In-situ sustained-release gel carrier, fat-soluble vitamin in-situ sustained-release gel and preparation method and application thereof

Abstract

The invention provides an in-situ slow-release gel carrier. The in-situ slow-release gel carrier comprises the following raw materials in parts by weight: 40-70 parts of egg yolk lecithin; 15 to 30 parts of tricaprylin; and 15 to 30 parts of normal propyl alcohol. According to the in-situ sustained-release gel disclosed by the invention, the egg yolk lecithin which has similar compatibility with fat-soluble vitamin D3 and can be used for better dissolving the vitamin D3 is taken as a carrier, and the prepared in-situ sustained-release gel can be used for slowly releasing the vitamin D3 under the combined action of the tricaprylin and the n-propyl alcohol, and meanwhile, the hypocalcemia of the dairy cow is prevented.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to an in-situ slow-release gel carrier, an in-situ slow-release gel of a fat-soluble vitamin, and a preparation method and application thereof. BACKGROUND

[0002] Low blood calcium of dairy cows is a metabolic disease that mainly occurs in the perinatal period and is dominated by negative calcium metabolism balance. It mainly occurs after delivery, and the incidence of subclinical hypocalcemia is about 30%, and the incidence of clinical hypocalcemia is about 3%. Affected cows have limb weakness, reduced feed intake, and stopped ruminating, and can also cause other diseases such as retained placenta, displacement of the abomasum, ketosis, and endometritis, and can even cause death in severe cases.

[0003] There are more than 6.7 million dairy cows, nearly 100 million beef cattle, and a large number of yaks and buffaloes in China, all of which are threatened by hypocalcemia after delivery. The market space for the prevention and control of such diseases is huge.

[0004] Vitamin D3 is one of the important members in the bone metabolism pathway, and it co-regulates the balance of calcium and phosphorus in the body with parathyroid hormone and calcitonin. Vitamin D3 mainly regulates the balance of plasma calcium and phosphorus by promoting the absorption of calcium and phosphorus in the intestinal tract, mobilizing bone calcium, and promoting the reabsorption of calcium and phosphorus in the kidney. Feeding or injecting vitamin D3 to dairy cows and dairy goats 10-14 days before delivery can promote the absorption of calcium in the intestinal tract and effectively reduce the incidence of hypocalcemia. Compared with the downstream activated components of vitamin D3, vitamin D3 is inexpensive and more gentle and controllable in blood concentration compared with calcium supplement preparations. However, the current treatment method is still a one-time large dose of vitamin D3, which can inhibit the concentration of parathyroid hormone (PTH) and leave a hidden danger of soft tissue calcium transfer.

[0005] Therefore, there is an urgent need for a method for slow release of vitamin D3 to reduce the harm of one-time large dose. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide an in-situ slow-release gel carrier, and the in-situ slow-release gel prepared by the present application can slow release vitamin D3 and prevent hypocalcemia in dairy cows.

[0007] The present application provides an in-situ slow-release gel carrier, which comprises the following raw materials by weight:

[0008] Egg yolk lecithin 40-70 parts;

[0009] Tricaprylin 15-30 parts;

[0010] n-Propyl alcohol 15-30 parts.

[0011] The in-situ sustained-release gel carrier provided by the application comprises 40-70 parts by weight of egg yolk lecithin; specifically, it can be 40 parts by weight, 41 parts by weight, 42 parts by weight, 43 parts by weight, 44 parts by weight, 45 parts by weight, 46 parts by weight, 47 parts by weight, 48 parts by weight, 49 parts by weight, 50 parts by weight, 51 parts by weight, 52 parts by weight, 53 parts by weight, 54 parts by weight, 55 parts by weight, 56 parts by weight, 57 parts by weight, 58 parts by weight, 59 parts by weight, 60 parts by weight, 61 parts by weight, 62 parts by weight, 63 parts by weight, 64 parts by weight, 65 parts by weight, 66 parts by weight, 67 parts by weight, 68 parts by weight, 69 parts by weight or 70 parts by weight.

[0012] The in-situ sustained-release gel carrier provided by the application comprises 15-30 parts by weight of tricaprylin; the application does not limit the above-mentioned tricaprylin, and a commercially available product can be used. Specifically, it can be 15 parts by weight, 16 parts by weight, 17 parts by weight, 18 parts by weight, 19 parts by weight, 20 parts by weight, 21 parts by weight, 22 parts by weight, 23 parts by weight, 24 parts by weight, 25 parts by weight, 26 parts by weight, 27 parts by weight, 28 parts by weight, 29 parts by weight or 30 parts by weight.

[0013] The in-situ sustained-release gel carrier provided by the application comprises 15-30 parts by weight of n-propanol. Specifically, it can be 15 parts by weight, 16 parts by weight, 17 parts by weight, 18 parts by weight, 19 parts by weight, 20 parts by weight, 21 parts by weight, 22 parts by weight, 23 parts by weight, 24 parts by weight, 25 parts by weight, 26 parts by weight, 27 parts by weight, 28 parts by weight, 29 parts by weight or 30 parts by weight.

[0014] The application provides application of the in-situ sustained-release gel carrier in preparation of an in-situ sustained-release preparation of a fat-soluble vitamin.

[0015] According to the application, the fat-soluble vitamin comprises vitamin D3, vitamin A and / or vitamin E. In particular, the fat-soluble vitamin is vitamin D3.

[0016] The application provides an in-situ sustained-release gel of a fat-soluble vitamin, which comprises the following raw materials in parts by weight:

[0017] Egg yolk lecithin 40-70 parts;

[0018] Fat-soluble vitamin 5-70 parts;

[0019] Tricaprylin 15-30 parts;

[0020] n-Propanol 15-30 parts

[0021] The vitamin includes vitamin D3, vitamin A and / or vitamin E.

[0022] The liposoluble vitamin in-situ sustained-release gel provided by the application includes 40-70 parts by weight of egg yolk lecithin; specifically, 40 parts by weight, 41 parts by weight, 42 parts by weight, 43 parts by weight, 44 parts by weight, 45 parts by weight, 46 parts by weight, 47 parts by weight, 48 parts by weight, 49 parts by weight, 50 parts by weight, 51 parts by weight, 52 parts by weight, 53 parts by weight, 54 parts by weight, 55 parts by weight, 56 parts by weight, 57 parts by weight, 58 parts by weight, 59 parts by weight, 60 parts by weight, 61 parts by weight, 62 parts by weight, 63 parts by weight, 64 parts by weight, 65 parts by weight, 66 parts by weight, 67 parts by weight, 68 parts by weight, 69 parts by weight or 70 parts by weight.

[0023] The liposoluble vitamin in-situ sustained-release gel provided by the application includes 5-70 parts of liposoluble vitamin.

[0024] The liposoluble vitamin in-situ sustained-release gel provided by the application includes 15-30 parts by weight of tricaprylin; the application does not limit the above-mentioned tricaprylin, which can be commercially available. Specifically, 15 parts by weight, 16 parts by weight, 17 parts by weight, 18 parts by weight, 19 parts by weight, 20 parts by weight, 21 parts by weight, 22 parts by weight, 23 parts by weight, 24 parts by weight, 25 parts by weight, 26 parts by weight, 27 parts by weight, 28 parts by weight, 29 parts by weight or 30 parts by weight.

[0025] The present inventors have creatively found that tricaprylin has better technical effects than MCT and other remaining medium-chain glycerides.

[0026] The liposoluble vitamin in-situ sustained-release gel provided by the application includes 15-30 parts by weight of n-propanol; specifically, 15 parts by weight, 16 parts by weight, 17 parts by weight, 18 parts by weight, 19 parts by weight, 20 parts by weight, 21 parts by weight, 22 parts by weight, 23 parts by weight, 24 parts by weight, 25 parts by weight, 26 parts by weight, 27 parts by weight, 28 parts by weight, 29 parts by weight or 30 parts by weight.

[0027] The application provides a preparation method of the liposoluble vitamin in-situ sustained-release gel.

[0028] The egg yolk lecithin, the liposoluble vitamin, the tricaprylin and the n-propanol are mixed, dissolved, ultrasonically treated, rotary evaporated, redissolved and filtered, and then the liposoluble vitamin in-situ sustained-release gel is obtained.

[0029] The application does not limit the source of the above-mentioned raw materials, which can be commercially available. The above-mentioned raw materials are described in detail above, and thus no further description is given here.

[0030] The mixing in the present application is mixing by vortex mixer, magnetic stirring at room temperature until complete dissolution, ultrasonic treatment for 10 min, then rotary evaporation in a rotary evaporation vessel at 37 DEG C and 0.1 MPa, then adding n-propanol for dissolution to obtain a clear and transparent liquid, filtering through a 0.22 mu m microporous filter to obtain a golden yellow transparent liquid containing a large amount of bubbles, standing until the bubbles basically disappear, and obtaining a completely prepared gel.

[0031] According to the present application, the mass ratio of the egg yolk lecithin, fat-soluble vitamin, tricaprylin and n-propanol is 4-7 g:4-7 g:1.5-3 mL:1.5 mL-3;

[0032] Particularly preferably, the mass ratio of the egg yolk lecithin, fat-soluble vitamin, tricaprylin and n-propanol is 7 g:7 g:1.5 mL:1.5 mL.

[0033] The present application provides the use of the above-mentioned fat-soluble vitamin in-situ sustained-release gel or the fat-soluble vitamin in-situ sustained-release gel prepared by the preparation method in the above technical solution in the preparation of a drug for preventing hypocalcemia of dairy cows.

[0034] The dairy cow in the present application is a perinatal period.

[0035] The purpose of the present application is to design a new dosage form of a sustained-release drug for treating perinatal diseases of dairy cows, and the emphasis on the sustained-release function is to improve the convenience of clinical operation of veterinarians in dairy farms and reduce the stimulation to perinatal cows (perinatal dairy cows are more sensitive to stimulation and more prone to stress).

[0036] The present application provides a drug for preventing hypocalcemia of dairy cows, which comprises the above-mentioned fat-soluble vitamin in-situ sustained-release gel or the fat-soluble vitamin in-situ sustained-release gel prepared by the preparation method in any one of the above technical solutions.

[0037] The dosage form of the drug in the present application includes injection, oral agent, spray;

[0038] The administration dose of the injection in the present application is 0.2 ml / animal / time.

[0039] The injection is administered by subcutaneous intramuscular injection, and the recommended injection site is the neck or the place with abundant muscle in the hip. Before injection, the injection site needs to be strictly disinfected, and 75% alcohol cotton ball is used to wipe the skin. After the alcohol evaporates, the injection operation is performed. The drug should be slowly injected during injection to avoid local irritation or stress reaction of the dairy cow caused by too fast injection. The specification of each injection is 0.2 ml / branch, and the appearance of the drug should be checked before use. If abnormal phenomena such as turbidity, precipitation or discoloration of the drug solution are found, the drug should not be used.

[0040] The adjuvant that can be selected by the medicine of the application includes but is not limited to: injection can add suspending agent (such as sodium carboxymethyl cellulose), osmotic pressure regulator (such as sodium chloride), pH regulator (such as phosphate buffer); oral agent can add diluent (such as microcrystalline cellulose), disintegrant (such as crosslinked sodium carboxymethyl cellulose), flavoring agent (such as sucrose); spray can add propellant (such as hydrofluoroalkane), surfactant (such as Tween-80). The above adjuvant should meet the quality standard of veterinary drugs, and the amount should meet the conventional range in the art.

[0041] When using the medicine of the application to prevent hypocalcemia of dairy cows, the reaction and health status of the dairy cow should be closely observed. If abnormal symptoms such as loss of appetite, listlessness, diarrhea, etc. are found in the dairy cow, the medicine should be stopped in time, and the veterinarian should be consulted. At the same time, the medicine record should be well kept, including the medicine time, dose, reaction of the dairy cow, etc. so as to evaluate and summarize the effect of the medicine.

[0042] The application provides an in-situ slow-release gel carrier, which comprises the following raw materials in parts by weight: egg yolk lecithin 40-70 parts; tricaprylin 15-30 parts; n-propanol 15-30 parts. The application can better dissolve the egg yolk lecithin of vitamin D3 by using the egg yolk lecithin having similar compatibility with the fat-soluble vitamin D3 as the carrier, and the prepared in-situ slow-release gel can slow release the vitamin D3 by the joint action of tricaprylin and n-propanol, and prevent hypocalcemia of dairy cows. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 . Metabolic results of the drug library in rats;

[0044] Figure 2 . Schematic diagram of the pharmacokinetic results of the solvent component ethanol group and n-propanol group in rat in-vivo experiment;

[0045] Figure 3 . Rabbit skin tissue HE staining result diagram;

[0046] Figure 4 . Schematic diagram of vitamin E and vitamin A phospholipid gel medicine;

[0047] Figure 5 . Schematic diagram of meloxicam medicine;

[0048] Figure 6 . Schematic diagram of meloxicam medicine;

[0049] Figure 7 From left to right, the drug medicine schematic diagram of egg yolk lecithin with 40%, 50% and 60% concentration;

[0050] Figure 8From left to right, respectively, 40%, 50%, 60% concentration of egg yolk lecithin subcutaneous drug depot medicine situation diagram;

[0051] Figure 9 Three different kinds of lecithin drug medicine diagram;

[0052] Figure 10 Egg yolk lecithin group in vitro sustained-release results;

[0053] Figure 11 Soybean lecithin group in vitro sustained-release results;

[0054] Figure 12 Hydrogenated soybean lecithin group in vitro sustained-release results;

[0055] Figure 13 From left to right, respectively, 25%, 10% and 5% solvent content of sustained-release system medicine diagram;

[0056] Figure 14 Ethanol composition medicine diagram;

[0057] Figure 15 n-propanol composition medicine diagram;

[0058] Figure 16 Glycerol composition medicine diagram;

[0059] Figure 17 1,2-propanediol composition medicine diagram;

[0060] Figure 18 Plasma total calcium content data results;

[0061] Figure 19 Plasma alkaline phosphatase content data results;

[0062] Figure 20 Plasma phosphorus content data results;

[0063] Figure 21 Plasma magnesium content data results;

[0064] Figure 22 Egg yolk lecithin sustained-release carrier dosage form of the nature, phase change process and after phase change diagram;

[0065] Figure 23 Plasma PTH data results;

[0066] Figure 24 Plasma CT data results;

[0067] Figure 25 Plasma 25 hydroxy vitamin D3 data results.

[0068] Figure 26 Fluorescence inverted microscope under the morphology of the drug

[0069] Figure 27 The morphology of the non-distilled drug group under the fluorescence inverted microscope DETAILED DESCRIPTION

[0070] The present application provides a kind of in situ sustained-release gel carrier, liposoluble vitamin in situ sustained-release gel and its preparation method and application, those skilled in the art can learn from the content herein, and appropriate improvement process parameters are realized.It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they all belong to the scope protected by the present application.The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application to realize and apply the present application technology.

[0071] It should be understood that the expression "one or more of" includes each object described after the expression and various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0072] The terms "comprising", "having" or "including", including the use of their grammatical synonyms, should generally be understood to be open-ended and non-limiting, for example, not excluding other non-recited elements or steps, unless otherwise specifically stated or understood from the context.

[0073] In this application, the term "and / or", describes the association relationship of associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent: A exists alone, A and B exist together, and B exists alone. Where A, B can be singular or plural.

[0074] In this application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single or multiple items.

[0075] It should be understood that the order of steps or the order of performing certain actions is not important as long as the present application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0076] The use of any and all examples, or exemplary language herein, such as "for example" or "including", is intended merely to better illustrate the present application and does not pose a limitation on the scope of the present application unless a claim is presented. Any language in this specification should not be interpreted as indicating any unclaimed element is essential to the practice of the present application.

[0077] In addition, the numerical ranges and parameters given in the description of the application are approximate values, and the numerical values in the specific examples have been presented as precisely as possible. However, any numerical value inherently involves a certain standard deviation due to the individual test method. Therefore, unless explicitly stated otherwise, all ranges, quantities, numerical values and percentages used in the present disclosure are to be understood as being modified by "about". Herein, "about" generally means within 10%, 5%, 1% or 0.5% of a specified value or range.

[0078] It should be understood that the size of the sequence number of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0079] Some cases are described in the examples and comparative examples of the present application, in which the examples show certain implementations of the present application. However, this does not mean that the effects of the present application can only be achieved in these cases.

[0080] In order to further illustrate the present application, the in-situ sustained-release gel carrier, the in-situ sustained-release gel of fat-soluble vitamins, and the preparation method and application thereof provided by the present application are described in detail below in combination with examples.

[0081] Example 1

[0082] Phospholipid in-situ gel formula loaded with vitamin D3: Weigh 7 g of egg yolk lecithin and 7 g of vitamin D3, add excess n-propanol (about 10 mL), 1.5 mL of glyceryl tricaprylate, mix uniformly using a vortex mixer, magnetically stir at room temperature until completely dissolved, ultrasonic for 10 min, then spin in a rotary evaporator container at 37℃, 0.1 MPa, then add 1.5 mL of n-propanol for dissolution, obtain a clear and transparent liquid, filter through a 0.22 μm microporous filter membrane, obtain a golden yellow transparent liquid containing a large amount of bubbles, stand until the bubbles basically disappear, and obtain a completely prepared gel.

[0083] Experimental design:

[0084] 1. Animal grouping:

[0085] SPF female SD rats (8 weeks old, body weight 200 g) were purchased from Sibeifeng (Beijing) Biotechnology Co., Ltd. with production license number SCXK (Jing) 2019-0010. The animal feeding conditions were under the conditions of temperature 25℃ and humidity 60%, following a 12 h cycle of day and night. The rats were randomly divided into 4 groups after being adaptively fed with mouse and rat maintenance feed for 3 days:

[0086] Control group: normal feeding without treatment;

[0087] Oral group: intragastric administration of vitamin D3 corn oil 0.2ml (containing VD3 200IU), a total of 1d;

[0088] Ethanol group: subcutaneous injection of vitamin D3 gel containing ethanol as solvent 0.2ml (containing VD3 800IU) per rat;

[0089] Propanol group: subcutaneous injection of vitamin D3 gel containing n-propanol as solvent 0.2ml (containing VD3 800IU) per rat;

[0090] 2 Determination of subcutaneous gel metabolic absorption

[0091] On the 5th day, the 10th day and the 14th day after injection of phospholipid in situ gel, one rat was randomly selected from each group (ethanol group and propanol group), and the skin of the neck and back of the gel injection site was cut after lindane anesthesia. The metabolic absorption of the in situ gel under the skin was observed, and the gel release time was evaluated.

[0092] The results are shown in Figure 1 The, Figure 1 The metabolic results of the drug depot in rats are shown in Figure 1 It can be seen that after selecting two different solvents (ethanol and n-propanol) to load vitamin D3 for release effect screening, the two groups of phospholipid in situ gels arranged in combination are injected subcutaneously into rats, and the phase change and release metabolism are observed. Autopsy was performed at 5 days, 10 days and 14 days after injection to observe the gel absorption at the injection site (see the above abc, the left side of each figure is the ethanol group and the right side is the propanol group). It is verified that the two groups of phospholipid gels can be completely metabolized and absorbed in 14 days.

[0093] 3 Determination of 25-(OH) vitamin D3 level in plasma

[0094] Because the status of vitamin D3 is evaluated by measuring 25-(OH) vitamin D3, the above experimental rats were used to collect 0.5mL of whole blood from the infraorbital vein of the rats using a capillary at the time nodes of 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 96, 144, 192, 264, 336h after injection of vitamin D3-loaded phospholipid in situ gel (note that the flat end of the capillary contacts the infraorbital vein of the rat) into a heparin anticoagulated ep tube, and centrifuged (8250xg, 5min, 4℃) within 2h using a refrigerated benchtop centrifuge (Multifuge™ X4 RPro CTS). The supernatant was taken and stored in a -80℃ freezer for determination of the content of 25-(OH) vitamin D3 in plasma to evaluate the release of vitamin D3 in rats.

[0095] Results are shown in Figure 2 Table 2, and Figure 2 The results of the rat in vivo experiment of the solvent component ethanol group and n-propanol group are shown in Figure 2. It can be observed from the analysis of the rat plasma that the two solvent experimental groups can avoid burst release compared with the oral group, and have vitamin D3 release compared with the control group. It can be observed that n-propanol is more stable than the ethanol group.

[0096] 4 New Zealand white rabbit subcutaneous irritation test

[0097] Select 3 female New Zealand white rabbits weighing 2 kg, purchased from Beijing Keyu Animal Breeding Center. Select injection grade soybean oil as the control group of the irritation test, and phospholipid in situ gel with propyl alcohol as the solvent as the experimental group. The rabbits were adaptively fed for 1 day, and the hair was removed using an electric shaver. After treatment, the skin was allowed to stand for 24 h to eliminate the irritation of the shaving process. Then, the control group of injection grade soybean oil was injected subcutaneously at 5 points on one side of the rabbit's back, and the experimental group of gel was injected subcutaneously on the opposite side of the back. The injection sites were observed for redness, swelling, and ulceration for 3 days after injection. Then, the rabbits were anesthetized with lumbosine and sacrificed, and the skin at the injection sites of the control and experimental groups was sampled. After fixation with paraformaldehyde, the samples were sent for sectioning and HE staining to observe whether there were tissue damage and inflammatory cell aggregation at the injection sites to evaluate the irritation of the phospholipid gel.

[0098] Results are shown in Figure 3 Table 2, and Figure 3 The results of the rabbit skin tissue HE staining are shown in Figure 3. The irritation of the phospholipid gel after subcutaneous injection in New Zealand white rabbits was verified. According to the above in vivo release results, n-propanol was selected as the solvent to prepare the phase change gel. The injection grade soybean oil of the control group (left 1) and the drug injection of the experimental group (left 2, left 3) did not produce strong irritation to the subcutaneous tissue, and no inflammatory cell enrichment and severe tissue damage were observed. The tissue morphology was complete in the section, and the phospholipid microsphere structure injected subcutaneously could be seen. The phospholipid microspheres were distributed in the tissue interstitium, and the compatibility was good. It was proved that the phospholipid gel had low irritation to the body.

[0099] Example 2

[0100] Vitamin A-loaded: Vitamin A 7 ml was added to 7 g egg yolk lecithin, 1.5 ml trioctanoin, and excess n-propanol (about 10 mL), stirred at room temperature using a magnetic stirrer, ultrasonicated for 10 min, then rotary evaporated in a rotary evaporation container at 37°C and 0.1 MPa. Then 1.5 mL of n-propanol was added for dissolution, and a clear oily liquid was obtained. After passing through a 0.22 um filter membrane, the foam was removed after standing, and the prepared gel was obtained.

[0101] Vitamin E: Vitamin E 7ml, added to 7g egg yolk lecithin and 1.5ml tricaprylin and excess n-propanol (about 10mL), ultrasonic 10min, then to a rotary evaporation container, rotary evaporation at 37℃, 0.1MPa, then 1.5mL n-propanol was added for dissolution, using a magnetic stirrer to stir at room temperature, a clear oily liquid was obtained, after 0.22um filter membrane, standing to defoam, the prepared gel was obtained. Figure 4 Vitamin E and Vitamin A phospholipid gel)

[0102] Then in vitro sustained release, take 25mm size dialysis bag (filtration 8KD-14KD), using a 1000uL pipette: each take 1mL medicine, add to the dialysis bag, clamp with dialysis clamp at both ends, put into a beaker. Add 100ml pre-warmed 37℃ PBS solution to the beaker, place the beaker on a multi-channel magnetic stirrer at 100rpm, 37℃, release the drug for 1 week. And at 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h, 168h after the start of release, change the solution, also add 100ml 37℃ PBS solution. Take 5mL of the changed solution into a 10ml centrifuge tube and freeze at -80℃ freezer for subsequent detection.

[0103] Comparative Example 1

[0104] Take meloxicam 7g, add to 7g egg yolk lecithin and 1.5ml tricaprylin and excess n-propanol (about 10mL), stir using a magnetic stirrer at room temperature, which cannot be well dissolved, after warming and dissolving, a viscous layered liquid is formed, the texture is unstable, and cannot be well formed into a medicine. Figure 5 , 6 ), Figure 5 , 6 Meloxicam group can see stratification.

[0105] Example 3

[0106] Design 40%, 50%, 60% concentration of phospholipid gel group, do rat in vivo phase change test

[0107] Prepare a group of 40%, 50%, 60% egg yolk lecithin content.

[0108] 40% group is 40% egg yolk lecithin, 30% tricaprylin, 30% n-propanol, i.e. 4g egg yolk lecithin, tricaprylin 3mL, n-propanol 3mL.

[0109] 50% group is egg yolk lecithin 50%, tricaprylin 25%, n-propanol 25%, namely 5g egg yolk lecithin, tricaprylin 2.5mL, n-propanol 2.5mL.

[0110] 60% group is egg yolk lecithin 60%, tricaprylin 20%, n-propanol 20%, namely 6g egg yolk lecithin, tricaprylin 2mL, n-propanol 2mL. Figure 7 Three groups of drug groups with different phospholipid concentrations,

[0111] The 40% group has good fluidity, the 50% group is less viscous, the 60% group is more viscous, and shaking the medicine bottle will have a wall hanging phenomenon.

[0112] 0.5ml was injected subcutaneously into the neck of a 200g female SD rat, and 3 days later, necropsy was performed, and it was observed that drug depots were formed in the subcutaneous tissue of the neck Figure 8 The subcutaneous drug depot was formed, from left to right, 40%, 50%, 60%, and the 40% group was relatively less, and the 40% group had better plasticity when the drug depot was touched, and the 50% group and the 60% group were more elastic. It is speculated that for the purpose of achieving long-term sustained release, a higher proportion of phospholipid concentration is more suitable.

[0113] Example 4

[0114] Egg yolk lecithin group: 7g of vitamin D3 was added to 7g of egg yolk lecithin, 1.5ml of tricaprylin and excess n-propanol (about 10mL), a magnetic stirrer was used to stir at room temperature, ultrasonic treatment was performed for 10min, then it was transferred to a rotary evaporation container and rotary evaporation was performed at 37℃ and 0.1MPa, then 1.5mL of n-propanol was added for dissolution, a clear oily liquid was obtained, after filtration through a 0.22um filter membrane, standing and defoaming, the prepared gel was obtained.

[0115] Soybean lecithin group: 7g of vitamin D3 was added to 7g of soybean lecithin, 1.5ml of tricaprylin and excess n-propanol (10mL), a magnetic stirrer was used to stir at room temperature, a clear oily liquid was obtained, after filtration through a 0.22um filter membrane, standing and defoaming, the prepared gel was obtained.

[0116] Hydrogenated soybean lecithin group (comparative example): 7g of vitamin D3 was added to 7g of hydrogenated soybean lecithin, 1.5ml of tricaprylin and excess n-propanol (about 10mL), a magnetic stirrer was used to stir at room temperature, it was observed that it could not be dissolved, it was in a semi-solid state, and heating to promote dissolution could not dissolve it, so a component with a hydrogenated soybean lecithin content of 50% was prepared, namely, the composition of hydrogenated soybean lecithin accounted for 50%, n-propanol accounted for 25%, and tricaprylin accounted for 25%, after heating to promote dissolution, it was in a paste state with high viscosity, and it was speculated that it could not be used as an injection drug. Figure 9).

[0117] Figure 9 From left to right are egg yolk lecithin group (70%), soybean lecithin group (70%) and hydrogenated soybean lecithin group (50%). It can be observed that the viscosity of the paste formed by the hydrogenated soybean lecithin group is too high.

[0118] In vitro sustained release was carried out under the same drug replacement conditions. After collecting the samples, Waters ARC-HPLC was used for detection. It can be observed that the egg yolk lecithin group (70%) showed a rapid release after 0.25h and then returned to a stable state, and slow release of vitamin D3 can be detected at subsequent time points; for the soybean lecithin group, the drug depot did not solidify rapidly at the beginning of the sustained release, and the release peak appeared at 0.5h, and vitamin D3 was basically undetectable at subsequent time points, suggesting that the drug release performance was poor during the solidification period, and only relied on the subsequent metabolism of soybean lecithin in the body to release the drug. Figure 10 The soybean lecithin group can be seen to solidify slowly, and a burst peak appears at 4h, and then it releases slowly, and vitamin D3 is basically no longer released at subsequent detection time points); for the hydrogenated soybean lecithin group, its drug depot solidification performance is similar to that of soybean lecithin, and a burst peak appears at 0.5h, and then it releases stably after 2h, but a burst release appears at 48h, and then the release decreases, and it cannot be detected at 120h. By evaluating the drug loading and sustained release ability of the three carriers, it is found that the carrier with egg yolk lecithin as the component has better sustained release performance. Figure 11 Figure 12 Example 5

[0119] Example 5

[0120] ① Egg yolk lecithin 70%, tricaprylin 5%, n-propanol 25%, i.e. 14g of egg yolk lecithin, 1mL of tricaprylin, 5mL of n-propanol.

[0121] ② Egg yolk lecithin 70%, tricaprylin 20%, n-propanol 10%, i.e. 14g of egg yolk lecithin, 4mL of tricaprylin, 2mL of n-propanol.

[0122] ③ Egg yolk lecithin 70%, tricaprylin 25%, n-propanol 5%, i.e. 14g of egg yolk lecithin, 5mL of tricaprylin, 1mL of n-propanol.

[0123] From left to right, the concentration of n-propanol solvent gradually decreases, and it can be seen that the egg yolk lecithin is not well dissolved and cannot form a uniform texture preparation. Compared with the previous egg yolk lecithin 70%, tricaprylin 15%, n-propanol 15% which can form a drug, it is determined that the concentration of the solvent should not be lower than 15%.( Figure 13 )

[0124] ​Figure 13 From left to right, the solvent concentration decreases in turn, and it is observed that the prescription of n-propanol 10% is difficult to dissolve.

[0125] Example 6

[0126] Preparation of egg yolk lecithin 70%, tricaprylin 15%, and change the type of solvent composition, respectively, ethanol group, n-propanol group, glycerol group and 1,2-propanediol group.

[0127] ① Ethanol group: egg yolk lecithin 70%, tricaprylin 15%, ethanol 15%, that is, 14g of egg yolk lecithin, tricaprylin 3mL, ethanol 3mL.

[0128] ② n-propanol group: egg yolk lecithin 70%, tricaprylin 15%, n-propanol 15%, that is, 14g of egg yolk lecithin, tricaprylin 3mL, n-propanol 3mL.

[0129] ③ Glycerol group: egg yolk lecithin 70%, tricaprylin 15%, glycerol 15%, that is, 14g of egg yolk lecithin, tricaprylin 3mL, glycerol 3mL.

[0130] ④ 1,2-propanediol group: egg yolk lecithin 70%, tricaprylin 15%, 1,2-propanediol 15%, that is, 14g of egg yolk lecithin, tricaprylin 3mL, 1,2-propanediol 3mL.

[0131] Observation of drug properties can see ethanol group (see Figure 14 Ethanol group, can see liquid texture golden yellow clear) and n-propanol group (see Figure 15 n-propanol group, can see liquid texture golden yellow clear) is a clear golden yellow slightly viscous liquid, shape stable, no delamination phenomenon.

[0132] And glycerol group after overnight stirring, it is observed that the system is wax-like, no flow, can not be pumped, it is inferred that glycerol can not be an effective solvent (see Figure 16 Glycerol group, can not be dissolved). At the same time, 1,2-propanediol group after overnight stirring, get can not be completely dissolved, have mass, texture too viscous can not reach the standard of injection of golden yellow liquid, no delamination, it is inferred that 1,2-propanediol may not be an ideal effective solvent (see Figure 17 1,2-propanediol group, can see there are solid block).

[0133] Verification example 1

[0134] In order to detect the clinical preventive effect of the vitamin D3-loaded egg yolk lecithin group, a test was conducted in a commercial dairy farm. In this test, 60 Holstein cows with similar expected delivery dates, actual delivery dates within ±3 days of the expected delivery date, more than 2 fetal times, and body weight of 700±50 kg were used as test objects. The cows were fed with the same prepartum and postpartum diets before and after delivery, and were randomly divided into 5 groups, namely, a negative control group, a positive control group, a test drug 0.5 dose group, a test drug 1 dose group, and a test drug 3 dose group.

[0135] Each experimental group was:

[0136] The negative control group (group A): The cows were not treated as the negative control group.

[0137] The positive control group (group B): A commercial vitamin D3 injection (1 ml: 7.5 mg) was used as the positive control.

[0138] The test drug 0.5 dose group (group C): 0.5 times the drug per 1 ml, containing 0.7 g of egg yolk lecithin, 0.15 ml of n-propanol, 0.15 ml of trioctanoin, and 0.35 g of vitamin D3.

[0139] The test drug 1 dose group (group D): 1 times the drug per 1 ml, containing 0.7 g of egg yolk lecithin, 0.15 ml of n-propanol, 0.15 ml of trioctanoin, and 0.7 g of vitamin D3.

[0140] The test drug 3 dose group (group E): 1 times the drug per 1 ml, containing 0.7 g of egg yolk lecithin, 0.15 ml of n-propanol, 0.15 ml of trioctanoin, and 0.7 g of vitamin D3. (Since the release time of the sustained-release system is related to the ratio of the drug depot carrier and the volume formed by the drug depot, the ratio of the drug was not changed to control the variables, and the test cows were injected with 1 dose group 3 times, so that the 3 dose group and the 1 dose group were consistent in metabolic time, but the 3 dose group released 3 times the amount of the 1 dose group in unit time)

[0141] The five groups of test cattle were treated and injected with drugs 7 days before the expected delivery date, according to the recommended usage and dosage, namely, the negative control group was not treated; 1 dose and 0.5 dose were subcutaneously injected with 1 dose and 0.5 dose of vitamin D3 sustained-release phase change gel at a dose of 0.2 mL / head / time, respectively; the positive control group was subcutaneously injected with vitamin D3 injection at a dose of 2 mL / head / time; the 3 dose group was subcutaneously injected at a dose of 0.2 mL / head / time for three times (total 0.6 mL). Tail vein blood samples were collected from the cows before injection, at delivery, and 1 day, 2 days, 3 days, 5 days, and 7 days after delivery, and centrifuged (8250 x g, 5 min, 4°C) within 2 hours using a refrigerated tabletop centrifuge (Multifuge™ X4 RPro CTS), and the supernatant was stored in a -20°C freezer for subsequent detection of total calcium, phosphorus, magnesium, and alkaline phosphatase (ALP). After delivery, the occurrence of clinical hypocalcemia was observed, and the number of subclinical hypocalcemic cows was counted.

[0142] The total calcium in the blood was used as the main indicator, and magnesium, phosphorus, and ALP were used as secondary indicators. The effect of the test drug, vitamin D3 sustained-release phase change gel, on the prevention of postpartum hypocalcemia in postpartum cows was evaluated by comparing it with the negative control group and the positive control drug (vitamin D3 injection), and the recommended dose was determined.

[0143] During the collection of samples: one cow in the negative control group was unable to stand and was eliminated from the lease due to clinical hypocalcemia, and another cow developed metritis due to retained placenta (classified as a complication of hypocalcemia), and two cows died in the disease group. One cow in the positive control group developed a true stomach displacement and was eliminated from the group due to poor recovery after using a blind needle to fix it, and another cow was eliminated from the group due to a fall injury caused by violent driving, and one cow developed metritis due to retained placenta in the 0.5 dose group, which was calculated as a complication of hypocalcemia, and one cow developed metritis due to retained placenta in the 1 dose group and the 3 dose group.

[0144] The calculated clinical hypocalcemia mortality rate of the negative control group was 16.67%; the clinical hypocalcemia mortality rate of the 0.5 dose group and the positive drug group was 8.33%.

[0145] The blood plasma was analyzed by using Roche Cobas 6000 c501 automatic biochemical analyzer to obtain the data of blood calcium, blood magnesium, blood phosphorus and ALP. It can be observed that the blood calcium of the control group is low, the blood calcium of the experimental group is high, and the effect of 0.5 times and positive control group is poor, 1 times dose group and 3 times dose group are close, and the blood calcium of 3 times dose group is higher after D2. On d5 and d7 of administration, the blood calcium level of 3 times dose group compared with negative control group is extremely significant, and the blood calcium level is above subclinical hypocalcemia (≤8.6mg / dL); 1 times dose group also has significant effect on the improvement of blood calcium level compared with negative control group. Although the two groups of cows are still in the condition of subclinical hypocalcemia after delivery, but the blood calcium level of D3 (3 times 8.88mg / dL, 1 times 8.76mg / dL) is already above subclinical hypocalcemia, and the whole compared with positive drug control group and negative drug control group has certain effect on the maintenance of blood calcium.

[0146] The alkaline phosphatase (ALP) of the experimental group is at a low level on d1 after delivery compared with the positive control group and the negative control group, and the 3 times dose group has significant difference, and the ALP of the 1 times dose group and the 3 times dose group also shows significant difference on d5 and d7 after delivery. It is speculated that the supplement of vitamin D3 reduces the dependence on ALP, ALP (especially bone-derived ALP, BALP) is a marker of osteoblast activity, which usually increases when bone calcium mobilization is enhanced (such as PTH stimulating bone resorption in hypocalcemia). For the vitamin D3 sustained release group, due to sufficient prepartum vitamin D3 reserves and slow release, the efficiency of intestinal calcium absorption is improved, the dependence on bone calcium mobilization is reduced, and the ALP activity is reduced. The ALP level is more stable, unlike the sharp increase of low calcium cows. It is speculated that the PTH level is more stable, so the stimulation of ALP is reduced. Compared with the positive drug control group of direct injection of vitamin D3, due to the one-time injection of vitamin D3 before delivery, the metabolic reserves after 7d are not as good as the sustained release experimental group, PTH responds to the low calcium environment, and mobilizes bone calcium into blood.

[0147] The blood phosphorus is increased compared with the negative control group, and it continues to increase after 24h after delivery, and it can be seen that the content of 1 times dose group, 3 times dose group and positive control group is higher, which may be due to the result of supplementing vitamin D3; 0.5 times dose group and negative control group have no great difference, which may be due to the reason that the released vitamin D3 per unit time cannot reach the threshold value, if the supplement is insufficient, the intestinal phosphorus absorption and bone phosphorus mobilization effect may be weak.

[0148] All groups showed elevated serum magnesium within 24 h after calving, and the 1-fold and 3-fold dose groups showed significantly higher serum magnesium levels than the negative control group at d1 after calving. We found that serum calcium and magnesium concentrations were negatively correlated because parathyroid hormone promotes magnesium reabsorption in the urine, leading to elevated serum magnesium levels. When vitamin D3 is sufficient, the increase in PTH is small, and PTH is a key hormone that promotes bone magnesium release and renal tubular magnesium reabsorption. Therefore, in vitamin D3 -prevented hypocalcemic cows, the secretion of parathyroid hormone may decrease because the serum calcium concentration is higher than that of the control group, which may lead to a decrease in serum magnesium concentration in experimental cows. Another study revealed that high blood magnesium content is the result of competition for intestinal calcium and magnesium absorption. Vitamin D3 significantly increases calcium absorption by upregulating intestinal TRPV6 calcium channels and calcium-binding protein (CaBP), while magnesium absorption depends on the TRPM6 / 7 channel. Both share some transporters, and a high-calcium environment inhibits magnesium absorption.

[0149] Table 1. Results of determination of Ca, ALP, P, and Mg in the plasma of cows

[0150]

[0151] Table 1 summarizes the results of each index in the plasma, comprehensively showing the significant comparisons of each index on the same day in different groups. Figure 18-21 The results are consistent with the significant results of the 3-fold dose group, the 1-fold dose group, and the negative control group in the table.

[0152] Therefore, the 1-fold dose and 3-fold dose vitamin D3 sustained-release carriers can both prevent hypocalcemia in cows to some extent. Considering that the 3-fold dose group and the 1-fold dose group have similar effects, and the 3-fold dose group is more cumbersome to use in clinical practice (requiring three injections on the animal), it increases the daily burden of veterinary management. Therefore, the 1-fold dose group is the most reasonable dosage form in terms of preventing low clinical hypocalcemia and in terms of manpower and cost.

[0153] Verification Example 2

[0154] The collected plasma samples were also subjected to Elisa detection. The Elisa kit was from Shanghai Langdon Company, and the data of parathyroid hormone (PTH), calcitonin (CT), and 25-(OH)VD3 were obtained. It can be observed that the parathyroid hormone level in the blood of cows injected with sustained-release vitamin D3 before calving was significantly lower than that of the control group after calving. The results of d2 detection showed that the PTH of the 1-fold dose group and the 3-fold dose group showed a significant and extremely significant trend compared with the negative control group. Because the blood calcium level was relatively stable, there was no serious hypocalcemia stimulation, and therefore the parathyroid gland did not need to secrete a large amount of PTH to mobilize bone calcium into the blood. Therefore, the measured PTH level after calving was lower than that of the control group. Figure 23Plasma PTH data results).

[0155] Because of the prenatal vitamin D3 reserve, the postpartum blood calcium decreased less, recovered faster, and was more stable and relatively higher. The negative control group had very low postpartum blood calcium and high PTH levels. Because the low blood calcium lacked the signal to stimulate CT secretion, the CT level remained at the base or low level. The PTH levels of the experimental and positive control groups were significantly lower than those of the control group. Although their blood calcium levels were more ideal than those of the control group, they were still at the lower or medium level of the physiological normal range, far from the "high calcium" state that could strongly stimulate the massive secretion of CT. None of the cows in the negative control group, the positive control group, and the experimental group had high blood calcium after giving birth. The CT levels of the two groups remained near the baseline, and there was no statistically significant difference between the groups. Figure 24 Plasma CT data results). This result did not detect the feedback between PTH and CT or the CT peak after calving, suggesting that the physiological pathway driving CT secretion was not activated, possibly because the calcium concentration was too low, which is consistent with the findings of Hollis et al. (1981).

[0156] It can be seen that compared with the positive control drug group and the negative control group, the 25-(OH) vitamin D3 content of the 1-fold dose group and the 3-fold dose group peaked at d1 and was significantly different from the negative control group, and there was no difference between the 0.5-fold dose group, the positive drug control group, and the negative control group. At the d2 time point, the 3-fold dose group and the 1-fold dose group still showed a significant trend compared with the negative control group, confirming that the drug was released in the body and there were differences between the experimental groups. Figure 25 Plasma 25-hydroxyvitamin D3 data results

[0157] Table 2: Determination results of PTH, CT, and 25-(OH) VD3 in the plasma of cows

[0158]

[0159] Table 2 summarizes the results of each indicator in the plasma, comprehensively displaying the significant comparison of each indicator on the same day in different groups. Figure 23-25 The results are consistent with the significant results of the 3-fold dose group, the 1-fold dose group, and the negative control group in the table.

[0160] A small amount of prepared rotary evaporation phospholipid and directly mixed and dissolved phospholipid were respectively pushed onto a glass slide to form a film, and a cover glass was placed on top to observe under a fluorescent inverted microscope. The rotary evaporation group can observe the phospholipid liposomes clustered into groups (see Figure 26 ), while the directly mixed and dissolved phospholipid group is blurred into large patches under the microscope, with unclear morphology (see Figure 27 ).

[0161] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. An in-situ sustained-release gel carrier, characterized in that, The ingredients include the following parts by weight: 40-70 parts egg yolk lecithin; 15-30 parts of trioctyl ethyl acetate; 15-30 parts of n-propanol.

2. The application of the in-situ sustained-release gel carrier according to claim 1 in the preparation of fat-soluble vitamin in-situ sustained-release formulations.

3. The application according to claim 2, characterized in that, The fat-soluble vitamins include vitamin D3, vitamin A, and / or vitamin E.

4. A fat-soluble vitamin in-situ sustained-release gel, characterized in that, The ingredients include the following parts by weight: 40-70 parts egg yolk lecithin; Fat-soluble vitamins: 5-70 parts; 15-30 parts of trioctyl ethyl acetate; 15-30 parts of n-propanol The vitamins include vitamin D3, vitamin A, and / or vitamin E.

5. A method for preparing the fat-soluble vitamin in-situ sustained-release gel according to claim 4, characterized in that, include: Egg yolk lecithin, fat-soluble vitamins, tricaprylic acid glyceride, and n-propanol are mixed, dissolved, sonicated, rotary evaporated, reconstituted, and filtered to obtain the final product.

6. The preparation method according to claim 5, characterized in that, The mass ratio of egg yolk lecithin, fat-soluble vitamins, tricaprylic acid glyceride, and n-propanol is 4~7g:4~7g:1.5~3mL:1.5mL~3; preferably, the mass ratio of egg yolk lecithin, fat-soluble vitamins, tricaprylic acid glyceride, and n-propanol is 7g:7g:1.5mL:1.5mL. The filtration is performed using a 0.22μm microporous membrane.

7. The use of the fat-soluble vitamin in-situ sustained-release gel according to claim 4 or the fat-soluble vitamin in-situ sustained-release gel prepared by the preparation method according to claim 5 or 6 in the preparation of drugs for preventing hypocalcemia in dairy cows.

8. The application according to claim 7, characterized in that, The dairy cows in question are in the peripartum period.

9. A drug for preventing hypocalcemia in dairy cows, characterized in that, This includes the fat-soluble vitamin in-situ sustained-release gel as described in claim 4, and the fat-soluble vitamin in-situ sustained-release gel prepared by the preparation method described in any one of claims 5 to 6.

10. The medicament according to claim 9, wherein the dosage form of the medicament includes injection, oral preparation, and spray; The dosage of the injection is 0.2 ml / head / dose.

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