Absorbable sustained-release agent for inhibiting fighting behavior of red deer during estrus

Abstract

The present application relates to the technical field of veterinary medicine, and discloses a kind of absorbable sustained-release agent for inhibiting the fighting behavior of male deer during estrus, comprising the following steps: stirring micronized medroxyprogesterone acetate with dextran sulfate and water for injection to obtain a homogeneous suspension paste; adding sodium hyaluronate dry powder without adding solvent to knead, and through competitive water absorption, dextran sulfate is dehydrated to form a physical coating layer on the surface of drug particles, obtaining a non-saturated hard rubber mass; after cooling, the remaining water for injection is pre-cooled and forced to pass through a microporous screen to achieve reciprocating convection extrusion, and then the product is obtained by swelling after standing.The present application utilizes the double retardation effect of interface physical coating layer and three-dimensional network of gel to inhibit the initial drug burst of long-acting preparation, prevent the gravity settlement of drug particles, achieve the long-term stable release of medroxyprogesterone acetate in animal body, and further reduce the fighting behavior of male deer during estrus.

Description

Technical Field

[0001] This invention relates to the field of veterinary pharmaceutical technology, specifically to an absorbable sustained-release agent that inhibits fighting behavior in male deer during estrus. Background Technology

[0002] Sika deer and red deer are important livestock genetic resources in my country, possessing significant medicinal and health benefits. Among them, the periodically regenerating antlers of male deer are the main economic source in the breeding process. Sika deer and red deer are seasonally estrous animals. During estrus, male deer experience increased levels of hormones such as testosterone, leading to increased libido, frequent fighting, shouting, and decreased appetite. This intense fighting can cause mechanical damage and excessive weight loss in male deer, affecting not only their recovery after fighting and the growth and yield of antlers the following year, but also posing a threat to the safety of breeders.

[0003] Currently, conventional methods for controlling estrus behavior in animals mainly include castration, daily physical restraint, and drug intervention. For deer used for antler production, since the cyclical growth of antlers is highly dependent on a complete reproductive endocrine system, castration methods that surgically or chemically destroy the testes will cause antler regeneration to stop; therefore, this approach is not feasible in deer farming. In daily feeding and management, farmers often use methods such as reducing protein feed intake, blindfolding, wearing anti-violence devices, or feeding anti-stress additives. However, because sika deer and red deer are currently highly wild and have a low degree of domestication, these management methods are difficult to effectively eliminate their hormone-driven instinctive behaviors.

[0004] In reproductive regulation, hormonal intervention is an effective approach to address this issue. For example, progesterone-like substances can be used to feedback-inhibit the hypothalamic-pituitary-gonadal axis, reducing gonadotropin secretion and thus lowering testosterone levels during estrus. However, existing intervention methods have limitations in terms of administration. Administering drugs through drinking water or feed cannot guarantee precise dosage for each male deer, making it difficult to achieve the desired inhibitory effect. If conventional short-acting injections are used, frequent restraint of the animals is required. Because deer experience strong stress responses, artificial restraint usually requires anesthesia, and repeated anesthesia can damage the deer's bodily functions and even lead to death. Furthermore, due to the relative scarcity of long-term baseline data on hormonal fluctuations during estrus in male deer, the release cycle of existing drugs is difficult to match with the actual estrus cycle of male deer.

[0005] Therefore, there is an urgent need to develop a long-acting sustained-release formulation that can be administered via a single injection to achieve long-term effects, precisely suppressing the fighting behavior of male deer during estrus without affecting the normal growth and ossification of antlers, while avoiding the risks of frequent anesthesia. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an absorbable sustained-release agent that inhibits fighting behavior in male deer during estrus. This solves the problem of intense fighting behavior caused by elevated testosterone levels during estrus in male deer, which not only leads to excessive physical exertion and mechanical damage but also affects the growth and yield of antlers the following year. Existing long-acting formulations suffer from problems such as excessively rapid initial drug release, short controlled-release period, and uneven drug release due to physical sedimentation.

[0007] To achieve the above objectives, the present invention provides the following technical solution: an absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, comprising the following steps:

[0008] S1. Add the micronized crystalline medroxyprogesterone acetate powder and dextran sulfate to a pressure-resistant mixing container, and add the corresponding amount of water for injection. Under the conditions of -80kPa to -100kPa vacuum and material core temperature of 60℃ to 65℃, centrifuge the mixture for 8 to 15 minutes to obtain a high-viscosity homogeneous suspension paste.

[0009] S2. Transfer the suspended paste into a water bath jacketed biaxial micro kneader, control the temperature to 38℃ to 42℃, and add sodium hyaluronate dry powder at a uniform speed under the meshing shear of the rotor; seal without adding solvent, and continue kneading for 30 to 45 minutes until a highly elastic unsaturated hard gel is formed.

[0010] S3. Quickly cool the hard rubber mass to 2°C to 8°C, and load it together with the remaining pre-cooled water for injection into a low-temperature convection extrusion device. Extrude it through a microporous mesh plate for 15 to 30 passes at a unidirectional extrusion pressure of 0.2MPa to 0.5MPa. After extrusion, allow it to stand at 2°C to 8°C for 10 to 14 hours to swell.

[0011] S4. The finished product is obtained by quantitative filling, sealing, and storage in the dark at an environment of 2℃ to 8℃.

[0012] By adopting the above technical solution, the absorbable sustained-release agent prepared by this invention can achieve the effect of inhibiting initial burst release and prolonging the controlled-release period. The specific working principle and process are as follows: In S1, centrifugal stirring under vacuum and controlled temperature conditions can eliminate air bubbles inside the system. The suitable temperature promotes the dissolution of dextran sulfate and its uniform mixing with medroxyprogesterone acetate particles to form a high-viscosity homogeneous suspension paste, providing a uniform material basis for subsequent interfacial coating. In S2, sodium hyaluronate powder is added to the high-viscosity suspension paste without adding solvent. Under the action of kneading shear force, sodium hyaluronate undergoes unsaturated hydration and actively absorbs free water in the system. The rapid reduction of free water in the system triggers competitive solvent deprivation, resulting in local solvent insufficiency of dextran sulfate, which then undergoes a dehydration phase transition, precipitates and deposits on the surface of medroxyprogesterone acetate particles, forming a physical coating layer. The polymer chain segments then undergo dense entanglement, and the material state is transformed into highly elastic unsaturated hard gel particles. In S3, the hard gel micelles are rapidly cooled to lock in the phase change structure and then mixed with pre-cooled water for injection for microporous convective extrusion. The mechanical shear force during extrusion breaks down the disordered entanglement of polymeric aggregates, promoting the entry of water into the gel network for uniform hydration. Forced convection causes macroscopic structural reconstruction of the system, forming a uniform three-dimensional blocking network, eliminating structural defects caused by local drug aggregation, and effectively reducing the initial burst release pathway of the drug. After the whole formulation is injected subcutaneously into animals, due to the dual blocking effect of the interfacial coating layer and the three-dimensional network, medroxyprogesterone acetate can maintain a long-term stable release, continuously inhibiting the function of the hypothalamus-pituitary-gonadal axis, reducing serum testosterone concentration, and thus effectively reducing fighting behavior in male deer during estrus.

[0013] Preferably, the medroxyprogesterone acetate is a micronized crystalline powder, and the dextran sulfate is coated on the surface of the medroxyprogesterone acetate to form a physical coating layer. The medroxyprogesterone acetate micronized powder with the physical coating layer is suspended and locked inside the three-dimensional gel network formed by the forced convection extrusion of the sodium hyaluronate after it has absorbed water and swelled. By adopting the above technical solution, the physical coating layer increases the mass transfer resistance of the drug particles to diffuse outward; the three-dimensional gel network has specific viscoelasticity, providing physical support space for the drug particles. The synergistic effect of the two stabilizes the drug release kinetics process and prolongs the duration of action of the drug in the animal body.

[0014] Preferably, based on the volume of the absorbable sustained-release agent, the mass concentrations of each raw material component are as follows: the concentration of medroxyprogesterone acetate is 340.0 mg / mL to 380.0 mg / mL; the concentration of dextran sulfate is 40.0 mg / mL to 60.0 mg / mL; the concentration of sodium hyaluronate is 180.0 mg / mL to 220.0 mg / mL; and the balance is water for injection. By adopting the above technical solution, the above concentration range defines the single effective dose for the drug to exert its physiological inhibitory effect, while providing the concentration threshold required for the formation of a continuous phase network of polymers. This formulation ensures that there is sufficient sodium hyaluronate in the system to consume water and trigger a dehydration phase transition, while also taking into account the syringe filling and extrusion permeability of the finished product.

[0015] Preferably, the purity of the medroxyprogesterone acetate is not less than 99.0%, and the particle size distribution D50 is 5.0 μm to 15.0 μm, and D90 is not greater than 30.0 μm. By adopting the above technical solution, the overall specific surface area of ​​the micropowder is controlled by limiting the particle size distribution range. A specific specific surface area can match the precipitation quality of dextran sulfate, promoting a uniform coating layer on the surface of the microparticles. This avoids the decrease in suspension stability caused by excessively large particles, and also prevents the phenomenon of excessively rapid drug release caused by excessively small particles and excessively large specific surface area.

[0016] Preferably, the sodium hyaluronate has a weight-average molecular weight range of 200,000 Da to 400,000 Da, and a glucuronic acid content of not less than 44.0%; the dextran sulfate has a weight-average molecular weight range of 36,000 Da to 50,000 Da, a sulfur content of 16.0% to 20.0% by mass, and an average of 2.0 to 2.5 free sulfate groups per glucosyl unit. By adopting the above technical solution, the molecular weight range of sodium hyaluronate ensures sufficient physical entanglement density between the long polymer chains after hydration to construct a robust gel framework. The molecular weight and free sulfate group density of dextran sulfate determine its solubility critical point, enabling it to undergo a phase transition and precipitate under conditions of reduced free water.

[0017] Preferably, the mass ratio of sodium hyaluronate to dextran sulfate is between 3:1 and 5.5:1. By adopting the above technical solution, this mass ratio establishes the dominant relationship between the two polymers in the system regarding water competition. Sodium hyaluronate, occupying a higher mass proportion, controls the hydration process and rapidly consumes the effective water in the liquid phase environment, causing dextran sulfate to lose its dissolution conditions and form a solid precipitate layer.

[0018] Preferably, in the low-field NMR transverse relaxation time (T2) distribution spectrum, the relative peak area ratio of bound water within the absorbable sustained-release agent is 94.81% to 97.02%, and the relative peak area ratio of free water is 2.98% to 5.19%. By employing the above technical solution, the high proportion of bound water indicates that the sodium hyaluronate network has formed tight hydrogen bonds with water molecules, restricting the free movement of water molecules. The low free water microphase environment limits the redissolution of the precipitated dextran sulfate, maintaining the structural integrity of the physical encapsulation layer during formulation storage and the initial stage of use.

[0019] Preferably, at a test temperature of 25°C, the yield stress of the absorbable sustained-release agent is within the range of 192.3 Pa to 238.9 Pa. By employing the above technical solution, the specific yield stress value indicates that the three-dimensional gel network possesses the mechanical strength to resist the gravitational settling of internal particles. After product storage and subcutaneous injection into animals, this mechanical strength can maintain the uniform distribution of drug particles in three-dimensional space, preventing particle aggregation and fusion that could alter release behavior.

[0020] Preferably, the physical coating layer is obtained under the following unsaturated hydration conditions: the water absorption and swelling process of the sodium hyaluronate is carried out without the addition of any additional solvent and accompanied by continuous meshing shear, until the material transforms from a high-viscosity homogeneous suspension paste into a highly elastic unsaturated hard gel. By adopting the above technical solution, the conventional dispersion path of sodium hyaluronate powder is forcibly altered without the addition of additional solvent. Shear force promotes full contact between the dry powder and the suspension paste and localized absorption of moisture, directly inducing a physical state transformation of the original liquid phase environment, thereby solidifying the interfacial barrier structure of the drug during the process.

[0021] Preferably, the forced convection extrusion refining refers to: extruding intermediate material in a highly elastic, unsaturated hard gel state along with the remaining water for injection through a mesh with micropores of 0.5 mm to 1.2 mm under unidirectional force. By employing this technical solution, the mechanical extrusion forces the unsaturated hard gel particles and the supplemented water to mix within the microporous channels. The mesh pore size forcibly limits the cross-sectional size of the extruded stream, dispersing the large polymer aggregates formed during the water-deficient stage. The reconstructed gel network possesses physical homogeneity, ensuring consistent degradation and release rates of the single-dose drug within the administration area.

[0022] This invention provides an absorbable, sustained-release agent for inhibiting fighting behavior in male deer during estrus. It has the following beneficial effects:

[0023] 1. This invention involves adding sodium hyaluronate powder under unsaturated hydration conditions. The sodium hyaluronate absorbs water, triggering a competitive solvent deprivation process that causes dehydration phase transition of dextran sulfate, which then deposits on the surface of medroxyprogesterone acetate particles, forming a physical coating layer. This coating layer increases the mass transfer resistance to the outward diffusion of the drug particles, thereby suppressing the burst release of long-acting formulations during the initial injection phase.

[0024] 2. This invention employs a microporous mesh forced convection extrusion process to physically refine hard micelles and pre-cooled water for injection, eliminating the polymer aggregates formed during the dehydration stage and reconstructing a three-dimensional gel network with physical homogeneity and specific yield stress. This gel network provides a stable physical support space for drug particles, preventing gravitational sedimentation of the formulation after storage and subcutaneous injection into animals, thus ensuring the consistency of single-dose drug release kinetics.

[0025] 3. This invention utilizes a dual-blocking system comprised of a specific particle size distribution of micronized drug particles, an interfacial physical coating layer, and a three-dimensional gel network to maintain a stable, long-term release of medroxyprogesterone acetate in animals. This stable drug release rate continuously inhibits the hypothalamic-pituitary-gonadal axis and reduces serum testosterone concentration, thereby decreasing the violent fighting behavior of male deer during estrus. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the preparation process and microscopic mechanism of the absorbable sustained-release agent of the present invention;

[0027] Figure 2 The rheological test curve of the sustained-release product of the present invention is shown in the figure.

[0028] Figure 3 Line graph showing the dynamic changes in serum testosterone (T) levels during estrus in each group of male deer;

[0029] Figure 4 This is a bar chart showing the changes in estrus behavior scores of male deer in each group according to the present invention;

[0030] Figure 5 This is a comparison chart of the average antler production of each group of sika deer in the following year according to the present invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products:

[0033] Medroxyprogesterone acetate (CAS No.: 71-58-9), molecular formula is C 24 H 34 O4, with a purity ≥99.0%, is a conventional commercially available crude drug with a particle size distribution D50 of 50.0 μm or higher. It needs to be pre-treated by ultrafine grinding in subsequent preparations to meet the requirements for long-acting sustained release.

[0034] Sodium hyaluronate (CAS No.: 9067-32-7) is a linear high-molecular-weight polyanionic polysaccharide sodium salt composed of alternating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine, with a weight-average molecular weight ranging from 200,000 Da to 400,000 Da and a glucuronic acid content ≥44.0%.

[0035] Glucuronium (CAS No.: 6556-12-3);

[0036] Dextran sulfate (CAS No.: 9011-18-1) is a polyanionic derivative of dextran polymers, formed by the esterification of hydroxyl groups on the dextran chain with sulfate and the combination with sodium ions. The weight-average molecular weight ranges from 36,000 Da to 50,000 Da, and the sulfur content by mass is 16.0% to 20.0%. Each glucosyl unit contains an average of 2.0 to 2.5 free sulfate groups.

[0037] Preparation Example 1:

[0038] This preparation example provides a micronized crystalline medroxyprogesterone acetate powder A, comprising the following steps:

[0039] S1. 100g of commercially available crude medroxyprogesterone acetate with a purity ≥99.0% is uniformly fed into the grinding chamber of a fluidized bed air jet mill. The grinding pressure is set to 0.6MPa and the classifier speed is set to 3000rpm for ultra-fine grinding. The ground material is collected by a cyclone collector.

[0040] S2. The particle size distribution of the micronized medroxyprogesterone acetate powder, as measured by a laser particle size analyzer, is D50 of 10.0 μm and D90 of 22.5 μm, thus obtaining medroxyprogesterone acetate powder A.

[0041] Preparation Example 2: This preparation example provides a micronized crystalline medroxyprogesterone acetate powder B, comprising the following steps:

[0042] S1. 100g of commercially available crude medroxyprogesterone acetate with a purity ≥99.0% is uniformly fed into the grinding chamber of a fluidized bed air jet mill. The grinding pressure is set to 0.8MPa and the classifier speed is set to 4500rpm for ultra-fine grinding. The ground material is collected by a cyclone collector.

[0043] S2. The particle size distribution of the micronized medroxyprogesterone acetate powder, as measured by a laser particle size analyzer, is D50 of 5.0 μm and D90 of 12.0 μm, thus obtaining medroxyprogesterone acetate powder B.

[0044] Preparation Example 3: This preparation example provides a micronized crystalline medroxyprogesterone acetate powder C, comprising the following steps:

[0045] S1. 100g of commercially available crude medroxyprogesterone acetate with a purity ≥99.0% is uniformly fed into the grinding chamber of a fluidized bed air jet mill. The grinding pressure is set to 0.4MPa and the classifier speed is set to 1500rpm for ultra-fine grinding. The ground material is collected by a cyclone collector.

[0046] S2. The particle size distribution of the obtained micronized medroxyprogesterone acetate powder, as measured by a laser particle size analyzer, is D50 of 15.0 μm and D90 of 30.0 μm, thus obtaining medroxyprogesterone acetate powder C.

[0047] Example 1:

[0048] This embodiment provides an absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, comprising the following steps:

[0049] S1. Add 366.0 mg of micronized crystalline medroxyprogesterone acetate powder A and 50.0 mg of dextran sulfate to a pressure-resistant mixing container, and add 0.60 mL of water for injection. Place the sealed container in a planetary centrifuge with a precision temperature control module to raise and stabilize the core temperature of the material at 62°C. At 62°C, set the revolution speed to 1750 rpm and the rotation speed to 650 rpm, maintain the system pressure at a vacuum of -90 kPa, and process for 12 minutes to obtain a high-viscosity homogeneous suspension paste.

[0050] S2. Transfer the suspension obtained in S1 without damage to the working chamber of a biaxial micro kneader equipped with a water bath jacket. Adjust the temperature of the water bath jacket to lower the temperature of the material inside the chamber to 40°C and maintain this temperature throughout the process. Start the kneader and set the biaxial speed to 45 rpm. Under continuous meshing and shearing of the rotors, uniformly sprinkle 200.0 mg of sodium hyaluronate powder over 3 minutes. Seal the kneading chamber and strictly do not add any additional solvent. Continue kneading at 45 rpm for 38 minutes until the system is transformed into a highly elastic, unsaturated, hard gel.

[0051] S3. The highly elastic hard gel pellets obtained in S2 are rapidly transferred to a cold storage at 5°C, calendered into sheets no more than 5 mm thick, and allowed to stand for 20 minutes for physical quenching and cooling. Approximately 0.21 mL of the remaining water for injection, pre-cooled to 5°C, is added to bring the total physical volume of the single-dose solid powder and water to a baseline of 1.0 mL. This volume is then loaded together with the cooled hard gel pellets into a convection extrusion apparatus equipped with a constant-temperature cold water jacket. This apparatus is internally fitted with a stainless steel microporous mesh plate with a pore size of 0.8 mm. The mechanical pusher is activated, and the unidirectional extrusion pressure is set to 0.35 MPa. 22 passes of reciprocating forced convection extrusion are performed to refine and homogenize the gel system. After extrusion, the gel is collected in a sterile, sealed container and allowed to swell at 5°C for 12 hours.

[0052] S4. Connect the gel after it has been allowed to stand and swell to the dispensing line. Under the condition of an ambient temperature of 5℃, fill it into a pre-filled syringe at a rate of 1.0 mL / vial. After sealing with a rubber piston, store it in the dark at a temperature of 2℃ to 8℃.

[0053] Example 2:

[0054] This embodiment provides an absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, comprising the following steps:

[0055] S1. Add 340.0 mg of micronized crystalline medroxyprogesterone acetate powder B and 40.0 mg of dextran sulfate to a pressure-resistant mixing container, and add 0.55 mL of water for injection. Place the sealed container in a planetary centrifuge equipped with a precision temperature control module to raise and stabilize the core temperature of the material to 60°C. At 60°C, set the revolution speed to 1500 rpm and the rotation speed to 500 rpm, maintain the system pressure at a vacuum of -80 kPa, and process for 8 minutes to obtain a high-viscosity homogeneous suspension paste.

[0056] S2. Transfer the suspension obtained in S1 without damage to the working chamber of a biaxial micro kneader equipped with a water bath jacket. Adjust the temperature of the water bath jacket to lower the temperature of the material inside the chamber to 38°C and maintain this temperature throughout the process. Start the kneader and set the biaxial speed to 30 rpm. Under continuous meshing and shearing of the rotors, uniformly sprinkle 180.0 mg of sodium hyaluronate powder over 2 minutes. Seal the kneading chamber, strictly without adding any additional solvent, and continue kneading at 30 rpm for 30 minutes until the system transforms into a highly elastic, unsaturated, hard gel.

[0057] S3. The highly elastic hard gel pellets obtained in S2 are rapidly transferred to a cold storage at 2°C, calendered into sheets no more than 5 mm thick, and allowed to stand for 15 minutes for physical quenching and cooling. Approximately 0.25 mL of the remaining water for injection, pre-cooled to 2°C, is added to bring the total physical volume of the single-dose solid powder and water to a baseline of 1.0 mL. This volume is then loaded together with the cooled hard gel pellets into a convection extrusion apparatus equipped with a constant-temperature cold water jacket. This apparatus is internally fitted with a stainless steel microporous mesh plate with a pore size of 0.5 mm. The mechanical pusher is activated, and the unidirectional extrusion pressure is set to 0.2 MPa. Fifteen passes of reciprocating forced convection extrusion are performed to refine and homogenize the gel system. After extrusion, the gel is collected in a sterile, sealed container and allowed to swell at 2°C for 10 hours.

[0058] S4. Connect the gel after it has been allowed to stand and swell to the dispensing line. Under an ambient temperature of 2°C, fill the gel into a pre-filled syringe at a rate of 1.0 mL / vial. After sealing with a rubber piston, store at 2°C to 8°C away from light.

[0059] Example 3:

[0060] This embodiment provides an absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, comprising the following steps:

[0061] S1. Add 380.0 mg of micronized crystalline medroxyprogesterone acetate powder C and 60.0 mg of dextran sulfate to a pressure-resistant mixing container, and add 0.65 mL of water for injection. Place the sealed container in a planetary centrifuge with a precision temperature control module to raise and stabilize the core temperature of the material at 65°C. At 65°C, set the revolution speed to 2000 rpm and the rotation speed to 800 rpm, maintain the system pressure at a vacuum of -100 kPa, and process for 15 minutes to obtain a high-viscosity homogeneous suspension paste.

[0062] S2. Transfer the suspension obtained in S1 without damage to the working chamber of a biaxial micro kneader equipped with a water bath jacket. Adjust the temperature of the water bath jacket to lower the temperature of the material inside the chamber to 42°C and maintain this temperature throughout the process. Start the kneader and set the biaxial speed to 60 rpm. Under continuous meshing and shearing of the rotors, uniformly sprinkle 220.0 mg of sodium hyaluronate powder over 5 minutes. Seal the kneading chamber, strictly without adding any additional solvent, and continue kneading at 60 rpm for 45 minutes until the system transforms into a highly elastic, unsaturated, hard gel.

[0063] S3. The highly elastic hard gel pellets obtained in S2 are rapidly transferred to a cold storage at 8°C, calendered into sheets no more than 5 mm thick, and allowed to stand for 30 minutes for physical quenching and cooling. Approximately 0.18 mL of the remaining water for injection, pre-cooled to 8°C, is added to bring the total physical volume of the single-dose solid powder and water to a baseline of 1.0 mL. This water is then loaded together with the cooled hard gel pellets into a convection extrusion apparatus equipped with a constant-temperature cold water jacket. This apparatus is internally fitted with a stainless steel microporous mesh plate with a pore size of 1.2 mm. The mechanical pusher is activated, and the unidirectional extrusion pressure is set to 0.5 MPa. Thirty passes of reciprocating forced convection extrusion are performed to refine and homogenize the gel system. After extrusion, the gel is collected in a sterile, sealed container and allowed to swell at 8°C for 14 hours.

[0064] S4. Connect the gel after it has been allowed to stand and swell to the dispensing line. Under the condition of an ambient temperature of 8°C, fill it into a pre-filled syringe at a rate of 1.0 mL / vial. After sealing with a rubber piston, store it in the dark at a temperature of 2°C to 8°C.

[0065] Comparative Example 1:

[0066] Compared with Example 1, the difference is that a conventional forward mixing process was used. 200.0 mg of sodium hyaluronate powder and 50.0 mg of dextran sulfate were completely dissolved in an appropriate amount of water for injection to prepare a high-viscosity blank hydrogel. Then, 366.0 mg of the micronized medroxyprogesterone acetate powder A obtained in Example 1 was added, and the remaining 0.21 mL of water for injection was added. The mixture was then mixed conventionally at 45 rpm for 50 minutes at room temperature (25°C) using a micro kneader of the same model. Low-temperature cold extrusion refining was not performed, and all other processes were the same.

[0067] Comparative Example 2:

[0068] The difference from Example 1 is that the unsaturated hydration intervention involving competitive solvent deprivation is not performed in step S2. When adding 200.0 mg of sodium hyaluronate powder in step S2, approximately 0.21 mL of the remaining required water for injection is simultaneously added to the kneading chamber, allowing the sodium hyaluronate to undergo saturated swelling and kneading under full water conditions. All other steps are the same.

[0069] Comparative Example 3:

[0070] Compared with Example 1, the difference is that dextran sulfate is not added in step S1. 366.0 mg of medroxyprogesterone acetate powder A is directly pre-dispersed with 0.60 mL of water for injection at 62°C by planetary centrifugation, and in step S3, 0.21 mL of the remaining water for injection, the same amount as in Example 1, is added. All other steps are the same.

[0071] Comparative Example 4:

[0072] The difference from Example 1 is that no high-temperature treatment is performed in step S1. The core temperature of the material pre-dispersed by planetary centrifugation is set to room temperature (25°C), and all other parameters are the same.

[0073] Comparative Example 5:

[0074] Compared to Example 1, the difference lies in that the mechanical network locking of "microporous mesh plate convection extrusion" is not performed in step S3. After transferring the highly elastic hard gel pellet obtained in S2 to a 5°C cold storage, approximately 0.21 mL of pre-cooled remaining water for injection was directly added. Without applying any mechanical extrusion pressure, it was allowed to swell and equilibrate at 5°C for 12 hours. All other steps were the same. Test Example 1:

[0075] Experimental steps:

[0076] The mixing process was recorded using a biaxial micro kneader equipped with a dynamic torque sensor. During the kneading phase of preparation, the instrument recorded rotor torque at a sampling rate of 1 Hz and extracted the maximum torque peak value for each group from the addition of sodium hyaluronate to the end of kneading.

[0077] Rheological tests were performed using a rotational rheometer equipped with a 20mm parallel plate testing system. The product, after constant-volume swelling, was placed on the testing stage, and the testing temperature was set to 25℃ with a gap of 1.0mm. An amplitude scanning mode was used, with a fixed angular frequency of 10 rad / s, and the shear stress scanning range was set from 0.1 Pa to 500 Pa. The storage modulus and loss modulus of the sample were recorded as curves of shear stress, and the shear stress value corresponding to the intersection of the two curves was taken as the yield stress. Before testing, the samples were allowed to equilibrate for 5 minutes, and each group of samples was tested in triplicate, with the average value taken.

[0078] The test results are shown in Table 1:

[0079] Table 1: Test results of maximum kneading torque and yield stress for each group of samples

[0080] Group Maximum torque during the kneading stage (N·m) Yield stress (Pa) of the final product Example 1 12.43 215.6 Example 2 10.87 192.3 Example 3 14.15 238.9 Comparative Example 1 2.31 45.8 Comparative Example 2 3.15 86.2 Comparative Example 3 11.62 164.5 Comparative Example 4 9.24 143.7 Comparative Example 5 12.38 75.4

[0081] According to the data in Table 1, the maximum torque during the kneading stage in Examples 1 to 3 ranged from 10.87 N·m to 14.15 N·m, and the yield stress of the products was greater than 190 Pa. With the addition of sodium hyaluronate powder under controlled initial moisture conditions, the total amount of available free water in the system was limited, and sodium hyaluronate absorbed moisture from the surrounding phase during hydration. This increased molecular chain entanglement within the system, transforming the material from a suspended paste to a high-molecular-weight semi-solid state, resulting in increased rotor resistance in the kneading equipment. Through low-temperature microporous forced extrusion, the high-molecular-weight agglomerates absorbed water and swelled to form a three-dimensional gel network, exhibiting high yield stress. This provided suspension support for the drug particles, preventing physical sedimentation of the formulation.

[0082] The maximum torques of Comparative Example 1 and Comparative Example 2 were 2.31 N·m and 3.15 N·m, respectively, with relatively low yield stress values. Comparative Example 1 used a process where the drug was mixed after complete water dissolution, while Comparative Example 2 added complete water simultaneously with the addition of sodium hyaluronate. The ample water content caused the sodium hyaluronate to expand and dissolve, without any local dehydration phase transition process, thus failing to create solvent deprivation conditions for the dextran sulfate. The system maintained a fluid state, failed to form a stagnant network, and exhibited low torque and low yield stress.

[0083] Comparative Example 3 lacked dextran sulfate, and Comparative Example 4 did not undergo high-temperature treatment. Both showed a certain increase in torque during the kneading stage, but the yield stress of the final products was lower than that of the examples, indicating that the absence of components and changes in temperature conditions affected the formation of the network structure. Comparative Example 5 did not undergo microporous mesh convection extrusion. After static swelling, the yield stress of the product decreased to 75.4 Pa, indicating that without mechanical extrusion, polymer agglomerates could not disentangle and form a network structure, resulting in a decrease in the suspension support capacity of the formulation. Test Example 2:

[0084] Experimental steps:

[0085] Adult male sika deer of similar age, weight, and health status, who were about to enter estrus, were selected. They were divided into a control group (group 0, no drug injection), Example 1 to 3 groups, and Comparative Examples 1 to 5 groups.

[0086] On September 15, each group of the examples and comparative examples underwent a single subcutaneous injection of the corresponding prepared sustained-release agent into the neck, with an injection volume of 1.2 mL. Group 0 received an equal volume of physiological saline.

[0087] Blood samples were collected from the jugular vein of each group on days 10, 20, 35, and 60 post-injection. Each blood sample was 5 mL and placed in a vacuum blood collection tube without a coagulant.

[0088] After blood collection, let it stand at room temperature for 30 minutes, then centrifuge at 3000 r / min for 15 minutes to separate the serum, aliquot it into cryovials and store at -20℃.

[0089] Testosterone concentration in serum samples was determined using radioimmunoassay. Samples at each time point were measured in triplicate, and data were recorded and the mean and standard deviation were calculated.

[0090] The test results are shown in Table 2:

[0091] Table 2: Dynamic monitoring results of serum testosterone (T) levels during estrus in male sika deer of each group (ng / mL)

[0092] Grouping 10d 20d 35d 60d 0 groups 18.52±0.83 18.78±0.74 19.61±1.08 11.85±0.61 Example 1 6.51±0.14 5.98±1.21 6.87±0.63 4.62±0.31 Example 2 7.89±0.32 7.15±1.08 8.31±0.82 5.51±0.12 Example 3 11.24±0.25 11.27±0.58 10.74±1.05 7.63±0.28 Comparative Example 1 5.14±0.47 9.38±0.92 16.42±1.15 11.37±0.44 Comparative Example 2 6.78±0.51 11.84±1.03 17.51±0.89 10.92±0.53 Comparative Example 3 4.05±0.22 8.65±1.14 15.28±1.21 11.14±0.68 Comparative Example 4 6.32±0.38 10.71±0.85 16.03±0.94 10.85±0.41 Comparative Example 5 8.16±0.65 13.52±1.27 18.24±1.02 11.64±0.55

[0093] According to the data in Table 2, the serum testosterone concentration in groups 1 to 3 of Examples was lower than that in the group 0 within 10 to 60 days. After entering the bloodstream, medroxyprogesterone acetate increases progesterone levels, triggering negative feedback regulation of the hypothalamus-pituitary-gonadal axis, inhibiting gonadotropin-releasing hormone secretion, reducing the release of luteinizing hormone and follicle-stimulating hormone, and decreasing the testosterone synthesis capacity of testicular interstitial cells. The decrease in testosterone concentration indicates that the drug maintains release for 60 days. The process in Examples involved unsaturated hydration intervention and convection extrusion using a mesh plate. Sodium hyaluronate absorbed free water in the system, and dextran sulfate underwent dehydration phase change to coat the surface of the medroxyprogesterone acetate micropowder. The network structure and physical coating layer increased the resistance to drug diffusion and mass transfer, reducing the initial burst release and prolonging the release time.

[0094] Comparative Example 1 used a full-volume water dissolution and mixing process. At 10 days, the testosterone concentration was 5.14 ng / mL, showing rapid initial release; at 35 days, it rose to 16.42 ng / mL, approaching the levels of Group 0, indicating drug depletion. Full-volume water dissolves the polymer backbone, preventing the formation of a blocking network.

[0095] In Comparative Example 2, all water was added simultaneously with sodium hyaluronate, and the dextran sulfate did not precipitate out to coat the drug, increasing the contact area between the drug and tissue fluid. Testosterone concentration rapidly increased from day 20, reaching 17.51 ​​ng / mL at day 35, and the inhibitory effect weakened.

[0096] Comparative Example 3, lacking dextran sulfate, showed a testosterone concentration of 4.05 ng / mL at 10 days, exhibiting an initial burst release. Lacking interfacial shielding, the sodium hyaluronate network could not effectively limit drug dissolution long-term, and the testosterone concentration rebounded to 15.28 ng / mL at 35 days, losing its long-term inhibitory effect.

[0097] Comparative Example 4, which was not subjected to high-temperature treatment, showed reduced polymer chain segment movement and entanglement, resulting in a looser network structure and accelerated degradation. At 35 days, the testosterone concentration recovered to 16.03 ng / mL, and the controlled-release period was shortened.

[0098] Comparative Example 5 did not involve mechanical extrusion, and the system was not refined and dispersed by shear force, resulting in internal structural defects. Local drug aggregation led to matrix rupture, and the testosterone concentration was 8.16 ng / mL at 10 days, rising to 13.52 ng / mL at 20 days. Unstable release kinetics led to a decrease in controlled-release efficacy.

[0099] Test Example 3:

[0100] Experimental steps:

[0101] Take 2.0 mL of each group of gel samples after volume adjustment and swelling, put them into a glass NMR tube with a diameter of 15 mm, remove air bubbles, and seal with a polytetrafluoroethylene stopper.

[0102] The NMR tube was placed in the isothermal probe of the low-field NMR analyzer. The probe temperature was set to 32℃, and the instrument frequency was set to 21MHz. CPMG pulse sequences were used to acquire the signal. The pulse duration for 90 degrees was set to 18μs, the pulse duration for 180 degrees to 35μs, the echo time to 0.2ms, the number of echoes to 8000, and the number of accumulations to 16.

[0103] After signal acquisition, the attenuation curve is inverted using analysis software to generate a transverse relaxation time (T2) distribution map. Based on the peak positions in the map, bound water peaks and free water peaks are distinguished, and the relative peak area ratio (%) of bound water and free water is calculated by integration.

[0104] The test results are shown in Table 3:

[0105] Table 3: Relative peak area test results of moisture state distribution of each group of samples

[0106] Group The percentage of the relative peak area of ​​bound water (%) Percentage of free water relative peak area (%) Example 1 96.34 3.66 Example 2 94.81 5.19 Example 3 97.02 2.98 Comparative Example 1 58.42 41.58 Comparative Example 2 62.15 37.85 Comparative Example 3 91.27 8.73 Comparative Example 4 82.59 17.41 Comparative Example 5 89.33 10.67

[0107] According to the data in Table 3, the relative peak area of ​​bound water in Examples 1 to 3 ranged from 94.81% to 97.02%, while the proportion of free water was less than 5.19%. The low-field NMR transverse relaxation time reflects the degree of binding between protons and the macromolecular backbone within the system; a short relaxation time indicates that the water is tightly bound. The high proportion of bound water in the Example group indicates that sodium hyaluronate forms hydrogen bonds with water molecules under kneading conditions, reducing the number of freely moving water molecules and converting them into a state bound to the polymer chains. The reduced free water content altered the liquid phase environment, causing dextran sulfate to precipitate on the surface of medroxyprogesterone acetate due to insufficient local solvent, forming an encapsulation layer.

[0108] The relative peak area ratios of free water in Comparative Examples 1 and 2 were 41.58% and 37.85%, respectively, higher than those in the Example. The process provided full water to allow sodium hyaluronate to swell and dissolve; the macromolecular network failed to bind all the water, and the system maintained a high proportion of free water. The free water environment caused dextran sulfate to remain in a dissolved state, without solvent removal, and no physical coating layer formed on the surface of the drug particles.

[0109] Comparative Example 3, without the addition of dextran sulfate, had a bound water content of 91.27%, reflecting the basic state of water bound to sodium hyaluronate. Comparative Example 4, using room temperature pre-dispersion, had a bound water content of 82.59%, indicating that room temperature limited the expansion of polymer molecular chains, affecting the binding of sodium hyaluronate with water molecules, and some water existed in a free state. Comparative Example 5, without the convection extrusion process, had a free water content of 10.67%, indicating that the hydration inside the polymer micelles without mechanical shearing was uneven, some water did not enter the gel network, and the uniformity of the network structure decreased.

[0110] Test Example 4:

[0111] Experimental steps:

[0112] Healthy male sika deer of similar age and weight who were about to enter estrus were selected and divided into group 0, Example 1 to 3, and Comparative Example 1 to 5.

[0113] On September 15, the sika deer in each group of the examples and comparative examples were injected subcutaneously into the neck with a sustained-release agent, with an injection volume of 1.2 mL. Group 0 was injected with 1.2 mL of physiological saline.

[0114] On days 10, 35, and 60 post-injection, the aggressive and mating behaviors of each group of sika deer were scored according to the estrus behavior scoring criteria. Individual behavior scores ranged from 0 to 5 points. The total score for each sika deer was recorded, and the average score for each group was calculated.

[0115] The following year, during the antler harvesting season, the weight of the antlers of each group of sika deer was measured, and the average antler yield was calculated.

[0116] The test results are shown in Table 4:

[0117] Table 4: Statistical analysis of male sika deer during estrus and antler production in the following year.

[0118] Group 10-day behavioral total score 35d Behavioral Total Score 60d behavioral total score Average antler yield in the following year (kg / head) 0 groups 7.6 9.5 7.2 1.62 Example 1 3.1 2.6 2.2 1.84 Example 2 3.5 3.2 2.5 1.81 Example 3 2.8 2.9 2.4 1.88 Comparative Example 1 2.4 8.6 6.9 1.66 Comparative Example 2 3.2 8.1 7.4 1.70 Comparative Example 3 2.1 7.9 6.8 1.69 Comparative Example 4 3.8 8.3 7.1 1.65 Comparative Example 5 4.5 8.8 7.5 1.67

[0119] According to the data in Table 4, the total behavioral scores of groups 1 to 3 in Examples 1 to 60 days ranged from 2.2 to 3.5, while the total behavioral score of group 0 ranged from 7.2 to 9.5. The average antler yield of groups 1 to 3 the following year was 1.81 kg to 1.88 kg, while the average antler yield of group 0 the following year was 1.62 kg. The process used in these examples employed unsaturated hydration intervention, where sodium hyaluronate absorbed moisture, and dextran sulfate underwent a dehydration phase transition on the surface of medroxyprogesterone acetate micropowder, resulting in its deposition. Combined with microporous mesh plate convection extrusion to form a gel network, this increased the mass transfer resistance of medroxyprogesterone acetate diffusion. Medroxyprogesterone acetate was released into the bloodstream, inhibiting the hypothalamic-pituitary-gonadal axis function, reducing serum testosterone concentration, and decreasing aggressive and mating behaviors in sika deer during estrus. The average yield of antlers in the following year in the example group (1.81 kg to 1.88 kg) was even higher than that in the control group (1.62 kg), proving that the absorbable slow-release agent, after degradation in the body, not only does not affect the ossification of deer antlers, but also effectively reduces the physical exertion and mechanical damage caused by fighting during the estrus period, which is beneficial to the growth and protection of deer antlers in the following year.

[0120] Comparative Example 1, using a water-dissolving mixing process, showed a total behavioral score of 2.4 at 10 days and increased to 8.6 at 35 days. Excessive water content led to premature dissolution of the gel network, shortened the release time of medroxyprogesterone acetate, and reduced behavioral inhibition.

[0121] In Comparative Example 2, all water was added simultaneously with sodium hyaluronate, and dextran sulfate did not form a dehydration phase transition layer on the surface of medroxyprogesterone acetate. The increased contact area with medroxyprogesterone acetate led to a behavioral score of 8.1 at 35 days.

[0122] Comparative Example 3, lacking dextran sulfate, had a total behavioral score of 2.1 at day 10. Sodium hyaluronate gel reduced the inhibitory effect on medroxyprogesterone acetate, accelerated medroxyprogesterone acetate dissolution, and the total behavioral score recovered to 7.9 at day 35.

[0123] Comparative Example 4, which was not subjected to high-temperature treatment, showed a reduced gel network entanglement density and a looser structure. Medroxyprogesterone acetate diffusion rate accelerated, and the total behavioral score rose to 8.3 at 35 days.

[0124] Comparative Example 5 did not undergo microporous mesh convection extrusion, resulting in structural defects within the gel. Medroxyprogesterone acetate release was unstable, with a total behavioral score of 4.5 at 10 days and rising to 8.8 at 35 days.

[0125] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, characterized in that, Includes the following steps: S1. Add the proportioned amount of micronized crystalline medroxyprogesterone acetate powder and dextran sulfate to a pressure-resistant mixing container, and add the corresponding amount of water for injection; under the conditions of -80kPa to -100kPa vacuum and material core temperature of 60℃ to 65℃, centrifuge the mixture for 8 to 15 minutes to obtain a high-viscosity homogeneous suspension paste. S2. Transfer the suspended paste into a water bath jacketed biaxial micro kneader, control the temperature to 38℃ to 42℃, and add sodium hyaluronate dry powder at a uniform speed under the meshing shear of the rotor; seal without adding solvent, and continue kneading for 30 to 45 minutes until a highly elastic unsaturated hard gel is formed. S3. Quickly cool the hard rubber mass to 2℃-8℃, and load it together with the remaining pre-cooled water for injection into a low-temperature convection extrusion device. Extrude it through a microporous mesh plate for 15 to 30 passes at a unidirectional extrusion pressure of 0.2MPa to 0.5MPa. After extrusion, let it stand at 2℃ to 8℃ to swell for 10 to 14 hours. S4. The finished product is obtained by quantitative filling, sealing, and storage in the dark at an environment of 2℃ to 8℃.

2. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, as described in claim 1, is characterized in that... The medroxyprogesterone acetate is a micronized crystalline powder, and the dextran sulfate is coated on the surface of the medroxyprogesterone acetate to form a physical coating layer; the medroxyprogesterone acetate micronized powder with the physical coating layer is suspended and locked inside the gel three-dimensional network formed by the sodium hyaluronate after absorbing water and swelling and then being refined by forced convection extrusion.

3. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, as described in claim 2, is characterized in that... Based on the volume of the absorbable sustained-release agent, the mass concentrations of each raw material component are as follows: the concentration of medroxyprogesterone acetate is 340.0 mg / mL to 380.0 mg / mL; the concentration of dextran sulfate is 40.0 mg / mL to 60.0 mg / mL; the concentration of sodium hyaluronate is 180.0 mg / mL to 220.0 mg / mL; and the balance is water for injection.

4. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, as described in claim 3, is characterized in that... The medroxyprogesterone acetate has a purity of not less than 99.0% and a particle size distribution D50 of 5.0 μm to 15.0 μm and D90 of not more than 30.0 μm; this particle size range is designed to provide a long-term stable interfacial drug release area in conjunction with the physical coating layer.

5. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus, as described in claim 2, is characterized in that... The sodium hyaluronate has a weight-average molecular weight range of 200,000 Da to 400,000 Da and a glucuronic acid content of not less than 44.0%; the dextran sulfate has a weight-average molecular weight range of 36,000 Da to 50,000 Da, a sulfur content of 16.0% to 20.0% by mass, and an average of 2.0 to 2.5 free sulfate groups per glucosyl unit.

6. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus according to claim 1, characterized in that, The mass ratio of sodium hyaluronate to dextran sulfate is between 3:1 and 5.5:

1. This mass ratio ensures, from the formulation level, that the limited free water in the system is preferentially absorbed by sodium hyaluronate, thereby directionally triggering the local dehydration and precipitation of dextran sulfate.

7. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus according to claim 1, characterized in that, In the low-field nuclear magnetic resonance transverse relaxation time T2 distribution spectrum, the relative peak area ratio of bound water inside the absorbable sustained-release agent is 94.81% to 97.02%, and the relative peak area ratio of free water is 2.98% to 5.19%; the low free water ratio environment forces the sulfated dextran to maintain a stable physical coating layer on the surface of the drug particles.

8. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus according to claim 1, characterized in that, At a test temperature of 25°C, the yield stress of the absorbable sustained-release agent is in the range of 192.3 Pa to 238.9 Pa. This stress threshold endows the three-dimensional gel network with the physicomechanical properties to provide drug particles with durable resistance to gravity settling.

9. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus according to claim 1, characterized in that, The physical coating layer is obtained under the following unsaturated hydration conditions: the water absorption and swelling process of the sodium hyaluronate is carried out without the addition of any additional solvent and accompanied by continuous meshing and shearing, until the material is transformed from a high-viscosity homogeneous suspension paste into a highly elastic unsaturated hard gel.

10. The absorbable sustained-release agent for inhibiting fighting behavior in male deer during estrus according to claim 1, characterized in that, The forced convection extrusion refining refers to: extruding intermediate material in a highly elastic, unsaturated, hard agglomerate state along with the remaining water for injection through a mesh plate with micropore diameters of 0.5 mm to 1.2 mm under unidirectional force; this extrusion process breaks up the disordered entanglement of macromolecular agglomerates and macroscopically reconstructs a retarded structure with uniform physical properties.

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