A Diclazuril Oral Nanosuspension with Intestinal Targeted Solubilization, Its Preparation Method and Application

Abstract

This invention discloses an intestinal-targeted solubilized diclazuril oral nanosuspension, its preparation method, and its application, relating to the field of pharmaceutical formulation technology. The diclazuril oral nanosuspension comprises the following components: diclazuril 2-200 g / L, stabilizer 15-25 g / L, solubilizer 2-25 g / L, preservative 1-20 g / L, defoamer 1-20 g / L, and the balance purified water. The diclazuril oral nanosuspension prepared by this invention achieves intestinal-targeted solubilization through the selection of stabilizers and solubilizers, specifically increasing the solubility and dissolution rate of diclazuril in intestinal fluid, while the increase in gastric juice and aqueous solution is not significant, effectively avoiding excessive absorption of diclazuril in the stomach. The diclazuril oral nanosuspension uses fewer pharmaceutical excipients, further improving the safety of the formulation and reducing material costs in the production process, demonstrating significant advantages in practical applications.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to an intestinal-targeted solubilized diclazuril oral nanosuspension, its preparation method, and its application. Background Technology

[0002] Coccidiosis in chickens is an intestinal disease characterized by emaciation, diarrhea, and bloody stools, caused by various coccidia genus parasitizing the intestinal epithelial cells. It is one of the major diseases seriously threatening the healthy development of the poultry industry. Globally, approximately 13 species of chicken coccidia are known, among which *Eimeria tenella*, *Eimeria bryonica*, *Eimeria toxicaria*, *Eimeria prematurei*, *Eimeria gianti*, *Eimeria spurulenta*, and *Eimeria lenientis* are considered the seven most harmful to the poultry industry. Eimeria tenella primarily infests the cecum, causing significant swelling of both cecums, which can be 3 to 5 times their normal size. Eimeria brucellae parasitizes the posterior segment of the small intestine, at the root of the cecum, and has some pathogenicity, causing punctate hemorrhages and catarrhal inflammation in the intestine. Eimeria toxicae damages the mid-section of the small intestine, causing intestinal wall dilation, thickening, and severe necrosis. Eimeria prematureii parasitizes the anterior third of the small intestine and has low pathogenicity. Eimeria giantii damages the mid-section of the small intestine, causing intestinal dilation and wall thickening. Eimeria clump-forming develops mainly in the epithelial surface, and worms at the same developmental stage often cluster together, resulting in numerous pale white spots on the damaged intestinal segment. Eimeria lenientis and Eimeria harveyi parasitize the anterior segment of the small intestine, have lower pathogenicity, and cause pinhead-sized hemorrhages on the intestinal wall, with severe mucosal hemorrhage. If multiple coccidia cause a mixed infection, the intestines will be enlarged, with numerous hemorrhages on the intestinal mucosa and a large amount of purplish-black blood containing sloughed intestinal epithelial cells. This demonstrates that chicken coccidia primarily parasitize various segments of the chicken's intestines.

[0003] Coccidiosis in chickens is primarily treated with medication for group control. Currently available anticoccidial drugs include sulfonamides, clopidogrel, mebendazole, salinomycin, and triazine phenylacetonitrile derivatives. Diclazuril, a triazine phenylacetonitrile anticoccidial drug, is characterized by high efficacy, low toxicity, and no residue, making it the lowest concentration anticoccidial drug currently available. Diclazuril is highly effective against Eimeria tenella, Eimeria burmannii, Eimeria toxicaria, Eimeria brucellosis, and Eimeria giantiformis. After administration, it not only effectively controls the occurrence and mortality of cecal coccidiosis but can even completely eliminate coccidial oocysts in infected chickens, making it an ideal anticoccidial drug.

[0004] Although diclazuril possesses good insecticidal activity, its solubility is extremely poor. It is only slightly soluble in dimethylformamide, sparingly soluble in tetrahydrofuran, and practically insoluble in water and most organic solvents. Low dosage concentrations pose a risk of uneven mixing during feed administration, and it is extremely difficult to dissolve in intestinal fluids. Diclazuril aqueous solutions solubilized with organic solvents have a short stability period and must be prepared and used immediately (stable in drinking water for no more than 4 hours), otherwise its efficacy is affected. These drawbacks limit its practical application. Therefore, developing convenient clinical formulations and stabilizing diclazuril's solubility in the chicken intestines is particularly urgent and important. Summary of the Invention

[0005] The purpose of this invention is to provide an intestinal-targeted solubilized diclazuril oral nanosuspension, its preparation method, and its application, thereby solving the problems existing in the prior art. The diclazuril oral nanosuspension prepared by this invention achieves intestinal-targeted solubilization through the selection of stabilizers and solubilizers, specifically increasing the solubility and dissolution rate of diclazuril in intestinal fluid, while the increase in gastric juice and aqueous solution is not significant, effectively avoiding excessive absorption of diclazuril.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an intestinal-targeted solubilized diclazuril oral nanosuspension, comprising the following components: diclazuril 2-200 g / L, stabilizer 15-25 g / L, solubilizer 2-25 g / L, preservative 1-20 g / L, defoamer 1-20 g / L, and the balance being purified water.

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

[0009] Furthermore, the stabilizer is composed of a first stabilizer and a second stabilizer;

[0010] The first stabilizer is at least one of polyoxyethylene 40 hydrogenated castor oil, polyvinylpyrrolidone, 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 co-solvent is at least one of sodium dodecyl sulfate and sodium dodecyl sulfonate.

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

[0015] Furthermore, the defoamer is simethicone.

[0016] This invention also provides a method for preparing the above-mentioned diclazuril oral nano-suspension, wherein each 1L of the diclazuril oral nano-suspension is prepared by the following steps:

[0017] (1) Add stabilizer and cosolvent to purified water and stir until homogeneous to obtain solution A;

[0018] (2) Add the preservative to the liquid A and stir until uniform to obtain liquid B;

[0019] (3) Add diclazuril to the liquid B and stir until homogeneous to obtain liquid C;

[0020] (4) Add the defoamer to the liquid C, stir evenly, and then add purified water to make up to 1L to obtain liquid D;

[0021] (5) Add the liquid D to a sand mill and grind it to obtain the diclazuril oral nano suspension.

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

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

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

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

[0026] The present invention also provides the application of the above-mentioned diclazuril oral nanosuspension in the preparation of a drug for treating coccidiosis in chickens.

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

[0028] Coccidia primarily parasitize the intestines, harming animal health. This invention utilizes drug nanotechnology and excipient screening to achieve intestinal-targeted solubilization, significantly increasing the solubility, dissolution rate, and drug content of diclazuril in intestinal fluid. In this invention, the drug in the nano-suspension mainly exists as nano-sized particles, thus minimizing sedimentation and stratification in drinking water, improving stability. Furthermore, the nano-sized, poorly soluble diclazuril particles increase adhesion to the mucosa and slow intestinal excretion, achieving a long-lasting effect. Due to its low concentration, diclazuril is prone to uneven mixing during feed preparation, affecting efficacy. The nano-suspension prepared in this invention exhibits good dispersibility in drinking water, and the selected stabilizers and solubilizers achieve intestinal-targeted solubilization, specifically increasing the solubility and dissolution rate of diclazuril in intestinal fluid, while the increase in gastric juice and aqueous solution is not significant, effectively preventing excessive absorption of diclazuril. Nanoparticle suspensions require fewer pharmaceutical excipients, which further improves the safety of the formulation and reduces the material costs in the production process, giving them significant advantages in practical applications. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 XRD patterns showing the effect of different treatments on the crystal form of the raw drug.

[0031] Figure 2 The graph shows the results of the determination of the content of diclazuril in intestinal fluid in different dosage forms. Detailed Implementation

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

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0037] This invention uses the anticoccidial drug diclazuril as the active pharmaceutical ingredient; one or more of the following as stabilizers: polyvinyl alcohol, poloxamer 188, polyoxyethylene 40 hydrogenated castor oil, Tween 80, polyvinylpyrrolidone, gellan gum, and sodium alginate; one of the following as a cosolvent: sodium dodecyl sulfate and sodium dodecyl sulfonate; at least one or more of the following as preservatives: methylparaben, sorbic acid, and benzyl alcohol; and simethicone as an antifoaming agent. The gellan gum added to the formulation forms a gel in the acidic environment of gastric juice, reducing the contact between drug particles and the stomach and preventing damage to nanoparticles. The slightly alkaline pH of the intestine causes the formulation to lose viscosity and become fluid after entering the body, increasing the contact area between drug particles and the intestinal wall, allowing the drug components to remain for a longer period and achieving a long-lasting effect. Intestinal fluid contains a large amount of bile salts (bile salts are the main components of bile involved in digestion and absorption). Hepatocytes continuously produce bile, which flows out through the hepatic ducts, into the duodenum via the common bile duct, or is transferred to the cystic duct and stored in the gallbladder. During digestion, it is then discharged from the gallbladder back into the duodenum. Bile salts themselves are a class of naturally occurring surfactants in the intestines. These surfactants can form micelles with poorly water-soluble drugs, thereby improving the solubility of poorly soluble drugs. Sulfonates are important components of bile salts and play an important role in the emulsification of intestinal contents. This invention additionally adds sodium dodecyl sulfate, which has a similar chemical structure, significantly increasing the solubility and dissolution rate of diclazuril. This invention utilizes the intense grinding process of a sand mill, namely the violent impact of the pins and stators on the ultrafine grinding media. The media then acts on the material to obtain a nanoscale suspension. The nanoparticles have a large specific surface area, increasing the contact area with the naturally occurring active substances in the intestines, further improving solubility. At the same time, it increases adhesion to the mucosa, reduces the intestinal excretion rate, and achieves a long-lasting effect. Tween 80, gellan gum, and sodium dodecyl sulfonate were screened to identify suspending and solubilizing agents, and an emulsifier carrier solution was prepared to ensure the stability of drug particles in the formulation system and enhance their solubility. The first stabilizer used in this invention—Tween 80—is an excellent O / W type emulsifier. High concentrations of electrolytes and changes in pH have minimal impact on its suspending ability, making it suitable as a pharmaceutical excipient for oral administration. As an amphiphilic active agent, its coating on the surface of drug particles enhances the binding of drug particles with abundant bile salts and phospholipids in the intestine, playing a positive role in achieving targeted solubilization in the intestine. Its anti-flocculation effect also prevents the aggregation of fine drug particles, increasing the stability of the diclazuril oral nanosuspension. The second stabilizer, gellan gum, alters the viscosity of the formulation by changing the pH of the gastrointestinal environment, resisting dissolution in the acidic environment of the stomach, while simultaneously allowing the active ingredients in the formulation to dissolve and fully exert their effects in the intestine, achieving targeted solubilization. The solubilizer—sodium dodecyl sulfonate—is a sulfonate salt, increasing the solubility and dissolution rate of diclazuril. When administered correctly, the drug disperses rapidly and evenly, and remains stable, reducing the risk that low drug concentrations may lead to uneven mixing and thus affect efficacy in clinical practice.The details are as follows:

[0038] Example 1

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

[0040] The preparation method of this intestinal-targeted solubilized diclazuril oral nanosuspension is as follows:

[0041] (1) Add 19g of the first stabilizer, 1g of the second stabilizer and 10g of the cosolvent to 250mL of purified water, stir at 30℃ and 700rpm for 10min to obtain solution A;

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

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

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

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

[0046] Example 2

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

[0048] The preparation method of this intestinal-targeted solubilized diclazuril oral nanosuspension is as follows:

[0049] (1) Add 14.25g of the first stabilizer, 0.75g of the second stabilizer and 2g of the cosolvent to 250mL of purified water, stir at 25℃ and 1000rpm for 30min to obtain solution A;

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

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

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

[0053] (5) Add liquid D to 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 nano suspension.

[0054] Example 3

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

[0056] The preparation method of this intestinal-targeted solubilized diclazuril oral nanosuspension is as follows:

[0057] (1) Add 23.75g of the first stabilizer, 1.25g of the second stabilizer and 2g of the cosolvent to 250mL of purified water, stir at 35℃ and 500rpm for 5min to obtain solution A;

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

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

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

[0061] (5) Add liquid D to 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 nano suspension.

[0062] Experimental Example 1

[0063] The first stabilizer in Example 1 was replaced with polyvinylpyrrolidone (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 with an electric stirrer at 700 rpm for 10 min.

[0065] (2) Add 50g of diclazuril raw material while stirring. After adding, use an electric mixer to stir at 700rpm for 10min.

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

[0067] (4) Take 50 mL of sample into a 50 mL stoppered graduated cylinder, let stand for 3 h, and observe and measure its sedimentation volume ratio and redispersibility respectively.

[0068] (5) Take another 100 μL sample and dilute it with purified water by about 10,000 times (or dilute it to meet the relevant requirements of CVP2020 "Pharmacopoeia of the People's Republic of China"). Use a visible light spectrophotometer to determine the shading degree so that it is between 8% and 20%.

[0069] (6) The diluted sample was tested for particle size and potential.

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

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

[0072]

[0073]

[0074] Note: " / / " indicates that the test could not be performed due to excessive viscosity or other reasons; redispersibility refers to the number of times the sample is fully dispersed by inverting the stoppered graduated cylinder 180° after standing for 3 hours.

[0075] Experiment Example 2

[0076] To achieve intestinal-targeted solubilization, two commonly used gelatable polysaccharides were screened, as follows:

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

[0078] (2) Add 0.25g gellan gum and sodium alginate respectively, add slowly and stir. After adding, use an electric mixer to stir at 700rpm for 10min.

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

[0080] (4) Take 20 glass bottles and divide them into a gellan gum group (simulated gastric juice (SGF), simulated intestinal juice (SIF), SIF + sodium lauryl sulfate, SIF + sodium lauryl sulfonate and drinking water, with two replicates in each group) and a sodium alginate group (SGF, SIF, SIF + sodium lauryl sulfate, SIF + sodium lauryl sulfonate and drinking water, with two replicates in each group). The amount of sodium lauryl sulfate and sodium lauryl sulfonate added is 10 g / L, and the amount of SGF, SIF and drinking water added is 50 mL.

[0081] (5) Take samples and determine solubility using the shake flask method.

[0082] The screening results of the targeted stabilizer and solubilizer are shown in Table 2. Table 2 shows that diclazuril's solubility in simulated intestinal fluid was higher than that in simulated gastric fluid. The addition of sodium alginate and gellan gum both improved the solubility of diclazuril in simulated intestinal fluid, but gellan gum showed a slightly better effect. Meanwhile, the addition of solubilizers, especially sodium dodecyl sulfate, significantly improved solubility. Therefore, the optimal formulation was determined to use gellan gum as the targeted stabilizer and sodium dodecyl sulfate as the solubilizer.

[0083] Table 2 Screening results of targeted stabilizers and cosolvents

[0084]

[0085] Note: " / / " indicates that the concentration was too low to be measured.

[0086] Experimental Example 3

[0087] Since parabens have a significant antagonistic effect with Tween 80, sorbic acid or benzyl alcohol was chosen as the preservative; the concentration was selected based on general industry standards with some fluctuation.

[0088] The specific experimental steps are as follows:

[0089] (1) Using sorbic acid or benzyl alcohol as preservatives, and adjusting the amount of preservatives added to 15 g / L, 20 g / L and 25 g / L respectively, prepare diclazuril oral nano suspension test samples 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 turbidity of 0.5 McFarland units (1×10⁻⁶). 8 A bacterial suspension (cfu / mL) was prepared. Fresh culture of *Aspergillus niger* was added to an appropriate amount of 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80 to elute the spores. The spore suspension was then aspirated into a sterile test tube using a suitable method. An appropriate amount of 0.9% sterile sodium chloride solution containing 0.05% (mL / mL) polysorbate 80 was added to prepare a solution containing 10 spores per mL. 8 CFU spore suspension:

[0091] (3) Take 5 intact test samples and inoculate them directly with the test bacteria. The inoculation amount per 1g or 1mL of test sample is 10. 5 ~10 6 CFU, the volume of the inoculum should 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℃ in the dark.

[0092] (4) On days 14 and 28 after inoculation, take 1 mL of the test sample from each of the above containers, dilute the test solution 10 times with PBS, and inoculate 100 μL onto two plates respectively. Tryptic-soybean agar is used to determine bacteria, and Sabouraud dextrose agar is used to determine fungi. At the same time, PBS is used as a negative control in place of the test solution and spread on two plates. Tryptic-soybean agar is incubated at 35°C for 3 days, and Sabouraud dextrose agar is incubated at 25°C for 5 days.

[0093] The results of the antibacterial efficacy tests for each group are shown in Tables 3-5. Based on Tables 3-5, it can be concluded that benzyl alcohol exhibits insufficient antibacterial efficacy at lower concentrations; however, sorbic acid meets the antibacterial efficacy standards at all three concentrations (low, medium, and high), demonstrating superior antibacterial ability. For cost-saving and formulation safety considerations, a lower concentration of 15 g / L was selected as the preservative concentration.

[0094] Table 3. Results of antibacterial efficacy test of diclazuril suspension with added 15 g / L preservative

[0095]

[0096]

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

[0098] Table 4. Results of antibacterial efficacy test of diclazuril suspension with added 20 g / L preservative

[0099]

[0100] Note: NI stands for No Increase, meaning that the number of test bacteria increased by no more than 0.5lg compared to the previous measurement time.

[0101] Table 5. Results of the antibacterial efficacy test of 525 g / L preservative diclazuril suspension

[0102]

[0103]

[0104] Note: NI stands for No Increase, meaning that the increase in the number of test bacteria compared to the previous measurement time does not exceed 0.5lg.

[0105] Experiment Example 4

[0106] The dosage of defoamer was adjusted to 2.5 g / L, 5 g / L, and 7.5 g / L, respectively. Diclazuril oral nano-suspension samples were prepared according to the method in Example 1. After vigorous shaking for 5 minutes, the time for complete foam disappearance was recorded. The sedimentation volume ratio and average particle size were determined according to the method in Example 1. The results are shown in Table 6. Table 6 shows that the 7.5 g / L concentration had the best defoaming effect, but severe sedimentation and a sedimentation volume ratio that did not meet the requirements. The defoaming time and sedimentation volume ratio at concentrations of 2.5 g / L and 5 g / L were relatively close. Considering all factors, the optimal defoamer concentration was 2.5 g / L.

[0107] Table 6. Effects of different concentrations of defoamer on formulation 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 remaining samples were prepared according to the method in Example 1. 50 mL of the sample was placed into a 50 mL stoppered graduated cylinder and allowed to stand for 3 hours. The sedimentation and properties were then observed.

[0111] The results showed that among the three samples prepared, the 10 g / L addition group precipitated severely and did not form a suspension; the 30 g / L addition group was too viscous and unsuitable for administration via drinking water, and both were discarded. The formulation with an addition of 20 g / L formed a white suspension, and the sedimentation volume ratio met the requirements.

[0112] Experimental Example 6

[0113] Based on the formulation of diclazuril oral nano-suspension and the physicochemical properties of the raw materials and excipients, the preparation process includes emulsifier carrier solution preparation, preservative mixing, active ingredient mixing, and material grinding. The following optimizes the preparation process of diclazuril oral nano-suspension:

[0114] 1. Preparation of solubilizer carrier solution

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

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

[0117]

[0118]

[0119] As shown in Table 7, Tween 80 and Gellan gum can be rapidly dispersed and miscible when the water temperature gradually rises to 25-35℃ and the mixture is stirred for 10 minutes. Considering practical production factors such as energy consumption and production efficiency, it is recommended to heat the water to 25-35℃ before mixing it with Tween 80 and Gellan gum, and then stir for 10 minutes.

[0120] 2. Preservative mixture

[0121] Based on the physicochemical properties of sorbic acid and experimental findings, at room temperature (25±5℃), adding 15 g / L of sorbic acid to the aforementioned Tween-80 aqueous solution results in rapid miscibility between the two solutions. After stirring for 1 min, 3 min, and 5 min, each solution is a clear aqueous solution. Therefore, to fully ensure the stability of all materials in the solution, the mixing time for the preservative sorbic acid was selected as 3 min, and the temperature for both sorbic acid and subsequent ingredients was determined to be room temperature (25±5℃).

[0122] 3. Mixing of active substances

[0123] Diclazuril active pharmaceutical ingredient is a solid powder. When added to the precursor solution, it should be fully wetted and evenly dispersed. The mixing procedure should be optimized as shown in Table 8.

[0124] Table 8. Effects of different stirring times and speeds on the mixing of active substances.

[0125]

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

[0127] 4. Grinding of materials and liquid

[0128] The mixture is ground to ensure that the active ingredient diclazuril in the final product is in 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 initial grinding media were selected with zirconium beads of 0.3 mm in diameter 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 criterion. The prescription used is shown in Table 9, and the grinding time screening is shown in Table 10.

[0131] Table 9. Grinding liquid formula

[0132]

[0133]

[0134] Detailed implementation rules:

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

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

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

[0138] (4) Add the diclazuril raw material to the above liquid while stirring. After the addition is complete, stir at 700 rpm for 10 minutes.

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

[0140] (6) Pour the prepared liquid into a clean sand mill. Set the temperature of the circulating cooler to 10℃ and the grinding speed to 2000rpm. Take 1mL of sample to test the particle size at 20, 25, 30, 35, 40, 45 and 50min respectively.

[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 and 50 minutes, the particle size of diclazuril in the liquid continuously decreases, and an inflection point appears when grinding reaches 45 minutes. Therefore, the grinding time for the liquid grinding process is selected as 45 minutes.

[0145] 4.2 Grinding speed screening

[0146] The initial grinding media, zirconium beads with a diameter of 0.3 mm, and the grinding time of 45 min were selected. The grinding speed was screened, and the particle size and redispersibility of the drug particles were used as the judgment criteria. The formulation 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 purified water at 25 °C to a 500 mL beaker, add Tween 80, sodium dodecyl sulfonate and gellan gum, stir at 700 rpm for 10 min to obtain an emulsifier carrier solution;

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

[0151] (4) Add the diclazuril raw material to the above liquid while stirring. After the addition is complete, stir at 700 rpm for 10 minutes.

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

[0153] (6) Pour the prepared liquid into a clean sand mill, set the temperature of the circulating cooler to 10℃, and set the grinding time to 45min. Grind 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 speeds (mean ± standard deviation, n = 3)

[0156]

[0157] As can be seen from Table 11, a grinding speed of 2000 rpm has a slight advantage in particle size compared to other grinding speeds, but a significant advantage in redispersibility. Therefore, a grinding speed of 2000 rpm is selected for the liquid grinding process.

[0158] 4.3 Grinding media size screening

[0159] The grinding speed was set at 2000 rpm and the grinding time was 45 min. The size of the grinding media was screened, and the particle size and redispersibility of the drug particles were used as the judgment criteria. The formulation 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 purified water at 25 °C to a 500 mL beaker, add Tween 80, sodium dodecyl sulfonate and gellan gum, stir at 700 rpm for 10 min to obtain an emulsifier carrier solution;

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

[0164] (4) Add the diclazuril raw material to the above liquid while stirring. After the addition is complete, stir at 700 rpm for 10 minutes.

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

[0166] (6) Pour the prepared liquid into a clean sand mill. Set the temperature of the circulating cooler to 10℃, the grinding speed to 2000rpm, the grinding time to 45min, and select grinding media sizes of 0.3-0.4mm and 0.6-0.8mm respectively for grinding.

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

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

[0169]

[0170] As can be seen from Table 12, compared with 0.6-0.8 mm, when the grinding medium is selected as 0.3-0.4 mm, the particle size of the drug particles and the redispersibility are significantly better. The grinding medium size of 0.3-0.4 mm is selected for the liquid grinding process.

[0171] Example 4

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

[0173] 1. Stability study in drinking water

[0174] The stability of diclazuril suspension in drinking water was investigated by simulating real-world application scenarios.

[0175] The specific implementation details are as follows:

[0176] Add 100 μL of diclazuril suspension to 1 L of drinking water. Set up three replicates and take 2 mL samples at 0, 3, 6, 9, 12 and 24 h. Take the upper sample 3-5 cm from the water surface and the lower sample 3-5 cm from the bottom. After sampling, evaluate its stability in drinking water according to 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] As can be seen from Table 13, the particle size of the drug particles in the water layer is similar at different times. After being placed under laboratory conditions for 24 hours, the distribution of this preparation is uniform and the stability is good.

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

[0181] The influence of excipients and preparation processes on the crystal form of the active pharmaceutical ingredient (API) will be characterized.

[0182] The specific implementation details are as follows: 30 mL each of the physically mixed sample (prescription amount of each component, stirred at 700 rpm for 10 min), the active pharmaceutical ingredient (only the active pharmaceutical ingredient and purified water were added, stirred at 300 rpm for 10 min), and the prepared diclazuril oral nano-suspension (stirred at 300 rpm for 10 min), centrifuged at 8000 rpm for 10 min, the supernatant was discarded and the sample was frozen at -20℃; after being placed in a freeze dryer for 48 h, the sample was sent for XRD analysis.

[0183] from Figure 1 It can be seen that the formulation excipients and preparation process have no effect on the crystal form of diclazuril. Currently, the excipients are reasonably matched and the production process is feasible.

[0184] 3. Influencing Factors Experiment

[0185] To study the inherent stability of drugs, understand the factors affecting their stability, and identify possible degradation pathways and products, this study aims to provide a scientific basis for formulation manufacturing processes, packaging, storage conditions, and the establishment of analytical methods for degradation products. Influencing factor experiments were conducted, including high-temperature and strong light exposure tests.

[0186] The specific steps are as follows:

[0187] Take one batch of test samples and conduct high temperature test and strong light irradiation test.

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

[0189] (2) Strong light irradiation test: Place the formulation in an illuminance of 4500 lx ± 500 lx, and the total illuminance of the light source should not be less than 1.2 × 10⁻⁶ lx. 6 Under conditions of lux·hr and near-ultraviolet lamp energy of not less than 200 W·hr / m, 4 mL samples were taken at 0d, 5d and 10d to study the stability of the drug by measuring changes in particle size, content and related substances.

[0190] Table 14. Experimental Results of Influencing Factors (Mean ± Standard Deviation, n = 3)

[0191]

[0192] As can be seen from Table 14, high temperature has a relatively limited effect on the formulation, but under strong light conditions, the particle size of the formulation increases slightly, indicating that the drug particles aggregate. This result is in line with expectations. Diclazuril needs to be stored in a light-proof and airtight container.

[0193] Example 5

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

[0195] The specific steps are as follows:

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

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

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

[0199] (4) Animal experiment on the pharmacokinetics of diclazuril in chickens: Sixty healthy white-feathered broilers were selected and divided into five groups of 12 birds each. They were administered diclazuril oral nano-suspension, diclazuril oral nano-suspension, diclazuril oral nano-suspension, diclazuril oral nano-suspension, diclazuril solution, and diclazuril premix, respectively, by gavage. Blood was collected from the wing vein at 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, 72h, 96h, 120h, and 144h after gavage. The samples were processed according to the established plasma pretreatment method and then tested by the instrument.

[0200] (5) Diclazuril retention test in intestinal fluid and intestinal tissue: Forty-eight healthy white-feathered broilers were selected and divided into two groups of 24 each. One group was administered 2 mg / kg bw diclazuril oral nano-suspension, and the other group was administered 2 mg / kg bw diclazuril premix. At 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration, three broilers from each experimental group were sacrificed at each time point, and their intestinal contents and intestinal tissue were collected. Samples were processed according to the established intestinal fluid pretreatment method and then analyzed. The measured sample data were fitted using Phoenix software.

[0201] Table 152 mg / kg.bw Diclazuril oral nano-suspension administered to chickens.

[0202]

[0203]

[0204] Table 162 mg / kg bw diclazuril premix administered orally to chickens

[0205]

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

[0207] Example 6

[0208] To verify the solubilizing effect of diclazuril oral nano-suspension, the solubility test in water, simulated intestinal fluid, and simulated gastric fluid was conducted using the diclazuril oral nano-suspension prepared in Example 1 as an example.

[0209] The specific steps are as follows:

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

[0211] (2) Shake the constant temperature shaker at 25℃ and 220rpm for 24 hours, and 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 then filter it through the membrane for use.

[0213] 1 μg / mL standard: Take 1 mg / mL standard stock solution, serially dilute to 1 μg / mL, dilute twice with water, filter through membrane and run on the instrument, repeat twice.

[0214] Table 17 Solubility of Samples with Different Solubilities (μg / mL)

[0215]

[0216] Note: " / / " indicates that the difference is too large and the value was discarded or that no value was measured.

[0217] As can be seen from Table 17, the solubility of diclazuril oral nano-suspension in simulated gastric juice was too low, and no effective value was determined. However, its solubility in simulated intestinal juice was more than twice that in water, indicating that the solubility of diclazuril in its main site of action—the intestine—was greatly improved by the process and excipients.

[0218] In summary, the diclazuril oral nano-suspension prepared by this invention can achieve intestinal targeting, significantly increase solubility and dissolution rate; moreover, the drug particles are small, have good dispersibility, and are easy to mix; fewer pharmaceutical excipients are used, which improves the safety of the formulation and reduces the material cost in the production process, and has significant advantages in practical applications.

[0219] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A diclazuril oral nanosuspension with intestinal-targeted solubilization, characterized in that, The oral nano-suspension of diclazuril consists of the following components: diclazuril 2-200 g / L, stabilizer 15-25 g / L, solubilizer 2-25 g / L, preservative 1-20 g / L, defoamer 2.5 g / L, and the balance being purified water; The stabilizer consists of a first stabilizer and a second stabilizer; The first stabilizer is Tween 80; The second stabilizer is gellan gum; The mass ratio of the first stabilizer to the second stabilizer is (19-20):1; The co-solvent is at least one of sodium dodecyl sulfate and sodium dodecyl sulfonate; The preservative is sorbic acid; The defoamer is simethicone.

2. The diclazuril oral nano-suspension according to claim 1, characterized in that, The oral nano-suspension of diclazuril consists of the following components: 100 g / L diclazuril, 20 g / L stabilizer, 10 g / L solubilizer, 15 g / L preservative, 2.5 g / L defoamer, and the balance being purified water. The mass ratio of the first stabilizer to the second stabilizer is 19:

1.

3. A method for preparing a diclazuril oral nanosuspension as described in any one of claims 1-2, characterized in that, The preparation method of each 1L of the diclazuril oral nano-suspension includes the following steps: (1) Add the stabilizer and cosolvent to the purified water and stir until homogeneous to obtain solution A; (2) Add the preservative to the liquid A and stir evenly to obtain liquid B; (3) Add diclazuril to the liquid B and stir evenly to obtain liquid C; (4) Add the defoamer to the liquid C, stir evenly, and then add purified water to make up to 1 L to obtain liquid D; (5) Add the liquid material D to a sand mill and grind it to obtain the diclazuril oral nano suspension; 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.

4. The use of the diclazuril oral nanosuspension as described in any one of claims 1-2 in the preparation of a drug for treating coccidiosis in chickens.

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