A kind of allylgestaltin nano preconcentrated solution and its preparation method and application

Abstract

The invention discloses an allergy-progesterone nano preconcentrate and a preparation method and application thereof. The nano preconcentrate is made of the following raw materials in mass percentage: 0.5-2.5% allergy-progesterone; 15-29% ethyl oleate; 45-65% polyoxyethylene sorbitan monooleate; 15-30% ethyl carbitol; and 1-5% ethanol. The allergy-progesterone nano preconcentrate of the invention is easy to use and can be arbitrarily miscible with water, thereby solving the disadvantage that the traditional allergy-progesterone oil solution is immiscible with water; at the same time, it can promote the absorption of allergy-progesterone through lymphatic circulation and improve bioavailability. The bioavailability of the nano preconcentrate after oral absorption after hydration is 497% of that of the allergy-progesterone oil solution, thereby reducing the clinical dosage of allergy-progesterone or reducing the number of administrations, thereby alleviating stress on animals.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drugs, and in particular relates to an allylgestalten nanometer preconcentrated solution and a preparation method and application thereof. Background Art

[0002] Oral administration is one of the most widely used administration routes in clinical practice. It has the advantages of flexible dosage form design, accurate dosage, simple process and convenient medication. The oral absorption of poorly soluble drugs is affected by many factors such as the physicochemical properties of the drug, the formulation factors of the preparation and the physiological condition of the digestive system of the body. Among them, the solubility of the drug in the gastrointestinal tract and the permeability to the intestinal mucosa are the main influencing factors. Alprogesterone (alprogesterone renogest, alprogesterone) is a synthetic steroidal progestin launched in France in the 1980s. It mainly adjusts the estrus cycle of female livestock by inhibiting the secretion of follicle-stimulating hormone and luteinizing hormone. It has been widely used in Europe to synchronize estrus in pigs and horses, and can also be used to maintain pregnancy in mares. Alprogesterone is almost insoluble in water. The clinical dosage form is an oil solution. The administration method is oral administration. It needs to be administered continuously for at least 14 days to delay estrus in sows, thereby achieving the purpose of synchronized estrus. Due to its poor solubility, alprogesterone may affect the absorption efficiency of the drug in the body. Therefore, it is of great clinical significance to improve the solubility and dissolution rate of allergaster by means of pharmaceutical preparation, promote the absorption of allergaster and thus improve its oral bioavailability. In recent years, domestic and foreign laboratories have conducted research on solid dispersions, cyclodextrin inclusion compounds and PLGA [Poly (lactic-co-glycolic Acid)] microspheres of allergaster to improve drug solubility, but the cost of the required excipients is high and the preparation process is complicated, and the industrial production technology requirements are high. Summary of the invention

[0003] The purpose of the invention is to provide an allylgestalten nanometer preconcentrated solution and a preparation method and application thereof.

[0004] In order to solve the problems existing in the prior art, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides an allylgestalten nanometer pre-concentrated solution, which is made of the following raw materials in percentage by mass:

[0006] Progesterone: 0.5-2.5%;

[0007] Ethyl oleate: 15-29%;

[0008] Polyoxyethylene sorbitan monooleate: 45-65%;

[0009] Ethyl carbitol: 15-30%;

[0010] Ethanol: 1-5%.

[0011] Preferably, the allylgestaltin nano preconcentrate is made of the following raw materials in percentage by mass:

[0012] Alprogestin: 2%;

[0013] Ethyl oleate: 21.5%;

[0014] Polyoxyethylene sorbitan monooleate: 53.1%;

[0015] Ethyl carbitol: 20.8%.

[0016] Ethanol: 2.6%.

[0017] In a second aspect, the present invention provides a method for preparing the allylgestaltin nano preconcentrate described in the first aspect, comprising the following steps:

[0018] ① placing the ethyl oleate, polyoxyethylene sorbitan monooleate, ethyl carbitol and ethanol in a centrifuge tube, vortexing and mixing them evenly to obtain a mixed solvent;

[0019] ② Then, the allergy-progesterone is added to the mixed solvent, and vortexed, ultrasonicated, and shaken in a water bath until the allergy-progesterone is completely dissolved to obtain an allergy-progesterone nano pre-concentrated solution.

[0020] Furthermore, in step ①, vortexing is performed for 3-5 minutes.

[0021] Furthermore, in the step ②, vortex for 3-5 minutes, preferably vortex for 3 minutes, and ultrasonicate for 2-3 minutes, preferably ultrasonicate for 3 minutes, and balance under 37° C. water bath shaking conditions until the allylprogesterone is completely dissolved to obtain the allylprogesterone nano preconcentrate.

[0022] In a third aspect, the present invention provides use of the allergyrin nano preconcentrated solution of the first aspect in the preparation of an allergyrin preconcentrated hydrated solution.

[0023] In a fourth aspect, the present invention provides a method for preparing a pre-concentrated hydrated solution of allylgestaltenone, comprising the following steps:

[0024] The allylpregestrin nano preconcentrated solution described in the first aspect is added into ultrapure water, and hydrated under 50-200 r / min magnetic stirring conditions to prepare a solution, and when the solution is clear and transparent with light blue opalescence, the allylpregestrin preconcentrated hydrated solution is obtained.

[0025] Furthermore, the solid-to-liquid ratio of the allylgestaltenone nano preconcentrate and ultrapure water is 1 g:10-1000 mL, preferably 1 g:100 mL.

[0026] In a fifth aspect, the present invention provides the use of the pre-concentrated hydrated solution of allylgestaltenone described in the fourth aspect in the preparation of a preparation for inhibiting the secretion of gonadotropin in livestock and preventing the development of ovarian follicles.

[0027] Furthermore, the livestock is a sow or a mare.

[0028] The advantages and beneficial effects of the present invention are:

[0029] The allylprogesterone nano preconcentrate prepared by the present invention has a clear and transparent appearance, good fluidity, is easily hydrated in water to form a nano solution, the hydration time is 35.76±1.10s, the droplets of the nano solution formed after hydration have a round and uniform appearance, no adhesion, an average particle size of 18.39±0.03nm, a PDI of 0.083±0.090, a Zeta potential of -1.12±0.12mV, and can remain stable within a 10-1000 times dilution range, and has dilution stability. The allylprogesterone nano preconcentrate can reach a cumulative release of 91.41% in vitro in 48h, which is significantly improved compared to 32.42% of the original drug. The allylprogesterone preparation is easy to use and can be mixed with water at will, thus solving the shortcoming that the traditional allylprogesterone oil solution is immiscible with water; at the same time, it can promote the absorption of allylprogesterone through lymphatic circulation and improve the bioavailability. The bioavailability of the nano preconcentrate after oral absorption after hydration is 497% of that of the allylprogesterone oil solution, thereby reducing the clinical dosage of allylprogesterone or the number of administrations, and alleviating stress on animals.

[0030] The pre-concentrated hydrated solution of allylgestaltin of the present invention has an obvious effect of regulating the concentrated estrus of sows or mares, realizing synchronous pregnancy, so as to realize the pre-delivery of livestock in groups and reduce the cost of artificial delivery care. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 a is the appearance of the mixed solvent without adding allylgestaltin;

[0032] Figure 1 b is the appearance of the allylgestaltin nano concentrated solution prepared in Example 1;

[0033] Figure 1 c is the appearance of the allylgestaltin nano preconcentrate prepared in Example 6 after hydration and irradiation by a red laser pen;

[0034] Figure 2 This is a TEM image of the hydrated allylgestaltenes nano-preconcentrate; Figure 2 a is (50nm), Figure 2 b is (100nm);

[0035] Figure 3 a is the particle size distribution of allylgestaltenes nano preconcentrate after hydration; Figure 3 b is Zeta potential;

[0036] Figure 4 The particle size distribution of the nano solution obtained by hydrating the nano preconcentrate of allylgestaltenes at different dilution multiples (n=3);

[0037] Figure 5 It is the cumulative release curve of allergy-progesterone nano-preconcentrate and allergy-progesterone oil solution;

[0038] Figure 6 This is the logarithmic remaining drug dose-time curve of allylgestaltenone (n=5);

[0039] Figure 7 The blood drug concentration-time curves after oral administration of three different preparations (alprogesterone oil solution, alprogesterone nano preconcentrate, and nano solution after hydration of alprogesterone nano preconcentrate);

[0040] Figure 8 This is the blood drug concentration-time curve after oral administration of the pre-concentrated hydrated solution of allergyprogesterone 1 hour after direct oral administration or intraperitoneal injection of lymph node blocking agent (CHX). DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application belong to the scope of protection of this application.

[0042] The experimental methods in the following embodiments and experimental examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following embodiments and experimental examples, unless otherwise specified, can be obtained from commercial channels.

[0043] Example 1

[0044] This embodiment provides a nano pre-concentrated solution of allylgestaltin, which is made of the following raw materials in percentage by mass:

[0045] Alprogestin: 2%;

[0046] Ethyl oleate: 21.5%;

[0047] Polyoxyethylene sorbitan monooleate: 53.1%;

[0048] Ethyl carbitol: 20.8%;

[0049] Ethanol: 2.6%.

[0050] The preparation method of the allylgestaltin nano pre-concentrated solution comprises the following steps:

[0051] ① Place the above-mentioned ethyl oleate, polyoxyethylene sorbitan monooleate, ethyl carbitol and ethanol in a centrifuge tube, and vortex and shake for 3 minutes to mix them evenly to obtain a mixed solvent;

[0052] ② Then add allylprogesterone to the mixed solvent, vortex for 3 minutes, ultrasonicate for 3 minutes, and balance under 37° C. water bath shaking conditions until allylprogesterone is completely dissolved to obtain allylprogesterone nano pre-concentrate.

[0053] Example 2

[0054] This embodiment provides a nanometer pre-concentrated solution of allylgestaltenes. The difference between this embodiment and embodiment 1 is only that the ratio of the nanometer pre-concentrated solution is different. This embodiment is made of the following raw materials in mass percentage:

[0055] Progesterone: 0.5%;

[0056] Ethyl oleate: 20%;

[0057] Polyoxyethylene sorbitan monooleate: 55%;

[0058] Ethyl carbitol: 22.5%;

[0059] Ethanol: 2%.

[0060] The rest is the same as in Example 1.

[0061] Example 3

[0062] This embodiment provides a nanometer pre-concentrated solution of allylgestaltenes. The difference between this embodiment and embodiment 1 is only that the ratio of the nanometer pre-concentrated solution is different. This embodiment is made of the following raw materials in mass percentage:

[0063] Progesterone: 2.5%;

[0064] Ethyl oleate: 24%;

[0065] Polyoxyethylene sorbitan monooleate: 45%;

[0066] Ethyl carbitol: 27.5%;

[0067] Ethanol: 1%.

[0068] The rest is the same as in Example 1.

[0069] Example 4

[0070] This embodiment provides a nanometer pre-concentrated solution of allylgestaltenes. The difference between this embodiment and embodiment 1 is only that the ratio of the nanometer pre-concentrated solution is different. This embodiment is made of the following raw materials in mass percentage:

[0071] Alprogestin: 2%;

[0072] Ethyl oleate: 21.5%;

[0073] Polyoxyethylene sorbitan monooleate: 53.1%;

[0074] Ethyl carbitol: 18.4%;

[0075] Ethanol: 5%.

[0076] The rest is the same as in Example 1.

[0077] Example 5

[0078] This embodiment provides a nanometer pre-concentrated solution of allylgestaltenes. The difference between this embodiment and embodiment 1 is only that the ratio of the nanometer pre-concentrated solution is different. This embodiment is made of the following raw materials in mass percentage:

[0079] Alprogestin: 2%;

[0080] Ethyl oleate: 21.5%;

[0081] Polyoxyethylene sorbitan monooleate: 53.1%;

[0082] Ethyl carbitol: 22.4%;

[0083] Ethanol: 1%.

[0084] The rest is the same as in Example 1.

[0085] Example 6

[0086] This embodiment provides an allergyrin preconcentrated hydrated solution, which is prepared from the allergyrin nano preconcentrated solution prepared in Example 1 and ultrapure water, wherein the solid-liquid ratio of the allergyrin nano preconcentrated solution to the ultrapure water is 1 g:100 mL.

[0087] The preparation method of the hydrated nano solution of the allylgestaltin nano pre-concentrate comprises the following steps:

[0088] Accurately weigh 1 g of the allylpregestrel nano preconcentrate prepared in Example 1, add it to 100 mL of ultrapure water, and form a nano solution under 37° C. and 100 r / min magnetic stirring conditions. When the solution is clear and transparent with a light blue opalescence, the allylpregestrel preconcentrated hydrated solution is obtained.

[0089] The hydration time of the solution was measured, and the timing was started when the allylgestaltin nano preconcentrate just touched the water surface. When the system was observed to be completely clear and transparent, and there were no lumps, the nano solution was completely hydrated, and the timing was stopped and the hydration time of the nano solution was recorded. The measured hydration time of the nano solution was 35.76s, and the nano solution time was short and the efficiency was high.

[0090] Example 7

[0091] This embodiment provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this embodiment and embodiment 6 is that this embodiment is prepared from the allergy-progesterone nano pre-concentrated solution prepared in embodiment 2 and ultrapure water, and the rest is the same as embodiment 6.

[0092] The hydration time of the solution was measured according to the method described in Example 6 and was 60.94 s.

[0093] Example 8

[0094] This embodiment provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this embodiment and embodiment 6 is that this embodiment is prepared from the allergy-progesterone nano pre-concentrated solution prepared in embodiment 3 and ultrapure water, and the rest is the same as embodiment 6.

[0095] The hydration time of the solution was measured according to the method described in Example 6 and was 58.32 s.

[0096] Example 9

[0097] This embodiment provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this embodiment and embodiment 6 is that this embodiment is prepared from the allergy-progesterone nano pre-concentrated solution prepared in embodiment 4 and ultrapure water, and the rest is the same as embodiment 6.

[0098] The hydration time of the solution was measured according to the method described in Example 6 and was 50.70 s.

[0099] Example 10

[0100] This embodiment provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this embodiment and embodiment 6 is that this embodiment is prepared from the allergy-progesterone nano pre-concentrated solution prepared in embodiment 5 and ultrapure water, and the rest is the same as embodiment 6.

[0101] The hydration time of the solution was measured according to the method described in Example 6 and was 45.70 s.

[0102] Embodiment 11

[0103] This embodiment provides an allergy-progesterone pre-concentrated hydrated solution. The solid-liquid ratio of the allergy-progesterone nano pre-concentrated solution and ultrapure water in this embodiment is 1 g:1000 mL, and the rest is the same as in Example 6.

[0104] The hydration time of the solution was measured according to the method described in Example 6 and was 41.68 s.

[0105] Example 12

[0106] This embodiment provides an allergy-progesterone pre-concentrated hydrated solution. The solid-liquid ratio of the allergy-progesterone nano pre-concentrated solution and ultrapure water in this embodiment is 1 g:10 mL, and the rest is the same as in Example 6.

[0107] The hydration time of the solution was measured according to the method described in Example 6 and was 43.84 s.

[0108] Comparative Example 1

[0109] This comparative example provides an allylgestaltenone preconcentrated solution, in which no ethanol is added, and the rest is the same as in Example 1.

[0110] Comparative Example 2

[0111] This comparative example provides an allylgestaltenone preconcentrated solution. In this comparative example, oleic acid is used instead of ethyl oleate, and the rest is the same as in Example 1.

[0112] Comparative Example 3

[0113] This comparative example provides an allylgestalten preconcentrate. In this comparative example, Span 80 is used instead of polyoxyethylene sorbitan monooleate, and the rest is the same as in Example 1.

[0114] Comparative Example 4

[0115] This comparative example provides an allylgestalten preconcentrated solution. In this comparative example, diethylene glycol diethyl ether is used instead of ethyl carbitol, and the rest is the same as in Example 1.

[0116] Comparative Example 5

[0117] This comparative example provides an allylgestaltenone preconcentrated solution, in which isopropanol is used instead of ethanol, and the rest is the same as in Example 1.

[0118] Comparative Example 6

[0119] This comparative example provides a nano-preconcentrated solution of allylgestaltin, in which the mass percentage of ethylcarbitol is 14% and the mass percentage of ethanol is 9.4%. The rest is the same as in Example 1.

[0120] Comparative Example 7

[0121] This comparative example provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this comparative example and Example 6 is that this comparative example is prepared from the allergy-progesterone nano pre-concentrated solution prepared in Comparative Example 1 and ultrapure water, and the rest is the same as Example 6.

[0122] The hydration time of the solution measured according to the method described in Example 6 was 140.31 s. Since no ethanol was added to this comparative example, its affinity with water was reduced, and thus the hydration time was greatly prolonged.

[0123] Comparative Example 8

[0124] This comparative example provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this comparative example and Example 6 is only that this comparative example is prepared from the allergy-progesterone nano pre-concentrated solution prepared in Comparative Example 2 and ultrapure water, and the rest is the same as Example 6.

[0125] The hydration time of the solution was 98.85 s as measured by the method described in Example 6. Compared with Example 6, the hydration time of the solution in this comparative example was greatly prolonged, which may be due to the different fat solubility of oleic acid and ethyl oleate, and their limited solubility for allylgestaltenes, resulting in prolonged hydration time.

[0126] Comparative Example 9

[0127] This comparative example provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this comparative example and Example 6 is only that this comparative example is prepared from the allergy-progesterone nano pre-concentrated solution prepared in Comparative Example 3 and ultrapure water, and the rest is the same as Example 6.

[0128] This comparative example cannot be hydrated with ultrapure water because Span 80 is too lipophilic to be hydrated.

[0129] Comparative Example 10

[0130] This comparative example provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this comparative example and Example 6 is only that this comparative example is prepared from the allergy-progesterone nano pre-concentrated solution prepared in Comparative Example 4 and ultrapure water, and the rest is the same as Example 6.

[0131] This comparative example cannot be hydrated with ultrapure water. Diethylene glycol diethyl ether is too lipophilic, which is not conducive to the stability of the allylgestaltenes nano pre-concentrate and cannot be hydrated.

[0132] Comparative Example 11

[0133] This comparative example provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this comparative example and Example 6 is only that this comparative example is prepared from the allergy-progesterone nano pre-concentrated solution prepared in Comparative Example 5 and ultrapure water, and the rest is the same as Example 6.

[0134] This comparative example cannot be hydrated with ultrapure water because isopropanol is highly lipophilic.

[0135] Comparative Example 12

[0136] This comparative example provides an allergy-progesterone pre-concentrated hydrated solution. The difference between this comparative example and Example 6 is only that this comparative example is prepared from the allergy-progesterone nano pre-concentrated solution prepared in Comparative Example 6 and ultrapure water, and the rest is the same as Example 6.

[0137] This comparative example cannot be hydrated with ultrapure water. The reason is that the hydration of the allylprogesterone nano pre-concentrate in water is closely related to the composition ratio of its prescription. The amount of ethyl carbitol used in this comparative example is too low to achieve uniform dispersion of the allylprogesterone oil solution in water. At the same time, the amount of ethanol used is too large, and the nanoparticles formed after hydration are also unstable. Therefore, this comparative example cannot be hydrated into a stable nano solution.

[0138] According to the comparison between Examples 6-10 and Comparative Examples 7-12, the hydration time of the allylgestaltenes nano-preconcentrated solution prepared according to specific ingredients and specific ratios in Examples 1-5 is much shorter than that in the comparative examples, among which the hydration time of Example 6 is only 35.76s, and the nano-solubilization efficiency is very high.

[0139] Experimental Example 1

[0140] The reagents and instruments involved in this experiment are as follows:

[0141] 1. Materials and Reagents

[0142] Allylgestalt standard (98%, Shanghai Yuanye Biotechnology Co., Ltd.); allylgestalt API (Ningbo Sansheng Biotechnology Co., Ltd.); monolinoleylglycerol (Maisine CC), propylene glycol monocaprylate (Capryol 90), caprylic capric acid polyethylene glycol glyceride (Labrasol), caprylic capric acid mono- and diglycerides (Labrafac MC60), monocaprylic acid propylene glycol ester (Capryol 90), and ethyl carbitol (Transcutol HP) were all donated by Guangzhou Tianrun Pharmaceutical Co., Ltd.; acetonitrile (chromatographic grade, Tianjin Komiou Chemical Reagent Co., Ltd.); oleic acid, ethyl oleate, polyoxyethylene sorbitan monooleate, Span 80, n-octanol, ethanol, and isopropanol were all analytical grade and purchased from Chengdu Kelong Reagent Co., Ltd.; caprylic capric acid glyceride (Shanghai Aladdin Biochemical Technology Co., Ltd.); purified water (Wahaha); MD44 dialysis bag (molecular weight cutoff 7000, Chengdu Pengshida Laboratory Supplies Co., Ltd.).

[0143] 2. Experimental equipment and analytical instruments

[0144] Waters Alliance high performance liquid chromatography system (HPLC, Waters Corporation, USA); MS-H280-ProScilogex magnetic stirrer (Serologex, USA); JD100-3 electronic analytical balance (Shenyang Longteng Electronic Instrument Co., Ltd.); HZS-H water bath oscillator (Harbin Dongming Medical Instrument Factory); Zetasizer Nano ZS Malvern laser particle size analyzer (Malvern, UK); JEM-1400FLASH transmission electron microscope (JEOL, Japan).

[0145] 3. Experimental Methods

[0146] 3.1 Quality evaluation of allylgestaltenone preparations

[0147] The appearance of the allergyrin nano preconcentrated solution prepared in Example 1 and the allergyrin preconcentrated hydrated solution prepared in Example 6 was evaluated by observing their color, viscosity and fluidity. The particle size and Zeta potential of the allergyrin preconcentrated hydrated solution prepared in Example 6 were measured by a Malvern laser particle size analyzer, and its morphology was photographed and observed using a transmission electron microscope. The solution was diluted 5, 10, 20, 50, 100, 500, and 1000 times, and its appearance properties were observed and its particle size was measured to investigate its dilution stability.

[0148] 3.2 Determination of drug loading and encapsulation efficiency of allylgestaltenone preparation

[0149] Accurately weigh 0.1g of allylpregestrel nano-preconcentrate, dilute it 100 times with acetonitrile, filter it after ultrasonication for 30 minutes, and detect it with HPLC to calculate the drug content W1. Repeat the operation three times to calculate the drug loading. Take another 0.1g of allylpregestrel nano-preconcentrate, dilute it with 10 times purified water, take 5mL and add it to a centrifuge tube, centrifuge it at 4000r / min for 10 minutes, accurately measure 1mL of the supernatant, dilute it 10 times, filter it after ultrasonication for 30 minutes, and detect it with HPLC to calculate the drug content W2. Calculate the encapsulation efficiency (EE) according to the following formula:

[0150] EE(%)=W2 / W1×100% (1)

[0151] 3.3 Study on the in vitro release of allylgestaltenone preparations

[0152] The in vitro release of allylprogesterone nano-preconcentrate was studied by dialysis, with allylprogesterone oil solution as a control. The preparation method of allylprogesterone oil solution is as follows:

[0153] Accurately weigh 10 mg of allylprogesterone raw material, completely dissolve in 10 mL of caprylic acid glyceride under 37 ° C water bath shaking conditions to obtain an allylprogesterone oil solution. Because allylprogesterone has extremely poor water solubility, in order to achieve the sink condition, a PBS solution of polyoxyethylene sorbitan monooleate with a mass volume concentration of 2g / 100mL is selected as the release medium. Take 1 mL of the allylprogesterone pre-concentrated hydrated solution prepared in Example 6 and the allylprogesterone oil solution respectively, put them into a dialysis bag, seal both ends and put them into 49 mL of the release medium, and release them in a constant temperature water bath oscillator at 37 ° C and 100r / min. Take 1 mL of the release medium as the test solution at 5, 10, 15, 30, 60min and 2, 4, 6, 8, 12, 24h, and then add 1 mL of the release medium. The test solution was filtered through a 0.22 μm microporous membrane and the content was determined by HPLC. The drug concentration and cumulative release rate were calculated. The in vitro release curve was drawn with the release time as the horizontal axis and the cumulative release rate as the vertical axis.

[0154] 3.4 Study on intestinal absorption characteristics of allylgestaltenone nano-preconcentrate in vivo

[0155] Eight-week-old SPF female SD rats weighing 220 g and fasted for 12 hours but not water were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. They were anesthetized and fixed by intraperitoneal injection of 20% urethane solution at 0.6 mL / 100 g.

[0156] Cut about 3cm incision along the midline of the abdomen to open the abdominal cavity, cut two places 1cm at the end of the pylorus and 5cm at the front of the cecum, wash the intestinal segment with 37℃ saline until there is no content, and then drain the liquid with air. Insert the silicone tube at both ends of the intestinal segment and tie it with thread, connect the constant flow pump, put the inlet and outlet silicone tubes together into a conical flask, balance the loop with perfusion fluid for 10min at a flow rate of 2mL / min, immediately collect 1mL of intestinal perfusion fluid from the conical flask as the 0h drug concentration and phenol red concentration, and supplement 1mL of phenol red KR buffer. Circulate perfusion at a flow rate of 2mL / min for 2h, collect 1.0mL of test solution from the conical flask into a 1.5mL centrifuge tube every 15min, and supplement 1mL of phenol red KR buffer. After the end, cut the intestinal segment of the test and measure the length and inner diameter.

[0157] The intestinal perfusion fluid was added with acetonitrile at a volume ratio of 1:1, vortexed for 3 minutes to precipitate possible proteins, centrifuged at 10000r / min and 4℃ for 15 minutes, and the supernatant was appropriately diluted with distilled water, filtered, and then injected into HPLC for determination. After the sample was diluted with NaOH (0.2mol / L), the absorbance value of phenol red was determined at 558nm.

[0158] 3.5 Pharmacokinetic study of allylgestaltenone preparation in rats

[0159] 20 6-8 week old SPF female SD rats, weighing 200-220g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. and randomly divided into 4 groups, 5 rats in each group, and fed with conventional feed for one week at 25±2°C, 12h light-dark alternation, and free drinking water. Fasting but not water for 12h before the adaptive feeding experiment, free drinking water during the experiment. Among them, three groups were given alprogesterone oil solution, alprogesterone nano pre-concentrated solution prepared in Example 1, and alprogesterone pre-concentrated hydrated solution prepared in Example 6 by gavage at 40mg / kg (in terms of alprogesterone). The fourth group was a lymphatic blocking group, which was first intraperitoneally injected with 3mg / kg of cycloheximide physiological saline injection, and then gavage at 40mg / kg (in terms of alprogesterone) after 1h to give the alprogesterone pre-concentrated hydrated solution prepared in Example 6. The preparation method of allylprogesterone oil solution is as follows: 200 mg of allylprogesterone is dissolved in 10 g of caprylic decanoic acid glyceride, which is used as the control group of the commercially available allylprogesterone oral solution; 0.5 mL of blood is collected from the orbital vein at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours after administration, and the blood sample is placed in a high-speed desktop refrigerated centrifuge at a speed of 5000 r / min and centrifuged at 4°C for 10 minutes, and the upper plasma is taken for sample processing and the allylprogesterone content in the plasma is determined, and the pharmacokinetic analysis is performed using DAS2.0 software.

[0160] 4 Results and discussion

[0161] 4.1 In vitro characterization of allylgestaltenone preparations

[0162] 4.1.1 Observation on the appearance of allylgestaltin nano-preconcentrate and the morphology of nano-solution after hydration

[0163] Under room temperature, the allylpregnenol nano preconcentrate prepared in Example 1 is a light yellow viscous liquid, and the allylpregnenol preconcentrate hydrated solution prepared in Example 6 is a transparent liquid with good fluidity. After irradiation with a red laser pen, the allylpregnenol nano preconcentrate hydrated solution exhibits an obvious Tyndall effect (such as Figure 1 The TEM results of the hydrated nanodroplets are shown in Figure 2 As shown, the droplets formed by diluting and emulsifying the allylgestaltenone nano-preconcentrate with water are spherical in shape with basically the same size and uniform distribution, and there is no adhesion.

[0164] 4.1.2 Particle size distribution and Zeta potential of preconcentrated hydrated solution of allylpregnenol

[0165] The allylpregnenol preconcentrated hydrated solution prepared in Examples 6-10 was heated at 37°C, and the particle size and PDI of the allylpregnenol nano preconcentrated hydrated solution were measured by a laser particle size analyzer. The average particle size of the allylpregnenol preconcentrated hydrated solution was 18.39±0.03nm (n=3), the PDI was 0.083±0.090 (n=3), and the Zeta potential was -1.12±0.12mV (n=3). The particle size distribution and Zeta potential were shown in Table 1. Figure 3 . The particle size of the pre-concentrated hydrated solution of allylprogesterone is about 20nm, which is relatively small, and the PDI is less than 0.2, indicating that the droplet size of the nano-pre-concentrated solution of allylprogesterone is small and uniform after in vitro emulsification; and the Zeta potential of the pre-concentrated hydrated solution of allylprogesterone is -1.12±0.12mV, and the absolute value of the potential is low, close to electrical neutrality, but according to the characteristics of the nano-pre-concentrated solution of allylprogesterone, after entering the body, it will quickly hydrate into nanoparticles in the gastrointestinal tract and be absorbed by the small intestine, and the drug will not aggregate and precipitate after a long time. In addition, the top of the intestinal mucosa and the mucus in the cavity of the body are negatively charged, and the excessive negative charge of the nano-droplets is not conducive to cell uptake, so the absolute low negative charge of the pre-concentrated hydrated solution of allylprogesterone will not affect the absorption of the drug in the intestine.

[0166] 4.1.3 Study on the dilution stability of allylgestaltenes nano-preconcentrate

[0167] After diluting the nano-preconcentrated solution of allergy-progesterone at different times, the particle size change of the preconcentrated hydrated solution of allergy-progesterone and whether drug precipitation occurs were investigated. When the dilution times were 10 to 1000 times, the results were as follows: Figure 4 As shown. The droplet size of the pre-concentrated hydrated solution of allylprogesterone did not change significantly, and no phase separation, turbidity or drug precipitation occurred within 12 hours after hydration. When the dilution multiple was less than 10 times, the droplet size of the pre-concentrated hydrated solution of allylprogesterone was greater than 25nm, and the PDI was greater than 0.2, indicating that the pre-concentrated hydrated solution of allylprogesterone could stably hydrate in the dilution range of 10 to 1000 times, and had a certain dilution stability.

[0168] 4.1.4 Drug loading and encapsulation efficiency of allylgestaltenone nanoparticle preconcentrate

[0169] The drug loading of the allylprogesterone nano preconcentrate prepared in Example 1 is 19.44±0.39 mg / g, n=3. The results of the encapsulation efficiency of allylprogesterone after hydration in Example 6 are shown in Table 1. The average encapsulation efficiency is 98.85%±0.69%, n=3, indicating that allylprogesterone has a higher encapsulation efficiency in the hydration solution.

[0170] Table 1 Allylgestaltenone pre-concentrated hydration solution encapsulation efficiency determination results

[0171]

[0172] 4.2 Study on the in vitro drug release of allylgestaltenone nano-preconcentrated solution

[0173] The experimental results using the forward dialysis method are as follows Figure 5 As shown in the figure, the pre-concentrated hydrated solution of allylprogesterone can quickly release to 47.96% within 4 hours, the cumulative release can reach 74.77% at 12 hours, and finally the cumulative release reaches 91.41% at 48 hours. However, the cumulative release of the oil solution of allylprogesterone can only reach 13.68% at 12 hours, and the cumulative release at 48 hours is only 32.42%. It can be seen that the pre-concentrated hydrated solution of allylprogesterone can increase the in vitro release of allylprogesterone. The main reason may be that the pre-concentrated hydrated solution of allylprogesterone increases the solubility of allylprogesterone, forming a high-concentration allylprogesterone solution in the dialysis bag, resulting in an increase in the concentration difference inside and outside the dialysis bag, thereby promoting drug release.

[0174] 4.3 Results of in vivo intestinal absorption studies

[0175] The logarithm of the remaining drug amount Xt (μg) of allylprogesterone in the intestinal perfusion solution was taken and linearly regressed with the sampling time t (h). According to the formula lnXt = x-Kat, the slope of the straight line was Ka. The logarithmic remaining drug amount-time curve of allylprogesterone at different time points of perfusion was taken every 15 minutes for 2 hours. Figure 6 . Ka represents the speed at which the component enters the systemic circulation from the absorption site, reflecting the speed of its absorption in the intestinal segment. The larger the Ka, the faster the component is absorbed in the intestinal segment; the absorption rate constant of the allergy-progesterone nano preconcentrate group is 0.52, lnXt = -0.5211t + 4.0209, and the absorption rate constant of the allergy-progesterone original drug group is 0.14, lnXt = -0.1449t + 3.9483. Peff represents the permeability of the component through the biological membrane, reflecting the strength of the absorption capacity of the component in different intestinal segments. Generally, when Peff>2.0×10-5cm / min, it indicates that the component is well absorbed in the intestinal segment. The Ka and Peff values ​​of the allergy-progesterone nano preconcentrate group are significantly greater than those of the allergy-progesterone original drug group, indicating that the allergy-progesterone nano preconcentrate can improve the absorption of the drug in the small intestine, thereby improving the oral bioavailability of allergy-progesterone.

[0176] 4.4 Pharmacokinetic studies in rats

[0177] After rats were orally administered with allergy-progesterone oil solution, allergy-progesterone nano-preconcentrated solution, and allergy-progesterone preconcentrated hydrated solution, the blood drug concentration-time data were analyzed by DAS2.0 software and showed that they were in line with the two-compartment model and exhibited rapid cross-intestinal absorption characteristics. The concentration-time curve and pharmacokinetic parameters of allergy-progesterone in plasma were respectively Figure 7As shown in Table 2. The peak concentration Cmax is the maximum blood concentration reached by the drug entering the blood after administration. The drug needs to reach a certain concentration in the body to exert its effect. From Cmax, it can be seen that the Cmax of the allergy tolerone nano preconcentrate group (1.33±0.49μg / mL) and the allergy tolerone preconcentrated hydrated solution group (6.83±1.06μg / mL) are significantly different from the allergy tolerone oil solution group (0.87±0.21μg / mL). Compared with the commercially available crude oil preparation, the allergy tolerone nano preconcentrate significantly increased the blood concentration of ALT in rats. AUC is the area under the drug-time curve, which is proportional to the total amount of drug absorbed and reflects the degree of drug absorption. The larger the AUC, the more completely the drug is absorbed by the organism. There is a significant difference in AUC0-t between the allylprogesterone nano-preconcentrated solution group (5.14±1.67μg / mL·h) and the allylprogesterone preconcentrated hydrated solution group (13.03±1.42μg / mL·h) and the allylprogesterone oil solution group (2.62±0.94μg / mL·h), indicating that the allylprogesterone nano-preconcentrated The dilution increased the absorption of the original drug ALT in the body; it is worth noting that the Cmax and AUC of the allergest preconcentrated hydrated solution group were also significantly different from those of the allergest nano preconcentrated solution group, indicating that the oral absorption of ALT can be improved after the allergest nano preconcentrated solution is hydrated and diluted. The reason may be that the rats are small in size and have less gastrointestinal fluid, so ALT-SMEDDS is not fully assimilated after entering the gastrointestinal tract and requires a longer hydration time, so the blood drug concentration is lower than that after direct dilution.

[0178] Table 2 Main pharmacokinetic parameters after oral administration of three different preparations (Mean±SD)

[0179]

[0180] Note: Compared with allylprogesterone oil solution: *, P < 0.05, **, P < 0.01; compared with allylprogesterone nano preconcentrate: b, P < 0.01.

[0181] 4.5 Verification of lymphatic absorption of allylgestaltenone nanoparticle preconcentrate

[0182] The plasma concentration-time curve and pharmacokinetic parameters of rats injected intraperitoneally with 3 mg / kg lymphatic blocker CHX and then orally administered with the rehydrated solution of allergy-progesterone nano-preconcentrate were as follows: Figure 8 As shown in the figure, it can be seen that after intraperitoneal injection of CHX, the blood concentration of allylprogesterone decreased significantly, and both Cmax and AUC0-2h were significantly different from those of the group not treated with CHX (p < 0.05), indicating that the pre-concentrated hydrated solution of allylprogesterone has a tendency of lymphatic transport ( Figure 8 ).

[0183] 5 Conclusion

[0184] In this experiment, an allylprogesterone nano-preconcentrate is prepared to encapsulate allylprogesterone to improve its water solubility. The allylprogesterone nano-preconcentrate prepared by the present invention has a clear and transparent appearance, good fluidity, and a hydration time of 35.76±1.10s to form a nano-solution. The emulsion droplets after nano-solution have a round and uniform appearance and no adhesion. The average particle size is 18.39±0.03nm, the PDI is 0.083±0.090, and the Zeta potential is -1.12±0.12mV. It can remain stable within a dilution range of 10 to 1000 times, and has a certain dilution stability. The cumulative release of allylprogesterone preconcentrated hydrated solution in vitro can reach 91.41% in 48 hours, which is significantly improved compared with 32.42% of the original drug. The allylprogesterone nano preconcentrated solution is easy to use and can be mixed with water at will, which solves the shortcoming that the traditional allylprogesterone oil solution is immiscible with water; at the same time, it can promote the absorption of allylprogesterone through lymphatic circulation and improve the bioavailability. The bioavailability of the nano preconcentrated solution after oral absorption after hydration is 497% of the allylprogesterone oil solution, thereby reducing the clinical dosage of allylprogesterone or reducing the number of dosing times, thereby alleviating stress on animals.

[0185] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. An allylgestalten nano preconcentrate, characterized in that: Made of the following raw materials in percentage by weight: Progesterone: 2%; Ethyl oleate: 21.5%; Polyoxyethylene sorbitan monooleate: 53.1%; Ethyl carbitol: 20.8%; Ethanol: 2.6 %.

2. The method for preparing the allylgestaltin nano preconcentrate as claimed in claim 1, characterized in that: The following steps are involved: ① placing the ethyl oleate, polyoxyethylene sorbitan monooleate, ethyl carbitol and ethanol in a centrifuge tube, and vortexing to mix them evenly to obtain a mixed solvent; ② Then, the allergy-progesterone is added to the mixed solvent, and vortexed, ultrasonicated, and shaken in a water bath until the allergy-progesterone is completely dissolved to obtain an allergy-progesterone nano pre-concentrated solution.

3. The method for preparing the allylgestaltin nano preconcentrate according to claim 2, characterized in that: In step ①, vortex and shake for 3-5 min.

4. The method for preparing the allylgestaltin nano preconcentrate according to claim 2, characterized in that: In the step ②, vortex for 3-5 min, ultrasonicate for 2-3 min, and balance under 37° C. water bath shaking conditions until the allergy to progesterone is completely dissolved to obtain the allergy to progesterone nano pre-concentrate.

5. A method for preparing a pre-concentrated hydrated solution of allylgestaltenone, characterized in that: The following steps are involved: The allylpregestrin nano preconcentrated solution according to claim 1 is added into ultrapure water, and hydrated to prepare a solution under 50-200 r / min magnetic stirring conditions, wherein the solid-liquid ratio of the allylpregestrin nano preconcentrated solution to ultrapure water is 1 g:10-1000 mL, and when the solution is clear and transparent with light blue opalescence, the allylpregestrin preconcentrated hydrated solution is obtained.

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