CN118425381A - In-vitro release detection method of in-situ gel injection - Google Patents

Abstract

The present application provides an in vitro release detection method for an in situ gel injection, using a dialysis bag and a mesh basket as a dissolution device, and using a dissolution instrument paddle method, which better simulates the state of phase transition to gel release after subcutaneous injection of an in situ gel preparation (especially a liquid crystal precursor injection). At the same time, the present application also provides an in vitro long-term release detection method and an in vitro accelerated release detection method, and establishes the correlation between the in vitro release curve of the drug and the in vivo release behavior. In the process of preliminary prescription screening and optimization process research, the in vitro long-term release effect can be predicted by the in vitro accelerated release test results, and then the in vivo release behavior of the drug can be predicted. Different component compositions/proportions and different process parameters can be quickly and accurately distinguished and evaluated and screened, greatly improving the efficiency of preliminary prescription process screening and optimization.

Description

In vitro release detection method for in situ gel injection

Technical Field

The present application belongs to the field of medicine, and specifically relates to an in vitro release detection method for an in situ gel injection.

Background technique

In situ hydrogel, also known as instant gel, is a type of preparation that undergoes phase transition at the site of administration after administration in a solution state, transforming from a liquid into a semi-solid gel. In situ hydrogel drug delivery systems have good tissue compatibility, excellent injectability, and long-term retention. They also have high drug loading, sustained-release drug delivery, and little impact on the body's environment. They have attracted much attention in the fields of pharmacy and biotechnology.

As a new type of sustained-release drug delivery system, many studies on in situ gel are still in the initial stage. Due to its sustained-release performance, in the early stage of prescription screening, if the prescription screening is carried out through animal in vivo PK tests, it will not only be expensive but also time-consuming. In vitro release testing is an important evaluation method for early prescription screening and process research, and is also a mid-control indicator after the prescription process is determined in the later stage. If the in vitro release method can be correlated with the in vivo release behavior, the efficiency of prescription screening will be greatly improved. Therefore, screening and optimizing the in vitro release method is an important task in the research of in situ gel injection.

"Study on Cubic Liquid Crystal Precursor Injection of Sinomenine Hydrochloride" (Li Qian, Master's thesis, "Anhui University of Traditional Chinese Medicine" 2018) The dynamic dialysis method was used to determine the in vitro release of cubic liquid crystal precursor injection of Sinomenine Hydrochloride, as follows: Accurately weigh about 0.5g of in-situ cubic liquid crystal of Sinomenine Hydrochloride, and then place the liquid crystal in a sterilized dialysis bag with a length of 6cm (dialysis molecular weight is 8000-14000D), clamp the two ends of the dialysis bag with a thin rope to make it sealed, and the drug will not leak out from the gaps at both ends, and then put the clamped dialysis bag into 6mL of The centrifuge tube was filled with a pH 7.4 PBS solution, and finally placed in a constant temperature air bath shaker with a speed of 60 r/min and a temperature of 37.0 ± 0.5 ° C. 1 mL of release medium was regularly drawn out using a pipette at 0.5, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192 and 216 h, and 1 mL of blank pH 7.4 PBS solution was added at the same time. The drawn sample solution was filtered through a 0.45 μm microporous membrane, and the sample was injected and measured according to the chromatographic method of sinomenine hydrochloride to calculate the cumulative release. However, the release method takes too long to operate and cannot be used for early prescription screening and process research.

Patent CN106491519A (publication date 2017.03.15) discloses an in vitro release experiment of bupivacaine hydrochloride cubic liquid crystal in situ gel injection. The method is as follows: weigh 0.3g of the above-mentioned different bupivacaine hydrochloride cubic liquid crystal in situ gel injections and add them dropwise to a centrifuge tube containing 10 ml of PBS buffer salt, and place the centrifuge tube in a shaker at 100rpm and 37°C. Take out the release medium for measurement at 1, 2, 4, 8, 12, 24, 36, 48, and 72h, respectively, and supplement with an equal amount of fresh release medium. Determine the drug concentration at different time points, and use high performance liquid chromatography to determine the drug concentration to obtain the in vitro release curve of the drug. Although the in vitro release time of this method is shortened to 72h, the patent only characterizes the in vitro release behavior of the preparation, does not conduct in vitro and in vivo correlation research, and does not establish a connection with the in vivo release behavior, so it is difficult to use this method to predict the in vivo release behavior.

Therefore, it is necessary to establish appropriate in vitro release experiments to distinguish the differences between different formulations and different processes, simulate the in vivo drug release behavior, and improve the efficiency of preliminary formulation and process research.

Summary of the invention

The present application optimizes and screens out in vitro release methods and conditions suitable for in situ gel preparations (especially liquid crystal precursor injections), establishes the correlation between in vitro long-term release curves and in vivo release behaviors, and further adjusts the in vitro release conditions to develop in vitro accelerated release conditions that can quickly distinguish the pros and cons of prescription processes during prescription screening, and can predict in vivo release behaviors based on in vitro accelerated release results, thereby greatly saving the time of in vitro release tests and improving the efficiency of prescription screening.

The present application provides an in vitro release detection method for an in situ gel injection, comprising the following steps: placing a mesh basket in a dialysis bag with one end closed, injecting an appropriate amount of release medium, then taking an injection sample and injecting it into the mesh basket, and after the sample is pelletized, closing the other end of the dialysis bag and placing it in the release medium for an in vitro release test.

In a preferred embodiment, the in vitro release test uses a shaking table method or a dissolution test paddle method, more preferably a dissolution test paddle method.

In a preferred embodiment, the present application provides an in vitro release detection method for an in situ gel injection, comprising the following steps: placing a mesh basket in a dialysis bag with one end closed, and injecting an appropriate amount of release medium, then taking an appropriate amount of injection sample and slowly injecting it into the mesh basket, and after the sample is spherical, closing the other end of the dialysis bag, and then placing the dialysis bag in a dissolution cup containing the release medium, using the dissolution instrument paddle method to conduct the test, taking samples at specified time points, using the high performance liquid phase method to determine the content, and calculating the cumulative release amount and/or cumulative release degree at different time points.

The mesh basket described in the present application is a hard container with holes on the surface. The mesh basket described in the present application can be made of any material suitable for release testing, such as stainless steel, ceramics, glass, plastic, resin materials, etc. The mesh basket can be any shape suitable for release experiments, such as cylindrical, spherical, rectangular, cubic, capsule-shaped, etc., preferably cylindrical, spherical, capsule-shaped, and more preferably capsule-shaped. The size of the mesh basket should be suitable for placement in the dissolution equipment. In a preferred embodiment, the mesh basket is a capsule-shaped mesh basket, the diameter of the capsule-shaped mesh basket is 20-40mm, and the height is 20-50mm; preferably, the diameter of the capsule mesh basket is 20-30mm, and the height is 30-50mm; preferably, the diameter of the capsule mesh basket is 20-25mm, and the height is 35-45mm; more preferably, the diameter of the capsule mesh basket is 22mm, and the height is 40mm. The mesh aperture of the capsule-shaped mesh basket should not be too large to prevent the gel from breaking and leaking out during the test; preferably, the mesh aperture of the capsule-shaped mesh basket is 1-2mm, and the aperture spacing is 1-3mm; preferably, the mesh aperture is 1.2-1.7mm, and the aperture spacing is 1.5-2.5mm; more preferably, the mesh aperture is 1.5mm, and the aperture spacing is 2mm.

The size of the dialysis bag described in the present application should at least be able to fit into the mesh basket, and after the two ends are closed, an appropriate space can be left between the mesh basket to facilitate the exchange of fluids inside and outside the dialysis bag. In some embodiments, the mesh basket described in the present application is a capsule-shaped mesh basket. The dialysis bag and the capsule-shaped mesh basket described in the present application are shown in Figure 1. Preferably, the diameter of the dialysis bag is 2mm-20mm larger than the diameter of the capsule-shaped mesh basket, preferably 5mm-20mm, more preferably 5mm-10mm; the length of the dialysis bag is 20mm-80mm larger than the length of the capsule-shaped mesh basket, preferably 30mm-80mm, preferably 50mm-70mm, more preferably 60mm. In a preferred embodiment, the diameter of the dialysis bag is 22mm-50mm, preferably 25mm-40mm in diameter, preferably 25mm-35mm in diameter, more preferably 28mm in diameter; the length of the dialysis bag is 40-120mm, preferably 60-120mm in length, preferably 80-110mm in length, more preferably 100mm in length.

In a preferred embodiment, the molecular weight cutoff of the dialysis bag is 8KD-14KD, preferably 10KD-14KD, and more preferably 12KD-14KD.

In a preferred embodiment, the release medium is a PBS solution, preferably, the pH value of the PBS solution is 6.5-7.5, preferably 6.8-7.5, or 7.0-7.5, more preferably 7.2-7.4. Preferably, the release medium may further comprise a surfactant, a preservative and/or a release regulator.

In a preferred embodiment, the rotation speed of the dissolution apparatus paddle method is 30-100 rpm, preferably 30-70 rpm, preferably 30-40 rpm, and more preferably 30 rpm.

In a preferred embodiment, the release temperature of the in vitro release detection method is 35°C-45°C, preferably 37°C-42°C, more preferably 37°C±0.5°C or 42°C±0.5°C.

In a preferred embodiment, the in situ gel injection is preferably a liquid crystal precursor injection, more preferably a leuprorelin liquid crystal precursor injection.

In a preferred embodiment, the present application provides an in vitro release detection method, which is an in vitro long-term release detection method. Preferably, the release duration of the in vitro long-term release test is substantially consistent with the in vivo release duration of the corresponding drug. Preferably, the in vitro long-term release behavior obtained by the method is highly correlated with the in vivo release behavior of the drug.

In a preferred embodiment, the release temperature of the in vitro long-term release detection method is 37°C±2°C, preferably 37°C±1°C, and more preferably 37°C±0.5°C.

In a preferred embodiment, the release medium of the in vitro long-term release detection method is a phosphate buffered saline solution, preferably a PBS solution. The pH value of the PBS solution is 6.5-7.5, preferably 6.8-7.5, or 7.0-7.5, more preferably 7.2-7.4, more preferably 7.2. In a preferred embodiment, the release medium contains a surfactant, and the surfactant is selected from sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate, Tween, preferably SDS. Preferably, the concentration of the SDS is 0.1%-1.0%, preferably 0.3%-0.8%, more preferably 0.5%. Preferably, the release medium may further contain a preservative.

In a preferred embodiment, the present application provides an in vitro release detection method, which is an in vitro accelerated release detection method. Preferably, the detection data obtained by the in vitro accelerated release detection method is highly correlated with the in vitro long-term release data. Further, the detection data obtained by the in vitro accelerated release detection method is highly correlated with the in vivo release behavior of the drug.

In a preferred embodiment, the method completes the in vitro accelerated release test within 48 hours; preferably, the in vitro accelerated release test is completed within 36 hours, and more preferably, the in vitro accelerated release test is completed within 24 hours.

In a preferred embodiment, the release temperature is 42°C±1°C, preferably 42°C±0.5°C, more preferably 42°C.

In a preferred embodiment, the release medium is a phosphate buffer solution, preferably a PBS solution. Preferably, the pH value of the PBS solution is 6.5-7.5, preferably 6.8-7.5, or 7.0-7.5, more preferably 7.2-7.4, more preferably 7.2. In a preferred embodiment, the release medium contains a surfactant, and the surfactant is selected from sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate, Tween, preferably SDS. Preferably, the concentration of SDS is 0.1%-1.0%, preferably 0.3%-0.8%, more preferably 0.5%. In a preferred embodiment, the release medium also contains a release regulator, and the release regulator is selected from DMSO, ethanol, glycerol, propylene glycol, etc.; preferably DMSO and ethanol, more preferably ethanol. Preferably, the concentration of ethanol is 0.5%-2%, preferably 0.7%-1.5%, more preferably 1%. In a preferred embodiment, the release medium may further contain a preservative.

Those skilled in the art should understand that the in vitro release detection method described in this application can solve the common problems of in vitro release of in situ gel preparations, and is applicable to different in situ gel preparations. This application takes liquid crystal precursor injection (especially leuprorelin fluid crystal injection) as an example to show the effect of the technical solution of this application. Those skilled in the art are familiar with the common raw materials and excipients of in situ gel preparations and their characteristics, and can obtain a variety of other formulation examples suitable for this application based on the prior art. For example, refer to Camurus' patent applications for liquid crystal precursor injection such as CN101014319B, CN104105479B, CN105055303A, etc., refer to Zhong Gen Tang Co., Ltd.'s patent applications CN113350480A, WO2022005169A1, etc., refer to IMD Pharmaceutical Co., Ltd. CN113490487A, etc.

The liquid crystal precursor injection described in the present application comprises an active pharmaceutical ingredient (API), an organic solvent, a glyceride, and a phospholipid. The glyceride is selected from monosubstituted glyceride, disubstituted glyceride, and trisubstituted glyceride, for example, selected from one or more of monoolein (GMO), diolein (GDO), dicaprin, triolein (GTO), sorbitol monooleate (SMO), medium chain triglyceride (MCT), etc., preferably monoolein (GMO), diolein (GDO), medium chain triglyceride (MCT). The phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, etc. The phospholipid can be of natural origin or artificially synthesized. For example, the phosphatidylcholine is selected from natural or synthetic phosphatidylcholine such as soybean phosphatidylcholine and egg yolk phosphatidylcholine. In a preferred embodiment, the weight ratio of glyceride to phospholipid is 30:70 to 70:30, preferably 40:60 to 60:40.

The organic solvent is usually an oxygen-containing organic solvent, has biocompatibility, and has moderate water solubility and low viscosity. The organic solvent can be a single component or a combination of different components. Applicable organic solvents can be selected from ethanol, isopropanol, ethyl acetate, isopropyl acetate, dimethylacetamide (DMA), n-methylpyrrolidone (NMP) and/or DMSO, etc.; preferably, the organic solvent is ethanol or an ethanol/DMSO mixed solvent.

In order to obtain better stability and injectability, the liquid crystal precursor injection may further contain a viscosity modifier and a stabilizer. The viscosity modifier can reduce the viscosity of the liquid crystal precursor injection within an appropriate dosage range and is selected from propylene glycol, water, etc. The viscosity modifier accounts for less than 15% of the total weight of the liquid crystal precursor injection, preferably less than 10%, and more preferably less than 5%. The stabilizer is selected from EDTA, fat-soluble acids, thiolated antioxidants, etc.; the fat-soluble acid is selected from benzoic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.; the thiolated antioxidant is selected from monothioglycerol, N-acetylcysteine or cysteine, etc.

The active pharmaceutical ingredient may be any compound having a desired biological or physiological effect, especially active agents that are used to treat chronic diseases and require long-term administration, but usually have a short residence time in the body due to rapid decomposition or excretion, and active agents with poor oral bioavailability. Those skilled in the art can select according to needs.

In a preferred embodiment, the liquid crystal precursor injection described in the present application is a leuprorelin liquid crystal precursor injection. The leuprorelin liquid crystal precursor injection described in the present application contains leuprorelin or a pharmaceutically acceptable salt thereof, an organic solvent, a glyceride, and a phosphatidylcholine. Preferably, the leuprorelin liquid crystal precursor injection also contains water. The leuprorelin or a pharmaceutically acceptable salt thereof is selected from mesylate, hydrochloride, acetate, pamoate, preferably leuprorelin acetate and leuprorelin mesylate, more preferably leuprorelin acetate. The glyceride is selected from one or more of monooleylglycerol (GMO), dioleylglycerol (GDO) and medium chain triglyceride (MCT); the phosphatidylcholine is soybean phosphatidylcholine; the organic solvent is selected from ethanol and an ethanol/DMSO mixed solvent. The weight ratio of ethanol to DMSO in the ethanol/DMSO mixed solvent is 1:(0.5-2), preferably 1:(0.8-1.2), more preferably 1:1.

In a preferred embodiment, the leuprorelin liquid crystal precursor injection described in the present application comprises the following ingredients in parts by weight: 0.01-50 parts by weight of leuprorelin or a pharmaceutically acceptable salt thereof, 200-600 parts by weight of glyceride, 200-600 parts by weight of phosphatidylcholine, 50-200 parts by weight of an organic solvent, and 10-100 parts by weight of water.

In a preferred embodiment, the leuprorelin liquid crystal precursor injection described in the present application comprises the following components in parts by weight: 10-50 parts by weight of leuprorelin or a pharmaceutically acceptable salt thereof, 250-500 parts by weight of glyceride, 300-550 parts by weight of phosphatidylcholine, 100-200 parts by weight of an organic solvent, and 10-70 parts by weight of water;

Preferably, the leuprorelin liquid crystal precursor injection comprises the following ingredients in parts by weight: 20-40 parts by weight of leuprorelin or a pharmaceutically acceptable salt thereof, 300-450 parts by weight of glyceride, 350-500 parts by weight of phosphatidylcholine, 125-175 parts by weight of an organic solvent, and 20-50 parts by weight of water.

In a preferred embodiment, the pH value of the leuprorelin liquid crystal precursor injection described in the present application is preferably 5.0-6.5, preferably 5.0-6.2, and more preferably 5.2-6.0.

For the in vitro release detection method described in this application, those skilled in the art should understand that the adjustment of the order of some steps will not significantly affect the technical effect of the method described in this application, and it also belongs to the protection scope of this application. For example, a mesh basket can be placed in the dialysis bag first and then the release medium can be injected, or the release medium can be injected into the dialysis bag first and then the mesh basket can be placed; the other end of the dialysis bag can be closed after the sample is pelletized, or the other end of the dialysis bag can be closed first and then the sample is pelletized. Those skilled in the art should understand that the dialysis bag described in this application can be a bag with one end closed and one end open; it can also be open at both ends, but one end is closed before use. The "closing" can be carried out using conventional methods in the art, such as tying with a thin rope, clamping with a clip, or other means that can achieve a closing effect. Those skilled in the art are familiar with these means and can expect that the replacement and adjustment of these conventional means will not have a significant effect on the overall effect. The technical solutions formed after these similar step sequence adjustments or replacement of conventional means also belong to the protection scope of this application.

Definition of Terms

In situ gel: In situ gel exists in liquid form in vitro. After administration, it undergoes a non-chemically cross-linked solid or semi-solid phase transition at the administration site due to contact with the external environmental conditions of the administration site (such as light, temperature, pH value, affinity, ionic strength, etc.), becoming a local drug storage reservoir. In situ gel is divided into: polymer precipitation system, sucrose ester precipitation system, critical solution temperature system, liquid crystal system, polymer cross-linking system according to different technical means/materials.

Liquid crystal in situ gel, also known as in situ liquid crystal system, refers to a type of preparation that can absorb water at the medication site after being administered in a solution state, and then undergo a phase transition, transforming from a flowable liquid state into a non-flowable highly viscous liquid crystal. The formation mechanism is to utilize the response of amphiphilic materials to external stimuli (such as temperature, water absorption, etc.) to complete the phase transition from a solution to a high-viscosity and high-strength liquid crystal state, among which the related research on cubic liquid crystals is the most. In situ liquid crystals can encapsulate drugs of various polarities. Water-soluble drugs (such as tetracycline) can be encapsulated in the water channel of cubic liquid crystals; oil-soluble drugs (such as vitamin E and aspirin, etc.) can be encapsulated in the bilayer film of cubic liquid crystals; amphiphilic drugs are encapsulated at the interface. In situ liquid crystals have excellent drug loading and release properties, and liquid crystal excipients have the characteristics of non-toxicity, biodegradability, and bioadhesion. It has become one of the research hotspots at this stage. However, in situ liquid crystals have high viscosity, which is not conducive to injection administration, so they are prepared into precursors with low viscosity and good fluidity for administration. Once the liquid crystal precursor enters the human body through subcutaneous or intramuscular injection, the mobile precursor will gradually absorb water from body fluids or peripheral tissues and spontaneously transform into liquid crystals, forming an in-situ reservoir and stably releasing drugs. Liquid crystal precursors are usually composed of active pharmaceutical ingredients, phospholipids, glycerides (such as GDO, GMO, SMO, etc.), solvents, etc., and are often administered in the form of injections.

In vitro release: It is a means of pharmaceutical research. Under simulated in vivo conditions (such as temperature, pH of the medium, stirring rate, etc.), the drug release rate test of the preparation is carried out, and finally a reasonable in vitro drug release is formulated to monitor the production process of the product and control the quality of the product. The in vitro release rate can reflect the comprehensive effect of various physical and chemical parameters such as drug solubility, particle size, dosage form rheology, etc., and can identify the impact of prescription and process changes on the preparation. It is an important quality control item for product development, quality control, stability investigation and post-approval changes of the product. Common in vitro release detection methods include shake bottle method (shaking table), dialysis method, paddle method (dissolution instrument), flow cell method, etc. Those skilled in the art are familiar with the specific operation of the above detection methods. The ability to accurately and precisely predict in vivo bioavailability from in vitro release curves is a long-term goal pursued in this field. In some cases, especially for sustained-release preparations, the release test can not only provide quality control in the preparation process, but also predict the behavior of the prescription in vivo. However, the in vivo environment is relatively complex and cannot be accurately simulated under current conditions. Therefore, it is very necessary to perform correlation analysis between in vitro release data and in vivo release data. If the two are significantly correlated, the in vitro release results can be used as an indicator of the quality of the product's in vivo bioavailability characteristics, and can also be used for prescription screening and batch-to-batch quality control.

Fitting: Fitting is to connect a series of points on a plane with a smooth curve. Because this curve has countless possibilities, there are various fitting methods. The fitted curve can generally be represented by a function. Curve fitting is often used in the drug development process. The appropriate curve type is selected to fit the observed data, and the relationship between the two variables is analyzed using the fitted curve equation (regression equation). Those skilled in the art are aware of the fitting software and fitting methods commonly used in this field, and have a clear understanding of the applicable scenarios and characteristics of different software and fitting methods, and have the ability to select appropriate software and fitting methods according to the characteristics of the data to be fitted. For example, software such as EXCEL, Minitab, Origin, etc. can be used for curve fitting.

The R value is the correlation coefficient of the regression equation, which measures the strength of the linear correlation between two variables. Its value ranges from -1 to 1, and the closer it is to 1 or -1, the stronger the relationship. R ² measures the overall fit of the regression equation, which expresses the overall relationship between the dependent variable and all independent variables. The R ² value ranges from 0 to 1, and the closer it is to 1, the stronger the relationship.

Preservatives: Ideally, the release duration of the in vitro long-term release test of the sustained-release preparation is substantially consistent with the in vivo release duration of the corresponding pharmaceutical preparation. In order to reduce the risk of deterioration of the release system during the long-term release test, a small amount of preservatives can be added to the release medium. Those skilled in the art are aware of the types and properties of conventional preservatives and are able to select the appropriate type and amount of preservatives. For example, the preservative is selected from benzalkonium bromide, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, etc. Preferably, the concentration of the preservative is 0.1%-0.5%, preferably 0.2%-0.4%.

Unless otherwise specified, the ethanol described in this application is anhydrous ethanol.

Unless otherwise stated, percentages and parts stated in this application are by weight.

In order to provide a more concise description, some quantitative data herein do not use the term "about". It should be understood that, regardless of whether the term "about" is clearly used or not, each numerical value given here not only includes the actual value (given value), and it is also meant to include the approximate value of this given value reasonably inferred based on a person of ordinary skill in the art, including the equivalent and approximate value of this given value produced due to experimental and/or measurement conditions. The approximate value is preferably ± 20%, ± 15%, ± 10%, ± 8%, ± 6%, ± 5%, ± 4%, ± 3%, 2%, ± 1% on the basis of the given value. In some embodiments, the approximate value is obtained by rounding.

This application compares the test results of different in vitro release methods, and creatively invents a detection method of dialysis bag + basket + dissolution instrument paddle method. This detection method can not only solve the problem of gel adhesion to the dialysis bag after the phase change of the in-situ gel preparation (especially the liquid crystal precursor injection), but also because the gel is attached to the basket in the dialysis bag, the basket has smaller holes, which will not hinder the release of the drug. At the same time, because the fluid movement of the release medium in the dialysis bag is relatively slow, the gel will not be dispersed into multiple small gel pieces after the phase change, which better simulates the state of the in-situ gel preparation (especially the liquid crystal precursor injection) after subcutaneous injection. The phase change to gel release.

The present application provides an in vitro long-term release detection method and an in vitro accelerated release detection method for in situ gel injection (especially leuprorelin liquid crystal precursor injection), establishes the correlation between the in vitro drug release curve and the in vivo release behavior, and in the early stage of prescription screening and process optimization research, the in vitro long-term release effect can be predicted by the in vitro accelerated release test results, and then the in vivo release behavior of the drug can be predicted. Different component compositions/proportions and different process parameters can be quickly and accurately distinguished and evaluated and screened, greatly improving the efficiency of early prescription process screening and optimization.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Dialysis bag and capsule basket.

Detailed ways

The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

1. In vitro release test method

This application refers to the dissolution and release rate determination method of the "Pharmacopoeia of the People's Republic of China" for in vitro release testing, and the specific operations are shown in the various embodiments.

Sampling and determination: 2 ml of sample was taken at set time points (2 ml of release medium was added after sampling), filtered through a 0.22 μm filter membrane, and the filtrate was taken and the content was determined by high performance liquid chromatography, and the cumulative release amount and cumulative release degree at different time points were calculated.

The content determination method is as follows: chromatographic conditions: octadecylsilane bonded silica gel chromatographic column (C18, 4.6mm×100mm, 3μm); mobile phase is triethylamine aqueous solution (take 15.2g of triethylamine, dissolve in 1000ml of water, and adjust the pH to 3.0 with phosphoric acid)-[acetonitrile: n-propanol (3:2)] (80:20); flow rate is 1.0-1.3ml per minute; detection wavelength is 220nm; column temperature is 30°C; injection volume is 20μl, and running time is 15 minutes.

2. In vivo PK test method

SD rats were used as a model to study the pharmacokinetic behavior of leuprorelin liquid crystal precursor injection. The rats were injected subcutaneously at a dose of 5 mg/kg. The injection site was the back of the rats. Blood was collected from the jugular venous plexus at the scheduled blood collection time: 0.5h, 1h, 2h, 4h, 6h, 24h, 2d, 4d, 7d, 9d, 11d, 14d, 16d, 18d, 21d, 23d, 25d, 28d, 31d, and 35d. Immediately after blood collection, the blood samples were placed in a precooled K2EDTA tube containing 3μL aprotinin (100TIU/mL) and mixed. The tubes were placed in an ice box and centrifuged at 2000g for 10 minutes at 4℃ to separate the plasma. The leuprorelin concentration C in rat plasma was determined by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and the AUC and in vivo cumulative release were calculated.

3. Preparation of Leuprorelin Liquid Crystal Precursor Injection Sample

Prescription composition Prescription 1 Prescription 2 Prescription 3 Prescription 4 Prescription 5 Leuprorelin acetate (calculated as leuprorelin) 20mg 20mg 20mg 20mg 20mg water 30mg 30mg 30mg 30mg 30mg Anhydrous ethanol 75mg 150mg 150mg 150mg 150mg DMSO 75mg - - - - Soybean Phosphatidylcholine (SPC) 480mg 420mg 360mg 400mg 480mg Glyceryl Monooleate (GMO) 320mg - 440mg - - Glyceryl dioleate (GDO) - 380mg - - - Medium Chain Triglycerides (MCT) - - - 400mg 320mg Adjust pH to 5.2-6.0 5.2-6.0 5.2-6.0 5.2-6.0 5.2-6.0

The preparation method is as follows:

Step 1: Mix water, anhydrous ethanol and DMSO (if any) evenly;

Step 2: Add leuprolide acetate and stir to dissolve;

Step 3: Add soybean phosphatidylcholine (SPC) and stir to dissolve;

Step 4: Add glycerol ester (GMO or GDO or MCT) and stir well;

Step 5: Adjust the pH value to the target value, filter and fill.

Example 1 In vitro release method screening

Common in vitro release methods for in situ gel injections include shake bottle method (shaking table), dialysis method, dissolution paddle method, and flow cell method. This example compares the experimental effects of different release methods.

1. Test materials

Liquid crystal precursor injection sample: Prescription 2 sample, the dosage for each test is 0.2g.

Release medium: PBS solution (pH 7.2, containing 0.5% SDS).

Dialysis bag: molecular weight cut-off 12KD-14KD, size: diameter 28mm, length 100mm.

2. Test methods

(1) Shake bottle method (shaking table): The liquid crystal precursor injection sample is directly injected into a conical flask containing the release medium. After it is allowed to stand for 10 minutes to form a ball, it is placed in a 37°C ± 0.5°C constant temperature incubator and shaken (shaking frequency 30 rpm, shaking amplitude 20 mm). Samples are taken at the sampling time point for testing.

(2) Dialysis method: Tie one end of the dialysis bag with a thin rope, inject 10 ml of release medium, and then slowly inject the liquid crystal precursor injection sample. Tie the other end of the dialysis bag and let it stand for 10 minutes to allow the liquid crystal precursor injection sample to form a ball in the release medium. Then place the dialysis bag in a conical flask containing the release medium and shake it in a constant temperature incubator shaker at 37°C ± 0.5°C (shaking frequency 30 rpm, shaking amplitude 20 mm). Take samples at the sampling time point for testing.

(3) Dissolution paddle method: The liquid crystal precursor injection sample is directly injected into the dissolution cup containing the release medium, and allowed to stand for 10 minutes until it forms a ball. Then, the paddle method (rotation speed 30 rpm) is used, and the release temperature is 37°C ± 0.5°C. Samples are taken at the sampling time point for testing.

(4) Flow cell method: The liquid crystal precursor injection sample is directly injected into the flow cell containing the release medium (with transparent beads at the bottom), and allowed to stand for 10 minutes until it forms balls. The flow rate is set to 8 ml·min ^-1 , the release temperature is 37°C ± 0.5°C, and samples are taken at the sampling time point for testing.

The sampling time points are: 0.25d, 1d, 2d, 4d, 7d, 11d, and 16d.

3. Test results

Table 1 Cumulative release of different in vitro release methods (%)

Sampling point (d) Shake bottle method Flow cell method Paddle method (dissolution apparatus) Dialysis 4 56.17 65.62 49.59 18.69 7 98.90 100.00 89.29 26.19 16 100.00 100.00 100 34.81

By comparing different in vitro release methods, it was found that the shake flask method, circulation cell method, and dissolution apparatus paddle method directly placed the sample in the release container, which is a completely open release environment. After the liquid crystal precursor injection sample initially contacts the aqueous medium and phase changes into a gel, the gel is easily dispersed into multiple small gel pieces under the release conditions of stirring or oscillating, resulting in a large change in the release surface area of the gel. The drug is released rapidly and is basically released completely within 7 days. In the dialysis method, the gel is released slowly. By observing its release process, it was found that the viscosity of the gel after the phase change of the liquid crystal precursor is relatively large and it is easy to adhere to the dialysis bag, which hinders the exchange of the internal and external media of the dialysis bag, so the release is relatively slow.

Considering that the release time of in situ gel injection is relatively long (usually 30 days), in order to better fit the in vivo release behavior, the dialysis method was selected and improved to solve the problems existing in the dialysis method.

Example 2 Dialysis Method Improvement

1. Test materials

Liquid crystal precursor injection sample: Prescription 2 sample, the dosage for each test is 0.2g.

Release medium: PBS solution (pH 7.2, containing 0.5% SDS).

Dialysis bag: molecular weight cut-off 12KD-14KD, size: diameter 28mm, length 100mm.

Capsule-shaped mesh basket: diameter 22mm, height 40mm; mesh aperture 1mm, hole spacing 1.5mm.

The combination of the dialysis bag and the capsule-shaped mesh basket is shown in FIG1 .

2. Test methods

(1) Dialysis bag + shaking table method: tie one end of the dialysis bag with a thin rope, inject 10 ml of release medium, and then slowly inject the liquid crystal precursor injection sample, tie the other end of the dialysis bag, let it stand for 10 minutes to allow the liquid crystal precursor injection sample to form a ball in the release medium, and then place the dialysis bag in a conical flask containing the release medium, and place it on a constant temperature incubation shaker at 37°C ± 0.5°C and shake (shaking frequency 30 rpm, amplitude 20 mm), and take samples at the sampling time point for testing.

(2) Dialysis bag + mesh basket + dissolution apparatus paddle method: Tie one end of the dialysis bag with a thin rope, place a capsule-shaped mesh basket in the dialysis bag, and inject 10 ml of release medium. Then take the liquid crystal precursor injection sample and slowly inject it into the capsule-shaped mesh basket. Tie the other end of the dialysis bag and let it stand for 10 minutes to allow the liquid crystal precursor injection sample to form a ball in the release medium. Then place the dialysis bag in the dissolution cup containing the release medium. Use the paddle method (rotation speed 30 rpm) and the release temperature 37°C ± 0.5°C. Take samples at the sampling time point and wait for testing.

(3) Dialysis bag + mesh basket + shaker method: Tie one end of the dialysis bag with a thin rope, place a capsule-shaped mesh basket in the dialysis bag, and inject 10 ml of release medium. Then take the liquid crystal precursor injection sample and slowly inject it into the capsule-shaped mesh basket. Tie the other end of the dialysis bag and let it stand for 10 minutes to allow the liquid crystal precursor injection sample to form a ball in the release medium. Then place the dialysis bag in a conical flask containing the release medium and shake it on a constant temperature incubation shaker at 37°C ± 0.5°C (shaking frequency 30 rpm, amplitude 20 mm). Samples are taken at the sampling time point for testing.

(4) Dialysis bag + dissolution apparatus paddle method: Tie one end of the dialysis bag with a thin rope and inject 10 ml of release medium. Take the liquid crystal precursor injection sample and slowly inject it into the dialysis bag. Tie the other end of the dialysis bag and let it stand for 10 minutes to allow the liquid crystal precursor injection sample to form a ball in the release medium. Then place it in the dissolution cup containing the release medium. Use the paddle method (rotation speed 30 rpm) and the release temperature 37°C ± 0.5°C. Take samples at the sampling time point and wait for testing.

The API content of the samples at each time point (sampling time point 0.25d, 1d, 2d, 4d, 7d, 11d, 16d) of the above methods (1)-(4) was detected to obtain the in vitro cumulative release curve. The in vitro cumulative release curves of the above methods (1)-(4) were fitted with the in vivo cumulative release curves of the same samples, specifically: linear fitting was performed with the in vitro cumulative release data as the horizontal axis and the in vivo cumulative release data as the vertical axis. If the R value is greater than 0.99, it is considered that the in vitro release curve is highly correlated with the in vivo release behavior.

3. Test results

Table 2 Fitting results of in vitro and in vivo cumulative release data of different dialysis methods

Release method 16d cumulative release (%) Fitting equation R Dialysis bag + shaker 34.81 Y＝3.9664X-26.843 0.9868 Dialysis bag + net + dissolution paddle method 78.31 Y＝1.1257X+2.5382 0.9977 Dialysis bag + basket + shaker 98.00 Y＝0.8786X+5.4453 0.9960 Dialysis bag + dissolution paddle method 46.81 Y＝1.9073X-5.6018 0.9776

Table 3 Comparison of in vitro release characteristics of different dialysis methods

Based on the 16-day in vitro cumulative release and the in vitro and in vivo fitting effects, and by comparing the release characteristics of different methods, the dialysis bag + basket net + dissolution paddle method was selected.

Example 3 Optimization of in vitro accelerated release conditions

In order to shorten the prescription screening time, an in vitro accelerated release test method was established, and the in vitro accelerated release time was controlled to 24 hours for rapid batch screening.

1. Test materials

Liquid crystal precursor injection sample: Prescription 2 sample, the dosage for each test is 0.2g.

The dialysis bag and the basket are the same as those in Example 2.

2. Test methods and results

2.1 Screening of release temperature

(1) In vitro long-term release test: Tie one end of the dialysis bag with a string, place a capsule basket in the dialysis bag, and inject 10 ml of release medium (PBS solution, pH 7.2, containing 0.5% SDS), then take the liquid crystal precursor injection sample and slowly inject it into the capsule basket, let it stand for 10 minutes to allow the liquid crystal precursor injection sample to form a ball in the release medium, tie the other end of the dialysis bag, place it in a dissolution cup containing 500 ml of release medium, and place it in a dissolution apparatus at 37°C ± 0.5°C, and use the paddle method (speed 30 rpm) to perform an in vitro release test. Take samples at 6h, 1d, 2d, 4d, 7d, 11d, 16d, 18d, 21d, 25d, 28d, 31d, and 35d, and use the high performance liquid chromatography method to determine the content, calculate the cumulative release amount and cumulative release rate at different time points, and obtain in vitro long-term cumulative release data.

(2) In vitro accelerated release test: adjust the release temperature (see Table 4), take samples at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h, respectively, and perform the other specific operations the same as (1) to obtain in vitro accelerated cumulative release data.

(3) Fit the obtained in vitro accelerated release data with the in vitro long-term release data of the same sample, specifically, perform logarithmic fitting with the in vitro accelerated cumulative release as the abscissa and the in vitro long-term cumulative release as the ordinate. If the R value is greater than 0.99, it is considered that the in vitro accelerated release curve is highly correlated with the in vitro long-term release behavior.

Table 4 Accelerated cumulative release in vitro at different release temperatures and its fitting results with in vitro long-term release

The results in Table 4 show that as the release temperature increases, the in vitro release rate of the sample continues to increase, but considering the drug stability, the release temperature cannot be too high.

By comparing the consistency of long-term release behavior and accelerated release behavior, comprehensively considering the release rates of 18h and 24h, and combining the correlation R comparison of the fitting equation, 42℃ was selected to continue the study.

2.2 Screening of release modifiers

The effects of release modifier types and concentrations on in vitro release were investigated.

(1) In vitro long-term release test: Same as 2.1(1), obtain in vitro long-term cumulative release data.

(2) In vitro accelerated release test: the release temperature was 42°C. Different release regulators (see Table 5) were added to the release medium. Samples were taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h. The rest of the specific operations were the same as (1). In vitro accelerated cumulative release data were obtained.

(3) The obtained in vitro accelerated cumulative release data were fitted with the in vitro long-term cumulative release data of the same sample, specifically, logarithmic fitting was performed with the in vitro accelerated cumulative release as the abscissa and the in vitro long-term cumulative release as the ordinate. If the R value is greater than 0.99, it is considered that the in vitro release curve is highly correlated with the in vivo release behavior.

Table 5 Accelerated cumulative release of different release modifiers in vitro and the fitting results of their long-term release curves in vitro

Note: The release medium is a PBS solution at pH 7.2 (containing 0.5% SDS) with the release regulator further added.

Adding a release modifier can accelerate the dissolution of the gel, forming multiple hydrophilic drug release channels and accelerating the release of the drug. The results in Table 5 show that compared with the absence of a release modifier, the in vitro release rate is improved to varying degrees after adding DMSO and ethanol as a release modifier. Considering the release completion of 18h and 24h, the R value of the fitting equation of the in vitro accelerated release curve and the in vitro long-term release curve, 1% ethanol is selected as a release modifier.

In summary, the preferred in vitro accelerated release conditions are an acceleration temperature of 42° C. and a release regulating solvent of 1% ethanol.

Example 4 Correlation verification of in vitro and in vivo release behaviors of different prescriptions

The in vitro and in vivo release of different prescriptions were investigated, and the correlation between in vitro and in vivo release was fitted. The ability of in vitro long-term release method and in vitro accelerated release method to distinguish different prescriptions was investigated.

1. Test materials

Liquid crystal precursor injection sample: Samples of prescriptions 1, 2, and 3, with a dosage of 0.2 g for each test.

The dialysis bag and the basket are the same as those in Example 2.

2. Test methods

(1) In vitro long-term release test: tie one end of the dialysis bag with a string, place a capsule-shaped mesh basket in the dialysis bag, and inject 10 ml of release medium, then take the liquid crystal precursor injection sample and slowly inject it into the capsule-shaped mesh basket, let it stand for 10 minutes to allow the liquid crystal precursor injection sample to form a ball in the release medium, tie the other end of the dialysis bag, place it in a dissolution cup containing 500 mL of release medium, and place it in a 37°C ± 0.5°C dissolution instrument (paddle method speed 30 rpm), take samples at 6h, 1d, 2d, 4d, 7d, 11d, 16d, 18d, 21d, 25d, 28d, 31d, and 35d, and use the high performance liquid chromatography method to determine the content, and calculate the cumulative release amount and cumulative release rate at different time points. Obtain in vitro long-term release data of different prescriptions. The release medium is PBS solution (pH 7.2, containing 0.5% SDS).

(2) In vitro accelerated release test: release temperature 42±0.5°C, release medium is PBS solution (pH 7.2, containing 0.5% SDS, 1% ethanol), samples are taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h, respectively, and other specific operations are the same as (1) to obtain in vitro accelerated cumulative release data.

(3) In vivo PK test: The method is the same as before to obtain in vivo PK data of different prescriptions.

(4) Curve fitting:

① Correlation between in vitro long-term release and accelerated release: Logarithmic fitting was performed with the in vitro accelerated cumulative release as the horizontal axis and the in vitro long-term cumulative release as the vertical axis to obtain the fitting equation, and the correlation coefficient R value was calculated to evaluate the correlation between the in vitro accelerated release data and the in vitro long-term release data.

② In vitro and in vivo correlation: Linear fitting was performed with the in vitro long-term cumulative release as the horizontal axis and the in vivo cumulative release as the vertical axis to obtain the fitting equation, and the correlation coefficient R value was calculated to evaluate the correlation between the in vitro long-term release data and the in vivo PK data.

3. Test results

Table 6 Accelerated cumulative release in vitro of different prescriptions (%)

Time point/h Prescription 1 Prescription 2 Prescription 3 6 37.06 31.72 23.9 12 76.34 62.01 56.55 twenty four 98.31 94.12 93.14

Table 7 In vitro long-term cumulative release of different prescriptions (%)

Time point/d Prescription 1 Prescription 2 Prescription 3 7 67.72 56.74 43.14 16 97.66 78.31 72.76 31 NA 99.87 98.07

Table 8 Fitting results of in vitro long-term release and accelerated release correlation, in vivo and in vitro correlation of different formulations

The results showed that the in vitro long-term release of different formulations had a good correlation with the in vitro accelerated release (R>0.99), and the in vitro long-term release also had a good correlation with the in vivo release (R>0.99). This suggests that in the formulation process screening of liquid crystal precursor injections, the in vivo release characteristics can be predicted by in vitro accelerated tests, thereby quickly screening and optimizing the formulation.

Example 5 Screening and Optimizing Prescriptions Using In Vitro Accelerated Tests

1. Test materials

Liquid crystal precursor injection sample: Samples of prescriptions 4 and 5, the dosage for each test is 0.2 g.

The dialysis bag and the basket are the same as those in Example 2.

2. Test methods

(1) In vitro accelerated release experiment: The samples of prescriptions 4 and 5 were subjected to an in vitro accelerated release experiment according to the method and conditions of "(2) In vitro accelerated release experiment" in Example 4, and the cumulative release amount and cumulative release degree at different time points were calculated to draw the release curves.

(2) Prediction of in vivo PK trend: Predict the in vivo PK trend based on in vitro accelerated release data.

(3) In vivo PK test verification: The pharmacokinetic study of the samples of Prescription 4 and Prescription 5 was carried out in rats. The samples were taken according to the in vivo PK test method, the blood drug concentration was measured, the AUC was calculated, the in vivo cumulative release amount and release rate at different time points were calculated, and the data were logarithmically fitted with the in vitro accelerated test data.

3. Test results

The results of the in vitro accelerated release test showed that the early release rates of Prescription 4 and Prescription 5 were similar, with a cumulative release of about 20% in 4 hours.

Prescription 5 has a fast release rate in the medium term (4h-12h), with a cumulative release of 86.39% in 12h. It is predicted that the cumulative release in the first 1/2 of the designed release cycle (30 days) will also be large, and the release in the second half of the cycle cannot be maintained. This prescription can be excluded in the process of prescription screening. The results of the in vivo PK test showed that the cumulative release of the preparation reached 96.09% at 16d, and the preparation of prescription 5 could not maintain the release in the second half of the cycle.

The release rate of prescription 4 in the middle release period (4h-12h) is similar to the early release rate. The cumulative release of prescription 4 in 12 hours is about 70%. Not only is the release rate in the first half of the cycle relatively stable, but it also leaves room for drug release in the second half of the cycle. The cumulative release in 24 hours is 95.38%. The results of the in vivo PK test show that the cumulative release in vivo is 78.43% at 16 days, and the release is basically complete at 31 days, which meets the requirements of the release cycle (1 month) of long-acting preparations.

The in vitro accelerated release curves of Prescription 4 and Prescription 5 were fitted with the in vivo release curves, and a good fit was achieved (R>0.99).

Table 9 Correlation between in vitro accelerated release and in vivo release of different formulations

prescription Fitting equation R Prescription 4 Y＝34.257ln(X)-67.248 0.9945 Prescription 5 Y＝38.832ln(X)-79.482 0.9942

It can be seen that the in vitro accelerated release method in the present application provides a convenient and reliable way for the early prescription screening of liquid crystal precursor injection, thereby improving the efficiency of prescription screening.

Claims (9)

1. An in-vitro release detection method of an in-situ gel injection comprises the following steps: placing a basket in a dialysis bag with one sealed end, injecting a proper amount of release medium, then taking an injection sample, injecting the injection sample into the basket, sealing the other end of the dialysis bag after the sample is balled, and placing the dialysis bag in the release medium for in-vitro release test.

2. The method of detection according to claim 1, wherein: the in vitro release test uses a shaking table method or a dissolution paddle method, preferably a dissolution paddle method.

3. The detection method according to any one of claims 1 to 2, wherein:

the basket is selected from cylinder, sphere, cuboid, cube, capsule, etc., preferably cylinder, sphere, capsule, more preferably capsule;

preferably, the basket is a capsule basket;

Preferably, the diameter of the capsule-shaped basket is 20-40mm, and the height is 20-50mm;

Preferably, the mesh screen of the capsule-shaped basket has a pore diameter of 1-2mm and a pore diameter interval of 1-3mm.

4. A detection method according to any one of claims 1 to 3, wherein:

The diameter of the dialysis bag is 2mm-20mm larger than that of the capsule-shaped basket, and the length of the dialysis bag is 20mm-80mm larger than that of the capsule-shaped basket; and/or

Preferably, the diameter of the dialysis bag is 22mm-50mm; the length of the dialysis bag is 40-120mm; and/or

Preferably, the dialysis bag has a molecular weight cut-off of 8KD-14KD, preferably 10KD-14KD, more preferably 12KD-14KD.

5. The method of any one of claims 1-4, wherein: the release medium is phosphate buffer salt solution, preferably PBS solution;

Preferably, the PBS solution has a pH of 6.5-7.5, preferably 6.8-7.5, or 7.0-7.5, more preferably 7.2-7.4;

Preferably, the PBS solution contains a surfactant selected from Sodium Dodecyl Sulfate (SDS), sodium dodecyl benzene sulfonate, tween, preferably SDS; preferably, the concentration of SDS is 0.1% -1.0%, preferably 0.3% -0.8%, more preferably 0.5%;

preferably, the release medium further comprises a preservative and/or a release modifier.

6. The method of any one of claims 1-5, wherein: the in-situ gel injection is preferably a liquid crystal precursor injection, more preferably a leuprorelin liquid crystal precursor injection;

Preferably, the liquid crystal precursor injection comprises a pharmaceutical active ingredient, an organic solvent, glyceride and phospholipid;

preferably, the liquid crystal precursor injection further comprises a viscosity regulator and/or a stabilizer.

7. The method of detection according to claim 6, wherein: the leuprorelin liquid crystal precursor injection comprises the following components in parts by weight: 0.01-50 parts by weight of leuprorelin or pharmaceutically acceptable salt thereof, 200-600 parts by weight of glyceride, 200-600 parts by weight of phosphatidylcholine, 50-200 parts by weight of organic solvent and 10-100 parts by weight of water;

Preferably, the leuprorelin liquid crystal precursor injection comprises the following components in parts by weight: 10-50 parts of leuprorelin or pharmaceutically acceptable salt thereof, 250-500 parts of glyceride, 300-550 parts of phosphatidylcholine, 100-200 parts of organic solvent and 10-70 parts of water;

preferably, the leuprorelin liquid crystal precursor injection comprises the following components in parts by weight: 20-40 parts of leuprorelin or pharmaceutically acceptable salt thereof, 300-450 parts of glyceride, 350-500 parts of phosphatidylcholine, 125-175 parts of organic solvent and 20-50 parts of water.

8. The detection method according to any one of claims 1 to 7, wherein: the method is an in vitro long-term release detection method;

Preferably, the in vitro long-term release detection method has a release temperature of 37 ℃ ± 2 ℃, preferably 37 ℃ ± 1 ℃, more preferably 37 ℃ ± 0.5 ℃;

Preferably, the release medium of the in vitro long-term release detection method is phosphate buffered saline, preferably PBS solution; preferably, the PBS solution has a pH of 6.5-7.5, preferably 6.8-7.5, or 7.0-7.5, more preferably 7.2-7.4, more preferably 7.2;

Preferably, the PBS solution contains a surfactant selected from Sodium Dodecyl Sulfate (SDS), sodium dodecyl benzene sulfonate, tween, preferably SDS; preferably, the concentration of SDS is 0.1% -1.0%, preferably 0.3% -0.8%, more preferably 0.5%;

preferably, the release medium further comprises a preservative.

9. The detection method according to any one of claims 1 to 7, wherein: the method is an in-vitro accelerated release detection method;

preferably, the release temperature is 42 ℃ ± 1 ℃, preferably 42 ℃ ± 0.5 ℃, more preferably 42 ℃;

Preferably, the release medium of the in vitro accelerated release detection method is phosphate buffered saline, preferably PBS solution;

Preferably, the PBS solution contains a surfactant selected from Sodium Dodecyl Sulfate (SDS), sodium dodecyl benzene sulfonate, tween, preferably SDS; preferably, the concentration of SDS is from 0.1% to 1.0%, preferably from 0.3% to 0.8%, more preferably 0.5%;

Preferably, the release medium further comprises a release modifier selected from DMSO, ethanol, glycerol, propylene glycol, and the like, preferably DMSO and ethanol, more preferably ethanol; preferably, the ethanol concentration is 0.5% to 2%, preferably 0.7% to 1.5%, more preferably 1%;

Preferably, the PBS solution has a pH of 6.5-7.5, preferably 6.8-7.5 or 7.0-7.5 or 7.2-7.4, more preferably 7.2;

preferably, the release medium further comprises a preservative.