WO2024056062A1 - Steroid hormone-phospholipid composition and preparation method therefor - Google Patents

Abstract

The present invention relates to a steroid hormone-phospholipid composition and a preparation method therefor. The composition comprises a steroid hormone and phospholipid, and may further comprise cholesterol. The composition is prepared into lyophilized powder, which is easy to store, good in stability and convenient to use. The preparation after reconstitution can maintain a long-acting stable release effect in vivo for 5-30 days.

Description

A steroid hormone phospholipid composition and its preparation method

This application claims priority from the following patent applications:

A prior application submitted to the State Intellectual Property Office of China on September 16, 2022, with patent application number 202211132233.8 and the invention title "A steroid hormone phospholipid composition and its preparation method";

A prior application submitted to the State Intellectual Property Office of China on August 31, 2023, with the patent application number 202311117793.0 and the invention title "A steroid hormone phospholipid composition and its preparation method";

The entirety of said prior application is incorporated into this application by reference.

Technical field

The invention belongs to the field of pharmaceutical preparations, and specifically relates to a long-acting steroid hormone phospholipid composition suitable for industrialization and a preparation method thereof.

Background technique

Phospholipids are substances with an amphiphilic structure with a hydrophilic head (phosphate group and quaternary ammonium salt) and a hydrophobic tail (long-chain hydrocarbon group), and have good biocompatibility. Cholesterol has a cyclopentane polyhydrophenanthrene core structure and is an important steroid compound in mammals. It not only participates in the formation of cell membranes, but is also an important substance in the synthesis of steroid hormones. Phospholipid bilayers and cholesterol are combined in a certain proportion to form liposomes, also known as lipid microvesicles, which are wrapped by a single amphiphilic bilayer and/or multiple amphiphilic bilayers separated by a series of concentric discontinuous It is composed of an aqueous compartment and has a fluid artificial membrane structure similar to that of a cell membrane. The phospholipid bilayer serves as the skeleton of the membrane. Cholesterol has a high melting point and can adjust the phase transition temperature and fluidity of the membrane. Water-soluble drugs are often contained in aqueous compartments, and lipophilic drugs are contained in the lipid bilayer of liposomes.

Steroid hormones, also known as steroid hormones, have a cyclopentane polyhydrophenanthrene core and can be divided into adrenocortical hormones and sex hormones according to their pharmacological effects. Among them, adrenocortical hormones include corticosterone, 11-dehydrocorticosterone, hydrocortisone and cortisone, which regulate glucose metabolism. They have good anti-inflammatory and anti-allergic effects and are also called steroidal anti-inflammatory drugs; and regulate Water and salt metabolism of 11-deoxycorticosterone, 17-hydroxy-11-deoxycorticosterone and aldehyde corticosterone.

Sex hormones include male hormones and estrogen and progesterone. Male hormones include methyltestosterone, testosterone propionate, and testosterone undecanoate; estrogen and progesterone include progesterone, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, and acetic acid. Chlormadinone, estradiol, estrone, estriol, ethinyl estradiol, estradiol benzoate, estradiol valerate, ethinyl estradiol, tibolone, dienogest, dydrogesterone, etc. . Steroid hormones are widely used clinically, but there are still many unmet clinical needs. Taking the steroidal progestogen progesterone as an example, its physical and chemical properties include low solubility and low permeability, poor water solubility, and low oral bioavailability. Clinically, progesterone is formed by using injectable vegetable oils such as castor oil and soybean oil to dissolve progesterone. When administered with an oil solution, the efficacy can be maintained for one day after a single administration. However, because the oily injection is not easily absorbed, it causes local muscle inflammation after injection, causing strong injection pain. In addition, frequent daily injections lead to obvious peaks and troughs in blood concentration. , many adverse reactions and low patient compliance.

Contents of the invention

In order to solve the above technical problems, in a first aspect, the present invention provides a pharmaceutical composition, which contains a steroid. Body hormones and phospholipids.

According to an embodiment of the present invention, the steroid hormone is one or more of adrenal hormones and sex hormones, preferably selected from sex hormones, preferably, the sex hormones are selected from progesterone, estradiol, dienogest One or more of hormones, dydrogesterone, tibolone, testosterone undecanoate, etc.

According to an embodiment of the present invention, the phospholipids include natural or synthetic phospholipids, the structural characteristics of which are a hydrophilic head composed of substituent groups (ammonium-containing bases or alcohols) connected to phosphoric acid and a hydrophobic tail composed of fatty acid chains. In some embodiments, the phospholipid is selected from one or more of glycerophospholipid and sphingomyelin, and the glycerophospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidyl One or more of glycerol and glycerophosphatidic acid. Examples of phospholipids suitable for use in the present invention include dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dierseroylphosphatidylcholine , egg yolk phosphatidylcholine, dilauroylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine One of ethanolamine, dioleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylserine, dimyristoylphosphatidylserine, distearoylphosphatidylserine One or more; preferably, the phospholipid is selected from dimyristoylphosphatidylcholine.

According to an embodiment of the present invention, the molar ratio of the phospholipid to the steroid hormone is 1:1000-1000:1, preferably 1:100-100:1, more preferably 10:1 to 1:10, For example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

According to embodiments of the present invention, the pharmaceutical composition may further comprise cholesterol.

According to an embodiment of the present invention, the molar ratio of the phospholipid to cholesterol is 10:1 to 1:10, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10, preferably, 4:1 to 1:1.

According to an embodiment of the present invention, the pharmaceutical composition may further comprise a solvent, the solvent is selected from one or a combination of organic solvents and/or water. Preferably, the organic solvent is selected from protic solvents, such as alcohols. kind.

According to an embodiment of the present invention, the protic solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, benzyl alcohol, formic acid, and acetic acid. kind.

According to an embodiment of the present invention, the alcohols are selected from one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, and benzyl alcohol.

In some embodiments, the solvent is selected from a mixed solution of a protic solvent and water (ie, an aqueous solution of a protic solvent). Preferably, the solvent is an aqueous solution of tert-butanol; preferably, the content of the protic solvent in the aqueous solution of the protic solvent is ≥50% (v/v), such as 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% (v/v), more preferably, in the aqueous solution of the protic solvent, the content of the protic solvent is ≥ 60%(v/v).

According to an embodiment of the present invention, the mass volume ratio of the steroid hormone to the solvent is 30-100g/L, for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100g/L.

According to an embodiment of the present invention, the composition contains 10-100 parts by weight of the steroid hormone, preferably 10-70 parts of the steroid hormone, for example, 10 ,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 , 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 parts; the composition, in parts by weight, contains all 5-300 parts of the phospholipid, for example, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 copies.

According to an embodiment of the present invention, when the composition further contains cholesterol, the composition contains 1-50 parts by weight of cholesterol, for example, 1, 2, 3, 4, 5, 6, 7, 8 ,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33 , 34, 35, 36, 37, 38, 39, 40, 45, 50 copies.

In some embodiments, the pharmaceutical composition includes progesterone, cholesterol and phospholipid, the phospholipid is selected from DMPC, and the molar ratio of the phospholipid to cholesterol is 5:1 to 1:1; preferably, the pharmaceutical combination The material further contains tert-butanol or a tert-butanol aqueous solution, and in the tert-butanol aqueous solution, the content of tert-butanol is ≥60% (v/v).

In some embodiments, the pharmaceutical composition includes, in parts by weight, 10-70 parts of progesterone; 1-30 parts of cholesterol; and 8-100 parts of phospholipids.

In some embodiments, the pharmaceutical composition includes estradiol, cholesterol and a phospholipid, the phospholipid is selected from DMPC, and the molar ratio of the phospholipid to cholesterol is 5:1 to 1:1; preferably, the drug The composition further includes tert-butanol or a tert-butanol aqueous solution, and the content of tert-butanol in the tert-butanol aqueous solution is ≥60% (v/v).

In some embodiments, the pharmaceutical composition contains 10-70 parts by weight of estradiol; 1-30 parts of cholesterol; and 8-100 parts of phospholipids.

In some embodiments, the pharmaceutical composition comprises dienogest, cholesterol and a phospholipid, the phospholipid is selected from DMPC, and the molar ratio of the phospholipid to cholesterol is 5:1 to 1:1; preferably, the The pharmaceutical composition further includes tert-butanol or a tert-butanol aqueous solution, and the content of tert-butanol in the tert-butanol aqueous solution is ≥60% (v/v).

In some embodiments, the pharmaceutical composition contains 10-70 parts by weight of dienogest; 1-50 parts of cholesterol; and 30-150 parts of phospholipids.

In some embodiments, the composition is a solid composition, which may be a lyophilized powder. In some embodiments, the present invention provides a solid composition obtained by mixing and drying the steroid hormone, phospholipid, and solvent. In other embodiments, the present invention provides a solid composition obtained by mixing and drying the steroid hormone, cholesterol, phospholipid, and solvent.

According to an embodiment of the present invention, the drying includes air drying, rotary evaporation, freeze drying, spray drying or freeze spray drying, preferably freeze drying.

According to an embodiment of the present invention, the solid composition can be reconstituted using one or more of a sterile aqueous solvent such as water for injection, physiological saline, PBS buffer, amino acid buffer, and the like.

In another aspect, the present invention provides a preparation method of the composition, comprising the following steps:

(i) Mix the steroid hormone, phospholipid, optionally added cholesterol and a solvent to obtain a solution;

In some embodiments, in step (i), the steroid hormone, phospholipid and solvent are mixed to obtain a solution; in some embodiments, in step (i), the steroid hormone is mixed with a solvent. , phospholipid, cholesterol and a solvent are mixed to obtain a solution; preferably, the steroid hormone and the cholesterol can be dissolved in the solvent first, and then the phospholipid is added for dissolution; the steroid hormone and the phospholipid can also be directly dissolved in the solvent; Steroid hormones, cholesterol and the phospholipids can also be directly dissolved in a solvent; the mixing/dissolving process can be carried out under mixing conditions such as stirring, ultrasonic, vortexing, etc., preferably with heating and stirring in a water bath; the mixing/dissolving process can be carried out in a At a suitable temperature, for example, 10°C-100°C, preferably 20°C-85°C, more preferably, 35-55°C; the stirring speed is 10rpm-20000rpm, preferably 100rpm-12000rpm.

The preparation method also includes the following steps:

(ii) preparing the solution into a solid composition.

According to an embodiment of the present invention, in the step (ii), the method for preparing the solid composition includes air drying, rotary evaporation, freeze drying, spray drying or freeze spray drying, preferably freeze drying.

According to an embodiment of the present invention, in the freeze-drying, the pre-freezing conditions maintain the plate temperature at preferably -20 to -50°C, and the holding time is between 10 min and 360 min. Sublimation drying conditions preferably have a vacuum degree of 0.01-10mbar, a plate temperature of -1 to -50°C, and a holding time of 30min-1500min. Analytical drying conditions preferably include a vacuum degree of 0.01-1mbar, a plate temperature of 0 to 50°C, and a holding time of 30-1500 minutes.

According to an embodiment of the present invention, the solid composition prepared in the present invention can be reconstituted to obtain a liquid, and the redissolution is: the solid composition is formulated into a suspension in an aqueous dispersion medium. The aqueous dispersion medium is selected from one or more of water for injection, physiological saline, PBS buffer, amino acid buffer, etc.; the mass concentration of the suspension is 0.01g/ml-10g/ml; the PBS Or the pH range of the amino acid buffer is 5.5-8.5, preferably 6.0-8.0, and the mass concentration is 0.001M-10M, preferably 0.01M-1M.

In another aspect, the solid composition obtained by the preparation method of the present invention.

According to an embodiment of the present invention, the present invention also provides a preparation, which is obtained by reconstituting the composition of the present invention (for example, the solid composition obtained according to the preparation method) in an aqueous dispersion medium.

According to an embodiment of the present invention, the particle size of the particles in the suspension obtained after the solid composition is reconstituted is:

D ₁₀ is 1-15 μm, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μm;

D ₅₀ is 5-50 μm; for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μm.

D ₉₀ is 20-150μm, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 , 125, 130, 135, 140, 145, 150μm.

According to the embodiment of the present invention, the preparation can be administered locally, preferably by subcutaneous injection and intramuscular injection.

According to embodiments of the present invention, the formulation can release the active ingredients in a long-lasting manner after administration. In some embodiments, it can be maintained in the body for 5-30 days (for example, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 30 days) active ingredient release effect.

beneficial effects

The invention provides a steroid hormone phospholipid composition. The composition is prepared into a freeze-dried powder, which is easy to store, has good stability, good safety, high solubility, and is easy to use. The preparation after reconstitution It can maintain long-term and stable release effect in the body for 5-30 days.

Description of drawings

Figure 1 illustrates the cryo-electron microscopy image of the blank control sample of D-0 reconstituted with physiological saline.

Figure 2 illustrates the cryo-electron micrograph of the physiological saline reconstituted prescription D-1.

Figure 3 illustrates the cryo-electron micrograph of the physiological saline reconstituted prescription D-2.

Figure 4 illustrates the cryo-electron micrograph of the physiological saline reconstituted prescription D-3.

Figure 5 illustrates the plasma concentration curves of the long-acting progesterone phospholipid composition and the reference substance in the effect example 1 in rats.

Figure 6 illustrates the plasma concentration curves of the long-acting progesterone phospholipid composition and the reference substance in the effect example 2 in rats.

Figure 7 illustrates the plasma concentration curves of the long-acting estradiol phospholipid composition and the reference substance in the effect example 3 in rats.

FIG8 shows the blood drug concentration curves of the long-acting dienogest phospholipid composition and the reference substance in rats in Effect Example 4

Detailed ways

The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustrative and explain the present invention and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention.

Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Substance name abbreviation:

DMPC dimyristoylphosphatidylcholine

DOPC dioleoylphosphatidylcholine

DPPG dipalmitoylphosphatidylglycerol

HSPC hydrogenated soy phosphatidylcholine

Preparation Example

Preparation Example 1. Preparation of long-acting progesterone phospholipid composition

Table 1

Preparation of solid compositions:

Weigh 200g progesterone and 92.4g cholesterol, add 3L 90% (v/v) tert-butyl alcohol, stir at room temperature until dissolved to form a mixed solution of steroid substances, add 324g DMPC, stir at room temperature until dissolved, and pack into 1000 10ml vials. Medium, 3.4ml per bottle. Place the sample in a freeze dryer and freeze dry it.

Preparation of reconstituted material:

The above freeze-dried product is reconstituted with an appropriate amount of physiological saline and mixed evenly to form a uniform suspension, which appears in an irregular, round, flowable shape under a microscope.

Preparation Example 2. Preparation of long-acting progesterone phospholipid composition

Table 2

Preparation of solid compositions:

Weigh 314.5g progesterone, add 3L 80% (v/v) tert-butyl alcohol, stir at 65°C until dissolved to form a mixed solution of steroid substances, add 6779g DMPC, stir at 65°C until dissolved, and divide into 1500 10ml vials. , 2.3ml per bottle. The sample is freeze-dried in a freeze dryer.

Preparation of reconstituted material:

The above freeze-dried product is reconstituted with an appropriate amount of physiological saline and mixed evenly to form a uniform suspension, which appears in an irregular, round, flowable shape under a microscope.

Preparation Example 3. Preparation of long-acting estradiol phospholipid composition

table 3

Preparation of solid compositions:

Weigh the estradiol and cholesterol according to the prescribed amount, add 0.5L 80% (v/v) tert-butanol, stir at room temperature until dissolved, add DMPC after weighing, stir at room temperature until dissolved, divide into 1000 vials, each bottle 0.53ml, aliquot and freeze-dry to obtain.

Preparation of reconstituted material:

The above freeze-dried product is reconstituted with an appropriate amount of physiological saline and mixed evenly to form a uniform suspension, which appears in an irregular, round, flowable shape under a microscope.

Preparation Example 4. Preparation of long-acting dienogest phospholipid composition

Table 4

Preparation of solid compositions:

Weigh 13.0g dienogest and 26.0g cholesterol, add 2.0L 80% (v/v) tert-butanol, stir at room temperature until dissolved to form a mixed solution of steroidal substances, add 91.0g DMPC, stir at room temperature until dissolved, and package. In 1000 vials, 2.2ml of each vial is packaged and lyophilized to obtain.

Preparation of reconstituted material:

The above freeze-dried product is reconstituted with an appropriate amount of physiological saline and mixed evenly to form a uniform suspension, which appears in an irregular, round, flowable shape under a microscope.

prescription research

(1) Types of phospholipids

In order to examine the pharmaceutical properties of different phospholipid excipients, the compositions of prescriptions 1-4 shown in Table A below were obtained with reference to the method of Preparation Example 1.

Table A

The results show that in the mixing and dissolving step of progesterone, phospholipids, cholesterol, and tert-butanol, the solution used in recipe 2 cannot form a solution using DPPG (dipalmitoylphosphatidylglycerol), which will result in the filtration and sterilization process being unachievable during the production process. The recipe 3 uses After HSPC (hydrogenated soybean phosphatidylcholine) is fully dissolved, there is still a small amount of visible foreign matter, and a gel precipitates after standing at room temperature; Recipe 1 uses DOPC (dioleoylphosphatidylcholine) to prepare a solid composition, but after reconstitution, After agglomeration, it cannot be evenly dispersed in the medium, affecting the needle penetration. Prescription 4 uses DMPC to form a homogeneous solution in the dissolution step. It remains clear and transparent for 24 hours at room temperature and is in a stable state. The reconstituted progesterone phospholipid composition is visually a homogeneous emulsion suspension, with particles in a spherical shape and multiple cross-sections. layer status. Therefore, DMPC is the preferred phospholipid excipient in the present invention.

(2)Solvent concentration

In order to examine the influence of solvent concentration, the compositions of prescriptions 5-10 shown in Table B below were obtained with reference to the method of Preparation Example 1. The results are shown in the table below.

Table B

(3)Drug loading capacity

As shown in Table C below, the drug loading of prescriptions C-1 to C-7 increases in the range of 40%-60%, DMPC:cholesterol=2:1 (molar ratio); the drug loading of prescriptions C-8 to C-14 is 40% The -60% interval increases, DMPC:cholesterol=3:1; prescriptions C-15 and C-16 are groups without cholesterol. The particle size of the DMPC:cholesterol=2:1 group was significantly higher than that of the DMPC:cholesterol=3:1 group, and the particle size of the group without cholesterol was the smallest. It shows that DMPC is the main excipient, which makes the progesterone phospholipid complex present a multi-layer structure, and cholesterol plays a role in regulating the particle size. The above prescriptions have good medicinal properties.

Table C

(4) Microstructure inspection

Examine the impact of the prescription on product form as shown in Table D below. According to the process of Preparation Example 1 or Preparation Example 2, the freeze-dried sample prepared according to the recipe in Table D below was reconstituted with physiological saline, and the microstructure of the reconstituted product was examined using a cryo-electron microscope. It can be seen from the cryo-electron microscopy images in Figures 1-4 that the products obtained have multi-layer structures and meet the requirements.

Form D

Effect example 1

Pharmacokinetic test of progesterone phospholipid composition in rats

Test product: Take a single tube of the long-acting progesterone phospholipid freeze-dried composition prepared according to Preparation Example 1, add an appropriate amount of physiological saline to reconstitute and suspend it to prepare a test product, with a specification of 67 mg/ml.

Reference substance: commercially available progesterone injection, specification 1ml: 25mg (Japan Mochida Pharmaceutical Co., Ltd.)

Experimental animals: Sprague Dawley rats (SPF grade), male SD rats, 6-8 weeks old, weight 180-250g, 6 rats per group, original source: Sprague (Beijing) Biotechnology Co., Ltd.; certificate No.: 1103242201003859385; Production license number: SCXK (Beijing) 2019-0010.

experiment procedure:

(1) For details on grouping and experimental design, see Table E-1 below

Table E-1

(2)Collect samples

Group 1: Subcutaneous injection, collect blood samples at 0, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 216h, 264h, 312h, 336h, and measure the corpus luteum Ketone blood concentration.

Group 2: Intramuscular injection, blood samples were collected at 0, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h to measure progesterone blood concentration.

Place the collected blood samples into heparin anticoagulated blood collection tubes, and centrifuge to separate the plasma (centrifugal force 6800g, centrifugation for 6 minutes, 2-8°C). Plasma samples were stored in a -80°C refrigerator before delivery. Drug concentrations in plasma were determined by HPLC-MS/MS.

Draw the blood drug concentration curve as shown in Figure 5.

Table E-2

The results are shown in Table E-2. After rats were subcutaneously injected with the progesterone phospholipid composition of the present invention, the half-life of the phospholipid composition was approximately 5.6 times that of the commercially available injection, proving that the progesterone phospholipid composition has an ideal sustained-release effect. . The dosage of the phospholipid composition is about 14 times that of the commercially available injection, but the peak concentrations (Cmax) of the two are similar, which proves that the safety of the phospholipid composition is good, and the clinical one-time dosage can at least reach that of the commercial preparation. 7 times. In summary, the rat PK data of the progesterone phospholipid composition shows that the product can take effect quickly, maintain a sustained-release effect for a long time, and is well tolerated, which is conducive to improving patients' medication compliance. sex.

Table E-3

As shown in Table E-3 and Figure 5, in rats, the blood concentration Cmax of the progesterone phospholipid composition of the present invention is slightly higher than that of the commercially available injection. At the same time, the average blood concentration can be maintained for about 11 days, both of which are on the market. It is above the Cmin of the injection solution and has been proven to be long-acting for 11 days, thus helping to improve medication compliance.

Effect example 2

According to the prescription shown in Table F-1 below, test sample 1-5 was prepared using the same method as Preparation Example 1.

Table F-1

(1) Reference substance: commercially available progesterone injection, specification 1ml: 25mg (Japanese Mochida Pharmaceutical Co., Ltd.)

(2) Experimental animals: Sprague Dawley rats (SPF grade), male SD rats, 6-8 weeks old, weight 180-250g, 6 rats per group, original source: Sprague (Beijing) Biotechnology Co., Ltd. ; Certificate number: 1103242201003859385; Production license number: SCXK (Beijing) 2019-0010.

(3)Test steps

The grouping and experimental design are detailed in Table F-2 below.

Table F-2

Collect samples

Test article 1-5: subcutaneous injection, collect blood samples at 0, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and measure progesterone blood drug concentration;

Control group: intramuscular injection, blood samples were collected at 0, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h to measure progesterone blood concentration.

Place the collected blood samples into heparin anticoagulated blood collection tubes, and centrifuge to separate the plasma (centrifugal force 6800g, centrifugation for 6 minutes, 2-8°C). Plasma samples were stored in a -80°C refrigerator before delivery. The drug concentration in plasma was measured by HPLC-MS/MS and the blood drug concentration curve was drawn as shown in Figure 6.

Table F-3

In the experiment of Effect Example 2, the dosage was administered for 7 days, and the average sustained release time was more than 5 days. Combined with the experimental results in Effect Example 1, the dosage was 14 days and the average sustained release was 11 days, it can be seen that the effective concentration of the long-acting progesterone phospholipid composition sustained release time ranges from 5 to 11 days, and its sustained release effect is related to the dose. It is estimated that the dosage of this dosage form is calculated based on 10 days, and the average sustained release time is 7 days.

Table F-4

Effect Example 3 Rat Pharmacokinetic Test of Estradiol Phospholipid Composition

Test product 1: Prepare a long-acting estradiol phospholipid freeze-dried composition according to prescription A in Preparation Example 3. Add an appropriate amount of physiological saline to a single tube to reconstitute and suspend the composition to prepare a test product, with a specification of 12 mg/ml.

Test product 2: Prepare a long-acting estradiol phospholipid freeze-dried composition according to Prescription B in Preparation Example 3. Add an appropriate amount of physiological saline to a single tube to reconstitute and suspend it to prepare a test product, with a specification of 12 mg/ml.

Reference substance: commercially available estradiol tablets (Abbott Healthcare Products B.V.), specification: 1 mg. Crush commercially available estradiol tablets into solid powder in a mortar, weigh and add an appropriate amount of physiological saline to reconstitute and suspend the tablets to prepare a reference solution with a concentration of 2 mg/ml.

Experimental animals: Sprague Dawley rats (SPF grade), male SD rats, 6-8 weeks old, weight 200-220g, 6 rats per group, original source: Sprague (Beijing) Biotechnology Co., Ltd.; certificate No.: 110324220107108468; Production license number: SCXK (Beijing) 2019-0010.

experiment procedure:

(1) For details on grouping and experimental design, see Table G-1 below

Table G-1

Note: The oral bioavailability of the reference substance is calculated as 5%.

(2)Collect samples

Group 1: Subcutaneous injection, blood samples were collected at 0h (before administration) and 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h after administration, and estradiol was measured Blood drug concentration.

Group 2: Subcutaneous injection, blood samples were collected at 0h (before administration) and 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after administration, and estradiol was measured Blood drug concentration.

Group 3: Gavage, blood samples were collected at 0h (before administration) and 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after administration to measure estradiol blood concentration.

Place the collected blood samples into heparin anticoagulated blood collection tubes, and centrifuge to separate the plasma (centrifugal force 4000 rpm, centrifugation for 10 min, 2-8°C). Plasma samples were stored in a -40°C refrigerator before delivery. Drug concentrations in plasma were determined by HPLC-MS/MS.

Table G-2

Table G-3

The pharmacokinetic parameters and drug-time curves of rats with a single subcutaneous injection of the estradiol phospholipid composition test products 1 and 2 of the present invention are as shown in Tables G-2, G-3, and Figure 7. The excipient molar ratio DMPC: Cholesterol=1:1 to 2:1 has a similar sustained-release effect. The half-life of the phospholipid composition is extended to about 12 times that of commercially available tablets. The estradiol phospholipid composition is administered in a single dose for test products 1 and 2. Can be relieved after taking medicine Release for at least 7 days. The rat PK data of the estradiol phospholipid composition shows that the product can take effect quickly and maintain stable sustained release for a long time, so it has development value.

Effect Example 4

Pharmacokinetic test of dienogest phospholipid composition in rats

Test product: Take a single tube of the long-acting dienogest phospholipid freeze-dried composition prepared according to Preparation Example 4, add an appropriate amount of physiological saline to reconstitute and suspend it to prepare a test product, with a specification of 13 mg/ml.

Reference substance: commercially available dienogest tablets, specification: 2 mg (Bayer Weimar GmbH und Co.KG). Crush commercially available dienogest tablets into solid powder in a mortar, weigh and add an appropriate amount of physiological saline to reconstitute and suspend the solution to prepare a reference solution with a concentration of 1 mg/ml.

Experimental animals: Sprague Dawley rats (SPF grade), female SD rats, 6-8 weeks old, weight 180-250g, 6 rats per group, original source: Sprague (Beijing) Biotechnology Co., Ltd.; certificate No.: 110324230103521638; Production license number: SCXK (Beijing) 2019-0010.

experiment procedure:

(1) For details on grouping and experimental design, see Table H-1 below

Table H-1

Note: The oral bioavailability of the control drug is 90%.

(2)Collect samples

Group 1: subcutaneous injection, blood samples were collected at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 72h, and 168h to measure dienogest blood concentration.

Group 2: intragastric administration, blood samples were collected at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h (blood collection time window ±1 minute), and blood concentration of dienogest was measured.

Place the collected blood samples in EP tubes and store them upright at 2-8°C. Centrifuge (4000 rpm, 2-8°C, 10 min) and then collect the plasma. Divide it into 2 parts, 100 μL + backup. Plasma samples were stored in a -80°C refrigerator before delivery. Drug concentrations in plasma were determined by HPLC-MS/MS.

Table H-2

Table H-3

The results are shown in Tables H-2, H-3 and Figure 8. After rats were subcutaneously injected with the dienogest phospholipid composition of the present invention, the half-life of the phospholipid composition was approximately 4.5 times that of the commercially available injection, proving that the dienogest phospholipid composition The progesterone phospholipid composition has ideal sustained release effect. The peak concentration (Cmax) of the phospholipid composition is approximately 1/6 of that of commercially available tablets, proving that the phospholipid composition is safe. In summary, the rat PK data of the dienogest phospholipid composition shows that this product It can take effect quickly, maintain a sustained release effect for a long time, and has good tolerance, which is helpful to improve the patient's medication compliance.

The above has provided an exemplary description of the embodiments of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the protection scope of the claims of this application.

Claims (10)

1. A pharmaceutical composition, characterized in that the pharmaceutical composition contains steroid hormones and phospholipids, wherein the steroid hormones are one or more of adrenal hormones and sex hormones; the phospholipids include natural or synthetic Phospholipids.
2. The pharmaceutical composition according to claim 1, wherein the sex hormone is selected from one of progesterone, estradiol, dienogest, dydrogesterone, tibolone, testosterone undecanoate, etc. or more;

   The phospholipid is selected from one or more of glycerophospholipid and sphingomyelin, and the glycerophospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and glycerophosphatidic acid. One or more; the phospholipid is, for example, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dierucylphospholipid Acylcholine, egg yolk phosphatidylcholine, dilauroylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, distearoylphosphatidylethanolamine, dipalmitate Acylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylserine, dimyristoylphosphatidylserine, distearoylphosphatidylserine One or more of them; preferably, the phospholipid is selected from dimyristoylphosphatidylcholine.
3. The pharmaceutical composition according to claim 1 or 2, characterized in that the molar ratio of the phospholipid to the steroid hormone is 1:1000-1000:1, preferably 1:100-100:1, more preferably The earth is 10:1 to 1:10, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.
4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that the pharmaceutical composition also contains cholesterol; preferably, the molar ratio of the phospholipid to cholesterol is 10:1 to 1:10, for example 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, more preferably, 4:1 to 1:1.
5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the pharmaceutical composition further contains a solvent, and the solvent is selected from one or a combination of organic solvents and/or water. Preferably, the The organic solvent is selected from protic solvents, such as alcohols;

   Preferably, the protic solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, benzyl alcohol, formic acid, and acetic acid;

   Preferably, the alcohols are selected from one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, and benzyl alcohol;

   Preferably, the solvent is selected from a mixed solution of a protic solvent and water (ie, an aqueous solution of a protic solvent). Preferably, the solvent is an aqueous solution of tert-butyl alcohol; preferably, in the aqueous solution of the protic solvent, the content of the protic solvent is ≥50% (v/v), and more preferably, the content of the protic solvent is ≥50% (v/v). In the aqueous solution, the content of protic solvent is ≥60% (v/v);

   Preferably, the mass-to-volume ratio of the steroid hormone to the solvent is 30-100 g/L.
6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the composition contains 10-100 parts by weight of the steroid hormone, preferably, the composition contains the 10-70 parts of steroid hormones; the composition, in parts by weight, contains 5-300 parts of the phospholipid; the composition, when further containing cholesterol, contains cholesterol in parts by weight 1-50 servings;

   Preferably, the pharmaceutical composition contains progesterone, cholesterol and phospholipid, the phospholipid is selected from DMPC, and the molar ratio of the phospholipid and cholesterol is 5:1 to 1:1; preferably, the pharmaceutical composition further Containing tert-butanol or a tert-butanol aqueous solution, the content of tert-butanol in the tert-butanol aqueous solution is ≥60% (v/v);

   Preferably, the pharmaceutical composition contains 10-70 parts by weight of progesterone; 1-30 parts of cholesterol; and 8-100 parts of phospholipids.

   Preferably, the pharmaceutical composition contains estradiol, cholesterol and phospholipid, the phospholipid is selected from DMPC, and the molar ratio of the phospholipid to cholesterol is 5:1 to 1:1; preferably, in the pharmaceutical composition Further comprising tert-butanol or tert-butanol aqueous solution, in the tert-butanol aqueous solution, the content of tert-butanol is ≥60% (v/v);

   Preferably, the pharmaceutical composition contains 10-70 parts by weight of estradiol; 1-30 parts of cholesterol; 8-100 parts of phospholipids;

   Preferably, the pharmaceutical composition contains dienogest, cholesterol and phospholipid, the phospholipid is selected from DMPC, and the molar ratio of the phospholipid and cholesterol is 5:1 to 1:1; preferably, the pharmaceutical composition further comprising tert-butanol or a tert-butanol aqueous solution, and in the tert-butanol aqueous solution, the content of tert-butanol is ≥60% (v/v);

   Preferably, the pharmaceutical composition contains 10-70 parts by weight of dienogest; 1-50 parts of cholesterol; and 30-150 parts of phospholipids.
7. The preparation method of pharmaceutical composition according to any one of claims 1-6, characterized in that it includes the following steps:

   (i) Mix the steroid hormone, phospholipid, optionally added cholesterol and a solvent to obtain a solution.
8. The preparation method according to claim 7, characterized in that the mixing process can be carried out under mixing conditions such as stirring, ultrasonic, vortexing, etc., preferably heating and stirring in a water bath; the mixing/dissolving can be performed at a suitable temperature, such as 10 °C-100°C, preferably 20°C-85°C, more preferably 35-55°C; the stirring speed is 10rpm-20000rpm, preferably 100rpm-12000rpm.
9. The preparation method according to claim 7 or 8, characterized in that the preparation method further includes the following steps:

   (ii) preparing the solution into a solid composition;

   Preferably, in step (ii), the method for preparing the solid composition includes air drying, rotary evaporation, freeze drying, spray drying or freeze spray drying, preferably freeze drying.
10. A preparation obtained by reconstituting the pharmaceutical composition according to any one of claims 1 to 6 or the pharmaceutical composition obtained according to the preparation method according to any one of claims 7 to 9 in an aqueous dispersion medium; preferably Alternatively, the preparation can be administered locally, more preferably, by subcutaneous injection and intramuscular injection.