Distribution of enrofloxacin within the bronchoalveolar region of healthy pigs

Abstract

This study aimed to elucidate the distribution of enrofloxacin (ERFX) within the bronchoalveolar region of pigs. Six clinically healthy pigs were allocated to intramuscular treatments with either a single dose of 5 mg/kg or 7.5 mg/kg ERFX. Samples of plasma and bronchoalveolar lavage fluid (BALF) were obtained from each pig 0 (before administration), 3, 8, and 24 hr after ERFX administration. The ERFX concentrations in pulmonary epithelial lining fluid (ELF) and BALF cells showed a similar pattern during the experimental period, whereby ERFX concentrations in both ELF and BALF cells were higher than those in the plasma. These results suggest that intramuscularly injected ERFX is well-distributed in the bronchoalveolar region.

Respiratory diseases, generally caused by infections with viruses, bacteria, and mycoplasma, are common in modern swine production. Bronchopneumonia and subsequent pleuritis due to bacterial or Mycoplasma spp. infections are frequent lesions of the lungs in pigs of all age groups [24]. Antimicrobials are generally used to treat respiratory diseases caused by bacteria and Mycoplasma spp. Therefore, information on the distribution of antimicrobials within the bronchoalveolar region of pigs is important and may aid in selecting appropriate antibacterial agents for the treatment of pneumonia, resulting in faster recovery from respiratory diseases.

Enrofloxacin (ERFX) is a quinolone-based agent with broad-spectrum antimicrobial activity against pathogens commonly associated with respiratory diseases in livestock [4, 21]. Several different types of ERFX formulations are commercially available, including single-dose formulations containing higher concentrations of ERFX to reduce labor and stress in livestock.

The pharmacokinetics and intrapulmonary distribution of ERFX must be characterized to determine whether it is an effective therapeutic agent for pneumonia. In particular, it is important to determine whether the intrapulmonary ERFX concentration reaches the upper limit of the minimum inhibitory concentration (MIC) for the target pathogen [16].

The distribution of ERFX within the bronchoalveolar region has been reported in dogs following oral administration [14] and in calves after intramuscular injection [16, 20]. Although some pharmacokinetic (PK) studies of ERFX have been performed to elucidate its biodistribution in pigs [1, 18, 27], no reports have been published concerning the concentrations of ERFX within the bronchoalveolar region.

This study aimed to elucidate the concentration of ERFX in bronchoalveolar lavage fluid (BALF) after intramuscular ERFX administration to healthy pigs.

Six clinically healthy female pigs were used in this study. Pigs were determined to be healthy if they exhibited good appetite and vitality and did not exhibit coughing, fever, or abnormalities in respiratory rate. The pigs used in the study were 12 weeks old and had a mean body weight of 44.7 ± 1.7 kg (mean ± SD, range: 42.0–47.2 kg). All protocols were approved by the Animal Care and Use Committee of Kagoshima University (VM16062). Animals were cared for according to the Guide for the Care and Use of Laboratory Animals of the Joint Faculty of Veterinary Medicine, Kagoshima University.

The pigs were randomized into two groups, with three pigs each, based on whether they were administered conventional or single-dose type ERFX formulation. Baytril^® 10% injectable solution (5 mg/kg; Bayer [Elanco], Tokyo, Japan) was used as the conventional type, and Baytril^® 1ject (7.5 mg/kg; Bayer [Elanco]) as the single-dose type. Both formulations were injected intramuscularly into each pig in a single dose.

Measurements of body temperature, heart rate, and respiratory rate, as well as peripheral venous blood samples, were obtained at 0 (before administration), 3, 8, and 24 hr after the injection. Blood samples were collected in heparinized tubes (VP-H100K, Terumo, Tokyo, Japan) and Vacutainer tubes (VP-NA052K, Terumo) containing dipotassium ethylenediaminetetraacetic acid (EDTA-2AK).

Blood samples collected in tubes containing EDTA-2AK were used to determine white blood cell (WBC) and red blood cell (RBC) counts, hemoglobin (Hb) levels, and hematocrit (Ht). Measurements were taken within 30 min of collection using an automated cell counter (Poch-100iV, Sysmex, Kobe, Japan). Plasma was separated from the blood collected in heparinized tubes by centrifugation (4°C, 2,000 g for 10 min) and stored at −80°C until analysis. The urea concentrations within the plasma were measured using a colorimetric method with assay kits (Quantichrom Urea Assay Kit, Bioassay Systems, Hayward, CA, USA). Bronchoalveolar lavage fluid (BALF) was collected using a flexible electronic endoscope (VQ TYPE 6112 B, Olympus, Tokyo, Japan) at 0 (before administration), 3, 8, and 24 hr after ERFX administration based on previously published reports [5, 17]. Prior to the procedure, pigs were administered 2 mg/kg of alfaxalone (Alfaxan, Meiji-Seika-Pharma, Tokyo, Japan) intramuscularly as a premedication. During BALF collection, anesthesia was maintained by inhalation of 2.5% isoflurane through the nose using a mask (FO-20A, Acoma Medical Industry, Tokyo, Japan). BALF was sampled from one of five randomly selected lobes; accessory lobe, middle lobe, right caudal lobe, left caudal lobe and left cranial lobe. Under general anesthesia, a flexible electronic endoscope was inserted into a subsegment of the lobe. Then, 30 mL of sterile 0.9% normal saline solution was infused into each lobe through the forceps channel of the endoscope, followed by immediate aspiration. This procedure was performed twice for each lobe. Material from the second aspiration was pooled with that of the first. Four samplings were performed per animal, all of which were sampled from different lobes. BALF specimens were immediately sent to a laboratory for cell counting, and 1.5 mL of BALF from each of the four samples was centrifuged at 400 g for 5 min. The supernatant and cell pellets were separated and frozen at −80°C until assays.

The concentrations of ERFX and ciprofloxacin (CPFX) were measured by high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) based on the previously established procedure reported by De Baere et al. [8]. Plasma samples (100 μL) were diluted 10-fold with distilled water. Each BALF cell pellet sample was mixed with 0.5 mL of 1 mol sodium hydroxide to lyse cells, and then each sample was mixed with 1.0 mL of 3% formic acid. Each sample (300 μL, diluted plasma, supernatant of BALF, or lysed BALF cell pellet) was mixed with 60 μL of internal standard (Lomefloxacin, Sigma-Aldrich, Tokyo, Japan; 300 ng/mL in 1% formic acid/methanol [4:1]) and 60 μL of methanol. The diluted sample (350 μL) was loaded into a solid-phase extraction column (Oasis HLB; Waters, Tokyo, Japan). The residue was dissolved in 250 μL of the mobile phase, and an aliquot (10 μL) of the extract was injected into the LC-MS/MS system (Prominence, Shimadzu, Kyoto, Japan; 4000 QTRAP, AB, Sciex, Tokyo, Japan). The detection limits for ERFX and CPFX were 0.001 μg/mL.

The ERFX concentration was determined in the pulmonary epithelial lining fluid (ELF) and cells in BALF [10, 11]. The concentration of ERFX in the ELF (ERFX_ELF) was calculated as follows:

ERFX_ELF=ERFX_BALF X urea_Plasma / urea_BALF

where ERFX_BALF was the concentration of ERFX in BALF, urea_Plasma was the concentration of urea in plasma, and urea_BALF was the concentration of urea in the BALF.

The concentration of ERFX in BALF cells (ERFX_Cell) was determined as follows:

ERFX_Cell=ERFX_PELLET / V_cell

where ERFX_PELLET was the concentration of ERFX in the BALF cell pellet supernatant and V_Cell was the mean volume of BALF cells. A volume of 1.28 μL/10⁶ BALF cells was used based on previous studies [10, 11]. The area under the ERFX concentration curve during the 0 to 24 hr timeframe (AUC_0–24) was calculated using a method based on the previously established procedure reported by Wang et al. [26].

Statistical analyses of the data were conducted using analysis of variance (one-way ANOVA) followed by the Tukey–Kramer multiple comparisons test to determine the differences in ERFX among the three types of samples at the same sampling time. All statistical analyses were performed using IBM SPSS Statistics 25 software (IBM, Tokyo, Japan), and P<0.05 was considered statistically significant.

The body temperature, heart rate, and respiratory rate of the pigs barely fluctuated, and abnormal clinical findings were not detected via visual inspection during the experiment. The WBC, RBC, Hb, and Ht values of the pigs barely fluctuated during the experiment.

The concentrations of CPFX, the primary metabolite of ERFX, were below the detection limits in all samples. Previous reports on the PK of ERFX also showed very low or below the detection limit of CPFX [19, 27], consistent with the results of this study. This suggests that ERFX is the main component responsible for drug activity in pigs.

The ERFX concentrations in the plasma, ELF, and BALF cells are shown in Fig. 1. Both groups showed the highest concentrations in the plasma, ELF, and BALF cells at 8 hr. In pigs administered conventional ERFX, the mean ERFX concentration in ELF and BALF cells at 3 hr and 8 hr was significantly higher than that in plasma (P<0.05). The dynamics of single-dose ERFX were similar; however, higher ERFX concentrations in the bronchoalveolar region remained up to 24 hr after administration. In addition, the mean ERFX concentration in BALF cells at 3 hr was significantly higher than that in the plasma and ELF (P<0.05). Fluoroquinolones, including ERFX, accumulate in phagocytic cells, such as alveolar macrophages and achieve high intracellular concentrations [23]. In this study, the single-dose group was administered a higher dose than the conventional group, suggesting that the excess accumulated in alveolar cells. These results demonstrate that intramuscularly administered ERFX in pigs was well distributed in the bronchoalveolar region.

Fig. 1.

Concentration of enrofloxacin determined within plasma, pulmonary epithelial lining fluid (ELF), and cells in bronchoalveolar lavage fluid (BALF cells) after intramuscular injection for pigs. (A) Baytril 10% injectable solution (Dose: 5 mg/kg) (B) Baytril 1ject (Dose: 7.5 mg/kg). Data are shown as mean ± SD. Values with different letters indicate significant differences between regions at the same sampling time (P<0.05).

In the present study, the plasma AUC was higher in proportion to the dose, which is consistent with a previous report [3]. The corresponding PK parameters in the plasma, ELF, and BALF cells are presented in Table 1. The mean value of peak ERFX concentration (Cmax) and AUC_0–24 in ELF and BALF cells were significantly higher than those in plasma (P<0.05). Additionally, in the single-dose group, the AUC in BALF cells was significantly higher than that in ELF.

Table 1. Pharmacokinetics (PK) parameters of enrofloxacine in plasma, pulmonary epithelial lining fluid and cells in bronchoalveolar lavage fluid (BALF cells) following single intramuscular injection in pigs.

	Conventional type (Baytril 10%, 5 mg/kg)			Single-dose type (Baytril 1ject, 7.5 mg/kg)
	Cmax (μg/mL)	Tmax (hr)	AUC0-24 (μg·hr/mL)	Cmax (μg/mL)	Tmax (hr)	AUC0-24 (μg·hr/mL)
Plasma	0.92 ± 0.03 a	6.33 ± 2.89	18.45 ± 0.62 a	1.27 ± 0.18 a	8.00 ± 0.00	24.30 ± 3.58 a
Pulmonary epithelial lining fluid	11.44 ± 1.56 b	8.00 ± 0.00	200.92 ± 12.52 b	13.46 ± 2.85 b	8.00 ± 0.00	238.36 ± 42.80 b
BALF cells	13.06 ± 5.88 b	8.00 ± 0.00	227.44 ± 105.29 b	17.22 ± 1.32 b	8.00 ± 0.00	309.45 ± 17.54 c

Data are shown as the mean ± SD. Cmax: peak concentration; Tmax: time to Cmax; AUC_0-24: area under the curve of enrofloxacin from 0 to 24 hr after intramuscular injection in pigs. Values with the different letter indicate the significant differences among each region in PK parameters (P<0.05).

To provide effective treatment of bacterial pneumonia, prediction of the antimicrobial effect of the agent is required, which is based on both effective PK and pharmacodynamic (PD) analyses. Such combined PK/PD approaches have been widely used in clinical practice [6]. Generally, the blood concentration of antimicrobial agents and the MIC against causative bacteria are used as indicators. In other words, it is considered important that antimicrobial components are distributed at the site of bacterial infection and exceed the MIC for that particular bacterium.

In previous surveys in Europe, the MIC₉₀ values of ERFX against porcine strains of Pasteurella multocida, Actinobacillus pleuropneumoniae, Streptococcus suis, and Mycoplasma hyopneumoniae were 0.03 µg/mL, 0.06 µg/mL, 0.5 µg/mL, and 0.5 µg/mL, respectively [9, 15]. According to a susceptibility study conducted in Japan using bovine or porcine clinical isolates, ERFX MIC₉₀ against P. multocida, Mannheimia haemolytica, and A. pleuropneumoniae have been reported to be 0.12 µg/mL, 0.5 µg/mL, and 0.5 µg/mL, respectively [13]. In the present study, ERFX concentrations in ELF and BALF cells remained well above 1.00 µg/mL from 3 to 24 hr after administering either formulation. Thus, ERFX concentrations in the bronchoalveolar region were higher than the MIC₉₀ for these bacteria.

Fluoroquinolones, such as ERFX exhibit concentration-dependent killing [22]. Therefore, the AUC_0–24 to MIC ratio (AUC/MIC) was used as an index of the microbiocidal activity [22, 25]. Although data are based on human clinical trials and laboratory animal infection models, for fluoroquinolones, AUC/MIC greater than 100–125 is generally associated with treatment efficacy [2, 22, 25, 28]. Assuming MIC of 0.5 µg/mL based on previous report on A. pleuropneumoniae [13], a typical swine respiratory disease-causing bacterium, the AUC/MIC in plasma, ELF, and alveolar cells were 37, 402, and 455, respectively, for the conventional type, and 49, 477 and 619, respectively, for the single-dose type. These data suggest that sufficient amounts of ERFX are likely to be distributed in the bronchoalveolar region at doses of 5 mg/kg and 7.5 mg/kg. Previous field studies have reported good therapeutic effects of ERFX in the treatment of respiratory diseases [12], and these results support our findings.

In this study, both formulations were expected to reach the bronchoalveolar region with sufficient concentrations of ERFX; however, the single-dose formulation maintained high ERFX concentrations in the bronchoalveolar region even 24 hr after administration. This would be beneficial for treating bacterial pneumonia in the two points below. First, efficacy can be achieved with a single dose, leading to labor savings. This is a significant advantage in large-scale swine rearing. However, because the present study only confirmed the pharmacokinetics up to 24 hr after administration, it may be necessary to confirm the concentration after that. The other is to minimize concerns about antimicrobial resistance. The concept of mutant prevention concentration (MPC) is important in this issue, and Damte et al. [7] reported an MPC₉₀ of 8 µg/mL for A. pleuropneumoniae isolated from pigs. In this study, the mean ERFX concentrations (mean± SD) in the ELF and BALF cells at 8 hr after administration were 11.44 ± 1.56 µg/mL and 13.06 ± 5.88 µg/mL for the conventional form and 13.46 ± 2.85 µg/mL and 17.22 ± 1.32 µg/mL for the single-dose form (Fig. 1). Furthermore, in the single-dose group, the ERFX concentration in ELF and alveolar cells remained above the MPC value until 24 hr after administration (8.3 ± 1.6 µg/mL and 10.7 ± 2.7 µg/mL, respectively). As a countermeasure against antimicrobial resistance, it is important to distribute drug components efficiently to the site of infection. The single-dose formulation allows a relatively large amount to be administered at once and is distributed at high concentrations at the site of infection, suggesting that it may be one way to prevent the emergence of resistant strains in respiratory disease-causing bacteria.

In the present study, we demonstrated the distribution of ERFX in the bronchoalveolar region after intramuscular injection in healthy pigs. In both conventional and single-dose formulations, ERFX was shown to be efficiently distributed into the bronchoalveolar region and maintain sufficient concentrations up to 24 hr after administration. Additional pharmacokinetic and pharmacodynamic studies, including microbiological research on other pathogens, are required to evaluate the efficacy of ERFX treatment for porcine respiratory diseases further.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare. This study was supported by grants from the Bayer Yakuhin Ltd (Elanco Japan K.K.).