogenous anti-opioid, through the antagonism peptide of three active fragmentss of intravenous injection, can reach and strengthens morphine analgesia, reverses the morphine tolerance and weaken or eliminate the Withrawal symptom effect.This medicine has the following advantages: 1) evident in efficacy: activeconstituents antagonism peptide is to reach the purpose that strengthens morphine analgesia, the tolerance of reverse morphine and eliminate or weaken junkies's Withrawal symptom through eliminating endogenous anti-opioid unnecessary in junkies's body and effect thereof, can eliminate Withrawal symptom from the source; 2) the antagonism peptide that obtains all is that some amino-acid residue residue quantity are no more than 25 small peptide, and is synthetic easily, is convenient to development research; 3) these small peptides get in the body and are degraded easily, do not possess the spinoff that produces antibody, and are safe; 4) medication is convenient, can be through multiple mode administrations such as intravenous injection mode or sublingual lozenges, to eliminate or to weaken the symptom of morphine abstinence syndrome; The antagonism peptide of anti-opioid 99A active fragments of the present invention will play a significant role in medical science and anti-additive medicament field, have a extensive future.
Below in conjunction with specific embodiment the present invention is explained further details.
Description of drawings
Fig. 1 is for detecting Pedis Canitis extract causes the mouse vas deferens shrinking effect to electricimpulse influence
Fig. 2 injects the influence of dog brain crude extract (AOS) to rat acupuncture analgesia effect for the detection side Intraventricular
Fig. 3 A and Fig. 3 B are the detected result of anti-opioid 99A to the retarding effect of morphine antinociceptic effect
The detected result that Fig. 4 is reversed the tolerance to morphine for the antiserum(antisera) that injects anti-opioid 99A in the morphine tolerance mouse tricorn.
Fig. 5 detects the positive position of anti-opioid 99A immunity on the mouse brain sheet for immunohistochemical methods
Fig. 6 is to the detected result by the caused morphine antinociceptic effect influence of grey matter micro-injection morphine around the homonymy cerebral aqueduct at rat habenular nucleus micro-injection 99A
Fig. 7 A-Fig. 7 D is anti-opioid peptide section 99A-14,99A-19, DBI-16 and the DBI-19 detected result to the influence of morphine (20mg/kgi.P.) antinociceptic effect
Fig. 8 A and Fig. 8 B are that 99A-14,99A-19 and DBI-16 antagonism morphine antinociceptic effect and DBI-19 strengthen the morphine antinociceptic effect respectively through dissimilar receptor-mediated qualification results
Fig. 9 is the detected result of nmda receptor and the influence of NOS in anti-opioid 99A and active fragments antagonism morphine antinociceptic effect thereof
Figure 10 A-Figure 10 C is antagonism peptide CP-99A-16, CP-99A-19 and the CP-DBI-16 detected result to the influence of morphine antinociceptic effect
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J.; Russell; DavidW., Molecular Cloning:A Laboratory Manual, 3
RdEdition, 2001, NY, Cold SpringHarbor).
The primer and dna sequence dna are given birth to worker's biotechnology ltd by Shanghai and are synthesized.
Used experiment mice is the male mouse of kunming (available from animal institute of AS) of age in 8-10 week, body weight 20-22g among the following embodiment
The acquisition of embodiment 1, anti-opioid 99A and functional verification thereof
One, the acquisition of pig brain anti-opioid 99A and dose-dependent effect thereof detect
1, the discovery of anti-opioid
The mouse vas deferens sample that is exsomatizing; (the 0.1MS ripple is wide to give square-wave pulse; 1 time/second) stimulate and can cause contraction (concrete grammar is referring to document: J.Hughes et al (1975) Effect of morphine on adrenergictransmission in the Mouse Vas deferens.Assessment of agonist and antagonistpotencies of Narcotic analgesics 53; 371-381); Then; Successively in perfusate, add Srm-Rhotaard (3.2nM) or Pedis Canitis extract (AOS, 200 μ g/mL) (for the dog brain extracts liquid concentrator elder generation after Sephadex G-50 gel (available from Pharmacia company) filters, behind ethyloic-Mierocrystalline cellulose (CM-C is available from H.Reeve-Angel&.Co.Ltd company) cation-exchange chromatography; Obtain sample), observe the influence that the electricity irritation mouse vas deferens is caused shrinking effect.It is as shown in Figure 1 that (M represents to morphine; MAP represents to Pedis Canitis extract), experiment is found in dog brain crude extract, to exist the composition that can strengthen the contraction of electricity irritation mouse vas deferens; And the effect that the enhancing shrinking effect of this sample and morphine inhibition electricity irritation mouse vas deferens shrink is on the contrary; Therefore, after in this dog brain crude extract injection mouse tricorn, the antinociceptic effect that can be observed morphine is suppressed significantly.
In addition; Above-mentioned dog brain crude extract is injected the tricorn (400 μ g/20 μ l/ only) of the effective rat of acupuncture analgesia (6); With saline water (Saline) is contrast (6), and the beginning acusector is induced after 10 minutes, and the shout reaction of rat to noxious stimulus observed at every interval in 10 minutes after 30 minutes.The result is as shown in Figure 2, and (I.C.V. represents through burying the conduit micro-injection sample in tricorn in advance, and on behalf of acusector, EA induce acupuncture analgesia; X-coordinate is the dog brain crude extract injection back time, and ordinate zou is the variation of vocalization threshold), (Saline) is contrast with saline water, after acupuncture induction, the acupuncture analgesia effect appears in rat; After the injection of dog brain crude extract, carry out acupuncture induction again, the acupuncture analgesia effect of rat then disappears, and the acupuncture analgesia effect of control rats still exists, and the AOS that exists in the above-mentioned experimental result prompting brain possibly be the infull internal factor of needle anaesthesia analgesia.
2, the acquisition of pig brain anti-opioid 99A and dose-dependent effect thereof detect
The method of separation and purification anti-opioid-99A from the pig brain; Be also to be improvement (the long people (1997) of grade of willow slightly equally with reference to the long people's of grade of willow method; The separation and purification of the anti-morphine analgesia peptide of pig brain and antiserum(antisera) thereof are to the preliminary study of morphine tolerance influence; Chinese biological The Chemicals 11 volumes 3 phase 322-326); Concrete grammar is: the homogenate in 0.1nM acetate of the full brain of fresh pig is extracted, with the liquid concentrator of Medulla sus domestica extract earlier after the Sephadex-G50 gel-filtration, after S-sepharoseFast Flow ion exchange chromatography (S-sepharose Fast Flow chromatography column is available from Pharmacia company) separates; Get the sample of the 1st elution peak; Separate through Q-sepharose Fast Flow ion exchange chromatography (Q-sepharoseFast Flow chromatography column is available from Pharmacia company), use the HPLC purifying at last, obtain purified anti-opioid material.
Detect the effect of the AOS of purifying to the antinociceptic effect of morphine; Method is: 10min after the abdominal cavity injects the 20mg/kg morphine, inject through tricorn or tail intravenous route the pig brain AOS of purifying respectively, wherein to mouse; The ID of tricorn is respectively 0.5,1.0,2.0nM/4 μ l; Tail venous ID is respectively 1.5,3.0,5.0mg/kg, is contrast with saline water (Saline), detects the antinociceptic effect of morphine then.(X-coordinate is represented the time behind the injection of morphia to the test-results of intracerebroventricular injection group, and ordinate zou is represented the variation (%) of vocalization threshold, and n is every group of mouse quantity shown in Fig. 3 A; I.p. represent abdominal injection; I.c.v. represent injection in the tricorn), the test-results of tail vein injection group is compared with control group shown in Fig. 3 B; The morphine antinociceptic effect of two groups of mouse is all suppressed by AOS in the pig brain significantly, and this retarding effect and dosage are closely related.This is called anti-opioid 99A (AOP-99A) by the albumen with antagonism morphine antinociceptic effect that extracts in the pig brain; This anti-opioid is carried out complete sequence determination; Sequencing result shows that this anti-opioid has the amino acid residue sequence of SEQ ID NO:1 in the sequence table; Be made up of 86 amino-acid residues, this sequence is consistent with the sequence of chitling DBI.The gene of coding anti-opioid 99A has the nucleotide sequence of SEQ ID NO:11 in the sequence table, by 258 based compositions.
Two, the detection of tolerance of anti-opioid 99A and morphine and dependence
1, the monoclonal antibody of preparation anti-opioid 99A
Extract the also anti-opioid 99A of purifying with step 1; By its monoclonal antibody of ordinary method preparation (preparing method's reference literature: J.M.Davis; J.E.Pennington; A.M Kubler., J.F.Consiene (1982) Asimple, single-step technique for selecting and cloning hybridomas forproduction of monoclonal antibodies.J Immunol Methods 50; 141-; Teruko Tumura; Heins Buer, Chriatian Birr and Rudiger Pipkom. (1983) Antibodies againstsynthetic peptides as a tool for function analysis of the transforming proteinPP
60srcCell 34587-596 Sep.1983; Xi Gou., Ai Wang., Xi Li.; But, belong to poor antigen Zh.Q.Gou and Y.Zhang (1991) Studies on monoclonal antibody against recombinant granulocytecolony-stimulating factor.Chinese Medical Sciences Journal (6) 5:212-214), because the molecular weight of anti-opioid 99A is about 10Kd; Immunogenicity is relatively poor, therefore, and before preparation antibody; Need earlier itself and the coupling of key hole maple hemocyanin (KLH) carrier as antigen; Then, the Balb/C mouse is carried out immunity (carrying out 2 subcutaneous multi-point injection immunity, two weeks of midfeather); Preceding 3 days of cytogamy, intrasplenic injection 13-20 μ g antigen booster immunization.During fusion, under aseptic condition, take out the splenocyte of immune mouse and the SP of mouse
20Cytogamy is used semi-solid culture technique, and the hybridoma through screening, clone obtain producing AOP-99A antibody is purified into monoclonal antibody at last from the ascites of collecting.
2, the detection of tolerance of anti-opioid 99A and morphine and dependence
Choose 20 healthy mices, every day three times, the Srm-Rhotaard of subcutaneous injection ascending-dose, ID is incremented to 60mg/kg by 10mg/kg, continues the mouse of formation in 10 days to the morphine tolerance.Mouse in morphine tolerance and dependence; After the abdominal injection morphine (50mg/kg); The antiserum(antisera) that contains anti-opioid 99A monoclonal antibody that step 1 is obtained dilutes in 1: 100,1: 10,1: 1 ratio respectively; Do injection in the tricorn respectively, ID is every 12 μ l (containing 13mg antibody approximately), makes blank (Blank) with non-tolerance mouse; Not inject sero-fast morphine tolerance mouse is contrast (Control), detects in the morphine tolerance mouse tricorn and injects the reverse effect of anti-opioid 99A antiserum(antisera) to the morphine tolerance.Detected result is as shown in Figure 4, and (X-coordinate is the different experiments group, and ordinate zou is the anti-injury of the morphine area under a curve (cm of unit
2); The * representative is compared P＜0.05 with tolerance mouse control group; * * representative is compared P＜0.01 with tolerance mouse control group); Compare with tolerance mouse control group; The morphine tolerance reverse in various degree occurs according to the difference of anti-opioid 99A antiserum(antisera) concentration, compares with the blank group of non-tolerance, and the antiserum(antisera) (1: 1) that injects 99A in the tricorn can reverse the tolerance of 50 milligrams of/kilogram doses of morphine fully.It is thus clear that as long as the dosage of anti-opioid 99A antibody is enough to combine with maincenter endogenous anti-opioid 99A, reduce anti-opioid 99A level, can make the morphine tolerance be able to reverse.
The therapeutic action of giving up effect-jump that the monoclonal antibody that adopts following dual mode to detect anti-opioid 99A is brought out Naloxone (10mg/kg).First kind of mode is to do subcutaneous injection with the ascending-dose morphine to mouse; Every day 2 times; Continue the tolerance and the dependence that formed morphine in 20 days, after the injection of morphine 6-8 hour the last time, the monoclonal antibody of the anti-opioid 99A of the above-mentioned acquisition of injection in tricorn; ID is every 12 μ l (containing 13 μ g antibody approximately), and abdominal injection opiate antagonist Naloxone (10mg/kg) is to bring out spontaneous jump symptom then.The result is as shown in table 1, and at control group (injection 12 μ l PBS in the tricorn), spontaneous jump symptom all appears in 5 mouse; In the treatment group, spontaneous jump symptom does not all appear in 5 mouse, explains that the monoclonal antibody of anti-opioid 99A can be eliminated the spontaneous jump symptom of being brought out by opiate antagonist Naloxone.
The detected result that the monoclonal antibody of table 1 anti-opioid 99A is brought out the therapeutic action of giving up effect-jump to Naloxone (10mg/kg)
| Control group ** | The treatment group ** |
| No.14 | No.20 |
| No.37 | No.40 |
| No.54 | No.60 |
| No.71 | No.80 |
| No.92 | No.100 |
* does subcutaneous injection with the ascending-dose morphine, and every day, secondary continued tolerance and the dependence of formation in 20 days to morphine
The second way is in the morphine tolerance and relies in the forming process of mouse; (ID is incremented to 80mg/kg by 10mg/kg to subcutaneous injection dosage escalation morphine; Inject every day 3 times, continue 12 days) time, the monoclonal antibody of in tricorn, injecting the anti-opioid 99A of above-mentioned acquisition once a day; ID is every 12 μ l (containing 13 μ g antibody approximately); As above-mentioned test, after the injection of morphine 6-8 hour the last time, detect the spontaneous jump symptom of being brought out with opiate antagonist (naloxone).The result is as shown in table 2, and the spontaneous jump symptom of being brought out by opiate antagonist Naloxone (10mg/kg i.P.) can eliminated or weaken to the monoclonal antibody of anti-opioid 99A.
The detected result that the monoclonal antibody of table 2 anti-opioid 99A is brought out the therapeutic action of giving up effect-jump to Naloxone (10mg/kg)
| Control group ** | ★ treatment group ** |
| No.10 | No.20 |
| No.310 | No.40 |
| No.520 | No.61 |
| No.725 | No.84 |
* does subcutaneous injection with the ascending-dose morphine, tolerance and dependence that lasting 12 days of every day three times formed morphine;
★ is when inject the ascending-dose morphine every day, and the monoclonal antibody of injecting anti-opioid 99A by tricorn once.
Infer according to above-mentioned test-results; Process in morphine tolerance and dependence formation; Increase along with dose of morphine; The synthetic of anti-opioid possibly increase in the brain, and the reverse of morphine tolerance or with the elimination of Withrawal symptom or weaken possibly be the cause that endogenous anti-opioid 99A extraordinary in the brain is offset by its monoclonal antibody.Recently, (Katsura M, Hara A such as Katsura M.; Higo A, Tarumi C, Hibino Y; Ohkuma S.Continuous treatmentwith morphine increases diazepam binding inhibitor mRNA in mouse brain.J.Neurochem 1998 dec.71 (6) 2638-41) and people such as Liu X.B. report [Liu X.B., Li Y., Zhou L.; Chen H.X.; Su.Zh.H., Hao W.Conditioned place preference associateswith the mRNA expression of diazepam binding inhibitor in brain regions of theaddicted rat during withdrawal.Mol.Brain Res 117 (2005) 47-54], when pharmacological dependence; The messenger RNA(mRNA) of Benzodiazepine binding inhibitors in the brain; Be that the expression amount of DBI mRNA in brain increases, thereby prove that above-mentioned supposition is reasonably, but because the molecular weight of anti-opioid 99A monoclonal antibody is about 130,000 dalton; Can not pass through hemato encephalic barrier, thereby not possess the prospect of clinical application.
Three, detect the significant points of anti-opioid 99A antagonism morphine antinociceptic effect in the mouse brain
Detect the distribution of anti-opioid 99A at the mouse brain with the immunohistochemical methods method: from the immunohistochemical staining result (as shown in Figure 5) of the crown section of mouse brain, observe habenular nucleus (Hb) position at the mouse brain stem, anti-opioid 99A immunity positive reaction is stronger.Known habenular nucleus has neurone to end at nuclei of median raphe, and it is the important post house of descending property inhibition in analgesia is regulated.Inject morphine 2 μ l (containing 20 μ g) through chronic burying in the conduit trace of rat periaqueductal gray (PAG) (position that opiate receptor distributes and concentrates); After 10 minutes again through burying the pure article of anti-opioid 99A that inject various dose in the conduit of homonymy habenular nucleus trace; ID is respectively 1,2,5nM/4 μ l, is contrast with saline water (Saline).As shown in Figure 6 [● represent the saline control group; ■ represents the 99A-1nM dose groups; ▲ represent the 99A-2nM dose groups;
represents the 99A-5nM dose groups; N is a mouse quantity; Ordinate zou is the variation (%) of vocalization threshold, and X-coordinate is the time after the injection of morphine], can significantly offset by the caused morphine antinociceptic effect of grey matter micro-injection morphine around the homonymy cerebral aqueduct at rat habenular nucleus micro-injection 99A; And the morphine antinociceptic effect is suppressed according to the difference of anti-opioid 99A ID to some extent, explains that habenular nucleus possibly be the significant points of anti-opioid 99A antagonism morphine antinociceptic effect.This result also points out, and is the same as opioid peptides, has different effects at the anti-opioid of maincenter different sites.
Four, anti-opioid 99A active peptide segment 99A-16,99A-19, the acquisition of DBI-16 and DBI-19 and active detection the thereof
Step 1 shows anti-opioid 99A complete sequence determination result; Its amino acid residue sequence is consistent with the amino acid residue sequence of chitling DBI; Show that they should belong to same family, but there is very big difference in their function, then opposite fully like ox DBI and anti-opioid 99A/ pig DBI to the effect of morphine antinociceptic effect; The former ox DBI has enhancement, and latter's anti-opioid 99A/ pig DBI then shows antagonistic action.In addition, the effect of chitling DBI also presents the release of the Regular Insulin that inhibition brought out by glucose.For disclosing secret wherein; Reference (Zh.W.Chen et alIsolation and characterization of porcine diazepam-binding inhibitor; Apolypeptide not only of cerebral occurrence but also common in intestinaltissues and with effects on regulation of insulin release Eur.J.Biochem 174; 239-245 (1988)) anti-opioid 99A is degraded with pancreatin; Therefrom find 99A-16 (SEQ ID NO:2 in the sequence table) and two active fragmentss of 99A-19 (SEQ ID NO:3 in the sequence table) through micro-performance liquid separation, they are DBI-16 (SEQ ID NO:4 in the sequence table) and DBI-19 peptide section (SEQ ID NO:15 in the sequence table) at the corresponding active fragments of ox DBI.The encoding sequence of 99A-16 has the nucleotide sequence of SEQ ID No:12, by 48 based compositions; The encoding sequence of 99A-19 has the nucleotide sequence of SEQ ID No:13, by 57 based compositions; The encoding sequence of DBI-16 has the nucleotide sequence of SEQ ID No:14, by 48 based compositions.
At anti-opioid 99A and the pairing amino-acid residue of ox DBI sequence, successively dodge the synthetic synthetic 99A-16 of ltd of brilliant biological polypeptide, 99A-19, DBI-16 and these four peptide sections of DBI-19 according to above-mentioned four peptide sections by U.S.'s couplet (Xi'an) bio tech ltd and Shanghai.Use the method identical to detect active peptide segment 99A-14 (0.5-10nM), 99A-19 (0.5-10nM), DBI-16 (10-40nM) and the DBI-19 (11-14nM) of various dose influence to morphine (20mg/kg i.P.) antinociceptic effect with step 1; With saline water (Saline) is contrast; (X-coordinate is represented the time behind the injection of morphia to active detected result shown in Fig. 7 A-Fig. 7 D; Ordinate zou is expressed as the variation (%) of vocalization threshold; N representes respectively to organize experiment mice quantity), can find out by figure, except that demonstrating in the effective dosage ranges of 11-14nM, DBI-19 strengthens (result who strengthens the morphine antinociceptic effect of mouse with ox DBI is consistent) the morphine antinociceptic effect; 99A-16,99A-19 and DBI-16 fragment all show antagonism morphine antinociceptic effect; But their effective dosage ranges is different, and the effective dose of 99A-16 and 99A-19 is 2.5-10nM, and the effective dose of DBI-16 is 10-40nM.Compare the amino acid residue sequence of 99A-16 and two peptide sections of DBI-16, though only deposit the difference of an amino residue, the effective dosage ranges of DBI-16 antagonism morphine antinociceptic effect is 4 times of 99A-16.And between 99A-19 and the DBI-19 peptide section, though only there is an amino residue that hydrophobicity is opposite, then opposite fully to the influence of morphine antinociceptic effect, thus disclosed the reason that causes anti-opioid 99A and ox DBI function to have very big difference.
Five, detect the acceptor of mediation antagonism morphine antinociceptic effect and enhancing morphine antinociceptic effect
Reference (M.Okubo; M.Kawaguchi (1998) Euro.J.Pharmacology 359; 243-249) earlier with the maincenter type antagonist Flumazinal of Benzodiazepine (BZ) acceptor (available from Sigma; Be dissolved among the 1%Tween 80); In injection of morphia preceding 30 minutes, mouse is carried out abdominal injection pre-treatment (2.0mg/kg i.p.), and then detect influence morphine (20mg/kg i.P.) antinociceptic effect with 99A (2nM), 99A-16 (10nM), 99A-19 (10nM) and DBI-16 (40nM) and DBI-19 (11.6nM).With saline water (Saline) is contrast (Control), and not use the pretreated mouse of Flumazinal to be contrast, (ordinate zou is the anti-injury of a morphine area under a curve, the cm of unit shown in Fig. 8 A simultaneously
2Undressed control group is represented in " 1 "; " 2 " representative is through the pretreated experimental group of antagonist; N representes respectively to organize experiment mice quantity); The result only observes DBI-19 the reinforcing effect of morphine antinociceptic effect is changed into retarding effect, and the antagonism morphine antinociceptic effect of other three peptide sections (99A-16,99A-19 and DBI-16) is then unaffected.Use the periphery type antagonist PK11195 (, being dissolved among the DMSO) of benzodiazepine receptors again, in injection of morphine preceding 5 minutes, mouse is carried out tail vein injection pre-treatment (10mg/kgi.v.) available from Sigma.Use 99A (2nM), 99A-16 (10nM), 99A-19 (10nM) and DBI-16 (40nM) to detect influence subsequently again to morphine (20mg/kg i.P.) antinociceptic effect.With saline water (Saline) is contrast (Control), and not use the pretreated mouse of PK11195 to be contrast, (ordinate zou is the anti-injury of a morphine area under a curve to the result, the cm of unit shown in Fig. 8 B simultaneously
2Undressed control group is represented in " 1 ", and " 2 " are represented through the pretreated experimental group of antagonist, and n representes respectively to organize experiment mice quantity), experiment shows that three peptide sections (99A-16,99A-19 and DBI-16) all are reversed the antagonistic effect of morphine antinociceptic effect.Above-mentioned experimental result shows, 99A-16, and the antagonism morphine antinociceptic effect of 99A-19 and DBI-16 is receptor-mediated through periphery type BZ, it then is receptor-mediated through maincenter type BZ that DBI-19 strengthens the morphine antinociceptic effect.
Six, detect nmda receptor and the effect of NOS in anti-opioid 99A and active fragments antagonism morphine antinociceptic effect thereof
Research shows; Excitatory amino acid nmda receptor and nitric oxide synthetase system (NOS) play crucial effects in morphine tolerance and dependence process; After with nmda receptor antagonist or no inhibitor mouse being carried out pre-treatment; The formation that can suppress the morphine tolerance and rely on prevents the appearance of Withrawal symptom.[Adams?ML，Kalicki?JM，MeyerER，Cicero?TJ(1993)Inhibition?of?the?morphine?withdrawal?syndrome?by?nitricoxide?synthase?inhibitor，NG-Nitro-L-arginine?methyl?ester.Life?Sci52：PL245-PL249；Cappendijk?SL，Vries?R，Dzoljic?MR(1993)Inhibitory?effectof?nitric?oxide(NO)synthase?inhibitors?on?naloxone-precipitated?withdrawalsyndrome?in?morphine-dependent?mice.Neurosci?Lett?142：97-100；Herman?BH，Vocci?F，Bridge?P(1995)The?effect?of?NMDA?receptor?antagonists?and?nitricoxide?synthase?inhibitors?on?opioid?tolerance?and?withdrawal.Neuropsychopharmacology?11：269-293；Vaupel?DB，Kimes?AS，London?ED(1995b)Nitric?oxide?synthase?inhibitors：preclinical?studies?of?potential?use?fortreatment?of?opiate?addiction.Neuropsychopharmacology?11：313-322；Trujillo?K.A.A?review?of?preclinical?studies：Are?NMDA?receptors?involvedin?opiate-induced?neural?and?behavioral?plasticity?Psychopharmacology(2000)131：121-121；Heinzen?EL，Pollack?G.M.(2004)The?development?of?morphineantinociceptive?tolerance?in?nitric?oxide?synthase-deficient?mice.Biochem.Pharmacology?vol?67(4)：735-741]。Using with the step 5 similar methods uses excitatory amino acid nmda receptor antagonist MK-801 (available from Sigma respectively; 0.1mg/kg i.p.) preceding 15 minutes in injection of morphine; Or with nitric oxide synthetase no inhibitor L-NAME (available from Sigma; 45mg/kg i.p.) in injection of morphine preceding 20 minute; Mouse is carried out pre-treatment, and then detect influence morphine (20mg/kg i.P.) antinociceptic effect with 99A (2nM), 99A-16 (10nM), 99A-19 (10nM) and DBI-16 (40nM). with saline water (Saline) is contrast (Control), simultaneously not use the pretreated mouse of MK-801 and L-NAME to be contrast; The result is as shown in Figure 9, and (ordinate zou is the anti-injury of a morphine area under a curve, the cm of unit
2Undressed control group is represented in " 1 "; " 2 " representative is through the pretreated experimental group of no inhibitor; " 3 " representative is through the pretreated experimental group of nmda antagonist; N representes respectively to organize experiment mice quantity), anti-opioid 99A and 99A-16,99A-19 and DBI-16 peptide section are disengaged the antagonistic action of morphine antinociceptic effect, explain that anti-opioid 99A and small peptide antagonism morphine antinociceptic effect thereof need the mediation of nmda receptor and NOS.Pushing away side thus, handle with nmda receptor antagonist and no inhibitor, can suppress the morphine tolerance and the formation that relies on maybe can prevent the appearance of Withrawal symptom, possibly be the cause that the effect because of anti-opioid is blocked.
The acquisition of embodiment 2, anti-opioid 99A antagonism peptide and active detection the thereof
One, the acquisition of the antagonism peptide of 99A-16,99A-19 and DBI-16
According to there being interactional principle between the protein molecule; (J.R.Heal et al Specificinteractions between sense and complementary peptides:The Basis for theproteomic code ChemBIOChem 2002,3 114-131) designs the antagonism peptide of 99A-16,99A-19 and three small peptides of DBI-16 to reference; Wherein, With the antagonism peptide called after CP-99A-16+5 of 99A-16, have the amino acid residue sequence of SEQ ID NO:5, form by 21 amino-acid residues; Its encoding sequence has the nucleotide sequence of the SEQ ID NO:8 in the sequence table, by 63 based compositions; With the antagonism peptide called after CP-99A-19 of 99A-19, have the amino acid residue sequence of SEQID NO:6, to form by 19 amino-acid residues, its encoding sequence has the nucleotide sequence of the SEQ ID NO:9 in the sequence table, by 57 based compositions; With the antagonism peptide called after CP-DBI-16+5 of DBI-16, have the amino acid residue sequence of SEQ ID NO:7, to form by 21 amino-acid residues, its encoding sequence has the nucleotide sequence of the SEQ ID NO:10 in the sequence table, by 63 based compositions.
Two, the activity of the antagonism peptide of 99A-16,99A-19 and DBI-16 detects
Choose 18 of normal kunming mices, divide three groups (6 every group, n=6); After injection of morphine (20mg/kgi.P.), inject CP-99A-16+5 (7.0mg/Kg), CP-99A-19 (6.6mg/Kg) and CP-DBI-16+5 (22mg/Kg) respectively, detect influence the morphine antinociceptic effect by the tail vein; [with saline water is contrast (Control; Saline 50 μ l i.V.)], result's [X-coordinate is the time (min) behind the injection of morphia, and ordinate zou is the variation (%) of vocalization threshold] shown in Figure 10 A-Figure 10 C; Compare with control group; The antinociceptic effect of morphine strengthens significantly behind injection CP-99A-16+5, CP-99A-19 and the CP-DBI-16+5, and obviously these antagonism peptides play a part the anti-opioid suppressor factor, have offset the effect of endogenous anti-opioid.In addition, because these antagonism peptide molecular weights less than 2000 dalton, are injected in the body and can be got into cns through hemato encephalic barrier by the tail vein, established antagonism peptide CP-99A-16+5, CP-99A-19 and CP-DBI-16+5 and had the prospect of clinical application.
Utilize the morphine tolerance of mouse and rely on model, also carried out the morphine abstinence syndrome effect experiment that Narlan (Naloxone) brings out, method is: with the Srm-Rhotaard of ascending-dose; Continue 12 days every day three times, makes mouse form tolerance and dependence to morphine to mouse do subcutaneous injection; After the 11st day last injection of morphine 6-8 hour; Give control mice (n=7) tail vein injection saline (Saline 50 μ l i.V.), abdominal injection Naloxone (10mg/kg is available from Sigma); The whole mouse of result all induce gives up effect-jump; But inject antagonism peptide CP-99A-19 (17mg/kg) respectively to morphine tolerance and dependence mouse tail vein, behind CP-99A-16+5 (17.5mg/kg) and the CP-DBI-16+5 (65mg/kg), the effect-jump of giving up that Naloxone brings out can be eliminated or obviously weaken (seeing table 3 and table 4).Rank test shows with control group to be compared; There is extremely significant difference; Further prove antagonism peptide CP-99A-16+5 of the present invention; CP-99A-19 and CP-DBI-16+5 have the drug effect that strengthens morphine analgesia, the tolerance of reverse morphine and weaken or eliminate the Withrawal symptom effect, show the prospect of clinical application.
Table 3 tail vein injection antagonism peptide CP-99A-16+5, CP-99A-19 and CP-DBI-16+5 bring out morphine abstinence syndrome symptom--the influence of jump to Narlan (Naloxone 10mg/kg i.P.)
* * rank test shows with control group to be compared, and difference is (P=1-0.025=0.975) extremely significantly
Table 4 tail vein injection antagonism peptide CP-99A-16+5, CP-99A-19 and CP-DBI-16+5 bring out morphine abstinence syndrome symptom--the influence of jump to Narlan (Naloxone 10mg/kg i.P.)
The * rank test shows with control group to be compared, significant difference (P=1-0.05=0.95).
Claims (7)
1. the antagonism peptide of an anti-opioid, its amino acid residue sequence is the SEQ ID NO:6 in the sequence table.
2. the gene of coding claim 1 described anti-opioid antagonism peptide.
3. gene according to claim 2 is characterized in that: the nucleotide sequence of said gene is the SEQ ID NO:9 in the sequence table.
4. contain claim 2 or 3 said expression carrier.
5. the transgenic cell line that contains claim 2 or 3 said genes.
6. the host bacterium that contains claim 2 or 3 said genes.
7. the antagonism peptide with the described anti-opioid of claim 1 is enhancing morphine analgesia, the tolerance of reverse morphine of activeconstituents and/or the medicine that weakens or eliminate the Withrawal symptom effect.
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| WO1995022557A1 (en) * | 1994-02-21 | 1995-08-24 | Astra Aktiebolag | Novel opioid peptides for the treatment of pain and use thereof |
| CN1626245A (en) * | 2003-09-16 | 2005-06-15 | 薛毅珑 | New application of chromaffin cell of adrenal medulla or opium peptide energy cell |
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| WO1995022557A1 (en) * | 1994-02-21 | 1995-08-24 | Astra Aktiebolag | Novel opioid peptides for the treatment of pain and use thereof |
| CN1626245A (en) * | 2003-09-16 | 2005-06-15 | 薛毅珑 | New application of chromaffin cell of adrenal medulla or opium peptide energy cell |
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| Title |
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| 杨树长等.猪脑抗吗啡镇痛肽的分离纯化及其抗血清对吗啡耐受影响的初步研究.《生物化学杂志》.1997,第13卷(第3期),第322-326页. * |
| 陈淑娟等.抗吗啡活性肽的分离纯化及一级结构测定.《生物化学杂志》.1996,第12卷(第2期),第211-214页. * |
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