Influence of chlorine, iodine, and citrate-based water sanitizers on the oral bioavailability of enrofloxacin in broiler chickens

Influence of chlorine, iodine, and citrate-based water sanitizers on the oral bioavailability of... Abstract This experiment was conducted to determine if the 3 most commonly used water sanitizers in commercial broiler chicken production affected the stability of enrofloxacin (ENR), when each was administered concurrently with ENR to broiler chickens via the drinking water. To that effect, the in vitro antibacterial activity of the solution of each ENR-sanitizer product (ESP) was compared to that of ENR alone. Also, bioavailability (F) studies of ESP were carried out in chickens and compared to the corresponding values of ENR in drinking water without sanitizer. Water sanitizers tested were iodine (as iodine-polyvinylpyrrolidone), chlorine (as sodium hypochlorite), and a citrate-based sanitizer from grapefruit extract. They were mixed with ENR in sterile de-ionized water, and the resulting substances were regarded as ESP. Then, the referred studies of ESP were carried out. Results showed that ESP of ENR/sodium hypochlorite decreased both the antimicrobial activity, as well as maximum serum concentration (Cmax) and F of ENR in chickens. ESP of ENR/citrate-based sanitizer increased both the in vitro antimicrobial activity and Cmax and F values of ENR at the 2 highest concentrations tested. ESP of ENR/iodine reduced both in vitro antimicrobial activity and Cmax values of ENR at the highest concentrations tested. This study demonstrated that interactions between water sanitizers and ENR must be considered when medicating chickens via the drinking water to meet pharmacokinetics/pharmacodynamics ratios. The use of a citrate-based sanitizer is recommended, as relative F was increased. DESCRIPTION OF PROBLEM The modern poultry industry requires precise dosing protocols and adequate equipment to ensure delivery of the chosen dose of drug to the flock and eventually to each chicken. This should be carefully taken into consideration when administering antibacterial drugs [1]. Based on ease of administration and theoretically better bioavailability (F), the preferred manner to deliver antibacterial drugs in commercial chickens, is through the drinking water [2]. However, in practice, considerable variations in F among flocks and even among individuals should be expected due to factors such as drinking habits in the flock and of each bird [3–5]; quality of drinking water [6, 7], including hard water [8]; adequacy of plumbing and placement of drinkers in the chicken house [9–11], and, possibly, the presence of sanitizers added to reduce bacterial load in water [1]. The possible interaction of a sanitizer added to the drinking water with a given antibacterial drug has been questioned by professionals in the field, but almost no formal literature to characterize such an interaction has been published. It is reasonable to assume that the water sanitizer chosen on a farm can influence the stability of the antibacterial drug when added to the water source, and consequently its F may be modified [2]. For example, iodophors (iodine-polyvinylpyrrolidone) are often utilized to disinfect piping and waterers and to reduce bacterial load in water. Their efficacy is based on their high reactivity with chemical and organic entities [12]. Thus, a chemical interaction with antibacterial drugs is feasible. Citrate-based sanitizers also have been used for the same purposes, and no information on their reactivity with antibacterial drugs is available. Furthermore, the most popular manner to maintain a reasonable bacterial count in water is by adding various products containing chlorine, such as hypochlorite preparations [13]. Chlorine-based products possess high oxidation reactivity [14]. However, chemical interactions that may occur with antimicrobial drugs and their possible consequences in terms of F, when chickens consume antibacterial drugs that have had time to interact with chlorine or other water sanitizers, are yet to be elucidated. Enrofloxacin (ENR) is an antibacterial drug often used in poultry medicine in Latin America and many parts of the world [15]. The molecule has an amino group that has been described as a likely target for oxidative reaction with reactive molecules [16], and in turn this interaction may modify F of ENR. Such interactions have not been characterized. Hence, the aim of this study was to assess whether the interactions of hypochlorite sodium, iodophor, and citrate-based sanitizers with ENR were capable of modifying the in vitro antibacterial action of the drug, and to determine the effects of such interactions on F of ENR in broiler chickens when administered through the drinking water. MATERIALS AND METHODS This study was conducted in 2 phases. The first phase (antibacterial activity) was carried out to identify possible changes in the in vitro antimicrobial activity of ENR in the presence of various water sanitizers; the second phase (bioavailability) was designed to identify if variations in F of ENR could be observed in chickens treated with various ESP. Antibacterial Activity Dilutions of ENR and of each sanitizer were mixed in a beaker and allowed to interact for 24 hours. The product(s) of these ENR-sanitizer interactions were inoculated into 80 agar wells prepared in a large plate, as suggested by Pillai et al. [17] and based on directions of CLSI [18]. Serial dilutions from 0.05 to 50 μg/mL were prepared to interact with free chlorine (5 to 40 μg/mL) from a commercially available preparation of Na-hypochlorite (pH 11) [19], with free iodine (8 to 64 μg/mL) from iodine-polyvinylpyrrolidone (pH 3.5) [20], and with the sanitizer Citrex® [21] (2 to 16 mg/mL, pH 2.3). Ranges tested covered the maximum concentrations recommended by manufacturers and approximately 8 times higher. Control wells included ENR alone (pH 11.5) [22] and the corresponding sanitizer, repeating the same serial dilutions as above. The test bacterium was Escherichia coli ATCC 10536. Data obtained were processed through the generalized linear model (GzLM) [23] for a continuous variable [24]. The Bonferroni statistical procedure was performed to determine if statistically significant differences existed at a significance level of P < 0.05. All data analyses were performed using the SPSS statistical package [25]. Bioavailability This study phase complied with Mexican regulations for use of experimental animals, as laid out by the Universidad Nacional Autónoma de Mexico (UNAM) and Mexican government regulations in NOM-062-ZOO-1999. Written permission was granted on January 13, 2015. In all, 1,365 healthy, 6-week-old, Cobb 500 female chickens, weighing 2.5 kg ± 20 g, were included in this trial. Cobb chickens were managed as guidelines recommend for this genetic-line [26], having a population density of 30 kg of biomass/m2, nipple-type drinkers (10 chickens/nipple), and manual hopper feeders (50 chickens/feeder). They were placed in an experimental chicken house with a concrete floor (8 m wide x 14.5 m long x 4 m high), which was covered with clean corn-straw bedding in groups of 35, and each group had 3 replicates (105 chickens per group), separated by wire mesh. Experimental chickens were not given access to feed and water 2 h before and 2 h after dosing. Then, they were allowed access to feed and water ad libitum. The finisher diet fed met all NRC requirements (Table 1) [11]. The drinking water was classified as having good quality, in terms of acceptable quantities of chemical elements and bacteria present [15]. The temperature of the room was kept at 21°C and a negative pressure ventilation system utilized. A dark:light cycle (23L:1D) was established (5 lux of intensity level). Table 1. Composition and calculated nutrient content of finisher diet. Ingredients (kg)  Phase finisher  Corn  660  Soybean meal  269  Soybean oil  32.10  Salt  2.10  Calcite limestone  6.50  Dicalcium phosphate  14.20  DL-methionine  1.65  L-lisyne  2.75  Vitamins1  2.50  Minerals2  5.71  Sodium bicarbonate  3.50  Total  1000  Calculated analysis (g/kg)    Metabolizable energy (Kcal/kg)  3147  Crude protein  183.00  Calcium  7.60  Available phosphorus  3.80  Metionine  4.50  Sulfur amino acids  7.40  Lysine  11.20  Potassium  6.80  Sodium  2.00  Chlorine  1.70  Linoleic acid  31.30  Ingredients (kg)  Phase finisher  Corn  660  Soybean meal  269  Soybean oil  32.10  Salt  2.10  Calcite limestone  6.50  Dicalcium phosphate  14.20  DL-methionine  1.65  L-lisyne  2.75  Vitamins1  2.50  Minerals2  5.71  Sodium bicarbonate  3.50  Total  1000  Calculated analysis (g/kg)    Metabolizable energy (Kcal/kg)  3147  Crude protein  183.00  Calcium  7.60  Available phosphorus  3.80  Metionine  4.50  Sulfur amino acids  7.40  Lysine  11.20  Potassium  6.80  Sodium  2.00  Chlorine  1.70  Linoleic acid  31.30  1Amount/kg: Retinol 0.9 g, cholecalciferol 0.019 g, d-alpha-tocopherol 0.004 g, phylloquinone 1.0 g, riboflavin 4.0 g, cyanocobalamin 0.060 g, pyridoxine 3.0 g, calcium pantothenate 13.0 g, niacin 25 g, biotin 0.063 g, choline chloride 250 g. 2Amount/kg: selenium 0.2 g, cobalt 0.1 g, iodine 0.3 g, copper 10 g, zinc 50 g, iron 100 g, manganese 100 g. View Large Four F studies were carried out for each ESP with 3 replicates per group (Table 2). Also, an F study with 3 replicates, administering only ENR in sterile de-ionized water (pH 7.6 with a resistivity of 18 MΩ·cm) as analyzed with a multiparametric apparatus [27] (at 20°C) was regarded as the reference value. Table 2. Description of treatments in which bioavailability of enrofloxacin was assessed after oral dosing the product(s) of the interaction of enrofloxacin plus a sanitizer. Treatments  Description (mg/kg body weight)  Percent inclusion of enrofloxacin and the sanitizer in water1  E  Enrofloxacin 10  Enrofloxacin 2.5  ECl+  Enrofloxacin 10 plus chlorine 0.002  Enrofloxacin 2.5 plus chlorine 0.00052  ECl++  Enrofloxacin 10 plus chlorine 0.004  Enrofloxacin 2.5 plus chlorine 0.0010  ECl+++  Enrofloxacin 10 plus chlorine 0.008  Enrofloxacin 2.5 plus chlorine 0.0020  ECl++++  Enrofloxacin 10 plus chlorine 0.016  Enrofloxacin 2.5 plus chlorine 0.0040  EI+  Enrofloxacin 10 plus iodine 0.0032  Enrofloxacin 2.5 plus iodine 0.00082  EI++  Enrofloxacin 10 plus iodine 0.0064  Enrofloxacin 2.5 plus iodine 0.0016  EI+++  Enrofloxacin 10 plus iodine 0.0128  Enrofloxacin 2.5 plus iodine 0.0032  EI++++  Enrofloxacin 10 plus iodine 0.0256  Enrofloxacin 2.5 plus iodine 0.0064  EC+  Enrofloxacin 10 plus citrate-based 0.8  Enrofloxacin 2.5 plus citrate-based 0.22  EC++  Enrofloxacin 10 plus citrate-based 1.6  Enrofloxacin 2.5 plus citrate-based 0.4  EC+++  Enrofloxacin 10 plus citrate-based 3.2  Enrofloxacin 2.5 plus citrate-based 0.8  EC++++  Enrofloxacin 10 plus citrate-based 6.4  Enrofloxacin 2.5 plus citrate-based 1.6  Treatments  Description (mg/kg body weight)  Percent inclusion of enrofloxacin and the sanitizer in water1  E  Enrofloxacin 10  Enrofloxacin 2.5  ECl+  Enrofloxacin 10 plus chlorine 0.002  Enrofloxacin 2.5 plus chlorine 0.00052  ECl++  Enrofloxacin 10 plus chlorine 0.004  Enrofloxacin 2.5 plus chlorine 0.0010  ECl+++  Enrofloxacin 10 plus chlorine 0.008  Enrofloxacin 2.5 plus chlorine 0.0020  ECl++++  Enrofloxacin 10 plus chlorine 0.016  Enrofloxacin 2.5 plus chlorine 0.0040  EI+  Enrofloxacin 10 plus iodine 0.0032  Enrofloxacin 2.5 plus iodine 0.00082  EI++  Enrofloxacin 10 plus iodine 0.0064  Enrofloxacin 2.5 plus iodine 0.0016  EI+++  Enrofloxacin 10 plus iodine 0.0128  Enrofloxacin 2.5 plus iodine 0.0032  EI++++  Enrofloxacin 10 plus iodine 0.0256  Enrofloxacin 2.5 plus iodine 0.0064  EC+  Enrofloxacin 10 plus citrate-based 0.8  Enrofloxacin 2.5 plus citrate-based 0.22  EC++  Enrofloxacin 10 plus citrate-based 1.6  Enrofloxacin 2.5 plus citrate-based 0.4  EC+++  Enrofloxacin 10 plus citrate-based 3.2  Enrofloxacin 2.5 plus citrate-based 0.8  EC++++  Enrofloxacin 10 plus citrate-based 6.4  Enrofloxacin 2.5 plus citrate-based 1.6  1Deionized water. 2Highest recommended concentration as recommended by manufacturer (%). View Large Each chicken was individually weighed and then received one of the ESP or ENR, as a single oral bolus dose by means of a plastic cannula [28] attached to a syringe, directed into the proventriculus. Once the cannula was ensured to be properly placed, the experimental solution was slowly delivered. In all cases, the dose of ENR was 10 mg/kg alone or as ESP and the volume adjusted to deliver 1 mL/2.5 kg of body weight. After treatment, blood samples were taken from the wing or jugular vein at time 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h, using 5 chickens per sampling period and drawing 3 mL of blood per chicken. Each bird was randomly bled only twice. To achieve a close time interval between administration of the drug and blood sampling, technical assistance and a timer were used. Differences between the target and the actual blood sampling times were never more than 5 minutes. Blood samples were immediately centrifuged, and approximately 1.0 mL of serum was collected, identified, and frozen until analyzed. The concentration of ENR in serum samples was measured by high performance liquid chromatography (HPLC) as proposed by Idowu and Peggins [29]. One mL of methylene chloride was added to each sample and centrifuged for 5 min at almost 18,000 x g. The aqueous phase was discarded, and the organic phase was evaporated. Residues were reconstituted in acetonitrile: methanol: water (17:3:80 mobile phase) with phosphoric acid (4% v/v) and trimethylamine (4% v/v). The aqueous extract was analyzed by HPLC (UV-visible detection at λ = 278 nm, with a symmetry-C18) [30]. Injection volume was 50 μL, and flow was 0.6 mL/min. Data were analyzed with the EZChrom [31]. This chromatographic method was validated and the analytical procedure was demonstrated to be specific [32]. The recovery was calculated with linear regression analysis [33]. The precision was demonstrated by inter-day coefficient of variance (<1.9) and inter-assay error (<1.8). The limit of quantification was 0.45 μg/mL; the limit of detection was 0.15 μg/mL. With regard to robustness and tolerance, an absolute difference of 1.54 and a coefficient of variance of 1.2% (<2.0%) were obtained. The serum concentrations of ENR vs. time relationships were analyzed using compartmental pharmacokinetics through the software from PKAnalyst [34]. The best fit was obtained in model 5 (r < 0.95) [35]. The following pharmacokinetics (PK) parameters were obtained: T1/2β = elimination half-life; Cmax = maximum serum concentration; Tmax = time to reach Cmax; AUC = area under the serum concentrations vs. time curve; AUMC = area under the moment curve; and MRT = mean residence time. Mean value of serum concentrations of ENR vs. time for all treatments was analyzed by means of Shapiro–Wilk [36] to test for normal distribution and PK parameters with normal distribution using a general linear model (GLM) [37]. Bonferroni multiple comparison tests for marginal means and standard errors were adjusted for the model considered and were performed at a significance level of P < 0.05. This model was analyzed by means of least squares, using the SPSS package. RESULTS AND DISCUSSION Antibacterial Activity In this trial, the concentrations of chlorine, iodine, or citrate tested were the maximum recommended by manufacturers [38, 39], and up to 8 times higher. These latter concentrations may be encountered because of empirical initiatives by workers in poultry houses or due to everyday use, which, in some cases, causes accidental accumulations of the sanitizer in the drinking water [40]. High concentrations of some sanitizers are often used for therapeutic purposes. For example, iodine has been used at concentrations of 13.5 ppm of free iodine to reduce mortality, due to hydropericardium caused by adenovirus [41], i.e., 1.6 times higher than the maximum concentration recommended for water sanitation. Table 3 shows the in vitro antibacterial activity measured by inhibition zone when allowing the interaction of ENR with chlorine, citrate-based, or iodine sanitizers. Table 3. In vitro antibacterial activity of various combinations of enrofloxacin and water sanitizers; Arithmetic mean ± SD of inhibition zones (mm).   Enrofloxacin  Sanitizer  50 μg/mL  5 μg/mL  0.5 μg/mL  0.05 μg/mL  0 μg/mL    Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Chlorine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  5.0 μg/mL  31.06b ± 0.47  23.11b ± 0.36  11.74b± 0.49  8.16b ± 0.46  9.09b ± 0.59  10.0 μg/mL  24.24c ± 0.54  14.82b,d ± 0.47  8.56c ± 0.58  9.84c,e ± 0.45  10.85c ± 0.55  20.0 μg/mL  21.00b,c± 0.68  13.68b-d ± 0.46  7.65b-d ± 0.27  16.98d-f ± 0.6  19.75d ± 0.19  40.0 μg/mL  19.78d ± 0.67  10.57c,d ± 0.59  7.48c,d ± 0.36  11.89e,f ± 0.26  25.31e ± 0.27  Citrate-based sanitizer            0.0 mg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  2.0 mg/mL  31.92b ± 0.70  22.47b ± 0.56  10.04a ± 0.64  0.00a ± 0.00  10.52b ± 0.38  4.0 mg/mL  32.05b,c ± 0.68  22.56b ± 0.50  10.19a ± 0.57  0.00a ± 0.00  11.17b ± 0.70  8.0 mg/mL  32.61c,d ± 0.37  22.60b ± 0.35  10.21a ± 0.34  0.00a ± 0.00  16.06c ± 0.27  16.0 mg/mL  33.48d ± 0.52  22.99b ± 0.19  11.74b ± 0.33  5.38b ± 0.35  21.74d ± 0.42  Iodine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  8.0 μg/mL  28.83a ± 0.59  22.17a ± 0.67  10.90a,b ± 0.68  7.54b ± 1.13  7.33b ± 0.38  16.0 μg/mL  29.09a ± 0.68  22.54a ± 0.42  9.00a,c ± 0.12  8.58b,c ± 0.6  7.52b,c ± 0.51  32.0 μg/mL  28.80a ± 0.67  21.69a ± 0.38  9.22a-d ± 0.47  9.43c ± 0.47  8.94c ± 0.63  64.0 μg/mL  25.16b ± 0.70  21.52a ± 0.35  10.13a–d ± 0.49  9.64c ± 0.35  10.92d ± 0.57    Enrofloxacin  Sanitizer  50 μg/mL  5 μg/mL  0.5 μg/mL  0.05 μg/mL  0 μg/mL    Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Chlorine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  5.0 μg/mL  31.06b ± 0.47  23.11b ± 0.36  11.74b± 0.49  8.16b ± 0.46  9.09b ± 0.59  10.0 μg/mL  24.24c ± 0.54  14.82b,d ± 0.47  8.56c ± 0.58  9.84c,e ± 0.45  10.85c ± 0.55  20.0 μg/mL  21.00b,c± 0.68  13.68b-d ± 0.46  7.65b-d ± 0.27  16.98d-f ± 0.6  19.75d ± 0.19  40.0 μg/mL  19.78d ± 0.67  10.57c,d ± 0.59  7.48c,d ± 0.36  11.89e,f ± 0.26  25.31e ± 0.27  Citrate-based sanitizer            0.0 mg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  2.0 mg/mL  31.92b ± 0.70  22.47b ± 0.56  10.04a ± 0.64  0.00a ± 0.00  10.52b ± 0.38  4.0 mg/mL  32.05b,c ± 0.68  22.56b ± 0.50  10.19a ± 0.57  0.00a ± 0.00  11.17b ± 0.70  8.0 mg/mL  32.61c,d ± 0.37  22.60b ± 0.35  10.21a ± 0.34  0.00a ± 0.00  16.06c ± 0.27  16.0 mg/mL  33.48d ± 0.52  22.99b ± 0.19  11.74b ± 0.33  5.38b ± 0.35  21.74d ± 0.42  Iodine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  8.0 μg/mL  28.83a ± 0.59  22.17a ± 0.67  10.90a,b ± 0.68  7.54b ± 1.13  7.33b ± 0.38  16.0 μg/mL  29.09a ± 0.68  22.54a ± 0.42  9.00a,c ± 0.12  8.58b,c ± 0.6  7.52b,c ± 0.51  32.0 μg/mL  28.80a ± 0.67  21.69a ± 0.38  9.22a-d ± 0.47  9.43c ± 0.47  8.94c ± 0.63  64.0 μg/mL  25.16b ± 0.70  21.52a ± 0.35  10.13a–d ± 0.49  9.64c ± 0.35  10.92d ± 0.57  a–fDifferent letters in each column indicate a statistically significant difference (P < 0.05). Note: Multiple statistical comparisons by means of Bonferroni tests were carried out using marginal means and standard error values adjusted by the statistical model (P < 0.05). View Large Sodium hypochlorite has been described as a potent oxidant xenobiotic, and, as such, it can easily react with many antibacterial drugs, even with fairly stable molecules like ENR [16]. In this trial, for ENR chlorine interaction, the lowest concentration of hypochlorite (5 μg/mL chlorine) increased its in vitro antibacterial activity (P < 0.05). However, as chlorine sanitizer concentrations increased, the antibacterial action of the interaction product(s) diminished in a linear manner [42]. For ENR-citrate-based sanitizer interaction(s), an increase in the antibacterial activity of the ESP was observed with the 2 highest sanitizer concentrations used (50 and 5 μg/mL) (P < 0.05 in both cases). In contrast, the highest concentration of the iodine-based sanitizer (64 μg/mL) diminished the antibacterial activity of the ESP when ENR was tested at 50 μg/mL (P < 0.05). Iodine, utilized as a water sanitizer, bases its antibacterial activity on rapid oxidation reactions and on the precipitation of proteins and nucleic acids [39, 43, 44]. It is likely that oxidation is also occurring with xenobiotics, such as ENR; yet, this interaction has not been described in the scientific literature, and it remains to be confirmed. Bioavailability Figure 1 presents serum profiles of ENR from ESP that differ from values obtained for the reference control (E), in a statistically significant manner (P < 0.05) and resulted in higher Cmax and/or area under the curve (AUC) values. Figure 2 depicts the opposite, i.e., lower Cmax and AUC values of ESP, as compared to the control. Table 4 presents PK parameters for ENR derived from ESP and the control PK parameters. Three treatments presented higher Cmax values than the control (E): ECl+, EC+++, and EC++++, which are ESP from the lowest concentration of chlorine combined with ENR, and the 2 highest concentrations of ESP derived from the citrate-based sanitizer combined with ENR (P < 0.05). However, AUC values were statistically different only in the ESP from the citrate-based sanitizer (P < 0.05). Relative bioavailability obtained was 127% for EC+++ and 154% for EC++++; only the latter treatment had a statistically significant longer T1/2β, Tmax, and mean residence time (MRT) values than the control (E) (P < 0.05). Aside from ECl+, all other treatments of the chlorine series, showed statistically lower Cmax values than the control (P < 0.05). Other variations in PK parameters were also noted, namely, a decrease in AUC and MRT and consequently an inferior relative bioavailability (Fr) (Table 4). There appears to be no clear explanation for the enhanced in vitro activity and higher Cmax of ENR obtained from the ECl+ treatment. A reduced oxidation process at low concentrations of chlorine and the sum of antibacterial actions of ENR and sodium hypochlorite may explain the enhanced in vitro antibacterial activity [45]; yet, a higher Cmax cannot be easily explained. A possible explanation of these results may be related to changes in the pH of the gastrointestinal contents caused by chlorinated water, which, in turn, may change the ratio of protonated to non-protonated ENR. Also, it is important to consider that the first product of the reaction between sodium hypochorite and ENR is ciprofloxacin [16]. Thus, at some point, it is theoretically possible to find a mixture of ENR and ciprofloxacin, as well as certain antibacterial activity of sodium hypochorite. The composite actions of these molecules may explain the higher antibacterial activity found with ECl+, as compared to ENR alone. This hypothesis requires further research. Figure 1. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered directly into the proventriculus (treatment E), and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL+: enrofloxacin 10 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite); treatment EC+++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 3.2 mg/kg; and treatment EC++++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 6.4 mg/kg. Figure 1. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered directly into the proventriculus (treatment E), and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL+: enrofloxacin 10 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite); treatment EC+++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 3.2 mg/kg; and treatment EC++++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 6.4 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered into the proventriculus (treatment E) and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL++: enrofloxacin 10 mg/kg plus chlorine 0.004 mg/kg (from sodium hypochlorite); treatment ECL+++: enrofloxacin 10 mg/kg plus chlorine 0.008 mg/kg; treatment ECL++++: enrofloxacin 10 mg/kg plus chlorine 0.016 mg/kg; and treatment EI++++: enrofloxacin 10 mg/kg plus iodine 0.0256 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered into the proventriculus (treatment E) and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL++: enrofloxacin 10 mg/kg plus chlorine 0.004 mg/kg (from sodium hypochlorite); treatment ECL+++: enrofloxacin 10 mg/kg plus chlorine 0.008 mg/kg; treatment ECL++++: enrofloxacin 10 mg/kg plus chlorine 0.016 mg/kg; and treatment EI++++: enrofloxacin 10 mg/kg plus iodine 0.0256 mg/kg. Table 4. Mean ± SD of pharmacokinetic variables for enrofloxacin administered at 10 mg/kg in broiler chickens after dosing them orally with the interaction products of enrofloxacin plus chlorine (from sodium hypochlorite), or a citrate-based or iodine-based water sanitizer.   Variable    T1/2β1  Tmax2  Cmax3  AUC4  AUCM5  MRT6  Fr7  Treatment  (h)  (h)  (μg/mL)  (μg/mL·h)  (μg/mL·h)  (h)  (%)  E  1.76a ± 0.11  2.54a ± 0.16  1.96a ± 0.18  13.60a ± 1.28  69.48a ± 9.61  5.09a ± 0.33  100.00a  ECl+  1.61a ± 0.02  2.32a ± 0.03  2.19b ± 0.05  13.85a ± 0.40  64.44a ± 2.47  4.65a ± 0.07  101.86a ± 2.94  ECl++  1.25b ± 0.05  1.81b ± 0.08  0.91c ± 0.03  4.50b ± 0.24  16.35b ± 1.57  3.62b ± 0.16  33.10b ± 1.83  ECl+++  1.28b ± 0.05  1.85b ± 0.08  0.82c ± 0.03  4.16b,c ± 0.27  15.45b ± 1.63  3.70b ± 0.16  30.66b ± 2.00  ECl++++  1.22b ± 0.18  1.76b ± 0.26  0.64d ± 0.01  3.12c ± 0.47  11.25b ± 3.56  3.53b ± 0.53  22.97c ± 3.47  EI+  1.81ª ± 0.06  2.59ª ± 0.10  1.81ª ± 0.03  12.85ª ± 0.66  66.91ª ± 6.08  5.19ª ± 0.20  94.53a ± 4.89  EI++  1.83a ± 0.09  2.64a ± 0.13  1.87a ± 0.06  13.49a ± 0.73  71.64a ± 7.07  5.29a ± 0.27  99.23a ± 5.38  EI+++  1.93a ± 0.06  2.79a ± 0.09  1.66b ± 0.07  12.61a ± 0.65  70.57a ± 5.59  5.58a ± 0.20  92.74a ± 4.84  EI++++  2.11b ± 0.09  3.05b ± 0.14  1.56b ± 0.02  12.98a ± 0.76  72.61a ± 5.00  6.10b ± 0.28  95.48a ± 5.63  EC+  1.92a ± 0.02  2.77a ± 0.03  1.96a ± 0.06  14.85a ± 0.47  82.50a ± 3.14  5.55a ± 0.07  109.20b ± 3.46  EC++  2.01a ± 0.15  2.90a ± 0.21  1.96a ± 0.03  15.53a ± 1.26  90.77a,b ± 13.8  5.81a ± 0.43  114.21b ± 9.30  EC+++  2.03a ± 0.09  2.94a ± 0.13  2.17b ± 0.07  17.33b ± 0.44  102.03b ± 6.51  5.88a ± 0.26  127.44c ± 3.27  EC++++  2.35b ± 0.08  3.39b ± 0.12  2.26b ± 0.04  20.95c ± 0.68  142.58c ± 9.63  6.79b ± 0.25  154.08d ± 5.02    Variable    T1/2β1  Tmax2  Cmax3  AUC4  AUCM5  MRT6  Fr7  Treatment  (h)  (h)  (μg/mL)  (μg/mL·h)  (μg/mL·h)  (h)  (%)  E  1.76a ± 0.11  2.54a ± 0.16  1.96a ± 0.18  13.60a ± 1.28  69.48a ± 9.61  5.09a ± 0.33  100.00a  ECl+  1.61a ± 0.02  2.32a ± 0.03  2.19b ± 0.05  13.85a ± 0.40  64.44a ± 2.47  4.65a ± 0.07  101.86a ± 2.94  ECl++  1.25b ± 0.05  1.81b ± 0.08  0.91c ± 0.03  4.50b ± 0.24  16.35b ± 1.57  3.62b ± 0.16  33.10b ± 1.83  ECl+++  1.28b ± 0.05  1.85b ± 0.08  0.82c ± 0.03  4.16b,c ± 0.27  15.45b ± 1.63  3.70b ± 0.16  30.66b ± 2.00  ECl++++  1.22b ± 0.18  1.76b ± 0.26  0.64d ± 0.01  3.12c ± 0.47  11.25b ± 3.56  3.53b ± 0.53  22.97c ± 3.47  EI+  1.81ª ± 0.06  2.59ª ± 0.10  1.81ª ± 0.03  12.85ª ± 0.66  66.91ª ± 6.08  5.19ª ± 0.20  94.53a ± 4.89  EI++  1.83a ± 0.09  2.64a ± 0.13  1.87a ± 0.06  13.49a ± 0.73  71.64a ± 7.07  5.29a ± 0.27  99.23a ± 5.38  EI+++  1.93a ± 0.06  2.79a ± 0.09  1.66b ± 0.07  12.61a ± 0.65  70.57a ± 5.59  5.58a ± 0.20  92.74a ± 4.84  EI++++  2.11b ± 0.09  3.05b ± 0.14  1.56b ± 0.02  12.98a ± 0.76  72.61a ± 5.00  6.10b ± 0.28  95.48a ± 5.63  EC+  1.92a ± 0.02  2.77a ± 0.03  1.96a ± 0.06  14.85a ± 0.47  82.50a ± 3.14  5.55a ± 0.07  109.20b ± 3.46  EC++  2.01a ± 0.15  2.90a ± 0.21  1.96a ± 0.03  15.53a ± 1.26  90.77a,b ± 13.8  5.81a ± 0.43  114.21b ± 9.30  EC+++  2.03a ± 0.09  2.94a ± 0.13  2.17b ± 0.07  17.33b ± 0.44  102.03b ± 6.51  5.88a ± 0.26  127.44c ± 3.27  EC++++  2.35b ± 0.08  3.39b ± 0.12  2.26b ± 0.04  20.95c ± 0.68  142.58c ± 9.63  6.79b ± 0.25  154.08d ± 5.02  a–dDifferent letters within each column indicate a statistically significant difference within a given treatment (P < 0.05). 1T1/2β = elimination half-life. 2Tmax = time to reach Cmax. 3Cmax = maximum serum concentration. 4AUC = area under the serum concentrations vs. time curve. 5AUMC = area under the moment curve. 6MRT = mean residence time. 7Fr = relative bioavailability (AUCinteraction/AUCE) * 100. 8Pharmacokinetic parameters were calculated with non-transformed data considering that the Shapiro–Wilk test indicated a normal distribution of the data (P < 0.05). View Large It is known that iodine is less reactive than chlorine [44], and this may explain why pharmacokinetic parameters could be altered only at high concentrations of iodine, yielding a decrease in Cmax for group EI+++ (32 μg/mL of free iodine) and group EI++++ (64 μg/mL of free iodine) (P < 0.05). Additionally, an increase of T1/2β, Tmax, and MRT was also obtained for the latter treatment (P < 0.05). As mentioned before, ESP from 2 of the highest concentrations of the citrate-based sanitizer produced statistically higher Cmax, AUC, and Fr values. The citrate-based sanitizer utilized is manufactured from extracts of orange, grapefruit seed, tangerine, and other vegetable sources [46]. Grapefruit seed extracts are known to be capable of interacting with glycoprotein G (gpG) in the GI epithelium, allowing better bioavailability of some drugs [47–49]. Enhanced F also has been demonstrated for capsicum, a potent gpG inhibitor, when given together with ENR, inducing a 60% increase in its Cmax value [50]. Additionally, it has been shown that grapefruit extracts also inhibit P-450 (CYP)-enzyme activity at the GI epithelial level [51–53], thus enhancing the absorption of some drugs. Consequently, it is likely that the active principles of citrate-based sanitizer contributed to the increased F of ENR obtained in this experiment. There is an increased awareness of the antibacterial-drug resistance issue in poultry medicine [54, 55]. Excessive use of antibacterial drugs [56], lack of bioequivalence of pharmaceutical preparations [57], and careless handling of antibacterial drugs [15] appear to be fueling this problem. The dose administered and the quality of the pharmaceutical preparations have to be adequate. However, the manner in which antibacterial drugs are delivered to poultry may result in less than adequate dosing. For example, for ENR pharmacokinetics/pharmacodynamics (PK/PD) ratios establish that a maximum Cmax value is required for optimal performance of this drug [58, 59]. To achieve this, it is advisable to withhold water for an h or so to increase thirst and enhance water intake. Also, if a single water tank is being primed with ENR, the water inlet should be closed to avoid further dilution of ENR [60]. Also water hardness, microbial density of water source, and presence of other chemicals in the water are not often taken into account [7, 8]. In summary, the water sanitizers (iodine, chlorine, or citrate)-ENR interaction products induce changes in the in vitro activity of this antibacterial drug and on its Fr. Such changes can enhance or diminish both the in vitro antibacterial activity and the Fr of the drug. These changes are dependent on both the nature of the sanitizer and its concentration. CONCLUSIONS AND APPLICATIONS This study showed that the products of the interaction of ENR with sodium hypochlorite in the drinking water decreased the antimicrobial activity, Cmax, and bioavailability of the drug in a directly proportional manner. This occurs when free chlorine concentrations are at or above 10 μg/mL, but not at concentrations of 5 μg/mL. The latter concentration increases Cmax from 1.96 to 2.19 μg/mL. This is the highest, most commonly recommended concentration of chlorine used for water sanitization. The products of the interaction of ENR with a citrate-based sanitizer in the drinking water increased the in vitro antimicrobial activity, Cmax, and Fr of ENR. This occurred when the sanitizer was added at 4 and 8 times the maximum recommended concentration for water sanitization. Hence, the use of a citrate-based sanitizer could be recommended, as relative F was increased by 54%. Detailed toxicity and water intake studies in chickens at these higher concentrations are needed. In general, there is little information on the role of the medium pH in the F of fluoroquinolones. Considering that they behave as switterions, it is reasonable to assume that the influence of pH is of little relevance. However, for trovafloxacin in acidic media in humans, and for rats receiving orange oily extract [61, 62], presence of citric compounds reduces F of these fluoroquinolones. Thus, in this trial, acidification of the medium does not appear to be responsible for the increase in F observed as it occurs with tetracyclines [63]. In any case, the increased F of ENR in this study is more likely linked to the chemical association of certain ions with the fluoroquinolone molecules [64], a hypothesis that requires further investigation. When free iodine reached concentrations of 32 μg/mL and 64 μg/mL in the drinking water, i.e., 4 to 8 times the maximum recommended dose, the products of interaction of ENR with free iodine reduced the Cmax. Also, the latter concentration reduced the in vitro antimicrobial activity of ENR. Interactions between water sanitizers and ENR must be considered during proper medication of chickens if PK/PD ratios, i.e., Cmax/minimum inhibitory concentration (MIC) ≥ 10 to 12 and AUC/MIC ≥ 125 are to be met. Footnotes Primary Audience: Growers, Producers, Veterinarians, Researchers REFERENCES AND NOTES 1. Vermeulen B. 2002. Drug administration to poultry. Adv. Drug. Deliv. Rev.  54: 795– 803. Google Scholar CrossRef Search ADS PubMed  2. Esmail S. 1996. Water: The vital nutrient. Poult. Int.  15: 72– 76. 3. Bailey M. 1999. The Water Requirements of Poultry. In: Dev. Poult. Nutr. 2 . Nottingham University Press, United Kindgom. Google Scholar CrossRef Search ADS   4. 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The following general formula: $${\rm{concentration(time)}} = \frac{{{\rm{Dose\,*}}\,{{\rm{K}}_{\rm{AE}}}\,{\rm{*\,time}}}}{{{\rm{Volume}}}}{{\rm{e}}^{{\rm{ - KAE}}}}\,{\rm{*\,time}}$$. 36. Shapiro S., Wilk M.. 1965. An analysis of variance test for normality. Biometrika 52: 591– 611. 37. The following formula: Where: Yij = individual pharmacokinetic parameter value in each dilution of the sanitizer; μ = general mean; Di = dilution of the sanitizer (i = 1, 2, 3, 4, 5); eij = random standard error N (μ, σe2). 38. Cooperative Extension Service. 1998. Sanitizing Poultry Drinking Water. Page 1 in Disaster Handb. National Edition Institute of Food and Agricultural Sciences . University of Florida, USA. 39. Sumano L. H., Ocampo C. L., Gutiérrez O. L.. 2015. Farmacología Veterinaria , 4th ed. Diseño e Impresiones Aranda SA de CV, Mexico. 40. Maharjan P., Clark T., Kuenzel C., Foy M., Watkins S.. 2016. 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Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Influence of chlorine, iodine, and citrate-based water sanitizers on the oral bioavailability of enrofloxacin in broiler chickens

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© 2017 Poultry Science Association Inc.
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Abstract

Abstract This experiment was conducted to determine if the 3 most commonly used water sanitizers in commercial broiler chicken production affected the stability of enrofloxacin (ENR), when each was administered concurrently with ENR to broiler chickens via the drinking water. To that effect, the in vitro antibacterial activity of the solution of each ENR-sanitizer product (ESP) was compared to that of ENR alone. Also, bioavailability (F) studies of ESP were carried out in chickens and compared to the corresponding values of ENR in drinking water without sanitizer. Water sanitizers tested were iodine (as iodine-polyvinylpyrrolidone), chlorine (as sodium hypochlorite), and a citrate-based sanitizer from grapefruit extract. They were mixed with ENR in sterile de-ionized water, and the resulting substances were regarded as ESP. Then, the referred studies of ESP were carried out. Results showed that ESP of ENR/sodium hypochlorite decreased both the antimicrobial activity, as well as maximum serum concentration (Cmax) and F of ENR in chickens. ESP of ENR/citrate-based sanitizer increased both the in vitro antimicrobial activity and Cmax and F values of ENR at the 2 highest concentrations tested. ESP of ENR/iodine reduced both in vitro antimicrobial activity and Cmax values of ENR at the highest concentrations tested. This study demonstrated that interactions between water sanitizers and ENR must be considered when medicating chickens via the drinking water to meet pharmacokinetics/pharmacodynamics ratios. The use of a citrate-based sanitizer is recommended, as relative F was increased. DESCRIPTION OF PROBLEM The modern poultry industry requires precise dosing protocols and adequate equipment to ensure delivery of the chosen dose of drug to the flock and eventually to each chicken. This should be carefully taken into consideration when administering antibacterial drugs [1]. Based on ease of administration and theoretically better bioavailability (F), the preferred manner to deliver antibacterial drugs in commercial chickens, is through the drinking water [2]. However, in practice, considerable variations in F among flocks and even among individuals should be expected due to factors such as drinking habits in the flock and of each bird [3–5]; quality of drinking water [6, 7], including hard water [8]; adequacy of plumbing and placement of drinkers in the chicken house [9–11], and, possibly, the presence of sanitizers added to reduce bacterial load in water [1]. The possible interaction of a sanitizer added to the drinking water with a given antibacterial drug has been questioned by professionals in the field, but almost no formal literature to characterize such an interaction has been published. It is reasonable to assume that the water sanitizer chosen on a farm can influence the stability of the antibacterial drug when added to the water source, and consequently its F may be modified [2]. For example, iodophors (iodine-polyvinylpyrrolidone) are often utilized to disinfect piping and waterers and to reduce bacterial load in water. Their efficacy is based on their high reactivity with chemical and organic entities [12]. Thus, a chemical interaction with antibacterial drugs is feasible. Citrate-based sanitizers also have been used for the same purposes, and no information on their reactivity with antibacterial drugs is available. Furthermore, the most popular manner to maintain a reasonable bacterial count in water is by adding various products containing chlorine, such as hypochlorite preparations [13]. Chlorine-based products possess high oxidation reactivity [14]. However, chemical interactions that may occur with antimicrobial drugs and their possible consequences in terms of F, when chickens consume antibacterial drugs that have had time to interact with chlorine or other water sanitizers, are yet to be elucidated. Enrofloxacin (ENR) is an antibacterial drug often used in poultry medicine in Latin America and many parts of the world [15]. The molecule has an amino group that has been described as a likely target for oxidative reaction with reactive molecules [16], and in turn this interaction may modify F of ENR. Such interactions have not been characterized. Hence, the aim of this study was to assess whether the interactions of hypochlorite sodium, iodophor, and citrate-based sanitizers with ENR were capable of modifying the in vitro antibacterial action of the drug, and to determine the effects of such interactions on F of ENR in broiler chickens when administered through the drinking water. MATERIALS AND METHODS This study was conducted in 2 phases. The first phase (antibacterial activity) was carried out to identify possible changes in the in vitro antimicrobial activity of ENR in the presence of various water sanitizers; the second phase (bioavailability) was designed to identify if variations in F of ENR could be observed in chickens treated with various ESP. Antibacterial Activity Dilutions of ENR and of each sanitizer were mixed in a beaker and allowed to interact for 24 hours. The product(s) of these ENR-sanitizer interactions were inoculated into 80 agar wells prepared in a large plate, as suggested by Pillai et al. [17] and based on directions of CLSI [18]. Serial dilutions from 0.05 to 50 μg/mL were prepared to interact with free chlorine (5 to 40 μg/mL) from a commercially available preparation of Na-hypochlorite (pH 11) [19], with free iodine (8 to 64 μg/mL) from iodine-polyvinylpyrrolidone (pH 3.5) [20], and with the sanitizer Citrex® [21] (2 to 16 mg/mL, pH 2.3). Ranges tested covered the maximum concentrations recommended by manufacturers and approximately 8 times higher. Control wells included ENR alone (pH 11.5) [22] and the corresponding sanitizer, repeating the same serial dilutions as above. The test bacterium was Escherichia coli ATCC 10536. Data obtained were processed through the generalized linear model (GzLM) [23] for a continuous variable [24]. The Bonferroni statistical procedure was performed to determine if statistically significant differences existed at a significance level of P < 0.05. All data analyses were performed using the SPSS statistical package [25]. Bioavailability This study phase complied with Mexican regulations for use of experimental animals, as laid out by the Universidad Nacional Autónoma de Mexico (UNAM) and Mexican government regulations in NOM-062-ZOO-1999. Written permission was granted on January 13, 2015. In all, 1,365 healthy, 6-week-old, Cobb 500 female chickens, weighing 2.5 kg ± 20 g, were included in this trial. Cobb chickens were managed as guidelines recommend for this genetic-line [26], having a population density of 30 kg of biomass/m2, nipple-type drinkers (10 chickens/nipple), and manual hopper feeders (50 chickens/feeder). They were placed in an experimental chicken house with a concrete floor (8 m wide x 14.5 m long x 4 m high), which was covered with clean corn-straw bedding in groups of 35, and each group had 3 replicates (105 chickens per group), separated by wire mesh. Experimental chickens were not given access to feed and water 2 h before and 2 h after dosing. Then, they were allowed access to feed and water ad libitum. The finisher diet fed met all NRC requirements (Table 1) [11]. The drinking water was classified as having good quality, in terms of acceptable quantities of chemical elements and bacteria present [15]. The temperature of the room was kept at 21°C and a negative pressure ventilation system utilized. A dark:light cycle (23L:1D) was established (5 lux of intensity level). Table 1. Composition and calculated nutrient content of finisher diet. Ingredients (kg)  Phase finisher  Corn  660  Soybean meal  269  Soybean oil  32.10  Salt  2.10  Calcite limestone  6.50  Dicalcium phosphate  14.20  DL-methionine  1.65  L-lisyne  2.75  Vitamins1  2.50  Minerals2  5.71  Sodium bicarbonate  3.50  Total  1000  Calculated analysis (g/kg)    Metabolizable energy (Kcal/kg)  3147  Crude protein  183.00  Calcium  7.60  Available phosphorus  3.80  Metionine  4.50  Sulfur amino acids  7.40  Lysine  11.20  Potassium  6.80  Sodium  2.00  Chlorine  1.70  Linoleic acid  31.30  Ingredients (kg)  Phase finisher  Corn  660  Soybean meal  269  Soybean oil  32.10  Salt  2.10  Calcite limestone  6.50  Dicalcium phosphate  14.20  DL-methionine  1.65  L-lisyne  2.75  Vitamins1  2.50  Minerals2  5.71  Sodium bicarbonate  3.50  Total  1000  Calculated analysis (g/kg)    Metabolizable energy (Kcal/kg)  3147  Crude protein  183.00  Calcium  7.60  Available phosphorus  3.80  Metionine  4.50  Sulfur amino acids  7.40  Lysine  11.20  Potassium  6.80  Sodium  2.00  Chlorine  1.70  Linoleic acid  31.30  1Amount/kg: Retinol 0.9 g, cholecalciferol 0.019 g, d-alpha-tocopherol 0.004 g, phylloquinone 1.0 g, riboflavin 4.0 g, cyanocobalamin 0.060 g, pyridoxine 3.0 g, calcium pantothenate 13.0 g, niacin 25 g, biotin 0.063 g, choline chloride 250 g. 2Amount/kg: selenium 0.2 g, cobalt 0.1 g, iodine 0.3 g, copper 10 g, zinc 50 g, iron 100 g, manganese 100 g. View Large Four F studies were carried out for each ESP with 3 replicates per group (Table 2). Also, an F study with 3 replicates, administering only ENR in sterile de-ionized water (pH 7.6 with a resistivity of 18 MΩ·cm) as analyzed with a multiparametric apparatus [27] (at 20°C) was regarded as the reference value. Table 2. Description of treatments in which bioavailability of enrofloxacin was assessed after oral dosing the product(s) of the interaction of enrofloxacin plus a sanitizer. Treatments  Description (mg/kg body weight)  Percent inclusion of enrofloxacin and the sanitizer in water1  E  Enrofloxacin 10  Enrofloxacin 2.5  ECl+  Enrofloxacin 10 plus chlorine 0.002  Enrofloxacin 2.5 plus chlorine 0.00052  ECl++  Enrofloxacin 10 plus chlorine 0.004  Enrofloxacin 2.5 plus chlorine 0.0010  ECl+++  Enrofloxacin 10 plus chlorine 0.008  Enrofloxacin 2.5 plus chlorine 0.0020  ECl++++  Enrofloxacin 10 plus chlorine 0.016  Enrofloxacin 2.5 plus chlorine 0.0040  EI+  Enrofloxacin 10 plus iodine 0.0032  Enrofloxacin 2.5 plus iodine 0.00082  EI++  Enrofloxacin 10 plus iodine 0.0064  Enrofloxacin 2.5 plus iodine 0.0016  EI+++  Enrofloxacin 10 plus iodine 0.0128  Enrofloxacin 2.5 plus iodine 0.0032  EI++++  Enrofloxacin 10 plus iodine 0.0256  Enrofloxacin 2.5 plus iodine 0.0064  EC+  Enrofloxacin 10 plus citrate-based 0.8  Enrofloxacin 2.5 plus citrate-based 0.22  EC++  Enrofloxacin 10 plus citrate-based 1.6  Enrofloxacin 2.5 plus citrate-based 0.4  EC+++  Enrofloxacin 10 plus citrate-based 3.2  Enrofloxacin 2.5 plus citrate-based 0.8  EC++++  Enrofloxacin 10 plus citrate-based 6.4  Enrofloxacin 2.5 plus citrate-based 1.6  Treatments  Description (mg/kg body weight)  Percent inclusion of enrofloxacin and the sanitizer in water1  E  Enrofloxacin 10  Enrofloxacin 2.5  ECl+  Enrofloxacin 10 plus chlorine 0.002  Enrofloxacin 2.5 plus chlorine 0.00052  ECl++  Enrofloxacin 10 plus chlorine 0.004  Enrofloxacin 2.5 plus chlorine 0.0010  ECl+++  Enrofloxacin 10 plus chlorine 0.008  Enrofloxacin 2.5 plus chlorine 0.0020  ECl++++  Enrofloxacin 10 plus chlorine 0.016  Enrofloxacin 2.5 plus chlorine 0.0040  EI+  Enrofloxacin 10 plus iodine 0.0032  Enrofloxacin 2.5 plus iodine 0.00082  EI++  Enrofloxacin 10 plus iodine 0.0064  Enrofloxacin 2.5 plus iodine 0.0016  EI+++  Enrofloxacin 10 plus iodine 0.0128  Enrofloxacin 2.5 plus iodine 0.0032  EI++++  Enrofloxacin 10 plus iodine 0.0256  Enrofloxacin 2.5 plus iodine 0.0064  EC+  Enrofloxacin 10 plus citrate-based 0.8  Enrofloxacin 2.5 plus citrate-based 0.22  EC++  Enrofloxacin 10 plus citrate-based 1.6  Enrofloxacin 2.5 plus citrate-based 0.4  EC+++  Enrofloxacin 10 plus citrate-based 3.2  Enrofloxacin 2.5 plus citrate-based 0.8  EC++++  Enrofloxacin 10 plus citrate-based 6.4  Enrofloxacin 2.5 plus citrate-based 1.6  1Deionized water. 2Highest recommended concentration as recommended by manufacturer (%). View Large Each chicken was individually weighed and then received one of the ESP or ENR, as a single oral bolus dose by means of a plastic cannula [28] attached to a syringe, directed into the proventriculus. Once the cannula was ensured to be properly placed, the experimental solution was slowly delivered. In all cases, the dose of ENR was 10 mg/kg alone or as ESP and the volume adjusted to deliver 1 mL/2.5 kg of body weight. After treatment, blood samples were taken from the wing or jugular vein at time 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h, using 5 chickens per sampling period and drawing 3 mL of blood per chicken. Each bird was randomly bled only twice. To achieve a close time interval between administration of the drug and blood sampling, technical assistance and a timer were used. Differences between the target and the actual blood sampling times were never more than 5 minutes. Blood samples were immediately centrifuged, and approximately 1.0 mL of serum was collected, identified, and frozen until analyzed. The concentration of ENR in serum samples was measured by high performance liquid chromatography (HPLC) as proposed by Idowu and Peggins [29]. One mL of methylene chloride was added to each sample and centrifuged for 5 min at almost 18,000 x g. The aqueous phase was discarded, and the organic phase was evaporated. Residues were reconstituted in acetonitrile: methanol: water (17:3:80 mobile phase) with phosphoric acid (4% v/v) and trimethylamine (4% v/v). The aqueous extract was analyzed by HPLC (UV-visible detection at λ = 278 nm, with a symmetry-C18) [30]. Injection volume was 50 μL, and flow was 0.6 mL/min. Data were analyzed with the EZChrom [31]. This chromatographic method was validated and the analytical procedure was demonstrated to be specific [32]. The recovery was calculated with linear regression analysis [33]. The precision was demonstrated by inter-day coefficient of variance (<1.9) and inter-assay error (<1.8). The limit of quantification was 0.45 μg/mL; the limit of detection was 0.15 μg/mL. With regard to robustness and tolerance, an absolute difference of 1.54 and a coefficient of variance of 1.2% (<2.0%) were obtained. The serum concentrations of ENR vs. time relationships were analyzed using compartmental pharmacokinetics through the software from PKAnalyst [34]. The best fit was obtained in model 5 (r < 0.95) [35]. The following pharmacokinetics (PK) parameters were obtained: T1/2β = elimination half-life; Cmax = maximum serum concentration; Tmax = time to reach Cmax; AUC = area under the serum concentrations vs. time curve; AUMC = area under the moment curve; and MRT = mean residence time. Mean value of serum concentrations of ENR vs. time for all treatments was analyzed by means of Shapiro–Wilk [36] to test for normal distribution and PK parameters with normal distribution using a general linear model (GLM) [37]. Bonferroni multiple comparison tests for marginal means and standard errors were adjusted for the model considered and were performed at a significance level of P < 0.05. This model was analyzed by means of least squares, using the SPSS package. RESULTS AND DISCUSSION Antibacterial Activity In this trial, the concentrations of chlorine, iodine, or citrate tested were the maximum recommended by manufacturers [38, 39], and up to 8 times higher. These latter concentrations may be encountered because of empirical initiatives by workers in poultry houses or due to everyday use, which, in some cases, causes accidental accumulations of the sanitizer in the drinking water [40]. High concentrations of some sanitizers are often used for therapeutic purposes. For example, iodine has been used at concentrations of 13.5 ppm of free iodine to reduce mortality, due to hydropericardium caused by adenovirus [41], i.e., 1.6 times higher than the maximum concentration recommended for water sanitation. Table 3 shows the in vitro antibacterial activity measured by inhibition zone when allowing the interaction of ENR with chlorine, citrate-based, or iodine sanitizers. Table 3. In vitro antibacterial activity of various combinations of enrofloxacin and water sanitizers; Arithmetic mean ± SD of inhibition zones (mm).   Enrofloxacin  Sanitizer  50 μg/mL  5 μg/mL  0.5 μg/mL  0.05 μg/mL  0 μg/mL    Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Chlorine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  5.0 μg/mL  31.06b ± 0.47  23.11b ± 0.36  11.74b± 0.49  8.16b ± 0.46  9.09b ± 0.59  10.0 μg/mL  24.24c ± 0.54  14.82b,d ± 0.47  8.56c ± 0.58  9.84c,e ± 0.45  10.85c ± 0.55  20.0 μg/mL  21.00b,c± 0.68  13.68b-d ± 0.46  7.65b-d ± 0.27  16.98d-f ± 0.6  19.75d ± 0.19  40.0 μg/mL  19.78d ± 0.67  10.57c,d ± 0.59  7.48c,d ± 0.36  11.89e,f ± 0.26  25.31e ± 0.27  Citrate-based sanitizer            0.0 mg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  2.0 mg/mL  31.92b ± 0.70  22.47b ± 0.56  10.04a ± 0.64  0.00a ± 0.00  10.52b ± 0.38  4.0 mg/mL  32.05b,c ± 0.68  22.56b ± 0.50  10.19a ± 0.57  0.00a ± 0.00  11.17b ± 0.70  8.0 mg/mL  32.61c,d ± 0.37  22.60b ± 0.35  10.21a ± 0.34  0.00a ± 0.00  16.06c ± 0.27  16.0 mg/mL  33.48d ± 0.52  22.99b ± 0.19  11.74b ± 0.33  5.38b ± 0.35  21.74d ± 0.42  Iodine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  8.0 μg/mL  28.83a ± 0.59  22.17a ± 0.67  10.90a,b ± 0.68  7.54b ± 1.13  7.33b ± 0.38  16.0 μg/mL  29.09a ± 0.68  22.54a ± 0.42  9.00a,c ± 0.12  8.58b,c ± 0.6  7.52b,c ± 0.51  32.0 μg/mL  28.80a ± 0.67  21.69a ± 0.38  9.22a-d ± 0.47  9.43c ± 0.47  8.94c ± 0.63  64.0 μg/mL  25.16b ± 0.70  21.52a ± 0.35  10.13a–d ± 0.49  9.64c ± 0.35  10.92d ± 0.57    Enrofloxacin  Sanitizer  50 μg/mL  5 μg/mL  0.5 μg/mL  0.05 μg/mL  0 μg/mL    Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  Chlorine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  5.0 μg/mL  31.06b ± 0.47  23.11b ± 0.36  11.74b± 0.49  8.16b ± 0.46  9.09b ± 0.59  10.0 μg/mL  24.24c ± 0.54  14.82b,d ± 0.47  8.56c ± 0.58  9.84c,e ± 0.45  10.85c ± 0.55  20.0 μg/mL  21.00b,c± 0.68  13.68b-d ± 0.46  7.65b-d ± 0.27  16.98d-f ± 0.6  19.75d ± 0.19  40.0 μg/mL  19.78d ± 0.67  10.57c,d ± 0.59  7.48c,d ± 0.36  11.89e,f ± 0.26  25.31e ± 0.27  Citrate-based sanitizer            0.0 mg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  2.0 mg/mL  31.92b ± 0.70  22.47b ± 0.56  10.04a ± 0.64  0.00a ± 0.00  10.52b ± 0.38  4.0 mg/mL  32.05b,c ± 0.68  22.56b ± 0.50  10.19a ± 0.57  0.00a ± 0.00  11.17b ± 0.70  8.0 mg/mL  32.61c,d ± 0.37  22.60b ± 0.35  10.21a ± 0.34  0.00a ± 0.00  16.06c ± 0.27  16.0 mg/mL  33.48d ± 0.52  22.99b ± 0.19  11.74b ± 0.33  5.38b ± 0.35  21.74d ± 0.42  Iodine            0.0 μg/mL  29.39a ± 0.59  21.52a ± 0.37  9.96a ± 0.55  0.00a ± 0.00  0.00a ± 0.00  8.0 μg/mL  28.83a ± 0.59  22.17a ± 0.67  10.90a,b ± 0.68  7.54b ± 1.13  7.33b ± 0.38  16.0 μg/mL  29.09a ± 0.68  22.54a ± 0.42  9.00a,c ± 0.12  8.58b,c ± 0.6  7.52b,c ± 0.51  32.0 μg/mL  28.80a ± 0.67  21.69a ± 0.38  9.22a-d ± 0.47  9.43c ± 0.47  8.94c ± 0.63  64.0 μg/mL  25.16b ± 0.70  21.52a ± 0.35  10.13a–d ± 0.49  9.64c ± 0.35  10.92d ± 0.57  a–fDifferent letters in each column indicate a statistically significant difference (P < 0.05). Note: Multiple statistical comparisons by means of Bonferroni tests were carried out using marginal means and standard error values adjusted by the statistical model (P < 0.05). View Large Sodium hypochlorite has been described as a potent oxidant xenobiotic, and, as such, it can easily react with many antibacterial drugs, even with fairly stable molecules like ENR [16]. In this trial, for ENR chlorine interaction, the lowest concentration of hypochlorite (5 μg/mL chlorine) increased its in vitro antibacterial activity (P < 0.05). However, as chlorine sanitizer concentrations increased, the antibacterial action of the interaction product(s) diminished in a linear manner [42]. For ENR-citrate-based sanitizer interaction(s), an increase in the antibacterial activity of the ESP was observed with the 2 highest sanitizer concentrations used (50 and 5 μg/mL) (P < 0.05 in both cases). In contrast, the highest concentration of the iodine-based sanitizer (64 μg/mL) diminished the antibacterial activity of the ESP when ENR was tested at 50 μg/mL (P < 0.05). Iodine, utilized as a water sanitizer, bases its antibacterial activity on rapid oxidation reactions and on the precipitation of proteins and nucleic acids [39, 43, 44]. It is likely that oxidation is also occurring with xenobiotics, such as ENR; yet, this interaction has not been described in the scientific literature, and it remains to be confirmed. Bioavailability Figure 1 presents serum profiles of ENR from ESP that differ from values obtained for the reference control (E), in a statistically significant manner (P < 0.05) and resulted in higher Cmax and/or area under the curve (AUC) values. Figure 2 depicts the opposite, i.e., lower Cmax and AUC values of ESP, as compared to the control. Table 4 presents PK parameters for ENR derived from ESP and the control PK parameters. Three treatments presented higher Cmax values than the control (E): ECl+, EC+++, and EC++++, which are ESP from the lowest concentration of chlorine combined with ENR, and the 2 highest concentrations of ESP derived from the citrate-based sanitizer combined with ENR (P < 0.05). However, AUC values were statistically different only in the ESP from the citrate-based sanitizer (P < 0.05). Relative bioavailability obtained was 127% for EC+++ and 154% for EC++++; only the latter treatment had a statistically significant longer T1/2β, Tmax, and mean residence time (MRT) values than the control (E) (P < 0.05). Aside from ECl+, all other treatments of the chlorine series, showed statistically lower Cmax values than the control (P < 0.05). Other variations in PK parameters were also noted, namely, a decrease in AUC and MRT and consequently an inferior relative bioavailability (Fr) (Table 4). There appears to be no clear explanation for the enhanced in vitro activity and higher Cmax of ENR obtained from the ECl+ treatment. A reduced oxidation process at low concentrations of chlorine and the sum of antibacterial actions of ENR and sodium hypochlorite may explain the enhanced in vitro antibacterial activity [45]; yet, a higher Cmax cannot be easily explained. A possible explanation of these results may be related to changes in the pH of the gastrointestinal contents caused by chlorinated water, which, in turn, may change the ratio of protonated to non-protonated ENR. Also, it is important to consider that the first product of the reaction between sodium hypochorite and ENR is ciprofloxacin [16]. Thus, at some point, it is theoretically possible to find a mixture of ENR and ciprofloxacin, as well as certain antibacterial activity of sodium hypochorite. The composite actions of these molecules may explain the higher antibacterial activity found with ECl+, as compared to ENR alone. This hypothesis requires further research. Figure 1. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered directly into the proventriculus (treatment E), and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL+: enrofloxacin 10 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite); treatment EC+++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 3.2 mg/kg; and treatment EC++++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 6.4 mg/kg. Figure 1. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered directly into the proventriculus (treatment E), and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL+: enrofloxacin 10 mg/kg plus chlorine 0.002 mg/kg (from sodium hypochlorite); treatment EC+++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 3.2 mg/kg; and treatment EC++++: enrofloxacin 10 mg/kg plus citrate-based sanitizer 6.4 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered into the proventriculus (treatment E) and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL++: enrofloxacin 10 mg/kg plus chlorine 0.004 mg/kg (from sodium hypochlorite); treatment ECL+++: enrofloxacin 10 mg/kg plus chlorine 0.008 mg/kg; treatment ECL++++: enrofloxacin 10 mg/kg plus chlorine 0.016 mg/kg; and treatment EI++++: enrofloxacin 10 mg/kg plus iodine 0.0256 mg/kg. Figure 2. View largeDownload slide Mean ± SD of serum concentrations of enrofloxacin after a single bolus administration of 10 mg/kg delivered into the proventriculus (treatment E) and serum concentrations of enrofloxacin derived from administering enrofloxacin plus different concentrations of water sanitizers treatment ECL++: enrofloxacin 10 mg/kg plus chlorine 0.004 mg/kg (from sodium hypochlorite); treatment ECL+++: enrofloxacin 10 mg/kg plus chlorine 0.008 mg/kg; treatment ECL++++: enrofloxacin 10 mg/kg plus chlorine 0.016 mg/kg; and treatment EI++++: enrofloxacin 10 mg/kg plus iodine 0.0256 mg/kg. Table 4. Mean ± SD of pharmacokinetic variables for enrofloxacin administered at 10 mg/kg in broiler chickens after dosing them orally with the interaction products of enrofloxacin plus chlorine (from sodium hypochlorite), or a citrate-based or iodine-based water sanitizer.   Variable    T1/2β1  Tmax2  Cmax3  AUC4  AUCM5  MRT6  Fr7  Treatment  (h)  (h)  (μg/mL)  (μg/mL·h)  (μg/mL·h)  (h)  (%)  E  1.76a ± 0.11  2.54a ± 0.16  1.96a ± 0.18  13.60a ± 1.28  69.48a ± 9.61  5.09a ± 0.33  100.00a  ECl+  1.61a ± 0.02  2.32a ± 0.03  2.19b ± 0.05  13.85a ± 0.40  64.44a ± 2.47  4.65a ± 0.07  101.86a ± 2.94  ECl++  1.25b ± 0.05  1.81b ± 0.08  0.91c ± 0.03  4.50b ± 0.24  16.35b ± 1.57  3.62b ± 0.16  33.10b ± 1.83  ECl+++  1.28b ± 0.05  1.85b ± 0.08  0.82c ± 0.03  4.16b,c ± 0.27  15.45b ± 1.63  3.70b ± 0.16  30.66b ± 2.00  ECl++++  1.22b ± 0.18  1.76b ± 0.26  0.64d ± 0.01  3.12c ± 0.47  11.25b ± 3.56  3.53b ± 0.53  22.97c ± 3.47  EI+  1.81ª ± 0.06  2.59ª ± 0.10  1.81ª ± 0.03  12.85ª ± 0.66  66.91ª ± 6.08  5.19ª ± 0.20  94.53a ± 4.89  EI++  1.83a ± 0.09  2.64a ± 0.13  1.87a ± 0.06  13.49a ± 0.73  71.64a ± 7.07  5.29a ± 0.27  99.23a ± 5.38  EI+++  1.93a ± 0.06  2.79a ± 0.09  1.66b ± 0.07  12.61a ± 0.65  70.57a ± 5.59  5.58a ± 0.20  92.74a ± 4.84  EI++++  2.11b ± 0.09  3.05b ± 0.14  1.56b ± 0.02  12.98a ± 0.76  72.61a ± 5.00  6.10b ± 0.28  95.48a ± 5.63  EC+  1.92a ± 0.02  2.77a ± 0.03  1.96a ± 0.06  14.85a ± 0.47  82.50a ± 3.14  5.55a ± 0.07  109.20b ± 3.46  EC++  2.01a ± 0.15  2.90a ± 0.21  1.96a ± 0.03  15.53a ± 1.26  90.77a,b ± 13.8  5.81a ± 0.43  114.21b ± 9.30  EC+++  2.03a ± 0.09  2.94a ± 0.13  2.17b ± 0.07  17.33b ± 0.44  102.03b ± 6.51  5.88a ± 0.26  127.44c ± 3.27  EC++++  2.35b ± 0.08  3.39b ± 0.12  2.26b ± 0.04  20.95c ± 0.68  142.58c ± 9.63  6.79b ± 0.25  154.08d ± 5.02    Variable    T1/2β1  Tmax2  Cmax3  AUC4  AUCM5  MRT6  Fr7  Treatment  (h)  (h)  (μg/mL)  (μg/mL·h)  (μg/mL·h)  (h)  (%)  E  1.76a ± 0.11  2.54a ± 0.16  1.96a ± 0.18  13.60a ± 1.28  69.48a ± 9.61  5.09a ± 0.33  100.00a  ECl+  1.61a ± 0.02  2.32a ± 0.03  2.19b ± 0.05  13.85a ± 0.40  64.44a ± 2.47  4.65a ± 0.07  101.86a ± 2.94  ECl++  1.25b ± 0.05  1.81b ± 0.08  0.91c ± 0.03  4.50b ± 0.24  16.35b ± 1.57  3.62b ± 0.16  33.10b ± 1.83  ECl+++  1.28b ± 0.05  1.85b ± 0.08  0.82c ± 0.03  4.16b,c ± 0.27  15.45b ± 1.63  3.70b ± 0.16  30.66b ± 2.00  ECl++++  1.22b ± 0.18  1.76b ± 0.26  0.64d ± 0.01  3.12c ± 0.47  11.25b ± 3.56  3.53b ± 0.53  22.97c ± 3.47  EI+  1.81ª ± 0.06  2.59ª ± 0.10  1.81ª ± 0.03  12.85ª ± 0.66  66.91ª ± 6.08  5.19ª ± 0.20  94.53a ± 4.89  EI++  1.83a ± 0.09  2.64a ± 0.13  1.87a ± 0.06  13.49a ± 0.73  71.64a ± 7.07  5.29a ± 0.27  99.23a ± 5.38  EI+++  1.93a ± 0.06  2.79a ± 0.09  1.66b ± 0.07  12.61a ± 0.65  70.57a ± 5.59  5.58a ± 0.20  92.74a ± 4.84  EI++++  2.11b ± 0.09  3.05b ± 0.14  1.56b ± 0.02  12.98a ± 0.76  72.61a ± 5.00  6.10b ± 0.28  95.48a ± 5.63  EC+  1.92a ± 0.02  2.77a ± 0.03  1.96a ± 0.06  14.85a ± 0.47  82.50a ± 3.14  5.55a ± 0.07  109.20b ± 3.46  EC++  2.01a ± 0.15  2.90a ± 0.21  1.96a ± 0.03  15.53a ± 1.26  90.77a,b ± 13.8  5.81a ± 0.43  114.21b ± 9.30  EC+++  2.03a ± 0.09  2.94a ± 0.13  2.17b ± 0.07  17.33b ± 0.44  102.03b ± 6.51  5.88a ± 0.26  127.44c ± 3.27  EC++++  2.35b ± 0.08  3.39b ± 0.12  2.26b ± 0.04  20.95c ± 0.68  142.58c ± 9.63  6.79b ± 0.25  154.08d ± 5.02  a–dDifferent letters within each column indicate a statistically significant difference within a given treatment (P < 0.05). 1T1/2β = elimination half-life. 2Tmax = time to reach Cmax. 3Cmax = maximum serum concentration. 4AUC = area under the serum concentrations vs. time curve. 5AUMC = area under the moment curve. 6MRT = mean residence time. 7Fr = relative bioavailability (AUCinteraction/AUCE) * 100. 8Pharmacokinetic parameters were calculated with non-transformed data considering that the Shapiro–Wilk test indicated a normal distribution of the data (P < 0.05). View Large It is known that iodine is less reactive than chlorine [44], and this may explain why pharmacokinetic parameters could be altered only at high concentrations of iodine, yielding a decrease in Cmax for group EI+++ (32 μg/mL of free iodine) and group EI++++ (64 μg/mL of free iodine) (P < 0.05). Additionally, an increase of T1/2β, Tmax, and MRT was also obtained for the latter treatment (P < 0.05). As mentioned before, ESP from 2 of the highest concentrations of the citrate-based sanitizer produced statistically higher Cmax, AUC, and Fr values. The citrate-based sanitizer utilized is manufactured from extracts of orange, grapefruit seed, tangerine, and other vegetable sources [46]. Grapefruit seed extracts are known to be capable of interacting with glycoprotein G (gpG) in the GI epithelium, allowing better bioavailability of some drugs [47–49]. Enhanced F also has been demonstrated for capsicum, a potent gpG inhibitor, when given together with ENR, inducing a 60% increase in its Cmax value [50]. Additionally, it has been shown that grapefruit extracts also inhibit P-450 (CYP)-enzyme activity at the GI epithelial level [51–53], thus enhancing the absorption of some drugs. Consequently, it is likely that the active principles of citrate-based sanitizer contributed to the increased F of ENR obtained in this experiment. There is an increased awareness of the antibacterial-drug resistance issue in poultry medicine [54, 55]. Excessive use of antibacterial drugs [56], lack of bioequivalence of pharmaceutical preparations [57], and careless handling of antibacterial drugs [15] appear to be fueling this problem. The dose administered and the quality of the pharmaceutical preparations have to be adequate. However, the manner in which antibacterial drugs are delivered to poultry may result in less than adequate dosing. For example, for ENR pharmacokinetics/pharmacodynamics (PK/PD) ratios establish that a maximum Cmax value is required for optimal performance of this drug [58, 59]. To achieve this, it is advisable to withhold water for an h or so to increase thirst and enhance water intake. Also, if a single water tank is being primed with ENR, the water inlet should be closed to avoid further dilution of ENR [60]. Also water hardness, microbial density of water source, and presence of other chemicals in the water are not often taken into account [7, 8]. In summary, the water sanitizers (iodine, chlorine, or citrate)-ENR interaction products induce changes in the in vitro activity of this antibacterial drug and on its Fr. Such changes can enhance or diminish both the in vitro antibacterial activity and the Fr of the drug. These changes are dependent on both the nature of the sanitizer and its concentration. CONCLUSIONS AND APPLICATIONS This study showed that the products of the interaction of ENR with sodium hypochlorite in the drinking water decreased the antimicrobial activity, Cmax, and bioavailability of the drug in a directly proportional manner. This occurs when free chlorine concentrations are at or above 10 μg/mL, but not at concentrations of 5 μg/mL. The latter concentration increases Cmax from 1.96 to 2.19 μg/mL. This is the highest, most commonly recommended concentration of chlorine used for water sanitization. The products of the interaction of ENR with a citrate-based sanitizer in the drinking water increased the in vitro antimicrobial activity, Cmax, and Fr of ENR. This occurred when the sanitizer was added at 4 and 8 times the maximum recommended concentration for water sanitization. Hence, the use of a citrate-based sanitizer could be recommended, as relative F was increased by 54%. Detailed toxicity and water intake studies in chickens at these higher concentrations are needed. In general, there is little information on the role of the medium pH in the F of fluoroquinolones. Considering that they behave as switterions, it is reasonable to assume that the influence of pH is of little relevance. However, for trovafloxacin in acidic media in humans, and for rats receiving orange oily extract [61, 62], presence of citric compounds reduces F of these fluoroquinolones. Thus, in this trial, acidification of the medium does not appear to be responsible for the increase in F observed as it occurs with tetracyclines [63]. In any case, the increased F of ENR in this study is more likely linked to the chemical association of certain ions with the fluoroquinolone molecules [64], a hypothesis that requires further investigation. 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Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc.

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Journal of Applied Poultry ResearchOxford University Press

Published: Mar 1, 2018

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