Diclazuril oral nano suspension for targeted solubilization of intestinal tracts as well as preparation method and application of diclazuril oral nano suspension

Abstract

The invention discloses an oral diclazuril nano suspension for targeted solubilization of intestinal tracts as well as a preparation method and application of the oral diclazuril nano suspension, and relates to the technical field of pharmaceutical preparations. The diclazuril oral nano suspension comprises the following components: 2-200 g / L of diclazuril, 15-25 g / L of a stabilizer, 2-25 g / L of a cosolvent, 1-20 g / L of a preservative, 1-20 g / L of a defoaming agent and the balance of purified water. According to the prepared diclazuril oral nano suspension, targeted solubilization of intestinal tracts can be achieved through the screened stabilizer and cosolvent, the solubility and the dissolution rate of diclazuril in intestinal juice are specifically increased, increase in gastric juice and an aqueous solution is not remarkable, and it can be effectively avoided that diclazuril is greatly absorbed in the stomach. The diclazuril oral nano suspension contains few medicinal auxiliary materials, the safety of the preparation is further improved, the material cost in the production process is reduced, and the diclazuril oral nano suspension has remarkable advantages in the practical application process.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to an intestinal tract targeted solubilization diclazuril oral nanosuspension and a preparation method and application thereof. Background Art

[0002] Chicken coccidiosis is an intestinal disease characterized by weight loss, diarrhea and bloody stools caused by various coccidia of the genus Eimeria parasitizing in intestinal epithelial cells. It is one of the major diseases that seriously threaten the healthy development of the poultry industry. There are about 13 known species of chicken coccidia in the world, of which seven species, including Eimeria tenella, Eimeria brucelli, Eimeria toxicophila, Eimeria precocious, Eimeria giant, Eimeria acervulina and Eimeria malaise, are considered to be the most harmful to the poultry industry. Eimeria tenella mainly attacks the cecum, and the two cecums are significantly swollen, which can be 3 to 5 times the normal size; Eimeria brunneri parasitizes in the posterior part of the small intestine, at the root of the cecum, and has a certain pathogenicity, which can cause intestinal punctate bleeding and catarrhal inflammation; Eimeria toxicophila damages the middle part of the small intestine, causing the intestinal wall to expand and thicken, with severe necrosis; Eimeria precociously parasitizes in the first third of the small intestine and has low pathogenicity; Eimeria giant damages the middle part of the small intestine, causing the intestinal tube to expand and the intestinal wall to thicken; Eimeria acuminate mostly develops on the epithelial surface, and the worms at the same developmental stage often gather together, and a large number of pale white spots appear in the damaged intestinal segment; Eimeria milde and Eimeria hardyii parasitize in the anterior part of the small intestine and have low pathogenicity, with pinhead-sized bleeding spots appearing on the intestinal wall and severe bleeding in the mucosa. If multiple coccidia are mixedly infected, the intestine will be thick, there will be a large number of bleeding spots on the intestinal mucosa, and there will be a large amount of purple-black blood with shed intestinal epithelial cells in the intestine. It can be seen that chicken coccidia mainly parasitize in various intestinal segments of chickens.

[0003] Chicken coccidiosis is mainly treated with drugs in groups. The existing anticoccidial drugs mainly include sulfonamides, chlorpheniramine, mebendazole, salinomycin, and triazine phenylacetonitrile. Diclazuril is a triazine phenylacetonitrile anticoccidial drug with the characteristics of high efficiency, low toxicity, and no retention. It is the drug with the lowest drug concentration among the anticoccidial drugs currently used. Diclazuril has excellent effects on chicken tender, acuminate, toxic, Brucella and giant Eimeria. After use, it can not only effectively control the occurrence and death of cecal coccidia, but can even make all the coccidia oocysts of sick chickens disappear. It is an ideal coccidial drug.

[0004] Although diclazuril has good insecticidal activity, its solubility is extremely poor. It is only slightly soluble in dimethylformamide, slightly soluble in tetrahydrofuran, and almost insoluble in water and most organic solvents. The drug concentration is low, there is a risk of uneven stirring when mixed feeding, and it is extremely difficult to dissolve in intestinal fluid; the diclazuril aqueous solution solubilized by organic solvents has a short stability period and must be prepared and used immediately (stable in drinking water for no more than 4 hours), otherwise it affects the efficacy. The above shortcomings limit its practical application process. Therefore, how to develop a clinical preparation that is convenient for administration and stably increase the solubility of diclazuril in the chicken intestine is particularly urgent and important. Summary of the invention

[0005] The purpose of the present invention is to provide an intestinal targeted solubilization diclazuril oral nanosuspension and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The diclazuril oral nanosuspension prepared by the present invention can achieve intestinal targeted solubilization through the screened stabilizer and cosolvent, specifically increase the solubility and dissolution rate of diclazuril in intestinal fluid, and the increase in gastric fluid and aqueous solution is not significant, which can effectively avoid the large-scale absorption of diclazuril.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides an intestinal tract targeted solubilization diclazuril oral nanosuspension, comprising the following components: 2-200 g / L of diclazuril, 15-25 g / L of a stabilizer, 2-25 g / L of a cosolvent, 1-20 g / L of a preservative, 1-20 g / L of a defoamer and the remainder of purified water.

[0008] Preferably, the diclazuril oral nanosuspension comprises the following components: 100 g / L diclazuril, 20 g / L stabilizer, 10 g / L cosolvent, 15 g / L preservative, 2.5 g / L defoamer and the balance purified water.

[0009] Further, the stabilizer consists of a first stabilizer and a second stabilizer;

[0010] The first stabilizer is at least one of polyoxyethylene 40 hydrogenated castor oil, polyvinyl pyrrolidone, polyvinyl alcohol, poloxamer 188 and Tween 80;

[0011] The second stabilizer is at least one of gellan gum and sodium alginate.

[0012] Furthermore, the mass ratio of the first stabilizer to the second stabilizer is 19:1.

[0013] Furthermore, the cosolvent is at least one of sodium dodecyl sulfate and sodium dodecyl sulfonate.

[0014] Furthermore, the preservative is at least one of methyl paraben, sorbic acid and benzyl alcohol.

[0015] Furthermore, the defoaming agent is simethicone.

[0016] The present invention also provides a method for preparing the above-mentioned diclazuril oral nanosuspension. The method for preparing each 1L of the diclazuril oral nanosuspension comprises the following steps:

[0017] (1) adding a stabilizer and a co-solvent into purified water and stirring the mixture to obtain a feed solution A;

[0018] (2) adding a preservative to the liquid A and stirring the mixture to obtain a liquid B;

[0019] (3) adding diclazuril to the feed solution B and stirring evenly to obtain feed solution C;

[0020] (4) adding the defoaming agent to the feed liquid C, stirring evenly, and adding purified water to make the volume to 1 L to obtain feed liquid D;

[0021] (5) Adding the liquid D into a sand mill and grinding it to obtain the diclazuril oral nanosuspension.

[0022] Furthermore, in step (1), the temperature of the purified water is 25-35° C.; and / or

[0023] In steps (1) to (4), the stirring speed is 500-1000 rpm and the stirring time is 5-30 min; and / or

[0024] The grinding speed of the grinding process is 500-2800rpm, and the stirring time is 0.2-1h; and / or

[0025] The diameter of the grinding media is 0.2-0.6 mm.

[0026] The present invention also provides application of the above-mentioned diclazuril oral nanosuspension in preparing a medicine for treating chicken coccidiosis.

[0027] The present invention discloses the following technical effects:

[0028] The main parasitic site of coccidia is in the intestinal tract, which endangers the health of animal bodies. The present invention realizes intestinal targeted solubilization through drug nano-technology and auxiliary material screening, and significantly increases the solubility, dissolution and drug content of diclazuril in intestinal fluid. In the nanosuspension of the present invention, the drug mainly exists in nano-scale particles, so it is not easy to settle and stratify in drinking water, and the stability of use can be improved. Secondly, the nano-sized insoluble diclazuril particles increase the adhesion to the mucosa, while reducing the intestinal discharge rate, achieving a long-term effect. Due to the low drug concentration of diclazuril, it is very easy to mix unevenly during the mixing and use process, thereby affecting the efficacy of the drug. The nanosuspension prepared by the present invention has good dispersibility in drinking water, and the intestinal targeted solubilization can be achieved by screening stabilizers and cosolvents, specifically increasing the solubility and dissolution rate of diclazuril in intestinal fluid, and the increase in gastric juice and aqueous solution is not significant, which can effectively avoid the large-scale absorption of diclazuril. Nanosuspensions use fewer pharmaceutical excipients, which further improves the safety of the preparation, reduces the material cost in the production process, and has significant advantages in actual application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The XRD patterns of the effects of different treatments on the crystal form of the raw drug;

[0031] Figure 2 This is a graph showing the results of determining the content of different dosage forms of diclazuril in intestinal fluid. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The present invention uses the anticoccidiosis drug diclazuril as a raw material drug; one or more of polyvinyl alcohol, poloxamer 188, polyoxyethylene 40 hydrogenated castor oil, Tween 80, polyvinyl pyrrolidone, gellan gum and sodium alginate as a stabilizer; one of sodium dodecyl sulfate and sodium dodecyl sulfonate as a cosolvent; at least one or more of methyl paraben, sorbic acid and benzyl alcohol as a preservative; and simethicone as a defoamer. The gellan gum added to the preparation is in a gel state in the acidic environment of gastric juice, which can reduce the contact between the drug particles and the stomach and prevent the nanoparticles from being damaged; the pH value of the intestine is slightly alkaline, and the viscosity of the preparation is reduced after entering, and it is in a flowing state, which increases the contact area between the drug particles and the intestinal wall, so that the drug components are retained for a long time and a long-term effect is achieved. There are a lot of bile salts mixed in the intestinal fluid (bile salts are the main components of bile involved in digestion and absorption), and the liver cells continue to produce bile. After the bile is produced, it flows out from the hepatic duct and is discharged into the duodenum through the common bile duct, or transferred to the cystic duct and stored in the gallbladder, and then discharged from the gallbladder to the duodenum during the digestion period. Bile salts themselves are a type of surfactant naturally present in the intestine. These surfactants can form gels with poorly water-soluble drugs, thereby increasing the solubility of poorly soluble drugs; sulfonates are important components of bile salts and play an important role in the emulsification of intestinal contents. The present invention additionally adds sodium dodecyl sulfate, which also has a similar chemical structure, and significantly increases the solubility and dissolution rate of diclazuril. The present invention utilizes the strong grinding process of the sand mill, that is, the violent impact of the rod pin and the stator on the ultrafine grinding medium, and the medium acts on the material to obtain a nano-level suspension preparation. The nano-sized particles have a large specific surface area, which increases the contact area with the natural active substances in the intestine, further improves the solubility, and at the same time increases the adhesion to the mucosa, reduces the intestinal discharge rate, and achieves a long-term effect. Tween 80, gellan gum and sodium dodecyl sulfate with suspending and solubilizing effects were screened out, and an emulsifier carrier solution was prepared to ensure the stability of the drug particles in the preparation system and enhance the solubility of the drug particles. The first stabilizer used in the present invention, Tween 80, is an excellent O / W type emulsifier. The high concentration of electrolytes and the change of pH value have little effect on its suspending ability, and it is suitable as a drug excipient for drinking water administration; as an amphiphilic active agent, it can be wrapped on the surface of drug particles to enhance the combination of drug particles with a large amount of bile salts, phospholipids, etc. in the intestine, and has a positive effect on achieving intestinal targeted solubilization; its anti-flocculation effect can also prevent the aggregation of fine drug particles and increase the stability of diclazuril oral nanosuspension. The second stabilizer, gellan gum, changes the viscosity of the preparation through the change of pH in the gastrointestinal environment, resists dissolution in the acidic environment of the stomach, and at the same time enables the effective ingredients in the preparation to fully play their role in the intestinal dissolution, achieving targeted solubilization. The cosolvent, sodium dodecyl sulfate, is a sulfonate, which increases the solubility and dissolution rate of diclazuril. After the drug is correctly administered, the drug disperses rapidly and evenly with stable properties, thereby reducing the risk of the drug being difficult to mix evenly due to too low a drug concentration in the clinic, thereby affecting the efficacy.The specific details are as follows:.

[0038] Example 1

[0039] The intestinal targeted solubilized diclazuril oral nanosuspension comprises the following components: 100 g / L diclazuril, 20 g / L stabilizer, 10 g / L cosolvent, 15 g / L preservative, 2.5 g / L defoamer and the remainder purified water; wherein the stabilizer is composed of a first stabilizer Tween 80 and a second stabilizer gellan gum, and the mass ratio of the first stabilizer to the second stabilizer is 19:1; the cosolvent is sodium dodecyl sulfate, the preservative is sorbic acid, and the defoamer is simethicone.

[0040] The preparation method of the intestinal tract targeted solubilization diclazuril oral nanosuspension is as follows:

[0041] (1) 19 g of the first stabilizer, 1 g of the second stabilizer and 10 g of the co-solvent were added to 250 mL of purified water, and stirred at 30° C. and 700 rpm for 10 min to obtain liquid A;

[0042] (2) Add 15 g of preservative to liquid A and stir at 700 rpm for 3 min to obtain liquid B;

[0043] (3) adding 100 g of diclazuril raw material to liquid B and stirring at 700 rpm for 10 min to obtain liquid C;

[0044] (4) Add 2.5 g of defoamer to liquid C, stir at 700 rpm for 3 min, add purified water to make the volume to 1 L, and obtain liquid D;

[0045] (5) Add liquid D into a clean sand mill and grind it at 2000 rpm using 0.3 mm zirconium beads for 45 min to obtain the intestinal targeted solubilized diclazuril oral nanosuspension.

[0046] Example 2

[0047] The intestinal targeted solubilized diclazuril oral nanosuspension comprises the following components: 200 g / L diclazuril, 15 g / L stabilizer, 2 g / L cosolvent, 20 g / L preservative, 1 g / L defoamer and the remainder purified water; wherein the stabilizer is composed of a first stabilizer Tween 80 and a second stabilizer gellan gum, and the mass ratio of the first stabilizer to the second stabilizer is 19:1; the cosolvent is sodium dodecyl sulfate, the preservative is sorbic acid, and the defoamer is simethicone.

[0048] The preparation method of the intestinal tract targeted solubilization diclazuril oral nanosuspension is as follows:

[0049] (1) 14.25 g of the first stabilizer, 0.75 g of the second stabilizer and 2 g of the co-solvent were added to 250 mL of purified water, and stirred at 25° C. and 1000 rpm for 30 min to obtain liquid A;

[0050] (2) Add 20 g of preservative to liquid A and stir at 1000 rpm for 30 min to obtain liquid B;

[0051] (3) adding 200 g of diclazuril raw material to liquid B and stirring at 1000 rpm for 30 min to obtain liquid C;

[0052] (4) Add 1 g of defoamer to liquid C, stir at 1000 rpm for 30 min, add purified water to make the volume to 1 L, and obtain liquid D;

[0053] (5) Add liquid D into a clean sand mill and grind it at 2800 rpm for 12 min using 0.3 mm zirconium beads to obtain the intestinal targeted solubilized diclazuril oral nanosuspension.

[0054] Example 3

[0055] The intestinal targeted solubilized diclazuril oral nanosuspension comprises the following components: 2 g / L diclazuril, 25 g / L stabilizer, 2 g / L cosolvent, 1 g / L preservative, 20 g / L defoamer and the remainder purified water; wherein the stabilizer is composed of a first stabilizer Tween 80 and a second stabilizer gellan gum, and the mass ratio of the first stabilizer to the second stabilizer is 20:1; the cosolvent is sodium dodecyl sulfate, the preservative is sorbic acid, and the defoamer is simethicone.

[0056] The preparation method of the intestinal tract targeted solubilization diclazuril oral nanosuspension is as follows:

[0057] (1) Add 23.75 g of the first stabilizer, 1.25 g of the second stabilizer and 2 g of the co-solvent into 250 mL of purified water, and stir at 35° C. and 500 rpm for 5 min to obtain liquid A;

[0058] (2) Add 1 g of preservative to liquid A and stir at 500 rpm for 5 min to obtain liquid B;

[0059] (3) adding 2 g of diclazuril raw material to liquid B and stirring at 500 rpm for 5 min to obtain liquid C;

[0060] (4) Add 20 g of defoamer to liquid C, stir at 500 rpm for 5 min, add purified water to make the volume to 1 L, and obtain liquid D;

[0061] (5) Add liquid D into a clean sand mill and grind it at 500 rpm for 60 min using 0.3 mm zirconium beads to obtain the intestinal targeted solubilized diclazuril oral nanosuspension.

[0062] Experimental Example 1

[0063] The first stabilizer of Example 1 was replaced with polyvinyl pyrrolidone (PVP), polyoxyethylene 40 hydrogenated castor oil, poloxamer 188 (P188) and polyvinyl alcohol (PVA) respectively to prepare a diclazuril oral nanosuspension. The specific preparation method is as follows:

[0064] (1) Add 100 mL of purified water at 25 °C to a 500 mL beaker, then add 5 g of stabilizer, and stir at 700 rpm for 10 min using an electric stirrer;

[0065] (2) Add 50 g of diclazuril API while stirring. After the addition is complete, use an electric stirrer to stir at 700 rpm for 10 min.

[0066] (3) Add the above-mentioned liquid into a clean sand mill and grind at 2000 rpm for 45 min to obtain a preliminary sample;

[0067] (4) Take 50 mL of the sample into a 50 mL stoppered measuring cylinder and let it stand for 3 h. Observe and measure its sedimentation volume ratio and redispersibility.

[0068] (5) Take another 100 μL sample and dilute it about 10,000 times with purified water (or dilute it to meet the requirements of the relevant content in CVP2020 "Pharmacopoeia of the People's Republic of China"), and use a visible light spectrophotometer to measure the shading degree to make it between 8% and 20%;

[0069] (6) The diluted sample is placed on a machine to measure the particle size and potential.

[0070] The test results of each formula group are shown in Table 1. As can be seen from Table 1, there is no obvious difference in sedimentation volume ratio and potential parameters among the groups, but Tween 80 has obvious advantages in redispersibility.

[0071] Table 1 Results of sedimentation volume ratio, redispersibility, average particle size and potential of each group

[0072]

[0073]

[0074] Note: “ / / ” indicates that the viscosity cannot be measured due to reasons such as high viscosity; redispersibility refers to the number of times the stoppered measuring cylinder is turned upside down 180° to fully disperse the sample after it has been allowed to stand for 3 hours.

[0075] Experimental Example 2

[0076] In order to achieve intestinal targeted solubilization, two commonly used gelling polysaccharides were screened as follows:

[0077] (1) Add 100 mL of purified water at 30 °C to two 1000 mL beakers, then add 5 g of Tween 80, and stir at 700 rpm for 10 min using an electric stirrer;

[0078] (2) adding 0.25 g of gellan gum and sodium alginate respectively, slowly adding and stirring, and stirring at 700 rpm for 10 min using an electric stirrer;

[0079] (3) Add the above-mentioned liquid into a clean sand mill and grind at 2000 rpm for 45 min to obtain a preliminary sample;

[0080] (4) Twenty glass bottles were divided into gellan gum group (simulated gastric fluid (SGF), simulated intestinal fluid (SIF), SIF + sodium dodecyl sulfate, SIF + sodium dodecyl sulfate and drinking water, two replicates in each group) and sodium alginate group (SGF, SIF, SIF + sodium dodecyl sulfate, SIF + sodium dodecyl sulfate and drinking water, two replicates in each group). The addition amount of sodium dodecyl sulfate and sodium dodecyl sulfate was 10 g / L, and the addition amount of SGF, SIF and drinking water was 50 mL;

[0081] (5) Using the shake flask method, take samples and determine the solubility.

[0082] The screening results of the targeted stabilizers and cosolvents are shown in Table 2. As can be seen from Table 2, the solubility of diclazuril in simulated intestinal fluid is higher than that in simulated gastric fluid, and the addition of sodium alginate and gellan gum both increase the solubility of diclazuril in simulated intestinal fluid, but the effect of gellan gum is slightly better; at the same time, the addition of cosolvents, especially sodium dodecyl sulfate, has a significant effect on improving solubility, so the preferred formula determines gellan gum as a targeted stabilizer and sodium dodecyl sulfate as a cosolvent.

[0083] Table 2 Targeted stabilizers and cosolvent screening results

[0084]

[0085] Note: “ / / ” means the concentration is too low to be determined.

[0086] Experimental Example 3

[0087] Since paraben active agents have a significant antagonistic effect with Tween 80, sorbic acid or benzyl alcohol is selected as a preservative; the concentration is selected based on the general standards in the industry.

[0088] The specific experimental steps are as follows:

[0089] (1) Sorbic acid or benzyl alcohol was used as a preservative, and the amount of the preservative added was adjusted to 15 g / L, 20 g / L and 25 g / L, respectively, and a diclazuril oral nanosuspension test sample was prepared according to the method of Example 1;

[0090] (2) Fresh cultures of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans were diluted with 0.9% sterile sodium chloride solution to a turbidimetric value of 0.5 McFarland units (1×10 8 Take a fresh culture of Aspergillus niger and add an appropriate amount of 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80 to wash out the spores. Then, use an appropriate method to aspirate the spore suspension into a sterile test tube, add an appropriate amount of 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80 to make a suspension containing 10 cfu / mL of spores per 1mL. 8 Spore suspension of cfu:

[0091] (3) Take 5 samples of the intact test sample and directly inoculate the test bacteria. The inoculation amount is 10 5 ~10 6 cfu, the volume of the inoculated solution shall not exceed 1% of the volume of the test sample. Mix thoroughly to ensure that the test bacteria in the test sample are evenly distributed, and then store at 20-25℃ away from light.

[0092] (4) On the 14th and 28th days after inoculation, take 1 mL of the test sample from each of the above containers, dilute the test solution 10 times with PBS, and take 100 μL to inoculate two plates respectively. Use tryptic soy peptone agar medium for bacterial determination and Sabouraud dextrose agar medium for fungal determination. At the same time, use PBS instead of the test solution to coat two plates as a negative control. Tryptic soy peptone agar medium is cultured at 35°C for 3 days, and Sabouraud dextrose agar medium is cultured at 25°C for 5 days.

[0093] The results of the antibacterial efficacy test of each group are shown in Table 3-Table 5. According to Table 3-Table 5, benzyl alcohol has insufficient antibacterial efficacy at lower concentrations; however, the low, medium and high concentrations of sorbic acid all meet the antibacterial efficacy standards and have better antibacterial ability. In consideration of cost saving and preparation safety, a lower concentration of 15g / L was selected as the preservative concentration.

[0094] Table 3 Antibacterial efficacy test results of suspensions with 15 g / L preservative diclazuril added

[0095]

[0096]

[0097] Note: NI means no increase, which means that the increase in the number of test bacteria does not exceed 0.5lg compared with the previous measurement time.

[0098] Table 4 Antibacterial efficacy test results of suspensions with 20 g / L preservative diclazuril added

[0099]

[0100] Note: NI stands for no increase, which means that the increase in the number of test bacteria does not exceed 0.5lg compared with the previous measurement time.

[0101] Table 525 Antibacterial efficacy test results of g / L preservative diclazuril suspension

[0102]

[0103]

[0104] Note: NI stands for no increase, which means that the increase in the number of test bacteria does not exceed 0.5lg compared with the previous measurement time.

[0105] Experimental Example 4

[0106] The addition amount of the defoamer was adjusted to 2.5 g / L, 5 g / L and 7.5 g / L respectively, and the diclazuril oral nanosuspension test sample was prepared according to the method of Example 1. After vigorous shaking for 5 minutes, the time when the foam completely disappeared was recorded, and the sedimentation volume ratio and the average particle size were measured according to the method in Experimental Example 1. The results are shown in Table 6. It can be seen from Table 6 that the defoaming effect is best at a concentration of 7.5 g / L, but the sedimentation is serious and the sedimentation volume ratio does not meet the requirements; the defoaming time and sedimentation volume ratio at concentrations of 2.5 g / L and 5 g / L are relatively close, and the optimal defoamer concentration is 2.5 g / L after comprehensive consideration.

[0107] Table 6 Effect of different concentrations of defoamers on preparation performance

[0108]

[0109] Experimental Example 5

[0110] The amount of stabilizer added was adjusted to 10 g / L, 20 g / L and 30 g / L respectively, and the rest was prepared according to the method of Example 1. 50 mL of the sample was taken into a 50 mL stoppered measuring cylinder, and allowed to stand for 3 h, and its sedimentation and properties were observed respectively.

[0111] The results showed that among the three samples, the 10g / L addition group had serious sedimentation and was not a suspension; the 30g / L addition group was too viscous and not suitable for drinking water administration, so both were discarded. The 20g / L addition formula was a white suspension, and the sedimentation volume ratio met the requirements.

[0112] Experimental Example 6

[0113] According to the prescription of diclazuril oral nanosuspension and the physical and chemical properties of raw materials and excipients, the preparation process includes preparation of emulsifier carrier solution, mixing of preservatives, mixing of active ingredients and grinding of liquid. The preparation process of diclazuril oral nanosuspension is optimized as follows:

[0114] 1. Preparation of Solubilizer Carrier Solution

[0115] Low water temperature may affect the solubility of Tween 80 and gellan gum, and thus affect the stability of the suspension. The mixing water temperature and stirring time were screened, see Table 7.

[0116] Table 7 Effect of different water temperatures on the preparation of solubilizer carrier solution

[0117]

[0118]

[0119] It can be seen from Table 7 that as the water temperature gradually rises to 25-35°C and the mixture is stirred for 10 minutes, Tween 80 and gellan gum can be quickly dispersed and dissolved. Considering the actual production factors such as energy consumption and production efficiency, it is chosen to heat the water to 25-35°C and then mix it with Tween 80 and gellan gum for 10 minutes.

[0120] 2. Preservative Mixture

[0121] According to the physical and chemical properties of sorbic acid and the test results, at room temperature of 25±5℃, when sorbic acid at a dosage of 15g / L is added to the Tween-80 aqueous solution, the two liquid solutions can be quickly mixed, and when stirred for 1min, 3min and 5min respectively, they are all clear aqueous solutions. Therefore, in order to fully ensure the stability of each material in the liquid, the mixing time of the preservative sorbic acid is selected as 3min, and the temperature of sorbic acid and subsequent ingredients is determined to be room temperature 25±5℃.

[0122] 3. Active substance mixing

[0123] The diclazuril API is a solid powder. When added to the precursor liquid, it should be fully wetted and evenly dispersed. The mixing procedure should be optimized, see Table 8.

[0124] Table 8 Effects of different stirring time and stirring speed on active material mixing

[0125]

[0126] It can be seen from Table 8 that after diclazuril is added to the above-mentioned liquid, it can be quickly wetted and is well dispersed after stirring at 500 rpm for 20 min, 700 rpm for 10 min, and 1000 rpm for 6 min. Considering the actual production factors such as energy consumption and production efficiency, the diclazuril mixing conditions are selected as a stirring speed of 700 rpm and a stirring time of 10 min.

[0127] 4. Liquid grinding

[0128] The mixed liquid is ground to ensure that the active ingredient diclazuril in the final product is nano-sized particles. The grinding process needs to be optimized, including the selection of grinding media size, grinding speed and grinding time.

[0129] 4.1 Grinding time screening

[0130] The preliminary selection of grinding media zirconium beads with a diameter of 0.3 mm and a grinding speed of 2000 rpm, the grinding time was screened, and the particle size of the drug particles was used as the judgment standard. The prescription used is shown in Table 9, and the grinding time screening is shown in Table 10.

[0131] Table 9 Grinding liquid prescription

[0132]

[0133]

[0134] Specific implementation details:

[0135] (1) Take 500 mL of purified water and heat it to 25°C;

[0136] (2) Add 300 mL of 25°C purified water into a 500 mL beaker, add Tween 80, sodium dodecyl sulfate and gellan gum, and stir at 700 rpm for 10 min to obtain;

[0137] (3) adding sorbic acid to the emulsifier carrier solution and stirring at 700 rpm for 3 min;

[0138] (4) Add diclazuril API to the above-mentioned liquid, stirring while adding, and after the addition is complete, stir at 700 rpm for 10 min;

[0139] (5) Add simethicone to the above solution, stir at 700 rpm for 3 min, and finally adjust the volume to 500 mL and mix well;

[0140] (6) Pour the prepared slurry into a clean sand mill, set the circulating cooler temperature to 10°C, set the grinding speed to 2000 rpm, and take 1 mL of sample to test the particle size at 20, 25, 30, 35, 40, 45, and 50 min of grinding;

[0141] (7) Repeat the above process three times and take the average value.

[0142] Table 10 Screening of grinding time (mean ± standard deviation, n = 3)

[0143]

[0144] As can be seen from Table 10, when the grinding time is between 20-50 min, the particle size of the diclazuril drug particles in the liquid feed continuously decreases, and an inflection point appears when the grinding time is 45 min. The grinding time selected for the liquid feed grinding process is 45 min.

[0145] 4.2 Grinding speed screening

[0146] The preliminary selection of grinding media zirconium beads with a diameter of 0.3 mm and a grinding time of 45 min was used. The grinding speed was screened, and the particle size and redispersibility of the drug particles were used as the judgment criteria. The prescription used is shown in Table 9, and the grinding speed screening is shown in Table 11.

[0147] The specific implementation details are as follows:

[0148] (1) Take 500 mL of purified water and heat it to 25°C;

[0149] (2) Add 300 mL of 25°C purified water into a 500 mL beaker, add Tween 80, sodium dodecyl sulfate and gellan gum, and stir at 700 rpm for 10 min to obtain an emulsifier carrier solution;

[0150] (3) adding sorbic acid to the emulsifier carrier solution and stirring at 700 rpm for 3 min;

[0151] (4) Add diclazuril API to the above-mentioned liquid, stirring while adding, and after the addition is complete, stir at 700 rpm for 10 min;

[0152] (5) Add simethicone to the above solution, stir at 700 rpm for 3 min, and finally adjust the volume to 500 mL and mix well;

[0153] (6) The prepared slurry was poured into a clean sand mill, the circulating cooler temperature was set to 10°C, the grinding time was set to 45 min, and the grinding was performed at 1500, 2000, and 2500 rpm respectively;

[0154] (7) Repeat the above process three times and take the average value.

[0155] Table 11 Screening of grinding speed (mean ± standard deviation, n = 3)

[0156]

[0157] It can be seen from Table 11 that when the grinding speed is 2000 rpm, it has a slight advantage in particle size compared with other grinding speeds, but has a significant advantage in redispersibility. The grinding speed selected for the liquid grinding process is 2000 rpm.

[0158] 4.3 Grinding media size screening

[0159] The grinding speed of the liquid was selected as 2000 rpm, the grinding time was 45 min, the grinding medium size was screened, and the particle size and redispersibility of the drug particles were used as the judgment criteria. The prescription used is shown in Table 9, and the grinding speed screening is shown in Table 12.

[0160] The specific implementation details are as follows:

[0161] (1) Take 500 mL of purified water and heat it to 25°C;

[0162] (2) Add 300 mL of 25°C purified water into a 500 mL beaker, add Tween 80, sodium dodecyl sulfate and gellan gum, and stir at 700 rpm for 10 min to obtain an emulsifier carrier solution;

[0163] (3) adding sorbic acid to the emulsifier carrier solution and stirring at 700 rpm for 3 min;

[0164] (4) Add diclazuril API to the above-mentioned liquid, stirring while adding, and after the addition is complete, stir at 700 rpm for 10 min;

[0165] (5) Add simethicone to the above solution, stir at 700 rpm for 3 min, and finally adjust the volume to 500 mL and mix well;

[0166] (6) The prepared slurry was poured into a clean sand mill, the circulating cooler temperature was set to 10°C, the grinding speed was set to 2000 rpm, the grinding time was set to 45 min, and the grinding media sizes were selected to be 0.3-0.4 mm and 0.6-0.8 mm respectively for grinding;

[0167] (7) Repeat the above process three times and take the average value.

[0168] Table 12 Screening of grinding speed (mean ± standard deviation, n = 3)

[0169]

[0170] It can be seen from Table 12 that when the grinding media is selected to be 0.3-0.4mm, the particle size and redispersibility of the drug particles are obviously superior to those of 0.6-0.8mm. The grinding media size selected for the liquid grinding process is 0.3-0.4mm.

[0171] Example 4

[0172] The quality of the diclazuril oral nanosuspension prepared in Example 1 was studied, including stability study in drinking water, effect of preparation on crystal form, and influencing factor test.

[0173] 1. Stability study in drinking water

[0174] The actual application situation was simulated and the stability of diclazuril suspension in drinking water was investigated.

[0175] The specific implementation details are as follows:

[0176] 100 μL of diclazuril suspension was added to 1L of drinking water. Three replicates were set up and 2 mL of samples were taken at 0, 3, 6, 9, 12 and 24 h. The upper layer was sampled 3-5 cm from the water surface and the lower layer was sampled 3-5 cm from the water bottom. After sampling, its stability in drinking water was evaluated according to the particle size. The particle size was determined and the results are shown in Table 13.

[0177] Table 13 Stability of different water layers (mean ± standard deviation, n = 3)

[0178]

[0179] It can be seen from Table 13 that the particle sizes of the drug particles in the water layer at different times are similar. After being placed under laboratory conditions for 24 hours, the preparation is evenly distributed and has good stability.

[0180] 2. Effect of preparation process on crystal form

[0181] Characterization study on the possible effects of excipients and preparation processes on the crystal form of the API.

[0182] The specific implementation details are as follows: 30 mL of each physically mixed sample (each component in the prescription amount, stirred at 700 rpm for 10 min), raw material drug (only raw material drug and purified water are added, stirred at 300 rpm for 10 min) and prepared diclazuril oral nanosuspension preparation (stirred at 300 rpm for 10 min), centrifuged at 8000 rpm for 10 min, discarded the supernatant and frozen at -20°C; placed in a freeze dryer for 48 hours and then sent for XRD detection.

[0183] from Figure 1 It can be seen that the preparation excipients and preparation process have no effect on the diclazuril crystal form. The current excipient combination is reasonable and the production process is feasible.

[0184] 3. Test of influencing factors

[0185] To study the inherent stability of drugs, understand the factors affecting their stability, possible degradation pathways and degradation products, and provide a scientific basis for the production process, packaging, storage conditions and the establishment of degradation product analysis methods. Conduct influencing factor tests, including high temperature and strong light tests.

[0186] The specific steps are as follows:

[0187] Take one batch of test products for high temperature test and strong light irradiation test.

[0188] (1) High temperature test: the preparation was placed under constant conditions of 50±5°C and 50% relative humidity, and 4 mL of samples were taken at 0d, 5d, and 10d to study the stability of the drug by changes in particle size, content, and related substances;

[0189] (2) Strong light test: place the preparation under an illumination of 4500 lx ± 500 lx, and the total illumination of the light source should not be less than 1.2 × 10 6 Under the conditions of 100 W·hr / m2 and near-ultraviolet lamp energy of not less than 200 W·hr / m2, 4 mL samples were taken at 0, 5 and 10 days to study the stability of the drug by analyzing the changes in particle size, content and related substances.

[0190] Table 14 Test results of influencing factors (mean ± standard deviation, n = 3)

[0191]

[0192] As can be seen from Table 14, the effect of high temperature on the preparation is quite limited. However, under strong light conditions, the particle size of the preparation increases slightly, indicating that the drug particles are aggregated. This result is in line with expectations. Diclazuril needs to be stored in a sealed container away from light.

[0193] Example 5

[0194] The intestinal absorption of the same dose of diclazuril oral nanosuspension (Example 1), commercially available diclazuril solution and commercially available diclazuril premix was compared by pharmacokinetic tests. The dissolution and retention of diclazuril oral nanosuspension and premix in the intestine were compared by measuring the diclazuril content in intestinal contents. The results are shown in Tables 15 and 16.

[0195] The specific steps are as follows:

[0196] (1) High performance liquid chromatography detection method: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile + 0.2% phosphoric acid (57 + 43, volume ratio); flow rate: 1 mL / min; wavelength: 278 nm; column temperature: 30°C; injection volume: 50 μL.

[0197] (2) Plasma pretreatment method: Pipette the plasma sample (0.5 mL) into a 4 mL polypropylene centrifuge tube. Add HCl solution (100 μL, 6 M) to the test tube. Extract the sample with 1.5 mL of 0.2% formic acid-acetonitrile solution; after vortexing for 30 seconds, centrifuge the test tube at 10,000 rpm for 10 minutes at 4°C. Pour the supernatant carefully into a 10 mL glass centrifuge tube and evaporate to near dryness under a gentle nitrogen flow at 40°C. Residues are reconstituted with 1 mL of the initial mobile phase and centrifuged at 12,000 rpm for 5 minutes at 4°C, then filtered through a 0.22 μm organic filter membrane, and the supernatant is analyzed by LC.

[0198] (3) Intestinal fluid pretreatment method: Weigh 1 g of intestinal content sample, add 5 mL of acetonitrile into a 50 mL centrifuge tube, vortex for 1 min, shake for 15 min, centrifuge at 6000 rpm for 10 min, collect the acetonitrile extract into a 50 mL centrifuge bottle, extract the residue repeatedly, combine the two extracts, add 5 mL of n-hexane, vortex for 10 s, discard the n-hexane layer, add 5 mL of n-propanol, and evaporate to near dryness under a gentle nitrogen flow at 40°C. The residue is reconstituted with 1 mL of the initial mobile phase, centrifuged at 12000 rpm for 10 min at 4°C, and then filtered through a 0.22 μm organic filter membrane for LC determination.

[0199] (4) Animal study on the pharmacokinetics of diclazuril in chickens: 60 healthy white-feathered broiler chickens were selected and divided into five groups, with 12 chickens in each group. They were gavaged with 1 mg / kg.bw diclazuril internal nanosuspension, 2 mg / kg.bw diclazuril internal nanosuspension, 5 mg / kg.bw diclazuril internal nanosuspension, 2 mg / kg.bw diclazuril solution and 2 mg / kg.bw diclazuril premix, respectively. Blood was collected from the wing vein at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, and 144 h after gavage. The samples were processed according to the established plasma pretreatment method and then tested on the machine.

[0200] (5) Diclazuril retention test in intestinal fluid and intestinal tissue: 48 healthy white broilers were selected and divided into two groups, each with 24 chickens. 2 mg / kg.bw diclazuril oral nanosuspension and 2 mg / kg.bw diclazuril premix were respectively administered orally. 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h after administration, 3 chickens from each test group were slaughtered at each time point, and their intestinal contents and intestinal tissues were collected. After the samples were processed according to the established intestinal fluid pretreatment method, they were tested on the machine. The measured real sample data were fitted with Phoenix software.

[0201] Table 15 2mg / kg.bw diclazuril oral nanosuspension administered orally to chickens

[0202]

[0203]

[0204] Table 16 2mg / kg.bw diclazuril premix gavage to chickens

[0205]

[0206] As can be seen from Tables 15 and 16, the peak time (Tmax) of diclazuril oral nanosuspension fluctuates within the range of 8-24h, the peak time of diclazuril solution fluctuates within the range of 12-36h, and the diclazuril premix fluctuates within the range of 12-72h. After the same dose of diclazuril oral nanosuspension premix is ​​administered, diclazuril can be detected in most chickens 2h after administration of the suspension, while the premix can be detected after 4h, indicating that diclazuril oral nanosuspension is absorbed faster than other diclazuril premixes. Figure 2 It can be seen that the highest concentration of the present invention in intestinal fluid is about 7 times that of the premix, which greatly improves the anti-coccidial efficacy; at 72h and later longer time points, the concentration of the diclazuril oral nanosuspension is significantly higher than that of the premix, indicating that it has a long-lasting effect.

[0207] Example 6

[0208] In order to verify the solubilization effect of the diclazuril oral nanosuspension, taking the diclazuril oral nanosuspension prepared in Example 1 as an example, a solubility test in water, simulated intestinal fluid, and simulated gastric fluid was carried out using a shake flask method.

[0209] The specific steps are as follows:

[0210] (1) Prepare solubility samples of diclazuril oral nanosuspension in water, simulated gastric fluid, and simulated intestinal fluid, and prepare three samples of each in parallel.

[0211] (2) Shake the culture medium in a constant temperature shaker at 25°C and 220 rpm for 24 h, then let it stand overnight.

[0212] (3) Take 1 mL of sample and filter it through a 0.22 μm filter membrane. Dilute it twice with DMF and filter it through the membrane.

[0213] 1μg / mL standard: Take 1mg / mL standard stock solution, dilute it to 1μg / mL, add water to dilute it twice, pass it through the membrane and put it on the machine, repeat twice.

[0214] Table 17 Solubility of samples with different solubility (μg / mL)

[0215]

[0216] Note: “ / / ” means the difference is too large to be discarded or the value cannot be determined.

[0217] As can be seen from Table 17, the solubility of diclazuril oral nanosuspension in simulated gastric fluid is too low and the effective value cannot be determined. The solubility in simulated intestinal fluid is more than two times higher than that in water, indicating that the solubility of diclazuril in its main site of action, the intestine, is greatly improved due to the effects of process and excipients.

[0218] In summary, the diclazuril oral nanosuspension prepared by the present invention can achieve intestinal targeting and significantly increase solubility and dissolution rate; the drug particles are small, have good dispersibility, and are easy to mix; less pharmaceutical excipients are used, which improves the safety of the preparation and reduces the material cost in the production process, and has significant advantages in practical application.

[0219] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An intestinal targeted solubilized diclazuril oral nanosuspension, characterized in that: The invention comprises the following components: 2-200 g / L of diclazuril, 15-25 g / L of a stabilizer, 2-25 g / L of a cosolvent, 1-20 g / L of a preservative, 1-20 g / L of a defoaming agent and the balance of purified water.

2. The diclazuril oral nanosuspension according to claim 1, characterized in that The diclazuril oral nanosuspension comprises the following components: 100 g / L of diclazuril, 20 g / L of a stabilizer, 10 g / L of a cosolvent, 15 g / L of a preservative, 2.5 g / L of a defoamer and the remainder of purified water.

3. The diclazuril oral nanosuspension according to claim 1, characterized in that The stabilizer consists of a first stabilizer and a second stabilizer; The first stabilizer is at least one of polyoxyethylene 40 hydrogenated castor oil, polyvinyl pyrrolidone, polyvinyl alcohol, poloxamer 188 and Tween 80; The second stabilizer is at least one of gellan gum and sodium alginate.

4. The diclazuril oral nanosuspension according to claim 3, characterized in that The mass ratio of the first stabilizer to the second stabilizer is 19:

1.

5. The diclazuril oral nanosuspension according to claim 1, characterized in that The co-solvent is at least one of sodium dodecyl sulfate and sodium dodecyl sulfonate.

6. The diclazuril oral nanosuspension according to claim 1, characterized in that: The preservative is at least one of methyl paraben, sorbic acid and benzyl alcohol.

7. The diclazuril oral nanosuspension according to claim 1, characterized in that: The defoaming agent is simethicone.

8. A method for preparing the diclazuril oral nanosuspension according to any one of claims 1 to 7, characterized in that: The preparation method of each 1L of the diclazuril oral nanosuspension comprises the following steps: (1) adding a stabilizer and a co-solvent into purified water and stirring the mixture to obtain a feed solution A; (2) adding a preservative to the liquid A and stirring the mixture to obtain a liquid B; (3) adding diclazuril to the feed solution B and stirring evenly to obtain feed solution C; (4) adding the defoaming agent to the feed liquid C, stirring evenly, and adding purified water to make the volume to 1 L to obtain feed liquid D; (5) Adding the liquid D into a sand mill and grinding it to obtain the diclazuril oral nanosuspension.

9. The preparation method according to claim 8, characterized in that: In step (1), the temperature of the purified water is 25-35°C; In steps (1)-(4), the stirring speed is 500-1000 rpm and the stirring time is 5-30 min; In step (5), the grinding speed of the grinding process is 500-2800 rpm, and the stirring time is 0.2-1 h; The diameter of the grinding media is 0.2-0.6 mm.

10. Use of the diclazuril oral nanosuspension according to any one of claims 1 to 7 in the preparation of a medicament for treating chicken coccidiosis.

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