A testosterone-cholesterol prodrug and its preparation method and application

Abstract

The invention discloses a testosterone-cholesterol prodrug and a preparation method and application, belonging to the field of medical technology. The present invention improves the fat solubility of testosterone by cholesterol modification of testosterone, and prepares a testosterone-cholesterol prodrug suspension of different particle sizes using wet grinding technology. It has a good sustained-release effect, and the sustained-release time is at least 40 days. Compared with testosterone undecanoate oil solution (prepared according to commercially available preparations), it does not cause lung damage, and provides new strategies and options for the development of long-acting testosterone ester injection.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a testosterone-cholesterol prodrug, a preparation method and an application thereof, and in particular to an injectable testosterone-cholesterol prodrug suspension, a preparation method thereof and an application thereof in a sustained-release drug delivery system. Background Art

[0002] Testosterone deficiency syndrome is a common clinical and biochemical disease that affects approximately 2-5.7% of men over 40 years old. The symptoms of testosterone deficiency worsen with age, affecting multiple organs and systems, manifesting as metabolic dysfunction, cognitive impairment, osteoporosis, anemia, and sexual dysfunction. Patients are prone to fatigue, mood swings, obesity, and an increased incidence of diabetes and Alzheimer's disease, which has a negative impact on their quality of life.

[0003] Testosterone replacement therapy is the most effective option so far. A variety of testosterone-related preparations have entered the market, such as Andriol (testosterone undecanoate oral tablets), 80 mg / tablet, 2-3 times a day, with the limitation that it needs to be taken with food to promote lymphatic absorption; Androgel (testosterone gel), 50-100 mg / d, its limitations are: (1) after the patient uses the gel, it is easy for others to come into contact with it, resulting in drug loss; (2) long-term use may cause local irritation; Striant TM (testosterone oral patch), specification 30mg / time, twice a day; Natesto (nasal gel), specification 11mg / time, three times a day, the limitation is that it causes irritation and gingivitis after use. In order to prolong the duration of drug action, a variety of testosterone sustained-release designs have been developed for the treatment of testosterone deficiency syndrome, for example, Androderm (testosterone transdermal patch), specification 5mg / tablet, twice a week, the limitation is that it may cause skin blisters, itching or irritation after use; Testopel (testosterone implant), specification 3-4 tablets / 4-6 months, has certain limitations, and may cause site infection, bleeding and implant site fibrosis; and related drugs esterified at the β-hydroxyl position of the 17th carbon atom of testosterone, such as Delastestryl (testosterone enanthate injection), specification 250mg / 2-4 weeks; Aveed (testosterone undecanoate injection oil solution), specification 750mg / 3mL, the limitations are: (1) there is a risk of causing oil microemboli in the patient's lungs; (2) causing pain and inflammation at the injection site. Based on the above limitations, how to develop a long-acting sustained-release delivery formulation of testosterone drugs with a long sustained-release time, convenient administration, fewer side effects, and greater safety is a problem that urgently needs to be solved.

[0004] Cholesterol is the most abundant steroid compound in mammals. It is a component of cell membranes and, as a steroidal compound, serves as a precursor to numerous steroid hormones, such as glucose, vitamin D, sex hormones, glucocorticoids, and testosterone. Cholesterol is insoluble in water. Studies have shown that modifying drugs with cholesterol can increase their lipid solubility and improve safety. Currently, there are no reports on cholesterol-modified testosterone to produce a less lipid-soluble prodrug. Summary of the Invention

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a testosterone-cholesterol prodrug, a preparation method and an application thereof, which solve the technical problems of the limited variety of existing long-acting testosterone preparations, the side effects caused by their use and the limitations during their use.

[0006] The present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a testosterone-cholesterol prodrug for treating testosterone deficiency syndrome, comprising a compound having a structure shown in formula (I):

[0008]

[0009] In a second aspect, the present invention provides a method for preparing a testosterone-cholesterol prodrug, comprising the following steps: under nitrogen protection, cholesterol chloroformate and testosterone are reacted under the catalytic action of DMAP and triethylamine, after the reaction is completed, the solvent is removed by reduced pressure distillation to obtain a solid 1; and the solid 1 is separated and purified by silica gel column chromatography technology to obtain a testosterone-cholesterol prodrug.

[0010] In a third aspect, the present invention provides a sustained-release pharmaceutical composition in the form of a suspension or a lyophilized powder, comprising a testosterone-cholesterol prodrug, a stabilizer, a salt, and a lyoprotectant; the concentration of the testosterone-cholesterol prodrug is 0.3%-6% (w / v); the weight ratio of the stabilizer to the testosterone-cholesterol prodrug is 1:3-1:100; the concentration of the salt is 1%-30% (w / v); and the concentration of the lyoprotectant is 2%-20% (w / v).

[0011] Furthermore, the stabilizer includes one or more of Tweens, Spans, poloxamers, celluloses, polyamides, and sulfates; the salt includes tartrate or phosphate; and the lyoprotectant includes one or more of sugars, polyols, polymers, amino acids, and inorganic salts.

[0012] Furthermore, the Tweens include Tween 20 (Tween 20), Tween 40 (Tween 40), Tween 60 (Tween 60), and Tween 80 (Tween 80); the Spans include Span 20 (Span 20), Span 40 (Span 40), Span 60 (Span 60), and Span 80 (Span 80); the Poloxamers include Pluronic F68 and Pluronic F127; the fibers include sodium carboxymethyl cellulose (CMC-Na) and hydroxyethyl cellulose (HEC); the polyamides include polyvinyl pyrrolidone (PVP K30); and the sulfates include sodium dodecyl sulfate (SDS).

[0013] Preferably, the stabilizer is poloxamer 407.

[0014] Furthermore, the sugars include glucose, sucrose, lactose, and trehalose; the polyhydroxy compounds include sorbitol and mannitol; the polymers include polyoxyethylene pyrrolidone and gelatin; the amino acids include glycine and tryptophan; and the inorganic salts include sodium chloride and citrate.

[0015] Preferably, the lyoprotectant is mannitol.

[0016] In a fourth aspect, the present invention provides a method for preparing a sustained-release pharmaceutical composition, wherein the method for preparing the suspension includes a media grinding method, a high-pressure homogenization method, an anti-solvent precipitation method or a supercritical fluid method.

[0017] Furthermore, the media grinding method comprises the following steps:

[0018] (1) mixing and dispersing a testosterone-cholesterol prodrug, a stabilizer, a salt, and a lyophilization protectant in an aqueous medium to obtain a suspension;

[0019] (2) Grinding beads are added to the suspension of step (1) for grinding to obtain a nanocrystal / micron crystal suspension with a particle size ranging from 100 nm to 50 μm, which is the sustained-release pharmaceutical composition.

[0020] In a fifth aspect, the present invention provides use of the testosterone-cholesterol prodrug or the sustained-release pharmaceutical composition in the sustained-release treatment of testosterone deficiency syndrome.

[0021] Furthermore, it is used for intramuscular injection or subcutaneous administration.

[0022] The injectable drug suspension of the testosterone-cholesterol prodrug of the present invention has the following advantages: (1) it adopts wet grinding technology, has a simple preparation process, is temperature-controllable, and is easy to scale up; (2) it does not require a carrier, requires a small amount of stabilizer, has a high drug loading capacity, and is safe; (3) the suspension is evenly dispersed and has good stability; (4) it is easy to freeze-dry, and after repeated suspension, it has good redispersibility, which is conducive to long-term storage; (5) it has good in vivo pharmacokinetic behavior, especially the testosterone-cholesterol prodrug nanocrystal suspension with a particle size of 300 nm has the best pharmacokinetic properties, stable blood drug concentration, and long sustained release time; (6) compared with commercially available preparations, the testosterone-cholesterol prodrug suspension does not cause lung inflammation and has great application potential.

[0023] The present invention has the following advantages and beneficial effects:

[0024] The present invention synthesizes lipophilic testosterone-cholesterol prodrug by cholesterol modification to testosterone, and then, prepares long-acting sustained-release nanocrystal / micron crystal suspension without carrier, good stability and high safety by wet grinding technology, after intramuscular injection, forms drug reservoir, produces long-acting sustained-release effect, and sustained-release time reaches at least 40 days, and blood drug concentration is stable, can reduce the number of administrations, improves patient compliance, and compared with testosterone undecanoate oil solution, testosterone-cholesterol prodrug suspension does not cause the adverse effect of pulmonary vascular embolism. Provide new ideas for the development of testosterone ester drugs, also provide strategy and selection for the development of long-acting injection preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The high resolution mass spectrum of the testosterone-cholesterol prodrug of Example 1 (A) and 1 H-NMR spectrum (B).

[0026] Figure 2 The X-ray powder diffraction patterns of the testosterone-cholesterol prodrug of Example 1, testosterone, cholesterol chloroformate, and the physical mixture of testosterone and cholesterol chloroformate.

[0027] Figure 3 The differential scanning calorimetry analysis spectra of testosterone (A) and the testosterone-cholesterol prodrug (B) of Example 1 are shown.

[0028] Figure 4 The figure is a particle size distribution diagram of the testosterone-cholesterol prodrug suspensions of different particle sizes in Example 5; wherein A is testosterone-cholesterol prodrug suspension 1, B is testosterone suspension 1, C is testosterone-cholesterol prodrug suspension 2, D is testosterone suspension 2, E is testosterone-cholesterol prodrug suspension 3, and F is testosterone suspension 3.

[0029] Figure 5The X-ray powder diffraction patterns of the stabilizer poloxamer 407 of Example 5, the physical mixture of the testosterone-cholesterol prodrug suspension lyophilized powder and poloxamer 407, the physical mixture of the testosterone suspension lyophilized powder and poloxamer 407, the testosterone-cholesterol prodrug suspension lyophilized powder of Example 6, and the testosterone suspension lyophilized powder.

[0030] Figure 6 The average plasma concentration of testosterone after intramuscular injection of 16.6 mg testosterone / kg of the testosterone-cholesterol prodrug suspension of different particle sizes of Example 5, testosterone suspension and testosterone undecanoate oil solution (prepared according to the commercial formulation) in rats.

[0031] Figure 7 Pathological sections of rat lung tissue after intramuscular injection of 16.6 mg testosterone / kg of the testosterone-cholesterol prodrug suspension of Example 5, a testosterone suspension, and a testosterone undecanoate oil solution (prepared according to a commercially available formulation) into the rat leg. DETAILED DESCRIPTION

[0032] The following examples are given to further illustrate the present invention, but are not intended to limit the present invention in any way.

[0033] Example 1 Synthesis of Testosterone-Cholesterol Prodrug

[0034] Cholesterol chloroformate (1.868 g, 4.16 mmol) was accurately weighed and dissolved in 10 mL of anhydrous dichloromethane. The mixture was placed in a reaction flask and stirred in an ice bath. DMAP (0.508 g, 4.16 mmol), triethylamine (289.2 μL, 2.08 mmol), and testosterone (0.6 g, 2.08 mmol) were then accurately weighed in sequence and dissolved in 10 mL of dichloromethane. The mixture was slowly added dropwise to the reaction flask and stirred in an ice bath for 2 h. The temperature was raised to room temperature and the reaction was allowed to proceed for 8 h. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column gradient chromatography (light yellow solid, yield 57%).

[0035] High-resolution mass spectrometry (A) and 1 H-NMR spectrum (B) was used to confirm the structure of the target compound. Figure 1The results of NMR analysis are as follows: δ 5.73 (s, 1H, H-1), 5.39 (d, J = 4.5 Hz, 1H, H-2'), 4.56 4.47 (m, 1H, H-2), 4.51 4.39 (m, 1H, H-1'), 1.19 (s, 3H, H-3), 1.01 (s, 3H, H-6'), 0.91 (d, J = 6.5 Hz, 3H, H-3'), 0.86 (dd, J = 6.2, 1.9 Hz, 9H, H-4, 4', 5'), 0.68 (s, 3H, H-7').

[0036] It has been verified that the structural formula of the target compound is as follows:

[0037]

[0038] Example 2 X-ray diffraction spectrum analysis

[0039] Each sample was scanned in the scanning range of 10-90° (2θ), with a step size of 0.05° and a tube voltage of 40 kV.

[0040] The X-ray diffraction patterns of testosterone, cholesterol chloroformate, a physical mixture of testosterone and cholesterol chloroformate, and a crystalline form of a testosterone-cholesterol prodrug were characterized. Figure 2 shown.

[0041] The results show that the physical mixture has strong diffraction peaks at 2θ angles of 7.85°, 8.43°, 12.26°, 14.10°, 14.42°, 14.89°, 17.25°, 17.88°, 19.98°, 23.24°, and 27.55°, which include the characteristic peaks of the raw materials testosterone and cholesterol chloroformate, indicating that the drug crystal form has not changed during the physical mixing of the two; the testosterone-cholesterol prodrug spectrum has obvious diffraction peaks at 2θ angles of 4.91°, 9.79°, 14.05°, 14.84°, 15.47°, 17.09°, 19.04°, and 20.51°, which are different from the positions of the characteristic peaks of the physical mixture, indicating that testosterone and testosterone-cholesterol prodrug are both crystalline drugs.

[0042] Example 3 Differential Scanning Calorimetry Analysis

[0043] 6 mg of testosterone powder and testosterone-cholesterol prodrug powder were weighed into a crucible, covered and pressed tightly, and alumina was used as a reference. Under nitrogen protection, the heating rate was 10℃ / min, and the scanning ranges were 30-210℃ and 30-230℃ respectively. The results are as follows Figure 3 shown.

[0044] Depend on Figure 3It can be seen that testosterone powder has a small endothermic peak at 114.7°C and an obvious endothermic peak at 155.80°C, indicating that testosterone is in a crystalline state; testosterone-cholesterol prodrug powder has an obvious endothermic peak at 225.36°C, indicating that testosterone-cholesterol prodrug powder is in a crystalline state.

[0045] Example 4 Solubility of Testosterone-Cholesterol Prodrug and Testosterone in Different Aqueous Media

[0046] An excess of testosterone API and testosterone-cholesterol prodrug were placed in 10 mL stoppered test tubes, and appropriate amounts of water, normal saline, pH 6.8 or pH 7.4 phosphate buffer solution were added. The stoppered test tubes were placed in a 37°C constant temperature shaker to ensure that the testosterone API and testosterone-cholesterol prodrug reached supersaturation in the above aqueous medium. The mixture was shaken continuously for 72 hours. The supernatant of each solution was filtered through a 0.22 μm microporous membrane, and the concentrations of testosterone and testosterone-cholesterol prodrug in different media were determined by high performance liquid chromatography injection. The results are shown in Table 1, which show that the aqueous solubility of testosterone-cholesterol prodrug is significantly lower than that of testosterone, and a suspension with better sustained-release effect is easy to prepare.

[0047] Table 1 Solubility of testosterone-cholesterol prodrug and testosterone in different media

[0048]

[0049] Example 5 Preparation of Testosterone-Cholesterol Prodrug Nano / Microcrystalline Suspensions of Different Particle Sizes

[0050] Testosterone-cholesterol prodrug and stabilizer are evenly dispersed in distilled water, and grinding beads are added and ground for a certain period of time.

[0051] The compositions and preparation methods of testosterone-cholesterol prodrug suspensions and testosterone suspensions of different particle sizes are shown in Tables 2 and 3, and the particle sizes and particle size distribution results measured by Malvern particle size analyzer and BT-9300s laser particle size distribution analyzer are shown in Tables 4 and 5, and the distribution diagram is shown in Table 5. Figure 4 As shown, the results show that testosterone-cholesterol prodrug suspension and testosterone suspension with uniform particle size and narrow particle size distribution can be prepared by media milling technology, respectively.

[0052] Table 2 Composition and grinding process of testosterone-cholesterol prodrug suspension

[0053]

[0054] Table 3 Composition and grinding process of testosterone suspension

[0055]

[0056] Table 4 Particle size and particle size distribution of testosterone-cholesterol prodrug suspension

[0057]

[0058] Table 5 Particle size and particle size distribution of testosterone suspension

[0059]

[0060] Example 6 Lyophilization of Testosterone-Cholesterol Prodrug Suspension

[0061] 10% (w / v) mannitol was evenly added to the testosterone-cholesterol prodrug suspension for lyophilization. The testosterone-cholesterol prodrug suspension freeze-dried powder had a smooth surface and a loose, round, cake-like structure. Adding distilled water and gently shaking easily dispersed the powder. The particle size and distribution after resuspension were almost unchanged compared to before lyophilization, indicating that the freeze-dried powder of the suspension prepared by this lyophilization technique has excellent properties. The particle size of suspensions of different particle sizes before and after lyophilization is shown in Tables 6 and 7.

[0062] Table 6 Particle size and particle size distribution of testosterone-cholesterol prodrug suspension before and after lyophilization

[0063]

[0064] Table 7 Particle size and particle size distribution of testosterone suspension before and after freeze-drying

[0065]

[0066] Example 7 Powder X-ray Diffraction Spectrum Analysis

[0067] Each sample was scanned in the scanning range of 10-90° (2θ) with a step size of 0.05° and a tube voltage of 40 kV.

[0068] The stabilizer poloxamer 407, the testosterone-cholesterol prodrug suspension 1 lyophilized powder of Example 6, the testosterone suspension 1 lyophilized powder (prepared by replacing the testosterone-cholesterol prodrug suspension of Example 6 with the testosterone suspension, and the remaining steps were the same), a physical mixture of poloxamer 407 and testosterone suspension 1 lyophilized powder (1:1, w:w), and a physical mixture of poloxamer 407 and testosterone-cholesterol prodrug suspension 1 lyophilized powder (1:1, w:w) were characterized by X-ray diffraction patterns. The results are shown as follows: Figure 5 shown.

[0069] Poloxamer 407 exhibits characteristic diffraction peaks at 19.27° and 23.42°. Physical mixtures of poloxamer 407 and testosterone suspension lyophilized powder and physical mixtures of poloxamer 407 and testosterone-cholesterol prodrug suspension lyophilized powder, respectively, contain characteristic peaks for poloxamer 407 and the respective drugs, indicating no interaction between the drugs and stabilizers. The presence of characteristic diffraction peaks for poloxamer 407 and the respective drugs in testosterone suspension lyophilized powder and testosterone-cholesterol prodrug suspension lyophilized powder indicates that the suspensions remain in crystalline form after grinding and lyophilization.

[0070] Example 8 Pharmacokinetic Study of Testosterone-Cholesterol Prodrug Suspension

[0071] Get 35 female healthy rats, body weight 200-220g, be randomly divided into 7 groups, 5 in every group, be injected intramuscularly respectively particle size be 300nm, 12μm, the testosterone-cholesterol prodrug suspension of 20μm, 300nm, 12μm, the testosterone suspension (control) of 20μm and the testosterone undecanoate oil solution prepared according to commercial prescription, equivalent dosage is 16.6mg / kg (by contained testosterone).Carry out rat orbital blood sampling at the prescribed time point, vortex 3min, 13000rpm centrifugal 5min, obtain plasma.Utilize HPLC-MS / MS to measure the concentration of testosterone in plasma, pharmacokinetic curve is as shown in Figure 2. Figure 6 The pharmacokinetic parameters processed by DAS software are shown in Table 8.

[0072] The results showed that the AUC of the testosterone-cholesterol prodrug suspension with a particle size of 300 nm was Tst Equivalent to testosterone undecanoate oil solution; C max It is 120ng / mL, indicating that the sustained-release time of intramuscular injection of 300nm Tst-Chol suspension can reach at least 40 days without causing a large jump in blood drug concentration, and it is highly safe.

[0073] Table 8 Pharmacokinetic parameters of testosterone-cholesterol prodrug suspensions of three particle sizes, testosterone suspensions, and testosterone undecanoate oil solutions (prepared according to commercially available prescriptions)

[0074]

[0075] Example 9 Lung Tissue Pathology Study of Intramuscular Injection of Testosterone-Cholesterol Prodrug Suspension

[0076] SD rats were randomly divided into four groups and injected into the lateral thigh muscle with 0.3 mL each of normal saline, testosterone-cholesterol prodrug suspension, testosterone suspension, and testosterone undecanoate oil solution prepared according to a commercial prescription. Lung tissues were collected from rats in each group on days 2, 7, and 14 after administration, washed with normal saline, and immersed in 4% paraformaldehyde tissue fixative for 48 hours. The fixed lung tissues were embedded, sectioned, stained with hematoxylin-eosin, and observed under a microscope. Figure 7 shown.

[0077] No pathological changes were observed in lung fibrosis after intramuscular injection of normal saline. No significant differences were observed in lung sections compared with the normal saline group after intramuscular injection of testosterone-cholesterol prodrug suspension or testosterone suspension 2, 7, and 14 days later. Pathological changes in the lungs (arrows) were observed 2, 7, and 14 days after intramuscular injection of testosterone undecanoate oil solution, indicating that intramuscular injection of testosterone undecanoate oil solution can cause lung inflammation. In contrast, testosterone-cholesterol prodrug suspension is safer and no adverse reactions to the lungs were found.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sustained-release pharmaceutical composition, characterized in that The invention is in the form of a suspension or a lyophilized powder, and includes a testosterone-cholesterol prodrug, a stabilizer, a salt, and a lyoprotectant; the concentration of the testosterone-cholesterol prodrug is 0.3%-10% (w / v); the weight ratio of the stabilizer to the testosterone-cholesterol prodrug is 1:3-1:100; the concentration of the salt is 1%-30% (w / v); and the concentration of the lyoprotectant is 2%-20% (w / v); The testosterone-cholesterol prodrug is a compound having a structure shown in formula (I): ； The stabilizer is poloxamer; the salt is tartrate or phosphate; and the freeze-drying protective agent is mannitol.

2. The sustained-release pharmaceutical composition according to claim 1, characterized in that The preparation method of the testosterone-cholesterol prodrug comprises the following steps: Under nitrogen protection, cholesterol chloroformate is reacted with testosterone under the catalysis of DMAP and triethylamine. After the reaction is completed, the solvent is removed by reduced pressure distillation, and then the product is separated and purified by silica gel column chromatography to obtain a testosterone-cholesterol prodrug.

3. The sustained-release pharmaceutical composition according to claim 1, characterized in that The poloxamer is poloxamer 188 or poloxamer 407.

4. The sustained-release pharmaceutical composition according to any one of claims 1 to 3, characterized in that The preparation method of the suspension includes a medium grinding method, a high-pressure homogenization method, an anti-solvent precipitation method or a supercritical fluid method.

5. The sustained-release pharmaceutical composition according to claim 4, characterized in that The media grinding method comprises the following steps: (1) mixing and dispersing a testosterone-cholesterol prodrug, a stabilizer, a salt, and a lyophilization protectant in an aqueous medium to obtain a suspension; (2) Grinding beads are added to the suspension of step (1) for grinding to obtain a nanocrystal / micron crystal suspension with a particle size ranging from 100 nm to 50 μm, which is the sustained-release pharmaceutical composition.

6. Use of the sustained-release pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a sustained-release drug for treating testosterone deficiency syndrome.

7. The use according to claim 6, characterized in that For intramuscular injection or subcutaneous administration.

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