Pharmacokinetics of Ceftiofur Sodium in Black-bone Silky Fowl after One Single Intravenous and Intramuscular Injection

Ceftiofur is a third-generation cephalosporin antibiotic developed exclusively for veterinary applications (Zhang et al., 2019). It is bactericidal in vitro, inhibiting cell wall synthesis (Yang et al., 2020b). In China, ceftiofur is only labeled to treat bacterial respiratory infections in ruminants and pigs. However, it is additionally approved by the US FDA to treat Escherichia coli infection in day-old chicks and day-old turkey poults. Although not approved in China, ceftiofur is being used extensively in an extra-label manner to treat poultry infections.
 
The pharmacokinetics profiles of ceftiofur have been determined in different avian species, such as chickens (Tell et al., 1998), ducks (Chung et al., 2007; Hope et al., 2012), geese (Chen et al., 2014) and turkeys (Tell et al., 1998). Ceftiofur has some similar pharmacokinetics profiles in these birds, such as a relatively poor distribution, but a quick and complete transformation to its active metabolites, desfuroylceftiofur (DCE) and desfuroylceftiofur conjugates (DCEC). The bioavailability data for ceftiofur, however, were rare following extravascular injections in avian. Additionally, a previous study (Tell et al., 1998) showed that the size and species of bird affect the pharmacokinetics of ceftiofur.
 
Black-bone silky fowl is a unique chicken breed which has been cultured in China for more than 2000 years. It has some health functions, including improving immunity, preventing feebleness, treating diabetes and anaemia and curing menstrual disturbance and postpartum complications (Muroya et al., 2000). Comparing with common chicken breeds, black-bone silky fowl has unique morphological features such as fluffy head feathers, rose comb, blue earlobes, silky feathers and black skin, meat and bone (Han et al., 2016). In addition to these morphological differences, other physiological variations were also found in black-bone silky fowl, such as lower expression of sialic acid receptors (Han et al., 2016) and the presence of melanosomes and melanin in various organs (Muroya et al., 2000). These differences may result in varied pharmacokinetic profiles.
 
To our knowledge, there are no pharmacokinetics studies which have been conducted with any cephalosporins in black-bone silky fowl. Given the paucity of bioavailability data of ceftiofur in birds, the potential effects of bird size and species on the pharmacokinetics of ceftiofur and the morphological and physiological differences between black-bone silky fowl and common chicken species, it is necessary to determine the pharmacokinetics of ceftiofur in black-bone silky fowl. Therefore, the present study aimed to investigate the pharmacokinetics of ceftiofur (measured by ceftiofur and its active metabolites concentrations) in black-bone silky fowl following a single intravenous or intramuscular injection of ceftiofur sodium.

Chemicals and reagents
 
The analytical standard for ceftiofur with a purity of 89.5% was provided by the China Institute of Veterinary Drugs Control (Beijing, China). The other chemicals and drugs used here are the same as those used in our previous studies (Yang et al., 2020b; Zhang et al., 2019). More details could be found there.
 
Animals
 
Six 90-day-old healthy crossbred black-boned silky fowls (3 males and 3 females) were purchased from Luoyang Nongfeng Agricultural Technology Co., Ltd. (Luoyang, Henan, China) and their average body weight (BW) was 1.6 ± 0.3 kg. All animals were housed in a temperature (25°C) and humidity (65%) controlled room and fed on antimicrobial-free diets with free access to water. They were acclimatized for 10 days before experiments were performed. All experiments were conducted at Henan University of Science and Technology in the spring of 2019.
 
Drug administration and sampling
 
A two-stage crossover design was applied in this study (Wu et al., 2018) and six chickens were equally allocated to the two crossover study groups. According to each individual BW, ceftiofur sodium solution was intravenously or intramuscularly injected to each bird at a dose of 10 mg/kg BW (calculated as pure ceftiofur). Intravenous injection was administered via the right wing vein within 30 s, while intramuscular dose was injected into the right pectoral muscle. After a 2-week washout period, the experiment was repeated by changing the treatments (Yang et al., 2018a).
 
Following each administration, blood samples (approximately 1 ml) were collected from the left wing vein and heparinized at 0 (before administration), 5, 10, 15, 30 min, 1, 1.5, 2, 2.5, 3, 5, 6, 8, 12, 16, 24 and 36 h. Plasma samples were collected after centrifugation at 3,000 × g for 10 min and stored at -20°C until further analysis (Jayachandran et al., 2018).
 
Drug assay
 
Ceftiofur and related metabolites (DCE and DCEC) were extracted from plasma samples and subsequently derivatized to a stable compound, desfuroylceftiofur acetamide (DCA), using a method previously validated for the feline and canine plasma (Yang et al., 2020b; Zhang et al., 2019). More details about the analytical method could be found in our previous study (Yang et al., 2020b; Zhang et al., 2019). The concentrations of ceftiofur and DCE-related metabolites (expressed as DCA) were determined using a Waters Alliance e2695 Series HPLC system (Waters Corporation, Milford, MA, USA) consisting of a 2487 ultraviolet detector. The wavelength was set at 265 nm.
 
Pharmacokinetic analysis
 
According to our previous studies (Li et al., 2013; Yang et al., 2015), pharmacokinetics parameters were determined for each individual bird using a non-compartmental method. Following both injections, the area under the concentration-time curve (AUC_0-∞) and the first moment curve (AUMC_0-∞) were calculated using the linear trapezoidal rule with extrapolation to time infinity (Sharma and Dumka, 2018); mean residence time (MRT) was then calculated as the ratio of AUMC_0-∞ to AUC_0-∞ (Yanget_al2013). After intravenous injection, volume of distribution (V_Z) was determined using equation V_Z = (Dose/AUC_0-∞)/lz, where Dose is the amount of drug intravenously administered (Yang et al., 2018b); total body clearance (Cl) was calculated as the radio of intravenous Dose to AUC_0-∞ and the volume of distribution at steady-state (V_SS) was calculated as V_SS = MRT × Cl (Yang et al., 2020a). Following intramuscular administration, mean absorption time (MAT) was calculated as the difference between the MRT values following both routes of administration; absorption half-life (t_1/2ka) was calculated as ln2×MAT (Yang et al., 2016).
 
Statistical analysis was performed to assess significant differences between the same pharmacokinetics parameters following both routes of administration using paired t tests (SPSS 20.0). A p value of < 0.05 was considered as significant (Yang et al., 2019).

Detection method
 
It was shown that the present method was selective for DCA. This method was linear for ceftiofur in the range of 0.1 to 50 μg/ml with a correlation coefficient of 0.998. The limits of detection (LOD) and quantitation (LOQ) were 0.05 and 0.1 μg/ml, respectively. The recoveries were between 80.44% and 88.56%; the intra-day coefficient of variation (CV) values were in the range of 1.38%-2.61%; and the inter-day CV ranged from 1.92% to 3.04%.
 
Pharmacokinetics results
 
Semi-logarithmic plots of the total concentrations of ceftiofur and DCE-related metabolites after one single intravenous or intramuscular injection are presented in Fig 1. The average concentration kept higher than LOQ (0.1 μg/ml) for 24 h following both injections. After intramuscular injection, the Cmax values ranging from 19.53 to 23.53 μg/ml were observed at 1.5 h in four chickens and 2 h in two subjects.
 

 
No significant differences were observed in all pharmacokinetic parameters except the MRT values following different routes of administration (Table 1).
 

 
After one single intramuscular injection, ceftiofur sodium exhibited a rapid absorption with a t_1/2ka of 0.40±0.13 h and a t_max of 1.67±0.26 h. The t_max value calculated here was longer than that in geese (calculated as 0.484 h; Chen et al., 2014), however it should be noted that there is a huge difference between the representations of current and previous t_max parameters. Based on the current analysis method, the metabolism pathway from ceftiofur to DCE was ignored and the parameters of t_1/2ka and t_max were used not only to describe the absorption of ceftiofur but also to describe the shortcut from ceftiofur to DCE. However, in the previous study (Chen et al., 2014), the t_1/2ka and t_max values were only applicable to ceftiofur because only the ceftiofur concentration was quantified. While in the present study, the concentrations of ceftiofur and DCE-related metabolites (expressed as DCA) were detectable within 24 h following an intramuscular injection at 10 mg/kg BW. Following one single intramuscular injection, the absolute bioavailability was calculated to be 93.03% in black-bone silky fowl, which was similar with that reported in geese (95.45%; Chen et al., 2014). These results indicated a complete absorption of ceftiofur following intramuscular administration.
 
A poor distribution was determined for ceftiofur in black-bone silky fowl with small values of V_Z and V_SS (0.338 and 0.319 L/kg, respectively). However, a smaller V_Z value (0.064 L/kg) was reported in geese (Chen et al., 2014). Poor distribution of ceftiofur sodium was also observed in mammals with the V_Z values ranging from 0.13 L/kg in camels (Goudah, 2007) to 0.51 L/kg in elephants (Dumonceaux et al., 2005). All these V_SS values reported in different species suggest limited penetration of ceftiofur sodium through biological membranes. The reason for this poor distribution may be due to the huge difference between the pKa value of ceftiofur sodium (3.7) and the actual pH value in the blood stream (7.4). In the blood stream, ceftiofur sodium would act as a weak acid with an insufficient lipid-soluble property to penetrate biological membranes (Fernandez-Varon et al., 2016).
 
Rapid elimination was observed for ceftiofur and related metabolites in black-bone silky fowl and similar half-lives (p=0.226) were calculated following a single intravenous and intramuscular injection (3.19 and 3.36 h, respectively). Tell et al., (1998) compared the pharmacokinetics profiles in different avian species after extravascular administration of ceftiofur sodium and shorter half-life (2.5 h) was reported in cockatiels; however, longer ones were reported in Amazon parrots (7.9 h), one-day chicks (5.33-7.50 h) and turkey poults (5.58-8.65 h). These inconsistencies may be due to differences in physiological status between species. Varied salts of ceftiofur also had different pharmacokinetics profiles.
 
Hope et al., (2012) determined the minimum inhibitory concentration (MIC) values of ceftiofur against various bacteria isolated from birds and found that the MIC₉₀ was below 1 μg/ml. Salmon et al., (1996) compared the in vitro activities of ceftiofur and DCE and found that their MIC₉₀ values against most bacteria were below 1.0 and 4.0 μg/ml, respectively. As time-dependent antibiotics, the time of cephalosporins concentration above MIC (T > MIC) is important to clinical success (Drusano, 2004). The optimal duration of plasma concentrations above the MIC has varied among studies, but a general assumption is that the drug concentration should be above the MIC for 50% of the dosing interval (Drusano, 2004). Assuming that ceftiofur and DCE have a same MIC value at 1.0 μg/ml against common avian organisms, the duration in which the total concentrations of ceftiofur and DCE are above 1.0 μg/ml is 12 h after both intravenous and intramuscular treatments (Table 1). Therefore, a total of 10 mg/kg BW ceftiofur sodium intravenously or intramuscularly given once daily to black-bone silky fowl is likely to result in plasma concentrations exceeding 1 μg/ml for 50% of the dosing interval. However, under such a multiple-dose regimen, there may be a risk of drug accumulation in edible tissues, which is worthy of further study.

In conclusion, this is the first pharmacokinetic study of ceftiofur sodium in black-bone silky fowl. The current results demonstrated their rapid and complete absorption, however, poor distribution and rapid elimination. Based on the T > MIC values calculated in the present study, an intravenous or intramuscular dose at 10 mg/kg of ceftiofur sodium once daily is predicted to be effective for treating avian bacteria with a MIC value of £ 1.0 μg/ml.

This study was financially supported by the National Natural Science Foundation of China (grant No. U1604107) and the Student Research Training Program in Henan University of Science and Technology (grant No. 2019402).