CN113999290B - Stable leuprorelin acetate and application thereof - Google Patents
Stable leuprorelin acetate and application thereof Download PDFInfo
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Abstract
The invention discloses stable leuprorelin acetate and application thereof, and relates to the technical field of polypeptide drug preparation. The method for improving the stability of the leuprorelin acetate comprises the following steps: modifying an imidazolyl group on 2-bit histidine of a leuprorelin structure to obtain a leuprorelin conjugate; salifying to obtain a polypeptide solution containing compensating ions, and freeze-drying at ultralow temperature in vacuum to obtain a stable polypeptide product; or modifying hydroxyl groups on the side chain of serine residues of the leuprorelin conjugate by adopting a PLA-co-PEG-Mal copolymer to obtain the conjugate. The leuprorelin prepared by the invention has more excellent stability, the sustained-release action time of the drug is obviously prolonged, and the half-life period is prolonged; and has higher biological activity; meanwhile, the traditional Chinese medicine composition has a certain treatment effect on hyperuricemia combined with insulin resistance.
Description
Technical Field
The invention belongs to the technical field of polypeptide medicine preparation, and particularly relates to stable leuprorelin acetate and application thereof.
Background
The polypeptide medicament has short half-life period in a human body, is unstable in product and is easy to degrade, most of the polypeptide medicaments are injections at present, the method for improving the stability of the polypeptide comprises chemical modification, cyclization and the like, and the method for preparing the polypeptide medicament into the sustained-release microspheres becomes a research hotspot in recent years, so that the aim of improving the action time of the medicament and reducing the decay rate of the medicament in the body is fulfilled. The amino acid structure of the polypeptide is easy to degrade and is sensitive to moisture, light, heat, acid, alkali and oxidation, and the polypeptide drug contains a small amount of moisture, compensating ions and the like, so that the stability of the drug is affected, and the drug is easy to degrade.
Leuprolide Acetate (LA) is a highly active analogue of Luteinizing Hormone Releasing Hormone (LHRH) produced in the hypothalamus (GnRH), is a synthetic water-soluble nonapeptide with two closed ends, has a relative molecular mass of 1269.47, is soluble in water, ethanol and propylene glycol, and has a pKa6.9. When the medicine is used for a short time, the medicine can promote the pituitary to secrete gonadotropin and the testis or the ovary to secrete steroid hormone due to the exciting effect of the medicine on a gonadotropin releasing hormone receptor. When the medicine is used for a long time, the secretion of gonadotropin is inhibited due to the influence of the down-regulation of a receptor, and further, the secretion of steroid hormones from reproductive organs is inhibited. Thus, leuprolide is used clinically to treat or ameliorate a variety of hormone-dependent diseases including: prostate cancer, endometriosis, uterine fibroids, precocious puberty, and the like.
However, LHRH-A is a water-soluble peptide drug, is not easy to permeate biological membranes, is unstable in gastrointestinal tracts, is easy to degrade by digestive enzymes, and has extremely low bioavailability which is less than 0.1 percent, 1 percent, less than 1 percent and 1 to 5 percent when being administrated by oral, nasal, rectal and vaginal routes. And the preparation is unstable in body fluid, has short half-life period of only 16h, so the preparation is not suitable for being prepared into oral preparations and common injections.
Disclosure of Invention
The invention aims to provide stable leuprorelin acetate and application thereof, wherein the leuprorelin has more excellent stability, the sustained-release action time of the drug is obviously prolonged, and the half-life period is prolonged; and has higher biological activity; meanwhile, the traditional Chinese medicine composition has a certain treatment effect on hyperuricemia combined with insulin resistance.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a method of increasing the stability of leuprolide acetate comprising: modifying an imidazolyl group on 2-bit histidine of a leuprorelin structure by adopting 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester to obtain a leuprorelin conjugate; salifying to obtain a polypeptide solution containing compensating ions, and freeze-drying at ultralow temperature in vacuum to obtain a stable polypeptide product;
wherein the chemical structure of the leuprorelin conjugate is shown as formula I or formula II,
the compensating ion is selected from acetic acid. The invention adopts 7-methoxy-2-oxo benzopyran-4-acetic acid N-succinimidyl ester to modify imidazolyl on 2-histidine in a leuprorelin structure to obtain a conjugate, and then the conjugate is subjected to salt transfer and ultra-low temperature vacuum freeze-drying to be freeze-dried, so that polypeptide solution forms more uniform ice crystals, the uniformity of water content and compensating ion content is further improved, and the long-term stability of the leuprorelin medicament is further enhanced. Secondly, the leuprorelin modified by 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester has higher biological activity, for example, the reduction effect of the testosterone concentration in the blood of a rat is obviously improved; the prepared leuprorelin conjugate has excellent effect of reducing blood uric acid, and effectively inhibits the expression of URAT 1; and can improve insulin resistance and relieve metabolic syndrome symptoms; has good effect of treating hyperuricemia and insulin resistance.
Preferably, the structure of the 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimide ester is shown as the formula III,
further, the preparation method of the compound shown in the formula I or the formula II comprises the following steps:
dissolving the protected leuprorelin in DMSO (the solid-to-liquid ratio is 1.5-2.5 g: 1mL), adjusting the pH to 5.0-6.0, adding 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester, reacting for 8-14 h at 30-40 ℃, and then carrying out cation exchange chromatography purification, dialysis and freeze-drying to obtain the protected leuprorelin conjugate;
taking the protected conjugate, adding the protected conjugate into the solution according to the volume ratio of TFA to Tis to H2Stirring the mixture for 4 to 6 hours at room temperature in a solution with O being 94-96: 2-3, precipitating by isopropyl ether, filtering, purifying by sephadex 200 column chromatography, concentrating, and freeze-drying to obtain the leuprorelin conjugate.
Preferably, the protected leuprolide has the structural formula shown below:
preferably, the preparation method of the leuprorelin with the protection is solid phase synthesis, and the preparation process is conventional operation.
Preferably, the molar ratio of the leuprorelin acetate to the 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester is 1: 1-1.4.
Preferably, the conditions for cation exchange chromatography purification are: 18-21 mM PBS pH 6-6.5, sodium chloride gradient: 0 to 0.5M.
Preferably, the salt formation method is reverse phase high performance liquid chromatography.
Preferably, the specific parameter settings of the ultra-low temperature vacuum freeze-drying include: the pre-freezing temperature is set to-160 to-120 ℃; setting the vacuum degree to be 0.2-0.5 mbar; the temperature in the sublimation drying stage is-35 to 0 ℃; the temperature of the desorption drying stage is 30-50 ℃.
More preferably, in the method, the PLA-co-PEG-Mal copolymer is adopted to modify the hydroxyl group on the side chain of the serine residue of the conjugate shown in the formula I or the formula II to obtain the conjugate, so as to replace the salt formation and ultralow temperature vacuum freeze drying operation. The prepared leuprorelin conjugate is directly conjugated with the PLA-co-PEG-Mal copolymer to obtain the conjugate based on the leuprorelin conjugate, so that the good biological activity of the leuprorelin is maintained, the stability of a polypeptide drug can be effectively improved, the sustained release effect of the drug is increased, the half-life period of the drug is prolonged, and the advantage of long-acting drug administration is achieved. The polypeptide drug conjugate prepared by the invention can be prepared into various dosage forms for direct administration, not only achieves the purpose of slow release as microspheres, but also can reduce the generation of side effects, and the use safety is obviously improved; and the preparation method is relatively simple and is more convenient for industrial production.
Further, the preparation method of the PLA-co-PEG-Mal copolymer specifically comprises the following steps:
s1: dehydrating polyethylene glycol, adding toluene into polyethylene glycol, refluxing and azeotroping for 3-5 h at 100-110 ℃, and evaporating under reduced pressure to remove toluene and water to obtain purified polyethylene glycol;
s2: adding levorotatory lactide into xylene serving as a reaction solvent, heating, stirring and dissolving, adding a catalyst stannous octoate, dropwise adding purified polyethylene glycol, controlling the reaction temperature to be 120-135 ℃, and stirring and reacting for 15-20 hours; then, carrying out reduced pressure distillation to remove xylene, adding chloroform for dissolution, methanol for precipitation, filtering, carrying out circulation operation for 2-4 times, and carrying out vacuum drying to obtain an intermediate M;
s3: dissolving the intermediate M in dichloromethane, adding acid-binding agent pyridine, adding maleic anhydride, reacting at 55-65 ℃ for 7-10 h, carrying out reduced pressure distillation and concentration, and adding isopropyl ether to precipitate crystals to obtain the PLA-co-PEG-Mal copolymer.
Preferably, the mass ratio of the levorotatory lactide to the polyethylene glycol in the step S2 is 1: 0.05-0.2; the adding amount of the catalyst stannous octoate is 1.0-2.0% of the mass of the monomer.
Preferably, the molar ratio of the intermediate M to the pyridine in the step S3 is 1: 2-2.1; the molar ratio of the intermediate M to the maleic anhydride is 1: 4-5.5.
Preferably, the PLA-co-PEG-Mal copolymer has a molecular weight of 20-50 kDa.
Further, the preparation method of the conjugate comprises the following steps:
(1) dissolving PLA-co-PEG-Mal copolymer in THF, adding HOSu and DCC, stirring, filtering, crystallizing with isopropyl ether, filtering, washing with water and isopropyl ether in sequence, and vacuum drying to obtain PLA-co-PEG-Mal-OSu;
(2) dissolving the protected leuprorelin conjugate in a PBS (PBS buffer solution with the concentration of 95-110 mM and the pH of 5.0-6.0) to obtain a leuprorelin conjugate solution with the concentration of 2.5-4 mg/mL, adding PLA-co-PEG-Mal-OSu, reacting for 24-36 h at the temperature of 0-4 ℃, adding 1M glycine to terminate the reaction, and then purifying, concentrating and freeze-drying by sephadex 200 column chromatography to obtain a protected conjugate;
(3) deprotection, taking the protected conjugate and adding the volume ratio TFA: Tis: H2Stirring the solution with O being 94-96: 2-3 at room temperature for 4-6 h, precipitating with isopropyl ether, filtering, purifying by sephadex 200 column chromatography, concentrating, and freeze-drying to obtain the conjugate.
Preferably, the molar ratio of the leuprorelin conjugate to the PLA-co-PEG-Mal-OSu in the step (2) is 1: 4.5-5.5.
More preferably, in the preparation process of the conjugate, a modified PLA-co-PEG-Mal copolymer is adopted to replace the PLA-co-PEG-Mal copolymer, and the preparation method of the modified PLA-co-PEG-Mal copolymer specifically comprises the following steps:
s1: dehydrating polyethylene glycol, adding toluene into polyethylene glycol, refluxing and azeotroping for 3-5 h at 100-110 ℃, and evaporating under reduced pressure to remove toluene and water to obtain purified polyethylene glycol;
s2: adding levorotatory lactide and 2',3' -dihydro-2 ',3' -dihydroxy safrole into xylene as a reaction solvent, heating, stirring and dissolving, adding a catalyst stannous octoate, dropwise adding purified polyethylene glycol, controlling the reaction temperature to be 120-135 ℃, and stirring and reacting for 15-20 hours; then, carrying out reduced pressure distillation to remove xylene, adding chloroform for dissolution, methanol for precipitation, filtering, carrying out circulation operation for 2-4 times, and carrying out vacuum drying to obtain an intermediate M;
s3: dissolving the intermediate M in dichloromethane, adding acid-binding agent pyridine, adding maleic anhydride, reacting at 55-65 ℃ for 7-10 h, carrying out reduced pressure distillation and concentration, and adding isopropyl ether to precipitate crystals to obtain the PLA-co-PEG-Mal copolymer. In the preparation process of the PLA-co-PEG-Mal copolymer, 2',3' -dihydro-2 ',3' -dihydroxy safrole is added to modify the PLA-co-PEG-Mal copolymer, so that the prepared modified copolymer has narrower molecular weight distribution; the material has lower thermal decomposition temperature, and completely meets the requirements of biomedical materials; the lower thermal degradation temperature can solve the problem of high treatment cost of the waste medical materials to a certain extent, and the pressure on the environment is reduced. The conjugate is conjugated with polypeptide medicines such as leuprorelin and the like, so that the stability of the polypeptide medicines can be further enhanced, the time of the sustained-release action of the medicines can be remarkably prolonged, the half-life period of the medicines is increased, and the utilization rate of the medicines is further improved. In addition, the existence of 2',3' -dihydro-2 ',3' -dihydroxy safrole in the copolymer ensures that the leuprorelin conjugate has certain efficacy of treating hyperuricemia and insulin resistance; and the leuprorelin conjugate is compounded for use, so that the treatment effect is better.
Preferably, the mass ratio of the levorotatory lactide to the 2',3' -dihydro-2 ',3' -dihydroxy safrole in the step S2 is 1: 0.6-0.8.
The invention also provides the application of the compound shown in the formula III in enhancing the bioactivity and stability of leuprorelin; the stability of leuprorelin and the efficacy of reducing testosterone levels are significantly enhanced.
The invention also provides the application of the compound shown in the formula I or II in preparing a medicament for treating hyperuricemia and insulin resistance. When the leuprorelin conjugate prepared by the invention is applied to animal model experiments, the serum uric acid content in rat serum, the HOMA-IR value and the expression content of renal urate reabsorption transporter are obviously reduced. The leuprorelin conjugate has excellent effect of reducing blood uric acid and can effectively inhibit the expression of URAT 1; and can improve insulin resistance and relieve metabolic syndrome symptoms; therefore, the traditional Chinese medicine composition has a certain treatment effect on hyperuricemia combined with insulin resistance.
The invention also provides the application of the conjugate obtained in the step of preparing a medicament for treating hyperuricemia and insulin resistance.
An agent for treating hyperuricemia complicated with insulin resistance, which comprises a compound of formula I or formula II.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester to modify imidazolyl on 2-histidine in a leuprorelin structure to obtain a conjugate, and then the conjugate is subjected to salt transfer and ultralow temperature vacuum freeze drying for freeze drying, so that the long-term stability of the leuprorelin medicament is further enhanced. The leuprorelin modified by 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester has higher biological activity, has excellent effect of reducing blood uric acid, and effectively inhibits the expression of URAT 1; and can improve insulin resistance and relieve metabolic syndrome symptoms; has good effect of treating hyperuricemia and insulin resistance. In addition, the leuprorelin or the conjugate thereof is directly conjugated with the PLA-co-PEG-Mal copolymer to obtain the conjugate thereof, so that the good biological activity of the leuprorelin is maintained, the stability of a polypeptide medicament can be effectively improved, the sustained release effect of the medicament is increased, the half-life period of the medicament is prolonged, and the medicament has the advantage of longer acting time; the existence of 2',3' -dihydro-2 ',3' -dihydroxy safrole in the copolymer can further enhance the stability of the polypeptide medicament and prolong the sustained-release action of the medicament; meanwhile, the leuprorelin conjugate has certain efficacy of treating hyperuricemia and insulin resistance; and the leuprorelin conjugate is compounded for use, so that the treatment effect is better.
Therefore, the invention provides the stable leuprorelin acetate and the application thereof, the leuprorelin has more excellent stability, the sustained-release action time of the drug is obviously prolonged, and the half-life period is prolonged; and has higher biological activity; meanwhile, the traditional Chinese medicine composition has a certain treatment effect on hyperuricemia combined with insulin resistance.
Drawings
FIG. 1 shows nuclear magnetic hydrogen spectra of leuprolide conjugate (a) and leuprolide acetate (b) prepared in example 1 of the present invention;
FIG. 2 shows the results of IR spectrum measurement in Experimental example 1 of the present invention (A-the copolymer obtained in example 2, B-the modified copolymer obtained in example 3);
FIG. 3 is a DTG curve (A-the copolymer obtained in example 2, B-the modified copolymer obtained in example 3) tested in test example 1 of the present invention;
FIG. 4 shows the results of the test of biological activity of the drug in test example 2 of the present invention;
FIG. 5 shows the results of the test of sustained drug release in Experimental example 3 of the present invention;
FIG. 6 shows the synthesis of conjugate of example 2 of the present invention (taking conjugate of formula I as an example).
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
the polyethylene glycol used in the invention is PEG-400, purchased from Zhengzhou Longyuan chemical Co.
Example 1:
a method for improving the stability of leuprorelin acetate comprises the following steps:
preparation of leuprorelin conjugate
Dissolving protected leuprorelin in DMSO (solid-to-liquid ratio of 2.1 g: 1mL), adjusting pH to 5.6, adding 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester, reacting at 36 deg.C for 13h, purifying by cation exchange chromatography (19.5mM PBS with pH of 6.4, sodium chloride gradient of 0.1, 0.3, 0.5M), dialyzing, and lyophilizing to obtain leuprorelin conjugate. Wherein the molar ratio of the leuprorelin acetate to the 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester is 1: 1.2;
taking the protected conjugate, adding the protected conjugate into the solution according to the volume ratio of TFA to Tis to H2Stirring at room temperature for 6h in a solution with O being 95.5: 2: 2.5, precipitating by isopropyl ether, filtering, purifying by sephadex 200 column chromatography, concentrating, and freeze-drying to obtain the leuprorelin conjugate.
Salt formation
Dissolving leuprorelin conjugate in acetonitrile/water (v/v, 2: 3) mixed solution to obtain leuprorelin conjugate solution (concentration, 0.05g/mL), and performing salt conversion by reverse phase high performance liquid chromatography (chromatographic column: 50 × 250mM specification ODS column, loading amount is 3g, mobile phase 1: 48mM ammonium acetate, mobile phase 2: acetonitrile, mobile phase 3: 0.05% acetic acid aqueous solution), specifically: injecting the leuprorelin conjugate solution into a chromatographic column, adding 4.5% of mobile phase 2/(1+2) for ion exchange, and then washing the target by using 52% of mobile phase 2/(2+ 3); the trans-salted leuprorelin conjugate was concentrated to 100 mg/mL.
Freeze-drying
Pre-freezing, starting a freeze dryer, setting the temperature to be-135 ℃, slowly adding the leuprorelin conjugate concentrated solution into a freeze-drying tray by using an injector, and maintaining the pre-freezing for 3.5 hours;
sublimation drying at vacuum degree of 0.4mbar and temperature of-5 deg.C for 24 hr;
analyzing and drying, setting the vacuum degree to 0.3mbar and the temperature to 32 ℃, and maintaining for 10 hours; then maintaining the temperature at 40 ℃ for 1.5h under the extreme vacuum condition to obtain the stable leuprorelin acetate.
Nuclear magnetism for testing leuprorelin conjugate and leuprorelin acetate1H spectrum (400MHz, CDCl)3) As shown in fig. 1. Compared with leuprorelin, the chemical shift of hydrogen on imidazole of the conjugate is shifted to high field, and the chemical shift value is reduced; and peaks of hydrogen on the benzene ring in the 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester structure at 7.47ppm and 7.35 ppm; the above results indicate that leuprolide conjugate was successfully prepared.
Testing the mass spectrum of the leuprorelin conjugate, the results obtained are: HRMS (ESI), Calcd for C71H92N16O16，m/z[M+H]+，1424.69。
Example 2:
preparation of PLA-co-PEG-Mal copolymer:
s1: dehydrating polyethylene glycol, adding toluene into polyethylene glycol, refluxing and azeotroping at 106 deg.C for 4.5h, and evaporating under reduced pressure to remove toluene and water to obtain purified polyethylene glycol;
s2: adding levorotatory lactide into xylene as a reaction solvent, heating, stirring and dissolving, adding a catalyst stannous octoate, dropwise adding purified polyethylene glycol, controlling the reaction temperature to be 128 ℃, and stirring and reacting for 18 hours; then, removing xylene through reduced pressure distillation, adding chloroform for dissolution, methanol for precipitation, filtering, performing circulation operation for 4 times, and performing vacuum drying to obtain an intermediate M; wherein the mass ratio of the levorotatory lactide to the polyethylene glycol is 1: 0.12; the adding amount of the catalyst stannous octoate is 1.7 percent of the mass of the monomer;
s3: dissolving the intermediate M in dichloromethane, adding acid-binding agent pyridine, adding maleic anhydride, reacting at 62 ℃ for 8.5h, carrying out reduced pressure distillation and concentration, adding isopropyl ether to precipitate crystals to obtain a PLA-co-PEG-Mal copolymer; wherein the molar ratio of the intermediate M to the pyridine is 1: 2.05; the molar ratio of the intermediate M to the maleic anhydride was 1: 4.9. The molecular weight of the copolymer was 32.4 kDa.
A method for improving the stability of leuprorelin acetate comprises the following steps:
leuprolide conjugate was prepared as in example 1.
Preparation of conjugates
(1) Dissolving PLA-co-PEG-Mal copolymer in THF, adding HOSu and DCC, stirring, filtering, crystallizing with isopropyl ether, filtering, washing with water and isopropyl ether in sequence, and vacuum drying to obtain PLA-co-PEG-Mal-OSu;
(2) dissolving the protected leuprorelin conjugate in a PBS (phosphate buffer solution) (the concentration is 105mM, and the pH value is 5.4) to obtain a leuprorelin conjugate solution with the concentration of 3.5mg/mL, adding PLA-co-PEG-Mal-OSu, reacting at 0 ℃ for 30h, adding 1M glycine to stop the reaction, and then carrying out sephadex 200 column chromatography purification, concentration and freeze drying to obtain a protected conjugate; the molar ratio of the leuprorelin conjugate to the PLA-co-PEG-Mal-OSu is 1: 5.1;
(3) deprotection, taking the protected conjugate and adding the volume ratio TFA: Tis: H2Stirring at room temperature for 5h in a solution of O95.5: 2: 2.5, precipitating with isopropyl ether, filtering, purifying by Sephadex 200 column chromatography, concentrating, and freeze-drying to obtain the conjugate.
The preparation route of the conjugate is shown in figure 6 (taking the form of the conjugate shown in formula I as an example).
Example 3:
the difference between the method for improving the stability of the leuprolide acetate and the method in the embodiment 2 is that: the PLA-co-PEG-Mal copolymer is replaced by the modified PLA-co-PEG-Mal copolymer.
The difference between the preparation of the modified PLA-co-PEG-Mal copolymer and the preparation of the PLA-co-PEG-Mal copolymer in example 2 is that: step S2, adding 2',3' -dihydro-2 ',3' -dihydroxy safrole; the mass ratio of the levorotatory lactide to the 2',3' -dihydro-2 ',3' -dihydroxy safrole is 1: 0.73. The molecular weight of the copolymer was 35.2 kDa.
Example 4:
the difference between the method for improving the stability of the leuprolide acetate and the method in the embodiment 3 is that: leuprolide is used instead of leuprolide conjugate.
Comparative example 1:
the difference between the method for improving the stability of the leuprolide acetate and the method in the embodiment 1 is that: leuprolide is used instead of leuprolide conjugate.
Comparative example 2:
the difference between the method for improving the stability of the leuprolide acetate and the method in the embodiment 2 is that: leuprolide is used instead of leuprolide conjugate.
Test example 1:
1. infrared characterization
Testing a sample by adopting a Fourier transform infrared spectrometer, dissolving the sample by using trichloromethane, tabletting and coating by using KBr, heating to completely volatilize the solvent, and then testing at 4000-500 cm-1And internally scanning and analyzing the structures of all groups in the sample.
The PLA-co-PEG-Mal copolymer obtained in example 2 and the modified PLA-co-PEG-Mal copolymer obtained in example 3 were subjected to the above-described tests, and the results are shown in FIG. 2. Analysis of the graph shows that compared with the spectrum of the PLA-co-PEG-Mal copolymer prepared in example 2, the infrared spectrum of the modified PLA-co-PEG-Mal copolymer prepared in example 3 is 1650-1500 cm-1The characteristic absorption peak of benzene ring skeleton vibration appears in the range, which indicates the existence of 2',3' -dihydro-2 ',3' -dihydroxy safrole in the modified PLA-co-PEG-Mal copolymer, and proves that the copolymer in example 3 is successfully prepared.
2. Molecular weight distribution test
The molecular weight (M) of the product was determined by Gel Permeation Chromatography (GPC)w). The sample was completely dissolved in spectral THF and filtered through a microfiltration membrane at a concentration of 14 mg/L. THF is used as a mobile phase in the test, the flow rate is 1.2mL/min, and the test temperature is (30 +/-1) DEG C.
The above tests were performed on the PLA-co-PEG-Mal copolymer prepared in example 2 and the modified PLA-co-PEG-Mal copolymer prepared in example 3, and the results are shown in Table 1:
TABLE 1 molecular weight distribution test results
| Test sample | Mw/Mn |
| Example 2 | 1.37 |
| Example 3 | 1.12 |
As can be seen from the data in Table 1, M of the copolymer obtained in example 3w/MnThe value is obviously less than that of example 2, which shows that the addition of 2',3' -dihydro-2 ',3' -dihydroxysafrole in the preparation process of the copolymer enables the obtained copolymer to have narrower molecular weight distribution, more uniform molecular weight distribution and molecular chain length, and further improves the comprehensive performance of the copolymer.
3. Thermal stability test
Sample 4mg in an aluminum sample pan, N2Under the protection of atmosphere, the temperature is raised from 25 ℃ to 600 ℃ at the speed of 10 ℃/min, and the temperature raising curve is recorded.
The above tests were performed on the PLA-co-PEG-Mal copolymer prepared in example 2 and the modified PLA-co-PEG-Mal copolymer prepared in example 3, and the DTG curve of the sample is shown in FIG. 3. As can be seen from the graph, the degradation temperature corresponding to one maximum weight loss rate of the copolymer prepared in example 3 is lower than that of example 2, indicating that the addition of 2',3' -dihydro-2 ',3' -dihydroxysafrole during the preparation of the copolymer lowers the degradation temperature of the copolymer, which is completely satisfactory in the biomedical field; the copolymer is applied to medical materials, the problem of high treatment cost of waste materials can be solved due to the lower thermal degradation temperature, and the pressure on the environment is reduced.
Test example 1:
biological activity assay
Subject: SD rats with weight of 280-300 g;
grouping experiments: 35 rats were randomly divided into 7 groups, including: experimental group, M1 group: high stability leuprolide acetate from example 1; group M2: the conjugate prepared in example 2; group M3: the conjugate prepared in example 3; m4: the conjugate prepared in example 4. Control, group D1: leuprorelin acetate; group D2: high stability leuprolide acetate prepared in comparative example 1. Blank group N: no drug was added.
Experimental treatment and test methods: the experimental group and the control group were injected subcutaneously with the drug daily, and the dose (measured by leuprorelin) was 0.08 mg/kg; the blank group was injected daily with an equal amount of saline subcutaneously. Blood was then taken from the rat fundus venous plexus at 1d, 3d, 6d, 12d, 15d, 18d, 21d, 24d, 30d, respectively, and testosterone concentrations were determined by radioimmunoassay.
And (4) analyzing results: the results are shown in fig. 4, from which it can be seen that the testosterone content in the plasma of rats is significantly reduced after the treatment of the experimental group and the control group compared to the blank group. The testosterone content in the plasma of the rats treated by the M1 group is obviously lower than that of D2, which shows that the inhibition effect of the leuprorelin on the testosterone content can be effectively enhanced after the leuprorelin is subjected to coupling treatment, and the biological activity is obviously improved; and the maintenance time of the biological activity of the M1 group after treatment is longer than that of the D2 group, which shows that the leuprorelin is modified by 7-methoxy-2-oxo benzopyran-4-acetic acid N-succinimidyl ester to obtain the conjugate thereof, and the stability of the polypeptide medicament can be enhanced to a certain degree. The maintenance time of the biological activity of the M2 group after treatment is longer than that of the D1 group and the M1 group, and the effect of the M4 group is better than that of the D2 group, which shows that the conjugation modification of the leuprorelin by adopting the PLA-co-PEG-Mal copolymer or the modified PLA-co-PEG-Mal copolymer can obviously improve the stability of the medicine and effectively prolong the maintenance time of the activity without reducing the biological activity of the leuprorelin. In addition, the effect of the M3 group is remarkably better than that of the M2 group, which shows that the improvement of the drug stability is enhanced by adopting the 2',3' -dihydro-2 ',3' -dihydroxy safrole modified PLA-co-PEG-Mal copolymer and then treating the leuprorelin.
Test example 2:
in vivo pharmacokinetic Studies
Subject: SD rats with weight of 280-300 g;
the experimental groups were grouped as in test example 1;
experimental treatment and test methods: after fasting the rats overnight, the drugs were injected subcutaneously at a dose (measured as leuprorelin) of 0.08 mg/kg; the blank group was injected daily with an equal amount of saline subcutaneously. Then blood is taken from the fundus venous plexus of the rat for 0, 0.5, 1, 2, 4, 6, 8, 12, 16 and 24 hours respectively, serum is taken by centrifugation, and the blood concentration is measured by a radioimmunoassay.
And (4) analyzing results: the test results are shown in fig. 5, and it can be seen from the analysis of the graph that the peak blood concentration is reached within 2h after the treatment of the experimental group and the control group. After the treatment of the M1 group, the change trend of the blood concentration is equivalent to that of the D1 group and the D2 group, and the blood concentration is rapidly reduced after reaching the peak value, after the treatment of the M2 group, the blood concentration is reduced to (5.94 +/-0.21) ng/mL within 2 hours after reaching the peak value, and the reduction trend of the blood concentration is obviously slowed down within 4 hours, which indicates that the PLA-co-PEG-Mal copolymer is adopted to carry out conjugation modification on the leuprorelin, so that the slow release time of the drug can be effectively prolonged. In addition, after the M3 group is treated, the blood concentration is reduced to (6.71 +/-0.18) ng/mL in 2 hours after reaching the peak value, and then the blood concentration is basically kept stable in 6 hours, which shows that the time of the drug slow release effect can be remarkably prolonged by adopting the 2',3' -dihydro-2 ',3' -dihydroxy safrole modified PLA-co-PEG-Mal copolymer and then treating leuprorelin.
Test example 3:
the experimental object for treating hyperuricemia and insulin resistance activity comprises the following components: SPF-grade Wistar male rats, 5-7 weeks old, 165-190 g in weight, were purchased from Beijing Wintolite laboratory animal technologies, Inc.
And (3) experimental modeling: 45 rats were bred adaptively for one week at 23 + -1 deg.C, humidity 56 + -5%, 12h/12h light/dark cycle, and were allowed to eat water ad libitum. Feeding the blank group (5) with D12450H purified feed, and freely drinking purified water; building a module (40) for D12451 high-fat purified diet feeding, drinking purified water and 10% fructose water freely, and intragastric gazing with 10% oteracil potassium suspension (dose, 750 mg/kg); after the breeding is continued for 8 weeks, blood is taken from the inner canthus after fasting for 1d without water prohibition, fasting Insulin (INS), fasting blood glucose (FPG) and blood Uric Acid (UA) are detected, and IR index (HOMA-IR) is calculated. After statistical analysis, the difference of the level of the model building group and the level of UA and HOMA-IR of the blank group has statistical significance, which indicates that the model building is successful.
Grouping experiments: after the rats were successfully modeled, 35 rats were randomly divided into 8 groups, including: experimental group, group K1: high stability leuprolide acetate from example 1; group M2: the conjugate prepared in example 2; group M3: the conjugate prepared in example 3; m4: the conjugate prepared in example 4. Control, group D1: leuprorelin acetate; group D2: the high stability leuprorelin acetate prepared in comparative example 1; group D3: conjugate prepared in comparative example 2. Model group: no drug was added.
Experimental treatment: high-fat feed, fructose diet and oteracil potassium are continuously given to each group for intragastric administration, and subcutaneous injection medicine is given to the experimental group and the control group, and the dosage (measured by leuprorelin) is 0.08 mg/kg; the model group was injected subcutaneously with an equivalent amount of physiological saline daily; each group was continuously intervened for 4 weeks. And then fasting without water prohibition for 1d, and administrating potassium oxonate to the other groups except the blank group after intragastric administration for 4 h. After administration for 2h, 3% pentobarbital is adopted to anaesthetize and draw materials, and the abdominal aorta is used for blood sampling to measure fasting blood glucose; the supernatant was extracted after centrifugation of the blood and stored in a refrigerator at-80 ℃. And the liver is taken and placed in liquid nitrogen for preservation, and the kidney is taken and placed in 4% formaldehyde solution for preservation.
Detection index and method
Detecting UA and FPG in serum by using a full-automatic biochemical analyzer;
HOMA-IR: HOMA-IR was calculated according to the international algorithm: HOMA-IR INS × FPG/22.5;
adding a diluted rat serum sample according to an ELISA kit operation instruction, placing the diluted rat serum sample at 37 ℃ for incubation for 30min, removing liquid in a hole, washing the rat serum sample for 5 times, adding a substrate working solution, incubating the rat serum sample at 37 ℃ in a dark place for 20min, adding a stop solution, reading a light absorption value at 450nm to prepare a standard curve, and finally calculating the corresponding INS concentration according to an OD value.
Renal urate reabsorption transporter (URAT1) expression: taking the kidney cortex as a paraffin section, measuring by an immunohistochemical SABC method, staining by a DAB method, and then carrying out the steps according to the instruction. Sections were observed and photographed under the mirror and semi-quantitative analysis was performed using Image-Pro Plus 6.0 Image analysis software, and the results were expressed as integrated optical density values (IOD SUM).
Analysis of results
The test results are shown in table 2:
table 2 results of various index tests
| Experimental group | UA(μmol/L) | HOMA-IR | IOD SUM |
| Blank group | 95.41±5.62 | 0.14±0.08 | 2210.41±451.43 |
| Model (model)Group of | 241.86±12.45 | 0.99±0.15 | 4107.10±516.13 |
| D1 group | 238.49±11.67 | 0.98±0.11 | 4099.47±473.69 |
| D2 group | 235.74±13.22 | 0.97±0.19 | 4090.65±379.94 |
| D3 group | 235.04±14.05 | 0.97±0.14 | 4089.55±401.82 |
| M1 group | 129.18±9.28 | 0.52±0.15 | 2591.69±297.11 |
| M2 group | 121.93±14.09 | 0.47±0.17 | 2586.36±325.52 |
| M3 group | 94.91±10.71 | 0.30±0.13 | 2294.63±251.23 |
| M4 group | 198.67±12.99 | 0.71±0.20 | 3457.17±386.56 |
As can be seen from the data in Table 2, compared with the blank group, the index levels of the model group are significantly different, indicating that the molding is successful. Compared with the model group, after the M1 group medicaments are treated, the blood uric acid content, the HOMA-IR value and the expression content of renal urate reabsorption transporters in the serum of rats are obviously reduced and are obviously lower than those of the D1-D2 group, and each index in the serum of the rats treated by the D1-D2 group is equivalent to that of the model group, so that the coupling compound is obtained by coupling the leuprorelin by adopting the 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester, the coupling compound has an excellent effect of reducing the blood uric acid, and the expression of URAT1 is effectively inhibited; and can improve Insulin Resistance (IR) status, relieve metabolic syndrome symptoms; has good effect of treating hyperuricemia and insulin resistance. The effect of the M3 group is obviously better than that of the M2 group, and the effect of the M4 group is better than that of the D3 group, which shows that the 2',3' -dihydro-2 ',3' -dihydroxy safrole modified PLA-co-PEG-Mal copolymer is adopted to treat leuprorelin, so that the leuprorelin has the effects of reducing uric acid and improving IR to a certain extent; and when the composition is compounded with leuprorelin, the composition has better treatment effect on hyperuricemia combined with insulin resistance.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (6)
1. A method of increasing the stability of leuprolide acetate comprising: modifying an imidazolyl group on 2-bit histidine of a leuprorelin structure by adopting 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimidyl ester to obtain a leuprorelin conjugate; salifying to obtain a polypeptide solution containing compensating ions, and freeze-drying at ultralow temperature in vacuum to obtain a stable polypeptide product;
the chemical structure of the leuprorelin conjugate is shown as a formula I or a formula II,
the compensating ion is selected from acetic acid.
2. The method of claim 1, wherein the leuprolide acetate stability is increased by: the structure of the 7-methoxy-2-oxobenzopyran-4-acetic acid N-succinimide ester is shown as a formula III,
3. the method of claim 1, wherein the leuprolide acetate stability is increased by: in the method, a PLA-co-PEG-Mal copolymer is adopted to modify a hydroxyl group on a side chain of a serine residue of the conjugate shown in the formula I or the formula II to obtain a conjugate of the conjugate, so that salt formation and ultralow-temperature vacuum freeze drying operations are replaced.
4. The method of claim 3, wherein the stability of leuprolide acetate is increased by: the PLA-co-PEG-Mal copolymer has a molecular weight of 20-50 kDa.
5. Use of a compound of formula I or formula II for the manufacture of a medicament for the treatment of hyperuricemia associated with insulin resistance, said compound of formula I or formula II having the structure:
6. an agent for treating hyperuricemia with insulin resistance, comprising a compound of formula I or formula II as described in claim 5.
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