Investigation of kinetic and tissue residues of cefquinome after its intramuscular administration in experimentally infected rabbits with Klebsiella pneumoniae

INTODUCTION

The economic importance of rabbits has increased dramatically for being raised for many objectives. Rabbits are considered a good source of palatable meat with high protein and low fat contents (Dalle Zotte, 2004).

Klebsiella pneumoniae (K. pneumoniae) is a G-ve bacilli belonging to the family of Enterobacteriaceae. It is a widespread colonizer of the animal intestine, and it is able to survive and multiply in moist environment. This opportunistic bacterium can cause pneumonia and infections in the urinary tract, soft tissue, and skin in healthy animals (Starlander & Melhus, 2012). K. Pneumoniae infection in rabbit is rare and usually associated with enteric form of infection (Sumitha & Sukumar, 2014). In certain cases, the use of some antibiotics resulted in a significant decrease in the K. pneumoniae numbers; nevertheless, treatment may be restricted if the bacterium produces an extended-spectrum ꞵ-lactamase (ESBL). ESBL- producing K. pneumoniae, which is reluctant to different antibacterial therapies (Lautenbach et al., 2001; Zhang et al., 2014), should be medicated by an extra ꞵ-lactamase-stable antibacterial.

Cefquinome is a 4^th generation cephalosporin antibiotic, which has been solely developed for veterinary use. It exhibits broad spectrum antimicrobial action and is stable against ꞵ-lactamases that are produced by a plurality of clinically important bacteria (CVMP, 1995; Bryskier, 1997), so cefquinome seems to have a potential for treating infections caused by bacteria in rabbits. It had excellent activity against all the Enterobacteriaceae with MIC₉₀ ranging from ≤ 0.12 to 2.0µg/mL (Murphy et al., 1994). It is mainly used for treatment of respiratory diseases in cattle, pigs, and horses, particularly in Europe (CVMP, 2003).

The pharmacokinetics of cefquinome is characterized by rapid absorption and elimination, low protein binding and limited distribution in healthy rabbits (Hwang et al., 2011; Shalaby et al., 2015). However, no published data are available on the pharmacokinetics of cefquinome in diseased rabbits, particularly the ones infected with K. Pneumoniae. Understanding the pharmacokinetics of cefquinome in K. Pneumoniae infected rabbits may allow for design of future studies to explore the potential use of cefquinome in the treatment of K. Pneumoniae as there are few antibiotics that are effective in treating it in rabbits. The choice of antibiotics in rabbits is limited, as many oral antibiotics affect by the intestinal flora, resulting in C. enterotoxaemia (Borriello & Carman, 1983). Therefore, alternative antibiotics, particularly the ones administered parentally, are needed to control this infection in rabbits. We aimed to investigate the influence of a moderate K. Pneumoniae infection on the pharmacokinetics and tissue residues of cefquinome in rabbits.

Figure (1): Chemical structure of cefquinome (Uney et al., 2011)

MATERIAL AND METHODS

Chemical reagents:

1)      All reagents used during extraction and analysis were analytical grade. Acetonitrile, methanol, tri-chloro acetic acid (TCA), isooctane and de-ionized water (D.W) were HPLC grade (Fisher)

2)      Tri-floro acetic acid (TFA) was spectrophotometric grade ≥ 99.9% (ALDRICH)

3)      Ammonium acetate buffer 0.05M; pH 5.

4)      Cefquinome sulfate reference standard: VETRANAL, analytical standard, 80.9% was provided by Sigma Aldrich Chemical Co. (St. Louis, MO).

5)      Cefquinome sulfate drug (COBACTAN®, 2.5%) was obtained from Intervet International Company, Cairo, Egypt. Cefquinome sulfate was administered I.M. at a dose of 2 mg ⁄ kg body weight (b.wt.) into the gluteal muscles (Elazab et al., 2018).

Instrument and chromatographic conditions:

The HPLC system (Agilent Technologies, 1200 series Japan) consists of a quaternary pump and a degasser to pump the mobile phase, an autosampler and a column oven. The detection was performed using a multi wave detector (MWD) set at 267 nm. The column temperature was kept at 40°C. The reverse-phase chromatography was performed with an analytical Agilent C18 column (250 mm by 4.6 mm; internal diameter, 5 µm: Agilent Technologies). The optimized method used an isocratic mobile phase consists of water containing 0.1% TFA and ACN (90:10) (Hwang et al., 2011). Mobile phase was filtered, degassed by passage through a 0.45µm nylon filter (Millipore, Bedford, MA) under a vacuum, and was sonicated for 30 min. The flow rate was 1 ml/min, and the injection volume was 10 µl. The HPLC 2D Chemstation software (Hewlett-Packard, Les Ulis, France) (Elazab et al., 2016).

Animals:

A total of 55 New Zealand white rabbits of both sexes was used in this study of mean (± SD) weight 1.5 ± 0.4 kg were used. Rabbits were housed in a specific pathogen-free room within the animal care facility, given free access to tap water and standard balanced rabbit chow. The animals were acclimated and did not receive any drug treatment for at least 15 days before the study. They were randomly divided into 5 groups, one group of 5 rabbits served as controls (they were used for preparation of blank sample and quality control samples for method validation); the remained 50 rabbits were grouped into 20 rabbits for pharmacokinetic study and 30 rabbits for residues depletion study.

Experimental Design: as in table (1)

Table (1):Grouping of experimental animals

Artificial infection:

Enteropathogenic Klebsiella Pneumoniae K1 (1×10⁹ CFU/ ml) were kindly supplied by Serology Department, Animal Health Research Institute, Dokki, Giza, Egypt. Infection was infested by endotracheal inoculation of 0.25 ml of bacterial suspension (Petraitiene et al., 2020).

After endotracheal inoculation, rabbits suffered from unkempt appearance anorexia, sneezing, dyspnea, coughing, ocuolonasal discharge, diarrhea and fever (≥1.5°C) were observed before administration of cefquinome and during the experiment course; The mean body temperatures of infected group before infection and before drug administration were 38.6 ± 0.3 and 41.5 ± 0.4°C, respectively (p < 0.05). Swabs from nasal discharges were cultured in beef infusion broth at 37^o C for 24 h and plated on MacConkey agar. Confirmation of K. pneumoniae was based on morphology, gram staining and standard biochemical tests (Chinedu et al., 2017).

Susceptibility testing:

The Minimum inhibitory concentration (MIC) of cefquinome against K. pneumoniaewas performed by a broth microdilution method as described by CLSI approved standards (CLSI, 2015).

Sample collection:

For pharmacokinetic study (gp. B&C); about 0.5 ml blood were taken from the saphenous vein at different time intervals; 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72- and 96-hours following injection. Samples were collected into heparinized tubes, and then centrifuged (1500xg for 10 min) and the serum was harvested and stored frozen at -20 °C until analyzed by HPLC assay.

For residue depletion study (gp. D&E); three rabbits were slaughtered at 1^st, 3^rd, 5^th, 7^th, 9^th and 14^th day following the last dose. Samples from liver, muscle and kidney were taken from slaughtered rabbits for drug assay. One grams of tissue specimens was weighed and stored frozen at -20 °C until assayed for concentrations of cefquinome (marker residues) using HPLC.

Stock solutions and standards preparation:

A standard stock solution of cefquinome was prepared by weighing of the standard with subsequent dissolution in water, obtaining a concentration of 1 mg/ml; this solution was stored at -20°C. The stock solution was diluted quantitatively with water for the preparation of calibration standards and quality control (QC) samples.

Cefquinome calibration standards were prepared fresh daily in blank rabbit serum at concentrations of 0.05, 0.10, 0.20, 0.50, 1, 2, 5 and 10µg/ml for serum analysis. The cefquinome calibration curve was prepared at concentrations of 0.05, 0.1, 0.2, 0.5, 1 and 2 µg/gm in blank muscle, liver and kidney for residue analysis. In the same manner, QC samples with cefquinome in serum at low (50 ng/ml), medium (500 ng/ml), and high (5000 ng/ml), for muscle (25, 50, 100 ng/gm), for liver (50, 100, 200 ng/gm) and for kidney (100, 200, 400 ng/gm) concentrations were freshly prepared to evaluate the recovery and precision of this HPLC method. Prior to HPLC analysis, these calibration standards and QC samples were processed according to the method that is summarized as follows.

Serum sample extraction:

Aliquots of calibrators, QC samples, and other serum samples (200 µl) were added to 100 μl of 5% (TCA) was added and mixed for 10 s, then centrifugation at 4,000 xg for 10 min. After centrifugation, the clear supernatant transferred into HPLC vial (Elazab et al., 2018).

Tissue sample extraction:

Weight 1 gm of tissue sample in 15 ml polypropylene centrifugal tube. Add 4 ml of 0.05 M ammonium acetate buffer (TAC5) pH 5 and 1 ml iso-octane, mix for few seconds, then centrifuge at 2400 xg/ 10 min. The isooctane was discarded and the supernatant was transferred to previously activated solid phase cartridges with 1 ml MEOH, 1 ml D.W and1ml TAC5; then cartridges was washed using 1 ml TAC5, 1 ml D.W, then 1 ml 10% ACN. Air was pass via the cartridge for 5 min. elution by 1 ml elution solution 20% ACN; at a flow rate of 3 ml/min, the elute was evaporated at 50°C under nitrogen stream till complete dryness, then reconstituted in 500 µl D.W, filtration with 0.45um Acrodiscs before injection into HPLC system (Elazab et al., 2016).

HPLC method validation (USP, 2019):

This method was validated following AOAC International guidelines (AOAC, 2013) for single-laboratory validation and United States Pharmacopeia Chapter Validation of Compendial Procedures and Chapter Chromatography (USP, 2019).

1)      Selectivity and specificity: It was evaluated by analyzing blank rabbit serum, muscle, liver, kidney and spiked rabbit serum, muscle, liver and kidney with cefquinome; chromatograms were visually examined for chromatographic interference from endogenous compounds.

2)      Linearity and range: These were evaluated by a calibration curve in the range of 0.02 to 10 µg/ml cefquinome in serum, and in the range of 0.05 to 2µg/gm in different matrixes. Triplicate injections of each level. Calibration curves were used to calculate the linear regression equation and the correlation coefficient.

The linear equation: y = a x +/- b, where y is the peak area, x is the concentration (ppm). a is the slope and b is intercept. The correlation coefficient (R²) was calculated for each standard curve.

3)      The limits of detection (LOD) and quantification (LOQ): were calculated using SD of intercept (S); [LOD= 3.3* S/a & LOQ= 10* S/a].

4)      Precision and recovery: QC samples were analyzed for the determination of intra-day precision, inter-day precision and recovery %.

The intra- and inter-day precision of the assay were determined by coefficient of variation (CV) = (mean/SD) * 100.

The recovery percent = (Obtained concentration/ Theoretical concentration) * 100.

5)      System Suitability Test (SST): It is generally performed to evaluate the suitability and effectiveness of the entire chromatographic system not only prior to use but also during the time of analysis. The SST parameters which are investigated are retention time (RT), symmetry, theoretical number of column (N) and tailing factor (T).

Statistical Analysis:

Data were expressed as mean ± S.E. The obtained data were evaluated by two-tailed T-Test using the commercially available software package (SPSS Inc., version 22.0, Chicago, IL, USA) to express the differences between groups (Morgan et al., 2019) The differences were considered significant when PZhang et al., 2010).

RESULTS

Susceptibility testing:

Minimum inhibitory concentration (MIC) of cefquinome was ≤ 0.125 μg/ml for K. pneumoniae.

Method validation and verification:

The analytical method linearity, range, LOD, LOQ, recovery and intraday & inter-day precision: the obtained results were summarized in table (2).

Table (2): Validation sheet of HPLC method

Specificity:The equilibrated chromatograms of cefquinome either in serum, muscle, liver, and kidney samples were demonstrated specific at retention time 14.2 min showing no interference between the extracted different spiked matrixes and pure standard Figure (2-6).

System suitability test (SST):The limits which are considered for the SST parameters and results of the present study are listed in table (3).

Table (3): SST parameters for cefquinome at a concentration of 0.1 ppm (n=6):

Pharmacokinetics results:

After intramuscular injection of cefquinome, there were no observed adverse effects such as signs of pain, tissue irritation, or lameness.

The cefquinome concentrations in healthy and K. pneumonia-infected rabbit’s serum following single IM administration are illustrated in table (4); the concentrations in serum of healthy rabbits were higher significantly at most time points than experimentally infected ones figure (7).

Table (4): Cefquinome levels in healthy- and K. pneumonia- infected rabbit’s serum (µg/ml) after single IM injection of 2 mg/kg of cefquinome Sulfate

ND; not detectable (< 0.0093 µg/ml).

Data are expressed as mean ± SE (n = 10).

* Significant at p < 0.05 using t- test.

Fig. (7): Cefquinome levels in healthy-and K. pneumonia-infected rabbit’s serum after single IM injection of 2 mg/kg of cefquinome Sulfate

The calculated pharmacokinetic parameters are shown in Tables (5); cefquinome was rapidly absorbed from injection sites in both healthy and K. pneumonia -infected rabbits with C_max (5.28 and 3.95 μg/ml, respectively) achieved at T_max 0.64 and 0.59 hr, respectively. Cefquinome was not detected in serum of healthy and K. pneumonia -infected rabbits at ≥ 8 hr after IM injection of cefquinome.

Table (5): The Pharmacokinetic parameters of cefquinome after single IM injection of (2 mg/kg) in healthy and K. pneumonia-infected rabbits

Data are expressed as mean ± SE (n = 10).

* Significant at p < 0.05 using t- test.

Results of tissue depletion study:

The cefquinome concentrations in healthy and K. pneumonia-infected rabbit’s tissue following single IM administration are illustrated in table (6).

Table (6): The tissue residues of cefquinome (µg/gm) after single IM injection of (2 mg/kg) in healthy and K. pneumonia-infected rabbits

Data are expressed as mean ± SE (n = 3).

* Significant at p < 0.05 using t- test.

Nd ˂ LOD.

DISCUSSION

Following IM administration of cefquinome in a single dose (2 mg/kg b.wt.) to healthy and experimentally infected rabbits, the drug serum concentration can be described by two-compartment model, this was previously reported by (Elazab et al., 2018) in rabbits, whileit was three-compartment in chickens (El-Sayed et al., 2015).

The values of distribution half-life (T_1/2alfa), maximum serum concentration (C_max), area under the curve (AUC_0–∞) and mean resident time (MRT) were significantly lower in infected rabbits than in healthy rabbits. Volume of distribution (V/F) and Total body clearance of drug (CL/F) was significantly higher in infected than in healthy rabbits. These changes in pharmacokinetic parameters in K. pneumonia-infected rabbits could be due to endotoxin- induced vasodilation and increased cardiac output which would lead to an increase in glomerular filtration rate. These cardiovascular changes could be due to the fever associated with K. pneumonia infection (Ganzinger and Haslberger, 1985; McColm et al., 1986; Goudah et al., 2006).

Similarly, Elazab et al., 2018 found that the T_{1/2 λz}, AUC0–∞, AUMC0–∞ and MRT were significantly lower in infected rabbits than in healthy rabbits. The CL/F was significantly higher in infected than in healthy rabbits.

Time to reach the maximum concentration T_max after IM administration in infected rabbits was 0.59 ± 0.01 h significantly lower than in healthy one 0.64 ± 0.001 h, and clearance CL/F was increased significantly in infected rabbits; Relatively lower T_max and higher CL/F demonstrate the rapid elimination of the drug in infected rabbits.

The present study showed that the T _1/2Beta of cefquinome following a single IM injection in healthy rabbits was 2.94 hr. In contrast, the T_{1/2 λz} of cefquinome in the present study was higher than its corresponding value in rabbits administered with the same dose IM (1.04 hr) by Hwang et al. (2011) and was lower than its corresponding value in rabbits administered with the same dose IM (0.722 hr) by Elazab et al. (2018). These differences could be related to the selected dose in this study, different rabbit’s breeds, species and age variations which could affecting the degree of protein binding of the drug and/or due to the difference in the method used for assaying of the drug.

In the present study, Minimum inhibitory concentration (MIC) of cefquinome was ≤ 0.125 μg/ml for K. pneumoniae more than 0.030 μg/ml obtained by (Zhang et al., 2014) and less than 0.25 μg/mlobtained by (Shan & Wang, 2017). In the present study, at 8 hr post IM administration of 2 mg/kg cefquinome sulfate in K. pneumoniae -infected rabbits, the serum cefquinome was less than the determined MIC, therefore, 8- hr dosing interval is recommended for K. pneumoniae -infected rabbits to achieve optimal effectiveness against these bacteria. This dose regimen would be inconvenient and impractical in the treatment of K. pneumoniae infection in rabbits. Therefore, further study is recommended to use higher IM doses of cefquinome sulfate than 2 mg/kg (e.g., 4 or 6 mg/kg) in rabbits to reach a more convenient and practical dose interval in this species to treat K. pneumoniae infection.

In the present investigation, repeated IM administration of cefquinome revealed that liver retains the highest drug concentrations in both healthy and infected rabbits while the lowest drug concentrations were found in muscle.

These results are slightly agreed with that reported by Gaber, 2005 & El-Sayed et al., 2015 inchickens, Elazab et al., 2018 in rabbits. These findings suggested that cefquinome is excreted mainly via the kidney (Limbert et al., 1991; San-Martin et al., 1998).

The experimental infection caused significant reduction in tissue concentrations of cefquinome in rabbits. The MRLs permitted by the European Agency for The Evaluation Of Medicinal Products Committee For Veterinary Medicinal Products, for cefquinome are 50, 100 and 200 µg/Kg in muscle, liver and kidney; respectively of bovine and porcine (CVMP, 1995.). The recommended withdrawal time is 7 in healthy rabbits and 3 days in K. pneumonia-infected rabbits to be safe for human consumption.

CONCLUSION

This study showed that K. pneumonia infection caused significant alterations in some pharmacokinetic parameters and tissue concentrations of cefquinome in rabbits. Based on these findings, it is recommended that in K. pneumonia infected rabbits, cefquinome sulfate could be injected IM at 2 mg/kg every 8 hr to treat the infection. This dose regimen would be inconvenient and impractical for treating this infection in rabbits. After repeated IM administrations of cefquinome (2 mg/kg b.wt.) once daily for 3 successive days, cefquinome was highly concentrated in the kidney followed by the liver, while dabs were detected in the muscle. Based on the MRLs established by regulatory agencies and statistical method suggested by EMEA, treated rabbits must not be slaughtered before 3 days from last dose of repeated administration of cefquinome to withdraw the drug residues from all tissues of treated rabbits.

ACKNOWLEDGMENT

We would like to thank Dr. Amany El- Bialy; Chief Researcher, Serology Unit, Animal Health Research Institute, Dokki, Giza on the supplying of K. pneumonia bacteria and confirmation of infection.