KR101811996B1 - A novel pegylated substance p - Google Patents
A novel pegylated substance p Download PDFInfo
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- KR101811996B1 KR101811996B1 KR1020150081555A KR20150081555A KR101811996B1 KR 101811996 B1 KR101811996 B1 KR 101811996B1 KR 1020150081555 A KR1020150081555 A KR 1020150081555A KR 20150081555 A KR20150081555 A KR 20150081555A KR 101811996 B1 KR101811996 B1 KR 101811996B1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/22—Tachykinins, e.g. Eledoisins, Substance P; Related peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/046—Tachykinins, e.g. eledoisins, substance P; Related peptides
Abstract
The present invention provides a novel PEGylation substructure P. The novel PEGylated Substance-P of the present invention has improved stability in vitro and in vivo, while maintaining Substance-P intrinsic activity, and in particular has an increased half-life in vivo, It shows a remarkable effect as an emulsion composition.
Description
The present invention relates to a novel pegylated substance P.
Substance-P (also called 'SP') is a neuropeptide that regulates the immune system and the nervous system. It has been known as a neurotransmitter in the past but recently it has been linked to the immune system and the nervous system. It has been found to play an important role. Substance P is a neuropeptide that can be secreted in the bone marrow. Like other growth factors, it binds to a specific receptor to transmit signals, and regulates bone marrow and lymphocyte functions.
Substance P is a peptide consisting of 11 amino acids and has the same amino acid sequence in human, mouse and rabbit, and is a species-free factor. It transmits a signal through neurokinin receptor -1, which is expressed in the cornea, skin, and immune cells. Functions of Substance P include pain induction and vasodilation. These results indicate that Substance P plays an important role in neuronal immune system regulation, myelofibrosis, cancer cell proliferation, wound healing, and the like in addition to the previously known function of neurotransmitter.
The structure of Substance P is represented by the following general formula (1).
[Chemical Formula 1]
Recently, Substance P has been used for wound healing. One of them has been developed as an eyedrop for accelerating the regeneration of epithelial cells during corneal injury, and the healing effect by Substance P in corneal injured animal models has been reported. In addition, development as an eyedropper is currently under way in Japan. In addition, research and development are underway to utilize Substance P in various fields.
However, since Substance P is decomposed by the neutral endopeptidase in the serum, there is a limit that the time for staying in the body is very short.
In this regard, research and development are needed on Substance P mutants with increased in vivo stability, and research and development related thereto have been hardly advanced. That is, techniques such as sialation, glycosylation, conjugation with other proteins, point mutation, and the like are applied to Substance P as a technique for increasing protein stability, The necessity to manufacture Substance P that has improved usability has been greatly increased, but related research and development using Substance P has not been accomplished so far.
Under such circumstances, research and development for improving the in vivo stability of Substance P is urgent.
The present invention seeks to provide a novel pegylating substance P.
The present invention provides a pharmaceutical composition comprising pegylated substance P as an active ingredient.
The present inventors have carried out research and development for development of Substance-P having enhanced in vitro and / or in vivo stability, and have found that pegylated Substance P has excellent in vitro and / or in vivo stability, particularly in vivo half life And thus the present invention has been completed.
Thus, the present invention provides a novel pegylated (PEGylated) subsequence P with increased in vitro and / or in vivo stability.
The present invention provides a novel pegylated substence P for use in a new form of pegylation subsequence P, e.g., Substance P, particularly in pharmaceutical formulations and methods of manufacture.
The present invention provides a pegylated substence P, which is covalently bound to at least one or more polyethylene glycol molecules. The pegylated Substance P according to the present invention has the advantage of greatly increasing the in vitro and / or in vivo stability, in particular the in vivo half life.
The pegylated substence P of the present invention is covalently bonded to at least one polyethylene glycol molecule and is preferably covalently bonded to one or two polyethylene glycol molecules by a covalent bond to a substance P (i. E., A mono- or di- P), and / or mixtures thereof.
Pegylation of the C-terminus is essential for the function of Substance P, since the activity of residues 7-11 of the C-terminus, a directly involved part of binding to the neurokinin receptor (NKR) There is a possibility of deteriorating the efficacy. Thus, the PEGylation subsequence P according to the present invention binds PEG to the amine group of the side chain of the lysine residue located between the N-terminus amine and the substance P sequence during pegylation, thereby restoring the C-terminus position required for receptor binding So that the activity of the substance P is largely maintained.
In certain embodiments of the invention, the pegylation substance P of the invention is a substance P that is covalently linked to one or two PEG molecules. For example, the Mono-PEGylated substence P may have the structure of Formula 2 or Formula 3 below.
(2)
(3)
The mono-PEG substance P is PEG-linked to the amine group of the side chain of the lysine residue located in the middle of the N-terminus amine or the substance P sequence during pegylation.
More preferably, the pegylated substence P has a structure represented by the following formula (4) as a substance P covalently bonded to two PEG molecules.
[Chemical Formula 4]
The structure of Formula 4, which is the most preferred example of the present invention, is a structure in which PEG is bonded to the amine group of the side chain of the lysine residue located in the middle of the N-terminus amine and the substance P sequence of Substance P.
As used herein, the term "PEG" refers to a polyethylene glycol molecule. In its exemplary form, PEG is a linear polymer having a terminal hydroxyl group, HO- (CH 2 CH 2 O) n -H has an (where, n is from about 8 to about 4,000). The terminal hydrogen may be replaced by a capping group, for example, an alkyl group or an alkanol group. Preferably, the PEG has at least one hydroxyl group, more preferably it is a terminal hydroxyl group.
Such a hydroxyl group is preferably attached to a linker moiety capable of reacting with the peptide to form a covalent bond. The average molecular weight of PEG molecules covalently bonded to Substance P of the present invention can be approximately 3000, 4000, 5000, 8000, 10,000, 20,000, 30,000, or 40,000 daltons. The PEG molecule is preferably from 3,000 to 12,000 Daltons. More preferably, it is from 4,000 to 12,000 daltons. Most preferably about 10,000 Daltons. PEGylation reagents can be linear or branched molecules and can be present singly or in tandem in series. The PEGylated substrons P of the present invention can be modified to have various reactive moities so that the PEG is attached to the amine groups of the side chain of the lysine residue located preferably in the middle of the N-terminus amine and / or the substance P sequence of the peptide have. More preferably, PEG is coupled to the amine group of the side chain of the lysine residue located in the middle of the N-terminus amine and the substance P sequence in tandem.
These activated polyethylene glycol (PEG) molecules can be conjugated to mPEG-succinimidyl succinate, mPEG-succinimidyl carbonate, mPEG-nitrophenyl carbonate, mPEG-imidate, mPEG- succinyl glutarate and mPEG- . Preferably, methoxypolyethylene glycol succinimidyl succinate can react with Substance P under a Tris solvent to produce PEGylated Substance P.
As used herein, the term "PEGylated" refers to covalently attaching one or more PEG molecules as described above to Substance P.
The present invention also provides a process for producing Di-PEGylated substence P.
The process according to the invention preferably comprises the following steps:
(a) dissolving Substance P in any one of the solvents selected from Tris, pH 6.0 to pH 8.5, PBS, sodium borate phosphate or distilled water;
(b) mixing methoxypolyethylene glycol succinimidyl succinate with the Substance P solution of step (a) and stirring at 2 ° C to 8 ° C for 10 to 30 hours; And
(c) separating the Di-PEGylated substance P from the solution of the step (b).
In the present invention, Substance-P has the amino acid sequence "RPKPQQFFGLM ", which is a peptide having the structure of Formula 1, and may be commercially available or synthesized chemically or genetically by methods known in the art.
In the present invention, methoxypolyethylene glycol succinimidyl succinate having a molecular weight of 5,000 to 40,000 Dalton is preferably used and those commercially available in the art can be used, and according to the most preferred embodiment of the present invention, Methoxypolyethylene glycol succinimidyl succinate having a molecular weight of 10,000 dalton may be used.
In the method for preparing Di-PEGylated substence P, the step (a) may be carried out in any one of the solvents selected from tris, pH, pH 8.5, PBS, sodium borate phosphate or distilled water, And dissolving. The process of the present invention has the advantage of performing the reaction at neutral pH conditions that do not cause structural modification of Substance P and affect its activity.
In the step (b), the step of mixing succinimidyl succinate with methoxypolyethylene glycol in the solution of Substance P does not change the activity of the Substance P peptide by performing the reaction at 2 ° C to 8 ° C, It is preferred to stir for a period of time.
The step of separating the Di-PEGylated substance P from the eluent of step (c) may be carried out in accordance with methods known in the art, for example, an amicon centrifugal filter, a hydrophobic interaction chromatography (HIC purification) IEC, ion exchange purification), and affinity chromatography.
The preparation of the Di-PEGylated Substance-P corresponds to a range of skill levels that are easily carried out by a person skilled in the art, and specific manufacturing methods can be referred to the embodiments of the present invention.
The present invention also provides a pharmaceutical composition comprising PEGylated Substance-P as an active ingredient. The PEGylated substence P according to the present invention may have the structure of the formulas (2) to (4), more preferably the structure of the formula (4).
The pharmaceutical composition of the present invention can be used for improving, preventing or treating diseases or conditions mediated by Substance-P.
In the present invention, the pharmaceutical composition is preferably used for the prevention or treatment of ischemic diseases. More preferably, in the present invention, the ischemic disease may be any one selected from the group consisting of ischemic ulcer, ischemic heart disease, myocardial infarction, angina pectoris, stroke, cerebral infarction, diabetic ulcer, diabetic lower limb ischemic disease or lower limb ischemic disease . Most preferably, the ischemic disease in the present invention is a lower limb ischemic disease, particularly a diabetic lower limb ischemic disease.
The pharmaceutical composition according to the present invention has a long half-life in the human body, promotes migration of stem cells, inhibits ischemic cell necrosis, increases blood flow, inhibits tissue fibrosis, Thereby exhibiting remarkable preventive and therapeutic effects on ischemic diseases.
The pharmaceutical composition of the present invention may be formulated into appropriate forms such as powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories, Can be used.
The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock and humans in various routes such as oral, sublingual, rectal, subcutaneous, intramuscular, intraperitoneal, intravenous, . The pharmaceutical composition may be prepared for storage or administration by mixing the PEGylated Substance P having the desired purity with a pharmaceutically acceptable carrier, excipient or stabilizing agent. Such materials are non-toxic to the recipient at the dosages and concentrations employed and include antioxidants polyarginine such as buffer ascorbic acid, such as phosphate, citrate, acetate and other organic acid salts, proteins such as serum albumin, gelatin or immunoglobulin (Having less than about 10 residues) peptide polyvinylpyrrolidinone, such as glycine, glutamic acid, aspartic acid or arginine, amino acid monosaccharides, disaccharides, and cellulose or derivatives thereof Other cationic surfactants such as chelating agents mannitol such as glucose, mannose or dextrin EDTA or sodium hydroxide of sugar alcohol such as sorbitol and / or non-ionic surfactants such as Tween, Pluronics or polyethylene glycols, .
The pharmaceutical compositions of the present invention can be prepared according to conventional methods in the art in the form of injectable sterile compositions. The injectable sterile composition may contain a solution or suspension of the active compound in a vehicle, for example a synthetic vegetable oil such as water or sesame oil, peanut oil or cottonseed oil, or a synthetic fatty vehicle such as ethyl oleate.
The injectable sterilized composition for injection may also contain a buffer, preservative, antioxidant or the like according to a state conventionally used in the art.
A "therapeutically effective amount" of the PEGylated Substance P used in the present invention is an amount of the active ingredient or pharmaceutical composition that induces a biological or medical response in an animal or human considered by a researcher, veterinarian, Amount, which includes an amount that induces symptomatic relief of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect.
Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, it is preferable to administer PEGylated Substance-P of the present invention once a day in a dose of 0.000005-500 mg / kg body weight, preferably 0.00005-50 mg / kg body weight .
The dosage of the pharmaceutical composition of the present invention may be determined by a physician insofar as it relates to a method of treating the above-described diseases, by factors that may modify the effectiveness of the drug, such as age, condition, weight, sex, The severity of the disease, the time of administration, and other clinical factors.
The pharmaceutical composition of the present invention can be administered once or continuously. However, it is preferable to continue administration to maintain a constant circulating concentration after the first administration. Effective dosing dosages and methods will be optimized based on the general techniques known in the art, and the number of repeated dosing will depend on the pharmacokinetic parameters of each patient and the route of administration. The dosage, usage, and frequency of administration may also be optimized according to the physical state, stability, rate of in vivo release, rate of in vivo clearance, etc. of the administered drug. In addition, the appropriate dosage depending on the route of administration can be calculated according to body weight, body surface area, and size of organs. Appropriate doses should be determined with appropriate dose-response data along with data on blood levels. The final dosage regimen will be decided by the physician and the physician should prescribe taking into account the inactivity of the drug, the severity of the patient, the patient's response to the drug, age, condition, weight, sex, diet and other clinical factors. do.
The novel PEGylated Substance-P of the present invention has improved stability in vitro and in vivo, while maintaining Substance-P intrinsic activity, and in particular has an increased half-life in vivo, It shows a remarkable effect as an emulsion composition.
1 is a schematic diagram illustrating a process for pegylating a substance P according to an embodiment of the present invention.
FIG. 2 is a graph showing chromatographic results before and after purification of Di-PEGylated Substance P by gel permeation chromatography (GPC). FIG.
3 is a diagram showing 1 H-NMR results of Di-PEGylated substence P;
4 is a diagram showing the MALDI-ToF results of the PEGylated substance P. FIG.
5 is a diagram showing MALDI-ToF results after digestion of Di-PEGylated substance P. FIG.
FIG. 6 is a diagram showing MALDI-ToF results after digestion of Di-PEGylated substance P. FIG.
Figure 7 shows the MALDI-ToF results after digestion of the PEGylated substance P mixture.
FIG. 8 is a graph showing the results of analysis of half-lives of Substance P and Pegylation Substance P in human serum.
FIG. 9 is a schematic diagram of an experiment for confirming the therapeutic effect of PEGylated Substance P using a lower limb ischemic mouse animal model.
FIG. 10 is a graph showing the results of confirming the promoting action of mesenchymal stem cells (MSC) and vascular endothelial progenitor (EPC) migration of pegylated Substance P through flow cytometry.
11 is a graph showing the results of confirming changes in the lower limb survival rate by administration of PEGylated Substance P in a non-diabetic lower limb ischemia animal model.
FIG. 12 is a graph showing the results of analysis of changes in blood flow by administration of PEGylated Substance P in an animal model of non-diabetic lower limb ischemia.
Figure 13 shows the histological changes of PEG-treated Substance P in non-diabetic ischemic animal models through H & E staining, Masson's trichrome staining, and CD31 (capillary: capillary) and SMA (arteriole) staining And the results of Fibrosis area analysis in ischemic tissues.
14 is a graph showing the results of analysis of changes in lower limb survival rate by administration of PEGylated Substance P in a diabetic lower limb ischemia animal model.
FIG. 15 is a diagram showing the results of confirming changes in blood flow due to administration of PEGylated Substance P in a diabetic lower limb ischemia animal model. FIG.
FIG. 16 is a graph showing the results of analysis of changes in blood flow rate due to administration of PEGylated Substance P through the ratio of necrosis and normal group in a diabetic ischemic ischemic animal model.
FIG. 17 is a graph showing the results of H & E staining and Masson's trichrome staining analysis of histological changes caused by administration of PEGylated Substance P in a diabetic ischemic ischemic animal model and fibrosis area analysis of ischemic tissues.
Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described hereinafter. However, the present invention is not limited to the embodiments disclosed below.
< Example 1> PEGylation Substance Preparation and analysis of P
One. PEGylation Substance Manufacture of P
A synthetic schematic diagram for the preparation of PEGylated Substance P is shown in Fig. Specifically, 100 mg of Substance P (source: Anygene) was dissolved in 20 ml of Tris buffer (10 mM, pH 7.5) and 5 equivalent of mPEG (10K) -succinimidyl succinate (source: sunbio) The reaction was allowed to proceed overnight at 4 ° C with stirring. Di-pegylated substence P (Di-PEGylated substance P) was isolated by performing centrifugation several times using Amicon centrifugal filter (10K MWCO).
2. Gel permeation chromatography ( GPC ) analysis
The synthesized PEGylated substence P was confirmed by gel permeation chromatography (GPC). The results are shown in Fig.
The results before the purification are shown in Fig. 2 (A), and the results after purification are shown in Fig. 2 (B).
The pink line before purification (Figure 2A) represents the PEGylated substance P mixture and the gray dotted line represents the substance P.
On the other hand, after the purification (FIG. 2B), gray dotted lines and gray solid lines indicate SP and PEG which did not participate in the reaction, respectively. Red lines were separated and purified by using Amicon centrifugal filter (10K) The remaining sample in the upper part shows diPEG-SP (Di-PEGylated substance P).
The separated Di-PEGylated substence P is represented by the following general formula (4).
[Chemical Formula 4]
3. Di - pegylated Substance P One H-NMR analysis
The Di-PEG-10K-Substance P separated from the above was confirmed by 1 H-NMR and the results are shown in FIG. As can be seen in FIG. 3, the analytical peak of the Di-PEGylated substance P was confirmed, which shows the same analytical result as the expected 1 H-NMR value.
4. MALDI - ToF analysis
Through the analysis of MALDI-ToF, the position of Pegylation of Substance P was confirmed.
The results of MALDI-ToF analysis of Di-PEGylated substance P and PEGylated substance P mixture before digestion are shown in FIG.
FIG. 4A shows the isolated Di-PEGylation subsequence P, and FIG. 4B shows the PEGylated substance P mixture.
As can be seen in FIG. 4A, a peak near 20,000 shows dipegylated SP and a peak near 10,000 shows dipegylated SP (z / z at 2) with +2 charge. Also, as shown in Figure 4B, the PEGylated substance P mixture is a mixture of Mono-PEGylated SP (11,386 peak), Di-PEGylated SP (22,582 peak), and unpurified free PEG (10,505) .
The DI-PEGylated SP and PEGylated substance P mixtures were identified for PEGylation by the digestion method. Samples were mixed with 8 M Urea in a ratio of 1: 1 (100 ul each), followed by the addition of 200 ul of 1% ammonium bicarbonate solvent. 10 ug trypsin was added thereto, and CaCl 2 was added to prepare a 5 mM CaCl 2 solution. The solution was reacted at 37 DEG C for 3.5 hours. ( N ) -RP K PQQFFGLM- (C) in the case of the digestion site, and two underlined ( N ) -RP K PQQFFGLM- (C) And the peptide fragment according to Digestion is shown in Table 1 below.
[Table 1] Peptide fragments
The analysis results are shown in Figs. 5 to 7.
FIG. 5 shows the results of Di-PEGylation substance P analysis, and 10489 peak (ΔMW: 11,735-10,489 = 1,246) was newly confirmed according to digestion. This can be interpreted as PEG + N-R / PKPQQFFGLM-C through the difference in molecular weight before and after digestion, indicating that the N-terminus is pegylated.
FIG. 6 also shows the result of Di-PEGylation substance P analysis, and 1192 peak (PKPQQFFGLM-C, 1192.44) was not confirmed. This is because PEG is attached to PKQQFFGLM-C (that is, because MW is increased due to PEG), indicating that the PEG is formed at the corresponding position.
That is, the PEGylation sites of Di-PEGylated substence P of Formula 4 according to the present invention were confirmed through the results of FIGS. 5 and 6.
FIG. 7 shows the analysis result of the PEGylated substance mixture, and it was confirmed that the PEG was formed on the N-RPK side as MW (ΔMW: 11386-10452 = 934) as much as PKQQFFGLM-C fell. That is, the mono-PEGylated substence P of
< Example 2> in human serum Substance P and PEGylation Substance Half-life analysis of P
The half-life of the di-PEGylated substence P separated from the substance P in human serum was analyzed using a parameter ELISA kit for quantitation of Substance P from R & D Biosystems.
The results are shown in Fig. As shown in Fig. 8, it was confirmed that Substance P completely disappeared within 10 to 20 minutes in human serum at 37 占 폚. On the other hand, the stability of PEG-modified Substance P was increased due to the effect of PEG, and the half-life was greatly increased. Thus, it was confirmed that the PEG-modified Substance P was stable for up to 48 hours in serum at 37 ° C.
From the above results, it was confirmed that the PEGylated Substance P according to the present invention has high stability in vivo, including in vitro.
< Example 3> Non-diabetic In the lower limb ischemic model PEGylation Substance Effect of P Confirm
One. Non-diabetic Manufacture of Ischemic Animal Models
A schematic diagram for the preparation of the lower limb ischemia animal model is shown in Fig.
After the anesthesia with the mouse (ICR, male, 5w), the hair at the surgical site was removed and the surgical site was sterilized three times using 70% ethanol and betadine. Then, the skin inside the thigh of the left hind paw was incised and the femoral artery was exposed. The proximal and distal portions of the femoral artery were ligated twice using a 6-0 prolene suture, and the femoral artery between the ligated portions was removed using microforcep or fine-point tweezers. The dissected skin was sutured with 6-0 prolene suture, and then stained with saline, Substance P (SP), or PEGylated substance P mixture (Di-PEG-SP, mono-PEG-SP mixture, ) Was intravenously injected (dose: 5 nmole per mouse kg) three times a week for 2 weeks.
After that, the results of the experiment were analyzed through 4 - week recovery observation.
2. Flow cell Through analysis PEGylation Substance Check the effect of P
In order to confirm that the stem cell migration promoting action of Substance P shows a further improved action when injected with PEGylated Substance P, the flow cytometry of MSCs and vascular endothelial progenitor cells (MSCs) of blood mononuclear cells (EPC) was investigated. After collecting the whole blood from the lower limb ischemic disease mice, the erythrocytes were removed using a Pichol gradient centrifugation, the mononuclear cells were separated, and resuspended in PBS buffer containing 1% bovine serum albumin. (MSC) marker, anti-CD-29 (Miltenyi Biotec Inc .; Cat # 130-096-309, 1:10), and the anti- Anti-CD-45 (Miltenyi Biotec Inc.; Cat # 130-B), which is not expressed in anti-CD-105 (Miltenyi Biotec Inc.; Cat # 130-092-930, 1:10) and mesenchymal stem cells (EPC) marker, anti-CD-31 (EPC) marker to investigate the ratio of EPCs to the mesenchymal stem cell (MSC) CD-309 (Miltenyi Biotec Inc.; Cat # 130-096-405, 1:10), and anti-CD-133 (Miltenyi Biotec Inc.; Cat # 130-097-422, 1:10) BioLegend Inc .; Cat # 141204) to treat vascular endothelial progenitor cells (EPC). The number of labeled cells was measured using a FACS Calibur (BD Bioscience) and analyzed using CELLQuest software (Becton Dickinson).
The results are shown in Fig.
The number of mesenchymal stem cells (MSC) and vascular endothelial progenitor cells (EPC) increased in the first day after injection of Substance P (SP) into non-diabetic ischemic disease mice, and PEG- SP), the same result as the substance P was obtained. On the other hand, this action was reduced by antagonist treatment (Ant + SP) on Substance P.
That is, it was confirmed that the stability of the substance was increased through the modification of Substance P by PEGylation, and the activity level of Substance P was equal to that of Substance P in activity. This confirms that the PEGylation contribution P according to the present invention can exhibit excellent pharmacological activity in the body as compared with the substance P when the stability effect is taken into consideration.
3. Non-diabetic Analysis of lower limb survival rate in mouse model of lower limb ischemic disease
The survival rate of the lower limb was analyzed in a mouse model of non-diabetic lower limb ischemic disease, and the results are shown in FIG. In the untreated non-diabetic ischemic mouse model (PBS group), about 40% of the limb amputation rate was observed and necrosis occurred in almost all mice. In contrast, the majority of the treatment group injected with Substance P or PEGylated Substance P It was confirmed that the legs survived, or the necrosis only partially occurred. In particular, PEGylated Substance P treatment group showed remarkable therapeutic effect compared to Substance P treatment group, in which all the lower limbs were preserved and only a few cell necrosis was observed. That is, it was confirmed that the enhanced half-life of the PEGylated substrate P improved the stability in the body and had a higher therapeutic effect than Substance P.
4. Non-diabetic Determination of blood flow opening in mouse model of lower limb ischemic disease
Doppler analysis was used to observe changes in blood flow for 4 weeks.
Specifically, each experimental animal was anesthetized on
The results are shown in Fig. In the control group (PBS group and antagonist + Substance P group), blood flow recovery was very low. PEGylated Substance P and Substance P treatment group showed significant improvement in blood flow. In particular, PEGylated Substance P showed a recovery of blood flow of 60% or more within one week of ischemic disease induction, It was confirmed that the body's stability was improved due to the enhanced half-life of P, and the blood flow was recovered faster than the substance P.
5. Non-diabetic Identification of Histologic Shape Analysis in Mouse Model of Lower Extra Ischemic Disease
After 4 weeks of the non - diabetic ischemic model, the ischemic muscle tissue from which blood vessels were removed was analyzed by histological analysis. For histological analysis, the muscles of the ischemic area were collected 4 weeks after the treatment and hematoxylin & eosin (H & E) staining and Masson 's trichrome staining were performed.
For H & E staining, the tissue was paraffin-sectioned to 4 μm thickness and then paraffin was removed from xylene. It was then hydrated through alcohol. Hematoxyline (Weigert, Sigma) was treated for 5 min and eosin Y for 1 min. The dyed slides were washed once with 95% alcohol and dehydrated for mounting.
For Masson's trichrome staining, paraffin sectioned samples were deparaffination, hydration, washing in xylene and alcohol. Bouin soln was sampled and placed at 56 ° C for 1 hour. Washing with secondary distilled water (until yellow disappears). Weigert iron hematoxylin was treated at room temperature for 10 minutes. After washing for 10 minutes with secondary distilled water, Biebrich scarlet-acid fuchsin soln was treated at room temperature for 10 minutes. After washing for 10 minutes with secondary distilled water, 5% phosphotungstic acid was treated for 10 minutes. Next, aniline blue was treated for 5 minutes. After washing with distilled water for 10 minutes, 1% acetic acid was treated for 3 minutes and then washed again. Finally, dehydration and mounting were carried out through alcohol.
The experimental results are shown in Fig.
In the control group (PBS injected group and antagonist + Substance P injected group), muscle necrosis was observed to occur extensively through H & E staining to confirm the degree of muscle necrosis in ischemic tissue. On the contrary, in PEG-treated Substance P treatment group, the necrosis of muscle was the greatest decrease, and the shape of muscle tissue was similar to that of normal leg muscle tissue.
In addition, the degree of fibrosis in the tissue was confirmed by Masson's trichrome staining to confirm the degree of fibrosis of the muscle tissue, and the results were compared with the control group (PBS injection group and antagonist + SP injection group) In contrast to the results of H & E staining, the shape of the muscle bundle was similar to that of the normal leg muscle tissue (Fig. Respectively. In particular, the PEGylation Substance P administration group showed significantly lower fibrosis level than the Substance P group, demonstrating improved therapeutic efficacy of PEGylated Substance P.
Staining of ischemic muscle tissue through co-staining of capillary staining (CD31) and smooth muscle alpha-actin (SMA) resulted in no treatment (PBS group and antagonist + (SP) and PEGylated Substance P (PEG-SP) treatment group, as compared to the control group (PEG-SP group). In particular, it was found that the necrosis of muscles was the greatest in the group treated with PEGylation Substance P, the degree of fibrosis was the lowest, and the shape of muscle bundle was similar to that of normal leg muscle tissue. That is, as shown in the results of performing CD31 and SMA staining, it was confirmed that the effect of regeneration of blood vessels was remarkably enhanced by PEG-SP treatment.
Example 4 Confirmation of Effect of PEGylated Substance P on Diabetic Short-limb ischemia Model
1. Preparation of diabetic dorsal ischemic model
Diabetes was induced by intraperitoneal administration of streptozotocin (Sigma) twice a week (dose: 100 mg / kg mouse) to produce a diabetic ischemic model. Two weeks after the injection of Streptozotocin, only rats with a blood glucose level of 500 mg / dL or more were selected and used for modeling diabetic lower limb ischemic disease mice.
After the mice (ICR, male, 5w) were anesthetized, the hair at the surgical site was removed and the surgical site was sterilized three times using 70% ethanol and betadine. Then, the skin inside the thigh of the left hind paw was incised and the femoral artery was exposed. The proximal and distal portions of the femoral artery were ligated twice using a 6-0 prolene suture, and the femoral artery between the ligated portions was removed using microforcep or fine-point tweezers. The dissected skin was sutured with 6-0 prolene suture, and intravenously injected with saline, Substance P (SP), or Di-PEG-SP (Substance P) per mouse kg). After that, the results of the experiment were analyzed through 4 - week recovery observation.
2. Analysis of lower limb survival rate in mouse model of diabetic lower limb ischemic disease
In the same manner as in Example 4-3, the survival rate was analyzed in the mouse model of diabetic lower limb ischemic disease prepared above, and the results are shown in Fig.
As shown in Fig. 14, untreated non-diabetic lower limb ischemic disease mouse model (PBS group) injected Substance P or PEGylated Substance P in contrast to 90% showing lower limb amputation and necrosis In the treatment group, it was confirmed that ischemic limb was preserved in over 60% lower limb ischemic mice. Especially, in the case of PEG-treated Substance P treatment group, it was confirmed that the treatment efficacy of PEGylated Substance P was remarkably higher than Substance P in the case of 80% model.
3. Identification of blood flow opening in mouse model of diabetic lower limb ischemic disease
The blood flow opening degree was confirmed in the same manner as in Example 4, 4, and the results are shown in Figs. 15 and 16. Fig.
FIG. 15 shows the result of checking the change in blood flow with time with a mouse. Experimental results showed that blood flow was the highest in the experimental group treated with PEGylation substance P.
The results of the experiment of FIG. 15 are quantified and shown in FIG. As is also apparent from Fig. 16, it was confirmed that the effect of the pegylation treatment P was remarkably higher than that of Substance P alone. In other words, increased treatment effect by pegylation of substance P was confirmed.
4. Identification of histological morphology in mouse model of diabetic lower limb ischemic disease
Hematoxylin & eosin (H & E) staining and Masson ' s trichrome staining were performed as in Example 3, 5, and the results are shown in Fig.
From these results, it was confirmed that the PEGylated substence P could have higher therapeutic efficacy in the human body through increased in vitro and / or in vivo stability improvement.
Claims (10)
[Chemical Formula 4]
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| WO2008095004A2 (en) * | 2007-01-31 | 2008-08-07 | Affymax, Inc. | Nitrogen-based linkers for attaching modifying groups to polypeptides and other macromolecules |
| WO2011133948A2 (en) | 2010-04-22 | 2011-10-27 | Longevity Biotech, Inc. | Highly active polypeptides and methods of making and using the same |
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| WO2008095004A2 (en) * | 2007-01-31 | 2008-08-07 | Affymax, Inc. | Nitrogen-based linkers for attaching modifying groups to polypeptides and other macromolecules |
| WO2011133948A2 (en) | 2010-04-22 | 2011-10-27 | Longevity Biotech, Inc. | Highly active polypeptides and methods of making and using the same |
| US20130096050A1 (en) | 2010-04-22 | 2013-04-18 | Longevity Biotech, Inc. | Highly active polypeptides and methods of making and using the same |
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