CN105153293B - A kind of Erythropoietin mimetic peptide and its dimer and preparation method and application - Google Patents
A kind of Erythropoietin mimetic peptide and its dimer and preparation method and application Download PDFInfo
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- CN105153293B CN105153293B CN201510530632.3A CN201510530632A CN105153293B CN 105153293 B CN105153293 B CN 105153293B CN 201510530632 A CN201510530632 A CN 201510530632A CN 105153293 B CN105153293 B CN 105153293B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/505—Erythropoietin [EPO]
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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
Abstract
The present invention relates to a kind of Erythropoietin mimetic peptides, which is characterized in that the amino acid sequence of the monomeric peptide of the simulating peptide is as shown in SEQ ID NO:1: GX1LYACHMGPITX2VCQPLRX3K；Wherein, X1For allylglycine (D-Allyiglycin), X2For 3- (1- naphthalene)-l-Alanine (Nal), X3For sarcosine (Sar), 6 and 15 cysteines (C) form intramolecular disulfide bond, N-terminal acetylation.The present invention also provides the simulating peptide and the preparation methods and its officinal salt of dimer.The present invention still further provides the pharmaceutical composition containing above-mentioned simulating peptide or dimer or its officinal salt.Erythropoietin mimetic peptide, dimer and its officinal salt of the invention can stimulate RBC acceptor garland rate, and significantly extend the half-life period of drug in vivo.
Description
Technical field
The invention belongs to fields of biomedicine, are related to a kind of Erythropoietin mimetic peptide, specifically, the present invention relates to
And one kind can combine and be activated with EPO Receipter EPO Receipter or can play promoting erythrocyte life
At the Erythropoietin mimetic peptide dimer and preparation method thereof of plain agonism, the invention further relates to the simulating peptides two
Aggressiveness preparation treatment by lack erythropoietin(EPO) or red cell population lack or defect characterized by disease drug in use
On the way.
Background technique
Hematopoietin (erythropoietin, EPO) is a kind of glycoprotein hormones, molecular weight about 34kD.Blood plasma
Present in hematopoietin be made of 165 amino acid, degree of glycosylation is very high, and sugared ingredient is mainly sialic acid.
According to carbohydrate content difference, naturally occurring hematopoietin is divided into two types i.e. α type and β type, wherein α
Type contains 34% carbohydrate, and β type contains 26% carbohydrate.Two types are in biological characteristics, antigenicity and clinic
It is all the same in application effect.Human erythropoietin gene is located at long 22nd area of No. 7 chromosomes.Its cDNA is by success within 1985
Clone, and start to produce recombinant human erythropoietin (recombinant human in enormous quantities using gene recombination technology
Erythropoietin, rHuEPO), it is widely used in clinic.Go out promoting erythrocyte using recombinant DNA technology biosynthesis to generate
Plain (Egrie, JC, Strickland, TW, Lane, J etc. (986) immuno-biology (Immunobiol) 72:213-224) is
It is inserted into the human erythropoietin gene of the clone in the ovarian tissue cells (Chinese hamster ovary celI) of Chinese hamster and expressed
Product.Naturally occurring human forcing erythrogenin is translated into first containing 166 amino acid and 166 are arginic more
Peptide chain.Upon translation in modification, fall 166 arginine with hydroxyl Isopeptidase cleavage.There is no the molecular weight of the polypeptide chain of the people EP0 of glycosyl
It is 18236Da, in complete erythropoietin molecule, glycosyl accounts for about the 40% of entire molecular weight
(J.Biol.Chem.262:12059)。
Hematopoietin is to be applied to clinical cell factor earliest, is that heretofore known effect is best single and safe
It is reliable to rise Homopure.For kidney anaemia, alpastic anemia, Huppert's disease and Paroxysmal Nocturnal blood urine etc.
There is certain curative effect；In addition, can also reduce the transfusion volume in operation using hematopoietin, and can be to a certain extent
Correct the anaemia as caused by malignant tumour, chemotherapy and rheumatoid arthritis.Since hematopoietin is mainly by renal tubule
Endothelial cell generates, and anaemia caused by renal illness is the preferred idicatio of hematopoietin；Hematopoietin entangles
The curative effect of positive renal anemia almost 100%, but renal function can not be improved.The treatment safety of hematopoietin, effectively, fit
Long-term treatment is closed, is also avoided that blood source anxiety.Global biotechnology medicines in 2006 in the market, hematopoietin class
Reconstituted drug accounts for 11,900,000,000 dollars, there is huge market capacity.
Early in 1989, U.S. FDA was used for the treatment of renal anemia with regard to official approval Recombinant Human Erythropoietin (EPOGEN), but
Until ability in 1992 lists in China.The annual morbidity of China's chronic nephritis is about 0.25%, and wherein quite a few patient is most
It can switch to renal failure, annual renal anemia patient about 50-60 ten thousand eventually.It is estimated according to conservative dosage, if pressing current valence
Lattice 30-40 member/, in addition the medication of other patients such as cancer associated anaemia, hundred million yuan of domestic market capacity about 12-16 is even more
(patient's average weight is calculated by 50Kg) greatly.From later period the 1990s, hematopoietin has entered China emphasis city
Hospital of city best-selling drugs conduct column, 2003 at national 62,130,000 yuan of key cities' sample hospital administration amount of money, ranking the 56th.
2004, national key cities' sample hospital money for drugs rose to 80,490,000 yuan, had increased by 30% on a year-on-year basis.
Hematopoietin acts on myeloid element as one kind, promotes erythroid progenitor cells hyperplasia, differentiation, finally
Mature endocrine hormone plays important regulating and controlling effect to body oxygen supply situation.Embryo early stage, hematopoietin by
Liver generates, and then gradually shifts to kidney, is mainly secreted by renal tubular interstitium cell after birth.
It is induced in red group of cell differentiation procedure in hematopoietin, globulin is induced, this can be such that cell absorbs more
More iron synthesizes functional hemoglobin, and this functional hemoglobin can be combined with the oxygen in mature red blood cell,
Therefore, red blood cell and hemoglobin play extremely important role in terms of providing body oxygen.This process is red thin by promoting
Caused by born of the same parents generate the interaction between element and the surface receptor of red group of cell.
When human body is in health status, tissue can absorb enough oxygen from already existing red blood cell, at this time
Intracorporal Erythropoietin is very low, and this normal lower Erythropoietin can irritate rush completely
The problem of into due to the age and the red blood cell of normal loss.When the horizontal quilt for carrying out oxygen conveying by red blood cell in the circulatory system
It reduces and then when there is anaerobic condition, the quantity of hematopoietin in vivo will will increase, and body anaerobic condition can be by
Caused by following reason: excessive radiation, because caused by high latitude or long-term coma oxygen uptake reduce, various types of anaemias
Etc..As the response for being in anaerobic stress to tissue, the raising of erythropoietin can stimulate red group of cell
Differentiation reaches the ability for improving RBC acceptor garland rate.When the quantity of intracorporal red blood cell is greater than the demand of normal tissue, cyclic system
The level of hematopoietin is lowered in system.Have the generation of red blood cell to pass just because of hematopoietin
Important role, therefore this parahormone is for treating and diagnosing the blood disease aspect by RBC acceptor garland rate lowly and characterized by defect
There is very extensive prospect.Nearest research is to speculate that hematopoietin therapy is different in a variety of diseases, disorder and hematology
Effectiveness in reason condition provides the foundation, these diseases include: in the treatment of chronic renal failure (CRF) anemia disease using rush
Erythropoietin(EPO) and in the treatment of AIDS and the cancer patient's anemia for receiving chemotherapy use hematopoietin
(Danna, RP, Rudnick, SA, Abels, RI, in: MB, Garnick write, the hematopoietin one in clinical application
International Prospect (Erythropoietin in Clinical Applications-An International
Perspective.Marcel Dekker；1990:p301-324).
The partial biological effect of hematopoietin can be by making to be used to adjust in the receptor with cell membrane surface
Section.At first, the promoting erythrocyte of cell surface combination is studied using the immature red blood cell isolated in the spleen of mouse
It is found when generating fibroin, this albumen is made of two kinds of polypeptides, and molecular weight is about 85000~100000KD
(Sawyer, et al. (1987) Proc.Natl.Acad.Sci.USA 84:3690-3694) is by more detailed narration.Promote red
The number of the binding site of erythropoietin is also computed, and about each cell membrane contains 800~1000 sites.?
The Kd level of about 300 binding sites is 90pM in these binding sites, and the binding force of remaining binding site compares
It is weak, about 570pM.Some researches show that the spleen red blood cells from the mouse for having infected friend virus anaemia strain to the sound of EP0
Situation is answered to find, about 400 binding sites, wherein Kd is horizontal high for 100pM, and Kd is horizontal low for 800pM.
Subsequent work is exactly two kinds of EPO Receipters by individual gene pirate recordings, this gene by gram
It is grand.For example, the DNA sequence dna of the EPO Receipter of mouse and people and the sequence of encoded peptide in WO90/08822
Through there is narration.Current model show hematopoietin be integrated to EPO Receipter result in two promote it is red thin
Born of the same parents generate the activation and dimerization of plain receptor, and this dimerization further causes the beginning of signal transduction.
The application of hematopoietin clone gene is more conducive to help to find the agonist of these important receptors and short of money
Anti-agent.The peptide that EPO Receipter can be acted in a way has been determined and has described.Especially it has been determined one
Group contains the peptide of main peptide fragment, these peptides can be combined with EPO Receipter and to irritate hematopoietin thin
The differentiation and proliferation of born of the same parents.But the EC50 for the peptide that erythrocyte proliferation can be stimulated to break up is but very low, between 20nM and 250nM, because
This these peptide has been more limited in clinical application.
Summary of the invention
For overcome the deficiencies in the prior art, that the present invention provides a kind of bioactivity is more preferable, bioavilability is higher
Erythropoietin mimetic peptide and its dimer and officinal salt and their preparation method.
Amino acid used in the present invention is other than including 20 kinds of common amino acids well-known to those skilled in the art
It further include the non-common amino acid in part, non-common amino acid structure and title are shown in Table 1
The title and structure of the non-common amino acid of table 1
The present invention provides a kind of Erythropoietin mimetic peptides, which is characterized in that the amino acid sequence of the simulating peptide
Column are as shown in SEQ ID NO:1:
SEQ ID NO:1GX1LYACHMGPITX2VCQPLRX3K；
Wherein, X1For allylglycine (D-Allyiglycin), X2For 3- (1- naphthalene)-l-Alanine (Nal), X3For
Sarcosine (Sar), 6 and 15 cysteines (C) form intramolecular disulfide bond, N-terminal acetylation.
The present invention also provides a kind of dimer of Erythropoietin mimetic peptide, the dimer is by SEQ ID
The D-Allyiglycine of Erythropoietin mimetic peptide shown in NO:1 polymerize to be formed by the free alkenyl of side chain, described more
The general formula of aggressiveness is as shown in following formula I；
The present invention also provides a kind of officinal salt of Erythropoietin mimetic peptide, the officinal salt is the mould
Peptidomimetic or dimer react the salt generated with acid compound or alkali compounds；
Preferably, the acid compound is selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, p-methyl benzenesulfonic acid, methylsulphur
Acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid or acetic acid；
Preferably, the alkali compounds is selected from ammonia, the hydroxide of alkali or alkaline earth metal and carbonate, carbon
Sour hydrogen salt；It is highly preferred that the alkali compounds is selected from sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate or potassium carbonate；
It is highly preferred that the officinal salt of the Erythropoietin mimetic peptide is selected from sulfate, pyrosulfate, trifluoro second
Hydrochlorate, sulphite, bisulfites, phosphate, hydrophosphate, dihydric phosphate, metaphosphate, pyrophosphate, hydrochloride,
Bromide, iodide, acetate, propionate, caprylate, acrylates, formates, isobutyrate, caproate, enanthate, third
Acetylenic acid salt, oxalates, malonate, succinate, suberate, fumarate, maleate, butine-l, 4- diacid salt, oneself
Alkynes -1,6- diacid salt, benzoate, chloro benzoate, methyl benzoic acid salt, dinitro-benzoate, hydroxy benzoate, first
P-methoxybenzoic acid salt, phenylacetate, phenpropionate, benzenebutanoic acid salt, citrate, lactate, gamma hydroxybutyrate, Glycolic acid
Salt, tartrate, mesylate, propane sulfonic acid salt, naphthalene-l- sulfonate, naphthalene-2-sulfonic acid salt or mandelate, it is therefore preferable to trifluoro
Acetate.
Erythropoietin mimetic peptide provided by the invention or its dimer pass through the usable acid or alkali of well-known technique
Property compound react into salt, generally use formation acid-addition salts acid are as follows: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphorus
Acid, p-methyl benzenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid.
Preferably, the officinal salt of the Erythropoietin mimetic peptide and its dimer is selected from sulfate, pyrosulfuric acid
Salt, trifluoroacetate, sulphite, bisulfites, phosphate, hydrophosphate, dihydric phosphate, metaphosphate, pyrophosphoric acid
Salt, hydrochloride, bromide, iodide, acetate, propionate, caprylate, acrylates, formates, isobutyrate, caproate,
Enanthate, propiolate, oxalates, malonate, succinate, suberate, fumarate, maleate, butine-l, 4-
Diacid salt, hexin -1,6- diacid salt, benzoate, chloro benzoate, methyl benzoic acid salt, dinitro-benzoate, hydroxyl
Benzoate, methoxy benzoic acid salt, phenylacetate, phenpropionate, benzenebutanoic acid salt, citrate, lactate, γ-hydroxyl fourth
Hydrochlorate, glycol hydrochlorate, tartrate, mesylate, propane sulfonic acid salt, naphthalene-l- sulfonate, naphthalene-2-sulfonic acid salt and mandelate etc.,
It is preferred that trifluoroacetate.
Alkali compounds can also be with Erythropoietin mimetic peptide of the invention or its dimer at salt, these alkalinity
Compound includes ammonia, hydroxide and carbonate, the bicarbonate of alkali or alkaline earth metal, typically have sodium hydroxide,
Potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate etc..
The present invention also provides a kind of Erythropoietin mimetic peptide or its dimer in preparation for treating to lack
Application in the drug for the disease that erythropoietin(EPO) or red cell population lack or defect is characterized.
Preferably, it is described by lack erythropoietin(EPO) or red cell population lack or defect characterized by disease be selected from latter stage
Renal failure or dialysis；AIDS related anemia, autoimmune disease or malignant tumour；Cystic fibrosis；Early stage is early
Maturity anaemia；Anaemia relevant to chronic inflammatory disease；Spinal cord injury；Acute bleeding；It aging and is generated with abnormal erythrocyte
Tumor disease.
The present invention also provides a kind of drug containing above-mentioned Erythropoietin mimetic peptide or dimer and its pharmaceutical salts
Composition, described pharmaceutical composition be used to treat by lack erythropoietin(EPO) or red cell population lack or defect characterized by disease
Disease.
Pharmaceutical composition of the invention includes one or more pharmaceutically acceptable auxiliary materials, and the auxiliary material is selected from water solubility
One of filler, pH adjusting agent, stabilizer, water for injection and osmotic pressure regulator are a variety of.
Water-soluble filler of the present invention is selected from the following one or more: mannitol, low molecular dextran, mountain
Pears alcohol, polyethylene glycol, glucose, lactose and galactolipin.
The pH adjusting agent is selected from the following one or more: citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid etc. are non-volatile
The acid and potassium hydroxide, sodium hydroxide or ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, saleratus of property
Or the physiologically acceptable organic or inorganic acid such as bicarbonate ammonium salt, alkali or salt etc..
The stabilizer is selected from the following one or more: EDTA-Na2, sodium thiosulfate, sodium pyrosulfite, sulfurous acid
Sodium, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, Macrogol 6000, Macrogol 4000, dodecane
Base sodium sulphate or trishydroxymethylaminomethane etc..Preferably sodium pyrosulfite, dipotassium hydrogen phosphate, arginine, Macrogol 6000
And trishydroxymethylaminomethane.
The osmotic pressure regulator is sodium chloride and/or potassium chloride.
Pharmaceutical composition of the invention can be by muscle, intravenous, subcutaneous injection by way of being administered, preferred dosage form
For freeze-dried powder or solution injection.
The preparation method of freeze drying injection: the Erythropoietin mimetic peptide dimerization liquid solution or described is taken
Erythropoietin mimetic peptide dimer officinal salt it is appropriate, be added water-soluble filler, stabilizer, osmotic pressure adjust
Agent etc., addition water for injection is appropriate, adjusts pH value and makes it dissolve to 4-8, be diluted with water to debita spissitudo, 0.1-0.5% is added
Active carbon stirs 10-20 minutes at 0-10 DEG C, and decarburization, using filtering with microporous membrane degerming, filtrate is dispensed, and freezing is dry
It is dry, white loose block is made, seals to obtain the final product.
The preparation method of injection: the Erythropoietin mimetic peptide or its dimer or its officinal salt solution are taken
Or appropriate freeze-dried powder, water-soluble filler, stabilizer, osmotic pressure regulator etc. is added, addition water for injection is appropriate, adjusts pH value
It is made it dissolve to 4-8, is diluted with water to debita spissitudo, 0.1-0.5% active carbon is added, is stirred 10-20 minutes at 0-10 DEG C,
Decarburization, using filtering with microporous membrane degerming, filtrate is dispensed, and is sealed to obtain the final product；Preferably, each specification contains the rush respectively
Erythropoetin mimetic peptides dimer 5 μ g, 100 μ g and 1mg.
Pharmaceutical composition of the invention can be by muscle, intravenous, subcutaneous injection by way of being administered, preferred dosage form
For freeze-dried powder or solution injection.Although dosage changes according to treatment object, administration mode, symptom and other factors, the present invention
Composition be effective in comparatively wide dosage range.In adult treatment, dosage range is in 50 μ g/ people -10mg/
People, once a day or per several days single administrations.Actual dose should determine by doctor according to related situation, these situation packets
Include the individual reaction of the physical condition, administration route, age, weight, patient of patient to drug, the serious journey of patient symptom
Degree etc., therefore above-mentioned dosage range is not to limit the scope of the invention in any way.
Compared with prior art, the Erythropoietin mimetic peptide and its dimer and officinal salt energy that this patent is related to
The raising of enough obvious stimulation mouse peripheral blood reticulocyte counts illustrates that they stimulate RBC acceptor garland rate, while can also be big
It is big to extend the half-life period of drug in vivo.Erythropoietin mimetic peptide and hematopoietin dimer of the invention and
Its officinal salt does not influence mature red blood cell, hematocrit, content of hemoglobin significantly, Number of Peripheral Blood Leucocyte
Counting also has not significant impact.
Specific embodiment
The present invention is further described in detail With reference to embodiment, the embodiment provided only for
The present invention is illustrated, the range being not intended to be limiting of the invention.
Below in an example, the various processes and method being not described in detail are conventional methods as known in the art.
Embodiment 1The synthesis of Erythropoietin mimetic peptide dimer
Erythropoietin mimetic peptide of the invention is prepared by Fmoc solid-phase peptide synthesis, is used
The CS 336X type instrument of CSBio company production carries out the synthesis of polypeptide of the invention.Synthetic method according to manufacturer instrument
Specification carries out.Fmoc solid-phase peptide synthesis as described herein refers to using fluoropolymer resin as solid phase reaction matrix, incites somebody to action
The fmoc-protected amino acid of aminoterminal is successively condensed in the presence of coupling reagent, thus the synthetic method of synthesis polypeptide.It has
Body method is referring to Fmoc solid phase peptide synthesis:a practical approach, and 2000, Oxford
University Press.And disulfide bond in monomer, such as 20%DMSO oxidizing process and iodine oxidation are formed by method for oxidation
Method.Polypeptide obtained is purified using HPLC C18 semi-preparative column, mobile phase is acetonitrile.It is obtained through desalination and freeze-drying
Polypeptide freeze-dried powder.
The alkenyl condensation reaction for being passed through allylglycine [(xb) G] side chain using monomer prepared by the above method, is formed
Following dimers.General formula is as follows
Wherein, two cysteines of each monomeric peptide form disulfide bond.
Embodiment 2Effect of the Erythropoietin mimetic peptide dimer to mouse
Using rat evaluation and to compare Erythropoietin mimetic peptide and erythropoietin protein red to mouse thin
The influence that born of the same parents generate.
Wherein, EPO drug is purchased from Shenyang Sansheng Pharmaceutical Co., Ltd.；
Kunming mouse is purchased from Chinese Academy of Sciences Shanghai Experimental Animal Center, and 25~30g of weight is female mice, in test
Groups of animals number: 10, it is divided into 3 groups.
Wherein, experimental mice skin injection Erythropoietin mimetic peptide dimer, the injection of positive controls mouse promote
Erythropoietin protein, blank control group mouse inject PBS buffer solution, and dosage is 4.5mg/kg, continuous three days, is then located
Dead mouse takes whole blood progress peripheral blood cells and reticulocyte count, blood count to be counted with full-automatic blood counting instrument.
The results are shown in Table 2, finds Erythropoietin mimetic peptide dimer and the equal energy of erythropoietin protein
The raising of obvious stimulation mouse peripheral blood reticulocyte count illustrates that they irritate RBC acceptor garland rate (being shown in Table 2).
Table 2, Erythropoietin mimetic peptide dimer influence Mouse Reticulocytes and generate
| Title | Dosage | Granulophilocyte number |
| Blank | PBS buffer solution | 113.65±2.75 |
| Experimental group | 4.5mg/kg | 758.85±4.65 |
| Positive controls | 4.5mg/kg | 675.9±3.47 |
Embodiment 3: effect of the Erythropoietin mimetic peptide dimer to macaque
EPO drug used in the present invention is purchased from Shenyang Sansheng Pharmaceutical Co., Ltd..
Using macaque for evaluating influence of the Erythropoietin mimetic peptide dimer to RBC acceptor garland rate, macaque, body
5.5~8.5kg is weighed, male and female are unlimited, are purchased from Hainan Experimental Animal Center.Macaque is grouped according to basic hemoglobin, is divided into two groups,
Every group three.Experimental group uses simulation peptide dimer of the invention, and intravenous injection is primary weekly, each 4.5mg/kg；Another group
EPO is used for positive controls, three times/week, 240 μ g/kg, successive administration five weeks, surveys weekly 1 hematological indices every time.
The results are shown in Table 3, and simulating peptide single intravenous injection causes Rhesus macaque peripheral blood content of hemoglobin to rise (32%),
Hematocrit increases, and illustrates that simulation peptide dimer of the invention can stimulate hemoglobin to generate.Promote in positive controls red
Erythropoietin can also increase Rhesus macaque peripheral blood content of hemoglobin (31%), hemocytes increasing hematocrit, but need to infuse weekly
It penetrates three times, it is clear that simulating peptide of the invention obviously has more preferably long-term effect with respect to EPO.
The effect of table 3, Erythropoietin mimetic peptide dimer to macaque
Effect of the 4 Erythropoietin mimetic peptide dimer of embodiment to rat
It is evaluated using rat and compares Erythropoietin mimetic peptide dimer and erythropoietin protein to big
The influence that Mice red cell generates.
Wherein, EPO drug is purchased from Shenyang Sansheng Pharmaceutical Co., Ltd.；
SD rat is purchased from Chinese Academy of Sciences Shanghai Experimental Animal Center, and 25~30g of weight is female rats, each group in test
Number of animals: 10, it is divided into 3 groups.
Wherein, experimental group rat skin injection Erythropoietin mimetic peptide dimer, the injection of positive controls rat promote
Erythropoietin protein, another group of rat are blank control, inject PBS buffer solution, the injection dosage of each group is 4.5mg/
Kg after single-dose, takes blood to carry out peripheral blood cells and reticulocyte count, the full-automatic blood of blood count for continuous three days
Ball count instrument meter number, and the long-term effect of Erythropoietin mimetic peptide is calculated, as a result, it has been found that Erythropoietin mimetic peptide
Dimer still has preferably stimulation hemopoiesis after single-dose in 2 weeks, it is clear that simulating peptide ratio of the invention
Erythropoietin protein (EPO) has more preferably long-term effect.The results are shown in Table 4.
Effect of the 4 Erythropoietin mimetic peptide dimer of table to rat
Embodiment 5The effect of Erythropoietin mimetic peptide dimer acid salt and basic salt to mouse
Sequence used in the present embodiment are as follows: simulation peptide dimer simulates peptide dimer methoxy benzoic acid salt, simulating peptide
Dimer trifluoroacetate.
Using rat evaluation and compare Erythropoietin mimetic peptide dimer and its methoxy benzoic acid salt, trifluoro second
The influence that hydrochlorate generates mouse red blood cell.
Wherein, positive control are as follows: EPO drug (recombinant human erythropoietin injection, lot number: 201405YC12) purchase
From Shenyang Sansheng Pharmaceutical Co., Ltd.；
Kunming mouse is purchased from Chinese Academy of Sciences Shanghai Experimental Animal Center, and 25~30g of weight is male mice, in test
Groups of animals number: 10, it is divided into 5 groups.
Wherein, Erythropoietin mimetic peptide dimer is subcutaneously injected in 5 groups of mouse respectively, simulates peptide dimer methoxyl group
Benzoate simulates peptide dimer trifluoroacetate, and EPO drug, PBS buffer solution (25mM) dosage is 4.5mg/kg, single
After administration, blood is taken within continuous three days to carry out peripheral blood cells and reticulocyte count, the full-automatic blood count of blood count
Instrument meter number.
The results are shown in Table 5, and the acid salt and basic salt of Erythropoietin mimetic peptide dimer of the invention can
It obtains and the comparable effect of dimer.
The acid salt of 5 Erythropoietin mimetic peptide of table and the effect of basic salt
Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right
It is required that range comprising the equivalent replacement of each factor.
Claims (26)
1. a kind of Erythropoietin mimetic peptide, which is characterized in that the amino acid sequence of the monomeric peptide of the simulating peptide such as SEQ
Shown in ID NO:1:
GX1LYACHMGPITX2VCQPLRX3K；
Wherein, X1For allylglycine (D-Allyiglycin), X2For 3- (1- naphthalene)-l-Alanine (Nal), X3For flesh ammonia
Sour (Sar), 6 and 15 cysteines (C) form intramolecular disulfide bond, N-terminal acetylation.
2. a kind of dimer of Erythropoietin mimetic peptide, which is characterized in that the dimer is as described in claim 1
The allylglycine of Erythropoietin mimetic peptide is polymerize and to be formed by the side chain alkenyl that dissociates, the general formula of the dimer
As shown in following formula I；
Wherein, X1For allylglycine (D-Allyiglycin), X2For 3- (1- naphthalene)-l-Alanine (Nal), X3For flesh ammonia
Sour (Sar).
3. a kind of officinal salt of Erythropoietin mimetic peptide, which is characterized in that the officinal salt is such as claim 1
The simulating peptide or dimer as claimed in claim 2 react the salt generated with acid compound or alkali compounds.
4. officinal salt as claimed in claim 3, which is characterized in that the acid compound is selected from hydrochloric acid, hydrobromic acid, hydrogen iodine
Acid, sulfuric acid, phosphoric acid, p-methyl benzenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid or
Acetic acid.
5. officinal salt as claimed in claim 3, which is characterized in that the alkali compounds is selected from ammonia, alkali metal or alkaline earth
Hydroxide and carbonate, the bicarbonate of metal.
6. officinal salt as claimed in claim 5, which is characterized in that the alkali compounds is selected from sodium hydroxide, hydroxide
Potassium, ammonium hydroxide, sodium carbonate or potassium carbonate.
7. officinal salt as claimed in claim 5, which is characterized in that the officinal salt of the Erythropoietin mimetic peptide
Selected from sulfate, pyrosulfate, trifluoroacetate, sulphite, bisulfites, phosphate, hydrophosphate, biphosphate
Salt, metaphosphate, pyrophosphate, hydrochloride, bromide, iodide, acetate, propionate, caprylate, acrylates, formic acid
Salt, isobutyrate, caproate, enanthate, propiolate, oxalates, malonate, succinate, suberate, fumaric acid
Salt, maleate, butine-l, 4- diacid salt, hexin -1,6- diacid salt, benzoate, chloro benzoate, methyl benzoic acid salt,
Dinitro-benzoate, hydroxy benzoate, methoxy benzoic acid salt, phenylacetate, phenpropionate, benzenebutanoic acid salt, citric acid
Salt, lactate, gamma hydroxybutyrate, glycol hydrochlorate, tartrate, mesylate, propane sulfonic acid salt, naphthalene-l- sulfonate, naphthalene -2-
Sulfonate or mandelate.
8. officinal salt as claimed in claim 7, which is characterized in that the officinal salt of the Erythropoietin mimetic peptide
For trifluoroacetate.
9. the preparation method of dimer as claimed in claim 2, which is characterized in that the method includes the steps:
1) amino acid is selected according to SEQ ID NO:1, passes through Fmoc solid phase polypeptide synthesis composition sequence such as SEQ ID NO:1 institute
The simulating peptide shown, and form intramolecular disulfide bond；
2) alkenyl to dissociate on the allylglycine side chain of the simulating peptide is subjected to intermolecular polymerization, obtains dimer.
10. preparation method as claimed in claim 9, which is characterized in that in step 1), make the simulation by method for oxidation
Peptide forms intramolecular disulfide bond.
11. preparation method as claimed in claim 10, which is characterized in that in step 1), the method for oxidation is 20%
DMSO oxidizing process or iodine oxidation method.
12. preparation method as claimed in claim 9, which is characterized in that the method also includes following steps:
Dimer purifying, desalination and freeze-drying are obtained into the freeze-dried powder of dimer.
13. preparation method according to claim 12, which is characterized in that the purifying is by preparing in HPLC C18 half
In column, realized using acetonitrile as mobile phase.
14. simulating peptide as described in claim 1 or any one of dimer as claimed in claim 2 or claim 3-8 institute
The officinal salt stated prepare for treat by lack erythropoietin(EPO) or red cell population lack or defect characterized by disease
Drug in application.
15. application as claimed in claim 14, which is characterized in that described to be lacked with lacking erythropoietin(EPO) or red cell population
Or the disease that defect is characterized is selected from latter stage renal failure or dialysis；AIDS related anemia, autoimmune disease, or dislike
Property tumour；Cystic fibrosis；Early stage prematureness anaemia；Anaemia relevant to chronic inflammatory disease；Spinal cord injury；Acute mistake
Blood；Aging and the tumor disease generated with abnormal erythrocyte.
16. one kind is containing simulating peptide as described in claim 1 or dimer as claimed in claim 2 or such as claim 3-8
Any one of described in officinal salt pharmaceutical composition.
17. pharmaceutical composition as claimed in claim 16, which is characterized in that described pharmaceutical composition includes one or more medicines
Acceptable auxiliary material on, the auxiliary material are selected from water-soluble filler, pH adjusting agent, stabilizer, water for injection and osmotic pressure tune
Save one of agent or a variety of.
18. pharmaceutical composition as claimed in claim 17, which is characterized in that the water-soluble filler auxiliary material is selected from sweet dew
One of alcohol, low molecular dextran, sorbierite, polyethylene glycol, glucose, lactose and galactolipin are a variety of.
19. pharmaceutical composition as claimed in claim 17, which is characterized in that the pH adjusting agent is selected from non-volatile sour Chinese holly
Rafter acid, phosphoric acid, lactic acid, tartaric acid and hydrochloric acid and potassium hydroxide, sodium hydroxide or ammonium hydroxide, sodium carbonate, potassium carbonate, carbon
One of sour ammonium, sodium bicarbonate, saleratus and ammonium hydrogen carbonate are a variety of.
20. pharmaceutical composition as claimed in claim 17, which is characterized in that the stabilizer is selected from EDTA-Na2, thiosulfuric acid
Sodium, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, Macrogol 6000,
Macrogol 4000, lauryl sodium sulfate and trishydroxymethylaminomethane.
21. pharmaceutical composition as claimed in claim 20, which is characterized in that the stabilizer is selected from sodium pyrosulfite, phosphoric acid
One of hydrogen dipotassium, arginine, Macrogol 6000 and trishydroxymethylaminomethane are a variety of.
22. pharmaceutical composition as claimed in claim 17, which is characterized in that the stabilizer is selected from the osmotic pressure regulator
For sodium chloride and/or potassium chloride.
23. pharmaceutical composition as claimed in claim 16, which is characterized in that described pharmaceutical composition is freeze drying injection
Or solution injection.
24. pharmaceutical composition as claimed in claim 23, which is characterized in that the freeze drying injection be by including under
State the method preparation of step:
It takes described in simulating peptide or any one of dimer as claimed in claim 2 or claim 3-8 described in claim 1
The solution of officinal salt is appropriate, and water-soluble filler, stabilizer, osmotic pressure regulator etc. is added, and addition water for injection is appropriate, adjusts
Section pH value makes it dissolve to 4-8, is diluted with water to debita spissitudo, and 0.1-0.5% active carbon is added, stirs 10- at 0-10 DEG C
20 minutes, decarburization, using filtering with microporous membrane degerming, filtrate was dispensed, freeze-drying, and white loose block, envelope is made
Mouth to obtain the final product.
25. pharmaceutical composition as claimed in claim 23, which is characterized in that the solution injection is by including following steps
Rapid method preparation:
It takes described in simulating peptide or any one of dimer as claimed in claim 2 or claim 3-8 described in claim 1
Water-soluble filler, stabilization is added in the solution or appropriate freeze-dried powder of the officinal salt of Erythropoietin mimetic peptide dimer
Agent, osmotic pressure regulator etc., addition water for injection is appropriate, adjusts pH value and makes it dissolve to 4-8, is diluted with water to debita spissitudo,
0.1-0.5% active carbon is added, is stirred 10-20 minutes at 0-10 DEG C, decarburization, using filtering with microporous membrane degerming, filtrate into
Row packing, is sealed to obtain the final product.
26. pharmaceutical composition as claimed in claim 25, which is characterized in that each specification of the solution injection contains respectively
There are Erythropoietin mimetic peptide dimer 5 the μ g, 100 μ g and 1mg.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101553242A (en) * | 2005-06-03 | 2009-10-07 | 阿费麦克斯公司 | Erythropoietin receptor peptide formulations and uses |
| CN103450348A (en) * | 2012-05-29 | 2013-12-18 | 中国人民解放军军事医学科学院毒物药物研究所 | Mimetic peptide of erythropoietin, preparation method and applications thereof |
| CN104231039A (en) * | 2013-06-08 | 2014-12-24 | 江苏豪森药业股份有限公司 | Functional micromolecules for synthesizing erythropoietin mimetic peptide derivatives, and preparation method thereof |
-
2015
- 2015-08-26 CN CN201510530632.3A patent/CN105153293B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101553242A (en) * | 2005-06-03 | 2009-10-07 | 阿费麦克斯公司 | Erythropoietin receptor peptide formulations and uses |
| CN103450348A (en) * | 2012-05-29 | 2013-12-18 | 中国人民解放军军事医学科学院毒物药物研究所 | Mimetic peptide of erythropoietin, preparation method and applications thereof |
| CN104231039A (en) * | 2013-06-08 | 2014-12-24 | 江苏豪森药业股份有限公司 | Functional micromolecules for synthesizing erythropoietin mimetic peptide derivatives, and preparation method thereof |
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