An in vitro accelerated release method for naltrexone injectable sustained-release microspheres

Abstract

The present application relates to a kind of naltrexone injection sustained-release microspheres in vitro accelerated release method, comprising the following steps: 1) loading flow cell;2) adding release mechanism, forming medium circulation;3) temperature is elevated, and release is accelerated;4) sampling detection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically the field of pharmaceutical preparation quality control, and specifically relates to an in vitro accelerated release method for naltrexone injection sustained-release microspheres. Background Technology

[0002] Naltrexone (NTX), an opioid receptor antagonist, is administered via intramuscular injection in its sustained-release microsphere formulation. This sustained-release mechanism utilizes the degradation of PLGA / PLG (lactic-co-glycolic acid copolymer) to achieve a slow release over a period of up to one month. The clinical dose of the commercially available drug Vivitrol is 380 mg, approximately four times the daily oral dose of naltrexone tablets over 28 days (50 mg). Because Vivitrol is a complex, long-acting injectable formulation, in vitro release rate is a core indicator for evaluating its critical quality attributes (CQAs).

[0003] Currently, pharmacopoeias specify that release testing for naltrexone injection sustained-release microspheres (e.g., Vivitrol) typically employs real-time release testing under simulated physiological conditions (37°C), such as the USP device 2 (paddle method) or device 4 (flow cell method). However, such conventional methods have significant technical drawbacks: 1. Long detection cycle (low efficiency): The real-time in vitro release of naltrexone microspheres often lasts 30-40 days. Such a long detection time makes this method unsuitable for rapid release of production batches, severely compressing the product's effective shelf life and increasing the company's warehousing and time costs.

[0004] 2. Limitations of method applicability (simulation of realism issues): Existing oscillation methods or dialysis bag methods not only have long release cycles, but the dialysis bag may also trap or adsorb naltrexone, introducing measurement errors; at the same time, long-term static release cannot simulate the dynamic mechanical environment (shear force, friction, body fluid flushing) of muscle tissue in the body.

[0005] 3. Drug stability risk (accuracy issues): During the incubation process, which can last for several weeks, the aqueous medium is highly susceptible to bacterial growth and naltrexone is easily oxidized and degraded, interfering with the accuracy of the test results.

[0006] Therefore, there is an urgent need in this field to establish a rapid, reliable, and effective in vitro accelerated release method that can simulate in vivo behavior in order to solve the above-mentioned technical problems. Summary of the Invention

[0007] The purpose of this invention is to provide an in vitro accelerated release method for naltrexone injection sustained-release microspheres. This method, through specific hydrodynamic design and temperature control, enables rapid release of the formulation while ensuring distinguishing power, thereby allowing for the detection and differentiation of sustained-release microspheres with different performance properties.

[0008] This invention provides a method for accelerated in vitro release of naltrexone injection sustained-release microspheres, comprising the following steps: 1) Loading the circulation pool A certain amount of naltrexone injection sustained-release microsphere formulation sample was placed in a flow cell device, and glass beads of a specific weight ratio were added to the flow cell as a turbulence medium. The flow cell is a closed-loop flow cell; 2) Add a release mechanism to form a medium circulation. A release medium is added to the flow pool to form a closed-loop circulation system; 3) Increase temperature to accelerate release The temperature of the entire circulation system is raised to 42-46°C to accelerate the drug release process; 4) Sampling and testing Sampling was performed according to a preset time gradient, and the drug concentration was determined and the cumulative release percentage was calculated using high-performance liquid chromatography.

[0009] The mass ratio of the glass beads to the formulation sample is 325:1.

[0010] The release medium includes a surfactant and an antibacterial agent.

[0011] The release medium is a 10mM isotonic phosphate buffer containing 0.02% Tween 20 and 0.02% ProClin™ 300.

[0012] The flow rate of the release medium is 8 ml / min.

[0013] The preferred temperature is 44°C.

[0014] The method for accelerated in vitro release of naltrexone injection sustained-release microspheres involved in this invention specifically includes the following steps: 1) Loading the flow cell: Weigh the naltrexone microsphere sample and place it in the flow cell device (USP device 4), and add glass beads in a specific weight ratio as a turbulence medium in the flow cell; Among them, the glass beads are 1mm in diameter, made of borosilicate, and the mass ratio of the filling amount to the microsphere sample is 325:1. They are fixedly filled, and the glass beads occupy the height of the cone part of the flow cell. 2) The flow cell device is filled with release medium and circulated in a closed loop, with the circulation rate controlled at 8 mL / min; the release medium is 200 mL of isotonic phosphate buffer containing 0.02% Tween 20 and 0.02% ProClin™ 300, pH 7.4; 3) Increase the temperature of the entire circulation system to 44°C to accelerate the drug release process; 4) Sampling was performed according to the preset time gradient at the following sampling points: 0.5, 2, 4, 8, 16, 24, 30, 36, 48, 56, 64, 72, 96, and 120 hours. The drug concentration was determined and the cumulative release percentage was calculated using high performance liquid chromatography.

[0015] The present invention aims to provide a method for accelerating the in vitro release of naltrexone injection sustained-release microspheres, which has the following technical advantages: (1) The in vitro accelerated release method for naltrexone injection sustained-release microspheres provided by the present invention can significantly improve detection efficiency. By increasing the test temperature from 37°C to 44°C and combining it with a dynamic circulation system of a flow cell, the present invention calculates a time scaling factor of 8 for the real-time release time and accelerated release time when testing the reference preparation under real-time and accelerated conditions. The correlation coefficient R2 of the curves of real-time release and accelerated release is 0.995. The present invention successfully shortens the real-time release test cycle, which originally required 30-40 days, to 5 days (120 hours), greatly improving the quality control efficiency and meeting the requirements for rapid batch release.

[0016] (2) The in vitro accelerated release method provided by this invention has excellent discriminative power and can keenly distinguish the microsphere quality fluctuations caused by process differences (e.g., different PLGA concentrations, different drug loadings). A specific amount of glass beads (1 mm) is added to the flow cell, and the slight impact and disturbance of the glass beads on the surface of the microspheres simulates the shear force and friction of muscle tissue in vivo; at the same time, the 8 ml / min flow rate in the pulseless mode can effectively simulate the flushing effect of blood circulation on the injection site. This combination of "dynamic + disturbance" makes the release curve closer to the real situation in vivo and can keenly distinguish the microsphere quality fluctuations caused by process differences (e.g., different PLGA concentrations, different drug loadings).

[0017] (3) All parameters of this method (flow rate, temperature, glass bead specifications, sample loading method) are controllable variables, which overcomes the randomness of traditional oscillation method or dialysis method. The RSD (relative standard deviation) of the experimental data is low and has excellent inter-laboratory reproducibility. Attached Figure Description

[0019] Figure 1 The cumulative release curves of naltrexone microspheres at different time points show the accelerated release.

[0020] Figure 2 The in vitro release curves of the naltrexone microsphere reference formulation at 37℃ and 44℃ are fitted (time scaling factor is 8).

[0021] Figure 3 Cumulative release curves of naltrexone microspheres with different PLGA concentrations in vitro.

[0022] Figure 4 Cumulative release curves of naltrexone microspheres with different drug loadings in vitro.

[0023] Figure 5 The cumulative release curves of naltrexone microspheres under different circulating flow rates in vitro.

[0024] Figure 6 The cumulative release curves of naltrexone microspheres under different release temperature conditions in vitro. Detailed Implementation

[0025] The present invention provides an in vitro accelerated release method for naltrexone injection sustained-release microspheres. The study investigated the leakage conditions, release medium, and acceleration conditions of the flow cell method for the accelerated release method, as well as the correlation between the accelerated release method of the present invention and existing real-time release methods.

[0026] The naltrexone injection sustained-release microspheres involved in this invention are prepared by incorporating anhydrous naltrexone into a biodegradable matrix PLGA. The preparation method includes the following steps: First, PLGA / PLG (lactic acid-glycolic acid copolymer) is dissolved in an organic solvent (ethyl acetate or dichloromethane), and naltrexone is dissolved in benzyl alcohol (30%, w / v) and added to the polymer solution to obtain a polymer solution containing naltrexone. Second, the organic phase is added to a 1% (w / v) polyvinyl alcohol aqueous solution pre-saturated with an organic solvent (to prevent rapid sedimentation of the polymer during preparation), and the droplet size is reduced by homogenization or magnetic stirring to obtain an o / w emulsion. Third, the emulsion is transferred to the aqueous phase and stirred at 220 rpm for 15 h under vacuum at room temperature to solidify the microspheres and remove the solvent. Fourth, the microspheres are sieved using a 212 μm upper sieve and a 25 μm lower sieve, and the microspheres on the 25 μm sieve are collected. Fifth, the microspheres are washed with a cold ethanol aqueous solution (25%, v / v, <5℃) and lyophilized to obtain naltrexone sustained-release microspheres.

[0027] Example 1. Leakage conditions of the flow cell in the accelerated release method (1) Configuration of the release medium The release medium is 10 mM isotonic phosphate buffer containing 0.02% Tween 20 (polysorbate 20) by weight / volume and 0.02% ProClin™ 300 (abbreviated as PC300, the main component of which is a mixture of isothiazolinones) by volume / volume.

[0028] Preparation steps: Weigh 0.46g of potassium dihydrogen phosphate, 1.2g of dipotassium hydrogen phosphate, and 8.0g of sodium chloride, add 1000ml of water, stir to dissolve, and adjust the pH to 7.4 with sodium hydroxide solution; then weigh approximately 200mg of polysorbate 20, measure 200µl of ProClin™ 300, add and stir well.

[0029] (2) Conditions of the flow tank Weigh a certain amount of naltrexone microsphere powder, add an appropriate amount of release medium, shake well and place in a shaker at 37°C. After shaking for 2 hours (undissolved raw material still exists), filter and take the filtrate to detect the API solubility (HPLC method).

[0030]

[0031] The sample was loaded with 20mg of microsphere powder. The drug loading of this product was approximately 33%, and the solubility of naltrexone API was approximately 0.824mg / ml.

[0032] According to the requirements of the leakage conditions of the flow cell method, the release medium should be greater than 40ml. Since the overall pipeline filling of the flow cell requires a volume of 30m, and the sampling volume of 14 sampling time points is set to 21ml, the total volume of the release medium in this invention is set to 200ml.

[0033] Example 2. Daytime precision study of accelerated release method This embodiment studies and verifies the accelerated release method of the present invention on the same batch of naltrexone microspheres, and verifies the daytime precision capability through the obtained release curve.

[0034] (1) Experimental methods Sample setup: The same batch of naltrexone microspheres were selected by the same researcher and accelerated in vitro release was carried out at two different times (January 26, 2026, and May 12, 2026).

[0035] Accelerated release conditions and methods: 1) Loading the flow cell: Weigh the naltrexone microsphere sample and place it in the flow cell device (USP device 4), and add glass beads in a specific weight ratio as a turbulence medium in the flow cell; The glass beads are 1mm in diameter and made of borosilicate. The mass ratio of the glass beads to the microsphere sample is 325:1. The glass beads are fixed in place, and the height of the conical portion of the flow cell is occupied by the glass beads.

[0036] 2) The flow cell device is filled with release medium and circulated in a closed loop, with the circulation rate controlled at 8 mL / min; the release medium is 200 mL of isotonic phosphate buffer (pH 7.4) containing 0.02% Tween 20 and 0.02% ProClin™ 300. 3) Increase the temperature of the entire circulation system to 44°C to accelerate the drug release process; 4) Sampling was performed according to a preset time gradient at the following sampling points: 0.5, 2, 4, 8, 16, 24, 30, 36, 48, 56, 64, 72, 96, and 120 hours. The drug concentration was determined and the cumulative release percentage was calculated using high performance liquid chromatography. Evaluation metrics: Calculate the cumulative release percentage at each time point and the similarity factor (f²) between the two tested samples. RSD is calculated for the results of three parallel samples from the same batch. Results from two different detection times are shown in Tables 1 and 2. Figure 1 As shown.

[0037] Table 1

[0038]

[0039] Table 2

[0040] (2) Conclusion Daytime precision verification: As shown in the table above, the relative standard deviation (RSD) of the test results for the two independent samples was controlled within 10% at each time point (less than 5% at most time points). This indicates that the method described in this invention has good repeatability and precision, and can eliminate random errors caused by differences in test time or operation.

[0041] Release curve similarity: Reference Figure 1 As shown, the similarity factor f2 of the accelerated release curves tested at different times was calculated to be 85.6%. Since f2 > 50%, this statistically demonstrates that the method can still maintain excellent intra-batch reproducibility under accelerated conditions (44℃).

[0042] Example 3. Correlation verification between accelerated release (44°C) and real-time release (37°C) This embodiment verifies the correlation between the accelerated release method and the real-time release method of naltrexone involved in this invention.

[0043] (1) Accelerated release experimental method Sample setup: Vivitrol (commercially purchased) was selected as the reference formulation of naltrexone microspheres, and the specific steps of the in vitro accelerated release method were the same as in Example 2.

[0044] Real-time release (37℃) conditions and methods: 1) Loading the flow cell: Weigh the naltrexone microsphere sample and place it in the flow cell device (USP device 4), and add glass beads in a specific weight ratio as a turbulence medium in the flow cell; The glass beads are 1mm in diameter and made of borosilicate. The mass ratio of the glass beads to the microsphere sample is 325:1. The glass beads are fixed in place, and the height of the conical portion of the flow cell is occupied by the glass beads.

[0045] 2) The flow cell device is filled with release medium and circulated in a closed loop, with the circulation rate controlled at 8 mL / min; the release medium is 200 mL of isotonic phosphate buffer (pH 7.4) containing 0.02% Tween 20 and 0.02% ProClin™ 300. 3) Raise the temperature of the entire circulation system to 37°C; 4) Sampling was carried out according to the preset time gradient. Sampling points were: 1, 2, 3, 4, 5, 6, 7, 9, 10.5, 12, 15, 16, 17, 18, 20, 22, 23, 24, 25, 26, 28, 30, 31, 33, and 35 days. The drug concentration was determined and the cumulative release percentage was calculated using high performance liquid chromatography.

[0046] The cumulative release percentage at each time point was calculated, and the cumulative release amount of the accelerated release curve and the real-time release curve were fitted point-to-point, and the linear regression equation was calculated; the results are shown in Table 3 (accelerated release) and Table 4 (real-time release), respectively.

[0047] Table 3

[0048]

[0049] Table 4

[0050]

[0051] (2) Experimental results Appendix Figure 2 The results from Tables 3-4 of Example 3 were plotted. After conversion using a time scaling factor (the ratio of the time point of 59% real-time release at 16 days to the time point of accelerated release at 2 days = 8), the real-time release curve and the accelerated release curve highly overlapped, and the linear correlation coefficient R was [missing value]. 2 It is 0.995.

[0052] Example 4: Validation of the discriminative power of the in vitro accelerated release method against different formulations and process parameters. This embodiment aims to verify the accelerated release detection of naltrexone microspheres with different mass properties using the in vitro accelerated release method described in this invention.

[0053] (1) Experimental methods Naltrexone microspheres with different PLGA concentrations (microsphere 1, microsphere 2) and different drug loadings (microsphere 3, microsphere 4, microsphere 5) were selected for in vitro accelerated release detection, and the specific steps were the same as in Example 2.

[0055]

[0056]

[0057] (2) Experimental results Table 5 and Figure 3 The results showed that differences in PLGA concentration affected the in vitro release of microsphere formulations 1 and 2 to varying degrees; Table 6 and Figure 4 The results showed that the in vitro release of microsphere formulations with high drug loading was significantly improved compared to formulations 3 and 4, with a significantly increased burst release at time 0, an overall faster release rate, and a longer total release period. The drug diffusion driving force of the high-drug-loading formulations was stronger than that of the low-drug-loading formulations.

[0058] These results demonstrate that the in vitro accelerated release method of the present invention can effectively differentiate the release of microspheres produced by different processes and has excellent discriminative power.

[0059] Table 5

[0060] Table 6

[0061] Example 5: Study on the circulation flow rate of the accelerated release method (1) Experimental methods Sample setup: Vivitrol (commercially purchased) was selected as the reference formulation of naltrexone microspheres. The specific steps of the in vitro accelerated release method were the same as in Example 2. The circulation flow rates of the two release media were set to 8 ml / min and 16 ml / min.

[0062] (2) Experimental results Refer to Table 7. Figure 5 As shown, the 48H release rate of the reference formulation was 83.6% at a circulation flow rate of 16 ml / min and 59.7% at a circulation flow rate of 8 ml / min. Increasing the flow rate would accelerate the overall release rate and reduce the distinguishing power of the accelerated release method of the present invention. Therefore, a circulation flow rate of 8 ml / min is preferred.

[0063] Table 7

[0064] Example 6 Temperature Study of Accelerated Release Method (1) Experimental methods Sample setup: The reference formulation of naltrexone microspheres, Vivitrol (commercially purchased), was used. The specific steps of the in vitro accelerated release method were the same as in Example 2, with three release temperatures set at 42°C, 44°C, and 46°C. (2) Experimental results According to Table 8, Figure 6As shown, the reference formulation showed poor accelerated release within 36 hours at 42°C, but its release rate was extremely fast within 48-72 hours. The excessively fast release rate would result in a lack of good discrimination. The reference formulation exhibited burst release (release rate above 80%) within 36 hours at 46°C, which could easily lead to changes in the release mechanism and make it impossible to simulate in vivo drug release behavior. Therefore, 44°C is preferred as the in vitro accelerated release temperature.

[0065] Table 8

[0066] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for accelerated release of naltrexone injectable sustained release microspheres in vitro, characterized by, Includes the following steps: 1) Loading the circulation pool A certain amount of naltrexone injection sustained-release microsphere formulation sample was placed in a flow cell device, and glass beads of a specific weight ratio were added to the flow cell as a turbulence medium. The flow cell is a closed-loop flow cell; 2) Add a release mechanism to form a medium circulation. A release medium is added to the flow pool to form a closed-loop circulation system; 3) Increase temperature to accelerate release The temperature of the entire circulation system is raised to 42-46°C to accelerate the drug release process; 4) Sampling and testing Sampling was performed according to a preset time gradient, and the drug concentration was determined and the cumulative release percentage was calculated using high-performance liquid chromatography.

2. The accelerated release method for naltrexone injectable sustained release microspheres according to claim 1, characterized by, The mass ratio of the glass beads to the formulation sample is 325:

1.

3. The accelerated release method of naltrexone injectable sustained release microspheres according to claim 1, wherein, The release medium contains surfactants and antibacterial agents.

4. The accelerated release in-vitro method for naltrexone injectable sustained release microspheres according to claim 3, characterized in that, The release medium is a 10 mM isotonic phosphate buffer containing 0.02% Tween 20 and 0.02% ProClin™ 300.

5. The method of accelerated release of naltrexone injectable sustained release microspheres according to claims 1-4, characterized in that, The flow rate of the release medium is 8 ml / min.

6. The accelerated release method of naltrexone injectable sustained release microspheres according to claim 1, wherein, The preferred temperature is 44°C.

7. The method for accelerated in vitro release of naltrexone injection sustained-release microspheres according to claim 1, characterized in that, Includes the following steps: 1) Loading the flow cell: Weigh the naltrexone microsphere sample and place it in the flow cell device (USP device 4), and add glass beads in a specific weight ratio as a turbulence medium in the flow cell; Among them, the glass beads are 1mm in diameter, made of borosilicate, and the mass ratio of the filling amount to the microsphere sample is 325:

1. They are fixedly filled, and the glass beads occupy the height of the cone part of the flow cell. 2) The flow cell device is filled with release medium and circulated in a closed loop, with the circulation rate controlled at 8 mL / min; the release medium is 200 mL of isotonic phosphate buffer containing 0.02% Tween 20 and 0.02% ProClin™ 300, pH 7.4; 3) Increase the temperature of the entire circulation system to 44°C to accelerate the drug release process; 4) Sampling was performed according to the preset time gradient at the following sampling points: 0.5, 2, 4, 8, 16, 24, 30, 36, 48, 56, 64, 72, 96, and 120 hours. The drug concentration was determined and the cumulative release percentage was calculated using high performance liquid chromatography.

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