CN104800829B - Oligopeptides combines or is preparing the purposes being used in the drug of analgesic with the combination of morphine - Google Patents
Oligopeptides combines or is preparing the purposes being used in the drug of analgesic with the combination of morphine Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Purposes of the combination in preparing for analgesic drug the present invention relates to oligopeptides joint or with morphine.Specifically, the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM NH of the present invention2Transcription and/or the expression of delta opiate receptor are improved in improving subject, and can improve the analgesic effect of morphine in turn.
Description
Technical field
Purposes of the combination in preparing for analgesic drug the present invention relates to joint or with morphine.Specifically, this
Invention is related to Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2The transcription of delta opiate receptor and/or the purposes of expression in improving subject, in turn
It can be used for combining or combining with morphine for treating or relieving pain.
Background technology
Tachykinin is that have common carboxyl terminal sequence（FXGLM-NH2）Peptide family, member most well known
Including：Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP), neurokinin A（NKA）With neurokinin B (NKB), they are from two different gene codes：
Ppta encodes SP, NKA；Pptb encodes NKB.2000 and 2002, tachykinin gene Pptc was identified before third, it is compiled
Code HK-1.HK-1 is initially to be accredited in mouse hematopoetic cell out, it is during ancestral's B cell is developed to pre B cell
It plays an important role.Later, it was also accredited out in rat and people.
Many evidences show that people and mouse HK-1 have similar Activation Activity with SP to neurokinin receptor, all to god
Through kinin receptor 1（NK1 receptors）There are very high compatibility and selectivity.However, unlike r/mHK-1 and SP, hHK-1
The compatibility of neurokinin receptor 1 is reduced many (being 14 times and 70 times respectively) with hHK-1 (4-11).Some researchers grind
Studied carefully people and mouse HK-1 immune system, cardiovascular system and pain sensation system some biological activities.It is passed in the regulation and control pain sensation
Aspect is passed, is found before the present inventor, r/mHK-1 and hHK-1 can generate dosage and time after intracerebroventricular injection
The analgesic effect of dependence, and the antagonist of neurokinin receptor 1 and mu opioid receptor antagonists can be blocked significantly by r/ respectively
Analgesic effect caused by mKH-1 and hHK-1.That is the analgesic effect of r/mKH-1 and hHK-1 respectively depends on neurokinin
Receptor 1 and mu opioid receptor.
Endogenic opioid peptides and opiate receptor take part in the adjustment process of pain.Currently, clinically using μ opiums mostly
The agonist of receptor relieves pain, but the side effects such as its tolerance and the dependence of the body ＆ mind easily generated are serious
Constrain its use.On the contrary, selectivity activation delta opiate receptor while generating potent analgesic effect, few side effects
It generates.Therefore, activation delta opiate receptor is presently believed to be a kind of ideal analgesic means.It has been reported that upper reconciliation increases ridge
The delta opiate receptor of marrow level can effectively enhance analgesic effect of the delta opiate receptor agonist in chronic inflammatory pain model
（C.M.Cahill,A.Morinville,C.Hoffert,D.O’Donnell,A.Beaudet.Up-regulation and
trafficking ofδopioid receptor in a model of chronic inflammation:
implications for pain control.Pain,101(2003)199 208.）.
Invention content
In order to raise delta opiate receptor, the present inventor has found Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM- by making great efforts long-term and unremittingly
NH2The expression of delta opiate receptor can be raised.
Therefore, one aspect of the present invention provides a kind of method raising delta opiate receptor expression in subject's body, packet
It includes to the subject（Such as mammal, such as people）Use the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of effective dose2。
Another aspect provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2It is preparing for raising subject（Such as lactation is dynamic
Object, such as people）The preparation of internal delta opiate receptor expression（Such as drug）In purposes.
The present invention also provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Joint is being prepared with the combination of morphine in subject（Example
Such as mammal, such as people）Middle analgesic preparation（Such as drug）In purposes.
The present invention further additionally provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Preparing the analgesic effect for enhancing morphine
Preparation（Such as drug）In purposes.
Some abbreviations are likely related in this specification and claims, the representative content of these abbreviations is ability
Well known to field technique personnel.But content to facilitate the understanding of the present invention, the content representated by relevant abbreviation is quoted from
It is as follows：
r/mHK-1：Rat/mouse hemokinin-1；FAM：Fluoresceincarboxylic acid；PPT：Preprotachykinin；MOR：μ-opium
Receptor；POMC：Proopiomelanocortin；KOR：κ-opiate receptor；PDYN：Prodynorphin；DOR：δ-opiate receptor；PENK：Enkephalins
It is former；GAPDH：Glyceraldehyde-3-phosphate dehydrogenase；HK-1（4-11）：ASQFFGLM-NH2。
Description of the drawings
Fig. 1:Mouse intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Afterwards, NK1 receptors and different preprotachykinins are in mRNA water
Flat expression situation of change.A, b and c are the representative NK1 receptors obtained with ABI7300 system softwares, PPT-A respectively
With the amplification curve of PPT-C.What A, B and C histogram were shown is from at least independent NK1 receptors repeated three times, PPT-A respectively
With the relative mRNA expression levels of PPT-C, reference gene is house-keeping gene GAPDH.It is all reaction every time it is triplicate, at least into
Row is independent three times to be repeated.The control group that each value is injected physiological saline is standardized, and the value of control group is set as 1, as a result with flat
Mean value ± SEM is presented.Compared with the control group, experimental group does not have significant difference.
Fig. 2:Mouse intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Endogenous opioid receptors and endogenous opiatepeptide exist afterwards
The expression situation of change of mRNA level in-site.A, b, c, d, e and f respectively be with ABI7300 system softwares obtain it is representative
MOR (μ-opiate receptor), POMC (Proopiomelanocortin), KOR (κ-opiate receptor), PDYN (prodynorphin), DOR (δ-opium by
Body) and PENK (proenkephalin) amplification curve.What A, B, C, D, E and F histogram were shown be respectively from least it is independent three times
The relative mRNA expression levels of the MOR, POMC, KOR, PDYN, DOR and the PENK that repeat, reference gene are house-keeping gene GAPDH.
All reactions are triplicate every time, at least carry out independent three times repeat.Item corresponds to standard error of the mean.***p≤0.001：
There is pole significant difference compared with respective control group.
Fig. 3：Sxemiquantitative Western Blotting measuring mouse intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2NK1 afterwards
Receptor, MOR, POMC and DOR protein expressions and control group protein expression situation.These pictures come from least independent heavy three times
Multiple representative picture.What column diagram indicated respectively is NK1 receptors (A), MOR (B), the opposite egg of POMC (C) and DOR (D)
White expression situation, reference gene are β-actin.*p≤0.05;**p≤0.01：It is significant compared with respective control group
Difference.
Fig. 4：After morphine is subcutaneously injected in intracerebroventricular injection people HK-1 (4-11) joints, in 48.5 DEG C of warm bath tail-flick test
It can dosage and time dependent generation synergic antalgic effect.* p≤0.01 p≤0.05, * * and p≤0.001 * * *, show people HK-1
(4-11) joint injection of morphia group has statistically significantly sex differernce compared with independent injection of morphia group.
Specific implementation mode
The present inventor is with ICR mouse（It is provided by Hangzhou Pedagogic University of Hangzhou, Zhejiang province city animal center）For research
Object has studied Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Influence to the expression of various opioid peptides receptors and its ligand.We test
As a result show the expression of NK1 receptors and preprotachykinin not by intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Influence.Phase
Correspond to our current work, compared with saline control group, intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Later, MOR（μ
Opiate receptor）[and the expression of KOR (kappa opioid receptor) and their own endogenic ligand POMC and PDYN almost do not have
There is change.It is interesting that in our current study, the transcription of DOR (delta opiate receptor) and protein level generation are extremely notable
Up-regulation, however PENK（The endogenic ligand of DOR）Expression significant changes do not occur.
Therefore, one aspect of the present invention provides a kind of side for improving the transcript and expression level of delta opiate receptor in subject
Method comprising apply a effective amount of Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH to the subject2.Present invention provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-
NH2In the preparation for preparing the transcript and expression level for improving delta opiate receptor in subject（Such as drug）In purposes.This
The another further aspect of invention provides a kind of pharmaceutical product（Such as drug or medicament）Or pharmaceutical composition comprising as activity at
That divides improves the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of the transcript and expression level of delta opiate receptor in subject2；And it is related to medicine including this
Learn the specification of product.
One aspect of the present invention provide it is a kind of raising subject in morphine analgesic effect method comprising to it is described by
Examination person applies a effective amount of Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2, the application be carried out before or after the application of morphine, or with
Coffee is administered simultaneously.Present invention provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Preparing the analgesia effect for improving the morphine in subject
The preparation of fruit（Such as drug）In purposes.Another aspect of the invention provides a kind of pharmaceutical product（Such as drug or medicament）
Or pharmaceutical composition comprising the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM- for improving the analgesic effect of morphine in subject as active constituent
NH2；And it is related to the specification of pharmaceutical product including this.
It is preferably mammal for subject of the present invention, is more preferably people.
For Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH in the raising subject of the present invention2Activity, those skilled in the art can be to it
Any modification is made, precursor is that the modification does not negatively affect its activity.For example, can be PEGylated to oligopeptides progress, to improve
Its half-life period in vivo；It can be connect with known penetrating peptide, to promote the Transdermal absorption etc. of oligopeptides of the present invention.In short, this
Field technology personnel can carry out the oligopeptides of the present invention various modifications to improve delivery efficiency and keep its activity.This kind of modification
Oligopeptides is also within the scope of the present invention.
The Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH as active constituent of the present invention2It can be together with pharmaceutically acceptable carrier
It uses.In addition to the active ingredient (s, method of the invention, purposes and product can also include suitable pharmaceutically acceptable carrier,
Including promoting reactive compound to be processed into the excipient and auxiliary agent of preparation.
The preparation for being for example suitable for injecting or being transfused includes aqueous and non-aqueous sterile injection liquid and aqueous and non-aqueous sterile
Suspension, the aseptic parenteral solution is optionally including antioxidant, buffer, bacteriostatic agent and can make preparation and purpose recipient
Blood equipressure solute, the sterile suspensions may include suspending agent and thickener.The preparation may be present in unit dose
Or in multi-dose container, for example, sealing ampoule, and can be stored in freeze-dried（Freeze-drying）Condition, before use immediately
It only needs that sterile liquid carrier, such as water for injection is added.
The active constituent of the present invention optionally can be combined with solid excipient, and optionally grinds obtained mixing
Object, and when needing, after suitable auxiliary agent is added, the mixture of particle is processed, to obtain required dosage form.Suitable excipient
Especially filler is for example sugared, including lactose, sucrose, mannitol or D-sorbite；Cellulose or starch formulation, gelatin, yellow alpine yarrow
Glue, methylcellulose, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone（PVP）.When needing,
Disintegrant, such as crosslinked polyvinylpyrrolidone, agar or alginic acid or its salt such as sodium alginate can be added.
The effective quantity of the active constituent of the present invention can be to improve the transcription of delta opiate receptor and/or expression in subject
Any amount, about 0.1-15mg Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH can be comparable to2, preferred 0.01-20mg oligopeptides
ASQFFGLM-NH2Dosage unit.It is highly preferred that dosage unit includes the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of about 1-4mg2.Most preferably
Ground, dosage unit include the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of about 2-3mg2.A effective amount of ability measured in those skilled in the art
It is interior, in particular according under the enlightenment of disclosure provided herein.
According to the present invention, pharmaceutical product of the invention（Drug, medicament）Or pharmaceutical composition can be with arbitrary effective dose
Number application administration subject.Preferably, pharmaceutical product of the invention（Drug, medicament）Or pharmaceutical composition can be with multidose
Administration, such as from about 2 to about 15 dosage, more preferably from about 4-10 dosage, most preferably from about 6 dosage.Particularly preferred
About primary frequency was administered by the pharmaceutical product of the present invention with every three weeks during the administration in embodiment（Drug, medicament）Or
Pharmaceutical composition is administered to subject, such as injection, infusion or oral.In particularly preferred embodiment, it is administered to pass through note
It penetrates.
It should be understood that the pharmaceutical product of the present invention（Drug, medicament）Or pharmaceutical composition can be by for passing through any suitable
The mode of any suitable of approach administration prepare.
The pharmaceutical product of the present invention（Drug, medicament）Or the dosage unit of pharmaceutical composition be based on it is conventional be administered by
Examination person.For example, dosage unit can be administered more than once a day, once a week, monthly etc..Dosage unit can be with two
It is administered based on times/week, i.e., twice a week, such as once every three days.
As it is used herein, "comprising" and " comprising ", " containing " or " being characterized in that " it is synonymous, and in being included in
Or open, and it is not excluded for the other element that do not state or method and step.Term "comprising" any table herein
State, especially in the method for the description present invention, purposes or when product, it is thus understood that including substantially by the component or element or
Step forms and those of forms product, method and purposes by the component or element or step.The sheet of description exemplified here
Invention suitably can be there is no any one or more of element not specifically disclosed herein, one or more limitations the case where
Under put into practice.
Following content can be contained by being related to the specification of the pharmaceutical product included in the pharmaceutical product of the present invention：It adapts to
Disease（Such as pain）, administration dosage（Such as above-mentioned exemplarily illustrate）And issuable side effect etc..
The term and statement used herein is used as descriptively rather than restrictive term, and in such term and statement
Use in it is not expected exclude shown in and described feature or part thereof any equivalent, it is appreciated that various modifications are being asked
It is possible in the scope of the present invention of protection.It is therefore understood that although the present invention by preferred embodiment and optionally
Feature specifically discloses, but the modification and transformation of concept disclosed herein, and such modification may be used in those skilled in the art
It is considered as in the scope of the present invention such as defined by accessory claim with variation.
It to be illustrated more clearly that the present invention, is described in detail in conjunction with following examples, but these embodiments are only
To the exemplary description of the present invention, the limitation to the application should not be construed as.
Embodiment 1
Material and method
Animal
The ICR mouse of male and female（20±1.0g）By Hangzhou Pedagogic University's animal center（Hangzhou China）It carries at random
For.All zoopery programs are ratified by Institutes Of Technology Of Zhejiang's animal feeding and using the committee, and meet in November, 1986
No. 24 committees of the European Community instruct (86/609/EEC).23 DEG C of -25 DEG C of rings of all animal feedings in 12 hours light dark cycles
In border, and carry out the food and water of preceding offer standard in experiment.It is reduced as far as possible suffered by mouse in all experimentations
Injury and pain.
Polypeptide
Fluo- Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2With Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Polypeptide is by Zhong Tai companies（Hangzhou China）Using solid
Phase polypeptide synthesis synthesizes, and uses efficient liquid phase（HPLC）Purifying, purity reach 98%.FAM is carboxyl fluorophor,
5-carboxyfluorescein is coupled on the amino acid group of Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2 peptides using standard coupling technique.Oligopeptides
ASQFFGLM-NH2It is dissolved in physiological saline, working solution concentration is 1.5mM.It is fluorescein-labeled（FAM）Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-
NH2It is dissolved in 50%DMSO（Dimethyl sulfoxide (DMSO)）In, the concentration of storing liquid is 15mM.It is with physiological saline that its is dilute before the experiments
Release the working concentration of 1.5mM.
Intracerebroventricular injection and tissue preparation
Intracerebroventricular injection is operated according to the Haley and McCormick operating methods in conscious mouse described.Often
The Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of mouse injection2Or fluo- Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Final dose be 6nmol, volume injected
It is 4 μ l.
The result of study of our early periods shows intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Afterwards, have in 20min apparent
Analgesic effect, therefore the time point of this experiment be selected as tri- time points of 5,10 and 20min.In injection FAM- oligopeptides
ASQFFGLM-NH2 or Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2（Every mouse 6nmol）Afterwards, at corresponding time point, the neck that breaks is put to death small
The accuracy of mouse, injection site is verified by micrometering.In control group, to 4 μ l physiological saline of mouse intracerebroventricular injection.So
Afterwards, Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH will be injected2Or the mouse brain of physiological saline takes out rapidly, be stored in -80 DEG C of refrigerators for into
One step is tested.It rapidly will injection FAM- Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Mouse brain take out, be fixed on containing 4% paraformaldehyde
（Sigma）0.1M pH7.4 phosphate buffer in for 24 hours, in frozen section (JUNG Tissue freezing
Medium, LEICA, Germany) it is dehydrated before with 30% sucrose solution.
Coronal section and micro- sem observation
Every mouse brain is frozen in -20 DEG C, and slice thickness is 10 μm.Then brain tissue slice is saved on slide.It cuts
Piece is stored in 4 DEG C, and microexamination is completed in 12h and is taken pictures.Sample differs and fluorescence microscope（NIKON TE2000-
U）.Anatomical structure is identified according to adult mice brain structure atlas.
Reverse transcription and real-time quantitative PCR
According to commodity operation instructions, using TRIzol reagents (Invitrogen, Carlsbad, CA) from each frost
Total serum IgE is extracted in brain tissue.In brief, brain tissue is ground with mortar, then according to the addition 1ml TRIzol per 100mg
TRIzol is added in dosage.In all experiments, Reverse Transcriptase kit is used（Gibco BRL）By 5 μ g total serum IgEs reverse transcriptions at cDNA,
Final volume is 50 μ l.
Primer sequence is as follows, this is identical [7] as the primer sequence in the work delivered before us：
NK1 receptors (F):5’-TGGACTCTGATCTCTTCCCCAACA-3’
NK1 receptors (R):5’-GGACCCAGATGACAAAGATGACCACTT-3’
PPT-C(F):5’-CGGGCCATCAGTGTGCACTA-3’
PPT-C(R):5’-GGAATCCCCGTCCCCAGCAT-3’
PPT-A(F):5’-GAAATCGATGCCAACGATGATC-3’
PPT-A(R):5’-AGGCTTGGGTCTTCGGGCGATTCT-3’
MOR(F):5’-ATCCTCTCTTCTGCCATTGGT-3’
MOR(R):5’-TGAAGGCGAAGATGAAGACA-3’
POMC(F):5’-AGATTCAAGAGGGAGCTGGA-3’
POMC(R):5’-CTTCTCGGAGGTCATGAAGC-3’
KOR(F):5’-CCGATACACGAAGATGAAGAC-3’
KOR(R):5’-GTGCCTCCAAGGACTATCGC-3’
PDYN(F):5’-CGGAACTCCTCTTGGGGTAT-3’
PDYN(R):5’-TTTGGCAACGGAAAAGAATC-3’
DOR(F):5’-AAGTACTTGGCGCTCTGGAA-3’
DOR(R):5’-GCTCGTCATGTTTGGCATC-3’
PENK(F):5’-AACAGGATGAGAGCCACTTGC-3’
PENK(R):5’-CTTCATCGGAGGGCAGAGACT-3’
GAPDH(F):5’-AGGAGCGAGACCCCACTAACAT-3’
GAPDH(R):5’-GTGATGGCATGGACTGTGGT-3’
The real-time PCR of relative quantification, which is carried out, with ABI7300 Real Time PCR Detection Systems equipment detects said gene in mRNA level in-site
Expression situation of change, using GAPDH as reference gene.Negative control replaces cDNA with distilled water.In each real-time quantitative PCR
Afterwards, a solubility curve analysis is done.The normalized expression level 2- of each target gene△△CtMethod calculates（Livak KJ,
Schmittgen TD(2001)Analysis of Relative Gene Expression Data Using Real-Time
Quantitative PCR and the2-ΔΔCTMethod.Methods25:402-408.）, formula be △ △ Ct=(Ct,
Target-Ct, GAPDH) Time x (Ct, Target-Ct, GAPDH) Time0.Time x indicate random time point（5,10 Hes
20min）, time0 represent target gene be normalized into control GAPDH（Physiological saline group）.Experiment is triplicate every time, and experiment is extremely
It is few to carry out independent repetition three times.All results are expressed as mean+SD（SEM）.
Western bloting
Total protein concentration is measured using Coomassie brilliant blue albuminimetry (Pierce Chemical Co.), uses ox blood
Pure albumen (BSA, Sigma) is used as standard protein.
Albumen carries out SDS denaturing polyacrylamide gel electrophoresis, and electricity is gone on nitrocellulose filter later.Then, by nitric acid
Cellulose membrane is placed on containing 1h is incubated at room temperature in 5% skim milk TBST, then primary antibody overnight incubation (β-actin (1/
10000, Bioworld), (1 POMC:10000, ABGENT), (1/10000, Epitomics) MOR, NK1 receptors (1/500,
Proteintech) and DOR (1/500, Bioworld)).After this, secondary antibody is incubated at room temperature 2h（β-actin, DOR, MOR and
The secondary antibody of TACR1 is HRP- goat anti-rabbit iggs (1/5000, Bioworld), POMC secondary antibodies be HRP- rabbit-anti sheep IgG (1/5000,
Bioworld)）.Finally develop the color on chemiluminescence imaging instrument (Sage Creation).Molecular weight of albumen size and pre-dyed albumen
Marker (Fermentas) is compared.
In order to analyze western bloing data, the gray scale of control is defined as 100%, and numerical value is by Quantity
One softwares (Bio-world) measure.The gray scale of sample is exported according to control gray scale in the form of percentage.All data
From at least independent three repeated experiments.
Data analysis
All data sources are at least independent three repeated experiments.Data are indicated in the form of average value ± SEM.It is all
Data with SPSS softwares (SPSS Inc., Chicago, IL, USA) analyze.P values ＜ 0.05 is considered to have significant difference.
As a result
1, mouse side room injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH is given2Afterwards, distribution sites in brain
Under fluorescence microscope, fluo- Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2 peptides show green light under blue light excitation.It is noted to telocoele
After penetrating drug 10min, fluo- Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2It is mainly distributed on the ventricles of the brain and several structures adjacent with the ventricles of the brain.So
And fluo- Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2It is with the main different distributions sites fluo-r/mHK-1, in fluo- oligopeptides
ASQFFGLM-NH2In processing group, green fluorescence is not found in aqueduct grey matter (PAG) and E/OV.
2, encode NK1 receptors and different preprotachykinins mRNA level in-site expression characteristic
We use the methed comparision of real-time quantitative RT-PCR NK1 receptors and preprotachykinin in mRNA level in-site
Expression variation（Fig. 1）.Wherein house-keeping gene GAPDH is used for doing internal reference.In real-time quantitative PCR experiment, 2-ΔΔCtMethod is used for
Analyze the opposite variation of gene expression.Compared with the control group（Physiological saline is standardized as 1）, intracerebroventricular injection oligopeptides
ASQFFGLM-NH2Afterwards the mRNA values of the NK1 receptors of 5min, 10min and 20min be 0.91 ± 0.05,0.93 respectively ±
0.04and0.87±0.06（Fig. 1 a, A）.The PPT-A mRNA values of 5min, 10min and 20min are 0.99 ± 0.06 respectively,
0.93 ± 0.17 and 0.96 ± 0.09 (Fig. 1 a, B).The PPT-C mRNA values of 5min, 10min and 20min are 0.96 respectively ±
0.08,1.10 ± 0.11 and 0.96 ± 0.03（Fig. 1 a, C）.Data analysis shows control and all Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Place
The Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of reason group2There was no significant difference, this shows in mouse, the mRNA tables of NK1 receptors and preprotachykinin
Up to level not by intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Influence.
3, the expression characteristic of the mRNA of encoding endogenous opiate receptor and opioid peptides
Utilize real-time quantitative RT-PCR technology, it has been found that compared with the control, Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Processing group
DOR is significantly increased in mRNA level in-site.Intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2The value at 5,10 and 20min time points afterwards
It is 601.7 ± 137.0,386.1 ± 83.1 and 254.3 ± 68.7 respectively（Fig. 2 e, E）.However, the endogenic ligand PENK of DOR
Significant changes do not occur in mRNA level in-site, after intracerebroventricular injection drug, the value at 5,10 and 20min time points is 1.08 respectively
± 0.11,0.94 ± 0.10 and 0.97 ± 0.08（Fig. 2 d, B）.MOR（Fig. 2 a, A）, POMC (endogenic ligand of MOR, figure
2b, B), KOR（Fig. 2 c, C）With PDYN (endogenic ligand of KOR, Fig. 2 d, D) in the expression water of mRNA after drug-treated
It is average that significant changes do not occur.
4, mouse intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2The expression of DOR receptor proteins is caused significantly to be increased afterwards
Finally, we have detected by intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Whether the up-regulation of caused DOR transcripts
It is related to the expression of DOR albumen increase.In fig. 3d it can be seen that, compared with the control group, through Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Processing
Afterwards, notable up-regulation has occurred in the expression of DOR albumen.At 5,10 and 20min time points, the expression quantity of DOR albumen is 1.78 respectively
± 0.28,3.26 ± 0.50 and 5.29 ± 0.86（Fig. 3 D）.In addition, we also have detected the transcription of NK1 receptors, MOR and POMC
The correlation with their own protein expression is expressed, by the visible these types of albumen of the result of Fig. 3 A-C in control group and hHK-1
Between without occur significant changes.
Embodiment 2
Material and method
Animal
The ICR mouse of male and female（20±1.0g）By Hangzhou Pedagogic University's animal center（Hangzhou China）It carries at random
For.All zoopery programs are ratified by Institutes Of Technology Of Zhejiang's animal feeding and using the committee, and meet in November, 1986
No. 24 committees of the European Community instruct (86/609/EEC).23 DEG C of -25 DEG C of rings of all animal feedings in 12 hours light dark cycles
In border, and carry out the food and water of preceding offer standard in experiment.It is reduced as far as possible suffered by mouse in all experimentations
Injury and pain.
Polypeptide
Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Polypeptide is by Zhong Tai companies（Hangzhou China）It is synthesized, and made using solid phase polypeptide synthesis
Use efficient liquid phase（HPLC）Purifying, purity reach 98%.Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2It is dissolved in physiological saline, working solution is dense
Degree is 1.5mM.
Intracerebroventricular injection and tail-flick test
Intracerebroventricular injection is operated according to the Haley and McCormick operating methods in conscious mouse described.Often
The Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of mouse injection2Final dose is 6nmol, and volume injected is 4 μ l.In intracerebroventricular injection people HK-1
After (4-11), the morphine of 2mg/kg is subcutaneously injected at once, then uses both drugs pair of warm bath tail-flick test detection joint injection
The influence of chmice acute pain.In warm bath tail-flick test, every mouse is only used primary.Mouse tail 1/3 is immersed 48.5 DEG C
Water bath with thermostatic control in, to the spontaneous time interval that tail portion is thrown away to water-bath of mouse be defined as flick latency since immersing.Often
Mouse first detects flick latency before experiment, and screens mouse of the flick latency in 2.5-4.5s and carry out experimental study,
Excessively blunt and excessively sensitive mouse is all discarded.In order to avoid mouse tail is burned, mouse flick latency is most
Big value is 15s.The time interval that every mouse surveys flick latency every time is 10s, and last four flick latencies are averaged
The flick latency being worth before being handled as medication, and the variation of this four flick latencies is no more than 10%.Joint injection people
After HK-1 (4-11) and morphine, 10,20,30,40,50 and 60 minutes respective flick latencies after drug injection are detected.
Analgesic activity is calculated with following equation：
The percentage change of flick latency=[（The basic flick latency before flick latency-medication after medication）/
Basic flick latency before medication] * 100%
Data analysis
All data sources are at least independent three repeated experiments.Data are indicated in the form of average value ± SEM.It is all
Data with SPSS softwares (SPSS Inc., Chicago, IL, USA) analyze.P values ＜ 0.05 is considered to have significant difference.
As a result
The synergic antalgic effect after morphine is subcutaneously injected in intracerebroventricular injection people HK-1 (4-11) joints
From fig. 4, it can be seen that intracerebroventricular injection people HK-1 (4-11) can significantly increase the analgesia effect of hypodermic morphine
Fruit, after injection in one hour time, the amplitude of the analgesia enhancing detected for each 10 minutes is about original morphine analgesia
Twice of effect.It can be seen that morphine, which is subcutaneously injected, in intracerebroventricular injection people HK-1 (4-11) joints has potent synergic antalgic
Effect.
It should be understood by one skilled in the art that can use in the practice of the invention except particular instantiation is in addition to those
Method and raw material etc. are without excessively experiment.All functions known in the art etc. of any such method and raw material etc.
The all expections of valence object are included in the invention.It should also be appreciated by one skilled in the art that can be in present specification and claims
The present invention of description carries out various changes and modifications, and the present invention includes all such changes and modifications.The invention also includes this
All steps, feature, composition and the compound and the step or feature for individually or simultaneously referring to or pointing out in specification
In any 2 or more any and all combinations.
Claims (10)
1. Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Combine with morphine and is preparing for the purposes in the analgesic preparation in subject.
2. according to the purposes described in claim 1, wherein the subject is mammal.
3. purposes according to claim 2, wherein the mammal is behaved.
4. purposes according to claim 1, wherein the preparation is drug.
5. according to the purposes described in any one of claim 1-4, wherein the oligopeptides is by PEGylated modification.
6. Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2Purposes in preparing the preparation for enhancing analgesic effect of the morphine in subject.
7. according to the purposes described in claim 6, wherein the subject is mammal.
8. purposes according to claim 7, wherein the mammal is behaved.
9. purposes according to claim 6, wherein the preparation is drug.
10. according to the purposes described in any one of claim 6-9, wherein the oligopeptides is by PEGylated modification.
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Non-Patent Citations (3)
| Title |
|---|
| Effects of rat mouse hemokinin-1, a mammalian tachykinin peptide, on the antinociceptive activity of pethidine administered at the peripheral and supraspinal level;cai yun fu et al.;《behavioural brain research》;20071231;摘要、fig.1 * |
| hemokinins and endokinins;N.M.Page;《CMLS》;20041231;1652-1663 * |
| In vivo characterization of the effects of human hemokinin-1 and human hemokinin-1(4-11), mammalian tachykinin peptides, on the modulation of pain in mice;cai y. fu et al.;《brain, behavior, and immunity》;20080208;摘要、第854页左栏第13-19行至右栏第2行，附图6B * |
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