CN104800835A - Uses of oligopeptide combination or combination of oligopeptide and enkephalin in preparation of drugs for analgesia - Google Patents
Uses of oligopeptide combination or combination of oligopeptide and enkephalin in preparation of drugs for analgesia Download PDFInfo
- Publication number
- CN104800835A CN104800835A CN201410034021.5A CN201410034021A CN104800835A CN 104800835 A CN104800835 A CN 104800835A CN 201410034021 A CN201410034021 A CN 201410034021A CN 104800835 A CN104800835 A CN 104800835A
- Authority
- CN
- China
- Prior art keywords
- lys
- phe
- gly
- sqffglm
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The present invention relates to uses of an oligopeptide combination or combination of an oligopeptide and enkephalin in preparation of drugs for analgesia, wherein specifically the oligopeptide ASQFFGLM-NH2 can increase the transcription and/or expression level of the delta opioid receptor in subjects so as to improve the analgesic effect of the enkephalin.
Description
Technical field
The present invention relates to oligopeptide associating or with being combined in for the preparation of the purposes in the medicine of analgesic of enkephalin.Specifically, the present invention relates to Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2improving transcribing and/or the purposes of expression of delta opiate receptor in experimenter, and then the analgesic effect of enkephalin can strengthened.
Background technology
Tachykinin has common carboxyl terminal sequence (FXGLM-NH
2) peptide family, its member the most well known comprises: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP), neurokinin A (NKA) and neurokinin B (NKB), and they to be encoded SP, NKA from two different gene codes: Ppta; Pptb encodes NKB.At 2000 and 2002, the 3rd front tachykinin gene Pptc was identified, and it is encoded HK-1.HK-1 is out identified in mouse hematopoetic cell at first, and it is grown in the process of pre B lymphocyte in ancestral's B cell and plays an important role.Afterwards, it was also out identified in rat and people.
Many evidences show, people and mice HK-1 and SP have similar Activation Activity to neurokinin receptor, all to neurokinin receptor 1(NK1 receptor) there are very high affinity and selectivity.But, unlike, hHK-1 and hHK-1 (4-11), many (being 14 times and 70 times respectively) is reduced for the affinity of neurokinin receptor 1 with r/mHK-1 and SP.Some researcheres have studied people and mice HK-1 and to unify at immune system, cardiovascular system some biologic activity of pain sensation system.In the transmission of the regulation and control pain sensation, find before the present inventor, r/mHK-1 and hHK-1 can produce dosage and time dependent analgesic effect after intracerebroventricular injection, and the antagonist of neurokinin receptor 1 and mu opioid receptor antagonists can significantly block the analgesic effect caused by r/mKH-1 and hHK-1 respectively.That is the analgesic effect of r/mKH-1 and hHK-1 depends on neurokinin receptor 1 and mu opioid receptor respectively.
Endogenic opioid peptide and opiate receptor take part in the adjustment process of pain.At present, mostly use the agonist of mu opioid receptor to carry out alleviating pain clinically, but the side effect such as its toleration and the dependency of body & mind that very easily produces seriously constrains it to be used.On the contrary, optionally activate delta opiate receptor while the potent analgesic effect of generation, seldom have side effect to produce.Therefore, activate delta opiate receptor and be considered to a kind of desirable pain relieving means at present.There is bibliographical information, the upper delta opiate receptor increasing spinal levels that is in harmonious proportion effectively can strengthen the analgesic effect (C.M.Cahill of delta opiate receptor agonist in chronic inflammatory pain model, A.Morinville, C.Hoffert, D.O ' Donnell, A.Beaudet.Up-regulation and trafficking of δ opioid receptor in a model of chronic inflammation:implicationsfor pain control.Pain, 101 (2003) 199 208.).
Summary of the invention
In order to raise delta opiate receptor, the present inventor finds Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH through long-term and unremitting effort
2the 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, and it comprises the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH such as, using effective dose to described experimenter (such as mammal, people)
2.
Another aspect provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2purposes in such as, preparation (such as medicine) for the preparation of delta opiate receptor expression in rise experimenter (such as mammal, people) body.
Present invention also offers Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2associating or with being combined in such as, for the preparation of the purposes in analgesic preparation (such as medicine) in the experimenter (such as mammal, people) of enkephalin.
The present invention additionally provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH further
2purposes in the preparation (such as medicine) of the analgesic effect for the preparation of enhancing enkephalin.
Likely relate to some abbreviations in this description and claims, these contents representated by abbreviation be those skilled in the art known.But, for the ease of understanding content of the present invention, by as follows for the content citation representated by relevant abbreviation:
R/mHK-1: rat/mouse hemokinin-1; FAM: CF 5(6)-Carboxyfluorescein; PPT: preprotachykinin; MOR: μ-opiate receptor; POMC: proopiomelanocortin; KOR: κ-opiate receptor; PDYN: prodynorphin; DOR: δ-opiate receptor; PENK: proenkephalin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HK-1(4-11): ASQFFGLM-NH
2.
Accompanying drawing explanation
Fig. 1: mice intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2after, NK1 receptor and different preprotachykinin are in the expression situation of change of mRNA level in-site.A, b and c are the representative NK1 receptor obtained with ABI7300 systems soft ware respectively, the amplification curve of PPT-A and PPT-C.What A, B and C rectangular histogram showed is that reference gene is house-keeping gene GAPDH from least independent NK1 receptor of three repetitions, the relative mRNA expression levels of PPT-A and PPT-C respectively.Institute responds each triplicate, at least carries out three independent repetitions.Each value is injected the matched group institute standardization of normal saline, and the value of matched group is set to 1, and result presents with meansigma methods ± SEM.Compared with matched group, experimental group does not have significant difference.
Fig. 2: mice intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2rear endogenous opioid receptors and endogenous opiatepeptide are in the expression situation of change of mRNA level in-site.A, b, c, d, e and f is the representative MOR (μ-opiate receptor) obtained with ABI7300 systems soft ware respectively, POMC (proopiomelanocortin), KOR (κ-opiate receptor), the amplification curve of PDYN (prodynorphin), DOR (δ-opiate receptor) and PENK (proenkephalin).That A, B, C, D, E and F rectangular histogram shows is the MOR repeated from least independent three times respectively, and the relative mRNA expression levels of POMC, KOR, PDYN, DOR and PENK, reference gene is house-keeping gene GAPDH.Institute responds each triplicate, at least carries out three independent repetitions.Bar corresponds to standard error of the mean.* * p≤0.001: have pole significant difference compared with respective matched group.
Fig. 3: sxemiquantitative Western Blotting measuring mice intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2rear NK1 receptor, MOR, POMC and DOR protein expression and matched group protein expression situation.These pictures come from least three independent representative pictures repeated.That bar diagram represents respectively is NK1 receptor (A), MOR (B), and the protein versus expression situation of POMC (C) and DOR (D), reference gene is β-actin.* p≤0.05; * p≤0.01: have significant difference compared with respective matched group.
Detailed description of the invention
The present inventor for object of study, have studied Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH with ICR mice (being provided by Hangzhou Pedagogic University of Hangzhou, Zhejiang province city animal center)
2on the impact of the expression of various opioid peptide receptor and part thereof.Our experimental result shows the expression of NK1 receptor and preprotachykinin not by intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2impact.Our current work corresponding, compared with saline control group, intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2afterwards, MOR(mu opioid receptor) [with KOR (kappa opioid receptor), and the expression of their respective endogenic ligand POMC and PDYN does not almost change.What is interesting is, in the research that we are current, DOR (delta opiate receptor) transcribe with protein level occur raise extremely significantly, but the endogenic ligand of PENK(DOR) expression not there is significant change.
Therefore, one aspect of the present invention provides a kind of method improving the transcript and expression level of delta opiate receptor in experimenter, and it comprises the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH using effective dose to described experimenter
2.Present invention provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2for the preparation of improve delta opiate receptor in experimenter transcript and expression level preparation (such as medicine) in purposes.Another aspect of the invention provides a kind of pharmaceutical product (such as medicine or medicament) or pharmaceutical composition, and it comprises the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of the transcript and expression level of delta opiate receptor in the raising experimenter as active component
2; And comprise this description relating to pharmaceutical product.
One aspect of the present invention provides a kind of method improving the analgesic effect of enkephalin in experimenter, and it comprises the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH using effective dose to described experimenter
2, this is used is carried out before or after the using of enkephalin, or uses with enkephalin simultaneously.Present invention provides Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2purposes in the preparation (such as medicine) for the preparation of raising analgesic effect of enkephalin in experimenter.Another aspect of the invention provides a kind of pharmaceutical product (such as medicine or medicament) or pharmaceutical composition, and it comprises the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of the analgesic effect of enkephalin in the raising experimenter as active component
2; And comprise this description relating to pharmaceutical product.
For experimenter of the present invention, it is preferably mammal, is more preferably people.
For Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH in raising experimenter of the present invention
2activity, those skilled in the art can make any modification to it, and precursor is that it is active in described modification not negative effect.Such as, carry out can PEGization to this oligopeptide, to improve its half-life in vivo; Can be connected with known penetrating peptide, to promote the Transdermal absorption etc. of oligopeptide of the present invention.In a word, those skilled in the art can carry out various modification to improve delivery efficiency and to keep it active to oligopeptide of the present invention.The oligopeptide of this kind of modification is also within the scope of the present invention.
Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH as active component of the present invention
2can use together with pharmaceutically acceptable carrier.In addition to the active ingredient (s, method of the present invention, purposes and product can also comprise suitable pharmaceutically acceptable carrier, comprise and promote that reactive compound is processed into excipient and the auxiliary agent of preparation.
Such as be suitable for injecting or the preparation of infusion comprises aqueous and non-aqueous sterile injection liquid and aqueous and non-aqueous sterile suspensions, described aseptic parenteral solution optionally comprises antioxidant, buffer agent, antibacterial and can make the solute of blood equipressure of preparation and object recipient, and described sterile suspensions can comprise suspending agent and thickening agent.Described preparation can be present in unit dose or multi-dose container, the ampoule such as sealed, and can be kept at freeze-dried (lyophilizing) condition, only needs to add sterile liquid carrier, such as water for injection before using immediately.
Active component of the present invention optionally can be combined with solid excipient, and optionally grind obtained mixture, and when needing, after adding suitable auxiliary agent, the mixture of processing granular, to obtain required dosage form.Suitable excipient particularly filler is such as sugared, comprises lactose, sucrose, mannitol or Sorbitol; Cellulose or starch formulation, gelatin, Tragacanth, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone (PVP).When needing, disintegrating agent can be added, such as crospolyvinylpyrrolidone, agar or alginic acid or its salt such as sodium alginate.
The effective dose of active component of the present invention can be improve delta opiate receptor in experimenter transcribe and/or expression any amount, it can be equivalent to about 0.1-15mg Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2, preferred 0.01-20mg Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2dosage unit.More preferably, dosage unit comprises the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of about 1-4mg
2.Most preferably, dosage unit comprises the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of about 2-3mg
2.Effective dose be determined in the ability of those skilled in the art, under the enlightenment particularly according to disclosure provided herein.
According to the present invention, pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition can use administration experimenter with any effective source strength.Preferably, pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition can with multidose administrations, such as from about 2 to about 15 dosage, more preferably from about 4-10 dosage, most preferably from about 6 dosage.In particularly preferred embodiment, in administration process, with administration in every three weeks frequency about once, pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition are administered to experimenter, such as injection, infusion or oral.In particularly preferred embodiment, administration is by injection.
Be to be understood that pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition can be prepared by the mode of any suitable for the administration by any suitable.
The dosage unit of pharmaceutical product of the present invention (medicine, medicament) or pharmaceutical composition carries out administration experimenter based on routine.Such as, dosage unit can administration more than once a day, once in a week, monthly etc.Dosage unit can be by twice/week based on administration, namely weekly twice, such as every three days once.
As used herein, " comprising " and " comprising ", " containing " or " being characterised in that " synonym, and be included or opening, and do not get rid of the other element of not stating or method step.Term " comprises " any statement in this article, particularly when describing method of the present invention, purposes or product, be understood to include substantially by described component or element or step forms and those products, method and the purposes that are made up of described component or element or step.The present invention of description exemplified here suitably can put into practice when there are not concrete disclosed any one or Various Components, one or more restrictions herein.
The description relating to this pharmaceutical product comprised in pharmaceutical product of the present invention can containing, for example lower content: indication (such as pain), application dosage (such as above-mentioned exemplified property illustrates) and issuable side effect etc.
The term adopted herein and statement are used as descriptive instead of restricted term; and do not expect shown in getting rid of in the use of this kind of term and statement and any equivalent of described feature or its part, but will be appreciated that in the various scope of the present invention being modified at request protection be possible.Therefore, although be to be understood that the present invention is specifically disclosed by preferred embodiment and optional feature, but those skilled in the art can adopt modification and the change of concept disclosed herein, and this type of is modified and change is regarded as in the scope of the present invention such as defined by accessory claim.
For being illustrated more clearly in the present invention, be now described in detail in conjunction with following embodiment, but these embodiments are only to exemplary description of the present invention, can not be interpreted as the restriction to the application.
Embodiment
Materials and methods
Animal
Male and female ICR mice (20 ± 1.0g) is provided at random by Hangzhou Pedagogic University's animal center (Hangzhou China).All zoopery programs are by Institutes Of Technology Of Zhejiang's animal feeding and use committee to ratify, and meet committee of the European Community on the 24th instruction November in 1986 (86/609/EEC).All animal feedings 12 little time/the dark cycle 23 DEG C of-25 DEG C of environment in, and carry out prerequisite for the food of standard and water in experiment.The injury suffered by mice and misery is reduced as far as possible in all experimentations.
Polypeptide
Fluo-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2with Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2polypeptide uses solid phase polypeptide synthesis to synthesize by Zhong Tai company (Hangzhou China), and uses efficient liquid phase (HPLC) purification, and its purity reaches 98%.FAM is carboxyl fluorophor, utilizes standard coupling technique to be coupled on the amino acid group of Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2 peptide by CF.Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2be dissolved in normal saline, working solution concentration is 1.5mM.Fluorescein-labeled (FAM) Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2be dissolved in 50%DMSO(dimethyl sulfoxide) in, the concentration of storage liquid is 15mM.The working concentration of 1.5mM is diluted to before the experiments with normal saline.
Intracerebroventricular injection and tissue preparation
The operational approach in conscious mouse that intracerebroventricular injection describes according to Haley and McCormick operates.The Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of every injected in mice
2or fluo-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2final dose be 6nmol, volume injected is 4 μ l.
The result of study display intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH in our early stage
2after, in 20min, have obvious analgesic effect, the time point of therefore this experiment is chosen as 5,10 and 20min tri-time points.At injection FAM-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2 or Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2after (every mice 6nmol), at corresponding time point, disconnected neck puts to death mice, and the accuracy of injection site verified by micrometering.In matched group, to mice intracerebroventricular injection 4 μ l normal saline.Then, Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH will be injected
2or the mouse brain of normal saline takes out rapidly, be kept in-80 DEG C of refrigerators for further experiment.To inject FAM-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH rapidly
2mouse brain take out, be fixed on 24h in the phosphate buffer of the pH7.4 of the 0.1M containing 4% paraformaldehyde (Sigma), dewater with 30% sucrose solution before the frozen section (JUNG Tissue freezingmedium, LEICA, Germany).
Coronal section and microscopic examination
Every mouse brain is chilled in-20 DEG C, and slice thickness is 10 μm.Then brain tissue slice is stored on slide.Section is kept at 4 DEG C, completes microexamination and take pictures in 12h.Sample difference and fluorescence microscope (NIKON TE2000-U).According to adult mice brain structure atlas qualification anatomical structure.
Reverse transcription and real-time quantitative PCR
According to commodity operation instructions, TRIzol reagent (Invitrogen, Carlsbad, CA) is used to extract total serum IgE from each freezing cerebral tissue.In brief, ground by cerebral tissue with mortar, the dosage then adding 1ml TRIzol according to every 100mg adds TRIzol.In all experiments, with Reverse Transcriptase kit (Gibco BRL), 5 μ g total serum IgE reverse transcriptions are become cDNA, final volume is 50 μ l.
Primer sequence is as follows, the primer sequence identical [7] in the work delivered before this and we:
NK1 receptor (F): 5 '-TGGACTCTGATCTCTTCCCCAACA-3 '
NK1 receptor (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’
Carry out relative quantification PCR in real time with ABI7300 Real Time PCR Detection System equipment and detect the expression situation of change of said gene in mRNA level in-site, using GAPDH as reference gene.Negative control distilled water replaces cDNA.After each real-time quantitative PCR, do a solubility curve analysis.The normalized expression level 2-of each target gene
△ △ Ctmethod calculates (Livak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression DataUsing Real-Time Quantitative PCR and the2-
Δ Δ CTmethod.Methods25:402-408.), formula is △ △ Ct=(Ct, Target-Ct, GAPDH) Timex (Ct, Target-Ct, GAPDH) Time0.Time x represents random time point (5,10 and 20min), and time0 represents target gene and is normalized into contrast GAPDH(normal saline group).Each experiment is triplicate, and experiment is at least carried out independently repeating for three times.All results are expressed as mean+SD (SEM).
Western bloting
Use Coomassie brilliant blue albuminimetry (Pierce Chemical Co.) to measure total protein concentration, use bovine serum albumin (BSA, Sigma) as standard protein.
Albumen carries out SDS denaturing polyacrylamide gel electrophoresis, and electricity forwards on nitrocellulose filter afterwards.Then, nitrocellulose filter is placed on containing incubated at room 1h in 5% skim milk TBST, then primary antibodie overnight incubation (β-actin (1/10000, Bioworld), POMC (1:10000, ABGENT), MOR (1/10000, Epitomics), NK1 receptor (1/500, Proteintech) and DOR (1/500, Bioworld)).After this, two anti-incubated at room 2h(β-actin, DOR, MOR and TACR1 two anti-be HRP-goat anti-rabbit igg (1/5000, Bioworld), POMC bis-anti-be the anti-sheep IgG (1/5000, Bioworld) of HRP-rabbit).Finally in the upper colour developing of chemiluminescence imaging instrument (Sage Creation).Molecular weight of albumen size and pre-dyed albumen marker (Fermentas) contrast.
In order to analyze western bloing data, the gray scale of contrast is defined as 100%, and its numerical value is measured by Quantity One software (Bio-world).The gray scale of sample is exported by the form with percentage ratio according to contrast gray scale.All data from least independently repeating experiment for three times.
Data analysis
All data from least independently repeating experiment for three times.Data represent with meansigma methods ± SEM form.All data SPSS software (SPSS Inc., Chicago, IL, USA) is analyzed.P value < 0.05 is considered to have significant difference.
Result
1, to mice side room injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2after, its distribution sites in brain
Under fluorescence microscope, fluo-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH2 peptide is at blue-light excited lower display green glow.After intracerebroventricular injection medicine 10min, fluo-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2mainly be distributed in the ventricles of the brain and several structure adjacent with the ventricles of the brain.But, fluo-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2with the different distributions site that fluo-r/mHK-1 is main be, at fluo-Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2in processed group, do not find green fluorescence in aqueduct grey matter (PAG) and E/OV.
2, to encode NK1 receptor and the different preprotachykinins expression characteristic in mRNA level in-site
We adopt the methed comparision of real-time quantitative RT-PCR NK1 receptor and preprotachykinin to change (Fig. 1) in the expression of mRNA level in-site.Wherein house-keeping gene GAPDH is used for doing internal reference.In real-time quantitative PCR experiment, 2-
Δ Δ Ctmethod is used to the relative change that analyzing gene is expressed.Compared with matched group (normal saline is standardized as 1), intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2the mRNA value of the NK1 receptor of rear 5min, 10min and 20min is 0.91 ± 0.05,0.93 ± 0.04and0.87 ± 0.06(Fig. 1 a, A respectively).The PPT-A mRNA value of 5min, 10min and 20min is 0.99 ± 0.06,0.93 ± 0.17 and 0.96 ± 0.09 (Fig. 1 a, B) respectively.The PPT-C mRNA value of 5min, 10min and 20min be respectively 0.96 ± 0.08,1.10 ± 0.11 and 0.96 ± 0.03(Fig. 1 a, C).Data analysis shows contrast and all Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2the Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH of processed group
2there was no significant difference, this shows in mice, and the mrna expression level of NK1 receptor and preprotachykinin is not by intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2impact.
3, the expression characteristic of the mRNA of encoding endogenous opiate receptor and opioid peptide
Utilize real-time quantitative RT-PCR technology, we find, compared with the control, and Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2the DOR of processed group occurs significantly to strengthen in mRNA level in-site.Intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2rear 5,10 and the value of 20min time point be respectively 601.7 ± 137.0,386.1 ± 83.1 and 254.3 ± 68.7(Fig. 2 e, E).But the endogenic ligand PENK of DOR significant change does not occur in mRNA level in-site, after intracerebroventricular injection medicine, 5,10 and the value of 20min time point be respectively 1.08 ± 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, Fig. 2 b, B), KOR(Fig. 2 c, C) and PDYN (endogenic ligand of KOR, Fig. 2 d, the D) expression at mRNA after drug treating all there is not significant change.
4, mice intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2after cause the expression of DOR receptor protein significantly to be increased
Finally, we have detected by intracerebroventricular injection Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2whether the rise of the DOR transcript caused increases relevant to the expression of DOR albumen.Can see in fig. 3d, compared with matched group, through Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2after process, the expression of DOR albumen there occurs remarkable rise.5,10 and 20min time point, the expression of DOR albumen be respectively 1.78 ± 0.28,3.26 ± 0.50 and 5.29 ± 0.86(Fig. 3 D).In addition, we also have detected the dependency of NK1 receptor, the transcriptional expression of MOR and POMC and their respective protein expressions, not significant change occur by the result of Fig. 3 A-C this several albumen visible between matched group and hHK-1.
5, Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2the analgesic effect of enkephalin can be strengthened.Enkephalin is the endogenic ligand of delta opiate receptor, and the current bibliographical information of enkephalin has significant analgesic effect.On the other hand, as above confirmed such, Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2the expression of delta opiate receptor can be significantly improved, the analgesic activity that can mediate stronger endogenic ligand enkephalin of the delta opiate receptor of the high expressed of so being induced by the present invention.
It should be understood by one skilled in the art that and can to adopt in the practice of the invention except the method and raw material etc. of particular instantiation except those and without the need to undo experimentation.All function equivalents known in the art of any this class methods and raw material etc. are all expected and are comprised in the present invention.Those skilled in the art it is also understood that and can carry out various variation and modification to the present invention described in this specification and claims book, and the present invention includes this type of variations all and modify.The present invention also comprise in this description the institute of mentioning individually or simultaneously or pointing out in steps, feature, compositions and compound, and any and all combinations of in described step or feature any 2 or more.
Claims (10)
1. Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2combine for the preparation of the purposes in analgesic preparation in experimenter with enkephalin.
2., according to the purposes described in claim 1, wherein said experimenter is mammal.
3. purposes according to claim 2, wherein said mammal is behaved.
4. purposes according to claim 1, wherein said preparation is medicine.
5. the purposes according to any one of claim 1-4, wherein said oligopeptide is modified by PEGization.
6. Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH
2purposes in the preparation for the preparation of enhancing enkephalin analgesic effect in experimenter.
7., according to the purposes described in claim 6, wherein said experimenter is mammal.
8. purposes according to claim 7, wherein said mammal is behaved.
9. purposes according to claim 6, wherein said preparation is medicine.
10. the purposes according to any one of claim 6-9, wherein said oligopeptide is modified by PEGization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410034021.5A CN104800835A (en) | 2014-01-23 | 2014-01-23 | Uses of oligopeptide combination or combination of oligopeptide and enkephalin in preparation of drugs for analgesia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410034021.5A CN104800835A (en) | 2014-01-23 | 2014-01-23 | Uses of oligopeptide combination or combination of oligopeptide and enkephalin in preparation of drugs for analgesia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104800835A true CN104800835A (en) | 2015-07-29 |
Family
ID=53686313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410034021.5A Pending CN104800835A (en) | 2014-01-23 | 2014-01-23 | Uses of oligopeptide combination or combination of oligopeptide and enkephalin in preparation of drugs for analgesia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104800835A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7235531B2 (en) * | 1999-12-21 | 2007-06-26 | Takeda Pharmaceutical Company | Tachykinin-like polypeptides and use thereof |
| CN102250251A (en) * | 2011-06-29 | 2011-11-23 | 河北师范大学 | Polyethylene glycol derivative of enkephalin analogue |
| EP2610619A1 (en) * | 2010-08-27 | 2013-07-03 | University of Miyazaki | Hemokinin-1 receptor and hemokinin-1-derived peptide |
-
2014
- 2014-01-23 CN CN201410034021.5A patent/CN104800835A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7235531B2 (en) * | 1999-12-21 | 2007-06-26 | Takeda Pharmaceutical Company | Tachykinin-like polypeptides and use thereof |
| EP2610619A1 (en) * | 2010-08-27 | 2013-07-03 | University of Miyazaki | Hemokinin-1 receptor and hemokinin-1-derived peptide |
| CN102250251A (en) * | 2011-06-29 | 2011-11-23 | 河北师范大学 | Polyethylene glycol derivative of enkephalin analogue |
Non-Patent Citations (5)
| Title |
|---|
| CAI Y. FU等: ""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"，第850-860页", 《BRAIN, BEHAVIOR, AND IMMUNITY》 * |
| CAI-YUN FU等: ""Rat/mouse hemokinin-1, a mammalian tachykinin peptide, markedly potentiates the antinociceptive effects of morphine administered at the peripheral and supraspinal level"，第293-301页", 《BEHAVIOURAL BRAIN RESEARCH》 * |
| MARC M. KURTZ等: ""Identification, localization and receptor characterization of novel mammalian substance P-like peptides"，第205-212页", 《GENE》 * |
| RUI-LONG XIA等: ""Study on the distribution sites and the molecular mechanism of analgesia after intracerebroventricular injection of rat/mouse hemokinin-1 in mice"，第113-120页", 《PEPTIDES》 * |
| 付彩云: ""新发现的哺乳动物速激肽hemokinin-1对痛觉的调节作用及其对吗啡和哌替啶镇痛作用的影响"", 《中国学位论文全文数据库》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yu et al. | Involvement of oxytocin in spinal antinociception in rats with inflammation | |
| Clarke et al. | Gonadotropin-inhibitory hormone is a hypothalamic peptide that provides a molecular switch between reproduction and feeding | |
| Argiolas et al. | ACTH-and α-MSH-induced grooming, stretching, yawning and penile erection in male rats: site of action in the brain and role of melanocortin receptors | |
| Champion et al. | Endomorphin 1 and 2, endogenous ligands for the μ-opioid receptor, decrease cardiac output, and total peripheral resistance in the rat | |
| Vaughan et al. | Characterization of a novel melanocortin receptor-containing node in the SNS outflow circuitry to brown adipose tissue involved in thermogenesis | |
| Ma et al. | Growth hormone secretagogues preserve the electrophysiological properties of mouse cardiomyocytes isolated from in vitro ischemia/reperfusion heart | |
| Clapper et al. | Effects of amylin and bupropion/naltrexone on food intake and body weight are interactive in rodent models | |
| Guan et al. | Peripheral administration of a novel diketopiperazine, NNZ 2591, prevents brain injury and improves somatosensory-motor function following hypoxia–ischemia in adult rats | |
| Elhabazi et al. | RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents | |
| Konya et al. | Activation of EP 4 receptors prevents endotoxin‐induced neutrophil infiltration into the airways and enhances microvascular barrier function | |
| JP2021130704A (en) | Compositions and Methods for Modulating AT2R Activity | |
| US10919939B2 (en) | Mu opioid receptor agonist analogs of the endomorphins | |
| Currie et al. | Urocortin I inhibits the effects of ghrelin and neuropeptide Y on feeding and energy substrate utilization | |
| Gonzalez et al. | Behavioral effects of neuropeptide EI (NEI) in the female rat: interactions with α-MSH, MCH and dopamine | |
| Hansen et al. | Feeding responses to a melanocortin agonist and antagonist in obesity induced by a palatable high-fat diet | |
| CN104436158A (en) | Application of Apelin-13 to treatment of diabetic nephropathy | |
| Cáceda et al. | Involvement of neuropeptide systems in schizophrenia: human studies | |
| KR20150048663A (en) | Use of thymosin alpha for the treatment of sepsis | |
| CN104800829A (en) | Uses of oligopeptide combination or combination of oligopeptide and morphine in preparation of drugs for analgesia | |
| Scoto et al. | Effects of intraplantar nocistatin and (±)-J 113397 injections on nociceptive behavior in a rat model of inflammation | |
| CN104800835A (en) | Uses of oligopeptide combination or combination of oligopeptide and enkephalin in preparation of drugs for analgesia | |
| CN104800827A (en) | Uses of oligopeptide in preparation of drugs for increasing expression of delta opioid receptor | |
| CN104800831A (en) | Uses of oligopeptide combination or combination of oligopeptide and pethidine in preparation of drugs for analgesia | |
| CN104800830A (en) | Oligopeptide combination or oligopeptide-pethidine combination product for cancerous pain treatment | |
| KR20230079126A (en) | Autophagy-inhibiting peptide and its organic acid salt to solve the problem of vascular permeability |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150729 |
|
| RJ01 | Rejection of invention patent application after publication |