CN105837681B - A kind of Erythropoietin mimetic peptide derivative, preparation method and use - Google Patents
A kind of Erythropoietin mimetic peptide derivative, preparation method and use Download PDFInfo
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Abstract
The present invention relates to a kind of Erythropoietin mimetic peptide derivative and its officinal salt with long-acting promoting erythrocyte systematic function.The present invention also provides the preparation method of above-mentioned Erythropoietin mimetic peptide derivative and its be used to prepare treatment by lack red blood cell lack erythropoietin(EPO) or red cell population lack or defect characterized by disease drug in purposes.Wherein, Erythropoietin mimetic peptide derivative provided by the invention monomeric peptide as shown in SEQ ID NO:1 or the polymer formed by lysine (K) side chain free amine group of monomeric peptide end；Wherein, two cysteines of each monomeric peptide form disulfide bond in the polymer, and the number of monomeric peptide is 2-10 in the polymer.
Description
Technical field
The invention belongs to fields of biomedicine, are related to a kind of Erythropoietin mimetic peptide derivative, specifically, this
Invention be related to one kind can combine and activate with EPO Receipter EPO Receipter or can rise rush it is red
Erythropoietin mimetic peptide derivative of erythropoietin agonism and preparation method thereof, the invention further relates to the moulds
Peptidomimetic derivative preparation treatment characterized by lacking erythropoietin(EPO) or by red cell population lack or defect characterized by disease
Purposes in the drug of disease.
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 entangle to a certain extent
The just anaemia as caused by malignant tumour, chemotherapy and rheumatoid arthritis.Since hematopoietin is mainly by renal tubule
Chrotoplast generates, and anaemia caused by renal illness is the preferred idicatio of hematopoietin；Hematopoietin is corrected
The curative effect of renal anemia almost 100%, but renal function can not be improved.The treatment safety of hematopoietin, effectively, be suitble to
Long-term treatment is also avoided that blood source anxiety.Global biotechnology medicines in 2006 in the market, the weight of hematopoietin class
Group 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, 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 derivative and its officinal salt and their preparation method.
On the one hand, the object of the present invention is to provide a kind of hematopoietin with long-acting promoting erythrocyte systematic function
Simulate peptide derivant and its officinal salt.
In another aspect, the present invention also provides the preparation method of Erythropoietin mimetic peptide derivative of the invention with
And its be used to prepare treat characterized by lacking erythropoietin(EPO) or by red cell population lack or defect characterized by disease
Drug in purposes.
Another aspect of the present invention additionally provides a kind of pharmaceutical composition, and described pharmaceutical composition includes that rush of the invention is red
Erythropoietin simulates peptide derivant.
Preferably, pharmaceutical composition of the invention is injection；It is highly preferred that pharmaceutical composition of the invention is freeze-dried powder
Needle or solution injection.
In a preferred embodiment, pharmaceutical composition of the invention also includes pharmaceutically acceptable adjunct ingredient.
The present invention provides a kind of Erythropoietin mimetic peptide derivative in vivo bioactivity and its can medicine
With salt, wherein the simulation peptide derivant is monomeric peptide or its polymer shown in SEQ ID NO:1；
SEQ ID NO:1:GGLYACHMGPIX1VCQPLRX2K；
Wherein, in the monomeric peptide, two cysteines (C) form intramolecular disulfide bond, X1Indicate Nal amino acid, X2Table
Show Sar amino acid；
The polymer is the polymer formed by the lysine (K) in SEQ ID NO:1 by side chain free amine group；
Wherein, two cysteines of each monomeric peptide form disulfide bond, monomer in the polymer in the polymer
The number of peptide is 2-10, i.e., the number of monomeric peptide is 2,3,4,5,6,7,8,9 in the described polymer
Or 10.
Wherein, the structural formula of the dimer are as follows:
The structural formula of the tripolymer are as follows:
When monomeric peptide number is 4-10 in the polymer, general structure is shown in formula I:
Wherein, integer of the n between 4-10；
Formulas I
The present invention also provides the preparation method of the Erythropoietin mimetic peptide derivative or its officinal salt, institutes
State method the following steps are included:
When the Erythropoietin mimetic peptide derivative is monomeric peptide, amino acid is selected according to SEQ ID NO:1,
Use Fmoc solid phase polypeptide synthesis synthon peptide；
When the Erythropoietin mimetic peptide derivative is polymer, amino acid is selected according to SEQ ID NO:1,
It, will then by the amino coupled dissociated on monomeric peptide lysine side-chain using Fmoc solid phase polypeptide synthesis synthon peptide
Monomeric peptide is polymerized to polymer；
Optionally, by obtained monomeric peptide or polymer at salt.
Preferably, the polymer is realized by method comprising the following steps:
Firstly, lysine is protected using DDE, it is dedicated using DDE after Fmoc solid phase polypeptide synthesis synthon peptide
Deprotection agent sloughs the DDE group of lysine, and the conjunction of Article 2 polypeptide is then carried out using the amino to dissociate on lysine side-chain
At, and so on synthesis polymer.
Wherein, Fmoc solid-phase peptide synthesis of the present invention refers to using fluoropolymer resin as solid phase reaction base
The fmoc-protected amino acid of aminoterminal is successively condensed by matter in the presence of coupling reagent, thus the synthesis side of synthon peptide
Method.
Preferably, the preparation method further includes purifying polymer obtained, obtains polypeptide through desalination and freeze-drying
The step of freeze-dried powder；Preferably, the purifying is carried out using HPLC C18 semi-preparative column, and mobile phase is acetonitrile.
Acid or alkalization can be used by well-known technique in Erythropoietin mimetic peptide derivative provided by the invention
It closes object and reacts into salt, the acid of the formation acid-addition salts generallyd use are as follows: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, right
Toluenesulfonic 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 derivative is selected from sulfate, pyrosulfate, three
Fluoroacetate, sulphite, bisulfites, phosphate, hydrophosphate, dihydric phosphate, metaphosphate, pyrophosphate, salt
Hydrochlorate, bromide, iodide, acetate, propionate, caprylate, acrylates, formates, isobutyrate, caproate, enanthic acid
Salt, 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, hydroxybenzoic acid
It is salt, methoxy benzoic acid salt, phenylacetate, phenpropionate, benzenebutanoic acid salt, citrate, lactate, gamma hydroxybutyrate, sweet
Alcohol hydrochlorate, tartrate, mesylate, propane sulfonic acid salt, how-l- sulfonate, how -2- sulfonate and mandelate etc., preferably three
Fluoroacetate.
Alkali compounds can also be with Erythropoietin mimetic peptide derivative at salt, these alkali compounds include
Ammonium, 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 the Erythropoietin mimetic peptide derivatives lack in preparation for treating it is red thin
Born of the same parents generate element be characterized or by red cell population lack or defect characterized by disease drug in application.
Preferably, it is described characterized by lacking erythropoietin(EPO) or by red cell population lack or defect characterized by disease
Selected from latter stage renal failure or dialysis；AIDS related anemia, autoimmune disease or malignant tumour；Cystic fibrosis
Property；Early stage prematureness anaemia；Anaemia relevant to chronic inflammatory disease；Spinal cord injury；Acute bleeding；Aging and with abnormal red
The tumor disease that cell generates.
The medicine group containing above-mentioned Erythropoietin mimetic peptide derivative and its pharmaceutical salts that the present invention also provides a kind of
Object is closed, described pharmaceutical composition lacks for treatment characterized by lacking erythropoietin(EPO) or with red cell population or defect is spy
The disease of sign.
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 auxiliary material of the present invention is selected from the following one or more: mannitol, low molecule dextrose
Glycosides, sorbierite, 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 salts, sodium bicarbonate, bicarbonate of property
The physiologically acceptable organic or inorganic acid such as potassium or bicarbonate ammonium salt, alkali or salt etc..
The stabilizer is selected from the following one or more: EDTA-2Na, 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: taking Erythropoietin mimetic peptide derivative solution appropriate, and water is added
Soluble filler, stabilizer, osmotic pressure regulator etc., addition water for injection is appropriate, adjusts pH value and makes it dissolve to 4-8, adds water
It is diluted to debita spissitudo, 0.1-0.5% active carbon is added, is stirred 10-20 minutes at 0-10 DEG C, decarburization, using miillpore filter
Filtration sterilization, filtrate are dispensed, and using freeze-drying, white loose block are made, seals to obtain the final product, each specification difference
Erythropoietin mimetic peptide derivative 5 the μ g, 100 μ g and 1mg contained.
The preparation method of injection: taking Erythropoietin mimetic peptide derivative solution or appropriate freeze-dried powder, and water is added
Soluble filler, stabilizer, osmotic pressure regulator etc., addition water for injection is appropriate, adjusts pH value and makes it dissolve to 4-8, adds water
It is diluted to debita spissitudo, 0.1-0.5% active carbon is added, is stirred 10-20 minutes at 0-10 DEG C, decarburization, using miillpore filter
Filtration sterilization, filtrate are dispensed, and are sealed to obtain the final product, 5 μ of Erythropoietin mimetic peptide derivative that each specification contains respectively
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 officinal salt that this patent is related to can be pierced obviously
The raising for swashing mouse peripheral blood reticulocyte count illustrates that they stimulate RBC acceptor garland rate, while can also greatly prolong medicine
The half-life period of object in vivo.Erythropoietin mimetic peptide derivative and erythropoietin protein are to the red thin of maturation
Born of the same parents, hematocrit, content of hemoglobin do not influence significantly, and Number of Peripheral Blood Leucocyte counts liquid and has not significant impact.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, 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.
The synthesis of 1 Erythropoietin mimetic peptide derivative of embodiment
Below in conjunction with specific embodiment, invention is further described in detail.
Erythropoietin mimetic peptide derivative of the invention is polymer, and wherein the preparation of monomer uses Fmoc solid phase
Polypeptide synthesis method, the CS 336X type instrument produced using CSBio company carry out the synthesis of polypeptide of the invention.The side of synthesis
Method is carried out according to the instrument specification of manufacturer.Fmoc solid-phase peptide synthesis as described herein refers to be made with fluoropolymer resin
For solid phase reaction matrix, the fmoc-protected amino acid of aminoterminal is successively condensed in the presence of coupling reagent, so that synthesis is more
The synthetic method of peptide.Its specific method is referring to Fmoc solid phase peptide synthesis:a practical
approach,2000,Oxford University Press.And disulfide bond in monomer, such as 20% are formed by method for oxidation
DMSO oxidizing process and iodine oxidation method.Polypeptide obtained is purified using HPLC C18 semi-preparative column, mobile phase is acetonitrile.
Polypeptide freeze-dried powder is obtained through desalination and freeze-drying.
The monomer prepared using the above method is reacted by the amido bond of K (lysine) side chain, forms following polymers.Tool
Body method are as follows: when preparing polymer, lysine is protected using DDE first, using Fmoc solid phase polypeptide synthesis synthon peptide,
The DDE group of lysine is sloughed using DDE special support protective agent, then carries out second using the amino to dissociate on lysine side-chain
The synthesis of polypeptide, and so on carry out the preparation of polymer；Wherein, in the polymer each monomeric peptide two and half Guangs
Propylhomoserin forms disulfide bond, and monomeric peptide number is 2-10, and X1 refers to Nal amino acid, and X2 refers to Sar amino acid.
Using the above method, inventor is prepared for ten kinds of Erythropoietin mimetic peptide derivatives, wherein the monomer
The number of peptide is respectively 1,2,3,4,5,6,7,8,9 and 10；
Its structural formula is as follows:
Erythropoietin mimetic peptide derivative 1, monomeric peptide number are 1:
GGLYACHMGPINalVCQPLRSarK
Erythropoietin mimetic peptide derivative 2, monomeric peptide number are 2:
Erythropoietin mimetic peptide derivative 3, monomeric peptide number are 3:
Erythropoietin mimetic peptide derivative 4, monomeric peptide number are 4:
Erythropoietin mimetic peptide derivative 5, monomeric peptide number are 5:
Erythropoietin mimetic peptide derivative 6, monomeric peptide number are 6:
Erythropoietin mimetic peptide derivative 7, monomeric peptide number are 7:
Erythropoietin mimetic peptide derivative 8, monomeric peptide number are 8:
Erythropoietin mimetic peptide derivative 9, monomeric peptide number are 9:
Erythropoietin mimetic peptide derivative 10, monomeric peptide number are 10:
The effect of 2. Erythropoietin mimetic peptide Derivatives In Mice of embodiment
Using rat evaluation and compare Erythropoietin mimetic peptide derivative and erythropoietin protein to small
The influence that Mice red cell generates.
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 12 groups.
Wherein, 10 groups of mouse skin injection Erythropoietin mimetic peptide derivative 1-10,1 group of mouse inject promoting erythrocyte
Fibroin is generated, 1 group of mouse is blank control, injects PBS buffer solution, dosage is 4.5mg/kg, continuous three days, is then put to death
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 1, finds Erythropoietin mimetic peptide derivative 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 1).
The influence that table 1, Erythropoietin mimetic peptide Derivatives In Mice granulophilocyte generate
| Title | Dosage | Granulophilocyte number |
| Blank | PBS buffer solution | 109.75±3.45 |
| Derivative 1 | 4.5mg/kg | 786.94±3.86 |
| Derivative 2 | 2.25mg/kg | 768.64±4.86 |
| Derivative 3 | 1.5mg/kg | 818.65±4.01 |
| Derivative 4 | 1.125mg/kg | 687.95±5.76 |
| Derivative 5 | 0.9mg/kg | 789.75±4.01 |
| Derivative 6 | 0.75mg/kg | 756.35±2.46 |
| Derivative 7 | 0.64mg/kg | 698.64±3.91 |
| Derivative 8 | 0.56mg/kg | 704.81±4.01 |
| Derivative 9 | 0.5mg/kg | 803.95±3.37 |
| Derivative 10 | 0.45mg/kg | 734.65±2.86 |
| EPO | 4.5mg/kg | 583.19±4.82 |
Embodiment 3: effect of the Erythropoietin mimetic peptide derivative to macaque
Using macaque for evaluating influence of the Erythropoietin mimetic peptide derivative 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.One group uses derivative 3, and intravenous injection is primary weekly, each 4.5mg/kg, and one group uses EPO, and three times/week, often
Secondary 240 μ/kg successive administration five weeks, survey weekly 1 hematological indices
As a result, it has been found that 3 single intravenous injection of derivative causes Rhesus macaque peripheral blood content of hemoglobin to rise (33%), blood is thin
Born of the same parents' hematocrit increases, and illustrates that derivative 3 stimulates hemoglobin to generate.Positive control hematopoietin equally increases Rhesus macaque peripheral
Blood content of hemoglobin (34%), hemocytes increasing hematocrit, but obviously do not have long-term effect, it needs to inject weekly three times.
Effect of the 4. Erythropoietin mimetic peptide derivative 3 of embodiment to rat
It is evaluated using rat and compares Erythropoietin mimetic peptide derivative 3 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, 1 group of rat skin injection Erythropoietin mimetic peptide derivative, 3,1 group of rat injection promoting erythrocyte generates
Fibroin, 1 group of rat are blank control, inject PBS buffer solution, and dosage is 4.5mg/kg, after single-dose, are taken within continuous three days
Blood carries out peripheral blood cells and reticulocyte count, and blood count is counted with full-automatic blood counting instrument, and calculates derivative
As a result the long-term effect of object 3 is shown in as a result, it has been found that derivative 3 still has stimulation hemopoiesis after single-dose in 2 weeks
Table 2.
Claims (18)
1. a kind of Erythropoietin mimetic peptide derivative or its officinal salt, wherein the simulation peptide derivant is SEQ
Monomeric peptide shown in ID NO:1 or its polymer；
SEQ ID NO:1:GGLYACHMGPIX1VCQPLRX2K；
Wherein, in the monomeric peptide, two cysteines (C) form intramolecular disulfide bond, X1Indicate Nal amino acid, X2It indicates
Sar amino acid；
The polymer is the polymer formed by the lysine (K) in SEQ ID NO:1 by side chain free amine group；
Wherein, two cysteines of each monomeric peptide form disulfide bond in the polymer, monomeric peptide in the polymer
Number is 2-10.
2. Erythropoietin mimetic peptide derivative according to claim 1 or its officinal salt, wherein the poly
Body is dimer, tripolymer or n aggressiveness, wherein integer of the n between 4-10；The structural formula of the dimer are as follows:
The structural formula of the tripolymer are as follows:
When monomeric peptide number is 4-10 in the polymer, general structure is shown in formula I:
Wherein, integer of the n between 4-10.
3. Erythropoietin mimetic peptide derivative according to claim 1 or its officinal salt, wherein it is described can medicine
It is that Erythropoietin mimetic peptide derivative reacts the salt generated with acid compound or alkali compounds with salt.
4. Erythropoietin mimetic peptide derivative according to claim 1 or its officinal salt, wherein the acidity
Compound is selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, p-methyl benzenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbon
Acid, succinic acid, citric acid, benzoic acid or acetic acid.
5. Erythropoietin mimetic peptide derivative according to claim 1 or its officinal salt, wherein the alkalinity
Compound is selected from ammonium, hydroxide and carbonate, the bicarbonate of alkali or alkaline earth metal.
6. Erythropoietin mimetic peptide derivative according to claim 1 or its officinal salt, wherein the alkalinity
Compound is selected from sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate or potassium carbonate.
7. Erythropoietin mimetic peptide derivative according to claim 1 or its officinal salt, wherein the rush is red
The officinal salt that erythropoietin simulates peptide derivant is selected from sulfate, pyrosulfate, trifluoroacetate, sulphite, sulfurous
Sour hydrogen salt, phosphate, hydrophosphate, dihydric phosphate, metaphosphate, pyrophosphate, hydrochloride, bromide, iodide, acetic acid
Salt, propionate, caprylate, acrylates, formates, isobutyrate, caproate, enanthate, propiolate, oxalates, the third two
Hydrochlorate, succinate, suberate, fumarate, maleate, butine-l, 4- diacid salt, hexin -1,6- diacid salt, benzene first
Hydrochlorate, chloro benzoate, methyl benzoic acid salt, dinitro-benzoate, hydroxy benzoate, methoxy benzoic acid salt, benzene second
Hydrochlorate, phenpropionate, benzenebutanoic acid salt, citrate, lactate, gamma hydroxybutyrate, glycol hydrochlorate, tartrate, methanesulfonic acid
Salt, propane sulfonic acid salt, how-l- sulfonate, how -2- sulfonate or mandelate.
8. Erythropoietin mimetic peptide derivative according to claim 1 or its officinal salt, wherein the rush is red
The officinal salt that erythropoietin simulates peptide derivant is trifluoroacetate.
9. Erythropoietin mimetic peptide derivative according to any one of claim 1 to 8 or its officinal salt
Preparation method the described method comprises the following steps:
When the Erythropoietin mimetic peptide derivative is monomeric peptide, amino acid is selected according to SEQ ID NO:1, is used
Fmoc solid phase polypeptide synthesis synthon peptide；
When the Erythropoietin mimetic peptide derivative is polymer, amino acid is selected according to SEQ ID NO:1, is used
Fmoc solid phase polypeptide synthesis synthon peptide, then by the amino coupled dissociated on monomeric peptide lysine side-chain, by monomer
Peptide is polymerized to polymer；
Optionally, by obtained monomeric peptide or polymer at salt.
10. according to the method described in claim 9, wherein, the polymer is realized by method comprising the following steps:
Firstly, lysine is protected using DDE, after Fmoc solid phase polypeptide synthesis synthon peptide, using the dedicated remove-insurance of DDE
The DDE group of lysine is sloughed in shield agent, and the synthesis of Article 2 polypeptide is then carried out using the amino to dissociate on lysine side-chain, and
And so on synthesis polymer.
11. method according to claim 9 or 10, wherein the preparation method further includes purifying polymer obtained,
The step of obtaining polypeptide freeze-dried powder through desalination and freeze-drying.
12. according to the method for claim 11, wherein the purifying is carried out using HPLC C18 semi-preparative column, mobile phase
For acetonitrile.
13. as Erythropoietin mimetic peptide derivative described in any item of the claim 1 to 8 or its officinal salt are being made
Be ready for use on treat characterized by lacking erythropoietin(EPO) or by red cell population lack or defect characterized by disease drug in
Application.
14. application as claimed in claim 13, wherein described to be lacked characterized by lacking erythropoietin(EPO) or with red cell population
Less or the disease that is characterized of defect is selected from latter stage renal failure or dialysis；AIDS related anemia, autoimmune disease, or
Malignant 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.
15. one kind contains such as Erythropoietin mimetic peptide derivative described in any item of the claim 1 to 8 or it can medicine
With the pharmaceutical composition of salt.
16. pharmaceutical composition as claimed in claim 15, wherein described pharmaceutical composition pharmaceutically may be used comprising one or more
The auxiliary material of receiving, the auxiliary material is in water-soluble filler, pH adjusting agent, stabilizer, water for injection and osmotic pressure regulator
It is one or more.
17. pharmaceutical composition as claimed in claim 16, wherein the water-soluble filler auxiliary material is one kind selected from the following
It is or a variety of: mannitol, low molecular dextran, sorbierite, 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, potassium hydroxide, hydrogen
Sodium oxide molybdena or ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate salts, sodium bicarbonate, saleratus and bicarbonate ammonium salt；
The stabilizer is selected from the following one or more: EDTA-2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, phosphorus
Sour hydrogen dipotassium, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, Macrogol 6000, Macrogol 4000, dodecyl sulphate
Sodium and trishydroxymethylaminomethane；
The osmotic pressure regulator is sodium chloride and/or potassium chloride.
18. pharmaceutical composition as claimed in claim 17, wherein the stabilizer is selected from the following one or more: Jiao Ya
Sodium sulphate, dipotassium hydrogen phosphate, arginine, Macrogol 6000 and trishydroxymethylaminomethane.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1826131A (en) * | 2003-05-30 | 2006-08-30 | 森托科尔公司 | Formation of novel erythropoietin conjugates using transglutaminase |
| 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 |
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2015
- 2015-01-13 CN CN201510015563.2A patent/CN105837681B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1826131A (en) * | 2003-05-30 | 2006-08-30 | 森托科尔公司 | Formation of novel erythropoietin conjugates using transglutaminase |
| 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 |
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