Melatonin implant and preparation method thereof

Abstract

The invention relates to the technical field of veterinary drug preparation production, and discloses a melatonin implant, the melatonin implant comprises an embedded core and a coating, the coating is sprayed on the surface layer of the embedded core, the embedded core comprises the following components by mass: 65-75% of melatonin, 8-12% of a carrier, 10-15% of a lubricant, and 7-9% of a first plasticizer; the coating comprises the following components: a film-forming agent, a second plasticizer and a solvent, the mass ratio of the film-forming agent to the second plasticizer is (1.5-2.5): 1, and the ratio of the mass of the film-forming agent to the volume of the solvent is (2.5-3.5): 100 (g / mL). The implant has large drug loading capacity and can be slowly released for a long time, and only 13% of the implant can be released in vitro for 28 days. The melatonin implant can be prepared by adopting a hot melt extrusion and coating method, and the preparation method has the advantages of simple process, continuous production, short and stable production period, less material loss, better drug content uniformity and high technical yield, and is beneficial to expanded production.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary drug preparation production, and in particular to a melatonin implant and a preparation method thereof. Background Art

[0002] Melatonin (MT) is a small molecule substance. It is a highly fat-soluble indoleamine hormone synthesized by the pineal gland using tryptophan as raw material under the action of a series of enzymes. It was first called the "hormone of darkness" in the West. Lerner et al. first extracted it from the pineal gland of cattle in 1958. Since it can lighten the black skin of frogs, the substance was named melatonin, that is, N-acetyl-5-methoxytryptamine. Melatonin has a wide range of physiological and pharmacological effects. It is not only related to biological rhythms, but also can remove reactive oxygen species, and has anti-aging, anti-inflammatory and body resistance enhancement functions. In fact, in the first 40 years after the discovery of melatonin, most of the research related to melatonin and the pineal gland was related to the hormone regulating the reproductive characteristics of animals that rely on photoperiod reproduction. Melatonin has been widely used to advance the breeding season of estrus females. A small amount of melatonin is added to the animal's body every day to maintain the melatonin concentration in the female's peripheral blood at a high enough level to simulate short days, without inhibiting the endogenous secretion of melatonin at night. As long as the melatonin concentration in the female's body during the day is maintained above 30-200pg / mL for 5 weeks, the breeding season can be effectively advanced to achieve the purpose of medication.

[0003] In clinical treatment, the half-life and duration of action of drugs are too short, which often makes the drugs unable to meet the requirements of sustained release and long-term effect. For example, melatonin tablets, capsules or injections, these dosage forms maintain blood drug concentration for a short time, have low bioavailability, and require repeated administration. In contrast, melatonin implants have become a more ideal choice due to their advantages such as constant sustained release rate, long release time, no first-pass effect and high bioavailability. In foreign countries, melatonin implants have been developed and marketed for 40 years, and the most representative product is 18mg implant, which gradually releases melatonin and maintains a sufficiently high concentration in the peripheral blood for up to 70 to 90 days, has been widely used in production and has achieved good results. Implantation of melatonin implants can reduce the number of empty ewes and increase the lambing rate of ewes; use in male livestock can increase sexual activity in the non-breeding season and improve the quality and quantity of sperm. In addition, the use of melatonin implants in fur animals such as minks and goats can promote the growth and development of fur, induce early maturity of fur, and increase cashmere production. It has been reported that melatonin has a good effect on treating canine X-type hair loss and controlling cat estrus in the short term. Adding 5mg melatonin per kilogram of feed can also reduce the incidence of gastric ulcers in piglets.

[0004] Currently, there are no veterinary melatonin implant products on the market in China, and the existing patents related to melatonin implants all have certain shortcomings.

[0005] A Chinese invention patent (publication number: CN111450043A, publication date: 2020.07.28) discloses a veterinary melatonin sustained-release implant and its preparation method and application. The implant is a melatonin implant prepared by hot-melt extrusion technology, but the implant has a low drug loading and a short sustained-release time of only 10 days. For the purpose of advancing the breeding season of seasonal estrus animals such as sheep, repeated administration is required, which not only causes discomfort and pain to the animals, violates animal welfare, but also increases the workload of breeders.

[0006] Melatonin implants have a long history of research and development and application abroad, but their product patents still have certain shortcomings. For example, in the Australian patent application number 38111 / .85, a subcutaneous implant was prepared, but the initial burst release of the implant was severe. After the burst release, the melatonin level in the peripheral blood reached 100-1000 times the normal nighttime level, which may have harmful effects. Secondly, the duration of action is short, only 10-14 days, and multiple repeated administrations are required during use, which will cause discomfort and pain to the animals and increase the workload of the breeders.

[0007] European Patent EP0246910B1 has improved the process to solve the problem of short sustained release time of melatonin implant in the above Australian Patent 38111 / .85. The published process flow is as follows:

[0008] Preparation of implant cores (8000 cores)

[0009] (1) Weigh 10 g of ethyl cellulose S100 and dissolve it in 100 mL of 95% ethanol.

[0010] (2) Weigh 200 g of melatonin and crush it in a mortar with a pestle.

[0011] (3) Weigh 0.2 g of quinoline yellow, mix with melatonin and transfer to a round-bottom container.

[0012] (5) Add 45 g of the ethanol solution prepared in step (1) to melatonin and mix thoroughly until a hard mixture is obtained.

[0013] (6) The mixture was manually sieved (mesh size #12) to form granules.

[0014] (7) Spread the granules on a tray and dry them at room temperature overnight.

[0015] (8) Crush the dried granules in a mortar with a pestle, transfer them to a round-bottom container, and add 45 g of the ethanol solution prepared in step (1) and mix well.

[0016] (9) Repeat steps (6), (7), and (8).

[0017] (10) The granules were sieved (sieve size #24) and the weight of the sieved granules was recorded.

[0018] (11) Calculate the weight of hydrogenated vegetable oil required (3% of the weight of the sieved particles).

[0019] (12) Weigh hydrogenated vegetable oil and 0.2 g of quinoline yellow and mix thoroughly with the sieved granules.

[0020] (13) The mixed granules are pressed into a core using a single-punch tablet press, and the hardness is about 10 kPa.

[0021] Embedded core coating

[0022] (1) Prepare 150 mL of a dichloromethane solution of 1.25% Ethocel M50 and 0.5% dibutyl phthalate (coating solution 1).

[0023] (2) Prepare 300 mL of a dichloromethane solution of 0.625% Ethocel M50 and 0.25% dibutyl phthalate (coating solution 2).

[0024] (3) Load 300 g of embedded cores into the Hi-Coater coating machine pan and set the pan speed to 5 rpm, inlet air temperature to 40° C., and exhaust air temperature to 50° C. Maintain these parameters for 10 minutes to preheat.

[0025] (4) Increase the coating pan speed to 30 rpm and spray 150 mL of coating solution 1.

[0026] (5) Maintain the speed and temperature of the coating pan and spray 100 mL of coating solution 2.

[0027] (6) Reduce the coating pan speed to 5 rpm and cool the implant to room temperature for 10 minutes. Coat the implant with 0.5 g of talc.

[0028] (7) Increase the coating pan speed to 30 rpm, increase the inlet air temperature to 40°C, and increase the exhaust air temperature to 30°C. Spray 100 mL of the solution prepared in step 2.

[0029] (8) Reduce the speed to 4 rpm and cool to room temperature for 10 minutes. Apply 0.5 g of talcum powder to the implant.

[0030] (9) Repeat steps (7) and (8).

[0031] (10) Transfer the prepared samples to a sealed light-proof container for storage.

[0032] Although the implant prolongs the sustained release time of the drug, the preparation steps are cumbersome and the production is discontinuous. Crushing with a pestle is inefficient, and the granulation needs to be dried overnight, which takes a long time; two coating solutions need to be prepared and coated twice. The coating solvent dichloromethane is highly toxic, harmful to human health and pollutes the environment. According to the "ICH Q3C Impurities: Guidelines for Residual Solvents" and the "Technical Guidelines for the Study of Residual Solvents in Chemical Drugs", dichloromethane is a Class II solvent and is a solvent that should be restricted. Summary of the invention

[0033] In view of the deficiencies of the prior art, the present invention provides a melatonin implant, which can be prepared by hot melt extrusion and coating method, has a simple and convenient preparation process, has a large drug loading capacity and can achieve long-term sustained release, and the in vitro release rate for 28 days is only 13%.

[0034] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0035] A melatonin implant, comprising an implant core and a coating, wherein the coating is sprayed on the surface of the implant core, and the components of the implant core include the following components by mass percentage:

[0036] Melatonin 65-75%,

[0037] Carrier 8-12%,

[0038] Lubricant 10-15%,

[0039] First plasticizer 7-9%;

[0040] The coating comprises a film-forming agent, a second plasticizer and a solvent, wherein the mass ratio of the film-forming agent to the second plasticizer is (1.5-2.5):1, and the mass ratio of the film-forming agent to the volume ratio of the solvent is (2.5-3.5):100 (g / mL).

[0041] Preferably, the carrier is one or more of ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose, and cellulose acetate, preferably ethyl cellulose, and more preferably ethyl cellulose N-50.

[0042] Preferably, the lubricant is one or more of hydrogenated vegetable oil, animal fat, beeswax, carnauba wax, stearyl alcohol, monoglyceride, preferably hydrogenated vegetable oil.

[0043] Preferably, the first plasticizer is one or more of polyethylene glycol, poloxamer, and povidone, and the molecular weight of polyethylene glycol is 1000-20000, preferably polyethylene glycol, and more preferably polyethylene glycol 6000.

[0044] Preferably, the film-forming agent is one or more of ethyl cellulose, cellulose acetate, acrylic resin, cellulose acetate titanate, hydroxypropyl methylcellulose titanate, and hydroxypropyl methylcellulose acetate succinate, preferably ethyl cellulose, and more preferably ethyl cellulose N-50.

[0045] Preferably, the second plasticizer is one or more of phthalates, glycerol, propylene glycol, polyethylene glycol, triacetin, castor oil, and acetylated glycerides, preferably phthalates, and more preferably diethyl phthalate.

[0046] Preferably, the solvent is one or more of water, ethanol, acetone, and isopropanol, preferably ethanol.

[0047] Preferably, the coating composition further includes a colorant, and the mass ratio of the colorant to the volume ratio of the solvent is 2.5-3.5:1000 (g / mL).

[0048] Preferably, the coating weight gain is 7.3% to 7.7% of the implanted core.

[0049] Furthermore, the present invention also provides a method for preparing the melatonin implant, comprising the following steps:

[0050] 1) Mixing: crush large particles and pass through a 50-100 mesh sieve; weigh the raw and auxiliary materials for embedding the core and mix them in a container;

[0051] 2) Preparation of embedded core by hot melt extrusion: the mixed materials are put into the feeder of the preheated hot melt extruder, and the preheating temperature is 50-160°C; after the materials are fed, they are mixed, extruded, pushed and transported by the rotating screw to the die with an inner diameter of 2.3mm for extrusion, and the screw speed is 5-35rpm; the die area is equipped with a cutter to cut the embedded core, and after cooling to room temperature, it is kept dry and protected from light for future use;

[0052] 3) Preparing the coating solution: weigh the raw materials of each coating component, place the solvent in a suitable container and stir it with a magnetic stirrer at a stirring speed of 600-800 rpm; slowly pour the film-forming agent into the solvent and stir until it is completely dissolved, add the plasticizer and continue stirring to obtain the coating solution;

[0053] 4) Preparation of implants: Place the implant core into a preheated coating pan and coat it with coating liquid spray. During the coating process, take samples and weigh them to calculate the coating weight gain. When the target weight gain is reached, stop spraying and dry the implant. After drying, take out and cool to room temperature and store in a sealed container away from light.

[0054] Among them, the hot melt extruder and coating machine need to be preheated for 15-20 minutes. The stirring time of the film-forming agent is 30-60 minutes, and the stirring time of the plasticizer is 15-25 minutes.

[0055] The coating machine pot speed is set to 5-10rpm, the fan speed is 1000-1500rpm, the inlet air temperature is 40-50℃, the outlet air temperature is 35-45℃, the atomization pressure is 0.10-0.20MPa, the fan pressure is 0.1-0.3MPa, the ejector pressure is 0.20-0.30MPa, and the peristaltic pump speed is 3-7rpm.

[0056] The specific steps for sampling and weighing during the period to calculate the coating weight gain are as follows: 1) Sampling before starting to spray: randomly draw samples from the coating pot, and determine the average tablet weight by weighing 3 groups of 30 tablets in each group; 2) Mid-control sampling: after about 80% of the coating liquid has been sprayed, start to draw coating samples from the coating pot every 15 minutes to check the weight gain of the coated tablets until the target weight gain is reached.

[0057] The obtained melatonin implant is cylindrical in shape, with a specific weight of (30±0.5) mg, a length of 6.2-6.4 mm, and an inner diameter of 2.2-2.4 mm.

[0058] The melatonin implant of the present invention only needs to be coated once to achieve the same sustained-release effect as the overseas commercial product Regulin, and the drug release characteristics are easy to control and the control range is large. The coating weight gain directly affects the release rate of the melatonin implant. As the coating weight gain increases, the coating film thickness increases, and the drug release rate slows down significantly. Therefore, the coating weight gain can be controlled to control the drug release rate. The present invention proves through in vitro release tests that the melatonin implant can achieve long-acting and sustained-release effects. The in vitro release is only 13% for 28 days, and the in vitro daily release is 0.1mg-0.2mg. Implanting melatonin in summer (June to July) can increase the estrus induction rate of ewes.

[0059] The hot melt extrusion adopted by the present invention is a solvent-free technology, which can be processed continuously with simple steps, reduces the difference between batches, and improves the repeatability and production efficiency of the process; secondly, it saves materials, and the material consumption for a single use is only tens of grams, which is conducive to prescription screening and process optimization; the technology has high output and is conducive to expanding production; the material loss is small and the degree of automation is high; the production cycle is short, and compared with the production cycle of the granulation tableting process that takes several days, the process of the present invention is shortened to a few hours, and it has a unique mixing mechanism, which can make the drugs reach molecular level mixing, and the uniformity of drug content is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the release curve of the melatonin implant core prepared in Test Examples 15, 20, 21, and 23.

[0061] Figure 2A The release curves of the melatonin implants prepared in Experimental Examples 48-50 and the foreign commercial product Regulin are shown in FIG. Figure 2BThe release rate curves of the melatonin implants prepared in Experimental Examples 48-50 and the foreign commercial product Regulin are shown.

[0062] Figure 3A The release curves of the melatonin implant prepared in Experimental Example 50 and the foreign commercial product Regulin are shown in FIG. Figure 3B The release rate curves of the melatonin implant prepared in Experimental Example 50 and the foreign commercial product Regulin are shown in FIG. Figure 3C This is a release curve of the melatonin implant prepared in Experimental Example 50 and the foreign commercial product Regulin. DETAILED DESCRIPTION

[0063] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] 1. Melatonin implant

[0065] First, based on the content and size of Regulin, a product sold abroad, it was determined that its main ingredient, melatonin, accounted for 90%, and auxiliary materials such as ethylcellulose (EC) and hydrogenated vegetable oil (HVO) accounted for 10%. Based on this formula, the melatonin loading was 90%, and ethylcellulose and hydrogenated vegetable oil were weighed in proportions of 9:1, 7:3, 5:5, 3:7, and 1:9, respectively, and mixed with melatonin and ethanol to prepare melatonin implant cores. The implant cores of each group were compared according to the extrusion process and parameters, extrudate morphology, and release.

[0066] The formulation and proportion of Test Example 1-5 are shown in Table 1-1.

[0067] The preparation method is as follows: accurately weigh melatonin, ethyl cellulose and hydrogenated vegetable oil according to the formula and place them in a container for manual premixing for 20 minutes. Add ethanol to the container 2 minutes before feeding and stir evenly. Set the extruder temperature to 50℃-65℃, more precisely, set the extruder temperature to 50-50-50-50-55-60-63-65℃. After the temperature rises to the set value and stabilizes for 20 minutes, put the mixed material into the feeder and extrude it through a die with an inner diameter of 2.3mm at a feed rate of 4rpm and a screw speed of 30rpm. The extrusion die area is equipped with a cutter, and the feed, screw and cutter speeds are coordinated to obtain a melatonin implant core. After the melatonin implant core is extruded and cooled, it is stored in a dry place away from light.

[0068] Table 1-1

[0069]

[0070] The evaluation results of Test Examples 1 to 5 are shown in Table 2.

[0071] Table 2

[0072]

[0073] The results of test examples 1-5 show that the implant cores prepared by these five groups of prescriptions have good formability and smooth surface, extrusion die pressure of 14-38 bar, and torque of 0.4-2.2 Nm, all within the normal range. The melatonin implant core of test example 3 is released the fastest, while the release of test example 1 is the slowest. However, since the prescriptions of test examples 1-5 contain ethanol, which is volatile, the extrusion pressure and torque will increase due to the volatilization of ethanol in the expanded test, causing the instrument to alarm and damage the instrument. It is not recommended to use this formula for hot melt extrusion preparation.

[0074] The formulation and proportion of Test Example 6-23 are shown in Tables 1-2 to 1-5.

[0075] Table 1-2

[0076]

[0077]

[0078] The preparation method of Test Example 6 is as follows: weigh the raw materials and auxiliary materials accurately according to the formula and place them in a container for manual premixing for 20 minutes. Set the extruder temperature to 136°C-145°C, more precisely, set the extruder temperature to 140-140-145-145-142-142-140-136°C. After the temperature rises to the set value and stabilizes for 20 minutes, put the mixed materials into the feeder and extrude them through a die with an inner diameter of 2.3mm under the condition of a feed-screw speed of 5-35rpm. The extrusion die area is equipped with a cutter, and the feed, screw and cutter speeds are coordinated to obtain a melatonin implant core. After the melatonin implant core is extruded and cooled, it is stored in a dark and dry place.

[0079] The preparation method of Experimental Example 7 is the same as that of Experimental Example 6, wherein the extruder temperature is 105°C-115°C, more precisely, the extruder temperature is set to 110-110-115-110-110-115-118-105°C.

[0080] The preparation method of Experimental Example 8 is the same as that of Experimental Example 6, wherein the extruder temperature is 90°C-108°C, more precisely, the extruder temperature is set to 90-90-100-100-100-105-105-108°C, and polyethylene glycol 6000 needs to be crushed and passed through a 50-mesh sieve before being weighed.

[0081] The preparation method of Experimental Example 9 is the same as that of Experimental Example 6, wherein the extruder temperature is 160°C-170°C, more precisely, the extruder temperature is set to 160-160-165-165-168-168-170-170°C.

[0082] Table 1-3

[0083]

[0084] The preparation method of Experimental Example 10 is the same as that of Experimental Example 6, wherein the extruder temperature is 129°C-145°C, more precisely, the extruder temperature is set to 140-142-145-145-145-138-132-129°C.

[0085] The preparation method of Experimental Example 11 is the same as that of Experimental Example 10, wherein the extruder temperature is 65°C-75°C, more precisely, the extruder temperature is set to 65-65-75-75-75-73-68-65°C.

[0086] The preparation method of Experimental Example 12 is the same as that of Experimental Example 10, wherein the extruder temperature is 129°C-145°C, more precisely, the extruder temperature is set to 140-142-145-145-145-138-132-129°C.

[0087] The preparation method of Experimental Example 13 is the same as that of Experimental Example 6, wherein the extruder temperature is 55°C, more precisely, the extruder temperature is set to 55-55-55-55-55-55-55-55°C, and polyethylene glycol 6000 needs to be crushed and passed through a 50-mesh sieve before being weighed.

[0088] Table 1-4

[0089]

[0090] The preparation method of Experimental Example 14 is the same as that of Experimental Example 13, wherein the extruder temperature is 132°C-140°C, more precisely, the extruder temperature is set to 140-140-140-140-135-135-132-135°C.

[0091] The preparation method of Experimental Example 15 is the same as that of Experimental Example 13, wherein the extruder temperature is 83°C-90°C, more precisely, the extruder temperature is set to 90-90-90-83-83-83-83-84°C.

[0092] The preparation method of Test Example 16 is the same as that of Test Example 6, wherein the extruder temperature is 136°C-145°C, more precisely, the extruder temperature is set to 140-140-145-145-142-142-140-136°C.

[0093] The preparation method of Experimental Example 17 is the same as that of Experimental Example 6, wherein the extruder temperature is 50°C-55°C, more precisely, the extruder temperature is set to 50-50-53-53-53-53-53-53-55°C.

[0094] The preparation method of Experimental Example 18 is the same as that of Experimental Example 13, wherein the extruder temperature is 84°C-88°C, more precisely, the extruder temperature is set to 88-88-88-84-84-86-86-88°C.

[0095] Table 1-5

[0096]

[0097] The preparation method of Experimental Example 19 is the same as that of Experimental Example 13, wherein the extruder temperature is 60°C-62°C, more precisely, the extruder temperature is set to 60-60-62-62-62-62-60-60°C.

[0098] The preparation method of Experimental Example 20 is the same as that of Experimental Example 13, wherein the extruder temperature is 150°C-160°C, more precisely, the extruder temperature is set to 150-150-155-155-157-157-160-160°C.

[0099] The preparation method of Experimental Example 21 is the same as that of Experimental Example 13, wherein the extruder temperature is 55°C-65°C, more precisely, the extruder temperature is set to 55-60-65-65-65-65-59-59°C.

[0100] The preparation method of Experimental Example 22 is the same as that of Experimental Example 13, wherein the extruder temperature is 50°C-55°C, more precisely, the extruder temperature is set to 50-50-53-53-53-53-53-55°C.

[0101] The preparation method of Experimental Example 23 is the same as that of Experimental Example 13, wherein the extruder temperature is 88°C-92°C, more precisely, the extruder temperature is set to 90-90-90-88-89-89-89-92°C.

[0102] The extrusion process and extrudate morphology of Test Example 6 23 were compared, and the evaluation criteria are shown in Table 3 and the results are shown in Table 4. The groups with higher scores were selected for release measurement.

[0103] Table 3

[0104] evaluate Extrusion process evaluation A Extrusion continuous, extrusion pressure 0-35bar, torque 0%-30% B Extrusion is continuous, extrusion pressure is 36-75bar, torque is 31%-65% C Extrusion is discontinuous, the extrusion pressure is too high, and the torque is too high. Section evaluation A smooth B Relatively flat C Uneven

[0105] Table 4

[0106]

[0107]

[0108] According to the extrusion process and extrudate state scoring results, test examples 15, 20, 21 and 23 were selected for release determination. The test results are shown in Figure 1 shown.

[0109] The release rate determination method is as follows: According to the dissolution and release rate determination method (General Rule 0931 Method 2), an in vitro release rate research test is conducted using an RC8MD dissolution tester, and the test is conducted under water tank conditions. Take 6 pellets of the sample to be tested, use 900 mL of 0.2% hexadecyltrimethylammonium bromide solution (dissolved in pH=6.8 phosphate buffer) as the release medium, the test temperature is 37.0±0.5°C, the speed is 100 revolutions per minute, operate according to the law, take an appropriate amount of dissolution solution at intervals (add an equal amount of release medium at the same time), filter through a 0.45μm water system membrane, and take the subsequent filtrate as the test solution. According to the chromatographic conditions under the content determination item, accurately measure 10μL of the reference solution and the test solution, respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the release rate of each pellet by the peak area according to the external standard method.

[0110] pass Figure 1 It can be seen from Tables 3 and 4 that Test Example 21 is the best test example.

[0111] The content and content uniformity of the melatonin implant core and Regulin prepared in Experimental Example 21 were measured, and the measurement results are shown in Table 5. The measurement method is as follows:

[0112] 1) Determination of melatonin content

[0113] Take 5 capsules of this product, grind and weigh about 30 mg accurately, place in a 200 mL volumetric flask, add 20 mL of methanol for ultrasonic treatment, wait for it to dissolve and cool, add diluent (0.5 g / L potassium dihydrogen phosphate solution (adjust pH to 3.5 with phosphoric acid)-acetonitrile (75:25)) to dilute to scale, shake well, filter, and take the filtrate as the test solution. Take another reference substance and weigh it accurately, add diluent to dissolve and dilute to a solution containing about 0.1 mg per 1 mL, as the reference solution. Accurately measure 10 μL, inject into the liquid chromatograph, determine according to the high performance liquid chromatography method (Appendix 0512), record the chromatogram, and calculate the content of melatonin in this product by peak area according to the external standard method. The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler; 0.5 g / L potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid)-acetonitrile (75:25) as mobile phase (diluent); detection wavelength 222 nm, column temperature 35°C; flow rate 1.0 mL per minute. The theoretical plate number calculated based on the melatonin peak is not less than 3000.

[0114] 2) Melatonin content uniformity determination method

[0115] Randomly select 10 samples from the same batch to be tested, and measure them according to the method under the content determination item. Measure the peak area of ​​each sample and calculate the relative content of the sample with the labeled amount as 100. i , find the average value X 10 and standard deviation S 10 and A 10 If A 10 +2.2S 10 ≤15, it is judged to be in compliance with the regulations. Otherwise, calculate the discriminant A 10 +S 10 , if A 10 +S 10 If the value is greater than 15, it is considered as not meeting the requirements.

[0116] Table 5

[0117]

[0118]

[0119] The content uniformity of Test Example 21 of the present invention is better than that of Regulin, a product sold overseas, and there is no significant difference in the content between the two.

[0120] 2. Coating liquid

[0121] The formulations and proportions of Test Examples 24-47 are shown in Tables 1-7 to 1-9.

[0122] Test Example 24 Preparation method is as follows: Accurately weigh ethyl cellulose according to the formula, and measure ethanol for use. Add ethanol to the liquid preparation container, and the liquid level of ethanol is preferably roughly the same as the inner diameter of the container. Start the agitator, and the stirring speed should be such that the liquid in the container is completely stirred, and the liquid surface just forms a vortex. Sprinkle the weighed ethyl cellulose on the vortex liquid surface at a steady speed, and the addition speed should be such that the powder is quickly stirred into the vortex. The feeding process should be completed within a few minutes. After the addition is completed, continue stirring until it is completely dissolved.

[0123] Test Example 25 Preparation method is as follows: Accurately weigh ethyl cellulose N-50 according to the formula, and measure ethanol for use. Add ethanol to the liquid preparation container, and the liquid level of ethanol is preferably roughly the same as the inner diameter of the container. Start the agitator, and the stirring speed should be such that the liquid in the container is completely stirred, and the liquid surface just forms a vortex. Sprinkle the weighed ethyl cellulose on the vortex liquid surface at a steady speed, and the addition speed should be such that the powder is quickly stirred into the vortex. The feeding process should be completed within a few minutes. After the feeding is completed, continue stirring until it is completely dissolved. Take the formula amount of diethyl phthalate and slowly add it dropwise into the vortex, and continue stirring until the coating liquid is completely mixed.

[0124] Among the remaining test examples, except for Test Examples 28, 32, 36, 40, and 44, the preparation methods are the same as Test Example 24, and the preparation methods of the remaining test examples are the same as Test Example 25.

[0125] Table 1-7

[0126]

[0127] In Test Examples 25 and 29, the mass ratio of ethyl cellulose to diethyl phthalate was 1:1.

[0128] In Test Examples 26 and 30, the mass ratio of ethyl cellulose to diethyl phthalate was 2:1.

[0129] In Test Examples 27 and 31, the mass ratio of ethyl cellulose to diethyl phthalate was 4:1.

[0130] Table 1-8

[0131]

[0132]

[0133] In Test Examples 33 and 37, the mass ratio of ethyl cellulose to diethyl phthalate was 1:1.

[0134] In Test Examples 34 and 38, the mass ratio of ethyl cellulose to diethyl phthalate was 2:1.

[0135] In Test Examples 35 and 39, the mass ratio of ethyl cellulose to diethyl phthalate was 4:1.

[0136] Table 1-9

[0137]

[0138] In Test Examples 41 and 45, the mass ratio of ethyl cellulose to diethyl phthalate was 1:1.

[0139] In Test Examples 42 and 46, the mass ratio of ethyl cellulose to diethyl phthalate was 2:1.

[0140] In Test Examples 43 and 47, the mass ratio of ethyl cellulose to diethyl phthalate was 4:1.

[0141] The coating solutions in the above test examples 24-47 were taken for viscosity measurement. The prepared coating solutions were quantitatively spread in a mold, placed in a 60°C oven to dry and form a film, and then placed under a relative humidity of about 75% for 2 hours before demoulding. After demoulding, the coating materials were cut into 800 mm long and 10 mm wide film materials, and the mechanical properties were measured. The film-forming effect evaluation criteria are shown in Table 6, and the measurement results are shown in Table 7.

[0142] Table 6

[0143] evaluate Film formation status A Easy to mold, easy to demould on all sides, the film is very soft B Easy to mold, easy to demould around, with pulling in the middle, the film is softer C Easy to mold, very hard film

[0144] Table 7

[0145]

[0146]

[0147] The viscosity of Test Examples 40-47 is too high, and the nozzle of the high-efficiency coating machine in the present invention is not suitable. Therefore, only the mechanical properties of the coating films prepared by the coating solutions in Test Examples 24-39 were tested subsequently. As a result, Test Example 34 had the best film-forming effect.

[0148] 3. Melatonin Implants

[0149] Test Example 48

[0150] A melatonin implant, the total weight of the melatonin implant core for coating is 300g, each pellet is (30±0.5)mg, the formula ratio of the melatonin implant core is the same as that of Test Example 21, the formula ratio of the coating liquid is the same as that of Test Example 34, and the specific formula is shown in Table 8.

[0151] Table 8

[0152]

[0153] The preparation method of the coating solution is as follows:

[0154] (1) Accurately weigh 30 g of ethyl cellulose N-50 and 3 g of sunset yellow, measure 1000 mL of ethanol for later use, and add ethanol to a liquid preparation container. The liquid level of ethanol should preferably be roughly the same as the inner diameter of the container;

[0155] (2) placing the container containing ethanol on a magnetic stirrer, starting the stirrer, and stirring at a stirring speed of 700 rpm / min;

[0156] (3) Sprinkle the weighed ethyl cellulose onto the vortex liquid surface at a steady speed. The speed of addition should be such that the powder is quickly stirred into the vortex. The addition process should be completed within a few minutes. After the addition is completed, continue stirring until it is completely dissolved;

[0157] (4) Dissolve the weighed Sunset Yellow in an appropriate amount of purified water, then slowly pour it into the vortex and continue stirring until it is completely blended.

[0158] (6) Pipette 13.4 mL of diethyl phthalate and slowly add it dropwise into the vortex, and continue stirring until the coating solution is completely mixed;

[0159] The core tablet coating method is as follows:

[0160] (1) Preheating the empty coating machine: set the coating machine parameters and keep preheating for 10 minutes;

[0161] (2) Material preheating: Place 300 g of melatonin implant core into the coating pan and preheat for 5 minutes;

[0162] (3) Sampling before spraying: Before starting spraying, randomly select samples from the coating pan and determine the average tablet weight by weighing three groups of 30 tablets each;

[0163] (4) Coating: spraying coating liquid for coating;

[0164] (5) In-process sampling: After about 80% of the coating solution has been sprayed, start taking coating samples from the coating pan every 15 minutes to check the weight gain of the coated tablets until the predetermined weight gain of 2.3%-2.7% is reached. Three groups of 30 coated tablets in each group are randomly selected and weighed, and the weight gain is recorded;

[0165] (6) Drying: After coating, dry for 30 minutes, remove from the container, cool to room temperature, and store in a sealed container away from light.

[0166] Test Example 49

[0167] Same as Test Example 48, the coating weight gain was 4.3%-4.7%.

[0168] Test Example 50

[0169] Same as Test Example 48, the coating weight gain was 7.3%-7.7%.

[0170] The melatonin implants prepared in Test Examples 48-50 and the foreign commercial product Regulin were tested according to the release rate determination method. The results are as follows: Figure 2A , 2B shown.

[0171] Results: The release curves of melatonin implants prepared in Test Examples 48-50 are similar to those of Regulin, and the release rate of Regulin within 120 hours is about 4%. In the present invention, the release rate of Test Example 48 within 120 hours is about 10%, that of Test Example 49 is about 7%, and that of Test Example 50 is about 4%. The release rate and release rate of the melatonin implant of Test Example 50 are closest to those of Regulin, and the melatonin implant prepared in Test Example 50 has the best in vitro release effect.

[0172] The melatonin implant prepared in Test Example 50 and the overseas commercial product Regulin were subjected to a long-term in vitro release test. The test results are as follows: Figure 3A-C, the test method is as follows: before the test, the vial is cleaned with ethanol to reduce microbial contamination in the bottle, and then dried in a 60°C blast drying oven. The long-term release performance of the melatonin implant is determined by the shaking table method. Take 12 100mL brown vials, add 50mL of 0.2% hexadecyltrimethylammonium bromide phosphate buffer (pH=6.8, hexadecyltrimethylammonium bromide has a preservative function) to each bottle, and cover with a rubber stopper. Place it in a constant temperature oscillator with a temperature of (37.0±0.5)℃ and a speed of 100r / min. After the temperature stabilizes, take 6 tablets of Regulin and the melatonin implant prepared in Example 7, respectively, and put them into the vial for constant temperature oscillation. When sampling, gently rotate the bottle to mix the solution evenly, sample 5mL at a fixed time, and supplement with an equal amount of 0.2wt.% hexadecyltrimethylammonium bromide-phosphate buffer solution (pH=6.8) at the same temperature. The peak area of ​​the sampled solution was measured by high performance liquid chromatography to determine the melatonin content for calculating the cumulative release percentage.

[0173] Results: The melatonin implant prepared in Test Example 50 of the present invention was compared with the foreign commercial product Regulin, and the two had similar release rates, 15%-20% in vitro release in 28 days; the release rate was similar at 0.5%-1.0%; and the daily release amount was similar at 0.1mg-0.2mg.

[0174] Therefore, the best formula in the above test is Test Example 50, which can achieve a release similar to that of Regulin, a product commercially available abroad, and can be prepared using the preparation method of the present invention.

[0175] Example 1

[0176] A melatonin implant, the formula and preparation method of which are the same as those of Test Example 50.

[0177] The melatonin implant obtained in Example 1 was subjected to clinical trials.

[0178] 1) Experimental animals

[0179] In the summer (June to July), 136 Tan sheep ewes, which were 0 to 3 days post-weaning and had similar body conditions and were between 2 and 5 years old, were selected. The test sheep were in good body conditions.

[0180] 2) Experimental flock management

[0181] The sheep used in the experiment were purebred Tan sheep, healthy, empty-breasted, similar in body condition, and aged between 2 and 5 years old. All experimental sheep were grouped according to the principle of age (parity) and balanced weight. During the experiment, breeding ewes were fed a complete mixed diet. Rams began to strengthen exercise, nutrition and provide sufficient protein feed 60 days before mating; semen collection training began at the end of March, and semen was collected and trained every 3 days for 20 days before the experiment to improve the semen quality of rams. The semen quality of each ram must be recorded every time semen is collected.

[0182] 3) Test methods

[0183] In June and July, 136 empty-pregnant ewes 0 to 3 days after weaning were selected and grouped according to the principle of balanced age (parity) and weight. The experimental group ewes (n = 77) were implanted with melatonin subcutaneously at the base of the ear, combined with the treatment of estrus induction and ovulation synchronization based on buserelin and the treatment of progesterone sustained-release agent assisted in estrus induction and ovulation synchronization. The control group ewes (n = 59) were not implanted with melatonin, and only the treatment of estrus induction and ovulation synchronization based on buserelin and the treatment of progesterone sustained-release agent assisted in estrus induction and ovulation synchronization were used to compare whether the use of melatonin can improve the reproductive performance of summer Tan sheep ewes.

[0184] 4) Observation of natural estrus of experimental ewes

[0185] Starting from the day when the melatonin implant was implanted subcutaneously at the ear base of the ewes in the experimental group, the ewes in the experimental group and the control group were tested for estrus by estrus rams every 12 hours. The estrus induction procedure was started when the test lasted for 28 days, at which time the test was stopped.

[0186] 5) Experimental ewes estrus induction treatment procedures

[0187] In this experiment, melatonin implants were implanted subcutaneously at the base of the ears of the experimental group of ewes. The estrus induction treatment procedure was started 28 days after the implantation of melatonin implants. The day of the estrus induction treatment procedure was 0 days. CIDR was placed in the vagina on the morning of 0 days, and buserelin was injected intramuscularly at the same time. CIDR was removed on the morning of 7 days, and D-PG-Cl and PMSG were injected intramuscularly at the same time. Starting 12 hours after the removal of CIDR, estrus rams were used to test estrus once in the morning and afternoon every day, and the ewes were injected intramuscularly for the second time within 12 hours after estrus. Starting 12 hours after the removal of CIDR, estrus rams were used to test estrus once in the morning and afternoon every day. The estrus was determined by the receiving ram mounting. Each estrus was observed to last for 12 hours. The estrus start time and the number of estrus observed for each sheep were recorded, and the time distribution from estrus to the removal of CIDR and the estrus duration were counted. The first artificial insemination and mating of estrus sheep was carried out 12 to 18 hours after estrus. In order to improve the pregnancy rate of estrus sheep, buserelin was injected at the same time as the first artificial insemination, and multiple mating methods were adopted with mating once every 12 hours, for a total of 2 to 3 times. 48 hours after the last artificial insemination and mating, a ram was put into the mixed group to supplement the estrus sheep. B-ultrasound pregnancy examination was carried out 45 days after artificial insemination. The artificial insemination estrus conception rate of estrus sheep was calculated according to the results of B-ultrasound examination; the total conception rate and lambing rate of ewes were calculated after lambing, and the data of abortion or premature birth during pregnancy were recorded and counted; lamb cultivation measures were taken for lambs, and the survival rate of lambs was counted after weaning.

[0188] 6) Results

[0189] The natural estrus rate and estrus duration of the ewes in the control group and the experimental group within 28 days of melatonin implantation are shown in Table 8. The natural estrus rate of the ewes in the control group and the experimental group in summer was 44.07% and 32.47%, respectively, with no significant difference (P>0.05). The estrus interval of the ewes in the control group and the experimental group was 35.08±14.27h and 32.64±11.24h, respectively, with no significant difference (P>0.05).

[0190] Table 8

[0191]

[0192] The estrus rate and estrus interval of the ewes in the control group and the experimental group were shown in Table 9. The estrus rates of the ewes in the control group and the experimental group were 79.66% and 93.51%, respectively, with significant differences (P < 0.05). The estrus intervals of the control group and the experimental group were 48.26 ± 7.29h and 46.17 ± 7.17h, and the estrus time was concentrated between 36 and 60h.

[0193] Table 9

[0194]

[0195]

[0196] The estrus duration of the ewes in the control group and the experimental group was shown in Table 10. The estrus duration of the ewes in the control group and the experimental group was 37.79±10.90h and 39.33±11.25h, respectively, with no significant difference (P>0.05).

[0197] Table 10

[0198]

[0199] It can be seen from this that melatonin implantation in ewes in summer (June to July) can significantly increase the induced estrus rate, but does not affect the estrus interval and estrus duration, nor does it have a significant effect on the natural estrus rate.

[0200] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A melatonin implant, characterized in that: The melatonin implant comprises an implant core and a coating, wherein the coating is sprayed on the surface of the implant core, and the components of the implant core include the following components by mass percentage: Melatonin 65-75%, Carrier 8-12%, Lubricant 10-15%, First plasticizer 7-9%; The coating comprises a film-forming agent, a second plasticizer and a solvent, wherein the mass ratio of the film-forming agent to the second plasticizer is (1.5-2.5):1, and the mass ratio of the film-forming agent to the solvent is (2.5-3.5):100 (g / mL).

2. The melatonin implant according to claim 1, characterized in that The carrier is one or more of ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose, and cellulose acetate, preferably ethyl cellulose, and more preferably ethyl cellulose N-50.

3. The melatonin implant according to claim 1, characterized in that The lubricant is one or more of hydrogenated vegetable oil, animal fat, beeswax, carnauba wax, stearyl alcohol, and monoglyceride, preferably hydrogenated vegetable oil.

4. The melatonin implant according to claim 1, characterized in that The first plasticizer is one or more of polyethylene glycol, poloxamer, and povidone. The molecular weight of polyethylene glycol is 1000-20000, preferably polyethylene glycol, and more preferably polyethylene glycol 6000.

5. The melatonin implant according to claim 1, characterized in that The film-forming agent is one or more of ethyl cellulose, cellulose acetate, acrylic resin, cellulose acetate titanate, hydroxypropyl methylcellulose titanate, and hydroxypropyl methylcellulose acetate succinate, preferably ethyl cellulose, and more preferably ethyl cellulose N-50.

6. The melatonin implant according to claim 1, characterized in that The second plasticizer is one or more of phthalates, glycerol, propylene glycol, polyethylene glycol, triacetin, castor oil, and acetylated glycerides, preferably phthalates, and more preferably diethyl phthalate.

7. The melatonin implant according to claim 1, characterized in that The solvent is one or more of water, ethanol, acetone, and isopropanol, preferably ethanol.

8. The melatonin implant according to claim 1, characterized in that The coating also includes a colorant, and the mass ratio of the colorant to the volume ratio of the solvent is 2.5-3.5:1000 (g / mL).

9. The melatonin implant according to claim 1, characterized in that The coating weight gain is 7.3%-7.7% of the embedded core.

10. The method for preparing the melatonin implant according to any one of claims 1 to 9, characterized in that: The following steps are involved: 1) Mixing: Crush large particles and pass through a 50-100 mesh sieve; weigh the raw and auxiliary materials for embedding the core and mix them in a container; 2) Preparation of implant core by hot melt extrusion: the mixed materials are put into the feeder of the preheated hot melt extruder, and the preheating temperature is 50-160°C; after the materials are fed, they are mixed, extruded, pushed and transported by the rotating screw to the die with an inner diameter of 2.3mm for extrusion, and the screw speed is 5-35rpm; the die area is equipped with a cutter to cut the implant core, and after cooling to room temperature, it is kept dry and away from light for future use; 3) Prepare coating solution: weigh the raw materials of each coating component, place the solvent in a suitable container and stir it with a magnetic stirrer at a stirring speed of 600-800 rpm; slowly pour the film-forming agent into the solvent and stir until it is completely dissolved, add the plasticizer and continue stirring to obtain the coating solution; 4) Preparation of implants: Place the implant core into the preheated coating pan and coat it with coating liquid spray. During the coating process, take samples and weigh them to calculate the coating weight gain. Stop spraying and dry the implant. After drying, take it out, cool it to room temperature, and store it in a sealed container away from light.

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