Testosterone microcrystals, lyophilized powder for injection, suspensions and methods for their preparation

Abstract

The present application relates to a kind of testosterone microcrystal, freeze-dried powder injection, suspension and its preparation method.The particle size range of testosterone microcrystal meets: D90≤20 μm.Testosterone microcrystal freeze-dried powder injection includes testosterone microcrystal, suspending agent and freeze-drying protective agent;The mass ratio of the suspending agent and the mass of the testosterone microcrystal meets (0.3-2):1.The particle size range of the testosterone microcrystal meets: D90≤20 μm.Testosterone microcrystal suspension includes water, testosterone microcrystal, suspending agent and freeze-drying protective agent;The concentration of the suspending agent is 0.3%-2%.The preparation of testosterone microcrystal prepared in the present application is short-acting preparation, has the advantages of small liver toxicity, dose accuracy and easy to use.

Description

Technical Field

[0001] This invention relates to a testosterone microcrystal, a lyophilized powder injection, a suspension, and a method for preparing the same. Background Technology

[0002] According to Merck's manual, adolescent boys with androgen deficiency should receive long-acting injections of 50 mg of testosterone enanthate or propionate every 2 to 4 weeks; the dose should be increased to 200 mg over 18 to 24 months. Secondary hypogonadism and any underlying pituitary or hypothalamic disorders should be treated. Hormone replacement therapy for androgen deficiency should be administered, with gradual increases in the small dose over 18-24 months. Treatment should begin at the appropriate age for the normal onset of puberty, usually around 12 years of age.

[0003] Constitutional delay of puberty can be treated with a 4-6 month course of testosterone therapy. After the course of treatment, treatment is stopped, and testosterone levels are measured several weeks or months later to distinguish between temporary and permanent deficiencies. If testosterone levels do not rise above initial values ​​and / or puberty does not occur after the completion of this treatment, a second course of low-dose therapy may be given. If endogenous puberty does not begin after two courses of treatment, the likelihood of permanent deficiency increases, and the patient needs to be re-evaluated for other causes of hypogonadism.

[0004] Currently, existing dosage forms of testosterone mainly include testosterone patches, methyltestosterone tablets, testosterone gel, testosterone undecanoate propionate injection, and testosterone undecanoate propionate soft capsules, etc. However, these dosage forms have problems such as long onset of action, high liver toxicity, inaccurate dosage, or inconvenience of use. Summary of the Invention

[0005] To overcome the technical defects of existing testosterone dosage forms, such as long onset of action, high hepatotoxicity, inaccurate dosage, or inconvenient use, this invention provides testosterone microcrystals, lyophilized powder for injection, suspensions, and their preparation methods. The formulation prepared from the testosterone microcrystals of this invention is a short-acting formulation with advantages such as low hepatotoxicity, accurate dosage, and convenient use.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides testosterone microcrystals with a particle size range satisfying: D90≤20 μm; and X-ray powder diffraction patterns expressed in 2θ angles showing diffraction peaks at 13.1º±0.2º, 15.4º±0.2º, 16.0º±0.2º and 18.3º±0.2º.

[0008] In this invention, the testosterone refers to 17β-hydroxyandrost-4-en-3-one propionate, CAS Registry No. 58-22-0.

[0009] In this invention, the D90 of the testosterone microcrystals can satisfy: 10.0 μm ≤ D90 ≤ 18.0 μm, preferably satisfy: 14.0 μm ≤ D90 ≤ 18.0 μm, for example, D90 = 14.3 μm, 14.8 μm, 15.1 μm, 15.3 μm, 15.4 μm, 15.7 μm, 15.8 μm, 15.9 μm or 17.4 μm.

[0010] In this invention, the particle size range of the testosterone microcrystals can satisfy: 5 μm ≤ D50 ≤ 20 μm; preferably, 5 μm ≤ D50 ≤ 10 μm, for example, D50 = 5.96 μm, 5.98 μm, 6.04 μm, 6.54 μm, 6.56 μm, 6.59 μm, 6.63 μm, 6.67 μm, 6.76 μm, 6.84 μm, 6.93 μm, 6.94 μm, 7.01 μm, 7.08 μm, 7.12 μm or 7.27 μm.

[0011] In this invention, the particle size range of the testosterone microcrystals can satisfy: 2 μm ≤ D10 ≤ 10 μm; preferably, 2 μm ≤ D10 ≤ 3 μm, for example, D10 = 2.51 μm, 2.55 μm, 2.58 μm, 2.60 μm, 2.77 μm, 2.80 μm, 2.81 μm, 2.82 μm, 2.83 μm, 2.84 μm, 2.86 μm, 2.87 μm or 2.90 μm.

[0012] In certain specific embodiments of the present invention, the testosterone microcrystals have diffraction peaks at 13.230º, 15.488º, 16.138º and 18.422º in the X-ray powder diffraction pattern expressed in 2θ angle.

[0013] In certain specific embodiments of the present invention, the testosterone microcrystals have diffraction peaks at 13.093º, 15.357º, 16.009º and 18.329º in the X-ray powder diffraction pattern expressed in 2θ angle.

[0014] In certain specific embodiments of the present invention, the testosterone microcrystals have diffraction peaks at 13.141º, 15.432º, 16.060º and 18.304º in the X-ray powder diffraction pattern expressed in 2θ angle.

[0015] In a specific embodiment of the present invention, the X-ray powder diffraction pattern of the testosterone microcrystals is as follows: Figure 1 As shown, as Figure 2 As shown or as Figure 3 As shown.

[0016] In a specific embodiment of the present invention, the testosterone microcrystals satisfy the following conditions: D10 = 2.82 μm, D50 = 6.63 μm, D90 = 14.8 μm, and the X-ray powder diffraction pattern expressed in 2θ angle is as follows. Figure 1 As shown.

[0017] In a specific embodiment of the present invention, the testosterone microcrystals satisfy the following conditions: D10 = 2.80 μm, D50 = 6.67 μm, D90 = 15.1 μm, and the X-ray powder diffraction pattern expressed in 2θ angle is as follows. Figure 2 As shown.

[0018] In a specific embodiment of the present invention, the testosterone microcrystals satisfy the following conditions: D10 = 2.86 μm, D50 = 6.84 μm, D90 = 15.4 μm, and the X-ray powder diffraction pattern expressed in 2θ angle is as follows. Figure 3 As shown.

[0019] Secondly, the present invention provides a method for preparing testosterone microcrystals, which includes the following steps: mixing and shearing an oil phase and an aqueous phase at a rotation speed of 6000-70000 rpm at 20-30°C;

[0020] The oil phase includes testosterone and an organic dispersant, wherein the concentration of testosterone is 50-90 mg / ml;

[0021] The aqueous phase includes water; the volume ratio of the oil phase to the aqueous phase is 1:(130-150).

[0022] In this invention, the preparation temperature can be 25±2℃.

[0023] In this invention, the rotational speed of the mixed shearing can be 10,000-12,000 rpm, for example, 10,300 rpm.

[0024] In this invention, the organic dispersant is a conventional choice in the art, such as organic solvents like ethanol and diethyl ether.

[0025] In this invention, the volume ratio of the oil phase to the water phase can be 1:150.

[0026] In this invention, the aqueous phase may also include conventional surfactants.

[0027] In this invention, after the mixed shearing, filtration can be performed conventionally.

[0028] Thirdly, the present invention provides a method for preparing testosterone microcrystals as described above, which yields testosterone microcrystals.

[0029] Fourthly, the present invention provides a testosterone microcrystalline lyophilized powder injection, which includes a suspending agent, a lyophilization protectant and the testosterone microcrystals as described above.

[0030] In this invention, the suspending agent is a conventional choice in the art, such as sodium hyaluronate. The molecular weight of the sodium hyaluronate can be 800,000 to 1,200,000.

[0031] In this invention, the mass ratio of the suspending agent to the mass of the testosterone microcrystals can be (0.3-2):1, preferably (0.3-0.6):1, for example 0.5:1.

[0032] In this invention, the freeze-drying protectant is a conventional choice in the art, such as sucrose.

[0033] In this invention, the mass ratio of the freeze-drying protectant to the mass of the testosterone microcrystals can be (5-15):1, preferably (7-9):1.

[0034] In this invention, after the testosterone microcrystalline lyophilized powder injection is reconstituted with water at a mass ratio of 100:1, the pH of the resulting reconstituted suspension is 5-7, preferably 5.8-6.2, for example, pH=6.

[0035] In this invention, the testosterone microcrystalline lyophilized powder injection may further include a pH adjuster.

[0036] The pH adjuster is a conventional choice in the art, such as a mixture of citric acid and sodium citrate, a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, a mixture of succinic acid and sodium succinate, or a mixture of histidine and histidine hydrochloride.

[0037] In a specific embodiment of the present invention, the pH adjuster is a mixture of citric acid and sodium citrate, wherein the mass of citric acid is 2.97% of the mass of the testosterone microcrystals, and the mass of sodium citrate is 23.51% of the mass of the testosterone microcrystals.

[0038] In a specific embodiment of the present invention, the testosterone microcrystalline lyophilized powder injection comprises testosterone microcrystals, sucrose, sodium hyaluronate, citric acid, and sodium citrate in a mass ratio of 10:90:5:0.297:2.351.

[0039] In a specific embodiment of the present invention, the testosterone microcrystalline lyophilized powder injection comprises testosterone microcrystals, sucrose, and sodium hyaluronate in a mass ratio of 10:90:5, and also includes succinic acid and sodium succinate. After the testosterone microcrystalline lyophilized powder injection is reconstituted with water in a mass ratio of 100:1, the pH of the resulting reconstituted suspension is 5.8-6.2.

[0040] In a specific embodiment of the present invention, the testosterone microcrystalline lyophilized powder injection comprises testosterone microcrystals, sucrose, and sodium hyaluronate in a mass ratio of 10:90:5, and also includes histidine and histidine hydrochloride. After the testosterone microcrystalline lyophilized powder injection is reconstituted in water at a mass ratio of 100:1, the pH of the resulting reconstituted suspension is 5.8-6.2.

[0041] Fifthly, the present invention provides a testosterone microcrystal suspension comprising water, a suspending agent, a lyophilization protectant, and testosterone microcrystals as described above.

[0042] In this invention, the concentration of the testosterone microcrystals can be 1%, and the percentage is the ratio of the mass of the testosterone microcrystals to the mass of the testosterone microcrystal suspension.

[0043] In this invention, the suspending agent is a conventional choice in the art, such as sodium hyaluronate. The molecular weight of the sodium hyaluronate can be 800,000 to 1,200,000.

[0044] In this invention, the concentration of the suspending agent can be 0.3%-2%, preferably 0.3%-0.6%, for example 0.5%, and the percentage is the ratio of the mass of the suspending agent to the mass of the testosterone microcrystalline suspension.

[0045] In this invention, the freeze-drying protectant is a conventional choice in the art, such as sucrose.

[0046] In this invention, the concentration of the freeze-drying protectant can be 5%-15%, for example 7%-9%, and the percentage is the ratio of the mass of the freeze-drying protectant to the mass of the testosterone microcrystalline suspension.

[0047] In this invention, the pH of the testosterone microcrystal suspension can be 5-7, preferably 5.7-6.2, for example 5.8-6.2.

[0048] In this invention, the testosterone microcrystal suspension may further include a pH adjuster.

[0049] The pH adjuster is a conventional choice in the art, such as a mixture of citric acid and sodium citrate, a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, a mixture of succinic acid and sodium succinate, or a mixture of histidine and histidine hydrochloride.

[0050] In a specific embodiment of the present invention, the pH adjuster is a mixture of citric acid and sodium citrate, wherein the mass of citric acid is 2.97% of the mass of the testosterone microcrystals, and the mass of sodium citrate is 23.51% of the mass of the testosterone microcrystals.

[0051] In a specific embodiment of the present invention, the testosterone microcrystal suspension comprises testosterone microcrystals, sucrose, sodium hyaluronate, citric acid, sodium citrate, and water in a mass ratio of 10:90:5:0.297:2.351:892.352.

[0052] In a specific embodiment of the present invention, the testosterone microcrystalline suspension comprises 1% testosterone microcrystals, 0.5% sodium hyaluronate, and 9% sucrose, the percentages being the ratio of the mass of each substance to the mass of the testosterone microcrystalline suspension, with the remainder being water, succinic acid, and sodium succinate; the pH of the testosterone microcrystalline suspension is 5.8-6.2.

[0053] In a specific embodiment of the present invention, the testosterone microcrystalline suspension comprises 1% testosterone microcrystals, 0.5% sodium hyaluronate, and 9% sucrose, the percentages being the ratio of the mass of each substance to the mass of the testosterone microcrystalline suspension, with the remainder being water, histidine, and histidine hydrochloride; the pH of the testosterone microcrystalline suspension is 5.8-6.2.

[0054] In a sixth aspect, the present invention provides a method for preparing the testosterone microcrystalline suspension as described above, comprising the following steps: mixing a suspending agent with the testosterone microcrystals as described above; wherein the suspending agent comprises water, a suspending agent and a lyophilization protectant.

[0055] In this invention, the suspending agent is a conventional choice in the art, such as sodium hyaluronate. The molecular weight of the sodium hyaluronate can be 800,000 to 1,200,000.

[0056] In this invention, the mass ratio of the suspending agent to the mass of the testosterone microcrystals can be (0.3-2):1, preferably (0.3-0.6):1, for example 0.5:1.

[0057] In this invention, the freeze-drying protectant is a conventional choice in the art, such as sucrose.

[0058] In this invention, the mass ratio of the freeze-drying protectant to the mass of the testosterone microcrystals can be (5-15):1, preferably (7-9):1.

[0059] In this invention, the pH of the testosterone microcrystal suspension can be 5-7, preferably pH=6.0±0.2.

[0060] In this invention, the testosterone microcrystal suspension may further include a pH adjuster.

[0061] The pH adjuster is a conventional choice in the art, such as a mixture of citric acid and sodium citrate, a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, a mixture of succinic acid and sodium succinate, or a mixture of histidine and histidine hydrochloride.

[0062] In a specific embodiment of the present invention, the pH adjuster is a mixture of citric acid and sodium citrate, wherein the mass of citric acid is 2.97% of the mass of the testosterone microcrystals, and the mass of sodium citrate is 23.51% of the mass of the testosterone microcrystals.

[0063] In this invention, the mass ratio of the water to the testosterone microcrystals can be 100:1.

[0064] In a sixth aspect, the present invention provides a method for preparing the testosterone microcrystalline suspension as described above, comprising the following steps: mixing water and the testosterone microcrystalline lyophilized powder for injection as described above.

[0065] In a seventh aspect, the present invention provides a method for preparing a testosterone microcrystalline lyophilized powder injection, which includes the following steps: freeze-drying the testosterone microcrystalline suspension as described above.

[0066] In this invention, the freeze-drying temperature can be -45~5℃.

[0067] In this invention, the heating or cooling rate of the freeze-drying process can be 1°C / min.

[0068] In a specific embodiment of the present invention, the freeze-drying process is as shown in the table below.

[0069]

[0070] In this invention, the pressure for freeze drying can be 0.1 mbar.

[0071] Eighthly, the present invention provides a testosterone microcrystalline lyophilized powder injection prepared by the method described above.

[0072] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0073] The reagents and raw materials used in this invention are all commercially available.

[0074] The positive and progressive effects of this invention are as follows:

[0075] (1) The testosterone microcrystalline formulation of the present invention does not require the removal of the ester chain by liver metabolism, does not produce 17-α-alkylated derivatives, and has less liver toxicity;

[0076] (2) The testosterone microcrystalline formulation of the present invention is a short-acting formulation, allowing for timely discontinuation of the drug when adverse reactions occur;

[0077] (3) The testosterone microcrystal formulation of the present invention is more accurate in dosage and more convenient to use than the topical gel formulation. Attached Figure Description

[0078] Figure 1 The image shows the XRD pattern of the lyophilized powder injection prepared in batch 1 of Example 1.

[0079] Figure 2 The image shows the XRD pattern of the lyophilized powder injection prepared in batch 2 of Example 1.

[0080] Figure 3 The image shows the XRD pattern of the lyophilized powder injection prepared in batch 3 of Example 1.

[0081] Figure 4 The image shows the appearance of the lyophilized powder injection prepared in Example 2;

[0082] Figure 5 This is a photograph of the appearance of the lyophilized powder injection prepared in Example 2 after one week of storage;

[0083] Figure 6 This is a photo of the lyophilized powder injection prepared in Example 3 after being reconstituted with water and left to stand for 5 minutes.

[0084] Figure 7 This is a photograph of the lyophilized powder injection prepared in Example 3 after being reconstituted with water and left to stand for 10 minutes.

[0085] Figure 8 This is a photo of the lyophilized powder injection prepared in Example 3 after being reconstituted with water and left to stand for 60 minutes. Detailed Implementation

[0086] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0087] The following preparation examples and implementation examples:

[0088] Testosterone was purchased from Hubei Gedian Renfu Pharmaceutical Co., Ltd.

[0089] Sodium hyaluronate, purchased from Bloomage Biotechnology Co., Ltd., model number HA-E2, with a molecular weight of 800,000-1,200,000.

[0090] Citric acid and sodium citrate: both purchased from Hunan Jiudian Hongyang Pharmaceutical Co., Ltd., model: pharmaceutical excipients.

[0091] Sucrose: Purchased from Jiangsu Hanstone Pharmaceutical Co., Ltd., type: for injection.

[0092] Anhydrous ethanol: purchased from Nanjing Chemical Reagent Co., Ltd., model: pharmaceutical excipient.

[0093] Example of preparation: Determining the preparation method of microcrystals

[0094] The four processes involved in experiments P1-P4 (all conducted at 20-30℃) in Table 1 below are compared.

[0095] Table 1. List of main experimental conditions for microcrystal preparation

[0096]

[0097] Based on the D50 and D90 values ​​of the product, the preparation method for the microcrystals is determined as follows:

[0098] Experiment P1

[0099] S1. Manually add 1 volume of 50 mg / ml testosterone-ethanol dropwise to 20 volumes of water;

[0100] S2. Microcrystals are obtained by vacuum filtration;

[0101] S3. Mix the microcrystals with the suspension to a testosterone concentration of 10 mg / ml; wherein the suspension consists of 7 wt% sucrose, 0.5 wt% sodium hyaluronate and a citrate-sodium citrate buffer to pH=6.5±0.5, with the remainder being water;

[0102] S4. Freeze-drying.

[0103] Experiment P2

[0104] S1. Mix testosterone with the suspension to a testosterone concentration of 10 mg / ml; wherein the suspension consists of 7 wt% sucrose, 0.5 wt% sodium hyaluronate and a citrate-sodium citrate buffer to pH=6.5±0.5, with the remainder being water;

[0105] S2, freeze-drying.

[0106] Experiment P3

[0107] S1. Manually add 1 volume of 50 mg / ml testosterone-ethanol dispersion to 100 volumes of water;

[0108] S2. Microcrystals are obtained by vacuum filtration;

[0109] S3. Mix the microcrystals with the suspension to a testosterone concentration of 10 mg / ml; wherein the suspension consists of 7 wt% sucrose, 0.5 wt% sodium hyaluronate and a citrate-sodium citrate buffer to pH=6.5±0.5, with the remainder being water;

[0110] S4. Filter using a 200-mesh sieve to remove clumps;

[0111] S5. The filtrate is subjected to high shear at 6000-7000 rpm for 5 minutes. After the shearing is completed, a sample can be taken to measure the particle size.

[0112] S6. Continue high shear for another 5 minutes. After the process is complete, samples can be taken for particle size measurement.

[0113] Experiment P4

[0114] S1. Testosterone-ethanol with a concentration of 100 mg / ml was mixed with water at a volume ratio of 1:125 and sheared online at a rotation speed of 10300 rpm.

[0115] S2. Microcrystals are obtained by vacuum filtration;

[0116] S3. Mix the microcrystals with the suspension to a testosterone concentration of 10 mg / ml; wherein the suspension consists of 7 wt% sucrose, 0.5 wt% sodium hyaluronate and a citrate-sodium citrate buffer to pH=6.5±0.5, with the remainder being water.

[0117] The freeze-drying process in the experiment is shown in Table 2 below. The absolute pressure during the freeze-drying process was kept constant at 0.1 mbar.

[0118] Table 2

[0119]

[0120] The particle sizes of the samples in experiments P1-P4 are shown in Table 3 below.

[0121] Table 3

[0122]

[0123] The results table for different preparation methods shows that the D50 and D90 of sample P4 are the smallest, and microcrystals will be prepared by online shearing in the future.

[0124] Example 1

[0125] In this embodiment, the formulation of the suspension is shown in Table 1-1 below. The pH of the suspension is 6.0±0.2. When preparing the lyophilized powder injection, the water for injection is removed during the process.

[0126] Table 1-1 Prescription Information Table

[0127]

[0128] In this embodiment, the process is carried out in three batches, and the specific production process is as follows:

[0129] (1) Preparation of oil phase: Weigh testosterone and dissolve it in a certain amount of anhydrous ethanol to obtain testosterone-ethanol with a concentration of 50 mg / ml, which is the oil phase;

[0130] (2) Preparation of suspension: According to Table 1-1, weigh 80% of the prescribed amount of water, add citric acid and sodium citrate, then add sucrose and sodium hyaluronate, stir to dissolve, then add the remaining water, weigh to the prescribed amount, stir to dissolve, disperse evenly, and finally sterilize at 121℃ for 15 min, cool to room temperature for use.

[0131] (3) Prepare the aqueous phase: Prepare water according to the volume ratio of oil phase to aqueous phase of 1:150;

[0132] (4) Online shearing: At room temperature (25±2℃), a shearing machine was used, and the flow rates of the oil phase and water phase were set. The water phase pump speed was 1250 rpm (flow rate of about 1250 ml / min), the oil phase pump speed was 75 rpm (flow rate of about 10 ml / min), and the shearing machine was used to mix and shear the oil phase and water phase at a speed of 10300 rpm to obtain testosterone microcrystals, which were then filtered.

[0133] (5) Concentration: The microcrystals obtained after filtration are directly mixed with an appropriate amount of the suspension obtained in step (2) until the testosterone concentration is about 20 mg / ml, and the concentration is measured.

[0134] (6) Diluting: Based on the concentration of the concentrated solution, add the suspension obtained in step (2) to dilute until the testosterone concentration is about 10 mg / ml, and measure the concentration;

[0135] (7) Filling: Add the remaining water according to the prescription in Table 1-1, fill the solution, and then dispense it into 2 ml borosilicate glass vials for injection, with a specification of 10 mg / vial, to obtain the suspension injection.

[0136] (8) Freeze-drying: The freeze-drying process is shown in Table 2 above, and freeze-dried powder injection is obtained;

[0137] (9) Crimping and labeling.

[0138] Example 2: pH screening of suspension buffer system before freeze-drying

[0139] Compare the various conditions involved in experiments P2-1 to P2-4 in Table 2-1 below.

[0140] Table 2-1 Main components of the suspension before freeze-drying

[0141]

[0142] In Experiment P2-1, the difference from Example 1 is that the total mass of the suspension remains unchanged, the mass of sodium hyaluronate is only 10% of that in Example 1, and the pH of the suspension is adjusted to 6.0 ± 0.2 by adjusting the mass of citric acid, sodium citrate and water.

[0143] In Experiment P2-2, the difference from Example 1 is that the total mass of the suspension remains unchanged, and the mass of sodium hyaluronate is only 10% of that in Example 1. By adjusting the mass of citric acid, sodium citrate, and water, the pH of the suspension is made to be 7.0 ± 0.2.

[0144] In Experiments P2-3, the difference from Example 1 is that the total mass of the suspension remains unchanged, and the mass of sodium hyaluronate is only 10% of that in Example 1. The pH of the suspension is adjusted to 8.0 ± 0.2 by adjusting the mass of sodium dihydrogen phosphate, disodium hydrogen phosphate, and water.

[0145] In Experiments P2-4, the difference from Example 1 is that the mass of sodium hyaluronate is only 10% of that in Example 1, and water is added to keep the total mass of the suspension constant.

[0146] Example 3: Screening of sodium hyaluronate concentration in suspension before freeze-drying

[0147] The concentration of sodium hyaluronate affects the suspension effect of the product. Using related substances, particle size, and sedimentation as evaluation indicators, the influence of different concentrations of sodium hyaluronate on the quality of the finished formulation was investigated. Suspensions with sodium hyaluronate concentrations of 0.05 wt%, 0.25 wt%, and 0.5 wt% were prepared, and the various conditions involved in experiments P3-1 to P3-3 in Table 3-1 were compared.

[0148] Table 3-1 Main components of the suspension before freeze-drying

[0149]

[0150] In Experiment P3-1, the difference from Example 1 is that the total mass of the suspension remains unchanged, the mass of sodium hyaluronate is only 10% of that in Example 1, and the pH of the suspension is made as consistent as possible with that in Example 1 by adjusting the mass of citric acid, sodium citrate and water.

[0151] In Experiment P3-2, the difference from Example 1 is that the total mass of the suspension remains the same, but the mass of sodium hyaluronate is only 50% of that in Example 1. By adjusting the mass of citric acid, sodium citrate, and water, the pH of the suspension is made as consistent as possible with that in Example 1.

[0152] Experiment P3-3 follows the same scheme as Example 1.

[0153] Example 4: Screening using suspension buffers before lyophilization

[0154] Compare the various conditions involved in experiments P4-1 to P4-3 in Table 4-1 below.

[0155] Table 4-1 Main components of the suspension before freeze-drying

[0156]

[0157] The scheme in Experiment P4-1 is the same as that in Example 1.

[0158] In Experiment P4-2, the difference from Example 1 is that the total mass of the suspension remains unchanged, and the pH of the suspension is adjusted to 6.0 ± 0.2 by adjusting the mass of succinic acid, sodium succinate and water.

[0159] In Experiment P4-3, the difference from Example 1 is that the total mass of the suspension remains unchanged, and the pH of the suspension is adjusted to 6.0 ± 0.2 by adjusting the mass of histidine, histidine hydrochloride, and water.

[0160] Example 5: Temperature Screening During Online Shearing

[0161] Based on previous research, the online shearing temperature was screened, and three batches of suspensions were prepared at room temperature, 2-8℃, and 40℃ respectively. The effects of different preparation temperatures on the particle size, related substances, and equipment status of the suspensions were investigated, and the various conditions involved in experiments P5-1 to P5-3 in Table 5-1 were compared.

[0162] Table 5-1 Conditions for online shearing and main components of the suspension before freeze-drying

[0163]

[0164] Experiment P5-1 follows the same scheme as Example 1.

[0165] In Experiment P5-2, the difference from Example 1 is that the temperature during online shearing is 2-8℃.

[0166] In Experiment P5-3, the difference from Example 1 is that the temperature during online shearing is 40°C, and there is abnormal noise from the equipment during sample preparation.

[0167] Effect Example

[0168] I. pH value

[0169] 1. Test subjects: the lyophilized powder injections in Examples 1-5.

[0170] 2. Test method: Add 1 ml of water to the lyophilized powder injection obtained by lyophilizing 1 bottle (1000 mg) of suspension to make a suspension, shake well, combine, and determine according to the method (General Chapter 0631, Part IV, Chinese Pharmacopoeia 2020).

[0171] 3. Indicator: pH value is 5.0-7.0.

[0172] II. Ethanol Residue

[0173] 1. Test subjects: the lyophilized powder injections in Examples 1-5.

[0174] 2. Test method: The residual solvent determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0861, Method II) shall be followed. The specific calculation formula is as follows:

[0175]

[0176] In the formula:

[0177] The average peak area of ​​the ethanol peak in reference solution 1 after continuous injection;

[0178] A SPL : Peak area of ​​ethanol in the test solution;

[0179] V STD : The dilution volume of the reference solution, in ml;

[0180] V SPL : The volume of the test solution diluted, in ml;

[0181] W STD1 The amount of ethanol in reference solution 1, in mg;

[0182] W SPL : Sample weight, mg;

[0183] P: Ethanol content,%.

[0184] Note: When the ethanol content is ≥95%, it is calculated as 100%.

[0185] 3. Indicator: Ethanol residue ≤0.5%.

[0186] III. Moisture

[0187] 1. Test subjects: the lyophilized powder injections in Examples 1-5.

[0188] 2. Test method: The moisture content was determined according to the method for moisture determination (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0832, Method 1). The specific calculation formula is as follows:

[0189]

[0190] W H2O Mass of water, mg;

[0191] V H2O : Volume of titrant consumed for calibrating water equivalent, in ml.

[0192]

[0193] WSPL Sample mass, mg;

[0194] V SPL : Volume of titrant consumed in calibrating the sample, in ml.

[0195] 3. Indicator: Moisture content not exceeding 3.0%.

[0196] IV. Content

[0197] 1. Test subjects: the lyophilized powder injections in Examples 1-5.

[0198] 2. Test method: Determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition). The specific calculation formula is as follows:

[0199]

[0200] In the formula:

[0201] W STD Reference standard sample weight, mg;

[0202] P: Content of reference standard, %

[0203] A STD : Average peak area of ​​the main peak in the reference solution (STD1) after continuous injection;

[0204] A SPL : Peak area of ​​the main peak in the test solution;

[0205] V STD : The dilution volume of the reference solution, in ml;

[0206] W: Average fill weight of the test sample, mg;

[0207] W SPL : Sample weight, mg;

[0208] V SPL : The dilution volume of the test solution, in ml;

[0209] S: Sample specification, mg.

[0210] 3. Indicators: The content is 90.0%-110.0% of the labeled amount.

[0211] V. Related Substances

[0212] 1. Test subjects: the lyophilized powder injections in Examples 1-5.

[0213] 2. Test method: Determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition). The specific calculation formula is as follows:

[0214]

[0215] In the formula:

[0216] W STD Reference standard sample weight, mg;

[0217] P: Content of reference standard, %

[0218] A STD : Average peak area of ​​the main peak in the reference solution (STD1) after continuous injection;

[0219] A SPL : Peak area of ​​impurities in the test solution;

[0220] V STD : The dilution volume of the reference solution, in ml;

[0221] W: Average fill weight of the test sample, mg;

[0222] W SPL : Sample weight, mg;

[0223] V SPL : The dilution volume of the test solution, in ml;

[0224] S: Sample specification, 10 mg.

[0225]

[0226] Note: Impurities with a content ≥0.02% are reported and included in the total impurities.

[0227]

[0228] In the formula:

[0229] W STD Reference standard sample weight, mg;

[0230] P: Content of reference standard, %

[0231] A STD : Average peak area of ​​the main peak in the reference solution (STD1) after continuous injection;

[0232] A SPL Peak area of ​​impurity I in the test solution;

[0233] V STD : The dilution volume of the reference solution, in ml;

[0234] W: Average fill weight of the test sample, mg;

[0235] WSPL : Sample weight, mg;

[0236] V SPL : The dilution volume of the test solution, in ml;

[0237] S: Sample specification, 10 mg.

[0238] 3. Specifications: Maximum single impurity: impurity C≤0.3%, impurity I (290 nm)≤0.2%, impurity K≤0.15%, unknown single impurity≤0.1%; total impurity≤0.6%.

[0239] VI. Particle size

[0240] 1. Test subject: the lyophilized powder injections in Examples 1-5, reflecting the particle size of testosterone microcrystals therein.

[0241] 2. Test method: Take one bottle of this product, add 1 ml of water, shake by hand for 1 minute to disperse evenly, and then test. Perform particle size and particle size distribution determination according to the method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0982, Method III), using a Malvern 3000 laser particle size analyzer, red light detection.

[0242] 3. Specifications: Based on volume distribution, D90≤20 μm.

[0243] VII. Dissolution

[0244] 1. Test subjects: the lyophilized powder injections in Examples 1-5.

[0245] 2. Test method: Dissolution and release rate determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II). The specific calculation formula is as follows:

[0246]

[0247] In the formula:

[0248] W STD Reference standard sample weight, mg;

[0249] P: Content of reference standard, %

[0250] A STD : Average peak area of ​​the main peak in the reference solution (STD1) after continuous injection;

[0251] A SPL : Peak area of ​​the main peak in the test solution;

[0252] V STD : The dilution volume of the reference solution, in ml;

[0253] V SPL: Medium volume, ml;

[0254] S: Sample specification, mg.

[0255] 3. Specifications: Dissolution rate ≥80% after 45 minutes.

[0256] 8. X-ray diffraction (XRD)

[0257] 1. Test subject: the lyophilized powder injections in Examples 1-5, reflecting the crystal form of testosterone microcrystals therein.

[0258] 2. Testing Method:

[0259] Instrument: X-ray diffractometer, manufacturer: BRUKER, model: D2 PHASER.

[0260] Parameters: X-ray power 30 kV, 10 mA, scanning angle 3°-45°, step size 0.02°, step time 0.30 s.

[0261] The test results for Example 1 are shown in Table 6-1-1 below.

[0262] Table 6-1-1

[0263]

[0264] The XRD patterns of testosterone microcrystalline lyophilized powder for injection prepared from batches 1, 2, and 3 are shown below. Figure 1 , Figure 2 and Figure 3 As shown.

[0265] The stability test results of Example 1 are shown in Table 6-1-2 below.

[0266] Table 6-1-2

[0267]

[0268] The results showed that the maximum single impurity and total impurities in the relevant substances increased under light conditions. The subsequent storage conditions will be set to avoid light. The small-scale test confirmed that the finished products prepared by the proposed formula process in three batches met the proposed quality standard requirements. There were no significant differences between the finished product batches, indicating that the proposed formula process has good reproducibility and can be further scaled up for production.

[0269] The detection results of Example 2 are shown in Table 6-2-1 below.

[0270] Table 6-2-1

[0271]

[0272] Based on the data from day 0, the particle size data and related substances of the four batches are basically consistent, with moisture content P2-1>P2-3>P2-2>P2-4. For details, please refer to [link / reference needed]. Figure 4 .

[0273] After being stored at 40±2℃ / 75%±10%RH for one week, P2-1, P2-2, and P2-3 all showed signs of collapse. The degree of collapse was generally consistent with the moisture content. For details, please refer to [link / reference needed]. Figure 5 (The two bottles with the same number on the left and right in the picture are two samples from the same batch), and the properties after two weeks are basically the same as those after one week.

[0274] After being placed at 40℃±2℃ / 75±10%RH for four weeks, the moisture content of P2-1 to P2-4 changed by 0.2%, 0.2%, 0.4% and 0.1%, respectively; the pH value changed by 0, 0, +0.1 and +1.1, respectively; the D90 changed by +4.2 μm, +3.5 μm, +3.1 μm and +5.1 μm, respectively; the total impurities changed by +0.01%, +0.05%, +0.01% and +0.03%, respectively; and the maximum single impurity remained almost unchanged.

[0275] Therefore, the collapse and hardening of the powder were caused by high moisture content and low sodium hyaluronate concentration. The moisture content will be controlled and the sodium hyaluronate concentration will be increased in the future.

[0276] The detection results of Example 3 are shown in Table 6-3-1 below.

[0277] Table 6-3-1

[0278]

[0279] The results show that the related substances and particle size data of the three batches of samples are basically consistent. Regarding sedimentation, P3-1 showed obvious stratification after standing for 5 minutes following reconstitution, while P3-2 and P3-3 did not show stratification. (See attached image for details.) Figure 6 After reconstitution and standing for 10 minutes, P3-2 began to separate into layers, while P3-3 still did not separate into layers. See details below. Figure 7 After reconstitution and standing for 60 minutes, P3-3 still did not separate into layers, as can be seen in the following figure. Figure 8 .

[0280] Therefore, the concentration of sodium hyaluronate is the key factor affecting the stability of testosterone microcrystalline lyophilized powder injection after reconstitution. In P3-1 and P3-2, due to the low concentration of sodium hyaluronate, stratification occurs after reconstitution, resulting in poor stability.

[0281] The stability test results of Example 4 are shown in Table 6-4-1 below.

[0282] Table 6-4-1

[0283]

[0284] Based on the data, the appearance, pH, moisture, total impurities, maximum single impurity, and D90 of the three batches of samples on day 0 were similar. After being placed at 40℃±2℃ / 75±10%RH for one week, P4-3 showed a tendency to yellow and shrink in appearance.

[0285] After being placed at 40℃±2℃ / 75±10%RH for 4 weeks, the pH values ​​of P4-1 to P4-3 changed by +0.2, +0.1, and +0.1, respectively, and the total impurities changed by +0.05%, +0.03%, and +0.04%, respectively. The maximum single impurity remained almost unchanged. D10 changed by +1.59 μm, +1.86 μm, and +1.69 μm, respectively; D50 changed by +4.19 μm, +5.04 μm, and +3.68 μm, respectively; and D90 changed by +8.2 μm, +10.9 μm, and +5.9 μm, respectively.

[0286] In summary, the citrate-sodium citrate buffer pair used as a pre-lyophilized suspension results in slightly better stability of the testosterone microcrystalline lyophilized powder injection.

[0287] The detection results of Example 5 are shown in Table 6-5-1 below.

[0288] Table 6-5-1

[0289]

[0290] Based on the particle size data of concentrated and diluted samples, the D10 and D50 of the three batches of samples are similar. Based on the D90 data, P5-2 > P5-3 > P5-1, and the D90 of sample P5-2 is very large, reaching 36.7 μm.

[0291] Based on the particle size data after freeze-drying, the D10 and D50 of the three batches of samples were similar. Based on the D90 and corresponding RSD data, P5-2 > P5-3 > P5-1. The D90 particle size of the freeze-dried samples increased compared to before freeze-drying, with P5-1, P5-2, and P5-3 increasing by 0.9 μm, 54.9 μm, and 11.7 μm, respectively.

[0292] Based on the relevant material data, the maximum single impurity in the three batches is similar, while the total impurity is P5-2>P5-1>P5-3.

[0293] It can be seen that the testosterone microcrystals prepared at different temperatures will affect the particle size of the testosterone microcrystal lyophilized powder injection. In P5-1 and P5-2, the testosterone microcrystals were obtained at excessively low and excessively high temperatures, respectively, which resulted in a significant increase in the D90 particle size of the testosterone microcrystal lyophilized powder injection.

[0294] Application examples

[0295] Reconstitute the lyophilized powder injections from Examples 1-5 with 1 ml of water for injection and administer subcutaneously once a day. This can improve patient compliance and alleviate delayed puberty development in males.

Claims

1. A testosterone microcrystal, characterized in that, Its particle size range satisfies: D90≤20.0 μm; the X-ray powder diffraction pattern expressed in 2θ angle has diffraction peaks at 13.1º±0.2º, 15.4º±0.2º, 16.0º±0.2º and 18.3º±0.2º.

2. The testosterone microcrystals according to claim 1, characterized in that, It satisfies at least one of the following conditions: (1) The D90 of the testosterone microcrystals satisfies: 10.0 μm≤D90≤18.0 μm, preferably 14.0 μm≤D90≤18.0 μm, for example, D90=14.3 μm, 14.8 μm, 15.1 μm, 15.3 μm, 15.4 μm, 15.7 μm, 15.8 μm, 15.9 μm or 17.4 μm; (2) The particle size range of the testosterone microcrystals satisfies: 5 μm ≤ D50 ≤ 20 μm; preferably, 5 μm ≤ D50 ≤ 10 μm, for example, D50 = 5.96 μm, 5.98 μm, 6.04 μm, 6.54 μm, 6.56 μm, 6.59 μm, 6.63 μm, 6.67 μm, 6.76 μm, 6.84 μm, 6.93 μm, 6.94 μm, 7.01 μm, 7.08 μm, 7.12 μm or 7.27 μm; (3) The particle size range of the testosterone microcrystals satisfies: 2 μm ≤ D10 ≤ 10 μm; preferably, 2 μm ≤ D10 ≤ 3 μm, for example, D10 = 2.51 μm, 2.55 μm, 2.58 μm, 2.60 μm, 2.77 μm, 2.80 μm, 2.81 μm, 2.82 μm, 2.83 μm, 2.84 μm, 2.86 μm, 2.87 μm or 2.90 μm; Preferably, it satisfies one of the following conditions: ①The testosterone microcrystals have diffraction peaks at 13.230º, 15.488º, 16.138º and 18.422º in the X-ray powder diffraction pattern expressed in 2θ angle; ② The testosterone microcrystals, when viewed in 2θ angle X-ray powder diffraction patterns, show diffraction peaks at 13.093º, 15.357º, 16.009º and 18.329º; ③ The testosterone microcrystals, in X-ray powder diffraction patterns expressed at 2θ angles, show diffraction peaks at 13.141º, 15.432º, 16.060º and 18.304º; ④ The X-ray powder diffraction pattern of the testosterone microcrystals is shown in Figure 1, Figure 2, or Figure 3; ⑤ The testosterone microcrystals meet the following conditions: D10=2.82 μm, D50=6.63 μm, D90=14.8 μm, and the X-ray powder diffraction pattern expressed in terms of 2θ angle is shown in Figure 1; ⑥ The testosterone microcrystals meet the following conditions: D10=2.80 μm, D50=6.67 μm, D90=15.1 μm, and the X-ray powder diffraction pattern expressed in terms of 2θ angle is shown in Figure 2. ⑦ The testosterone microcrystals meet the following conditions: D10=2.86 μm, D50=6.84 μm, D90=15.4 μm, and the X-ray powder diffraction pattern expressed in 2θ angle is shown in Figure 3.

3. A method for preparing testosterone microcrystals, characterized in that, It includes the following steps: At 20-30°C, the oil phase and the aqueous phase are mixed and sheared at a speed of 6000-70000 rpm; the oil phase includes testosterone and an organic dispersant, and the concentration of the testosterone is 50-90 mg / ml; the aqueous phase includes water; the volume ratio of the oil phase to the aqueous phase is 1:(130-150). Preferably, it satisfies at least one of the following conditions: ① The preparation temperature is 23-27℃; ② The rotational speed of the mixed shearing is 10000-12000 rpm, for example 10300 rpm; ③The organic dispersant is ethanol or diethyl ether; ④ The volume ratio of the oil phase to the water phase is 1:150; ⑤ The aqueous phase also includes surfactants; ⑥ After the mixed shearing, the mixture is filtered.

4. Testosterone microcrystals prepared by the method described in claim 3.

5. A testosterone microcrystalline lyophilized powder for injection, characterized in that, It includes suspending agents, lyophilization protectants, and testosterone microcrystals as described in any one of claims 1, 2, and 4; Preferably, it satisfies at least one of the following conditions: ①The suspending agent is sodium hyaluronate; the molecular weight of the sodium hyaluronate is preferably 800,000 to 1,200,000; ② The mass ratio of the suspending agent to the mass of the testosterone microcrystals is (0.3-2):1, preferably (0.3-0.6):1, for example 0.5:1; ③The freeze-drying protectant is sucrose; ④ The mass ratio of the freeze-drying protectant to the mass of the testosterone microcrystals is (5-15): 1, preferably (7-9): 1; ⑤ After the testosterone microcrystalline lyophilized powder for injection is reconstituted with water at a mass ratio of 100:1, the pH of the resulting reconstituted suspension is 5-7, preferably 5.8-6.2, for example, pH 6. ⑥ The testosterone microcrystalline lyophilized powder injection further includes a pH adjuster; the pH adjuster is preferably a mixture of citric acid and sodium citrate, a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, a mixture of succinic acid and sodium succinate, or a mixture of histidine and histidine hydrochloride; the pH adjuster is preferably a mixture of citric acid and sodium citrate, wherein the mass of citric acid is 2.97% of the mass of the testosterone microcrystals, and the mass of sodium citrate is 23.51% of the mass of the testosterone microcrystals; ⑦ The testosterone microcrystalline lyophilized powder injection comprises testosterone microcrystals, sucrose, sodium hyaluronate, citric acid, and sodium citrate in a mass ratio of 10: 90: 5: 0.297: 2.351; ⑧ The testosterone microcrystalline lyophilized powder injection comprises testosterone microcrystals, sucrose, and sodium hyaluronate in a mass ratio of 10:90:5, and also includes succinic acid and sodium succinate. After the testosterone microcrystalline lyophilized powder injection is reconstituted with water in a mass ratio of 100:1, the pH of the resulting reconstituted suspension is 5.8-6.

2. ⑨ The testosterone microcrystalline lyophilized powder injection comprises testosterone microcrystals, sucrose, and sodium hyaluronate in a mass ratio of 10:90:5, and also includes histidine and histidine hydrochloride. After the testosterone microcrystalline lyophilized powder injection is reconstituted in water at a mass ratio of 100:1, the pH of the resulting reconstituted suspension is 5.8-6.

2.

6. A testosterone microcrystalline suspension, characterized in that, It comprises water, a suspending agent, a lyophilization protectant, and testosterone microcrystals as described in any one of claims 1, 2, and 4; Preferably, it satisfies at least one of the following conditions: ① The concentration of the testosterone microcrystals is 1%, and the percentage is the ratio of the mass of the testosterone microcrystals to the mass of the testosterone microcrystal suspension; ②The suspending agent is sodium hyaluronate; the molecular weight of the sodium hyaluronate is preferably 800,000 to 1,200,000; ③ The concentration of the suspending agent is 0.3%-2%, preferably 0.3%-0.6%, for example 0.5%, and the percentage is the ratio of the mass of the suspending agent to the mass of the testosterone microcrystalline suspension; ④ The freeze-drying protectant is sucrose; ⑤ The concentration of the freeze-drying protectant is 5%-15%, for example 7%-9%, and the percentage is the ratio of the mass of the freeze-drying protectant to the mass of the testosterone microcrystalline suspension; ⑥ The pH of the testosterone microcrystal suspension is 5-7, preferably 5.7-6.2, for example, 5.8-6.2; ⑦ The testosterone microcrystalline suspension further includes a pH adjuster; the pH adjuster is preferably as defined in claim 5; ⑧ The testosterone microcrystal suspension comprises testosterone microcrystals, sucrose, sodium hyaluronate, citric acid, sodium citrate, and water in a mass ratio of 10: 90: 5: 0.297: 2.351: 892.352; ⑨ The testosterone microcrystalline suspension comprises 1% testosterone microcrystals, 0.5% sodium hyaluronate, and 9% sucrose, the percentages being the ratio of the mass of each substance to the mass of the testosterone microcrystalline suspension, with the remainder being water, succinic acid, and sodium succinate; the pH of the testosterone microcrystalline suspension is 5.8-6.2; ⑩ The testosterone microcrystalline suspension comprises 1% testosterone microcrystals, 0.5% sodium hyaluronate, and 9% sucrose, with the percentages being the ratio of the mass of each substance to the mass of the testosterone microcrystalline suspension. The remainder consists of water, histidine, and histidine hydrochloride. The pH of the testosterone microcrystalline suspension is 5.8-6.

2.

7. A method for preparing the testosterone microcrystalline suspension as described in claim 6, characterized in that, It includes the following steps: The suspension is mixed with testosterone microcrystals as described in any one of claims 1, 2 and 4; the suspension comprises water, a suspending agent and a lyophilization protectant; Preferably, it satisfies at least one of the following conditions: ①The suspending agent is sodium hyaluronate; the molecular weight of the sodium hyaluronate is preferably 800,000 to 1,200,000; ② The mass ratio of the suspending agent to the mass of the testosterone microcrystals is (0.3-2):1; preferably (0.3-0.6):1, for example 0.5:1; ③The freeze-drying protectant is sucrose; ④ The mass ratio of the freeze-drying protectant to the mass of the testosterone microcrystals is (5-15): 1, preferably (7-9): 1; ⑤ The pH of the testosterone microcrystalline suspension is 5-7, preferably 5.8-6.2; ⑥ The testosterone microcrystalline suspension further includes a pH adjuster; the pH adjuster is preferably as defined in claim 5; ⑦ The mass ratio of the water to the testosterone microcrystals is 100:

1.

8. A method for preparing the testosterone microcrystalline suspension as described in claim 6, characterized in that, It includes the following steps: Mix water with the testosterone microcrystalline lyophilized powder for injection as described in claim 5.

9. A method for preparing a testosterone microcrystalline lyophilized powder injection, characterized in that, It includes the following steps: Freeze-dry the testosterone microcrystalline suspension as described in claim 6; Preferably, it satisfies at least one of the following conditions: ① The freeze-drying temperature is -45~5℃; ②The heating or cooling rate of the freeze-drying process is 1℃ / min; ③ The freeze-drying process is shown in the table below. ， ④ The pressure for freeze drying is 0.1 mbar.

10. A testosterone microcrystalline lyophilized powder for injection prepared by the method described in claim 9.

Images