Polyorthoester in-vivo analysis method and application thereof
By tracking the degradation process and drug release behavior of polyorthoester in vivo, using experimental animal models and efficient chromatographic analysis technology, the problems of drug safety and effectiveness in the existing technology are solved, and the drug release system is optimized.
Patent Information
- Application Number
- CN202410786410.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-18
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art is difficult to accurately track and evaluate the degradation process and drug release behavior of polyorthoster in vivo, affecting the safety and effectiveness of drugs.
By selecting experimental animal models, injecting and sampling at different time points, polyorthoesters and their degradation products are extracted and separated, and qualitative and quantitative analysis is performed using liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry to establish a standard curve and mass concentration, and histological and biological indicators are tested.
Accurate tracking of the degradation process of polyorthoesters in vivo and evaluation of drug release behavior is achieved, which improves the safety and effectiveness of drugs and optimizes the drug release system.
Smart Images
Figure CN119125335A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a polyorthoester in vivo analysis method and application thereof. Background Art
[0002] Biomaterials are widely used in the biomedical field. Poly(ortho esters) POE, a biodegradable polymer material first synthesized in the 1960s, has developed rapidly due to its good performance. Moreover, the application of poly(ortho esters) pharmaceutical excipients in the medical field has made certain progress. This type of material is mainly used in drug delivery systems, especially in the delivery system of biological drugs. For example, they are used to prepare injectable in situ gels, which can form a semi-solid state after being injected into the body, thereby achieving a sustained release effect of the drug. These in situ gel products have an irreplaceable position in disease treatment, especially in the treatment of mental illness, pain and other diseases.
[0003] At present, a variety of in situ gel injections based on polyorthoesters have been approved worldwide, including drugs for analgesia, psychiatric treatment and local anesthesia. The therapeutic advantage of these drugs is that they can provide a long-term sustained-release effect, reduce the frequency of injection, and thus improve patient compliance. Polyorthoesters are hydrophobic polymers prepared by condensing polyorthoacids or polyorthoesters with polyols under anhydrous conditions to form orthoester bonds. They are insoluble in water and do not swell in aqueous solutions. They are soluble in organic solvents such as cyclohexane and tetrahydrofuran. Its degradation in the body is caused by the hydrolysis reaction at the orthoester bond. The final degradation product is a water-soluble small molecule with little toxicity and side effects, and is easily metabolized by the body.
[0004] In addition to the excellent properties of surface dissolution, polyorthoesters also have acid sensitivity and adjustability of glass transition temperature. The acid sensitivity of polyorthoesters makes it possible to be used as a drug delivery system, and the drug release rate is mainly regulated by the potential acid excipients or acidic environment in the polymer. The hydrolysis of POE is mainly divided into three processes. The first is to hydrolyze the polymer fragments containing carboxyl terminals, which can catalyze the hydrolysis of orthoesters. After that, the free lactic acid released will further catalyze the hydrolysis of orthoesters. Then, the orthoesters are hydrolyzed in two steps to first produce diols and pentaerythritol dipropionate, and finally hydrolyze to pentaerythritol and propionic acid. Using electron paramagnetic resonance to monitor the degradation of polyorthoesters, some studies have found that the degradation process of POE is mainly surface erosion, but it also includes the overall erosion process.
[0005] The applicant has synthesized and developed a series of polyorthoester materials. In order to study the degradation of polyorthoesters in vivo and in vitro simulating in vivo, an analytical method was developed to simultaneously detect polyorthoesters and their degradation products. Summary of the invention
[0006] In view of this, the present invention proposes an in vivo analysis method of polyorthoesters and its application to study the degradation of polyorthoesters in vivo and in vitro in a simulated in vivo environment, and to detect polyorthoesters and their degradation products.
[0007] In one aspect, the present invention provides a polyorthoester in vivo analysis method, comprising:
[0008] S1: Determine and select the experimental animal model;
[0009] S2: administering the drug by injection, and taking samples from the experimental animal model at different time points after administration, and tracking the degradation process of the polyorthoester through the sampling;
[0010] S3: extracting and separating the polyorthoester and its degradation products, and performing qualitative and quantitative analysis on the degradation products;
[0011] S4: Establish the standard curve and mass concentration relationship through analysis results.
[0012] Optionally, the different time points in S2 are 0, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours.
[0013] Optionally, the qualitative and quantitative analysis of the degradation products in S3 is performed by liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry.
[0014] Optionally, S2 further includes: performing histological analysis on the tissues of the experimental animal model by sampling to evaluate the effect of the in vivo degradation of the poly(orthoester) on the tissues.
[0015] Optionally, the histological analysis includes pathological examination and immunohistochemical staining.
[0016] Optionally, S2 further includes detecting biological indicators in the experimental animal model to evaluate the effect of the in vivo degradation of the polyorthoester on the biological process.
[0017] Optionally, the biological indicators include inflammatory factor levels and cell apoptosis.
[0018] Optionally, the degradation products include triethylene glycol, pentaerythritol, ethylene glycol, glycolide, glycolic acid and propionic acid.
[0019] Compared with the prior art, the beneficial effects of the present invention are: by selecting an appropriate experimental animal model and sampling at the same time point after administration, the degradation process of polyorthoester in the body can be tracked more accurately, thereby optimizing the drug release system. By extracting and separating polyorthoester and its degradation products, and using liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry for qualitative and quantitative analysis, the degradation products of polyorthoester can be detected more sensitively and specifically. By establishing a standard curve and mass concentration relationship, the degradation rate and drug release behavior of polyorthoester can be more accurately evaluated. By performing histological analysis on the tissues of the experimental animal model, the effect of the in vivo degradation of polyorthoester on the tissue can be evaluated, thereby improving the safety of the drug. By detecting the biological indicators in the experimental animal model, the effect of the in vivo degradation of polyorthoester on the biological process can be evaluated, thereby improving the effectiveness of the drug.
[0020] On the other hand, the present application also provides an application of an in vivo analysis method for poly(orthoester), including the application of the above-mentioned analysis method in the preparation of ophthalmic disease drugs, pain relief drugs, polypeptide and protein drugs, and vaccines.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: the polyorthoester in vivo analysis method provided by the present application has significant advantages in the preparation of ophthalmic disease drugs, pain relief drugs, peptide and protein drugs, and vaccines. First, the method can effectively track and evaluate the degradation process and drug release behavior of polyorthoester in vivo through precise qualitative and quantitative analysis, thereby optimizing the drug release system and improving drug efficacy. Secondly, through the detection of histological and biological indicators, the effect of polyorthoester in vivo degradation on tissues and biological processes can be comprehensively evaluated to ensure the safety and effectiveness of the drug. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:
[0023] Figure 1 This is a weight change curve diagram of the auxiliary degradation experiment of an embodiment of the present invention;
[0024] Figure 2 This is a schematic diagram of the tissue compatibility test results of an embodiment of the present invention;
[0025] Figure 3 This is a schematic diagram of the hemolytic test results of an embodiment of the present invention;
[0026] Figure 4 It is a DETOSU gas phase diagram of an embodiment of the present invention. DETAILED DESCRIPTION
[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0028] Studying the in vivo degradation of pharmaceutical polymer excipients, poly(orthoesters), has important theoretical value and practical significance.
[0029] First, as a hydrophobic polymer, polyorthoester has unique drug controlled release properties. By adjusting its molecular structure, the drug release rate can be precisely controlled, thereby improving the efficacy of the drug and reducing side effects. Therefore, a deep understanding of the degradation behavior of polyorthoester in the body is crucial to optimizing its drug controlled release performance.
[0030] Secondly, polyorthoesters have broad application prospects in the medical field. It can be used as a drug carrier to accurately deliver drugs to the lesion site and improve the therapeutic effect of the drug. At the same time, the biocompatibility and degradability of polyorthoesters also make it an important candidate for biomedical materials. Therefore, studying the in vivo degradation mechanism of polyorthoesters is important for promoting its application in the medical field. In addition, by studying the in vivo degradation of polyorthoesters, we can gain a deep understanding of its interaction process with the organism. This helps to predict and evaluate the safety and effectiveness of polyorthoesters in the body, and provides an important theoretical basis for drug development and clinical application. Finally, the in vivo degradation study of polyorthoesters can also promote scientific and technological progress in related fields. For example, by improving the synthesis method of polyorthoesters and optimizing their degradation conditions, their drug controlled release performance and biocompatibility can be further improved. At the same time, this will also promote the cross-integration of multiple disciplines such as polymer science, biomedical engineering, and pharmaceutical preparation, and promote innovation and development in related fields.
[0031] In summary, studying the in vivo degradation of pharmaceutical polymer excipients poly(orthoesters) is of great significance for optimizing drug controlled release performance, promoting its application in the medical field, gaining a deeper understanding of its interaction with organisms, and promoting scientific and technological progress.
[0032] In some embodiments of the present application, this embodiment provides a polyorthoester in vivo analysis method, comprising:
[0033] S1: Determine and select the experimental animal model;
[0034] S2: administering the drug by injection, and taking samples from the experimental animal model at different time points after administration, and tracking the degradation process of the polyorthoester through the sampling; the sampling includes blood samples or in vitro simulated biological fluids to track the degradation process of the polyorthoester;
[0035] S3: extracting and separating the polyorthoester and its degradation products, and performing qualitative and quantitative analysis on the degradation products;
[0036] S4: Establish the standard curve and mass concentration relationship through analysis results.
[0037] In this preferred embodiment, the different time points in S2 are 0, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours.
[0038] It is understandable that this embodiment provides a method for analyzing polyorthoesters in vivo, which implements tracking of the degradation process of polyorthoesters by administering drugs to experimental animal models and selecting samples at different time points after administration. By extracting and separating polyorthoesters and their degradation products and performing qualitative and quantitative analysis on them, the degradation of polyorthoesters in vivo can be more accurately understood. In addition, by establishing a standard curve and a mass concentration relationship through the analysis results, an important reference basis is provided for subsequent research and application. In a preferred embodiment, the selected time points are relatively dense, so that the degradation process of polyorthoesters can be more comprehensively monitored, thereby improving the accuracy and reliability of the analysis.
[0039] In this preferred embodiment, the qualitative and quantitative analysis of the degradation products in S3 is performed by liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry.
[0040] In this preferred embodiment, the step S2 further includes: performing histological analysis on the tissues of the experimental animal model by sampling to evaluate the effect of the in vivo degradation of the polyorthoester on the tissues.
[0041] In this preferred embodiment, the histological analysis includes pathological examination and immunohistochemical staining.
[0042] In this preferred embodiment, the step S2 further includes detecting biological indicators in the experimental animal model to evaluate the effect of the in vivo degradation of the polyorthoester on the biological process.
[0043] In this preferred embodiment, the biological indicators include inflammatory factor levels and cell apoptosis.
[0044] It can be understood that in this preferred embodiment, a more comprehensive evaluation of the in vivo degradation process of polyorthoesters is performed. After step S2, histological analysis of experimental animal model tissues is added, which helps to observe and evaluate the effects of polyorthoesters on surrounding tissues when they degrade in vivo. Histological analysis includes pathological examination and immunohistochemical staining, which can provide detailed information on changes in tissue structure and expression of specific proteins, thereby providing a deeper understanding of the potential effects of polyorthoester degradation on tissues.
[0045] In addition, this embodiment also involves the detection of biological indicators in the experimental animal model, which further supplements the evaluation of the degradation effect of polyorthoesters in vivo. The biological indicators detected include inflammatory factor levels and cell apoptosis, which can reflect the effects of polyorthoester degradation on biological processes, such as inflammatory response and cell survival rate. Through these tests, the overall impact of the degradation products of polyorthoesters on organisms can be more comprehensively evaluated, providing important safety and effectiveness information for the application of polyorthoesters. These additional analysis steps enhance the design of the overall study and ensure a comprehensive understanding of the in vivo behavior of polyorthoesters, thereby providing a solid foundation for future applications and further research.
[0046] In this preferred embodiment, the degradation products include triethylene glycol, pentaerythritol, glycolic acid and propionic acid.
[0047]
[0048] In summary, it can be seen that the present application can more accurately track the degradation process of polyorthoesters in vivo by selecting appropriate experimental animal models and sampling at the same time after administration, thereby optimizing the drug release system. By extracting and separating polyorthoesters and their degradation products, and using liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry for qualitative and quantitative analysis, the degradation products of polyorthoesters can be detected more sensitively and specifically. By establishing a standard curve and a mass concentration relationship, the degradation rate and drug release behavior of polyorthoesters can be more accurately evaluated. By performing histological analysis on the tissues of the experimental animal model, the effect of the in vivo degradation of polyorthoesters on the tissues can be evaluated, thereby improving the safety of the drug. By detecting the biological indicators in the experimental animal model, the effect of the in vivo degradation of polyorthoesters on the biological process can be evaluated, thereby improving the effectiveness of the drug.
[0049] Furthermore, the above detection also includes the purity detection of the intermediate 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane, and the detection method includes:
[0050] The purity of 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane was determined by gas chromatography and area normalization.
[0051] Example 1 Biological Sample Acquisition
[0052] Pharmacokinetics test: Prepare polyorthoester I injection at an appropriate concentration. 12 hours before the experiment, fast 10 male SD rats (age about 8 weeks, weight about 200g) but not water, and give the above injection. After administration, about 0.5mL of blood was collected from the eye socket at 0.5, 2, 6, 12, 24, 48, 72, 96 and 120h, placed in a 1.5mL heparinized centrifuge tube, and quickly centrifuged at 4000rpm for 5min in a centrifuge. Take the upper plasma and freeze it at -20℃. The plasma sample was processed according to the plasma sample processing method under "Plasma sample processing" and then measured.
[0053] Treatment of plasma samples: 100 μL of rat plasma sample was placed in a 1.5 mL centrifuge tube, 25 μL of methanol and 100 μL of sodium hydroxide (0.5 mol / L) were added, vortexed for 30 s, 1 mL of acetonitrile was added, vortexed for 1 min, centrifuged at room temperature for 5 min (5000 r / min), the supernatant was separated and placed in a blank 1.5 mL centrifuge tube, and the organic solvent was evaporated under nitrogen flow at 40°C. 400 μL of mobile phase was added to the residual material, vortexed for 1 min to fully dissolve, centrifuged again at room temperature for 5 min (5000 r / min), and 200 μL of supernatant was aspirated into the sample injection plate for testing.
[0054] Embodiment 2
[0055] The purity of 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane is tested by a specific example below:
[0056] Instrument used: Trace-1300 gas chromatograph;
[0057] Detector: Hydrogen flame ionization detector;
[0058] Chromatographic column: DB-1 chromatographic column (30m×0.320mm×0.25μm);
[0059] Column temperature: programmed temperature, initial temperature: 40°C, hold for 4 min; increase the temperature to 200°C at 20°C / min, hold for 18 min;
[0060] Detection time: 30 min; injection port temperature: 200-°C; detector temperature: 250-°C;
[0061] Sample concentration: 2 mg / mL; weigh 20 mg of 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane sample, dissolve it in 10 mL of n-heptane, filter it through a 0.22 μm nylon filter membrane, and take the filtrate into a liquid phase vial for sampling and detection.
[0062] Result: Purity: 98.35%.
[0063] It can be understood that the detection process using Trace-1300 gas chromatograph and hydrogen flame ionization detector to detect the purity of 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane has significant advantages. First, the selection of DB-1 chromatographic column and optimized temperature program can achieve effective separation and detection of the sample to be tested, ensuring the accuracy of the results. Secondly, by setting appropriate sample concentration and injection volume, combined with a high-sensitivity detector, the sensitivity and reproducibility of the detection can be improved. In addition, the use of 0.22μm nylon filter membrane to filter the sample helps to remove impurities and further ensure the purity of the test results. Finally, the method has moderate detection time, simple operation, and reliable results. It is suitable for the purity detection of 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane, providing strong support for its application in drug preparation and quality control. In general, the detection process has the advantages of high efficiency, accuracy, reliability, and easy operation, and has important application value.
[0064] Among them, Example 2 refers to the general chapter "0521 Gas Chromatography Determination Method (3) Area Normalization Method" of the 2020 edition of the "Chinese Pharmacopoeia" to detect the purity of DETOSU.
[0065] 1) Instrument
[0066] Chromatograph: Trace1300 gas chromatograph;
[0067] Detector: flame ionization detector (FID);
[0068] Chromatographic column: DB624 chromatographic column (30mx0.25mmx1.4um).
[0069] 2) Gas chromatography conditions
[0070] Chromatographic column: DB624 chromatographic column (30mx0.25mmx1.4um);
[0071] Column temperature: programmed temperature, initial temperature 40°C, hold for 4 min, increase to 200°C at 20°C / min and hold for 10 min;
[0072] Detector: F ID detector;
[0073] Inlet temperature: 200°C; Detector temperature: 250°C;
[0074] Injection method: direct liquid injection;
[0075] Carrier gas: nitrogen;
[0076] Carrier gas flow rate: 0.8 mL / min;
[0077] Injection volume: 4uL;
[0078] Split injection: split ratio 19:1.
[0079] 3) Sample determination
[0080] Weigh 20 mg of intermediate I accurately into a 10 mL volumetric flask, dilute to the mark with DMSO as solvent, shake well, and inject for testing. The substance with a retention time of 22.028 min is identified as impurity I, the substance with a retention time of 23.645 min is the main peak of DETOSU, and the substance with a retention time of 24.860 min is impurity II. Figure 4 This is the gas phase diagram of DETOSU.
[0081] Example 3 Residual Monomer (Glycolide) Detection
[0082] 1) Instrument
[0083] Chromatograph: Trace 1300 gas chromatograph;
[0084] Detector: flame ionization detector (FID);
[0085] Chromatographic column: DB-5 chromatographic column (15mx0.32mmx1 um).
[0086] 2) Chromatographic conditions
[0087] Chromatographic column: DB-5 chromatographic column (15mx0.32mmx1 um);
[0088] Column temperature: programmed temperature, starting at 30°C, increasing to 170°C at 10°C / min, and maintaining for 2 min;
[0089] Inlet temperature: 180°C; Detector temperature: 300°C;
[0090] Detector: F ID detector;
[0091] Inlet temperature: 200°C; detector temperature: 250°C;
[0092] Injection method: liquid injection;
[0093] Carrier gas flow rate: 0.8 mL / min;
[0094] Hydrogen flow rate: 30.0mL / min; compressed air flow rate: 400mL / min; nitrogen gas flow rate: 35mg / mL; injection volume: 1mL; split injection: split ratio 40:1;
[0095] Weigh about 50 mg of the intermediate accurately, add it to a 10 mL volumetric flask, dissolve it with a small amount of dichloromethane and dilute to the mark. Inject the sample according to the chromatographic conditions and detect the peak area.
[0096] 3) Results
[0097] The contents calculated according to the standard curve were 11.34%, 10.78% and 10.14% respectively;
[0098] Next, we conducted an excipient degradation test to investigate the stability of the excipient in PBS and in the presence of lipase, and calculated the weight change, such as Figure 1 shown.
[0099] Meanwhile, the glycolide content was detected by mass spectrometry. The mass spectrometer was ACQUITY UPLC I Class and Xevo TQ-S micro LC-MS (Waters Technology (Shanghai) Co., Ltd.), and the detection was performed by electrospray ionization source ESI (21 eV).
[0100] Embodiment 4
[0101] The following is a histological analysis, specifically a tissue compatibility test. The polymer was injected into the subcutaneous tissue behind the neck of rats. The experimental animals were killed and dissected at different times after injection (7d, 14d, and 21d). The drug-administered site and major organs were sliced and observed under a microscope after HE staining. Compared with the control group (normal saline), no obvious abnormalities were found in the relevant tissue slices. The experimental results are shown in Figure 2 .
[0102] The following biological index tests were conducted, specifically the hemolysis test. In order to preliminarily evaluate the safety of the polymer as an injection excipient, the hemolysis of the material to SD rat blood cells was evaluated using the gel extract by extraction method. The microplate reader detected that the hemolysis rate of the series of polymers was less than 5% in the range of 0.5-2.5g / L, and the blood compatibility was good. There was no significant difference between the phenomena in each experimental group and the negative control group (normal saline). The experimental results are shown in Figure 3 .
[0103] In some embodiments of the present application, a method for preparing a polyorthoester having acid-sensitive degradation and positive temperature-sensitive properties is also provided:
[0104] The method comprises
[0105] S1: Prepare oligomer monomers, select glyceryl monostearate (GMS) and glycolide (GA), and the molar ratio of the two components is 1:1. Weigh glyceryl monostearate (GMS) (17.928g, 0.05mol) and glycolide (GA) (5.8035g, 0.05mol) in a pressure-resistant reaction tube, and heat and stir at 180℃ for 24h under the protection of inert gas. The obtained product is glyceryl monostearate diglycolide (GMS-d i GL).
[0106] S2: 3,9-Di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane (DETOSU), triethylene glycol (TEG) and glyceryl monostearate diglycolide (GMS-d i GL) were selected to prepare polyorthoesters with acid-sensitive degradation and positive thermosensitive properties.
[0107] The preparation conditions are as follows: under strict anhydrous conditions, 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane is dissolved in anhydrous tetrahydrofuran (THF) and triethylene glycol (TEG), and glyceryl monostearate diglycolide (GMS-d i GL) is dissolved in 5 ml of anhydrous tetrahydrofuran (THF), and then the two groups of solutions are mixed to initiate a polymerization reaction, the solution is cooled to room temperature, and then concentrated by rotary evaporation at 50°C, and then rotary evaporated at 80°C to obtain a polyorthoester with acid-sensitive degradation and positive temperature-sensitive properties.
[0108] It is understandable that the present embodiment provides a method for preparing a polyorthoester with acid-sensitive degradation and positive temperature-sensitive properties. The method ensures that the polymer has the desired characteristics through specific steps and conditions. First, in step S1, the oligomer monomer glyceryl monostearate diglycolide (GMS-d i GL) is prepared by a copolymerization reaction in which the molar ratio of glyceryl monostearate (GMS) and glycolide (GA) is 1:1. This step is carried out under high temperature and inert gas protection to ensure the purity and efficiency of the reaction.
[0109] Next, in step S2, 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane (DETOSU), triethylene glycol (TEG) and previously prepared GMS-d iGL were used to synthesize polyorthoesters. The key to this step is the anhydrous condition and specific reaction temperature control. The polymerization reaction was initiated by mixing the solution of DETOSU and TEG with the solution of GMS-d iGL, and then cooled at room temperature and concentrated by rotary evaporation at 50°C and 80°C, finally obtaining polyorthoesters with acid-sensitive degradation and positive thermosensitive properties.
[0110] The advantage of this method is that it can precisely control the structure and properties of the polymer so that it degrades under specific environmental conditions (such as acidity or temperature changes), which is of great significance in drug delivery, biomedical engineering and other applications. Through delicate chemical synthesis and strict process control, this method provides an effective way to prepare high-performance polymers with specific functions.
[0111] The proportion of raw materials is described in detail below by Embodiment 5-7:
[0112] The oligomer monomer of this embodiment is prepared from glyceryl monostearate (GMS) and glycolide (GA). The molar ratio of the two components is 1:1. Glyceryl monostearate (GMS) (17.928 g, 0.05 mol) and glycolide (GA) (5.8035 g, 0.05 mol) are weighed in a pressure-resistant reaction tube, and heated and stirred at 180°C for 24 hours under the protection of inert gas. The resulting product is glyceryl monostearate diglycolide (GMS-d i GL).
[0113] Embodiment 5
[0114] The polymer with acid-sensitive degradation and positive temperature-sensitive properties of the present embodiment is prepared from 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane (DETOSU), triethylene glycol (TEG) and glyceryl monostearate diglycolide (GMS-d i GL). The molar ratio of the three components (DETOSU:TEG:GMS-d i GL) is 90:80:20. Under strict anhydrous conditions, DETOSU (1.910 g, 0.009 mol) is dissolved in 15 ml of anhydrous tetrahydrofuran (THF) and TEG (1.2014 g, 0.008 mol) in a 50 ml flask, and GMS-d i GL (0.9493 g, 0.002 mol) is dissolved in 5 ml of anhydrous THF. The GMS-d i GL solution is added to the solution of DETOSU and TEG to initiate polymerization. Within a few minutes, the solution reached boiling point. The solution was allowed to cool to room temperature and then concentrated by rotary evaporation at 50°C and subsequently rotary evaporation at 80°C to yield a polyorthoester with acid-sensitive degradation and positive thermosensitive properties.
[0115] Embodiment 6
[0116] The polymer with acid-sensitive degradation and positive temperature-sensitive properties of the present embodiment is prepared from 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane (DETOSU), triethylene glycol (TEG) and glyceryl monolaurate diglycolide (GML-d i GL). The molar ratio of the three components (DETOSU:TEG:GML-d i GL) is 95:80:20. Under strict anhydrous conditions, DETOSU (2.0164 g, 0.0095 mol) is dissolved in 15 ml of anhydrous (tetrahydrofuran) THF and TEG (1.2014 g, 0.008 mol) in a 50 ml flask, and GML-d i GL (0.7807 g, 0.002 mol) is dissolved in 5 ml of anhydrous THF. The GML-d i GL solution is added to the solution of DETOSU and TEG to initiate polymerization. Within a few minutes, the solution reached boiling point. The solution was allowed to cool to room temperature and then concentrated by rotary evaporation at 50°C and subsequently rotary evaporation at 80°C to yield a polyorthoester with acid-sensitive degradation and positive thermosensitive properties.
[0117] Embodiment 7
[0118] The polymer with acid-sensitive degradation and positive temperature-sensitive properties of the present embodiment is prepared from 3,9-di(ethylene)-2,4,8,10-tetraoxaspiro[5,5]undecane (DETOSU), triethylene glycol (TEG) and glyceryl monooleate diglycolide (GMO-diGL). The molar ratio of the three components (DETOSU:TEG:GMO-d iGL) is 100:85:15. Under strict anhydrous conditions, DETOSU (2.1225g, 0.0100mol) is dissolved in 15ml of anhydrous ethyl acetate and TEG (1.2750g, 0.0085mol) in a 50ml flask, and GMO-d i GL (0.7088g, 0.0015mol) is dissolved in 5ml of anhydrous ethyl acetate. The GMO-d iGL solution is added to the solution of DETOSU and TEG to initiate polymerization. Within a few minutes, the solution reaches boiling point. The solution was allowed to cool to room temperature and then concentrated by rotary evaporation at 50°C and subsequently rotary evaporation at 80°C to yield a polyorthoester with acid-sensitive degradation and positive thermosensitive properties.
[0119] It is understood that by precisely controlling the ratio of monostearate glyceryl, glycolide, DETOSU, TEG and different types of diglycolide, polyorthoesters with specific acid sensitivity and temperature sensitivity can be customized to meet different drug release requirements. Carrying out the reaction under anhydrous conditions and using inert gas protection can help improve the purity and yield of the polymerization reaction while reducing the occurrence of side reactions. The prepared polyorthoester has acid-sensitive degradation characteristics and can be decomposed under a specific pH environment in the body, thereby achieving the fixed-point release of the drug. The positive temperature-sensitive nature of the polyorthoester enables it to undergo structural changes at body temperature, further regulating the drug release rate and enhancing the therapeutic effect. Through rotary evaporation concentration and heating steps, the target polymer can be efficiently obtained, the production process is simplified, and the production efficiency is improved. Due to the potential of polyorthoesters in drug release systems, the polymers prepared by this method can be used for the controlled release of various drugs such as ophthalmic disease drugs, pain relief drugs, peptides and protein drugs, vaccines, etc., and have broad application prospects.
[0120] Moreover, on the other hand, the present application also provides an application of a poly(orthoester) in vivo analysis method, including the application of the above-mentioned analysis method in the preparation of ophthalmic disease drugs, pain relief drugs, polypeptide and protein drugs, and vaccines.
[0121] It can be seen that the poly(orthoester) in vivo analysis method provided above has significant advantages in the preparation of ophthalmic disease drugs, pain relief drugs, peptide and protein drugs, and vaccines. First, the method can effectively track and evaluate the degradation process and drug release behavior of poly(orthoester) in vivo through precise qualitative and quantitative analysis, thereby optimizing the drug release system and improving the drug efficacy. Secondly, through the detection of histological and biological indicators, the effect of poly(orthoester) in vivo degradation on tissues and biological processes can be comprehensively evaluated to ensure the safety and effectiveness of the drug. Furthermore, the high sensitivity and specificity of the method enable it to provide accurate data support in drug release kinetics research and accelerate the development of new drugs. In general, the poly(orthoester) in vivo analysis method provided in the present application provides an accurate, comprehensive and efficient evaluation method for the preparation of ophthalmic disease drugs, pain relief drugs, peptide and protein drugs, and vaccines, and has great application value.
[0122] In summary, the present invention provides a polyorthoester in vivo analysis method and its application, which can more accurately track the degradation process of polyorthoester in vivo by selecting an appropriate experimental animal model and sampling at the same time point after drug administration, thereby optimizing the drug release system. By extracting and separating polyorthoester and its degradation products, and using liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry for qualitative and quantitative analysis, the degradation products of polyorthoester can be detected more sensitively and specifically. By establishing a standard curve and mass concentration relationship, the degradation rate and drug release behavior of polyorthoester can be more accurately evaluated. By performing histological analysis on the tissues of the experimental animal model, the effect of the in vivo degradation of polyorthoester on the tissue can be evaluated, thereby improving the safety of the drug. By detecting the biological indicators in the experimental animal model, the effect of the in vivo degradation of polyorthoester on the biological process can be evaluated, thereby improving the effectiveness of the drug. Moreover, the method can effectively track and evaluate the degradation process and drug release behavior of polyorthoester in vivo through accurate qualitative and quantitative analysis, thereby optimizing the drug release system and improving the efficacy. Secondly, through the detection of histological and biological indicators, the impact of the in vivo degradation of polyorthoesters on tissues and biological processes can be comprehensively evaluated to ensure the safety and effectiveness of the drug. Furthermore, the high sensitivity and specificity of the method enable it to provide accurate data support in drug release kinetics research and accelerate the development of new drugs. In general, the in vivo analysis method of polyorthoesters provided in this application provides an accurate, comprehensive and efficient evaluation method for the preparation of ophthalmic disease drugs, pain relief drugs, peptide and protein drugs, and vaccines, and has great application value.
[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A poly(orthoester) in vivo analysis method, characterized in that: The method comprises: S1: Determine and select the experimental animal model; S2: administering the drug by injection, and taking samples from the experimental animal model at different time points after administration, and tracking the degradation process of the polyorthoester through the sampling; S3: extracting and separating the polyorthoester and its degradation products, and performing qualitative and quantitative analysis on the degradation products; S4: Establish the standard curve and mass concentration relationship through analysis results.
2. The in vivo analysis method of polyorthoester according to claim 1, characterized in that: The different time points in S2 are 0, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours.
3. The in vivo analysis method of polyorthoester according to claim 1, characterized in that: In S3, the degradation products are qualitatively and quantitatively analyzed by liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry.
4. The in vivo analysis method of polyorthoester according to claim 1, characterized in that: The step S2 further includes performing histological analysis on the tissues of the experimental animal model by sampling to evaluate the effect of the in vivo degradation of the polyorthoester on the tissues.
5. The in vivo analysis method of polyorthoester according to claim 4, characterized in that: The histological analysis included pathological examination and immunohistochemical staining.
6. The in vivo analysis method of polyorthoester according to claim 4, characterized in that: The step S2 also includes detecting biological indicators in the experimental animal model to evaluate the effect of the in vivo degradation of the polyorthoester on the biological process.
7. A polyorthoester in vivo analysis method according to claim 6, characterized in that: The biological indicators include inflammatory factor levels and cell apoptosis.
8. The in vivo analysis method of polyorthoester according to claim 1, characterized in that: The degradation products include triethylene glycol, pentaerythritol, ethylene glycol, glycolide, glycolic acid and propionic acid.
9. An application of a polyorthoester in vivo analysis method, characterized in that: Use of the analytical method according to any one of claims 1 to 8 in the preparation of ophthalmic disease drugs, pain relief drugs, polypeptide and protein drugs, and vaccines.