CN104800827A - Uses of oligopeptide in preparation of drugs for increasing expression of delta opioid receptor - Google Patents

Abstract

The present invention relates to the application of oligopeptide in the preparation of medicine for increasing the expression of delta opioid receptor in vivo. Specifically, the present invention relates to the use of oligopeptide ASQFFGLM-NH ₂ in increasing the transcription and/or expression level of δ opioid receptors in a subject.

Description

Use of oligopeptides in the preparation of drugs for improving the expression of delta opioid receptors

technical field

The present invention relates to the application of oligopeptide in the preparation of medicine for increasing the expression of delta opioid receptor in vivo. Specifically, the present invention relates to the use of oligopeptide ASQFFGLM-NH ₂ in increasing the transcription and/or expression level of δ opioid receptors in a subject.

Background technique

Tachykinins are a family of peptides with a common carboxy-terminal sequence (FXGLM-NH ₂ ), the most well-known members of which include: substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), They are encoded from two different genes: Ppta encodes SP, NKA; Pptb encodes NKB. In 2000 and 2002, a third pro-tachykinin gene, Pptc, was identified, which encodes HK-1. HK-1 was originally identified in mouse hematopoietic cells, where it plays an important role in the development of pro-B cells to pre-B cells. Later, it was also identified in rats and humans.

Many evidences show that human and mouse HK-1 and SP have similar activating activity on neurokinin receptors, and both have high affinity and selectivity for neurokinin receptor 1 (NK1 receptor). However, unlike r/mHK-1 and SP, hHK-1 and hHK-1(4-11) have much lower affinity for neurokinin receptor 1 (14-fold and 70-fold, respectively). Some researchers have studied some biological activities of human and mouse HK-1 in immune system, cardiovascular system and pain system. In terms of regulating pain transmission, the inventors of the present invention have previously found that both r/mHK-1 and hHK-1 can produce dose- and time-dependent analgesic effects after intracerebroventricular injection, and antagonists of neurokinin receptor 1 and μ opioid receptor antagonists could significantly block the analgesic effects induced by r/mKH-1 and hHK-1, respectively. That is to say, the analgesic effect of r/mKH-1 and hHK-1 depended on neurokinin receptor 1 and μ opioid receptor, respectively.

Endogenous opioid peptides and opioid receptors are involved in the regulation of pain. At present, mu opioid receptor agonists are mostly used clinically to relieve pain, but their side effects such as tolerance and easy physical and mental dependence seriously restrict their use. In contrast, selective activation of δ-opioid receptors produces potent analgesic effects with few side effects. Therefore, activation of δ-opioid receptors is currently considered an ideal means of analgesia. It has been reported in the literature that up-regulating and increasing δ-opioid receptors at the level of the spinal cord can effectively enhance the analgesic effect of δ-opioid receptor agonists in chronic inflammatory pain models (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.).

Contents of the invention

In order to up-regulate the δ-opioid receptor, the inventors of the present invention have found through long-term unremitting efforts that the oligopeptide ASQFFGLM-NH ₂ can up-regulate the expression of the δ-opioid receptor.

Therefore, one aspect of the present invention provides a method for up-regulating the expression level of δ opioid receptors in a subject, which comprises administering an effective dose of oligopeptide ASQFFGLM-NH ₂ to said subject (such as a mammal, such as a human) .

Another aspect of the present invention provides the use of the oligopeptide ASQFFGLM-NH ₂ in preparing a preparation (such as a drug) for up-regulating the expression level of δ-opioid receptors in a subject (such as a mammal, such as a human).

There may be some abbreviations involved in the specification and claims, and the contents represented by these abbreviations are well known to those skilled in the art. However, in order to facilitate understanding of the content of the present invention, the content represented by relevant abbreviations is quoted as follows:

r/mHK-1: rat/mouse hemokinin-1; FAM: carboxyfluorescein; PPT: pre-tachykininogen; MOR: μ-opioid receptor; POMC: proopiomelanocortin; KOR: κ-opioid Receptor; PDYN: prodynorphin; DOR: δ-opioid receptor; PENK: proenkephalin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HK-1(4-11): ASQFFGLM-NH ₂ .

Description of drawings

Figure 1: Changes in the expression of NK1 receptors and different pre-tachykininogens at the mRNA level after intracerebroventricular injection of oligopeptide ASQFFGLM-NH ₂ in mice. a, b and c are the amplification curves of representative NK1 receptors, PPT-A and PPT-C obtained with ABI7300 system software, respectively. A, B, and C histograms show the relative mRNA expression levels of NK1 receptor, PPT-A, and PPT-C from at least three independent replicates, respectively, and the internal reference gene is the housekeeping gene GAPDH. All reactions were performed in triplicate with at least three independent replicates. Each value was normalized by the saline-injected control group, the value of the control group was set to 1, and the results are presented as mean ± SEM. Compared with the control group, the experimental group had no significant difference.

Figure 2: Changes in the expression of endogenous opioid receptors and endogenous opioid peptides at the mRNA level after intracerebroventricular injection of oligopeptide ASQFFGLM-NH ₂ in mice. a, b, c, d, e and f are representative MOR (μ-opioid receptor), POMC (proopiomelanocortin), KOR (κ-opioid receptor), respectively obtained by ABI7300 system software Amplification curves of PDYN (prodynorphin), DOR (delta-opioid receptor) and PENK (proenkephalin). A, B, C, D, E, and F histograms show the relative mRNA expression levels of MOR, POMC, KOR, PDYN, DOR, and PENK from at least three independent replicates, respectively, and the internal reference gene is the housekeeping gene GAPDH. All reactions were performed in triplicate with at least three independent replicates. Bars correspond to standard deviation of the mean. ***p≤0.001: There is a very significant difference compared with the respective control groups.

Figure 3: The expression of NK1 receptor, MOR, POMC and DOR protein and the protein expression of the control group were determined by semi-quantitative Western Blotting experiment after intracerebroventricular injection of oligopeptide ASQFFGLM-NH ₂ . These pictures are representative pictures from at least three independent replicates. The bar graphs represent the relative protein expression levels of NK1 receptor (A), MOR (B), POMC (C) and DOR (D), respectively, and the internal reference gene is β-actin. *p≤0.05; **p≤0.01: Significant difference compared with respective control groups.

Detailed ways

The inventors of the present invention took ICR mice (provided by the Animal Center of Hangzhou Normal University, Hangzhou City, Zhejiang Province) as the research object, and studied the influence of oligopeptide ASQFFGLM-NH ₂ on the expression levels of various opioid peptide receptors and their ligands . Our experimental results showed that the expression levels of NK1 receptor and pre-tachykininogen were not affected by intracerebroventricular injection of oligopeptide ASQFFGLM-NH ₂ . Corresponding to our present work, MOR (μ opioid receptor)[ and KOR ₍ κ opioid receptor), as well as their respective endogenous The expression levels of the ligands POMC and PDYN were barely changed. Interestingly, in our current study, the transcriptional and protein levels of DOR (delta opioid receptor) were extremely significantly upregulated, whereas the expression level of PENK (endogenous ligand of DOR) was not significantly changed.

Therefore, one aspect of the present invention provides a method for increasing the transcription and expression level of δ opioid receptors in a subject, which comprises administering an effective amount of oligopeptide ASQFFGLM-NH ₂ to the subject. The present invention also provides the use of the oligopeptide ASQFFGLM-NH ₂ in the preparation of a preparation (such as a medicament) for increasing the transcription and expression levels of δ opioid receptors in a subject. Yet another aspect of the present invention provides a pharmaceutical product (such as a drug or a medicament) or a pharmaceutical composition comprising, as an active ingredient, an oligopeptide ASQFFGLM-NH ₂ that increases the transcription and expression levels of delta opioid receptors in a subject ; and include instructions for the pharmaceutical product concerned.

The subject of the present invention is preferably a mammal, more preferably a human.

For the oligopeptide ASQFFGLM-NH ₂ of the present invention, those skilled in the art can make any modification, provided that the modification does not negatively affect its activity. For example, the oligopeptide can be PEGylated to increase its half-life in vivo; it can be linked with known penetrating peptides to promote the transdermal absorption of the oligopeptide of the present invention, etc. In conclusion, those skilled in the art can make various modifications to the oligopeptides of the present invention to improve delivery efficiency and maintain their activity. Such modified oligopeptides are also within the scope of the present invention.

The oligopeptide ASQFFGLM-NH ₂ as an active ingredient of the present invention can be used together with a pharmaceutically acceptable carrier. In addition to the active ingredients, the methods, uses and products of the present invention may contain suitable pharmaceutically acceptable carriers, including excipients and auxiliaries which facilitate processing of the active compounds into preparations.

For example, preparations suitable for injection or infusion include aqueous and non-aqueous sterile injection solutions and aqueous and non-aqueous sterile suspensions, which may optionally contain antioxidants, buffers, bacteriostats and Solutes that render the formulation isotonic with the blood of the intended recipient, the sterile suspensions may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, eg, water for injection, immediately prior to use.

The active ingredient of the invention may optionally be combined with a solid excipient, and the resulting mixture optionally ground, and the mixture of granules processed, if desired, after adding suitable auxiliaries, to obtain the desired dosage form . Suitable excipients are especially fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose or starch preparations, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxylated Sodium methylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate may be added.

The effective amount of the active ingredient of the present invention can be any amount that improves the transcription and/or expression level of δ opioid receptors in the subject, which can be equivalent to about 0.1-15 mg oligopeptide ASQFFGLM-NH ₂ , preferably 0.01- Dosage unit of 20 mg oligopeptide ASQFFGLM-NH ₂ . More preferably, the dosage unit comprises about 1-4 mg of oligopeptide ASQFFGLM- _NH2 . Most preferably, the dosage unit comprises about 2-3 mg of oligopeptide ASQFFGLM- _NH2 . Determination of an effective amount is within the capabilities of those skilled in the art, particularly in light of the disclosure provided herein.

According to the present invention, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention can be administered to a subject in any number of effective doses. Preferably, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention may be administered in multiple doses, for example from about 2 to about 15 doses, more preferably from about 4-10 doses, most preferably about 6 doses dose. In a particularly preferred embodiment, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention is administered to the subject at a frequency of about once every three weeks during the course of administration, e.g. injection, infusion or orally. In particularly preferred embodiments, administration is by injection.

It should be understood that the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention may be formulated in any suitable manner for administration by any suitable route.

The dosage unit of the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention is administered to a subject on a routine basis. For example, dosage units may be administered more than once daily, weekly, monthly, etc. Dosage units may be administered on a bi-weekly basis, ie twice a week, for example every three days.

As used herein, "comprising" is synonymous with "comprising", "comprising" or "characterized by", and is inclusive or open-ended and does not exclude additional unstated elements or method steps. The term "comprising" in any expression herein, especially when describing the method, use or product of the present invention, should be understood as including consisting essentially of said components or elements or steps and consisting of said components or elements or Those products, methods and uses that consist of steps. The invention exemplarily described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The instructions related to the pharmaceutical product included in the pharmaceutical product of the present invention may contain the following contents: indication (such as pain), administration dosage (such as the above-mentioned exemplification), possible side effects and the like.

The terms and expressions which have been employed herein are used as terms of description rather than limitation, and in the use of such terms and expressions it is not intended to exclude any equivalents of the features shown and described, or parts thereof, and various modifications are to be recognized. It is possible within the scope of the claimed invention. Accordingly, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be employed by those skilled in the art and that such modifications and variations are considered to be defined within the scope of the invention.

In order to illustrate the present invention more clearly, the following examples are now described in detail, but these examples are only exemplary descriptions of the present invention and should not be construed as limitations on the present application.

Example 1

Materials and methods

animal

Male and female ICR mice (20±1.0 g) were randomly provided by the Animal Center of Hangzhou Normal University (Hangzhou, China). All animal experimental procedures were approved by the Committee on the Care and Use of Animals of Zhejiang Sci-tech University and complied with Council Directive No. 24, 1986 (86/609/EEC). All animals were housed at 23°C-25°C with a 12-hour light/dark cycle, and provided with standard food and water before the experiments were conducted. During all experiments, the mice suffered as little injury and pain as possible.

polypeptide

Fluo-oligopeptide ASQFFGLM-NH ₂ and oligopeptide ASQFFGLM-NH ₂ peptides were synthesized by Zhongpei Company (Hangzhou, China) using solid-phase peptide synthesis and purified by high-performance liquid phase (HPLC), with a purity of 98%. FAM is a carboxyfluorophore, and 5-carboxyfluorescein was coupled to the amino acid group of the oligopeptide ASQFFGLM-NH2 peptide using standard coupling techniques. The oligopeptide ASQFFGLM-NH ₂ was dissolved in saline, and the concentration of the working solution was 1.5mM. Fluorescein-labeled (FAM) oligopeptide ASQFFGLM-NH ₂ was dissolved in 50% DMSO (dimethyl sulfoxide) at a stock solution concentration of 15 mM. It was diluted to a working concentration of 1.5 mM with saline before the experiment.

Intraventricular injection and tissue preparation

Intracerebroventricular injections were performed as described by Haley and McCormick in conscious mice. The final dose of oligopeptide ASQFFGLM-NH ₂ or fluo-oligopeptide ASQFFGLM-NH ₂ injected per mouse was 6 nmol, and the injection volume was 4 μl.

Our previous research results showed that intracerebroventricular injection of oligopeptide ASQFFGLM-NH ₂ had obvious analgesic effect within 20 minutes, so the time points of this experiment were selected as 5, 10 and 20 minutes. After injection of FAM-oligopeptide ASQFFGLM-NH2 or oligopeptide ASQFFGLM-NH ₂ (6 nmol per mouse), mice were sacrificed by neck dislocation at corresponding time points, and the accuracy of the injection site was verified by microscopic measurements. In the control group, 4 μl of normal saline was injected into the lateral ventricle of the mice. Then, the brains of mice injected with oligopeptide ASQFFGLM-NH ₂ or saline were quickly removed and stored in a -80°C refrigerator for further experiments. The mouse brain injected with FAM-oligopeptide ASQFFGLM-NH ₂ was quickly taken out, fixed in 0.1M pH7.4 phosphate buffer containing 4% paraformaldehyde (Sigma) for 24h, and frozen section (JUNG Tissue freezingmedium , LEICA, Germany) before dehydration with 30% sucrose solution.

Coronal Sectioning and Microscopic Observation

Each mouse brain was frozen at -20°C and sectioned at a thickness of 10 μm. The brain tissue sections were then preserved on slides. The slices were stored at 4°C, and microscopic observation and photographing were completed within 12 hours. Samples were observed with phase contrast and fluorescence microscopy (NIKON TE2000-U). Anatomical structures were identified from the Atlas of Adult Mouse Brain Structures.

Reverse transcription and real-time quantitative PCR

Total RNA was extracted from each frozen brain tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the commercial instructions. Briefly, brain tissue was ground in a mortar and pestle, and TRIzol was added at a dose of 1 ml TRIzol per 100 mg. In all experiments, 5 μg of total RNA was reverse-transcribed into cDNA with a reverse transcription kit (Gibco BRL) in a final volume of 50 μl.

The primer sequences are as follows, which are identical to those in our previous published work [7]:

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'

ABI7300 real-time PCR detection system equipment was used for relative quantitative real-time PCR to detect the expression changes of the above genes at the mRNA level, and GAPDH was used as an internal reference gene. For negative controls, distilled water was used instead of cDNA. After each real-time quantitative PCR, a melting curve analysis was performed. The standard expression level of each target gene was calculated using the 2- ^ΔΔCt method (Livak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the2- ^ΔΔCT Method.Methods25:402-408.), The formula is △△Ct=(Ct,Target-Ct,GAPDH)Timex(Ct,Target-Ct,GAPDH)Time0. Time x represents any time point (5, 10 and 20 min), and time0 represents the normalization of the target gene to the control GAPDH (saline group). Each experiment was performed in triplicate and experiments were performed in at least three independent repetitions. All results are presented as mean ± standard deviation (SEM).

Western blotting

Total protein concentration was determined using the Coomassie brilliant blue protein quantification method (Pierce Chemical Co.), using bovine serum albumin (BSA, Sigma) as a standard protein.

Proteins were subjected to SDS denaturing polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. Next, place the nitrocellulose membrane in TBST containing 5% skimmed milk and incubate for 1 h at room temperature, then incubate overnight with the primary antibody (β-actin (1/10000, Bioworld), POMC (1:10000, ABGENT), MOR (1/ 10000, Epitomics), NK1 receptor (1/500, Proteintech) and DOR (1/500, Bioworld)). After that, the secondary antibody was incubated at room temperature for 2h (the secondary antibody of β-actin, DOR, MOR and TACR1 is HRP-goat anti-rabbit IgG (1/5000, Bioworld), the POMC secondary antibody is HRP-rabbit anti-goat IgG (1/ 5000, Bioworld)). Finally, the color was developed on a chemiluminescence imager (Sage Creation). The molecular weight of the protein was compared with the prestained protein marker (Fermentas).

For the analysis of western bloing data, the gray scale of the control was defined as 100%, and its value was determined by Quantity One software (Bio-world). The grayscale of the sample is output as a percentage of the grayscale of the control. All data are from at least three independent replicate experiments.

data analysis

All data are from at least three independent replicate experiments. Data are presented as mean ± SEM. All data were analyzed with SPSS software (SPSS Inc., Chicago, IL, USA). A P value <0.05 was considered to have a significant difference.

result

1. After injecting oligopeptide ASQFFGLM-NH ₂ into the lateral chamber of mice, its distribution site in the brain

Under a fluorescence microscope, the fluo-oligopeptide ASQFFGLM-NH2 peptide showed green light under blue light excitation. After 10 minutes of injection into the lateral ventricle, the fluo-oligopeptide ASQFFGLM-NH ₂ was mainly distributed in the ventricle and several structures adjacent to the ventricle. However, the main different distribution sites of fluo-oligopeptide ASQFFGLM-NH ₂ and fluo-r/mHK- ₁ were not in the aqueductal gray matter (PAG) and E/ OV found green fluorescence.

2. Expression characteristics of encoding NK1 receptors and different pre-tachykininogens at the mRNA level

We used the method of real-time quantitative RT-PCR to compare the expression changes of NK1 receptor and pre-tachykininogen at the mRNA level (Figure 1). The housekeeping gene GAPDH was used as an internal reference. In real-time quantitative PCR experiments, the 2 ^-ΔΔCt method was used to analyze relative changes in gene expression. Compared with the control group (normal saline, normalized to 1), the NK1 receptor mRNA values at 5min, 10min and 20min after intracerebroventricular injection of oligopeptide ASQFFGLM-NH ₂ were 0.91±0.05, 0.93±0.04and0.87±0.06, respectively (Fig. 1a, A). The PPT-A mRNA values at 5 min, 10 min and 20 min were 0.99±0.06, 0.93±0.17 and 0.96±0.09, respectively (Fig. la, B). The PPT-C mRNA values at 5 min, 10 min and 20 min were 0.96±0.08, 1.10±0.11 and 0.96±0.03, respectively (Fig. 1a,c). Data analysis showed no significant difference in oligopeptide ASQFFGLM-NH ₂ between the control and all oligopeptide ASQFFGLM-NH ₂ treated groups, suggesting that in mice, the mRNA expression levels of NK1 receptors and pre-tachykininogens were not affected by the lateral ventricles. Effect of injection of oligopeptide ASQFFGLM- _NH2 .

3. Expression characteristics of mRNA encoding endogenous opioid receptors and opioid peptides

Using real-time quantitative RT-PCR technology, we found that the DOR of the oligopeptide ASQFFGLM-NH ₂ treated group was significantly enhanced at the mRNA level compared with the control. The values at time points 5, 10, and 20 min after intracerebroventricular injection of the oligopeptide ASQFFGLM-NH ₂ were 601.7 ± 137.0, 386.1 ± 83.1, and 254.3 ± 68.7, respectively (Fig. 2e, E). However, the endogenous ligand of DOR, PENK, did not undergo significant changes at the mRNA level, with values of 1.08±0.11, 0.94±0.10 and 0.97±0.08 at 5, 10 and 20 min time points after intracerebroventricular injection of the drug, respectively (Fig. 2d , B). MOR (Fig. 2a, A), POMC (an endogenous ligand of MOR, Fig. 2b, B), KOR (Fig. 2c, C) and PDYN (an endogenous ligand of KOR, Fig. 2d, D) in There was no significant change in the expression level of mRNA after drug treatment.

4. After intracerebroventricular injection of oligopeptide ASQFFGLM-NH ₂ in mice, the expression of DOR receptor protein was significantly increased

Finally, we examined whether the upregulation of DOR transcripts induced by intracerebroventricular injection of the oligopeptide ASQFFGLM-NH ₂ was associated with increased expression of DOR proteins. As can be seen in Figure 3D, the expression of DOR protein was significantly upregulated after treatment with oligopeptide ASQFFGLM-NH ₂ compared with the control group. At 5, 10 and 20 min time points, the expression levels of DOR protein were 1.78±0.28, 3.26±0.50 and 5.29±0.86, respectively (Fig. 3D). In addition, we also detected the correlation between the transcriptional expression of NK1 receptor, MOR and POMC and their respective protein expressions. From the results of Figure 3A-C, it can be seen that these proteins did not occur between the control group and hHK-1. Significant changes.

Those of ordinary skill in the art will appreciate that methods, materials, etc. other than those specifically exemplified may be employed in the practice of the present invention without undue experimentation. All art-known functional equivalents of any such methods and materials, etc., are intended to be encompassed by the present invention. It should also be understood by those skilled in the art that various variations and modifications may be made to the invention described in the specification and claims, and that the present invention includes all such variations and modifications. The present invention also includes all the steps, features, compositions and compounds mentioned or indicated in this specification individually or simultaneously, and any and all combinations of any two or more of said steps or features.

Claims (6)

1. Gly-Lys-Ala-Phe-Val-Lys-Lys SQFFGLM-NH ₂for the preparation of raise delta opiate receptor transcribe and/or expression preparation in purposes.

2. purposes according to claim 1, wherein said rise occurs in subject.

3., according to the purposes described in claim 2, wherein said experimenter is mammal.

4. purposes according to claim 3, wherein said mammal is behaved.

5. purposes according to claim 1, wherein said preparation is medicine.

6. the purposes according to any one of claim 1-5, wherein said oligopeptide is modified by PEGization.