Fat chain modified human granulocyte colony stimulating factor derivative and preparation method thereof

Abstract

The present invention relates to a human granulocyte colony stimulating factor derivative modified by a fat chain, which is derived from the site-specific modification of the free cysteine ​​residue of the human granulocyte colony stimulating factor by a fat chain-maleimide, and the preparation method thereof comprises the synthesis of the fat chain-maleimide, the synthesis of the human granulocyte colony stimulating factor derivative modified by the fat chain, and the separation, purification and characterization of the product. Compared with the unmodified human granulocyte colony stimulating factor, the human granulocyte colony stimulating factor derivative modified by the fat chain has a high in vitro activity retention rate, a prolonged sustained action time in the body of the experimental animal, a reduced frequency of administration, and an improved drug bioavailability and efficacy.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a fatty chain-modified human granulocyte colony stimulating factor derivative and a preparation method thereof. Background Art

[0002] Neutrophils are the main white blood cells in the blood and are considered the first natural line of defense against bacteria. Since they circulate in the blood for 8-12 hours and only take 2-5 days to move into tissues, they need to be constantly replaced in the organism, and their proliferation is mainly controlled by granulocyte colony-stimulating factor (G-CSF). Chemotherapy-induced neutropenia (CIN) increases the risk of infection, which can manifest as fever, namely febrile neutropenia (FN), thus increasing the possibility of serious consequences. FN and severe or prolonged CIN can lead to delays or interruptions in chemotherapy, which can affect the patient's optimal treatment.

[0003] At present, studies have found that G-CSF can promote the proliferation, differentiation and migration of granulocytes and enhance the function of mature granulocytes by binding to the granulocyte colony stimulating factor receptor (G-CSFR). Recombinant human granulocyte colony stimulating factor (rhG-CSF) is a non-glycosylated protein composed of 175 amino acids, with a free cysteine ​​residue Cys 18 and a molecular weight of 18.8kDa. Compared with the amino acid sequence of natural G-CSF, there is an additional methionine at the N-terminus. rhG-CSF has been successful in treating bone marrow suppression associated with cancer chemotherapy and has been approved for the following clinical indications: cancer patients receiving bone marrow suppressive chemotherapy; patients with acute myeloid leukemia receiving induction or curing chemotherapy; cancer patients receiving bone marrow transplantation (BMT); patients receiving peripheral blood progenitor cell collection and treatment; patients with severe chronic neutropenia. Some researchers have also found that rhG-CSF has some good effects in ischemic cerebral infarction, immune regulation, and acute oral mucositis caused by radiotherapy for nasopharyngeal carcinoma. rhG-CSF is also used in all patients who have received massive radiation exposure resulting in hematopoietic acute radiation syndrome (ARS).

[0004] There are currently three main short-acting rhG-CSFs for clinical use, namely, non-glycosylated methionyl recombinant human G-CSF (r-Met Hu G-CSF) such as Filgrastim, glycosylated G-CSF such as Lenograstim, and mutant gene products of G-CSF such as Nartograstim. They have good clinical effects on neutropenia in cancer patients, but as a protein drug, rhG-CSF is rapidly degraded in the body or cleared by the kidneys, resulting in a short plasma half-life and the need for repeated and frequent administration, which not only increases the pain of patients, but also repeat injections are very likely to cause some adverse reactions. Therefore, it is very necessary to use long-acting technology to conduct secondary development of rhG-CSF to improve its in vivo medication effect.

[0005] Fat chain modification technology is a new type of protein drug long-acting technology developed in recent years. Compared with the current common long-acting technologies, especially PEG, fat chain modification has the following advantages: (1) Fat chain modification can shield the areas of protein polypeptide molecules that are vulnerable to enzyme attack, thereby delaying or inhibiting the destructive effect of proteases and prolonging the in vivo action time; in addition, fat chain modification mainly improves the half-life of drugs by binding to human serum albumin (HSA). Since fatty acids are the natural ligands of plasma HSA and can reversibly bind to HSA, due to the large molecular weight of HSA and its ability to bind to Fc receptors (FcRn), when fat chain modified drugs bind to HSA, they limit the entry of proteases into the drug, greatly reduce renal clearance, and utilize FcRn-mediated circulation, thereby prolonging the drug half-life. Studies have shown that increasing the fat chain length of fat chain modified drugs generally leads to tighter HSA binding and increased therapeutic half-life. In some cases, fatty acid chain modification can also induce drug self-assembly to form polymers, further prolonging its half-life in the body; (2) Fatty acids are important components of human fat, lipids and cell membrane phospholipids. On the one hand, they are not easy to produce immunogenicity. On the other hand, they help to improve the lipid solubility of hydrophilic drugs and increase intestinal mucosal permeability. Therefore, fatty chain modification is more helpful to improve the lipid solubility, intestinal mucosal permeability and absorption efficiency of drugs, and it is possible to achieve oral, topical and pulmonary administration. For example, the fatty chain-modified GLP-1 drug, semaglutide, has been approved by the FDA for oral administration; (3) Studies have shown that fatty chain modification can interfere with T cell activation because it can reduce binding to MHC II molecules and reduce immunogenicity. Currently, several fatty acid-modified drugs such as detemir, degludec, liraglutide and semaglutide have been launched on the market, with global sales reaching US$10 billion in 2020, and the market prospects are broad. There are no reports of fatty chain-modified hG-CSF. Summary of the invention

[0006] One object of the present invention is to provide a novel fatty chain modified human granulocyte colony stimulating factor derivative, which prolongs the duration of action of human granulocyte colony stimulating factor in the body, reduces the number of administrations, increases the bioavailability of the drug and improves patient compliance.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned fatty chain modified human granulocyte colony stimulating factor derivative, so as to obtain a product with high yield, high purity and high uniformity, and to facilitate industrial production.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] A fatty chain-modified human granulocyte colony-stimulating factor (hG-CSF) derivative as shown in formula (I),

[0010]

[0011] Among them, R 1 It is a fatty chain with a molecular formula of C n H 2(n-m)+1 (n=integer of 1-32, m=integer of 0-6, n>m); R 2 It is the part of human granulocyte colony stimulating factor except the free thiol group of cysteine ​​residue, and the human granulocyte colony stimulating factor is a naturally derived human granulocyte colony stimulating factor, a genetically recombinant human granulocyte colony stimulating factor or a human granulocyte colony stimulating factor mutant.

[0012] Preferably, R 1 C 15 Straight chain alkyl.

[0013] The present invention also provides a method for preparing the fatty chain-modified human granulocyte colony stimulating factor derivative represented by the above formula (I), the method comprising:

[0014] In a dimethyl sulfoxide-sodium phosphate buffer, the fatty chain-maleimide of formula (II) and the human granulocyte colony stimulating factor of formula (III) are reacted at 4-40° C. for 1-120 min (preferably at 20° C. for 5-30 min, particularly preferably 30 min), and the resulting reaction solution is separated and purified to obtain the fatty chain-modified human granulocyte colony stimulating factor derivative of formula (I); the dimethyl sulfoxide-sodium phosphate buffer is prepared in a volume ratio of 10-100:90-0 (preferably 90:10) (0 means that the buffer is pure dimethyl sulfoxide without sodium phosphate buffer), and the pH of the sodium phosphate buffer is 6.5-8.0 (preferably 7.0); the molar ratio of the fatty chain-maleimide of formula (II) to the human granulocyte colony stimulating factor of formula (III) is 10-0.1:1 (preferably 5:1);

[0015]

[0016] Among them, R 1 It is a fatty chain with a molecular formula of C n H 2(n-m)+1 (n=integer of 1-32, m=integer of 0-6, n>m); R 2 It is the part of human granulocyte colony stimulating factor except the free thiol group of cysteine ​​residue, and the human granulocyte colony stimulating factor is a naturally derived human granulocyte colony stimulating factor, a genetically recombinant human granulocyte colony stimulating factor or a human granulocyte colony stimulating factor mutant.

[0017] Preferably, R 1 C 15 Straight chain alkyl.

[0018] Furthermore, the volume of the dimethyl sulfoxide-sodium phosphate buffer is 0.1-10 mg / mL (preferably 1 mg / mL) based on the mass of the human granulocyte colony stimulating factor represented by formula (III).

[0019] Furthermore, the post-separation and purification treatment is as follows: the reaction solution is purified by reverse phase liquid chromatography, the chromatographic column is Bio-C8 (21.2×250mm, 10μm), the mobile phases A and B are 100% pure water (adjusted to pH 4.2 with TFA) and 100% acetonitrile (adjusted to pH 4.2 with TFA), respectively, 40%-55% mobile phase B linear gradient elution for 5min, 55%-75% mobile phase B linear gradient elution for 20min, the flow rate is 10mL / min, the detection wavelength is 280nm, the eluate containing the target compound is collected, the acetonitrile is removed by rotary evaporation, and the dialysis is performed with 10mmol / L pH 4.2 acetate buffer as the dialysate, and the retentate is ultrafiltered and concentrated to obtain the fatty chain modified human granulocyte colony stimulating factor derivative (present in acetate buffer) shown in the formula (I).

[0020] Preferably, the fatty chain modified human granulocyte colony stimulating factor derivative represented by formula (I) is stored in the following manner: the ultrafiltration concentrated retentate is prepared into a solution containing 5% sorbitol and 0.0033% Tween 20 and stored in a 4°C refrigerator.

[0021] Further, the fatty chain-maleimide represented by the formula (II) is prepared by the following method: the fatty chain-maleimide is obtained by reacting a fatty amine with maleic anhydride, and is prepared by a generally accepted method reported in the literature. The fatty chain-maleimide is separated and purified using a reversed-phase high-performance liquid chromatography preparative column. The purified fatty chain-maleimide is analyzed for purity using reversed-phase high-performance liquid chromatography. Its molecular weight is determined by mass spectrometry. The correctness of its chemical structure is determined using NMR detection.

[0022] Preferably, the fatty acid is acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignin acid, cerotic acid, montanic acid, melisic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid.

[0023] The present invention provides a pharmaceutically acceptable salt of a human granulocyte colony stimulating factor derivative modified with a fat chain as shown in the above formula (I).

[0024] The present invention provides a preparation of a human granulocyte colony stimulating factor derivative modified with a fat chain as shown in formula (I) or a pharmaceutically acceptable salt thereof (if loaded on a human body acceptable carrier, it can also be prepared into a preparation).

[0025] The present invention also provides a human granulocyte colony stimulating factor derivative modified by a fat chain as shown in formula (I), a pharmaceutically acceptable salt of the human granulocyte colony stimulating factor derivative modified by a fat chain as shown in formula (I), or a preparation of the human granulocyte colony stimulating factor derivative modified by a fat chain as shown in formula (I) for use in the preparation of a drug for treating neutropenia caused by radiotherapy, chemotherapy, etc.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0027] Compared with unmodified human granulocyte colony stimulating factor, the novel fatty chain modified human granulocyte colony stimulating factor derivative provided by the present invention has a high in vitro activity retention rate, a prolonged sustained action time in experimental animals, reduces the frequency of administration, and improves the bioavailability and efficacy of the drug. The preparation method provided by the present invention is used to obtain a single fatty chain modified human granulocyte colony stimulating factor derivative with high yield, high purity and high uniformity, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 RP-HPLC Analysis of Purity of C15-MAL

[0029] Figure 2 Structural Analysis of C15-MAL by Time-of-Flight Mass Spectrometry

[0030] Figure 3 Purity analysis of C15-rhG-CSF after purification by reverse phase preparative HPLC

[0031] Figure 4 MALDI-TOF MS Analysis of C15-rhG-CSF

[0032] Figure 5 MALDI-TOF MS analysis of rhG-CSF

[0033] Figure 6 Circular dichroism analysis of rhG-CSF and C15-rhG-CSF

[0034] Figure 7 In vitro activities of rhG-CSF, PEG-rhG-CSF and C15-rhG-CSF

[0035] Figure 8 Plasma drug concentration-time curves of subcutaneous injection of C15-rhG-CSF or rhG-CSF in mice

[0036] Fig. 9 The bar graph of the effect of different doses of rhG-CSF and C15-rhG-CSF on the peripheral blood leukocyte count of mice treated with CTX (C15-rhG-CSF was a single injection, and rhG-CSF was divided into 5 days, with 0.1 mg / kg or 0.2 mg / kg injected every day, totaling 0.5 mg / kg or 1.0 mg / kg. "*" indicates that compared with the normal control group, (*) p<0.05, (**) p<0.01, and (***) p<0.001.)

[0037] Fig.10 The bar graph of the effect of different doses of rhG-CSF and C15-rhG-CSF on peripheral blood red blood cells of mice treated with CTX (C15-rhG-CSF was a single injection, and rhG-CSF was divided into 5 days, with 0.1 mg / kg or 0.2 mg / kg injected every day, totaling 0.5 mg / kg or 1.0 mg / kg. "*" indicates that compared with the normal control group, (*) p<0.05, (**) p<0.01, and (***) p<0.001.) DETAILED DESCRIPTION

[0038] The following is a detailed description of the specific implementation of the present invention by way of examples in combination with the technical scheme, which should not be construed as limiting the present invention. Unless otherwise specified, the experimental methods used are all conventional methods, and the reagents used can be purchased from chemical reagent companies, and the chemical reagents used are all analytically pure.

[0039] Example 1 Synthesis of fatty chain-maleimide

[0040] Add 60mL THF solution containing 34.17mmol palmitic acid to a round-bottom flask, then add 34.17mmol TEA and 34.17mmol DPPA and react for 48h at room temperature and nitrogen environment. After the reaction, use a rotary evaporator at a temperature of 40±1℃ to remove the solvent, add 60mL t-BUOH and mix with nitrogen and reflux at 90℃ for 5h. After the reaction, the solvent is removed by rotary evaporation at 60±1℃ to obtain N-Boc-pentadecanamine, add 4mol / L HCl and stir at room temperature overnight, then add an equal volume of ethyl acetate for extraction, and finally rotary evaporation to obtain n-pentadecanamine. Take 23.45mmol maleic anhydride and dissolve it in 5mL anhydrous dichloromethane and add it to a 50mL three-necked flask. Dissolve 23.45mmol n-pentadecanamine in 10mL anhydrous dichloromethane and gradually add it to a three-necked flask, reflux at 40±1℃ for 30 minutes (partial solidification). After cooling, filter and wash twice with cold anhydrous dichloromethane, dry to obtain a white solid (N-pentadecyl maleic acid). Take 12.8mmol N-pentadecyl maleic acid and 8.8mmol sodium acetate, dissolve in 15mL acetic anhydride, add to a 100mL round-bottom flask, heat at 100±1℃ for two hours, cool, add 50mL water, put into a separatory funnel and let stand, wash twice with ether (2×30mL), wash twice with 2.0% KOH solution (2×30mL), and wash once with 30mL water, take out the organic phase, spin dry and dissolve in 20mL dichloromethane, use MgSO 4 Dry, filter, and spin dry to obtain pentadecyl-maleimide (C 15 -MAL) crude product. The reaction equation is as follows:

[0041]

[0042] C was analyzed using a YMC preparative column (YMC-Pack ODS-A, 20 mm × 250 mm, 10 μm) in a Shimadzu LC-6AD system. 15 -MAL was used for separation and purification experiments. The liquid chromatography conditions were: injection volume 2 mL, flow rate 15 mL / min, detection wavelength 210 nm, mobile phases of pure water (A) and pure acetonitrile (B), and elution conditions of 90% (v / v) B isocratic elution for 60 min.

[0043] After purification, C 15-MAL purity. Liquid chromatography conditions are: chromatographic column Phenomenex C18 (250mm×4.6mm, 5μm), flow rate 1mL / min, detection wavelength 210nm. Mobile phases A and B are 100% pure water and 100% acetonitrile, respectively. Inject 20μL under the equilibrium condition of 90% (v / v) B, and elute condition is 90% (v / v) acetonitrile isocratic elution for 60min.

[0044] Thermo Scientific ITQ 1100TM from ThermoFisher, USA, was used to analyze the C 15 -MAL was subjected to mass spectrometry analysis to determine its molecular weight.

[0045] use 1 H NMR detection of C 15 -MAL chemical shift of each hydrogen atom to determine its chemical structure. The sample to be tested is dissolved in deuterated methanol and the experimental data is recorded by a 500MHz nuclear magnetic resonance instrument.

[0046] The synthesized C 15 The yield of -MAL was 41.3% and the liquid phase purity was 98.0% ( Figure 1 ). Synthesized C 15 -MAL uses mass spectrometry and 1 H NMR confirmed the correctness of the structure. Figure 2 It can be seen that the synthesized C 15 The molecular weight of -MAL is 307Da, which is consistent with the theoretical value. As can be seen from Table 1, 1 The main chemical shifts of the H-NMR spectrum are 0.91, 1.30, 1.57, 3.49, and 6.81, and the chemical groups to which they belong are consistent with the target product C 15 -MAL, indicating that the structure is correct.

[0047] Table 1 C 15 -MAL 1 H-NMR data

[0048]

[0049]

[0050] Example 2 Synthesis of fatty chain modified human granulocyte colony stimulating factor derivatives

[0051] Using synthetic C 15 -MAL was used to modify the cysteine ​​thiol group of rhG-CSF (recombinant human granulocyte stimulating factor, Hangzhou Jiuyuan Gene Engineering Co., Ltd., liquid chromatography purity greater than 98%). 15-MAL (PEG to rhG-CSF molar ratio of 1:1, 3:1, 5:1 or 8:1) was reacted in a mixed solution of 2 mL DMSO and phosphate buffer (DMSO volume ratio of 10%-100%, temperature of 4, 20, 30 or 40 ° C, the reaction time was 30min, 60min, 90min and 120min respectively. The pH was 7.0, the temperature was 20 ° C, 15 The reaction conditions of a molar ratio of -MAL to rhG-CSF of 5:1 and a reaction time of 30 min were used as control standards for the analysis of each factor to determine the optimized conditions. After the reaction was completed, samples (0.5 mL each) were taken, and 50 μL of 1 mol / L DTT was added to each sample to terminate the reaction, and the reaction mixture was analyzed by liquid chromatography. The liquid chromatography analysis conditions were as follows: the chromatographic column was TSKgel Protein C4-300 (4.6 mm × 150 mm, 3 μm), and the mobile phases A and B were 100% pure water (containing 0.1% TFA) and 100% acetonitrile (containing 0.1% TFA), respectively. The injection volume was 20 μL. 40%-55% mobile phase B linear gradient elution for 5 min, 55%-75% mobile phase B linear gradient elution for 20 min, the flow rate was 1 mL / min, and the detection wavelength was 280 nm.

[0052] The optimization determined C 15 The reaction conditions for rhG-CSF modification by -MAL were as follows: the volume ratio of DMSO in the DMSO-phosphate buffer solution was 90%, pH 7.0, temperature 20°C, C 15 -MAL / rhG-CSF molar ratio was 5:1, reaction time was 30 min, and the conversion rate of single modified product could reach 89.1%.

[0053] Example 3 Isolation and Purification of Fat Chain Modified hG-CSF Derivatives

[0054] C15-rhG-CSF was separated and purified using a reverse phase preparative column. The preparative liquid chromatography conditions were as follows: the chromatographic column was Bio-C8 (21.2×250mm, 10μm), the mobile phases A and B were 100% pure water (adjusted to pH 4.2 with TFA) and 100% acetonitrile (adjusted to pH 4.2 with TFA), respectively. The injection volume was 500μL. 40%-55% mobile phase B linear gradient elution for 5min, 55%-75% mobile phase B linear gradient elution for 20min, the flow rate was 10mL / min, and the detection wavelength was 280nm.

[0055] The purified C15-rhG-CSF was subjected to rotary evaporation at 30±1°C to remove acetonitrile, and then the solution was replaced with 10mmol / L pH 4.2 acetate buffer by overnight dialysis using a dialysis bag with a molecular weight cutoff of 3500Da, and then concentrated using an ultrafiltration tube with a molecular weight cutoff of 3000Da, and then analyzed for purity by reverse phase high performance liquid chromatography, with the same operating conditions as in Example 2. Finally, the C15-rhG-CSF solution was prepared into a solution containing 5% sorbitol and 0.0033% Tween 20 and stored in a 4°C refrigerator.

[0056] The yield of purified C15-rhG-CSF was 84.6% and the liquid phase purity was 99.0% ( Figure 3 ).

[0057] Example 4 Characterization of fatty chain modified hG-CSF derivatives

[0058] Example 4-1: Molecular weight analysis

[0059] MALDI-TOF was used to determine the molecular weight of C15-rhG-CSF and rhG-CSF before modification. The operation steps are as follows: (1) Spotting: Take 1μL of protein sample and spot it on the sample target. After natural drying, take 0.6μL of CHCA matrix solution and spot it on the corresponding target position and dry it naturally. Use the same method to spot the standard at the adjacent position of the sample target. (2) Calibration: Select the linear method in the positive ion mode to calibrate the sample test range. (3) The relevant parameters are as follows: The calibration range of the standard material is: The calibration range of the linear high molecular weight calibration material is: 66430±100; (4) Test sample: Select the linear method in the positive ion mode to test the sample molecular weight. (5) Mass spectrometry data and spectrum processing: The raw data and spectrum generated by 5800MALDI-TOF / TOF are exported by 4000Series Explorer V3.5 software.

[0060] The measured relative molecular mass of C15-rhG-CSF is 19056.762Da, and the relative molecular mass of rhG-CSF is 18819.998Da, which is consistent with the theoretical value ( Figure 4 and 5 ).

[0061] Example 4-2: Secondary structure analysis

[0062] The secondary structure of C15-rhG-CSF and rhG-CSF was analyzed using a J-815 circular dichroism spectrometer (CD) from JASCO. Using pH 4.2 acetate buffer as the solvent, C15-rhG-CSF and rhG-CSF were prepared into a solution with a concentration of 0.2 mg / mL and a volume of 1 mL. The secondary structure of the protein was determined by the positive and negative values ​​of the absorption of the corresponding points of the CD spectrum at different wavelengths. The specific parameter settings selected a cuvette of 0.1 mm, a scanning step of 0.2 nm, and a scanning speed of 100 nm / min. The CD spectra obtained by three scans were averaged.

[0063] from Figure 6 It can be seen that the secondary structure of C15-rhG-CSF has no significant change compared to rhG-CSF. This indicates that after removing DMSO from the reaction system, C15-rhG-CSF can restore the natural conformation of rhG-CSF, and the fatty chain modification has little effect on the secondary structure of rhG-CSF.

[0064] Example 4-3: In vitro activity analysis

[0065] The in vitro activity of rhG-CSF and C15-rhG-CSF was analyzed in NFS-60 cells using the MTT method.

[0066] (1) Preparation of cell suspension: Take a bottle of well-grown NFS-60 cells and transfer them to a sterile centrifuge tube with a sterile pipette. Collect the cells by centrifugation and discard the supernatant. Wash three times with basal culture medium, pipetting evenly each time, centrifuge at 1000 rpm for 5 minutes each time, discard the supernatant, resuspend the cells in basal culture medium and mix well. Count the cells using a hemocytometer under a microscope at 10×10 magnification. Finally, adjust the cell concentration to 1.0×10 with basal culture medium based on the counting results. 5 ~2.0×10 5 Each plate requires >5 mL of cell suspension.

[0067] (2) Preparation of standard and test solution: The standard and test solution are pre-diluted to a concentration of about 2000 IU / mL or 0.03 μg / mL using basal culture medium according to their labeled amount or protein content. If the basal culture medium becomes darker in color during the dilution process, it should be replaced with fresh culture medium. Each dilution step should not exceed 10 times. It is recommended to use a dilution of 50 μL → 450 μL. The recombinant human granulocyte stimulating factor stock solution intermediate needs to be filtered with a 0.22 μm filter membrane before dilution.

[0068] (3) Detection: Add the test solution and standard solution to a 96-well cell culture plate, and make a 2-fold serial dilution from high concentration to low concentration, 50μL→50μL, for a total of 10 dilutions. Make at least 2 replicates for each dilution. Keep 50μL of the test solution and standard solution in each well, discard the excess solution in the well, and add 50μL of the diluted cell suspension to each well. Add 100μL of basal culture fluid as a blank control and 50μL of basal culture fluid plus 50μL of cell suspension as a negative control. All the above operations must be performed under sterile conditions; the 96-well cell culture plate after sample addition should be placed at 37°C and 5% CO 2 , and cultured in a saturated humidity incubator for 40-48h. Under the microscope, cells with high concentration of G-CSF grew well, but cells with low concentration almost died; 20μL of MTT solution was added to each well, and the cells were placed at 37℃, 5% CO 2 Incubate for another 4-5 hours under saturated humidity; add 100 μL of lysis buffer to each well and place at 37°C, 5% CO 2 , incubate in a saturated humidity incubator overnight; observe under a microscope, and after the crystals are completely dissolved, measure the OD value at 570nm with 630nm as the reference wavelength.

[0069] (4) Result processing and judgment: Call the set template, set the relevant parameters, and read the data; if the subsequent dilution multiple is used as the horizontal axis and the OD value is used as the coordinate, A represents the maximum OD value of the corresponding curve, D represents the minimum OD value, B represents the slope value, and C represents the EC value of each curve. 50 (half-effective dose dilution multiple), R 2 Represents the square of the correlation coefficient. R 2 Should be ≥ 0.98; the data is processed according to the four-parameter regression method, and the results are calculated according to the following formula:

[0070] Biological activity of the test article Ps 1 (IU / mL) = Pr 1 ×Ds×Es / (Dr×Er)(1)

[0071] Standard biological specific activity Pr 2 (IU / mg) = Pr 1 / Cr;(2)

[0072] Biological specific activity Ps of the test sample 2 (IU / mg)=Ps 1 / Cs; (3)

[0073] Relative biological activity of the test product (%) = Ps 2 / Pr 2 *100% (4)

[0074] Where Pr 1 is the biological activity of the standard, IU / mL;

[0075] Ds is the pre-dilution multiple of the test product;

[0076] Dr is the pre-dilution multiple of the standard;

[0077] Es is the dilution multiple of the test sample equivalent to the half-effective dose of the standard sample;

[0078] Er is the dilution multiple of the half-effective dose of the standard;

[0079] Cr is the protein content of the standard, mg / mL;

[0080] Cs is the protein content of the test sample, mg / mL;

[0081] The in vitro activities of rhG-CSF and C15-rhG-CSF were characterized. Figure 7 The in vitro activity of C15-rhG-CSF was 6.29×10 7 IU / mg, compared with the in vitro activity of rhG-CSF before modification (7.21×10 7 IU / mg), the activity retention rate of C15-rhG-CSF was 87.2%. The in vitro activity retention rate of C15-rhG-CSF was higher than that of the reported PEG-rhG-CSF modification, because the molecular weight of the fatty chain was small, and the steric hindrance to the binding of rhG-CSF to the receptor was small.

[0082] Example 4-4: Pharmacokinetic analysis in animals

[0083] Male Kunming mice (16-20 g) were purchased from Shandong Experimental Animal Center (Jinan, China) with license number SCXK2014-0007. The animal husbandry system was maintained under standard laboratory conditions. All animal operations were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Shandong University of Technology and approved by the Animal Ethics Committee of Shandong University of Technology. Normal mice were randomly selected for grouping and sampling, as shown in Table 2. After subcutaneous administration of the mice, orbital blood samples were collected from 6 animals at each time point according to Table 2, and then the mice were killed by cervical dislocation. After blood sample collection, serum was separated after centrifugation for 30 min and stored in a refrigerator at -20 °C for testing. The concentration of rhG-CSF or C15-rhG-CSF in the blood sample was measured using a 96-well ELISA detachable strip (Coming, USA) and a Human G-CSF DuoSet kit (purchased from R&D, USA) on a Model 450 ELISA reader (Bio-RAD, USA). For specific operations, please refer to the operating manual of the Human G-CSF DuoSet kit.

[0084] Table 2 Grouping and administration of rhG-CSF and C15-rhG-CSF

[0085]

[0086] from Figure 8 It can be seen that the blood concentration of C15-rhG-CSF in mice reaches its peak in 0.5 hours, which is shorter than rhG-CSF (2h), indicating that the fatty chain modification is also conducive to the transmembrane absorption of rhG-CSF and can exert its efficacy more quickly. In addition, after reaching the peak, the blood concentration of C15-rhG-CSF in mice decreases more slowly than rhG-CSF, indicating that its half-life is longer and can exert its efficacy for a longer time.

[0087] Example 4-5: Animal in vivo efficacy analysis

[0088] Male Kunming mice (16-20 g) were purchased from Shandong Experimental Animal Center (Jinan, China) with license number SCXK2014-0007. The animal husbandry system was maintained under standard laboratory conditions. All animal operations were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Shandong University of Technology and approved by the Animal Ethics Committee of Shandong University of Technology. Except for the normal control group injected with an equal amount of normal saline, the other mice were injected with cyclophosphamide (CTX) 1 mg / kg body weight for 3 consecutive days. Five normal mice and modeling mice were randomly selected to measure the white blood cell (WBC) and red blood cell (RBC) counts to determine whether the modeling was successful. The mice with successful modeling were divided into 5 groups, with 12 mice in each group. The specific groups were as follows: normal control group, model control group, rhG-CSF high-dose group (0.2 mg / kg), rhG-CSF low-dose group (0.1 mg / kg), C15-rhG-CSF high-dose group (1.0 mg / kg), and C15-rhG-CSF low-dose group (0.5 mg / kg). The specific administration is shown in Table 3. The normal control group and the model control group were subcutaneously injected with normal saline on D1, D2, D3, D4 and D5. On D6 after administration, the mice were killed by cervical dislocation and blood was collected. Blood was collected three times from each mouse for testing, and the number of WBC and RBC was recorded using a hemocytometer.

[0089] Statistical analysis method: All data are expressed as mean ± standard error (mean ± SD). The significance of the difference between the two groups was analyzed using Students' t-test in GraphPad Prism 6 software. P < 0.05 was considered significant. "*" indicates that compared with the normal control group, (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001.

[0090] Table 1 Grouping and administration of rhG-CSF and C15-rhG-CSF

[0091]

[0092]

[0093] from Fig. 9 and 10 It can be seen that the WBC and RBC counts of mice treated with CTX were significantly reduced (P<0.001, compared with the control group), which indicates that the modeling of mice was successful. After the CTX-treated mice were subcutaneously injected with rhG-CSF for 5 consecutive days, the peripheral blood WBC counts of the mice were also improved. However, when 0.1 mg / kg rhG-CSF was administered for 5 consecutive days, the peripheral blood WBC counts of the mice did not return to normal levels, which was significantly different from the normal control group (P<0.05, compared with the control group). When 0.2 mg / kg rhG-CSF was administered for 5 consecutive days, the peripheral blood WBC counts of the mice returned to normal levels. A single subcutaneous injection of C15-rhG-CSF significantly increased the WBC and RBC counts of CTX-treated mice. When 0.5 mg / kg C15-rhG-CSF was administered, the peripheral blood WBC counts returned to the level of the blank control group. When 1.0 mg / kg C15-rhG-CSF was administered, the peripheral blood WBC count returned to normal levels and even exceeded normal levels. In summary, a single subcutaneous injection of C15-rhG-CSF was more effective on peripheral blood cells in mice than multiple subcutaneous injections of rhG-CSF.

Claims

1. A fatty chain-modified human granulocyte colony-stimulating factor derivative as shown in formula (I), in, R1 is C 15 A straight chain alkyl group; R2 is the part of human granulocyte colony stimulating factor excluding the free thiol of the cysteine ​​residue, wherein the human granulocyte colony stimulating factor is a genetically recombinant human granulocyte colony stimulating factor, and the genetically recombinant human granulocyte colony stimulating factor is rhG-CSF, which is a non-glycosylated protein composed of 175 amino acids, has a free cysteine ​​residue Cys 18, and has an additional methionine at the N-terminus compared with the amino acid sequence of natural G-CSF.

2. A method for preparing a fatty chain-modified human granulocyte colony stimulating factor derivative as shown in formula (I), characterized in that The method is: In a dimethyl sulfoxide-sodium phosphate buffer, the fatty chain-maleimide of formula (II) and the human granulocyte colony stimulating factor of formula (III) are reacted at 4-40° C. for 1-120 min, and the obtained reaction solution is separated and purified to obtain the fatty chain-modified human granulocyte colony stimulating factor derivative of formula (I); the dimethyl sulfoxide-sodium phosphate buffer is prepared in a volume ratio of 10-100:90-0, and the pH of the sodium phosphate buffer is 6.5-8.0; the molar ratio of the fatty chain-maleimide of formula (II) to the human granulocyte colony stimulating factor of formula (III) is 10-0.1:1; Among them, R1 is C 15 A straight chain alkyl group; R2 is the part of human granulocyte colony stimulating factor excluding the free thiol of the cysteine ​​residue, wherein the human granulocyte colony stimulating factor is a genetically recombinant human granulocyte colony stimulating factor, and the genetically recombinant human granulocyte colony stimulating factor is rhG-CSF, which is a non-glycosylated protein composed of 175 amino acids, has a free cysteine ​​residue Cys 18, and has an additional methionine at the N-terminus compared with the amino acid sequence of natural G-CSF.

3. The method for preparing the fatty chain modified human granulocyte colony stimulating factor derivative represented by formula (I) as claimed in claim 2, characterized in that: The dimethyl sulfoxide-sodium phosphate buffer solution is prepared in a volume ratio of 90:

10.

4. The method for preparing the fatty chain modified human granulocyte colony stimulating factor derivative represented by formula (I) as claimed in claim 2, characterized in that: The molar ratio of the fatty chain-maleimide represented by formula (II) to the human granulocyte colony stimulating factor represented by formula (III) is 5:

1.

5. The method for preparing the fatty chain modified human granulocyte colony stimulating factor derivative represented by formula (I) as claimed in claim 2, characterized in that: The volume of the dimethyl sulfoxide-sodium phosphate buffer is 0.1-10 mg / mL based on the mass of the human granulocyte colony stimulating factor represented by the formula (III).

6. The method for preparing the fatty chain modified human granulocyte colony stimulating factor derivative represented by formula (I) as claimed in claim 2, characterized in that The post-treatment of separation and purification is as follows: the reaction solution is purified by reverse phase liquid chromatography, the chromatographic column is Bio-C8, the mobile phases A and B are 100% pure water and 100% acetonitrile respectively, 40%-55% mobile phase B linear gradient elution for 5 minutes, 55%-75% mobile phase B linear gradient elution for 20 minutes, the flow rate is 10mL / min, the detection wavelength is 280nm, the eluate containing the target compound is collected, the acetonitrile is removed by rotary evaporation, and dialysis is performed with 10mmol / L pH 4.2 acetate buffer as the dialysate, and the obtained retentate is ultrafiltered and concentrated to obtain the fatty chain modified human granulocyte colony stimulating factor derivative represented by the formula (I).

7. A pharmaceutically acceptable salt of the fatty chain modified human granulocyte colony stimulating factor derivative represented by formula (I) as claimed in claim 1.

8. A preparation of the fatty chain-modified human granulocyte colony-stimulating factor derivative represented by formula (I) as claimed in claim 1.

9. Use of a fatty chain modified human granulocyte colony stimulating factor derivative of formula (I) as claimed in claim 1, a pharmaceutically acceptable salt of a fatty chain modified human granulocyte colony stimulating factor derivative of formula (I) as claimed in claim 7, or a preparation of a fatty chain modified human granulocyte colony stimulating factor derivative of formula (I) as claimed in claim 8 in the preparation of a medicament for treating neutropenia caused by radiotherapy or chemotherapy.

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