CN109419771B - Testosterone undecanoate sustained-release pharmaceutical composition, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, and relates to testosterone undecanoate suspension, and a preparation method and application thereof. Specifically, the invention relates to a testosterone undecanoate suspension comprising testosterone undecanoate, a stabilizer and water, wherein: the content of testosterone undecanoate is 10% -50%; the particle size of the testosterone undecanoate meets the following requirements: d10 is 0.2-0.5 μm, D50 is 0.5-1.5 μm, and D90 is 2.2-6.0 μm; the stabilizer comprises a first stabilizer and a second stabilizer, and the weight ratio of the first stabilizer to the second stabilizer is (1-10): (1-20), and the weight ratio of the testosterone undecanoate to the stabilizer is (1-20): (1-20). The testosterone undecanoate suspension has good stability, good redissolution condition after sedimentation, high bioavailability and small irritation, and can stably maintain the hormone level in the body for a long time.

Description

Testosterone undecanoate sustained-release pharmaceutical composition, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and relates to testosterone undecanoate suspension, a freeze-dried preparation thereof, a preparation method and application thereof.

Background

Male hypogonadism is characterized by insufficient endogenous testosterone production, resulting in abnormally low levels of circulating testosterone, i.e. serum testosterone levels below 10 nmol/l. The clinical manifestations of male hypogonadism patients are various. Testosterone deficiency is accompanied by symptoms of varying severity such as sexual dysfunction, decreased muscle mass and strength, depressed mood, and osteoporosis. In a specific period when some men step from middle age to elderly, climacteric syndrome occurs in men, which is caused by a decrease in androgen levels and an abnormality in androgen receptors due to various factors. Nowadays, with the increasing social pressure, a part of young people also develop hypogonadal symptom groups.

The current primary treatment for male hypogonadism is androgen replacement therapy. The natural testosterone half-life is very short, only 10-20 minutes. Oral administration of testosterone does not achieve clinically relevant levels of testosterone. Therefore, testosterone structures must be chemically modified or administered by other routes in order to treat male hypogonadism. Of these, 17 β -hydroxylated esters such as testosterone propionate and Testosterone Enanthate (TE) represent the most important compounds marketed for the treatment of male hypogonadism. Among these testosterone esters, testosterone undecanoate is the only commercially available oral testosterone ester in China. Testosterone undecanoate has obvious effect of treating male climacteric syndrome as a prodrug, supplements male androgen, relieves the illness state of patients, and has important practical significance. The structural formula of testosterone undecanoate is shown in formula I below:

however, testosterone undecanoate is a poorly water-soluble drug, and oral testosterone undecanoate soft capsules (trade name ANDRIOL) and intramuscular testosterone undecanoate oil solutions (trade name smolon) are mainly used clinically at present. When the soft capsule is orally taken, the soft capsule needs to be eaten together with a large amount of fat, and the fat and the lipid are directly absorbed through intestinal lymph, so that the deactivation caused by the first-pass effect of the liver is avoided; the soft capsule is substantially not absorbed when administered with empty stomach. In addition, because the half-life period of the medicine is short (testosterone undecanoate is rapidly hydrolyzed by serum esterase after entering blood to be absorbed), patients take (3-4 capsules in total) soft capsules for multiple times every day, and the compliance is poor.

The prescription of the long-acting testosterone undecanoate injection used clinically comprises benzyl benzoate and oil for injection, wherein the benzyl benzoate may cause anaphylactic reaction, and the oil may generate adverse reactions such as pulmonary oil embolism, injection pain and the like.

Therefore, there is a need to develop new testosterone undecanoate formulations that are safe, effective, of controlled quality and convenient to use clinically.

Disclosure of Invention

The inventor of the present invention has made intensive studies and extensive experiments to obtain a testosterone undecanoate sustained-release pharmaceutical composition, such as an aqueous testosterone undecanoate suspension. The inventor further prepares a freeze-dried preparation of the suspension. The testosterone undecanoate sustained-release pharmaceutical composition provided by the invention is appropriate in particle size and good in stability. There was a certain reduction in local irritancy of intramuscular injections compared to the commercial oil solutions. Rat in vivo experiments prove that the suspension has a certain slow release effect, and the muscle local tissue residue of the novel preparation is less than that of a commercially available oil solution preparation; the bioavailability is improved to a certain extent. The following invention is thus provided:

one aspect of the invention relates to a testosterone undecanoate suspension comprising testosterone undecanoate, a stabilizer, and water, wherein:

the content of testosterone undecanoate is 10% -50% (w/v, g/ml);

the particle size of the testosterone undecanoate meets the following requirements: d10 is 0.2-0.5 μm, D50 is 0.5-1.5 μm, and D90 is 2.2-6.0 μm;

the stabilizer comprises a first stabilizer and a second stabilizer, and the weight ratio of the testosterone undecanoate to the stabilizer is (1-20): (1-20) wherein, in the formula,

a first stabilizer selected from any one or more of soy lecithin, hydroxypropyl cellulose, polysorbates such as polysorbate 80 or polysorbate 20, poloxamer 188, poloxamer 407, povidone, hypromellose, acacia, tragacanth, sodium alginate, methylcellulose, sodium carboxymethylcellulose, alkyl polyglycoside, inulin lauryl carbamate, polyethylene glycol, hydroxypropyl cellulose, and sugars such as glucose, fructose or sucrose,

a second stabilizer selected from any one or more of polyethylene glycol succinate, sodium dodecyl sulfate, phenethylamine, arginine salt or docusate sodium; and is

The weight ratio of the first stabilizer to the second stabilizer is (1-10): (1-20).

Testosterone undecanoate is the active ingredient or active ingredient of the pharmaceutical composition of the present invention.

The terms "first" and "second" in the first stabilizer and the second stabilizer are merely for convenience of distinguishing or referring to different stabilizers, and do not have an ordinal meaning, if not specifically stated.

In one embodiment of the invention, the testosterone undecanoate sustained-release pharmaceutical composition, when it is a testosterone undecanoate aqueous sustained-release suspension, wherein the testosterone undecanoate is present in an amount of 10% to 50% (w/v, g/ml), preferably 10% to 40% (w/v, g/ml), particularly preferably 10% to 25% (w/v, g/ml), such as 10% to 15% (w/v, g/ml), 10% (w/v, g/ml), 12% (w/v, g/ml), 12.5% (w/v, g/ml), 15% (w/v, g/ml), 20% (w/v, g/ml) or 25% (w/v, g/ml).

Without being bound by theory, if the concentration of the main drug is higher than the upper limit of 50%, the early-stage quick release amount is increased, and the effective treatment concentration may be exceeded. If the concentration of the principal drug is less than the lower limit of 10%, the release amount is small, and an effective therapeutic concentration may not be achieved or a long-lasting effect may not be maintained.

In one embodiment of the invention, the testosterone undecanoate sustained-release pharmaceutical composition is characterized in that the weight ratio of testosterone undecanoate to the stabilizer is (1-20): (1-20); preferably (1-15): (1-10); more preferably (1-10): (1-5). For example, 15:1, 10:1, 5:1, and any two specific ratios.

In one embodiment of the present invention, characterized by any one or more of the following items (1) to (8):

(1) the content of testosterone undecanoate is 10-30% (g/ml);

(2) the D10 is 0.2-0.3 μm; preferably 0.2 to 0.25 μm;

(3) the D50 is 0.9-1.4 μm;

(4) the first stabilizer is selected from one or more of polysorbates, hydroxypropyl cellulose, soybean lecithin, sodium carboxymethyl cellulose and hydroxypropyl cellulose;

(5) the second stabilizer is selected from any one or more of polyethylene glycol succinate (TPGS) and docusate sodium;

(6) the weight ratio of the testosterone undecanoate to the stabilizer is (1-15): (1-15); preferably (1-15): (1-10), more preferably (1-10): (1-5);

(7) the weight ratio of the first stabilizer to the second stabilizer is (1-5): (1-20), preferably (1-5): (1-10); more preferably (1-2): (1-5) or 1: (1-2); e.g., 1:1, 1:1.5, 1:1.6, 1:1.8, 1:2, 1:3, 1:4, 1:5, and any two specific ratios;

(8) the testosterone undecanoate suspension further comprises a bacteriostatic agent and/or a pH adjusting agent.

Preferably, D10 is 0.2-0.3 μm; more preferably 0.2 to 0.25. mu.m. Without being bound by theory, a smaller D10 favors a more rapid release of testosterone undecanoate to achieve effective therapeutic concentrations in a short time to exert drug efficacy.

In one embodiment of the present invention, preferably, the testosterone undecanoate sustained-release pharmaceutical composition is one in which D10 is 0.2 to 0.4 μm or 0.2 to 0.3 μm. For example, D10 is 0.2 μm, 0.3 μm, or 0.4 μm, and ranges of values between any two of these specific values.

In one embodiment of the present invention, preferably, the testosterone undecanoate sustained-release pharmaceutical composition is one wherein D50 is 0.9 μm to 1.4 μm; more preferably, D50 is 0.9 μm to 1.2. mu.m. For example, D50 is 0.9 μm, 1.0 μm, 1.1 μm, 1.2 μm, 1.3 μm, or 1.4 μm, and any two specific values are within the numerical range.

In one embodiment of the present invention, preferably, the testosterone undecanoate sustained-release pharmaceutical composition is one in which D90 is 2.5 to 5.0 μm, 2.5 to 4.5 μm, 2.5 to 4.0 μm, 3.0 to 5.0 μm, 3.0 to 4.5 μm, 3.0 to 4.0 μm, or 3.0 to 3.5 μm. For example, D90 is 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, or 5.0 μm, and any two specific values are within the numerical range. .

D10 needs to be controlled in a smaller nano-particle size range to achieve faster release after administration to reach the target blood concentration. D50 and D90 are controlled in the micron particle size range, so that the small particle size is dissolved and released firstly to quickly reach the effective treatment concentration, and the large particle size is dissolved and released later to reach the sustained release effect.

The present inventors have found that if D10 is larger than 0.5 μm, the immediate release effect is deteriorated and a relatively rapid release after administration to a therapeutically effective concentration cannot be achieved. If the D10 is less than 0.2 μm, the particle size in the preparation is small overall, a burst effect is generated, the drug is released too much, the release exceeds 20% in 2 minutes, the ultra-effective treatment concentration can be achieved, and the maintaining time is shortened.

The present inventors have also found that the larger the particle diameter of D50 larger than 1.4. mu.m, the more irritating, i.e., the less in vivo compatibility, the less safe the preparation.

In one embodiment of the invention, the testosterone undecanoate suspension comprises the following components:

preferably, the testosterone undecanoate suspension comprises the following components:

the aqueous sustained release suspension of testosterone undecanoate can be administered directly to the human body, for example by intramuscular injection. Before the testosterone undecanoate aqueous sustained-release suspension is used, the suspension needs to be shaken up to improve the uniformity of the suspension. Without being limited by theory, the intramuscular injection of the suspension can agglomerate at the injection site after injection to form a drug reservoir, and then the drug is dissolved in sequence according to the size of the drug particles, and the dissolved drug enters the systemic circulation to achieve the effect of slow release. Meanwhile, testosterone undecanoate with an ester structure is combined, and the result of slow release can be caused by the existence of the esterase hydrolysis process.

Another aspect of the invention relates to a testosterone undecanoate lyophilized preparation, which is prepared by lyophilizing any one of testosterone undecanoate suspensions according to the invention;

preferably, the lyoprotectant is selected from one or more of glucose, mannitol, sucrose, lactose;

preferably, the lyoprotectant is used in an amount of 10% to 40%, preferably 10% to 30%, particularly preferably 15% to 25%, e.g. 15%, 20% or 25% of the solids content.

The testosterone undecanoate freeze-dried preparation can be re-dissolved by adding normal saline or glucose injection, and can be used for intramuscular injection.

Yet another aspect of the invention relates to a method of preparing a testosterone undecanoate suspension of the present invention, comprising the steps of:

(1) uniformly mixing a first stabilizer, a second stabilizer and water to obtain a first mixture;

(2) adding testosterone undecanoate to the first mixture in step (1) to obtain a second mixture;

(3) uniformly dispersing the second mixture, preferably, uniformly dispersing the second mixture through a dispersion emulsification homogenizer;

(4) homogenizing the product of the step (3) by a high-pressure homogenizer.

In one embodiment of the present invention, the homogenizing in step (4) comprises the steps of:

(4-1) at least 5 cycles at 200bar to 300 bar; preferably, 5-15 cycles; e.g., 8-12 cycles; for example 10 cycles;

(4-2) 5-15 cycles at 400 bar-600 bar; e.g., 8-12 cycles; for example 10 cycles;

(4-3) 5-15 cycles at 700-900 bar; e.g., 8-12 cycles; for example 10 cycles;

(4-4)1050 bar-1150 bar, 5-15 cycles; e.g., 8-12 cycles; for example 10 cycles.

In one embodiment of the present invention, preferably, in step (4-1), the cycle is performed at 200bar to 300bar until there is no noticeable impact sound of the ceramic beads with the valve seat.

In one embodiment of the present invention, the homogenizing in step (4) comprises the steps of:

(4-1) 8-12 cycles at 200 bar-300 bar; for example 10 cycles;

(4-2) 8-12 cycles at 450-550 bar; for example 10 cycles;

(4-3) 8-12 cycles at 750 bar-850 bar; for example 10 cycles;

(4-4)1050 bar-1150 bar, 8-12 cycles; for example 10 cycles.

In one embodiment of the present invention, the homogenizing in step (4) comprises the steps of:

(4-1) 10 cycles at 200 bar-300 bar;

(4-2) 10 cycles at 500 bar;

(4-3) 10 cycles at 800 bar;

(4-4)1100bar, 10 cycles.

Yet another aspect of the invention relates to the use of any one of the testosterone undecanoate suspensions or lyophilized formulations of testosterone undecanoate of the invention in the manufacture of a medicament for the treatment or prevention of a disorder resulting from insufficient endogenous testosterone production, such as male hypogonadism; preferably, the male hypogonadism is selected from at least one of male sexual dysfunction, a reduction in male muscle mass and strength, male depressed mood and male osteoporosis.

In the present invention,

d10, D50 and D90 respectively refer to the corresponding particle sizes at 10%, 50% and 90% of the cumulative distribution map of the particle sizes. The particle size cumulative distribution diagram is a diagram automatically generated by an instrument through the Malvern 2000 laser diffraction principle and gives data.

In the present invention, testosterone undecanoate aqueous suspension, testosterone undecanoate crystal suspension, testosterone undecanoate aqueous sustained-release suspension, and testosterone undecanoate suspension have the same meaning, if not specifically mentioned.

Advantageous effects of the invention

The invention has at least one of the following technical effects:

(1) the testosterone undecanoate suspension prepared by the invention has good stability and good redissolution condition after sedimentation;

(2) in vivo pharmacokinetic experiments of rats show that testosterone undecanoate suspension and testosterone undecanoate oil injection sold in the market have similar slow release effect, can stably maintain the hormone level in vivo for a long time, meanwhile, the local tissue residue of muscle is reduced to a certain extent compared with that of oil solution sold in the market, and the bioavailability is higher than that of oil solution sold in the market;

(3) the muscle irritation test of the rabbit shows that the muscle irritation of the suspension is obviously reduced compared with the muscle irritation of the commercial oil solution preparation.

Drawings

FIG. 1: and (5) observing by a scanning electron microscope. FIG. 1A: raw material medicaments. FIG. 1B: 300nm crystal suspension. FIG. 1C: 1.2 μm crystal suspension. FIG. 1D: 4.8 μm crystal suspension.

FIG. 2: x-ray powder diffraction patterns of 3 raw materials with different particle sizes. The samples from top to bottom in FIG. 2 are bulk drug, 4.8 μm crystal suspension, 1.2 μm crystal suspension, 300nm crystal suspension in that order. Counts in the abscissa represents peak intensity, and 2theta in the ordinate represents diffraction angle 2 theta.

FIG. 3: results of muscle stimulation experiments. Fig. 3A, 3C, 3E, 3G, 3I, 3K, and 3M show experimental results of a muscle transection diagram, and fig. 3B, 3D, 3F, 3H, 3J, 3L, and 3N show experimental results of a muscle transection diagram.

Wherein:

fig. 3A, fig. 3B, positive control group.

Fig. 3C, fig. 3D, negative control group.

Fig. 3E, fig. 3F, blank adjuvant group.

FIG. 3G, FIG. 3H, 300nm suspension (sample 3).

FIG. 3I, FIG. 3J, 1.2 μm suspension (sample 2).

FIG. 3K, FIG. 3L, 4.8 μm suspension (sample 1).

Fig. 3M, fig. 3N, oil solution formulation.

FIG. 4: and (3) a curve chart of the change of the in vivo drug concentration of the testosterone undecanoate nano-micron crystal suspension and the testosterone undecanoate oil injection with time.

FIG. 5: and (3) a curve chart of the change of the in vivo drug concentration of the testosterone undecanoate nano-micron crystal suspension and the testosterone undecanoate oil injection with time.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Preparation examples 1 to 3: preparation of Testosterone undecanoate suspension (samples 1-3)

The prescription composition is as follows:

the preparation method comprises the following steps:

adding soybean lecithin and docusate sodium into water at room temperature, stirring, dispersing uniformly, adding screened testosterone undecanoate bulk drug, dispersing uniformly again (grade A, 2min) in a dispersing emulsifying homogenizer (Shanghai Hengchuan mechanical equipment Co., Ltd., C25), pouring into a sample cup of a high-pressure homogenizer with a condensing device, circulating for several times at low pressure of 200 bar-300 bar, pressurizing to 500bar 10 after no striking sound is generated between the ceramic beads and the valve seat to obtain 4.8 μm crystal suspension (corresponding to sample 1), continuing to 800bar 10 and 1100bar 10 to obtain 1.2 μm crystal suspension (corresponding to sample 2), continuing to 1300bar 10, and then 800bar 10 to obtain 300nm crystal suspension (corresponding to sample 3).

Then adding a proper amount of benzalkonium chloride as a bacteriostatic agent and a proper amount of sodium carbonate into each product respectively, and adjusting the pH value to 7.35-7.45.

Samples 1-3 were prepared.

According to the measurement method in example 1 below, it was actually measured that D50 of sample 2 was 1.150. mu.m, and D50 of sample 3 was 293.3 nm.

Preparation examples 4 to 8: preparation of Testosterone undecanoate suspension (samples 4-8)

Sample 4 recipe composition: 12.5g of testosterone undecanoate, 1g of tween 20 and 0.5g of docusate sodium.

Sample 5 recipe composition: 12.5g of testosterone undecanoate, 2g of polyethylene glycol 4000 and 1g of docusate sodium.

Sample 6 recipe composition: 12.5g of testosterone undecanoate, 1g of hydroxypropyl cellulose and 1g of docusate sodium.

Sample 7 recipe composition: 12.5g of testosterone undecanoate, 1g of hypromellose and 0.5g of TPGSS.

Sample 8 recipe composition: 12.5g of testosterone undecanoate, 1g of polysorbate 80 and 1g of TPGS.

The preparation method comprises the following steps:

according to the proportion, the two stabilizers are sequentially added into water according to the prescription amount under the condition of room temperature and stirred, after being dispersed uniformly, the screened testosterone undecanoate bulk drug is added, and the mixture is fully and uniformly dispersed again (grade A, 2min) under a dispersion emulsification homogenizer (Shanghai Hengchuan mechanical equipment Co., Ltd., C25). Pouring the uniformly dispersed suspension into a sample cup of a high-pressure homogenizer with a condensing device, circulating for several times at low pressure of 200-300 bar, pressurizing to 500bar 10 after no obvious impact sound is generated between the ceramic beads and the valve seat, and continuing to 800bar 10 and 1100bar 10.

Then adding proper bacteriostatic agent benzalkonium chloride and proper amount of sodium carbonate into each product to adjust the pH value to 7.35-7.45.

Samples 4-8 were prepared.

Preparation examples 9 to 11: preparation of lyophilized powder of Testosterone undecanoate (lyophilized samples 1-3)

Respectively taking 2ml of each of the samples 1-3 prepared in the preparation examples 1-3, placing the samples in penicillin bottles, adding mannitol with 20% of solid content, uniformly mixing, pre-freezing at-60 ℃, slowly heating at-40 ℃, subliming under vacuum condition, and finally heating and subliming under vacuum condition of +25 ℃ to obtain testosterone undecanoate freeze-dried powder, namely freeze-dried samples 1-3. The specification was 0.25 g/bottle.

Comparative preparation examples A-B: preparation of Testosterone undecanoate suspension (control samples A-B)

Control sample a recipe composition: 12.5g of testosterone undecanoate, 1g of tween 20 and 0.5g of docusate sodium.

Control sample B recipe composition: 12.5g of testosterone undecanoate, 2g of polyethylene glycol 4000 and 1g of docusate sodium.

The above two formulations were the same as those of the previous preparation 4 and preparation 5, respectively.

The preparation method comprises the following steps:

under the condition of room temperature, two stabilizers in the corresponding formula compositions are respectively taken and sequentially added into water to be stirred, after the two stabilizers are uniformly dispersed, the screened testosterone undecanoate bulk drug is added, and the mixture is fully and uniformly dispersed again (grade A, 2min) under a dispersion emulsification homogenizer (Shanghai Hengchuan mechanical equipment Co., Ltd., C25). Pouring the uniformly dispersed suspension into a sample cup of a high-pressure homogenizer with a condensing device, circulating for a plurality of times under the low pressure of 200-300 bar, and gradually pressurizing after the ceramic beads and the valve seat have no obvious impact sound.

Comparative preparation a pressurization process: gradually pressurizing to 500bar 10, 800bar 10, 1000bar 10.

Comparative preparation B pressurization process: gradually pressurizing to 500bar 10, 800bar 10, 1000bar 10, 1200bar 10.

Then adding a proper amount of benzalkonium chloride as a bacteriostatic agent and a proper amount of sodium carbonate into each sample respectively to adjust the pH value to be between 7.35 and 7.45.

Control samples A-B were obtained.

Example 1: particle size distribution measurement experiment

1. Laboratory sample and instrument

Preparation example 2the testosterone undecanoate suspension (sample 2) obtained.

Testosterone undecanoate lyophilized powder prepared in preparation example 10 (lyophilized sample 2).

Testosterone undecanoate suspensions from control preparations A-B (control samples A-B)

Malvern 2000 laser granulometer.

2. Experimental methods

Cleaning an instrument, adding testosterone undecanoate suspension into dispersion medium pure water to enable the light shading rate to reach 5% -20%, and then measuring the particle size by using a Malvern 2000 laser particle sizer.

Taking a bottle of testosterone undecanoate freeze-dried powder product with the specification of 0.25 g/bottle, adding 2ml of pure water for redissolving, performing ultrasonic treatment to uniformly disperse, and measuring the particle size by using a Malvern 2000 laser particle size analyzer.

3. Results of the experiment

As shown in table 1 below.

Table 1: results of particle size distribution

Sample name	D10(μm)	D50(μm)	D90(μm)
				Sample 2	0.260	1.150	3.051
Lyophilized sample 2	0.456	1.268	3.343
				Control sample A	0.563	1.895	4.674
Control sample B	0.132	0.981	2.241

As can be seen from table 1:

the cumulative particle size distribution in the suspension of sample 2 to 10% corresponds to a particle size of 0.260. mu.m. The cumulative particle size distribution in the suspension is 50% corresponding to a particle size of 1.150. mu.m. The cumulative particle size distribution in the suspension reached 90% corresponding to a particle size of 3.051 μm.

After freeze-drying and redissolving, only D10 of the suspension of the sample 2 is increased a little, and D50 and D90 values are not increased obviously. The stability of the suspension is better.

The D10 of control sample A was greater than 0.5 μm and the D10 of control sample B was less than 0.2 μm.

Example 2: observation by scanning electron microscope

1. Laboratory sample and instrument

Samples 1 to 3 obtained in production examples 1 to 3.

Testosterone undecanoate as a raw material (Zhejiang Xianju pharmaceutical Co., Ltd., lot: 20160301).

Scanning electron microscope (Hitachi S4800) with cold field emission.

2. Experimental methods

Each sample was diluted to an appropriate concentration, dropped on a tin foil paper, naturally dried, and then placed on a sample holder, sputtered with a 20 × 80 gold coating, and set at 20 kv.

3. Results of the experiment

As shown in fig. 1A-1D.

As can be seen from the figure, the particle size distribution of the sample prepared by the invention is relatively uniform, and the particle size is consistent with the measured value of the instrument.

Example 3: x-ray diffraction experiments

1. Laboratory sample and instrument

Samples 1 to 3 obtained in production examples 1 to 3.

Testosterone undecanoate as a raw material (Zhejiang Xianju pharmaceutical Co., Ltd., lot: 20160301).

D/MAX2000 rotary target anode X-ray diffractometer, Rigaku, Japan.

2. Experimental methods

Diluting each sample to a proper concentration, dripping the diluted sample on tin foil paper, naturally drying the tin foil paper, then placing the sample on a sample rack of an instrument, and automatically measuring the sample.

3. Results of the experiment

As shown in fig. 2.

The results show that the diffraction angle positions and the number of characteristic absorption peaks of suspensions with various particle sizes and bulk drugs are not changed, so that the crystal forms of the suspensions are not changed.

Example 4: stability test (1)

1. Experimental Material

Sample preparation: testosterone undecanoate suspension prepared in preparation example 2 (sample 2), purified water.

The instrument comprises the following steps: malvern 2000 laser granulometer.

2. Experimental methods

Preparation example 2the suspension of testosterone undecanoate was left at room temperature for 0, 30, 60, 90, 120, 150 and 180 days, respectively, and then the particle size was measured using a malvern 2000 laser particle sizer.

3. Results of the experiment

As shown in table 2 below.

Table 2: experimental data on stability of testosterone undecanoate suspension

The results show that the particle size is kept stable, the prescription is better, and the used stabilizer is more applicable.

Example 5: stability test (2)

1. Experimental Material

Sample preparation: preparation examples 4-8 prepared testosterone undecanoate suspensions samples 4-8.

The instrument comprises the following steps: malvern 2000 laser granulometer.

2. Experimental methods

Preparation examples 4-8 the testosterone undecanoate suspensions prepared were allowed to stand at room temperature for 0, 1, 3, 5, and 10 days, respectively, and then the particle size was measured using a malvern 2000 laser particle sizer.

3. Results of the experiment

As shown in table 3 below.

Table 3: stability test data for samples 4-8

The data in table 3 show that for sample 4, 5 and 10 days, 5 days, 6, 7, and 8 days (indicated in italics), significant growth, agglomeration, etc. of the measured particle size has occurred, i.e., the suspension is unstable, and the selection of stabilizer is not suitable.

Because the stability of the nanocrystal suspension is a main obstacle to popularization and application, the stability of the suspension is mainly investigated by taking the particle size change as an index in the prescription screening process.

In addition, comparing the particle size of samples 4 and 5 at day 0 with control sample a and control sample B of the same formulation as in example 1 above, it was found that the impact of the manufacturing process on the particle size, particularly D10, was greater. When the last cycle of homogenization is at a lower pressure, for example 1000bar x 10, the value of D10 is higher, possibly higher than 0.5 μm; when the last cycle of homogenization is at a higher pressure, for example 1200bar 10, the value of D10 is lower, possibly below 0.2 μm.

Example 6: muscle irritation test

1. Experimental samples and experimental animals

The experimental samples are shown in table 3 below. Wherein, the testosterone undecanoate oil injection sold in the market is purchased from Zhejiang Xianju pharmaceutical Co., Ltd, and the specification is as follows: 2 ml: 250 mg.

Male rabbits, New Zealand rabbits, weighing 2.5-3.0 kg, 7 rabbits, purchased from Beijing Jinmuyang laboratory animals Breeding, Inc.

2. Experimental groups and dosing regimens

As shown in table 4 below.

Table 4: experimental grouping and dosing

Note: in table 3, the right side refers to the right quadriceps femoris muscle, and the left side refers to the left quadriceps femoris muscle.

3. Experimental methods

Sterilizing the right hind limb of each rabbit with strong iodine, injecting 2mL of testosterone undecanoate suspension into the quadriceps femoris muscle, injecting 1cm of testosterone undecanoate suspension into the central part of the quadriceps femoris muscle of the rabbit, injecting slowly, taking 10s for 2mL of testosterone suspension, and rapidly withdrawing the testosterone suspension after injection. The left corresponding site was treated with an equal volume of sterile saline as a control. At 48 hours after administration, each group of animals was anesthetized with sodium pentobarbital, then sacrificed by bleeding via the femoral artery, and visually observed and recorded for the presence of redness, swelling, bleeding, degeneration or necrosis at the injection site. Further histopathological examination of the injection site was also performed. The materials are taken, dehydrated, embedded, sliced and subjected to HE staining, and the histopathological examination and observation conditions are carried out under a microscope after the slices are prepared. The muscle stimulation response grading was performed with reference to the muscle stimulation response grading criteria shown in the following table 5.

Table 5: grading standard of muscle stimulation response

4. Results of the experiment

As shown in fig. 3A-3N.

After the injection of the crystal suspension liquid with each grain size of testosterone undecanoate is given to the rabbits, no relevant change in the weight and the weight increment of the animals is seen; visual inspection during dissection revealed that the testosterone undecanoate oil solution formulation resulted in a small amount of hyperemia at the injection site, while there was no significant change at the site of intramuscular injection of testosterone undecanoate crystal suspensions of various particle sizes. The examination under the microscope shows that the contrast of the crystal suspension of testosterone undecanoate in each grain size and the blank auxiliary material solution thereof mainly takes a small amount of inflammatory cell exudation as a main part, and the muscle stimulation response level is 0 level; the testosterone undecanoate oil solution as a commercial medicine is mainly characterized by fibrous connective tissue hyperplasia, vacuole degeneration and a small amount of inflammatory infiltration, and the muscle stimulation response grade is 1 grade.

The above experimental results show that compared with testosterone undecanoate oil solution, testosterone undecanoate has slightly less muscle irritation in crystal suspensions of different particle size specifications. There was no significant difference in irritation between the suspensions of different D50.

Example 7: in vivo pharmacokinetics research of testosterone undecanoate crystal suspensions with different particle sizes

1. Experimental Material

Test samples:

examples 1 to 3 samples 1 to 3 were prepared.

Testosterone undecanoate injection (from Zhejiang Xianju pharmaceutical Co., Ltd., specification: 2 ml: 250mg) is commercially available.

Experimental animals: provided by the experimental animal center of the military medical science institute, the animal experiment qualification number: SCXK 2007 & 004. Female SD rats weighing 180-220 g, 20 in total, were randomly divided into four groups.

2. Dosing regimens

Injecting the rat intramuscularly with the dosage of 26mg/kg, and adjusting the osmotic pressure of the sample to 280 mmol/L-320 mmol/L before administration. The sampling time points are 0min, 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, 60h, 72h, 96h, 120h, 144h and 168h, about 0.3ml of blood is taken from the orbit every 48h after 169h, and the blood taking period is about 20 days in total. Taking 0.2-0.3 ml of blood each time, placing in heparinized EP tube, centrifuging at 14000rpm for 10min, taking supernatant plasma, freezing in a refrigerator at-80 deg.C, and testing.

3. Method for processing plasma samples

Precisely sucking 100 μ L of plasma, adding into 1.5ml of EP tube, adding 100 μ L of methanol and 100 μ L of L-phencynonate hydrochloride internal standard solution (2ng/ml) into the EP tube, finally adding 100 μ L of methanol, vortexing for 1min, centrifuging at 14000rpm for 10min, taking supernatant into a sample injection cannula, and performing sample injection LC-MS/MS analysis.

4. Method for measuring plasma sample

Adopting a liquid chromatography-mass spectrometry method, wherein the internal standard is levorotatory phencynonate hydrochloride, and the mobile phase is as follows: a (containing 0.05% formic acid and 5mM ammonium formate) -B (methanol) at a flow rate of 0.45ml/min, a column temperature of 40 ℃ and a sample size of 20. mu.L.

The elution conditions are shown in Table 6 below.

Table 6: elution conditions

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	90	10
1.8	10	90
			2.5	5	95
2.8	90	10
			5	90	10

5. Results of the experiment

When a suspension of testosterone undecanoate is injected intramuscularly, testosterone esters are gradually released from the injection site into the blood circulation and are broken down into testosterone and free fatty acids by serum esterases. After intramuscular injection administration in rats, the blood concentration was measured at each time point, and the testosterone concentration of each testosterone undecanoate crystal suspension and testosterone undecanoate oil injection in vivo as a function of time is shown in fig. 4 and fig. 5.

In vivo experiment results show that the testosterone undecanoate crystal suspension has a long-acting slow-release effect.

Tmax, Cmax, AUC And MRT were calculated by Drug metabolism calculation software DAS2.0(Drug And Statistics). Tmax means the time required for the drug to reach the maximum concentration, and determines the speed of the drug effect or adverse reaction generated by the drug. Cmax means the concentration of drug that peaks in the body and determines whether the drug is effective or has adverse effects. AUC (0- ∞), which represents the area under the time curve, represents the relative amount of drug entering the systemic circulation, reflecting the magnitude of drug absorption. MRT is the mean residence time, a parameter indicative of the elimination process of a drug, and as with clearance, the MRT value is independent of the dosage.

As shown in table 7 below.

Table 7: DAS2.0 statistical results

The results show that:

(1)300nm suspension released faster in vivo, higher Cmax values and shorter in vivo retention time.

(2) Comparing the oil solution group with the other three groups, the AUC value of the oil solution group is lower than that of the other three groups, and the blood concentration of the oil solution group is close to 0ng/ml, which indicates that the oil is remained in the muscle and can not be completely released, and the bioavailability is slightly lower than that of the suspension group.

(3) The 4.8 mu m suspension has larger particle size, and is prepared into proper particle size distribution by improving the process, but the blood concentration of the suspension has multimodal phenomenon, probably because the blood concentration fluctuates because the particle size distribution in the suspension is wider, and simultaneously, the blood concentration of the preparation is lower and can not reach effective treatment concentration.

Example 8: in vitro Release test 1

The aim of this experiment was to control the particle size range of the suspension by the cumulative release of the in vitro release test.

1. Laboratory sample and instrument

Preparation example 2the testosterone undecanoate suspension (sample 2) obtained.

Model ZRS-8G Intelligent dissolution tester (Tianjin Xinzhou technical Co.).

2. Experimental methods

The release rate is measured by taking 125mg/ml testosterone undecanoate nano suspension, taking PH 7.4 phosphate buffer salt and 0.5% sodium dodecyl sulfate as release medium 900ml according to the 2015 version Chinese pharmacopoeia dissolution determination slurry method, the medium temperature is 37 +/-0.5 ℃, the rotating speed is 25 revolutions per minute, and 2ml uniform suspension samples are added into each dissolution cup.

The preparation method of phosphate buffer salt with pH 7.4 comprises the following steps: taking 1.36g of monopotassium phosphate, taking 79ml of 0.1mol/l sodium hydroxide solution, and diluting the solution to 200ml by using water to obtain the potassium phosphate.

Sample adding mode: or mixing about 2ml of injection in advance, placing into 2ml syringe with needle, precisely weighing, adding the suspension into each dissolution cup when the paddle rotates, precisely weighing empty syringe with needle, drug-containing syringe and residual liquid after administration to obtain actual dosage.

Sampling time: at 2min, 30min and 120 min, respectively taking 5ml of release medium, immediately filtering with 0.22 μm filter membrane, discarding at least 3ml of initial filtrate, and taking 1.5ml of subsequent filtrate as sample solution;

if the release at 2 minutes, 30 minutes and 120 minutes per cup should be 10-20%, 45-60% and 65-90%, respectively, this is considered satisfactory. The release medium does not need to be supplemented in the operation container during the test process, and the volume of the release medium does not need to be corrected in the calculation formula.

Measuring the content of testosterone undecanoate by adopting a high performance liquid chromatography:

mobile phase: acetonitrile: isopropyl alcohol: 20 parts of water: 70: 10

Sample introduction amount: 20 μ L

Detection wavelength: 240nm

Column temperature: 40 deg.C

Flow rate: 1.0ml/min

3. Results of the experiment

As shown in table 8 below.

Table 8: cumulative amount of Release (%)

Time (minutes)	Cumulative amount of Release (%)
		0	0
2	15.2
		5	25.9
15	35.2
		30	49.1
60	65.3
		120	81.4

The results show that the cumulative release of this product per cup at 2min, 30min and 120 min should be 15.2%, 49.1% and 81.4%, respectively.

The results show that the particle size distribution and the release of the suspension prepared in preparation example 2 are both ideal and meet the set requirements.

Example 9: in vitro Release test

1. Laboratory sample and instrument

Control sample A and control sample B prepared in previous control preparations A-B.

Model ZRS-8G Intelligent dissolution tester (Tianjin Xinzhou technical Co.).

2. Experimental methods

As in previous example 8.

3. Results of the experiment

As shown in tables 9-10 below.

Table 9: cumulative Release amount (%)

Table 10: cumulative Release amount (%)

Time (minutes)	Cumulative amount of Release (%)
		0	0
2	26.8
		5	35.3
15	43.1
		30	53.4
60	67.3
		120	85.6

From the above experimental results, it can be seen that when D10 of the control sample a is greater than 0.5 μm, the cumulative release amount at each sampling time point is reduced, the release amount at 2 minutes is less than 10%, the release amount is less, and the in vitro release quality standard is not reached to 10-20%. If the D10 of the control sample B is less than 0.2 μm, the overall particle size in the formulation is small, a burst effect is produced, and the drug release is excessive, with over 20% being released in 2 minutes to 26.8%, exceeding the in vitro release quality standard by 10-20%.

The results show that D10 above 0.5 μm is less effective in immediate release and does not achieve a more rapid release to a therapeutically effective concentration after administration. If the D10 is less than 0.2 μm, the particle size in the preparation is small overall, a burst effect is generated, the drug is released too much, the release exceeds 20% in 2 minutes, the ultra-effective treatment concentration can be achieved, and the maintaining time is shortened.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (24)

1. A testosterone undecanoate suspension comprising testosterone undecanoate, a stabilizer, and water, wherein:

the content of testosterone undecanoate is 10-50% (g/ml);

the particle size of the testosterone undecanoate meets the following requirements: d10 is 0.2-0.5 μm, D50 is 1.0-1.3 μm, and D90 is 2.2-6.0 μm;

the stabilizer comprises a first stabilizer and a second stabilizer, and the weight ratio of the testosterone undecanoate to the stabilizer is (1-20): (1-20) wherein, in the formula,

the first stabilizing agent is soybean lecithin,

the second stabilizer is docusate sodium; and is

The weight ratio of the first stabilizer to the second stabilizer is (1-10): (1-20).

2. Testosterone undecanoate suspension according to claim 1, characterized by any one or more of the following items (1) to (5):

(1) the content of testosterone undecanoate is 10-30% (g/ml);

(2) the D10 is 0.2-0.3 μm;

(3) the weight ratio of the testosterone undecanoate to the stabilizer is (1-15): (1-15);

(4) the weight ratio of the first stabilizer to the second stabilizer is (1-5): (1-20);

(5) the testosterone undecanoate suspension further comprises a bacteriostatic agent and/or a pH adjusting agent.

3. Testosterone undecanoate suspension according to claim 2, wherein in item (2) said D10 is 0.2-0.25 μ ι η.

4. Testosterone undecanoate suspension according to claim 2, wherein in item (3) the weight ratio of testosterone undecanoate to stabilizer is (1-15): (1-10).

5. Testosterone undecanoate suspension according to claim 2, wherein in item (3) the weight ratio of testosterone undecanoate to stabilizer is (1-10): (1-3).

6. Testosterone undecanoate suspension according to claim 2, item (4), wherein the weight ratio of the first stabilizer to the second stabilizer is (1-5): (1-10).

7. Testosterone undecanoate suspension according to claim 2, item (4), wherein the weight ratio of the first stabilizer to the second stabilizer is (1-2): (1-5).

8. Testosterone undecanoate suspension according to claim 1 or 2, comprising the following components in the ratio:

9. testosterone undecanoate suspension according to claim 1 or 2, comprising the following components in the ratio:

10. a lyophilized formulation of testosterone undecanoate made by lyophilizing a testosterone undecanoate suspension of any one of claims 1-9.

11. The lyophilized formulation of testosterone undecanoate according to claim 10, wherein the lyoprotectant is selected from one or more of glucose, mannitol, sucrose, lactose.

12. The lyophilized formulation of testosterone undecanoate according to claim 11, wherein the lyoprotectant is present in an amount of 10% to 40% of solid content.

13. The lyophilized formulation of testosterone undecanoate according to claim 11, wherein the lyoprotectant is present in an amount of 10% to 30% of the solid content.

14. The lyophilized formulation of testosterone undecanoate according to claim 11, wherein the lyoprotectant is present in an amount of 15% to 25% of solid content.

15. A method of preparing a testosterone undecanoate suspension of any one of claims 1-9, comprising the steps of:

(1) uniformly mixing a first stabilizer, a second stabilizer and water to obtain a first mixture;

(2) adding testosterone undecanoate to the first mixture in step (1) to obtain a second mixture;

(3) uniformly dispersing the second mixture;

(4) homogenizing the product of the step (3) by a high-pressure homogenizer.

16. The method according to claim 15, wherein in the step (3), the second mixture is dispersed uniformly by a dispersion emulsification homogenizer.

17. The method of claim 15, wherein the homogenizing in step (4) comprises the steps of:

(4-1) at least 5 cycles at 200bar to 300 bar;

(4-2) 5-15 cycles at 400 bar-600 bar;

(4-3) 5-15 cycles at 700-900 bar;

(4-4)1050 bar-1150 bar, 5-15 cycles.

18. The method according to claim 17, wherein, in the step (4-1), 5 to 15 cycles are carried out at 200 to 300 bar.

19. The production method according to claim 17, wherein in the step (4-1), the cycle is performed at 200bar to 300bar until no striking sound is made from the ceramic beads to the valve seat.

20. The method of any one of claims 15 to 19, wherein the homogenizing in step (4) comprises the steps of:

(4-1) 8-12 cycles at 200 bar-300 bar;

(4-2) 8-12 cycles at 450-550 bar;

(4-3) 8-12 cycles at 750 bar-850 bar;

(4-4)1050 bar-1150 bar, 8-12 cycles.

21. The method of any one of claims 15 to 19, wherein the homogenizing in step (4) comprises the steps of:

(4-1) 10 cycles at 200 bar-300 bar;

(4-2) 10 cycles at 500 bar;

(4-3) 10 cycles at 800 bar;

(4-4)1100bar, 10 cycles.

22. Use of a testosterone undecanoate suspension according to any one of claims 1 to 9 or a testosterone undecanoate lyophilized formulation according to any one of claims 10 to 14 for the manufacture of a medicament for the treatment or prevention of a disease caused by insufficient endogenous testosterone production.

23. The use according to claim 22, wherein the disorder resulting from insufficient endogenous testosterone production is male hypogonadism.

24. The use of claim 23, wherein the male hypogonadism is selected from at least one of male sexual dysfunction, decreased muscle mass and strength in men, depressed mood in men, and male osteoporosis.

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