Pharmacokinetics of Marbofloxacin Following Oral Administration in Piperine, Quercetin Alone and Both in Combination Pretreated Broiler Chickens

Marbofloxacin is a third generation fluorinated quinolone compound, which has broad spectrum of antimicrobial activity against gram-negative and gram-positive bacteria (Shan et al., 2014). However, low oral bioavailability of marbofloxacin (56.82%) reduces its therapeutic effectiveness in chickens (Anadon et al., 2002). Presence of drug efflux pumps/permeability glycoproteins (P-glycoprotein/multi drug resistance 1) at the apical surface of enterocytes might be responsible for reduced bioavailability of fluoroquinolones, oxytetracycline and doxycycline after oral administration (Haritova et al., 2010). Cytochrome P-450 isoenzyme (CYP3A37) and P-glycoprotein (MDR1) activities at duodenum and liver had affected the bioavailability of substrate drugs in broiler chickens (Patel et al., 2019).
       
Piperine, an important alkaloid compound present in black pepper (Piper nigrum Linn.) and long pepper (Piper longum Linn.) has reduced the gastric first pass effect by inhibiting cytochrome P450 enzymes and P-glycoprotein activities in gut epithelial cells (Yurdakok-Dikmen et al., 2018). Piperine treatment had shown to increase the oral bioavailability of various antibacterial drugs like pefloxacin (Madhukar et al., 2008), gatifloxacin (Patel et al., 2011), amoxicillin (Barve and Ruparel, 2015) and rifampicin (Singh et al., 2018). Quercetin, a flavonoid compound present in many plants and foods could also competitively inhibit the drug metabolizing enzyme CYP3A4 (Choi et al., 2011) and the members of MDR family MDR1, MRP2 and BCRP (Liu et al., 2016). Quercetin treatment had also shown to increase the bioavailability of ciprofloxacin in rat (Devi et al., 2016). Pharmacokinetics of marbofloxacin after oral administration in broiler chickens and other birds were studied earlier. However, pharmacokinetics of marbofloxacin following oral administration in piperine and quercetin pretreated broiler chickens have not been evaluated so far. Hence, the present study was planned to investigate the pharmacokinetic profile of  marbofloxacin following oral administration in piperine, quercetin alone and both in combination pretreated broiler chickens.

Experimental animals
 
The experiment was conducted on 18 broiler chickens of ‘Cobb 400’ strain, at College of Veterinary Science and Animal Husbandry, Junagadh Agricultural University, Junagadh. Day old chicks were procured Venky’s India Ltd. Anand, Gujarat and maintained as per standard CPCSEA guidelines for laboratory animal facility (CPCSEA, 2003). During entire study period (October to December-2017) the broiler chickens were reared in winter season with 20 to 30°C temperature and relative humidity 40 to 55%. Broiler starter and finisher feed of BIS (9862-1992) specification (Simran Feeds Pvt. Ltd, Indore, India) and reverse osmosis drinking water were provided ad libitum to chickens. Experiment protocol (JAU/JVC/IAEC/SA/22/2017; date: 26/05/2017) was approved by Institutional Animal Ethics Committee (IAEC), College of Veterinary Science and Animal Husbandry, Junagadh Agricultural University, Junagadh.
 
Drugs and chemicals
 
Marbofloxacin powder for HPLC standard assay, piperine and quercetin for oral administration were purchased from Sigma Aldrich, Bangalore, India. Marbofloxacin tablet 50 mg (Marbomet®, Intas Pharmaceuticals Ltd., Ahmedabad, India) was used for oral administration to broiler chickens. HPLC grade water, acetonitrile, per chloric acid and formic acid were procured from Merck India Ltd., Mumbai, India.
 
Experimental design
 
At the age of 3 weeks, eighteen broiler chickens were randomly allocated into three groups (n=6); group I (pretreatment of piperine 10 mg/kg, PO, for 3 days), group II (pretreatment of quercetin 10 mg/kg, PO, for 3 days) and group III (pretreatment of piperine and quercetin 10 mg/kg, each, PO, for 3 days). Exposure doses of piperine and quercetin were selected as per earlier reported bio-enhancing effects of these drugs (Swathilatha and Lakshmi, 2014). Broiler chickens were fasted for 12 h before oral administration of marbofloxacin. After 3 days of piperine and quercetin pretreatment, at 4^th day, marbofloxacin (5 mg/kg, PO, single dose) was administered to all chickens. Blood samples (approximately, 0.50 ml) were collected through IV catheter (26 G × ¾; 0.6 × 0.19 mm; flow 15 ml/min) fixed in wing vein at 0 (before drug administration), 0.083 (5 min), 0.166 (10 min), 0.25 (15 min), 0.5 (30 min), 1, 2, 4, 8, 12 and 24 h and transferred to sterile heparinized test tubes. Plasma samples were collected after centrifugation of blood samples at 10000 rpm for 10 min at 4°C (Eppendorf 5430 R, Germany) and stored at -86°C until assayed for marbofloxacin concentration (usually within 48 h of collection).
 
HPLC analysis of marbofloxacin concentration
 
Marbofloxacin concentration in plasma samples was analyzed with minor modifications in method described earlier (Carpenter et al., 2006). The HPLC apparatus (LC-2010 CHT, Schimadzu, Japan) with reverse phase Zorbax Eclipse XDB-C₁₈ analytical column (4.6 mm × 250 mm; ID 5 µm; Agilent Technologies, USA) was used for marbofloxacin assay. The mobile phase was a mixture of 10 mM formic acid in HPLC water and acetonitrile (80:20), which delivered into column at a flow rate of 0.75 ml/min with column oven temperature of 30°C. Effluents were detected at 295 nm wavelength in UV detector. Plasma samples (250 µl) were precipitated with addition of 250 µl of 0.8 M perchloric acid and vortexed for 10 min followed by centrifugation at 12000 rpm for 10 min. Clear supernatant was collected in HPLC vials and 20 μl was injected in to HPLC system through auto sampler. The marbofloxacin was detected from plasma at the retention time of 8.2±0.2 min as depicted in Fig 1. Quantification of marbofloxacin in plasma samples was done by reference to the resultant standard curve. The lower limit of quantification of the drug was 0.049 µg/ml. The assay was sensitive and reproducible and linearity was observed from 0.049 to 50 μg/ml with mean correlation coefficient (R²) >0.999. Precision and accuracy were estimated by analyzing six replicates at three different concentrations of plasma standards, i.e. 0.049, 6.25 and 50 μg/ml. The intra-day and inter- day coefficients of variation for 6 samples were satisfactory, with relative standard deviations (RSD) less than 6.90%.
 

 
Pharmacokinetic analysis
 
The appropriate pharmacokinetic model was selected by visual examination of individual concentration-time curves and by application of Akaike’s Information Criterion (Yamaoka et al., 1978). After oral administration, the marbofloxacin serum concentration time data were best fitted in one compartment open model. Different pharmacokinetic parameters were estimated as per standard methods (Baggot, 1977; Gibaldi and Perrier, 1982).
 
Statistical analysis
 
The pharmacokinetic data were presented as Mean±S.E. and analyzed statistically by using (Microsoft Office Excel® 2013) software for Windows. Difference among the various treatment groups were analyzed by one way analysis of variance (ANOVA) followed by post hoc Tukey high significant difference test. Where p<0.05 was considered as statistically significant.

The semilogarithmic plots of marbofloxacin plasma concentrations following single dose (5 mg/kg) oral administration in piperine, quercetin alone and both in combination pretreated (10 mg/kg each, PO, for 3 days) broiler chickens are depicted in Fig 2 and pharmacokinetic variables are presented in Table 1. After single oral administration of marbofloxacin in piperine, quercetin and both in combination pretreated broiler chickens, maximum plasma concentrations (C_max) were 1.96±0.20, 1.95±0.15 and 1.64±0.11  µg/ml, respectively. The C_max values were non-significantly different among the various treatment groups. However, the C_max values observed in the present study were higher than earlier reported C_max of 1.05±0.15 µg/ml after alone oral marbofloxacin administration in chickens (Anadon et al., 2002). Similarly, piperine pretreated rats showed increased C_max value of marbofloxacin as compared to control rats (Chauhan et al., 2020).  In present study, plasma concentration at 24 h (last observed concentration) in combination pretreated chickens was 0.18±0.02 μg/ml, which significantly (p<0.05) higher than the respective values of 0.09±0.02 and 0.16±0.02 μg/ml in piperine and quercetin pretreated broiler chickens.
 

 

       
In present study, the mean elimination half-life (t_1/2β) of the drug in combination pretreated broiler chickens was (7.71±0.59 h) significantly (p<0.05) higher than piperine (6.23±1.01 h) and quercetin (5.69±0.39) alone treatment groups. In combination pretreated broiler chickens, lower elimination rate constant (b) and higher elimination half-life (t_1/2β) indicated reduced rate of drug elimination from body. Lower t_1/2β of 4.62±0.42 h was reported earlier after single oral administration of marbofloxacin in broiler chickens (Patel et al., 2018). In support to our findings, higher values of t_1/2β were recorded after piperine combination with oxy tetracycline (normal: 4.93±0.42 h; piperine treated: 6.38± 0.44 h) in hens (Singh et al., 2005) and gatifloxacin (normal: 3.74±0.073 h; piperine treated: 4.03±0.097 h) in layers (Patel et al., 2011). Similarly, after quercetin treatment, t_1/2β of ciprofloxacin was increased (normal: 1.85±0.22 h; quercetin treated: 2.00±0.38 h) in rats (Devi et al., 2016). After single dose oral administration of marbofloxacin in piperine, quercetin and both in combination pretreated broiler chickens the area under the curve (AUC) values were 17.66±1.94, 18.36±2.12 and 18.60±1.31 µg.h/ml, respectively. After alone marbofloxacin oral administration, lower value of AUC (11.51±1.94 µg.h/ml) was observed in ducks (Goudah and Hasabelnaby, 2010). In present study, mean AUC value was higher in combination pretreated chickens indicated higher systemic exposure of the drug as compared to piperine and quercetin alone groups.
       
It has been documented that the efficacy of concentration-dependent antibacterial agents like fluoroquinolones mostly depends on AUC/MIC and C_max/MIC ratios. The AUC/MIC₉₀ ratio of 100-125 h and C_max/MIC₉₀ ratio of 8-12 are optimum for achieving maximum clinical cure and to prevent emergence of bacterial resistance (Toutain and Lees, 2004; Marín et al., 2009). In present study, pharmacodynamic efficacy of marbofloxacin following single dose oral administration was evaluated with earlier reported In vitro MIC₉₀ value of 0.125- 0.20 µg/mL against bacterial pathogens of animals and birds (Spreng et al., 1995; Haritova et al., 2006). After oral administration of marbofloxacin, AUC/MIC ratios at MIC₉₀ levels of 0.05, 0.10 and 0.2 µg/ml were 372.00, 186.60 and 93.00 h, respectively in combination pretreated broiler chickens, which significantly higher than respective values of 353.20, 176.60 and 88.30 h (in piperine pretreated) and 367.20, 183.60 and 91.80 h (in quercetin pretreated) broiler chickens. The C_max/MIC ratios were not altered among different treatment groups, however they were in the optimum range of 8.20- 39.20 at above mentioned MIC₉₀ values. Present findings suggested that oral administration of marbofloxacin (5 mg/kg) showed better efficacy in combination pretreated broiler chickens as compared to other groups.
       
Mean apparent volume of distribution (Vd_(area)/F) in combination pretreated broiler chickens was (2.30±0.27 l/kg) which significantly (p<0.05) higher than piperine (1.71±0.39 l/kg) and quercetin (1.65±0.26 l/kg) alone groups. Patel et al., (2018) has observed lower Vd_(area) value of 1.32±0.10 l/kg after oral administration of marbofloxacin in broiler chickens. Piperine and quercetin in combination shown largest volume of distribution suggesting more drug penetration in deeper body tissues as compared to alone pretreatment groups. In similar to this, Vd_(area) was significantly (p<0.05) increased from 1.11±0.08 to 1.37±0.11 l/kg in trikatu (piperine containing formulation) treated goats as compared to normal goats (Madhukar et al., 2008).
       
The renal, biliary or hepatic metabolic pathways are important mechanisms involved in the excretion of fluoroquinolones from body. Marbofloxacin is excreted in unchanged form up to 30% in urine of broiler chickens (Anadon et al., 2002). In present study, the total body clearance values were similar in all groups of broiler chickens. Piperine, quercetin and combine pretreatment had no significant effect on body clearance rate of orally administered marbofloxacin. In similar to this, trikatu treatment did not altered the pefloxacin clearance (normal: 0.29±0.02 l/h/kg; trikatu treated: 0.29±0.02 l/h/kg) in goats (Madhukar et al., 2008). In the present study, clearance rate was not altered among different treatment groups, whereas, half-life was higher in combination pretreatment group that might be due to higher volume of distribution and reduced metabolism of drug in the body. Mean residence time in combination pretreated broiler chickens was (10.71±0.70 h) significantly (p<0.05) higher than piperine (7.67±0.48 h) and quercetin (8.88±0.28 h) alone groups.
       
Nuclear pregnane X receptor (PXR) is an important transcriptional regulator factor which controls the expression of drug transporter MDR1 and drug metabolizing enzyme CYP3A4 and regulates the drug clearance in the liver and intestine (Tebbens et al., 2018). After piperine and quercetin combined treatment, drug metabolizing enzyme CYP3A37 and drug efflux protein MDR1 mRNA expressions were significantly (p<0.05) down regulated in liver and duodenum as compared to normal chickens (Patel et al., 2019). The CYP3A37 is the major drug metabolizing enzyme in broiler chickens. In present study, increased volume of distribution and half-life of marbofloxacin were observed in combination pretreatment group which might be due to reduced activity of CYP3A37 and MDR1 at intestine and liver of broiler chickens.

In conclusion, the piperine and quercetin combination pretreatment has delayed the excretion of drug in broiler chickens. Mean elimination half-life (t_1/2β), volume of distribution (Vd_area /F), the mean residence time (MRT) and AUC/MIC₉₀ ratio were increased in combination pretreated broiler chickens as compared to piperine and quercetin alone pretreatment groups. Piperine and quercetin combined pretreatment has improved the pharmacokinetic profile of marbofloxacin following single dose oral administration in broiler chickens. Further detailed pharmacokinetics and pharmacogenomics interaction studies are required to design appropriate therapeutic dosage regimens of piperine and quercetin like bio-enhancers with routinely used antibiotics in broiler chickens.