Comparative Study on Pharmacokinetics of Four Long-Acting Injectable Formulations of Enrofloxacin in Pigs

A comparative study on pharmacokinetics of four long-acting enrofloxacin injectable formulations was investigated in 36 healthy pigs after intramuscular injection according to the recommended single dose @ 2.5 mg/kg body weight. The drug concentrations in the plasma were computed using high-performance liquid chromatography (HPLC) with fluorescence detection. WinNonLin5.2.1 software was used to analyze the experimental data and compared it under one-way ANOVA using SPSS software with a 95% confidence interval (CI). The main pharmacokinetic parameters, that is, the maximum plasma concentrations (Cmax), the time to maximum concentration (Tmax), area under the time curve concentration (AUCall) and Terminal half-life (T1/2) were 733.84 ± 129.87, 917.00 ± 240.13, 694.84 ± 163.49, 621.98 ± 227.25 ng/ml, 2.19 ± 0.0.66, 1.50 ± 0.37, 2.89 ± 0.24, 0.34 ± 0.13 h, 7754.43 ± 2887.16, 8084.11 ± 1543.98, 7369.42 ± 2334.99, 4194.10 ± 1186.62 ng h/ml, 10.48 ± 2.72, 10.37 ± 2.38, 10.20 ± 2.81, and 10.61 ± 0.86 h for 10% enrofloxacin (Alkali), 20% enrofloxacin (Acidic), Yangkang and control drug Nuokang® respectively. There were significant differences among Cmax, Tmax, and AUCall of three formulations compare with that of the reference formulation. No significant differences were observed among the T1/2 for tested formulations compare with the reference formulation. The pharmacokinetic parameters showed that the tested formulations were somewhat better compared to the reference one. The calculated PK/PD indices were effective for bacteria such as Actinobacillus pleuropneumoniae and Pasteurella multocida with values higher than the cut-off points (Cmax/MIC90≥10–12 and AUC/MIC90 ≥ 125). However, they were not effective against bacteria like Haemophilus parasuis, Streptococcus suis, E. coli, and Bordetella bronchiseptica where lower values were obtained.


INTRODUCTION
Fluoroquinolones are essential therapeutic agents used for the treatment of animal infection (1) against a wide variety of microorganisms, including Mycoplasma species (2). Enrofloxacin (ENR) is a third-generation fluoroquinolone having broad-spectrum antimicrobial activity used in the veterinary field (3) that enhances the possibility of selecting resisting bacteria (4). They are widely used in farm animals because of their antimicrobial activity against a wide range of pathogens, favorable pharmacokinetic properties, and little toxicity (5). Quinolones are not allowed to use in poultry, where eggs are consumed by humans (6). A documentary at the University of Georgia showed enrofloxacin sensitivity for gramnegatives was 89%, and gram-positive was 38% (7). Enrofloxacin is signified for the treatment of respiratory and alimentary tract infections (8). Enrofloxacin is accepted for the treatment and control of swine respiratory disease caused by Pasteurella multocida, Streptococcus suis, Actinobacillus pleuropneumoniae, and Haemophilus parasuis (9).
After administration, enrofloxacin partially metabolizes into ciprofloxacin (CIP) in some species including pigs (16). The metabolic conversion of enrofloxacin to ciprofloxacin varies in different animal species, that is, 59% in dairy cows (17), 36% in sheep (18), 47% in buffalo calves (19), 64% in beef steers (17), and 51.5% in healthy pigs (16). CIP is also effective against Gram-positive, negative, aerobes, and mycoplasmas. Although CIP has limited usage in the veterinary field, as a metabolite of ENR in animals, it reduces animal mortality and enhances growth (20,21). Enrofloxacin shows moderate bioavailability, reasonable protein binding, and better binding to tissues (4,22). Although ENR is the classical antibiotics of the quinolone family, the misuse and overuse of antibiotics have been recognized as a significant cause of developing resistant pathogens (23) and may have adverse effects on human health from its residues in animals, such as allergy and ENR-resistant strains (20).
The long-acting enrofloxacin injection for livestock and poultry optimizes various components. It does not affect the curative effect while reducing the total dose, the treatment cost, and has the advantages of long-lasting effects, highefficiency, safe and reliable preparation. Long-acting injections of enrofloxacin are suitable for various infectious diseases in the respiratory system, digestive system, urinary system, and soft tissues of the skin caused by susceptible bacteria and mycoplasmas (24).
Generally, PK/PD modeling is used to assess the clinical efficacy of antimicrobial agents (25). The most commonly used PK parameters are the maximum plasma concentration (C max ) and the area under the plasma concentration-time curve (AUC) (26). Fluoroquinolones are known as concentration-dependent drugs, and AUC/MIC and C max /MIC are better interpreters for the antibacterial effect (27,28). Therefore, it is crucial to calculate the C max and AUC value of enrofloxacin against the pathogens in swine.
Our study aimed to investigate the pharmacokinetic profiles of four long-acting ENR injectable formulations in pigs after intramuscular administration at a single dose of 2.5 mg per kg body weight. To establish the safe and effective therapeutic management of drugs in pigs, this pharmacokinetics study will establish appropriate clinical treatment for the new formulations of enrofloxacin injection for farm animals, especially for pigs.

Methodology Establishment
Established a sensitive, specific, accurate, and reliable method for quantitative analysis of biological samples and confirmed the method. In this study, the protein was precipitated by methanol to establish an HPLC method for the determination of ENR in pig plasma.

Standard Solution Preparation
First stock solution of 1 mg/ml ENR was prepared by adding 25 mg ENR into 25 ml acetonitrile (HPLC Grade) in a 25 ml capacity volumetric flask. The stock solution was stored at 4 • C for the preparation of 2 nd stock solutions and further use. Second stock solutions of ENR of 500 µg/ml, 50µg/ml, 5 µg/ml, and 1 µg/ml were made by adding the required amount of mobile phase. The same procedure was followed to prepare the working solutions of CIP. All the prepared solutions were kept at 4 • C for further use and kept at room temperature before use.

Extraction Procedure
An aliquot (400 µl) of plasma containing ENR was placed in a 2 ml centrifuge tube. 400 µl of methanol was added to the mixture. The mixture was vortexed for 1 min at high speed and then sonicated for 5 min. After then, the mixture was centrifuged for 10 min at 12,000 rpm and 4 • C. The upper, aqueous layer was transferred into an auto-sampler vial through a 0.22 µm microporous membrane using a 1 ml syringe. Plasma extracts were then analyzed for ENR and its metabolite CIP using the described HPLC conditions.

HPLC Method Validation
The effectiveness of the HPLC method was validated by evaluating sensibility, specificity, linearity, stability, accuracy, and precision. Eight different blank plasma samples with corresponding standard plasma with ENR standard were evaluated to justify specificity. The stability of plasma samples under three different storage conditions was assessed by determining six replicates of quality control samples (0.1, 1, 5 µg/ml) such as storage at room temperature for 24 h, at 4 • C for 24 h, and freeze-thaw cycles (from −20 • C to room temperature, three times). Accuracy and precision of intra-day and inter-day were investigated by six replicates of quality control samples on the same day, and for 3 consecutive days, respectively.

Experimental Animals
All animal experiments were approved by the Animal Administration and Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences. The certificate number was SCXK (Gan) 2019-002. Total 32 healthy pigs (Duroc × Changbai × Dabai) were taken, an average weight of about 27 kg and age 15-16 weeks, randomly divided into four groups, eight pigs in each group. Half female and half male. During the study, the pigs were housed in a clean, quiet environment and fed balanced food, and the water supply was ad-libitum. The average environment temperature and relative humidity were 20 • C and 60%, respectively. The test site was GLP/GCP Management Center for Veterinary Drugs, Standard Experimental Animal Field of the Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences.

Administration of Drugs, Blood Sample Collection and Blood Sample Processing
The pigs were kept to adapt to the environment for 7 days before the administration of drugs. The pigs were kept fasted for 12 h, and weight was measured before drug administration. According to the clinical recommendation, a dose of 2.5 mg per kg of body weight was administered once intramuscularly. The blood collection site was the anterior vena cava. Ten percent ENR injection (alkaline) was administered in group-1, 20% ENR injection (acidic) was administered in group-2, 10% ENR injection (Yangkang) was administered in group-3, 20% ENR injection (Nuokang R ) was administered in group-4. The blood collection schedule was to be 0 (before administration of drug), 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 8, 12, 16, 24, 36, 48 60, and 70 th h. The blood collection volume was 5 ml. The blood was collected in a heparinized tube and centrifuged at 4,500 rpm for 10 min, and the supernatant was aspirated in another tube. The plasma was kept at −20 • C to analyze.

Data Processing and Statistical Analysis
Linear Trapezoidal with Linear Interpolation calculation method of WINNONLIN noncompartmental analysis program (Version 5.2.1) was used to analyze the experimental data. We obtained the most important pharmacokinetic parameters, that is, C max , T max AUC all, and T 1/2 weighting by the 1/Y scheme of the software. The parameters were compared under one-way ANOVA using SPSS software with a 95% confidence interval (CI). The p-value of <0.05 was considered statistically significant.

Method Validation
Eight points (0.025, 0.05, 0.1, 0.2, 0.5 1, 2, 5 µg/ml) were considered for establishing a standard curve of enrofloxacin and ciprofloxacin in plasma. Calculations of the standard curve were based on the peak area with the respective concentrations of ENR and CIP. They showed a good selectivity and linear relationship with a correlation coefficient (R 2 ) = 0.999 in plasma. The mean recovery was more than 94% and 90% for ENR and CIP, respectively. The limit of quantitation (LOQ) was 50 ng/ml in plasma. The intra-day and inter-day precisions (RSD) were <12.7 and <7.3% respectively. The chromatogram (Figure 1) shows (A) control blank plasma; (B) ENR in pig plasma measured at 7.5 min; (C) ciprofloxacin in standard plasma measured at 5.8 min, and (D) ENR and ciprofloxacin in standard plasma measured at 7.5 min and 5.7 min respectively which shows the suggested method for the detection of ENR and its active metabolites CIP is specific and accurate.

Pharmacokinetic Analysis
A semi-logarithmic plot of the mean plasma concentration (ng/ml) in pigs at various time points following IM  administration of 10% enrofloxacin (Alkali), 20% enrofloxacin (acidic), 10% enrofloxacin (Yangkang), and reference formulations (Nuokang R ) at a single dose of 2.5 mg/kg body weight is shown (Figure 2). The main descriptive pharmacokinetic parameters, that is, the maximum plasma concentrations (C max ), the time to maximum concentration (T max ), area under the time curve concentration (AUC all ), and Terminal half-life (T 1/2 ) of tested and reference formulations of ENR in pigs are presented in a table (Table 1).

Pharmacodynamic Analysis
The PK/PD indices C max /MIC 90 , and AUC/MIC 90 were calculated ( Table 3) for the most prevalent pathogens associated with swine diseases, that is, Streptococcus suis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Actinobacillus pleuropneumoniae, and Bordetella bronchiseptica using the mean values of C max and AUC for plasma and the respective minimum inhibitory concentration (MIC) values reported by different authors ( Table 2). These data concern the inhibitory activity of various formulations of ENR against some pathogens, causing severe diseases in pigs.

DISCUSSION
Enrofloxacin reveals a concentration-dependent bactericidal activity (26). This drug is very lipophilic and shows amphoteric properties due to the addition of a carboxylic acid and a tertiary amine (32). It is bactericidal and has outstanding activity against both Gram-positive as well as Gram-negative pathogens (33). This antibiotic also can be used to control some intracellular pathogens (34). The extremely poor water solubility and wettability of ENR cause difficulties in the design of pharmaceutical formulations and lead to variable bioavailability (35). In the two formulations, 10% ENR (Alkali) and 20% ENR (Acidic), 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) was used. HP-β-CD is a safe and effective drug carrier commonly used as a cyclodextrin analog. It improves the physicochemical and pharmacokinetic properties of drugs, forming inclusion complexes with drug substrates (36). In 10% ENR (Yangkang) formulations, arginine was used for mild alkalinity. In ENR injection, arginine can improve its bacteriostatic function (37). Arginine provides good stability, safety and efficient, long-acting and anti-inflammatory action; maintain certain stability when storing at low-temperature, etc. (38)(39)(40)(41).
It has been reported that large inter-species differences occur in the half-life of ENR. It depends on the age and development of the liver and kidneys of the host animals (46). Breed or physiological state is also considered for the variability (42). Sometimes ENR converts into ciprofloxacin in the body, which also acts as an antimicrobial agent (45). In this study, we did not find CIP in plasma in contradiction to the findings of other authors who injected 2.5 mg/kg body weight, intramuscularly, for 3 days in older pigs (76-86 kg) (16). It may be due to the younger pigs; we used in our experiment that supports other studies (3,42). A negligible amount of ciprofloxacin, an active metabolite of enrofloxacin were detected in a study where the authors administered enrofloxacin to younger pigs at a dose of 7.5 mg/kg, subcutaneously (22). The low doses that we administered   may be another reason why we didn't find ciprofloxacin. We found longer T 1/2 in our study compared to other studies. The longer T 1/2 may interrupt the primary metabolic pathways to metabolize enrofloxacin to ciprofloxacin (22). Most common pathogens isolated from swine are reported as Streptococcus suis (16.9%), Haemophilus parasuis (9.7%), Escherichia coli (6.3%), Pasteurella multocida (3.4%), Actinobacillus pleuropneumoniae (0.3%), Bordetella bronchiseptica (1.5%), Salmonella enteria (2.3%), and Erysipelothrix rhusiopathiae (0.9%) (47).
To select the dosage regimens for therapeutic use, three criteria should be satisfied, that is, (a) bacteriological and clinical cure; (b) Least possibility for the strains becoming resistant; (c) No adverse effects on the host (48). Clinically C max /MIC for plasma is generally considered for the measurement of treatment efficiency (43). MIC values of the most common gram-negative pathogens are below 60 ng/ml including Actinobacillus and Pasteurella species, but some species like Salmonella and E. coli have MIC levels in ranges of 30-125 ng/ml (43). Fluoroquinolone antibacterial agents show concentration-dependent effects, that is, killing rate and killing degree. The killing of bacteria depends on the drug concentration. Pharmacodynamics and Pharmacokinetic properties of fluoroquinolones show the key breakpoint that determines the efficacy of these drugs is C max /MIC≥10-12 and AUIC (AUC/MIC) ≥125 (49). This breakpoint also prevents the development of resistant bacteria against fluoroquinolones (50). These findings mainly come from the study of gram-negative bacteria. But recently, researchers evaluated the efficacy of various fluoroquinolones for Streptococcus pneumoniae and proposed that the AUC: MIC need to successfully treat Gram-positive bacteria somewhat lower (i.e.,   (51).
MIC 90 values of enrofloxacin for pathogens in swine ranges from 16 ng/ml to 500 ng/ml even up to 1,000 ng/ml for some resistant pathogens ( Table 2). The mean C max /MIC and AUC/MIC ratios of all tested and reference formulations of enrofloxacin against Actinobacillus pleuropneumoniae and Pasteurella multocida showed the breakpoints more than reported values (C max /MIC 90 ≥ 10-12 and AUC/MIC 90 ≥ 125) indicating that the administration of 2.5mg/kg enrofloxacin of these formulations may have an adequate antibacterial effect and could be considered as an appropriate dose for treatment against these pathogens. But the C max /MIC 90 and AUC/MIC 90 of all tested and reference formulations of enrofloxacin with the recommended doses were not satisfactory for the key breakpoint against Haemophilus parasuis, Streptococcus suis, E. coli, and Bordetella bronchiseptica in swine ( Table 3).
Some manufacturing companies recommend enrofloxacin injections as a single dose of 2.5 mg/kg body weight, that is, Nuokang R produced by Tianjin Zhongsheng Tiaozhan Biotechnology Co., Ltd. China. This drug is known as a longacting drug. In this experiment, we have used this drug as a reference drug. Another companies 'Shandong Dezhou Shenniu Pharmaceutical Co., Ltd. China also started to produce three new formulations of enrofloxacin injection with the same dose. After testing all these new formulations and reference formulation, this study ensured longer T 1/2, indicating a long-term effect, but the obtained C max and AUC were insufficient to kill some microorganisms.
On the other hand, the low antibiotic concentrations can develop antibiotic-resistant bacteria (52) and these bacteria may create side effects on humans and animals (53). The infections caused by resistant organisms are more challenging to treat than infections caused by the non-resistant organism (54). Antibiotic resistance leads to higher treatment costs, prolonged curation, and increased mortality (55). The optimization of the dosage regimen is essential not only for the treatment but also for reducing antimicrobial resistance (26). As enrofloxacin shows a concentration-dependent bactericidal action, and the peak concentration/MIC and AUC/MIC ratios are considered the indicators of efficacy (56), it is essential to optimize the dose regimen by considering both the pharmacokinetics and pharmacodynamics parameters of the tested and reference drugs. The administration of ENR should ensure correspondingly effective concentrations in plasma against the pathogens, causing diseases in swine (44).
The values of PK/PD indices are preliminary used to optimize dosing regimens and dosing intervals on a rational base, followed by validation in clinical studies for systemically acting antimicrobial agents (57). A valued dosing strategy for infectious diseases requires a thorough understanding of the complex connections among germs, drugs, and the immune system of the host (58). Fluoroquinolone antibiotics have a rapid bactericidal effect and show a significant postantibiotic impact (59). Post-antibiotic effects also affect dosing strategies (60). The existence of post-antibiotic effects, that is, the inhibition of bacterial growth after limited exposure of microorganisms to antibiotics (61), can improve the therapeutic effect when the marginal pharmacokinetic/pharmacodynamic index value is high and can extend the dosing interval (60). Factors that influence the accuracy of efficacy predictions based on PK/PD indices are related to inoculation effect, microbial growth rate (generation time), the growth phase of an invading organism, the response of the host to the pathogen (immune system, drug diffusivity, pH at the site of the infection), infection site (exudate nature, tissue perfusion, natural barriers), etc. (62). For this reason, the calculated ratios are surrogate markers of efficacy. Further studies in a clinical context would be necessary to evaluate the results obtained in this research.

CONCLUSION
The pharmacokinetic parameters showed the tested formulations 10% enrofloxacin (Alkali), 20% enrofloxacin (Acidic), and 10% enrofloxacin (Yangkang) is somewhat better comparing to the reference formulation 20% enrofloxacin (Nuokang R ) in the swine model. All tested and reference formulations of ENR, administered at a single dose of 2.5 mg/kg IM, could be used to treat swine diseases caused by Actinobacillus pleuropneumoniae and Pasteurella multocida. But the dose of these formulations would not be effective against some important pathogens like Haemophilus parasuis, Streptococcus suis, E. coli, and Bordetella bronchiseptica because the C max /MIC 90 values were nearly 3-16 times lower than 10 and the AUC/MIC 90 values were nearly 4-30 times lower than 125. It can be concluded that the dose of both tested and reference formulations was not sufficient to treat the pigs infected by the pathogens having more MIC 90 scores. Further studies are required to optimize the dosage regimen and establish the safety of the dosage of tested and reference formulations in clinical applications.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Administration and Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences. The certificate number was SCXK (Gan) 2019-002.