Pharmacokinetics of Ethanamizuril Dispersed by Different Solvents In Chickens After Oral Administration

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Pharmacokinetics of Ethanamizuril Dispersed by Different Solvents In Chickens After Oral Administration

https://doi.org/10.21203/rs.3.rs-3230017/v1

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In this study Ethanamizuril in different dispersants were administrated orally to target animal, chickens for pharmacokinetic study. An ultra-performance liquid chromatography method was established for sample detection and the extraction method was validated. Internal standard was used to calculate the drug concentration in chicken plasma. The results showed that the peak time of Ethanamizuril in chicken under the three dispersants was 4 ~ 5 hours, and the half-life (t_1/2) of Ethanamizuril in sodium carboxymethyl cellulose solution (CMC-Na), PEG300 and Ethanamizuril solution diluted by water had a significant difference (P < 0.05) and AUC and C_max had no significant difference which also showed no significant difference in drug exposure. AUC and C_max were important indicators of drug exposure in toxicity studies. The study showed that different dispersants had little effect on the evaluation of toxicity studies of the drug.

Biological sciences/Drug discovery/Drug delivery

Biological sciences/Drug discovery/Toxicology

Ethanamizuril

Pharmacokinetics

Dispersants

Oral Administration

The results of the pharmacokinetic study of the drug in animals can be used as the basis for clinical and toxicity studies. In this study, Ethanamizuril was dispersed in sodium carboxymethylcellulose solution and polyethylene glycol 300, and Ethanamizuril solution was diluted with water. The three kinds of solution were administered orally to chickens for pharmacokinetic studies. Study the differences in pharmacokinetic behavior of drugs in different dispersants to guide clinical and toxicological studies.

Chicken coccidiosis is a worldwide epidemic chicken intestinal disease caused by a variety of unicellular coccidia parasitic in the intestine. Chemical drugs have been playing a leading role in the prevention and treatment of coccidiosis. According to different chemical structures, anticoccidial drugs are mainly divided into polyether ionophore antibiotics and chemically synthesized anticoccidial drugs¹. In addition, there are some non-specific anti coccidiosis drug preparations, such as Chinese herbal medicine, as a supplement to the prevention and treatment of coccidiosis. These drugs have broad-spectrum resistance to coccidia and are not easy to develop resistance. At the same time, they can also promote animal growth and development and improve feed utilization. Chemically synthesized coccidial drugs can be divided into polyether ionic antibiotics according to their chemical structure, including Maduramycin, monensin, lassamycin, salinomycin, nalaxine, etc; Sulfa drugs, including sulfaquinoline, sulfachlorpyrifos, sulfamethazine, etc; Amides include nitrobenzamide and nitrochlorobenzamide; Quinolines include phenoxyquinoline, decaoxyquinoline and butoxyquinoline; Pyridines, such as chloropropylpyridine; Triazines include diclazuride and tocotrizuride². Triazine anticoccidial drugs are the most effective anticoccidial drugs with the lowest concentration known at present. They are cheap and widely used. For more than half a century, the long-term use of drugs has led to the existence of heavy or light resistance in almost all drugs. In the case of poor therapeutic effect of existing drugs, people are eager to find new anti coccidial drugs with high efficiency, low toxicity and not easy to drug resistance^3,4.

Ethanamizuril(EZL) belongs to triazine anticoccidial compounds. The study shows that its main action stage is the schizore production and gamete reproduction stage of the life cycle of coccidia. The peak period of action is 3 to 4 days after coccidia infection. The drug can cause obvious outer membrane fusion, invagination and convexity of coccidia, as well as the abnormalities of multiple organelles. The characteristic action site is the membranous structure of coccidia. This phenomenon has not been reported in the ultrastructural effects of similar drugs. It was found that the insect proteins sensitive to EZL are mainly macromolecular complexes, cells, organelles and cell membrane compots, including multiple surface antigens and proteins related to intracellular metabolic processes. EZL down regulates the expression of enolase, LDH, u6l233 and q70cdo at mRNA and protein levels. Enolase and LDH participate in coccidial invasion and play a key role in coccidial infection. They may be potential targets of drugs, which need to be further verified^5–8.

In the research of new veterinary drugs, during various clinical and toxicological tests of drugs used for oral administration, in order to facilitate the administration and measurement, it is necessary to disperse the drugs with various mediums before oral administration to animals, including preparing high viscosity suspension with sodium carboxymethyl cellulose, or selecting pharmaceutical grade solvent to disperse or dissolve the raw materials before relevant tests. Therefore, it is necessary to investigate the effects of various dispersants on the pharmacokinetics and exposure of oral drugs. In this study, EZL was dispersed in sodium carboxymethyl cellulose solution (CMC-Na) and PEG300, and EZL solution was diluted with water, and then administered orally to the target animals, chickens, for pharmacokinetic study. The absorption, metabolism, and exposure of EZL in different dispersants were analyzed to provide basis for clinical application and related research^5,9,10.

2.1 Materials

2.1.1 Chemicals and Reagents

EZL (purity ≥ 99.5%) was synthesized, identified and determined by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science (SHVRI). EZL solution(1%) also was prepared and determined by SHVRI, including solubilizer, stabilizer and other pharmaceutical excipients to ensure the stability of the water after 1000 times dilution, and was used for adding to animal drinking water for drug administration.

Toltrazuril sulfoxide (TSO,99.4%±0.2%), as internal standard was purchased from WTTEGA, Laboratorien. Acetonitrile and formic acid were HPLC grade solvent, obtained from Fisher Scientific Co. (Santa Clara, CA, USA). Analytical ethyl acetate, acetonitrile was obtained from Sinopharm Chemical (Shanghai, China). The water used in the experiments was purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA).

2.1.2 Apparatus

Ultra-performance liquid chromatography (UPLC) equipment is Acquity UPLC H-Class system equipped with Acquity Quaternary Binary Solvent Manager, Acquity Sample Manager and PDA detector. The Empower III chromatography software (Waters) is used for data acquisition. A highspeed blender (IKA MS3) was purchased from IKA-werk (Germany). An ultrasonic instrument (KQ-500DE; Kunshan Ultrasonic Instrument Ltd. CO., China), a vortex mixer (Genie-2; Scientific Industries, Inc., Bohemia, NY, USA), and a centrifuge (Shanghai Anting Scientific Instruments, Shanghai, China) were used for samples of cecal contents preparation. The cages used in this experiment were 81 cm long, 60 cm high and 70 cm deep were purchased from Shanghai Changshuo Biotechnology Co., Ltd (Shanghai, China).

2.1.3 Animals and Experimental Design

Yellow broiler chicken, weighing 1.5-2.0 kg, was purchased from hatchery in Pudong New Area, Shanghai. The animals were kept in clean and hygienic conditions at 22 ± 2 ° C and 60 ± 10% relative humidity. The light and dark were alternated every 4 hours. These birds can drink and eat freely without any medicine. The cage size is 600 mm × 500 mm × 430 mm. Use air conditioning and ventilation fans to maintain the recommended temperature and humidity. The protocol complies with the guidelines of the Chinese institutional animal care and use committee and was approved by the ethics committee of the institute.

EZL was administered orally to chickens at 0.67 mg/kg in water, 0.5%CMC-Na or PEG300 (n = 6 for each sampling time point). 0.5 mL blood samples were collected from chicken wing vein to anticoagulant tube with heparin at 0、0.5、2、4、6、8、12、24、36、48、72 h, and tubes were centrifuged at 3000 rpm for ten minutes. Plasma was got for upside and stored at -40°C until determination.

2.1.3 Standard solution

Accurately weigh 20 mg EZL dissolved in methanol, sonicate to completely dissolve the drug, configure 1000 µg/mL of EZL stock solution, dilute with methanol into 2, 4, 10, 20, 40, 100, 200, 400, 400 µg/mL of EZL standard, 4°C refrigerator storage. Accurately weigh 10 mg of TSO dissolved in 10 mL of methanol, configure 1000 µg/mL of TSO standard stock solution, dilute with methanol to 200 µg/mL, and store in a 4°C refrigerator.

2.2. Sample Preparation

Before the experiment, blood samples were taken out from the refrigerator and placed at room temperature. 100 µL plasma was placed into the centrifuge tube, adding 10 µL internal standard solution precisely, mixed for 30 s, and then adding 1 ml of ethyl acetate, centrifuged at 15000 rpm for 10 min. The supernatant was suck up and blown dry with nitrogen. 100 µL mobile phase (acetonitrile-water, 10:90, V/V) was added and solubilized the residue, vortex for 30 s. Finally, the supernatant solution was filtered through a 0.22 µm filter membrane before analyzing by HPLC.

2.3 HPLC Analysis

An ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 µm) column was used to detect EZL and TSO equipped with a precolumn, Van guard BEH C18(5 × 2.1 mm, 1.7µm)

The mobile phase was a mixture of acetonitrile(A) and water (B) as a gradient elution at a flow rate of 0.2 mL/min. Initially, the gradient was held for 2 min at 10% of phase A, then the mobile phase composition was changed gradually to 80% of A until 7 min, 80% of A from 7 to 10 min, and to 10% of A at 11 min. The total analysis time was 15 minutes when the column temperature is 30 ± 5 ℃. The injection volume is 10 µ L, and the ultraviolet detector is set at the wavelength of 251nm.

2.4 Method Validation

Before the pharmacokinetic experiment of different dispersants in chicken, the performance parameters of the chromatographic system were evaluated by calculating the specificity, resolution, selectivity, and column efficiency of EZL under gradient elution program. The validation of the development method was carried out in accordance with the guidelines established by European Commission Decision 2021 (EU) or Directive 808.

Selectivity was evaluated by analyzing 20 blank chicken plasma samples to study potential interfering peaks of about 2.5% around the peak edge of the target analyte.

The matrix addition standard calibration curve was prepared by using a series of 7 standard working solutions of EZL concentration. Take blank plasma, add standard solution to the concentration of standard koji, and treat it as described in Item 2.2. The standard curve was obtained by plotting the relationship between the ratio of peak area of EZL to the area of internal standard peak (y-axis) and the corresponding concentration of EZL in plasma (x-axis), with the concentration range of 0.04 to 10 µg/mL. Linear regression analysis was used to calculate slope, intercept and correlation coefficient (r2). When r2 was greater than 0.99, it indicates good linearity.

The limit of detection (LOD) and limit of quantification (LOQ) of EZL in plasma are estimated as concentrations, which can produce signal to noise ratios (S/N) higher than 3 and 10, respectively.

In the analysis process, the stability test of EZL was conducted at the concentrations of 0.04, 0.4 and 4 µ g/mL in 5 repeated plasma samples only under specific conditions; (a) Place the treated sample at room temperature for 8 hours; (b) Store the plasma sample at − 20 ℃ and freeze it for 24h. Take it out and place it at room temperature for 1h to melt into a cycle. After three consecutive cycles, measure the stability of EZL in the plasma. Then, the plasma sample was processed and analyzed, and the concentration obtained was compared with the concentration added at 0 hour.

Three concentrations of 0.04, 0.4, and 4 µg/mL were selected for the experiment to investigate the recovery and precision. 100 uL blank plasma was added to the corresponding standard solution and processed according to the sample processing method under 2.2. Three batches of samples were processed and measured continuously, and each concentration of each batch of samples was repeated five times. The relative recovery and intra batch and inter batch variation coefficients were calculated.

2.5 Quality Control

The quality control sample refers to the sample prepared by adding the standard solution of the known amount of the substance to be tested into the blank sample. In ensure the accuracy and reliability of the experimental results, the quality control sample with known concentration needs to be added when testing the unknown sample. The quantity of quality control samples is generally required to be no less than 5% of the number of unknown samples, and should include low, medium, and high concentrations, which should be analyzed simultaneously with the unknown samples at a certain interval according to the total number of unknown samples. The deviation of the quality control sample measurement should be controlled within 15%. If the deviation of more than 1/3 of the quality control samples is greater than 15%, the measurement result is invalid and needs to be re measured.

2.6. Data Analysis

The statistical analyses and the pharmacokinetic parameters of EZL were analyzed by Drug and Statistics, or Drug Analysis System software (DAS, Version 3.0; Shanghai BioGuider Pharmaceutical Technology Co., Ltd, China).

3.1. Optimization of the Chromatographic Separation Conditions

According to previous experiments, TSO and EZL are triazine compounds with similar chemical structure, and the retention behavior of TSO was similar to that of EZL, so TSO was selected as the internal standard. The composition of the liquid phase mobile phase would affect the response value and peak shape of the analyte in UPLC detection. Using water containing 0.1% formic acid as the water phase improved the peak shape and response of EZL. The signal response of EZL and TSO was also the best at 251nm. After the sample was processed and analyzed, the chromatogram is shown in Fig. 1. The retention time of EZL and TSO has no interference from other matrix components, and the specificity was good.

3.2Method Validation

3.2.1 Limit of detection and lower limit of quantification

According to 2.4, the analysis results show that the signal-to-noise ratio (S/N) was greater than 3 when the concentration of EZL was 0.02 µg/mL, so the detection limit of this experimental method was 0.02 µg/mL; When the concentration of EZL was 0.04 µg/mL, the signal-to-noise ratio (S/N) is greater than 10. According to the requirements of the Guidelines for Non-clinical Pharmacokinetic Experiment of Veterinary Chemicals, the limit of quantitation should meet the requirements of detecting the blood drug concentration of the sample with 3–5 half-lives or the maximum blood drug concentration of 1/10 to 1/20. This detection method meets the detection requirements.

3.2.2 Standard curve drawing

Prepare the standard plasma sample according to the method described in 2.4. With the ratio of EZL/TSO as the Y value and the standard concentration as the X value, the standard curve was obtained by data processing calculation: Y = 4.9641X-0.0969, R² = 0.9999, which meets the detection requirements.

3.2.3 Accuracy and precision

In this study, the sample recoveries and inter-batch and intra-batch precision of 0.04, 0.4 and 4 µg/mL were investigated. The accuracy was expressed by the relative recovery of each concentration, and the precision was expressed by the relative standard deviation (RSD) between batches and within batches.

According to the requirements of the Guidelines for Non-clinical Pharmacokinetic Experiment of Veterinary Chemicals, the recovery rate of the added concentration near the limit of quantification should be controlled within 80% − 120%, and the RSD range should be within 20%; The recovery of other added concentrations should be within 85% − 115%, and the RSD should be within 15%. The test results of the accuracy and precision of the analytical method in this experiment were shown in Table 1. The recovery rate of added concentration was between 91.4% and 110%, and the coefficient of variation within and between batches was within 10%. The test results showed that the method had good repeatability, and the coefficient of variation and recovery met the test requirements.

Table 1

Accuracy and precision results of EZL in chicken plasma
Spiked Level (µg/mL)	No.	Intra-level Mean Recovery (%,n = 5)			Intra-level RSD (%,n = 5)	Inter-level Mean Recovery (%,n = 15)			Inter-level RSD (%,n = 15)
0.04	1	99.50	±	5.79	5.82
	2	96.00	±	8.60	8.96	101.83	±	8.54	8.38
	3	110.00	±	2.24	2.03
0.4	1	104.95	±	3.16	3.01
	2	103.60	±	4.28	4.13	99.97	±	7.52	7.53
	3	91.35	±	5.41	5.92
4	1	100.78	±	3.34	3.31
	2	102.48	±	5.71	5.57	100.82	±	5.21	5.17
	3	99.22	±	5.70	5.74

Table 2

Stability of chicken plasma spiked samples in 8 h at room temperature
Spiked Level (µg/mL)	Detected Value (µg/mL)					Mean Recovery (%,n = 5)
0.4	0.430	0.392	0.443	0.441	0.390	104.8 ± 6.3
4	3.875	3.896	4.210	4.125	4.335	102.2 ± 4.9

3.2.4 Stability inspection

In this experiment, the stability of two sample concentrations of 0.4 and 4µg/mL at room temperature for 8 hours and repeated freeze-thaw for three times was investigated. The sample pretreatment was described in 2.2, and the test results were shown inTable2 and Table 3. It was seen from the above tables that the average relative recoveries of 0.4 and 4 µg/mL of the two investigated concentrations were 104.8% ± 6.3% and 102.2% ± 4.9% respectively when placed at room temperature for 8 hours. The average relative recoveries of the two concentrations of 0.4 and 4 µg/mL were 94.5% ± 4.3% and 92.7% ± 0.7% respectively after repeated freeze-thaw for three times. The test results showed that the drug content in plasma was stable after the sample was placed at room temperature for 8 hours and repeated freeze-thaw.

Table 3

Stability of chicken plasma spiked after freezing-thawing three times
Spiked Level (µg/mL)	Detected Value (µg/mL)					Mean Recovery (%,n = 5)
0.4	0.373	0.389	0.400	0.366	0.361	94.5 ± 4.3
4	3.750	3.709	3.702	3.696	3.682	92.7 ± 0.7

3.3 Pharmacokinetic experiment results

3.3.1 Blood drug concentration-time test data

According to the UPLC-UV analysis method described in the methodology, the plasma samples collected from three groups of experimental chickens at each time point after administration were analyzed, and the quality control samples were added evenly during the analysis to ensure the reliability and authenticity of the experimental results. The dosage of three groups of experimental chickens under different dispersions was 1.33 mg/kg. The drug concentration-time curves were shown in Fig. 2.

3.3.2 Pharmacokinetic parameters

It could be seen from Table 4 that the peak time of EZL in chicken under the three dispersants was 4–5 hours, and the half-life (t_1/2) of the drug had a significant difference (P < 0.05). Compared with beagles, the absorption of EZL in chicken is slower and its half-life is longer.¹¹

Table 4

Pharmacokinetic parameters of EZL of diverse dispersants
Pharmacokinetic Parameter	Unit	Dispersant
Pharmacokinetic Parameter	Unit	Water	0.5%CMCNa	PEG300
AUC_(0−t)	mg/L*h	82.85 ± 35.68	82.19 ± 17.08	71.42 ± 16.04
MRT_(0−t)	h	16.28 ± 5.52	15.68 ± 1.22	12.75 ± 3.15
t_1/2z	h	11.31 ± 4.08^a	9.47 ± 1.75^b	7.16 ± 0.73^c
T_max	h	4.67 ± 1.03	4.67 ± 1.03	4.33 ± 0.82
V_z/F	L/kg	0.27 ± 0.057	0.23 ± 0.07	0.20 ± 0.036
CL_z/F	L/h/kg	0.018 ± 0.007	0.017 ± 0.004	0.019 ± 0.004
C_max	mg/L	3.78 ± 0.52	3.79 ± 0.88	4.15 ± 0.76

T test was used to analyze the difference of main pharmacokinetic parameters. It was found that the differences of the exposure of EZL in water, 0.5% CMC-Na and PEG200 were not significant in target animal chickens (P > 0.05), as shown in Table 5.

Table 5

Statistical analysis of pharmacokinetic parameters of diverse dispersants
Pharmacokinetic Parameter	Unit	Water	0.5%CMCNa	PEG300	Difference
AUC_(0−t)	mg/L*h	82.85 ± 35.6	82.19 ± 17.08	71.42 ± 16.38	P > 0.05
C_max	mg/L	3.78 ± 0.52	3.79 ± 0.88	4.15 ± 0.76	P > 0.05

This article described the pharmacokinetics of three different dispersants of EZL in chickens after oral administration. The pharmacokinetic parameters were obtained by processing with the pharmacokinetic analysis software DAS 3.0. The results showed that there was no difference of AUC and C_max in the effects of dispersants water, CMC-Na, and PEG300 used in the study on the exposure of EZL to experimental chickens. This study provided a reference for the toxicological, pharmacological, and clinical applications of EZL.

Author Contributions: Conceptualization, T.J. Hang and F.Q. Xue, writing-original draft preparation, X.Y. Wang.; writing-review and editing, Z.H. Zhang, methodology, L.F. Zhang and C.Z. Fei; Validation, C.M. Wang, project administration, K.Y. Zhang; All authors have read and agreed to the published version of the manuscript.

Funding: The study was supported by the grants of National Natural Science Foundation of China (31602113).

Institutional Review Board Statement: The study was approved by the Institutional Animal Care and Use Committee at Institute of Shanghai Veterinary Research Institute, CAAS. The protocol complies with the guidelines of the Chinese institutional animal care and use committee and was approved by the ethics committee of the institute. This study was in accordance with ARRIVE guidelines.

Informed Consent Statement: Not applicable.

Data Availability Statement: All data are available in the main text or supplementary materials. All data, code, and materials used in the analysis are available for academic and nonprofit purposes by contacting the corresponding authors ([email protected]).

Acknowledgments: The study was supported by the grants of National Natural Science Foundation of China (31602113).

Conflicts of Interest: The authors declare no conflict of interest.

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No competing interests reported.

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Pharmacokinetics of Ethanamizuril Dispersed by Different Solvents In Chickens After Oral Administration

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Version 1

Abstract

Figures

Simple Summary

1. Introduction

2. Materials and Method

2.1 Materials

2.1.1 Chemicals and Reagents

2.1.2 Apparatus

2.1.3 Animals and Experimental Design

2.1.3 Standard solution

2.2. Sample Preparation

2.3 HPLC Analysis

2.4 Method Validation

2.5 Quality Control

2.6. Data Analysis

3. Results and Discussion

3.1. Optimization of the Chromatographic Separation Conditions

3.2Method Validation

3.2.1 Limit of detection and lower limit of quantification

3.2.2 Standard curve drawing

3.2.3 Accuracy and precision

3.2.4 Stability inspection

3.3 Pharmacokinetic experiment results

3.3.1 Blood drug concentration-time test data

3.3.2 Pharmacokinetic parameters

4. Conclusion

Declarations

References

Additional Declarations

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