Composition loaded with PEG-EPO (polyethylene glycol-erythropoietin) and mesenchymal stem cells, medicine and preparation method of composition

Abstract

The invention provides a pegylation modification method of erythropoietin. According to the invention, EPO is modified by adopting different modified PEGs, so that the EPO molecule with prolonged plasma half-life period is obtained, and the problems of short retention time and low activity of the EPO molecule in plasma are solved. According to the invention, the prepared mPEG-SPA-EPO is further subjected to purification and identification. The composition is further prepared by adopting the prepared modified EPO, and the application of the composition is identified. The EPO modified by PEG is prepared into the composition to be applied to diabetic rats, compared with recombinant EPO, the composition has better effects of reducing blood sugar and body weight, and mesenchymal stem cells are added into the composition, so that the blood sugar reducing effect of the rats is further enhanced. The composition prepared by the invention can be applied to preparation of medicines for reducing blood sugar and treating diabetes, has a relatively long half-life period in vivo, and has a relatively good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to a composition loaded with PEG-EPO and mesenchymal stem cells, a medicine and a preparation method thereof. Background Art

[0002] The revolution in biotechnology has also led to the discovery of a variety of therapeutic proteins and peptides. The discovery of these proteins and peptides has brought hope for the treatment of some dangerous diseases that have long been considered incurable. With the continuous advancement of biotechnology, proteins and peptides have gradually achieved large-scale production. Protein drugs have become an important part of the drugs launched on the market every year and are widely used to treat or alleviate symptoms related to many metabolic and tumor diseases. Even many protein drugs have become the first choice for the treatment of a variety of diseases.

[0003] Natural and recombinant protein and peptide drugs play an increasingly important role in the treatment of difficult diseases, but their poor stability, short half-life and strong immunogenicity greatly reduce their efficacy. Polyethylene glycol (PEG) is one of the most widely used non-ionic hydrophilic polymers. PEG modification can prolong the blood circulation time of the target drug, thereby increasing the possibility of the drug reaching the site of action before being identified as a foreign body and cleared by the body. Polyethylene glycol modification enhances the anti-enzymatic stability of the protein, significantly prolongs the blood half-life, and reduces immunogenicity.

[0004] Polyethylene glycol (PEG) is a water-soluble high molecular weight inert polymer, composed of repeated oxyethylene groups, containing two terminal hydroxyl groups, the weight of which can range from hundreds to tens of thousands, with a molecular formula of HO-CH2-CH2-(CH2-CH2-O)n-OH, and a linear or branched chain structure. In order to obtain a single-function PEG, one of the terminal hydroxyl groups of PEG is often activated to a methoxy mPEG form, i.e., CH3-(CH2-CH2-O)n-OH, and then mPEG is activated to a modifier for drug modification.

[0005] PEG is degradable in vivo, has very low antigenicity, and is non-toxic. As a hydrophilic, non-charged macromolecule, when it is covalently bound to the non-essential groups of proteins, it can act as a barrier to block the antigenic determinants on the surface of protein molecules, avoid or reduce the production of antibodies, or prevent the binding of antigens and antibodies to inhibit the occurrence of immune responses. After the protein is modified with PEG, the molecular weight increases and the filtration of the glomerulus decreases; the barrier effect of PEG protects the protein from being easily hydrolyzed by proteases, thereby enhancing its stability; all of these contribute to the extension of the circulation half-life of protein drugs and the increase of the effective concentration and duration of action of the drug. Studies have shown that many enzymes and proteins modified with polyethylene glycol have significantly reduced antigenicity, significantly prolonged in vivo duration of action, and greatly improved efficacy in vivo. After administration, the relatively stable blood drug concentration and long half-life of PEG drug protein conjugates greatly increase the medication interval, and the toxicity of the drug is reduced due to the reduction of fluctuations in blood drug concentration caused by multiple daily administration or short dosing intervals.

[0006] Over the years, PEG has been proven to be non-toxic in food, cosmetics and pharmaceutical applications. The low immunogenicity of PEG has been supported by many studies. After studying the clearance of PEG monomers of different molecular weights, it was found that the clearance of PEG does not involve structural changes, and its clearance rate is proportional to the molecular weight. Among them, molecules with a molecular weight below 20kDa are cleared by the kidneys and excreted in the urine (the critical value of molecular weight clearance by the kidneys is known to be 70kDa. PEG molecules with higher molecular weights are cleared very slowly by the kidneys, and as the molecular weight of PEG increases, the clearance rate of the liver will also increase. The properties of PEG protein conjugates are similar to many natural polymers, so they can show many exciting properties.

[0007] PEGylation technology has gradually gained attention with the extensive use of protein drugs and has become a hot topic for medical researchers. At present, PEGylation modification technology is relatively mature. After modifying a considerable number of macromolecular protein drugs, this technology can extend the half-life of the drug in vivo, thereby reducing the dosage, reducing toxic side effects, and improving patient compliance while maintaining bioequivalence. PEG modification of drug proteins and peptides has achieved the purpose of reducing their immunogenicity, extending their half-life, and retaining their biological activity to the maximum extent.

[0008] The process of covalently modifying biological molecules such as proteins or peptides with polyethylene glycol (PEG) derivative modifiers is called PEGylation. The PEG modification technology of drugs is formed on the basis of the gradual development of PEG chemistry technology. PEG chemistry technology is to activate the non-reactive PEG into a modifier with different active functional groups to facilitate the reaction with proteins or peptides under relatively mild conditions.

[0009] The ideal PEGylation technology is to select appropriate PEG according to the site to be modified to obtain a uniform product, that is, site-specific PEG modification. Effective PEG modification includes a single high molecular weight linear or branched PEG molecule modifying a single site or multiple sites of the drug. In theory, single-site PEG-modified protein drugs can retain higher biological activity. According to the structure-activity relationship analysis of proteins, amino acid residues on the surface of proteins that do not bind to receptors should be selected as modification sites as much as possible.

[0010] Since the 1950s, PEG has been widely used as a separation and purification aid, embedding matrix, antifreeze, medical device lubricant, food additive, and carrier for dermatological applications, suppositories, parenteral medications, tablets and pills. In the late 1970s, the concept of PEGylation began to emerge, that is, activated PEG is covalently coupled to protein or peptide molecules by chemical methods.

[0011] After protein or peptide drugs are properly pegylated, their main biological functions remain unchanged and they can acquire the following favorable properties: improved solubility; reduced or eliminated immunogenicity; reduced likelihood of degradation by hydrolases, reduced rate of renal clearance, and prolonged half-life of the drug; changes in the drug's distribution and kinetic behavior in the body; reduced frequency of medication, improved patient compliance, improved quality of life, and reduced treatment costs.

[0012] At present, PEG modification technology has gone through two generations of development. The first generation of PEG modification was limited to low molecular weight mPEG (<12kDa), and the widely used modifier was mPEG-succinic acid succinimidyl ester.

[0013] (mPEG-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG-benzotriazole carbonate (mPEG-BTC), etc., are mainly connected to drug molecules through amide bonds. These modifiers preferentially react with lysine residues, but also react with histidine and tyrosine. This non-specific connection method may cause different subunits of an enzyme to be connected to different numbers of PEG molecules, resulting in conjugates with different degrees of modification and different modification sites.

[0014] Amino modification: There are generally more free amino groups in proteases, and they have higher nucleophilic reactivity, so they become the most commonly used modified groups in chemical modification, mainly the α-amino group, ε-amino group and N-terminal amino group of lysine. The modification methods are alkylation and acylation. Alkylation is mainly connected by imine bonds, and acylation is mainly connected by amide bonds. The main modifiers are mPEG-SS, mPEG-SC, mPEG-SPA, mPEG-NHS, mPEG-CHO and mPEG-ALD.

[0015] PEG modification of protein drugs will cause a decrease in biological activity. PEG is coupled to specific groups on the surface of proteins, which affects the spatial structure of proteins and inevitably shields the active sites on the surface of proteins to a certain extent. PEG modification conditions, reaction byproducts, etc. can all cause a decrease in biological activity. There are many reasons why PEGylation causes a decrease in the biological activity of protein drugs. Therefore, for a certain drug, it is necessary to establish the best method for its PEG modification. The optimization modification method mainly includes the selection of PEG activation mode (modifier) ​​and appropriate coupling conditions to achieve the retention of biological activity of protein drugs, improvement of general performance, reduction of product inertness and high recovery rate of target modifications. People often focus on the number of PEG couplings and PEG molecular weight in this area. In fact, each step from PEG activation to protein coupling has an important influence on the change in the activity of the final product.

[0016] The protein coupling conditions include the pH value of the modification reaction, the molar ratio of drug to PEG, drug concentration, reaction time and temperature, which are the main factors affecting the PEG chemical modification reaction. By orthogonal experimental design, suitable modification reaction conditions can be obtained, one or several of the influencing factors can be controlled to obtain the target modified product with high yield.

[0017] At present, more than ten kinds of PEG-modified proteins and peptide drugs have been approved by the FDA for marketing, including bovine adenosine deaminase, asparaginase, interferon α-2a, interferon α-2b, etc. These PEGylated proteins, while retaining the original biological activity of natural proteins, overcome the shortcomings of the original proteins in clinical applications, such as instability, easy degradation by proteases, short half-life, strong immunogenicity, and fast renal clearance rate.

[0018] Erythropoietin (EPO) is a sialic acid-containing, heat-stable (80°C) glycoprotein, composed of 165 amino acids, with a relative molecular weight of 34,000 Daltons. It originates from the kidney and fetal liver. Under hypoxic conditions, EPO can be expressed in more organs outside the kidney. In 1985, recombinant human erythropoietin (rhEPO) was produced using genetic recombination technology and widely used in clinical practice.

[0019] The most well-known effect of EPO is its hematopoietic effect. It can bind to the EPO receptor (EPOR) on the surface of erythroid precursor cells in the bone marrow, promote the proliferation, differentiation, maturation of erythroid precursor cells and release them into the peripheral blood. Interestingly, EPO also has certain physiological effects in non-hematopoietic tissues. Studies have found that EPOR is expressed in non-hematopoietic tissues (endothelium, retina, lung, liver, kidney, myocardium, adipocytes, macrophages and pancreatic cells), and is also expressed in the central (hypothalamus, hippocampus, cortex and spinal cord) and peripheral (dorsal root ganglia, nerve axons and Schwann cells) nervous systems. In addition to promoting hematopoiesis, EPO is also involved in processes such as cell proliferation, immune modification, metabolic homeostasis, vascular function and cell protection.

[0020] In recent years, there have been many studies on EPO and diabetes. Some studies have shown that EPO can regulate glucose metabolism in diabetic rats, improve glucose tolerance and insulin sensitivity, and reduce pancreatic β-cell damage. EPO not only affects glucose metabolism, but studies have also shown that EPO is closely related to diabetic microvascular complications. In vivo and in vitro studies and some small-scale clinical research results have confirmed that EPO has a potential protective effect on early DR and diabetic macular edema. At the same time, the DPN rat model has also confirmed that EPO can increase glutathione levels, reduce intracellular reactive oxygen species, reduce oxidative stress damage, inhibit the expression of apoptotic proteins, reverse mechanical and thermal damage, and restore the conduction velocity of nerve fibers.

[0021] Therefore, based on the broad application prospects of EPO, it is of great significance to provide modified EPO polypeptide drug molecules, increase their blood half-life, improve the stability and activity of drug molecules, and establish a suitable PEG-EPO modification method.

[0022] Existing studies have shown that mesenchymal cells have a certain regulatory effect on the differentiation of pancreatic islet cells in diabetic patients, and can improve the therapeutic effect of pancreatic β cells on diabetes to a certain extent.

[0023] In order to better apply protein drugs in clinical treatment, scholars have modified a series of peptides and protein drugs with PEG. Through PEGylation, the half-life of protein drugs in the body is extended, thereby improving their bioavailability and reducing the frequency and dosage of the drug, which not only facilitates drug use but also reduces the cost of clinical treatment. Summary of the invention

[0024] The purpose of the present invention is to provide a composition loaded with PEG-EPO and mesenchymal stem cells, a drug and a preparation method thereof in view of the short serum half-life of EPO and the limited drugs for treating diabetes in the prior art.

[0025] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0026] The present invention discloses a method for pegylation modification of erythropoietin, comprising:

[0027] 1) preparing phosphate buffer;

[0028] 2) dissolving the modified PEG molecule in the phosphate buffer to obtain solution A;

[0029] 3) dissolving erythropoietin in the phosphate buffer to obtain solution B;

[0030] 4) Mix solution A and solution B, add NaBH3CN, and shake overnight to react;

[0031] 5) Add glycine solution to terminate the reaction, and the PEG-modified EPO product is prepared.

[0032] Preferably, the modified PEG molecule is selected from polyethylene glycol propionaldehyde (mPEG-ALD), polyethylene glycol succinimidyl propionate (mPEG-SPA) and polyethylene glycol succinimidyl ester (mPEG-SC).

[0033] Preferably, the modified PEG molecule is selected from polyethylene glycol succinimidyl propionate (mPEG-SPA).

[0034] Preferably, the concentration of the phosphate buffer is 50-100 mmol / L, and the pH value is 5.0-7.0.

[0035] Preferably, in step 4), the solute mass ratio of solution A to solution B is 5:1 to 10:1.

[0036] Preferably, the reaction condition in step 4) is 4° C. and the reaction time is 8-10 hours.

[0037] Preferably, the reaction time is 10 hours.

[0038] The invention discloses PEG-modified erythropoietin, which is prepared by the method described above.

[0039] Preferably, the PEG-modified erythropoietin is mPEG-SPA-EPO.

[0040] The invention discloses a composition, which comprises the PEG-modified erythropoietin and a pharmaceutically acceptable carrier.

[0041] Preferably, the composition may further include mesenchymal stem cells.

[0042] The invention discloses application of the PEG-modified erythropoietin and / or the composition in preparing medicine for treating diabetes.

[0043] The invention discloses application of the PEG-modified erythropoietin and / or the composition in preparing a drug for lowering blood sugar.

[0044] The present invention discloses a method for pegylation modification of erythropoietin and a composition prepared by using the modified EPO prepared by the method and its use. The present invention obtains an EPO molecule with a prolonged plasma half-life by modifying EPO with different modified PEGs, thereby solving the problem that the EPO molecule has a short residence time and low activity in plasma. The PEG-modified EPO is prepared into a composition and administered to diabetic rats, which has a better hypoglycemic effect than recombinant EPO. Mesenchymal stem cells are added to the composition, which further enhances the hypoglycemic effect of rats. The composition prepared by the present invention can be used to prepare drugs for hypoglycemia and treatment of diabetes, has a long half-life in the body, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 .Determination of the stability of different types of PEG-modified EPO in rat plasma in vitro.

[0046] Figure 2 . Identification of the prepared mPEG-SPA-EPO product by reversed phase high performance liquid chromatography; wherein Figure 2 A is the chromatogram of recombinant EPO. Figure 2 B is the chromatogram of unpurified mPEG-SPA-EPO prepared by the method of Example 1, Figure 2 C is the chromatogram of mPEG-SPA-EPO purified by molecular sieves.

[0047] Figure 3 .The hypoglycemic effect of recombinant EPO and related modified compositions, and the changes in blood sugar in rats after subcutaneous injection of recombinant EPO, mPEG-SPA-EPO, and a combination of mPEG-SPA-EPO and mesenchymal stem cells.

[0048] Figure 4 .The effects of recombinant EPO and related modified compositions on the body weight of diabetic model rats, and the changes in body weight of rats after subcutaneous injection of recombinant EPO, mPEG-SPA-EPO, and a combination of mPEG-SPA-EPO and mesenchymal stem cells. DETAILED DESCRIPTION

[0049] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0050] Example 1 Establishment of the method for PEG modification of EPO

[0051] Prepare phosphate buffer (50 mmol / L, pH 7.0):

[0052] Preparation of 50 mmol / L disodium hydrogen phosphate solution: weigh 7.098 g Na2HPO4 and add ultrapure water to make up to 1 L.

[0053] Preparation of 50 mmol / L sodium dihydrogen phosphate solution: weigh 5.999 g NaH2PO4 and add ultrapure water to make up to 1 L.

[0054] Mix 50 mmol / L disodium hydrogen phosphate solution and 50 mmol / L sodium dihydrogen phosphate solution in a volume ratio of 6:4.

[0055] The modified PEG molecules (polyethylene glycol propionaldehyde (mPEG-ALD), polyethylene glycol succinimidyl propionate (PEG-SPA) and polyethylene glycol succinimidyl ester (mPEG-SC)) were dissolved in phosphate buffer solution (50 mmol / L, pH 7.0) to prepare solution A at a concentration of 0.3 mg / mL.

[0056] The recombinant EPO protein (5 mg / mL) was dissolved in a phosphate buffer solution (50 mmol / L, pH 7.0) to obtain solution B, and the protein concentration was prepared to be 1.5 mg / mL. Solution A and solution B containing different modified PEG molecules were mixed at a mass ratio of 10:1, 200 microliters of 1 mg / mL NaBH3CN were added, and the reaction was shaken overnight at 4°C (10 hours), and 500 microliters of 1M glycine solution were added to terminate the reaction, and PEG-modified EPO products were prepared: mPEG-ALD-EPO, mPEG-SPA-EPO and mPEG-SC-EPO.

[0057] Example 2 Analysis of the in vitro rat plasma stability of PEG-modified EPO products

[0058] Since there are many proteases in rat plasma, EPO faces the problem of being decomposed by proteases. Whether the modified EPO has a higher anti-protease function. Therefore, we further tested the stability of the modified conjugate in rat plasma in vitro.

[0059] 1) The protein concentrations of the PEG-modified EPO products prepared in Example 1: mPEG-ALD-EPO, mPEG-SPA-EPO and mPEG-SC-EPO were determined by the BCA (Binconic Acid) method, and then the above products and recombinant EPO protein were diluted to the same protein concentration of 0.02 mg / mL according to the determined concentrations for later use.

[0060] 2) The protein solution prepared in step 1) was added to the newly prepared rat anticoagulated plasma at the same EPO concentration. Each protein solution was measured using plasma from 5 rats. The mixture was incubated at 37° C., and plasma samples were taken 2, 4, and 6 hours after incubation. The EPO content in the serum was detected using an EPO enzyme-linked immunosorbent assay kit, and the EPO retention rate was calculated based on the measured EPO content. The measurement results are shown in Figure 1 .

[0061] from Figure 1 According to the measurement results, in vitro rat plasma, the stability of several modified EPO molecules prepared by the method of Example 1 is different. Among them, the stability of mPEG-ALD-EPO, mPEG-SPA-EPO and mPEG-SC-EPO in plasma is significantly improved compared with the recombinant unmodified EPO molecule, and the stability of mPEG-SPA-EPO is the highest among the three modifications, and can still achieve 80% retention rate after incubation in rat plasma in vitro for 6 hours.

[0062] Example 3 Purification and Identification of mPEG-SPA-EPO Product

[0063] From the experimental results of Example 2, the mPEG-SPA-EPO prepared by modifying EPO has higher stability and higher retention time in rat plasma. Therefore, we further purified and identified the mPEG-SPA-EPO prepared by the method of Example 1.

[0064] The purification was carried out using a molecular sieve Superdex 7510 / 300GL column, specifically as follows:

[0065] 1) Collect the mixed solution of mPEG-SPA-EPO reaction in a 1.5 mL centrifuge tube, transfer to a centrifuge balanced at 4°C and centrifuge at 1500 rpm for five minutes to remove tiny bubbles in the reaction solution.

[0066] 2) The Superdex 7510 / 300GL column was washed with a running buffer of 20 mM sodium phosphate and 50 mM NaCl at pH 7.0. After equilibration for 2 column volumes (48 mL), the reaction mixture (0.5 mL each time) was added to the Superdex 7510 / 300GL column via a syringe.

[0067] 3) The mixed solution was purified using a molecular sieve Superdex 7510 / 300GL column, with a mobile phase of Na2SO4 buffer system (0.1M, pH=7.0) containing 20mMPB, a flow rate of 0.5mL / min, and the solution of the first chromatographic peak was collected at an absorbance of 220nm. The collected solution was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, dialyzed overnight in a system of 20mM PB and 5% mannitol, and the concentrate was stored at -4°C after ultrafiltration and concentration.

[0068] The purified mPEG-SPA-EPO was identified by reverse phase high performance liquid chromatography (RP-HPLC), and the specific operation was as follows.

[0069] Recombinant EPO, unpurified mPEG-SPA-EPO reaction solution, and mPEG-SPA-EPO separated by molecular sieve Superdex7510 / 300GL column chromatography were used as test samples. 150 μL of sample was collected into a 1.5 mL centrifuge tube and stored at 4°C until the modification process was completed. All components were tested uniformly;

[0070] 2) Use a 100 μL microsyringe to draw 20 μL of the sample from each centrifuge tube, add it to the HPLC in batches, and pass it through the stationary phase, i.e., Xbr idge Peptide BEH C18 chromatographic column (100 mm×4.6 mm, 3.5 μm particle size);

[0071] 3) Run the gradient elution program, perform UV detection at 220nm, and the flow rate is 0.5mL / min. Select 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution as solution A and solution B. The gradient elution scheme is as follows: 0-0.5 minutes (10-10% B), 0.5-1 minutes (10-90% B), 1-1.4 minutes (90-90% B), 1.4-1.5 minutes (90-10% B) and 1.5-2 minutes (10-10% B). The running time is 45 minutes.

[0072] 4) The retention time of the chromatogram of peptides in different states can be used as the qualitative test result. Figure 2 .

[0073] from Figure 2From the test results, the peak positions of the three samples are consistent. From the peak area, the content of recombinant EPO in the solution after mPEG-SPA-EPO purification has been significantly reduced.

[0074] Example 4 Establishment and grouping of diabetic rat model

[0075] The experimental animals were fed in a standard laboratory room, with sufficient water and feed, 12H light / 12H no light day and night cycle, and the feeding and use of rats were in accordance with China's "Regulations on the Administration of Experimental Animals". 33 rats were adaptively fed for one week and randomly divided into a normal control group (n = 3) and a diabetic group (n = 30). After the rats were fasted for 12 hours, the diabetic group was intraperitoneally injected with a single 0.1% STZ solution 50 mg / kg, and the control group was intraperitoneally injected with the same dose of sodium citrate buffer. After 24 hours, the rat tail vein blood sugar was measured. If the tail vein blood sugar was greater than 16.5mM / L for three consecutive days, and compared with normal rats, it was obviously more than 16.5mM / L, and the typical clinical signs such as polydipsia, polyphagia, and polyuria, that is, "three more and one less", it was determined to be a diabetic rat, and the diabetic model was considered to be successfully established. The rat tail vein blood sugar was monitored once a week to ensure that the blood sugar of the model rats was greater than 16.5mM / L, and the rats in each group were continued to be fed with sufficient water and feed. The rats with successful modeling were divided into 4 groups, with 5 rats in each group for each treatment group experiment.

[0076] Example 5: Hypoglycemic effect of mPEG-SPA-EPO and mesenchymal cell composition

[0077] Current studies have shown that mesenchymal stem cells can improve the therapeutic effect of pancreatic β cells on diabetes to a certain extent. Therefore, we further investigated the effect of combined administration of mesenchymal stem cells and EPO on hypoglycemia. Rat mesenchymal stem cells were prepared and isolated using conventional methods, and markers were identified.

[0078] In order to study the hypoglycemic effect of recombinant EPO, mPEG-SPA-EPO and the combination of mPEG-SPA-EPO and mesenchymal stem cells, referring to Example 2, the recombinant EPO protein and mPEG-SPA-EPO were diluted to the same protein concentration of 0.2 mg / mL using phosphate buffer (50 mmol / L, pH 7.0), and the separated mesenchymal stem cells were added to the obtained mPEG-SPA-EPO solution to 5×10 2 The four groups of blank control (PBS buffer), recombinant EPO, mPEG-SPA-EPO and the combination of mPEG-SPA-EPO and mesenchymal stem cells were used to study the hypoglycemia in diabetic rats. The specific operation is as follows.

[0079] 1) Fasting diabetic rats were weighed and 300 μg / kg (based on the weight of EPO protein) of the above-mentioned drug was injected subcutaneously into the rats.

[0080] 2) Immediately after the injection, blood was collected through the rat tail vein, and after the first drop of blood was removed, the next drop of blood was collected and the blood glucose concentration was immediately measured using a one-button blood glucose monitoring system, and this point was recorded as 0 minute.

[0081] 3) At 30 minutes after the subcutaneous injection of the polypeptide, blood was collected from the tail vein in the same manner and the blood sugar level was measured and recorded as 30 minutes. At the same time, a glucose solution (2 g / kg) was orally administered intragastrically.

[0082] 4) At 30, 60, 90, 120, and 180 minutes after oral gavage of glucose, blood samples were collected from the rat tail vein. After the first drop of blood was discarded, the next drop of blood was collected and the blood glucose level was immediately measured and recorded using a one-button blood glucose monitoring system. The test results are shown in Figure 3 .

[0083] from Figure 3 According to the results, compared with the control group, the three groups of recombinant EPO, mPEG-SPA-EPO and the combination of mPEG-SPA-EPO and mesenchymal stem cells can reduce the blood glucose level in rat serum. Among them, the mPEG-SPA-EPO group has a better blood glucose lowering effect than the recombinant EPO group, indicating that the mPEG-SPA-EPO group has a higher half-life in serum and maintains a better blood glucose lowering effect. The combination of mPEG-SPA-EPO and mesenchymal stem cells has a better blood glucose lowering effect than the mPEG-SPA-EPO group because mesenchymal stem cells are further added, which have a certain regulatory effect on pancreatic β cells.

[0084] Example 6 Effects of mPEG-SPA-EPO and mesenchymal cell composition on body weight of diabetic rats

[0085] We further investigated the effects of mPEG-SPA-EPO and mesenchymal cell compositions on the body weight of model rats. In order to study the effects of recombinant EPO, mPEG-SPA-EPO, and the combination of mPEG-SPA-EPO and mesenchymal stem cells on the body weight of diabetic rats, referring to Example 2, recombinant EPO protein and mPEG-SPA-EPO were diluted to the same protein concentration of 0.02 mg / mL using phosphate buffer (50 mmol / L, pH 7.0), and separated mesenchymal stem cells were added to the obtained mPEG-SPA-EPO solution to 5×10 2The blank control (PBS buffer), recombinant EPO, mPEG-SPA-EPO, and the combination of mPEG-SPA-EPO and mesenchymal stem cells were injected into diabetic rats.

[0086] 1) The diabetic rats were fed a normal diet every day;

[0087] 2) During the entire treatment period, the model rats were treated with 300 μg / kg (according to the concentration of EPO protein) injected once a day, including the PBS group, the recombinant EPO group, the mPEG-SPA-EPO group, and the combination group of mPEG-SPA-EPO and mesenchymal stem cells;

[0088] 3) The body weight of rats in each group was measured at different time points of treatment. The test results are shown in Figure 4 .

[0089] from Figure 4 According to the body weight measurement results, compared with the PBS group, the other recombinant EPO group, the mPEG-SPA-EPO group and the combination group of mPEG-SPA-EPO and mesenchymal stem cells can all control the body weight of rats well. Moreover, the mPEG-SPA-EPO group and the combination group of mPEG-SPA-EPO and mesenchymal stem cells have better weight control effects than the recombinant EPO group.

[0090] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for pegylation of erythropoietin, characterized in that: include: 1) preparing phosphate buffer; 2) dissolving the modified PEG molecule in the phosphate buffer to obtain solution A; 3) dissolving erythropoietin in the phosphate buffer to obtain solution B; 4) Mix solution A and solution B, add NaBH3CN, and shake overnight to react; 5) Add glycine solution to terminate the reaction, and the PEG-modified EPO product is prepared.

2. The method according to claim 1, characterized in that The modified PEG molecule is selected from polyethylene glycol propionaldehyde, polyethylene glycol succinimidyl propionate and polyethylene glycol succinimidyl ester.

3. The method according to claim 1, characterized in that The concentration of the phosphate buffer is 50-100 mmol / L, and the pH value is 5.0-7.

0.

4. The method according to claim 1, characterized in that: In step 4), the solute mass ratio of solution A to solution B is 5:1 to 10:

1.

5. The method according to claim 1, characterized in that In step 4), the reaction condition is 4° C. and the reaction time is 8-10 hours.

6. A PEG-modified erythropoietin, characterized in that: The PEG-modified erythropoietin is prepared by the method described in any one of claims 1-5.

7. The PEG-modified erythropoietin according to claim 6, characterized in that The PEG-modified erythropoietin is mPEG-SPA-EPO.

8. A composition, characterized in that The composition comprises the PEG-modified erythropoietin according to any one of claims 6-7 and a pharmaceutically acceptable carrier.

9. The composition according to claim 9, characterized in that The composition also includes mesenchymal stem cells.

10. Use of the PEG-modified erythropoietin according to any one of claims 6-7 and / or the composition according to any one of claims 8-9 in the preparation of a drug for treating diabetes.

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