Pharmacokinetic Behavior and Pharmacokinetic/Pharmacodynamic Integration of Danofloxacin Following Single or Co-Administration with Meloxicam in Healthy Lambs and Lambs with Respiratory Infections

The aim of this study was to determine the pharmacokinetics and pharmacodynamics of danofloxacin (DAN; 6 mg/kg) following subcutaneous administration alone or co-administration with meloxicam (MLX; 1 mg/kg) in healthy lambs and lambs with respiratory infections. The study was carried out using a total of four groups: HD (healthy; n = 6) and ID (infected; n = 7) groups who were administered DAN only, and HDM (healthy; n = 6) and IDM (infected; n = 7) groups who were administered DAN and MLX simultaneously. The plasma concentrations of DAN were determined using high-performance liquid chromatography–UV and analyzed by the non-compartmental method. DAN exhibited a similar elimination half-life in all groups, including both the healthy and infected lambs. The total clearance in the HDM, ID and IDM groups and volume of distribution in the HDM and IDM groups were significantly reduced. MLX in the IDM group significantly increased the area under the curve (AUC) and peak concentration (Cmax) of DAN compared to the HD group. The Mannheimia haemolytica, Escherichia coli, and Streptococcus spp. strains were isolated from bronchoalveolar lavage fluid samples of the infected lambs. When co-administration with meloxicam, DAN at a 6 mg/kg dose can provide optimum values of ƒAUC0–24/MIC (>56 h) and ƒCmax/MIC (>8) for susceptible M. haemolytica isolates with an MIC90 value of 0.25 µg/mL and susceptible E. coli isolates with an MIC value of ≤0.125 µg/mL.


Introduction
Respiratory diseases, which are common in all countries where lamb breeding is carried out, are considered to be complex diseases that include interactions between multiple agents such as the host's immune system and physiological state, viruses, bacteria, Mycoplasma, and environmental factors. They cause serious financial losses due to low body weight gain, treatment costs, and death [1][2][3]. Bacterial agents frequently isolated from lambs with pneumonia are Mannheimia haemolytica and Pasteurella multocida [4,5]. Antibiotics are used to treat acute pneumonia, to reduce the effects of chronic nonprogressive pneumonia, and to treat the remainder of the flock to prevent more deaths from acute pneumonia. The quick detection of infected lambs is very important for the success of antibiotic treatment [3,6]. The antibiotic selection for the treatment of infection depends on many factors, including the infective microorganism, the susceptibility of the microorganism to antibiotics, the site of infection, local factors (pH, inoculum size, etc.), host factors, the potential side effects of antibiotics, and antibiotic resistance. Following the selection of the Table 1. The mean (±SD) age, body weight, rectal temperature, and respiratory rate of the healthy lambs (n = 6) and those with respiratory infections (n = 7) selected for the pharmacokinetic study. HD, healthy group who received danofloxacin alone; HDM, healthy group who received danofloxacin and meloxicam; ID, infected group who received danofloxacin alone; IDM, infected group who received danofloxacin and meloxicam.

Danofloxacin Analysis
The plasma concentration of danofloxacin was analyzed using the high-performance liquid chromatography (HPLC)-UV system (Shimadzu, Tokyo, Japan) as per a previously reported method [41][42][43][44]. First, 200 µL of acetonitrile was added to 200 µL of the plasma sample. The mixture was vortexed for 30 s and centrifuged at 9167× g for 10 min at 15 • C. Then, 20 µL of supernatant was injected into the HPLC-UV system, comprising a pump (LC-20AT controlled CBM-20A), degasser (DGU-14A), autosampler (SIL-20A), column oven (CTO-10A), and ultraviolet detector (SPD-10A), which was set to 278 nm. Chromatographic separation was performed using a Gemini ™ C18 column (250 × 4.6 mm; internal diameter, 5 µm; Phenomenex, Torrance, CA, USA). The column oven and autosampler temperatures were kept at 30 and 22 • C, respectively. The mobile phase comprised orthophosphoric acid of 0.4% in water, including 0.4% triethylamine and acetonitrile (87:13, v/v), pumped at a flow rate of 1 mL/min. HPLC system control and data analysis were performed with LC solution software (Shimadzu, Japan). To prepare the calibration standards (0.04-10 µg/mL) and the quality control samples (0.05, 0.5, and 5 µg/mL), control plasma from animals that had received no treatment was spiked with working standards of danofloxacin (98%, analytical purity; Sigma-Aldrich, St. Louis, MO, USA) prepared in water. The calibration standards and quality control samples were run with each assay. The linearity of the standard curve created with the calibration standards was r 2 > 0.999. The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coefficient of variation of less than 20% and a bias of ±15%. To determine the recovery, precision, and accuracy of the assay, five replicates for quality control samples were tested for five consecutive days. The recovery from the plasma was >91%. The percent coefficient of variation for the intra-and interday precision was <7.9%, while the percent bias for the intra-and interday accuracy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consulting Inc., Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of danofloxacin for each lamb and to calculate the pharmacokinetic parameters by the non-compartmental method using the formulae described by Gibaldi and Perrier [45]. After SC administration, the elimination rate constant ( Antibiotics 2021, 10,1190 4 of 17

Danofloxacin Analysis
The plasma concentration of danofloxacin was analyzed using the high-performance liquid chromatography (HPLC)-UV system (Shimadzu, Tokyo, Japan) as per a previously reported method [41][42][43][44]. First, 200 µL of acetonitrile was added to 200 µL of the plasma sample. The mixture was vortexed for 30 s and centrifuged at 9167× g for 10 min at 15 °C. Then, 20 µL of supernatant was injected into the HPLC-UV system, comprising a pump (LC-20AT controlled CBM-20A), degasser (DGU-14A), autosampler (SIL-20A), column oven (CTO-10A), and ultraviolet detector (SPD-10A), which was set to 278 nm. Chromatographic separation was performed using a Gemini ™ C18 column (250 × 4.6 mm; internal diameter, 5 µ m; Phenomenex, Torrance, CA, USA). The column oven and autosampler temperatures were kept at 30 and 22 °C, respectively. The mobile phase comprised orthophosphoric acid of 0.4% in water, including 0.4% triethylamine and acetonitrile (87:13, v/v), pumped at a flow rate of 1 mL/min. HPLC system control and data analysis were performed with LC solution software (Shimadzu, Japan). To prepare the calibration standards (0.04-10 µg/mL) and the quality control samples (0.05, 0.5, and 5 µg/mL), control plasma from animals that had received no treatment was spiked with working standards of danofloxacin (98%, analytical purity; Sigma-Aldrich, St. Louis, MO, USA) prepared in water. The calibration standards and quality control samples were run with each assay. The linearity of the standard curve created with the calibration standards was r 2 > 0.999. The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coefficient of variation of less than 20% and a bias of ±15%. To determine the recovery, precision, and accuracy of the assay, five replicates for quality control samples were tested for five consecutive days. The recovery from the plasma was >91%. The percent coefficient of variation for the intraand interday precision was <7.9%, while the percent bias for the intra-and interday accuracy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consulting Inc., Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of danofloxacin for each lamb and to calculate the pharmacokinetic parameters by the non-compartmental method using the formulae described by Gibaldi and Perrier [45]. After SC administration, the elimination rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), area under the curve (AUC), total body clearance (ClT/F), volume of distribution (Vdarea/F), and mean residence time (MRT) were calculated. The volume of distribution at a steady state (Vdss/F) after SC administration was calculated as MRT * ClT/F. The AUC was determined by the linear up/log down method. The ʎz was determined from the linear portion of the terminal phase by linear regression analysis. The peak concentration (Cmax) and the time to reach the Cmax (Tmax) were determined by direct observation from the individual plasma concentration-time curves plotted for each lamb that received danofloxacin.

In Vitro Protein Binding Assay of Danofloxacin
In plasma samples obtained from healthy lambs and lambs with respiratory infections, the plasma protein binding ratio of danofloxacin alone or in combination with meloxicam was evaluated using ultrafiltration [46]. Studies were carried out at physiologic temperature and pH (7.4). Pooled plasma was separately collected from healthy lambs (n = 12, HD and HDM groups) and lambs (n = 14, ID and IDM groups) with respiratory infections that had received no drug treatment. Then, the obtained plasma samples were divided into 4 groups: PBHD and PBID groups who were spiked with danofloxacin only, and PBHDM and PBIDM groups who were spiked with danofloxacin and meloxicam simultaneously. Plasma samples were spiked with danofloxacin and meloxicam to make a 0.05, 0.5, and 5 µg/mL concentration. Three replicates of each concentration of danofloxacin were made. To achieve equilibrium between drugs and plasma proteins, the spiked plasma samples were incubated at 37 °C for 30 min prior to ultrafiltration. Samples The plasma concentration of danofloxacin was analyzed using liquid chromatography (HPLC)-UV system (Shimadzu, Tokyo, Jap reported method [41][42][43][44]. First, 200 µL of acetonitrile was added to sample. The mixture was vortexed for 30 s and centrifuged at 9167 Then, 20 µL of supernatant was injected into the HPLC-UV system (LC-20AT controlled CBM-20A), degasser (DGU-14A), autosamp oven (CTO-10A), and ultraviolet detector (SPD-10A), which was s tographic separation was performed using a Gemini ™ C18 colum nal diameter, 5 µ m; Phenomenex, Torrance, CA, USA). The column temperatures were kept at 30 and 22 °C, respectively. The mobile p phosphoric acid of 0.4% in water, including 0.4% triethylamine a v/v), pumped at a flow rate of 1 mL/min. HPLC system control a performed with LC solution software (Shimadzu, Japan). To prepar ards (0.04-10 µg/mL) and the quality control samples (0.05, 0.5, plasma from animals that had received no treatment was spiked w of danofloxacin (98%, analytical purity; Sigma-Aldrich, St. Louis, M water. The calibration standards and quality control samples wer The linearity of the standard curve created with the calibration sta The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coe less than 20% and a bias of ±15%. To determine the recovery, prec the assay, five replicates for quality control samples were tested for The recovery from the plasma was >91%. The percent coefficient of and interday precision was <7.9%, while the percent bias for the in racy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scie Sunnyvale, CA, USA) was used to plot the plasma concentration-t acin for each lamb and to calculate the pharmacokinetic parameter mental method using the formulae described by Gibaldi and Perrie istration, the elimination rate constant (ʎz), terminal elimination ha the curve (AUC), total body clearance (ClT/F), volume of distributio residence time (MRT) were calculated. The volume of distribution a after SC administration was calculated as MRT * ClT/F. The AUC w linear up/log down method. The ʎz was determined from the linea nal phase by linear regression analysis. The peak concentration reach the Cmax (Tmax) were determined by direct observation from concentration-time curves plotted for each lamb that received dan

In Vitro Protein Binding Assay of Danofloxacin
In plasma samples obtained from healthy lambs and lambs tions, the plasma protein binding ratio of danofloxacin alone o meloxicam was evaluated using ultrafiltration [46]. Studies were logic temperature and pH (7.4). Pooled plasma was separately c lambs (n = 12, HD and HDM groups) and lambs (n = 14, ID and ID atory infections that had received no drug treatment. Then, the ob were divided into 4 groups: PBHD and PBID groups who were spi only, and PBHDM and PBIDM groups who were spiked with dan cam simultaneously. Plasma samples were spiked with danofloxa make a 0.05, 0.5, and 5 µg/mL concentration. Three replicates of danofloxacin were made. To achieve equilibrium between drugs an spiked plasma samples were incubated at 37 °C for 30 min prior to u z ), area under the curve (AUC), total body clearance (Cl T /F), volume of distribution (Vd area /F), and mean residence time (MRT) were calculated. The volume of distribution at a steady state (Vd ss /F) after SC administration was calculated as MRT * Cl T /F. The AUC was determined by the linear up/log down method. The The plasma concentration of danofloxacin was analyzed using the high-performance liquid chromatography (HPLC)-UV system (Shimadzu, Tokyo, Japan) as per a previously reported method [41][42][43][44]. First, 200 µL of acetonitrile was added to 200 µL of the plasma sample. The mixture was vortexed for 30 s and centrifuged at 9167× g for 10 min at 15 °C. Then, 20 µL of supernatant was injected into the HPLC-UV system, comprising a pump (LC-20AT controlled CBM-20A), degasser (DGU-14A), autosampler (SIL-20A), column oven (CTO-10A), and ultraviolet detector (SPD-10A), which was set to 278 nm. Chromatographic separation was performed using a Gemini ™ C18 column (250 × 4.6 mm; internal diameter, 5 µ m; Phenomenex, Torrance, CA, USA). The column oven and autosampler temperatures were kept at 30 and 22 °C, respectively. The mobile phase comprised orthophosphoric acid of 0.4% in water, including 0.4% triethylamine and acetonitrile (87:13, v/v), pumped at a flow rate of 1 mL/min. HPLC system control and data analysis were performed with LC solution software (Shimadzu, Japan). To prepare the calibration standards (0.04-10 µg/mL) and the quality control samples (0.05, 0.5, and 5 µg/mL), control plasma from animals that had received no treatment was spiked with working standards of danofloxacin (98%, analytical purity; Sigma-Aldrich, St. Louis, MO, USA) prepared in water. The calibration standards and quality control samples were run with each assay. The linearity of the standard curve created with the calibration standards was r 2 > 0.999. The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coefficient of variation of less than 20% and a bias of ±15%. To determine the recovery, precision, and accuracy of the assay, five replicates for quality control samples were tested for five consecutive days. The recovery from the plasma was >91%. The percent coefficient of variation for the intraand interday precision was <7.9%, while the percent bias for the intra-and interday accuracy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consulting Inc., Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of danofloxacin for each lamb and to calculate the pharmacokinetic parameters by the non-compartmental method using the formulae described by Gibaldi and Perrier [45]. After SC administration, the elimination rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), area under the curve (AUC), total body clearance (ClT/F), volume of distribution (Vdarea/F), and mean residence time (MRT) were calculated. The volume of distribution at a steady state (Vdss/F) after SC administration was calculated as MRT * ClT/F. The AUC was determined by the linear up/log down method. The ʎz was determined from the linear portion of the terminal phase by linear regression analysis. The peak concentration (Cmax) and the time to reach the Cmax (Tmax) were determined by direct observation from the individual plasma concentration-time curves plotted for each lamb that received danofloxacin.

In Vitro Protein Binding Assay of Danofloxacin
In plasma samples obtained from healthy lambs and lambs with respiratory infections, the plasma protein binding ratio of danofloxacin alone or in combination with meloxicam was evaluated using ultrafiltration [46]. Studies were carried out at physiologic temperature and pH (7.4). Pooled plasma was separately collected from healthy lambs (n = 12, HD and HDM groups) and lambs (n = 14, ID and IDM groups) with respiratory infections that had received no drug treatment. Then, the obtained plasma samples were divided into 4 groups: PBHD and PBID groups who were spiked with danofloxacin only, and PBHDM and PBIDM groups who were spiked with danofloxacin and meloxicam simultaneously. Plasma samples were spiked with danofloxacin and meloxicam to make a 0.05, 0.5, and 5 µg/mL concentration. Three replicates of each concentration of danofloxacin were made. To achieve equilibrium between drugs and plasma proteins, the spiked plasma samples were incubated at 37 °C for 30 min prior to ultrafiltration. Samples z was determined from the linear portion of the terminal phase by linear regression analysis. The peak concentration (C max ) and the time to reach the C max (T max ) were determined by direct observation from the individual plasma concentration-time curves plotted for each lamb that received danofloxacin.

In Vitro Protein Binding Assay of Danofloxacin
In plasma samples obtained from healthy lambs and lambs with respiratory infections, the plasma protein binding ratio of danofloxacin alone or in combination with meloxicam was evaluated using ultrafiltration [46]. Studies were carried out at physiologic temperature and pH (7.4). Pooled plasma was separately collected from healthy lambs (n = 12, HD and HDM groups) and lambs (n = 14, ID and IDM groups) with respiratory infections that had received no drug treatment. Then, the obtained plasma samples were divided into 4 groups: PBHD and PBID groups who were spiked with danofloxacin only, and PBHDM and PBIDM groups who were spiked with danofloxacin and meloxicam simultaneously. Plasma samples were spiked with danofloxacin and meloxicam to make a 0.05, 0.5, and 5 µg/mL concentration. Three replicates of each concentration of danofloxacin were made. To achieve equilibrium between drugs and plasma proteins, the spiked plasma samples were incubated at 37 • C for 30 min prior to ultrafiltration. Samples of 1 mL were transferred to an Amicon Ultra Centrifugal Filter (Ultracel 10 kD; Millipore Corporation, Bedford, MA, USA) Antibiotics 2021, 10, 1190 5 of 17 and centrifuged at 4000× g for 15 min. The ultrafiltrate was analyzed directly by HPLC for determination of free danofloxacin concentrations. The protein binding of danofloxacin was determined the following formula: Protein binding (%) = [(Total drug−Free drug)/Total drug] × 100. In addition, a preliminary study was performed to identify danofloxacin nonspecific binding to the ultrafiltration device.

Statistical Analysis
All values for the pharmacokinetic parameters and pharmacokinetic/pharmacodynamic (PK/PD) data are expressed as the geometric mean (min-max). Other values are presented as the mean ± SD. The normality of the data distribution was assessed with the Shapiro-Wilk test and the homogeneity of variance with the Levene's test. A two-way between groups ANOVA was used to evaluate the interaction of health/infection condition and meloxicam administration for the pharmacokinetic parameters. The interaction of health/infection condition, meloxicam and concentrations of danofloxacin for the protein binding of danofloxacin was evaluated by a three-way ANOVA. The intergroup differences of the pharmacokinetic parameters and protein binding were analyzed using the post hoc Tukey's test following the ANOVA (SPSS 22.0, SPSS for Windows, SPSS Inc., Chicago, IL, USA). A p-value of <0.05 was considered statistically significant.

Collection of Bronchoalveolar Lavage Fluid Samples
BAL fluid samples were taken from 98 lambs whose clinical scoring results were 1, 2 or 3 in order to isolate and identify the bacterial agents caused infection in lambs with respiratory infections. The trans-nasal method was used for the collection of BAL fluid. Before the collection of BAL fluid, the nostrils of the lambs were cleaned with an alcohol swab. Following extension of the head and neck of the lamb, a sterile nasogastric probe (3.33 × 1210 mm; Bıcakcilar, Istanbul, Turkey) was advanced trans-nasally until a weak resistance was encountered in the trachea. With the recurrent cough reflex, it was understood that the hull area was reached. Then, the catheter was withdrawn 1-2 cm, and 10 mL of sterile saline (0.9% NaCl) was injected into the trachea with the help of an injector placed at the tip of the catheter, followed by immediate aspiration. The BAL fluid (1-2 mL) was kept at 4 • C until microbiological analysis. The microbiological isolation process was started within 12 h following the sampling.

Bacterial Isolation and Identification
Direct streaking was performed on 5% sheep blood agar and MacConkey agar (Merck, Darmstadt, Germany) plates from the BAL fluid samples. The plates were incubated aerobically at 37 • C for 24-48 h. For the Mycoplasma culture, the samples were inoculated on Mycoplasma-selective agar (Mycoplasma agar base, Oxoid, CM0401 + Mycoplasma-selective supplement G, Oxoid, SR0059C). The Mycoplasma agar plates were incubated at 37 • C for 2-7 days in an environment with 5% CO 2 . The Mycoplasma-selective agar plates were examined daily under a microscope after 48 h incubation and were evaluated for growth. Isolated bacteria were identified by colony morphology, growth characteristics, and biochemical tests [47]. The isolated strains were stored at −80 • C for antimicrobial susceptibility tests.

Minimum Inhibitory Concentration
In this study, the broth microdilution method described by the Clinical and Laboratory Standards Institute [48] was used to determine the MIC values of danofloxacin on the bacteria isolated from lambs with respiratory infections. E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as quality control isolates. Danofloxacin was tested in a range from 0.016 to 16 µg/mL in doubling dilutions. The final test concentration of bacteria was 5 × 10 5 colony forming units/mL in Mueller-Hinton broth (Merck, Germany). The MICs were read after 24 h incubation at 37 • C. The lowest concentration of drug that visibly Antibiotics 2021, 10, 1190 6 of 17 inhibited bacterial growth was accepted as the MIC. The concentrations that inhibited 50% (MIC 50 ) and 90% (MIC 90 ) of the isolates were calculated for danofloxacin. The MIC 50 and MIC 90 were calculated using "n × 0.5" and "n × 0.9", respectively, in which n was the number of test strains. Then, if the resulting number was not an integer, the next integer following the respective value was taken as the MIC 50 or MIC 90 value [49].

Pharmacokinetic/Pharmacodynamic Integration
The PK/PD surrogate indices used to determine the inhibitory activity or effect of danofloxacin against susceptible pathogens were AUC 0-24 /MIC and C max /MIC ratios [50][51][52][53]. In this study, the ƒAUC 0-24 /MIC and ƒC max /MIC ratios were calculated using the ƒAUC and ƒC max obtained from the free concentrations of danofloxacin in the plasma of the ID and IDM groups after danofloxacin administration and the MIC values determined for the bacterial strains isolated from the lambs with respiratory infections.
Antibiotics 2021, 10, 1190 6 of 17 bacteria was 5 × 10 5 colony forming units/mL in Mueller-Hinton broth (Merck, Germany). The MICs were read after 24 h incubation at 37 °C. The lowest concentration of drug that visibly inhibited bacterial growth was accepted as the MIC. The concentrations that inhibited 50% (MIC50) and 90% (MIC90) of the isolates were calculated for danofloxacin. The MIC50 and MIC90 were calculated using "n × 0.5" and "n × 0.9", respectively, in which n was the number of test strains. Then, if the resulting number was not an integer, the next integer following the respective value was taken as the MIC50 or MIC90 value [49].

Pharmacokinetic/Pharmacodynamic Integration
The PK/PD surrogate indices used to determine the inhibitory activity or effect of danofloxacin against susceptible pathogens were AUC0-24/MIC and Cmax/MIC ratios [50][51][52][53]. In this study, the ƒAUC0-24/MIC and ƒCmax/MIC ratios were calculated using the ƒAUC and ƒCmax obtained from the free concentrations of danofloxacin in the plasma of the ID and IDM groups after danofloxacin administration and the MIC values determined for the bacterial strains isolated from the lambs with respiratory infections.

Pharmacokinetics
Semi-logarithmic plasma concentration-time curves of danoflaxacin alone or co-administered with meloxicam in healthy lambs and lambs with respiratory infections are presented in Figure 1. In the HD, HDM, ID and IDM groups, the plasma concentration of danofloxacin following administration of a 6 mg/kg dose of SC was 1.72 ± 0.64, 1.90 ± 0.77, 2.01 ± 0.84, and 2.56 ± 0.85 µ g/mL, respectively, at the first observational point (15 min). Then, it dropped to 0.057 ± 0.015, 0.086 ± 0.026, 0.075 ± 0.027, and 0.108 ± 0.026 µ g/mL, respectively, at 24 h. Semi-logarithmic plasma concentration-time curves after subcutaneous administration of danofloxacin alone or co-administered with meloxicam in healthy lambs (n = 6) and lambs with respiratory infections (n = 7). HD, healthy group who received danofloxacin alone; HDM, healthy group who received danofloxacin and meloxicam; ID, infected group who received danofloxacin alone; IDM, infected group who received danofloxacin and meloxicam. Data are presented as mean ± SD. Semi-logarithmic plasma concentration-time curves after subcutaneous administration of danofloxacin alone or co-administered with meloxicam in healthy lambs (n = 6) and lambs with respiratory infections (n = 7). HD, healthy group who received danofloxacin alone; HDM, healthy group who received danofloxacin and meloxicam; ID, infected group who received danofloxacin alone; IDM, infected group who received danofloxacin and meloxicam. Data are presented as mean ± SD.
The pharmacokinetic parameters of danofloxacin alone or co-administered with meloxicam in healthy lambs and lambs with respiratory infections are presented in Table 2. A two-way ANOVA revealed no significant interaction between the healthy/infected conditions and meloxicam administration of any of the pharmacokinetic parameters (p > 0.05), but significant main effects were found for the healthy/infected conditions (p < 0.05) on Cl T /F and AUC and for meloxicam administration (p < 0.05) on the Cl T /F, Vd area /F, Vd ss /F, C max , and AUC, indicating that these factors acted independently. Danofloxacin exhibited similar t 1/2 The linearity of the standard curve created with the calibration standards was r 2 > 0.999. The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coefficient of variation of less than 20% and a bias of ±15%. To determine the recovery, precision, and accuracy of the assay, five replicates for quality control samples were tested for five consecutive days. The recovery from the plasma was >91%. The percent coefficient of variation for the intraand interday precision was <7.9%, while the percent bias for the intra-and interday accuracy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consulting Inc., Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of danofloxacin for each lamb and to calculate the pharmacokinetic parameters by the non-compartmental method using the formulae described by Gibaldi and Perrier [45]. After SC administration, the elimination rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), area under the curve (AUC), total body clearance (ClT/F), volume of distribution (Vdarea/F), and mean residence time (MRT) were calculated. The volume of distribution at a steady state (Vdss/F) after SC administration was calculated as MRT * ClT/F. The AUC was determined by the linear up/log down method. The ʎz was determined from the linear portion of the terminal phase by linear regression analysis. The peak concentration (Cmax) and the time to reach the Cmax (Tmax) were determined by direct observation from the individual plasma concentration-time curves plotted for each lamb that received danofloxacin.

In Vitro Protein Binding Assay of Danofloxacin
In plasma samples obtained from healthy lambs and lambs with respiratory infections, the plasma protein binding ratio of danofloxacin alone or in combination with meloxicam was evaluated using ultrafiltration [46]. Studies were carried out at physiologic temperature and pH (7.4). Pooled plasma was separately collected from healthy lambs (n = 12, HD and HDM groups) and lambs (n = 14, ID and IDM groups) with respiratory infections that had received no drug treatment. Then, the obtained plasma samples were divided into 4 groups: PBHD and PBID groups who were spiked with danofloxacin only, and PBHDM and PBIDM groups who were spiked with danofloxacin and meloxicam simultaneously. Plasma samples were spiked with danofloxacin and meloxicam to make a 0.05, 0.5, and 5 µg/mL concentration. Three replicates of each concentration of danofloxacin were made. To achieve equilibrium between drugs and plasma proteins, the spiked plasma samples were incubated at 37 °C for 30 min prior to ultrafiltration. Samples z and MRT in all groups (p > 0.05). However, the Cl T /F and Vd area /F of danofloxacin showed significant differences between the groups (p < 0.05). The Cl T /F decreased significantly (p < 0.05) upon meloxicam administration (HDM and IDM groups) and the effect of infection (ID and IDM groups). The administration of meloxicam to healthy (HDM) and infected lambs (IDM) significantly increased the Vd area /F and Vd ss /F of danofloxacin compared to the HD group (p < 0.05). In the HD, HDM, ID, and IDM groups, danofloxacin reached different C max values of 3.36, 3.73, 3.61, and 4.59 µg/mL (p < 0.05), respectively, with T max values of 1.0 h (p > 0.05). In particular, the administration of meloxicam (IDM) to infected lambs significantly increased the C max of danofloxacin compared to the HD group (p < 0.05). The AUC increased significantly in the HDM and IDM groups compared to the HD and ID groups (p < 0.05). In the ID and IDM groups, the ƒAUC was 10.36 (7.43-14.99) h·µg/mL and 14.80 (11.25-20.33) h·µg/mL, respectively, and ƒC max was 2.42 (2.00-3.00) µg/mL and 3.60 (2.58-5.05) µg/mL, respectively. Table 2. The pharmacokinetic parameters of danofloxacin (6 mg/kg; SC) alone or co-administered with meloxicam (1 mg/kg; SC) in healthy lambs (n = 6) and lambs with respiratory infections (n = 7). t 1/2 reported method [41][42][43][44]. First, 200 µL of acetonitrile was added to 200 µL of the sample. The mixture was vortexed for 30 s and centrifuged at 9167× g for 10 min a Then, 20 µL of supernatant was injected into the HPLC-UV system, comprising a (LC-20AT controlled CBM-20A), degasser (DGU-14A), autosampler (SIL-20A), oven (CTO-10A), and ultraviolet detector (SPD-10A), which was set to 278 nm. C tographic separation was performed using a Gemini ™ C18 column (250 × 4.6 mm nal diameter, 5 µ m; Phenomenex, Torrance, CA, USA). The column oven and autos temperatures were kept at 30 and 22 °C, respectively. The mobile phase comprised phosphoric acid of 0.4% in water, including 0.4% triethylamine and acetonitrile v/v), pumped at a flow rate of 1 mL/min. HPLC system control and data analys performed with LC solution software (Shimadzu, Japan). To prepare the calibration ards (0.04-10 µg/mL) and the quality control samples (0.05, 0.5, and 5 µg/mL), plasma from animals that had received no treatment was spiked with working sta of danofloxacin (98%, analytical purity; Sigma-Aldrich, St. Louis, MO, USA) prep water. The calibration standards and quality control samples were run with each The linearity of the standard curve created with the calibration standards was r 2 The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coefficient of varia less than 20% and a bias of ±15%. To determine the recovery, precision, and accu the assay, five replicates for quality control samples were tested for five consecutiv The recovery from the plasma was >91%. The percent coefficient of variation for th and interday precision was <7.9%, while the percent bias for the intra-and interda racy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consultin Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of da acin for each lamb and to calculate the pharmacokinetic parameters by the non-co mental method using the formulae described by Gibaldi and Perrier [45]. After SC istration, the elimination rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), are the curve (AUC), total body clearance (ClT/F), volume of distribution (Vdarea/F), an residence time (MRT) were calculated. The volume of distribution at a steady state after SC administration was calculated as MRT * ClT/F. The AUC was determined linear up/log down method. The ʎz was determined from the linear portion of the nal phase by linear regression analysis. The peak concentration (Cmax) and the reach the Cmax (Tmax) were determined by direct observation from the individual concentration-time curves plotted for each lamb that received danofloxacin.

In Vitro Protein Binding Assay of Danofloxacin
In plasma samples obtained from healthy lambs and lambs with respirator tions, the plasma protein binding ratio of danofloxacin alone or in combinatio meloxicam was evaluated using ultrafiltration [46]. Studies were carried out at logic temperature and pH (7.4). Pooled plasma was separately collected from lambs (n = 12, HD and HDM groups) and lambs (n = 14, ID and IDM groups) with atory infections that had received no drug treatment. Then, the obtained plasma s were divided into 4 groups: PBHD and PBID groups who were spiked with danof only, and PBHDM and PBIDM groups who were spiked with danofloxacin and cam simultaneously. Plasma samples were spiked with danofloxacin and melox make a 0.05, 0.5, and 5 µg/mL concentration. Three replicates of each concentra danofloxacin were made. To achieve equilibrium between drugs and plasma prote spiked plasma samples were incubated at 37 °C for 30 min prior to ultrafiltration. S z , terminal half-life; Cl T /F, total clearance; Vd area /F, volume of distribution; Vd ss /F, volume of distribution in a steady state; T max , time to reach the peak concentration; C max , peak plasma concentration; AUC, area under the concentration-time curve; MRT, mean residence time; HD, healthy group who received danofloxacin alone; HDM, healthy group who received danofloxacin and meloxicam; ID, infected group who received danofloxacin alone; IDM, infected group who received danofloxacin and meloxicam. d ultraviolet detector (SPD-10A), which was set to 278 nm. Chromawas performed using a Gemini ™ C18 column (250 × 4.6 mm; interhenomenex, Torrance, CA, USA). The column oven and autosampler ept at 30 and 22 °C, respectively. The mobile phase comprised ortho-0.4% in water, including 0.4% triethylamine and acetonitrile (87:13, ow rate of 1 mL/min. HPLC system control and data analysis were olution software (Shimadzu, Japan). To prepare the calibration stand-) and the quality control samples (0.05, 0.5, and 5 µg/mL), control s that had received no treatment was spiked with working standards , analytical purity; Sigma-Aldrich, St. Louis, MO, USA) prepared in n standards and quality control samples were run with each assay. standard curve created with the calibration standards was r 2 > 0.999. uantitation (LLOQ) was 0.04 µg/mL with a coefficient of variation of bias of ±15%. To determine the recovery, precision, and accuracy of ates for quality control samples were tested for five consecutive days. he plasma was >91%. The percent coefficient of variation for the intraon was <7.9%, while the percent bias for the intra-and interday accus ±5.3%. tic Analysis .0.173 software (Pharsight Corporation, Scientific Consulting Inc., ) was used to plot the plasma concentration-time curve of danofloxnd to calculate the pharmacokinetic parameters by the non-compartg the formulae described by Gibaldi and Perrier [45]. After SC admintion rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), area under al body clearance (ClT/F), volume of distribution (Vdarea/F), and mean ) were calculated. The volume of distribution at a steady state (Vdss/F) ion was calculated as MRT * ClT/F. The AUC was determined by the method. The ʎz was determined from the linear portion of the termiregression analysis. The peak concentration (Cmax) and the time to were determined by direct observation from the individual plasma curves plotted for each lamb that received danofloxacin.
n Binding Assay of Danofloxacin ples obtained from healthy lambs and lambs with respiratory infecrotein binding ratio of danofloxacin alone or in combination with luated using ultrafiltration [46]. Studies were carried out at physiond pH (7.4). Pooled plasma was separately collected from healthy d HDM groups) and lambs (n = 14, ID and IDM groups) with respirhad received no drug treatment. Then, the obtained plasma samples groups: PBHD and PBID groups who were spiked with danofloxacin nd PBIDM groups who were spiked with danofloxacin and meloxi-. Plasma samples were spiked with danofloxacin and meloxicam to d 5 µg/mL concentration. Three replicates of each concentration of ade. To achieve equilibrium between drugs and plasma proteins, the les were incubated at 37  Data are presented as the geometric mean (min-max) and were analyzed by two-way ANOVA with Tukey's test. a,b,c Different letters in the same row are statistically different (p < 0.05).

In Vitro Protein Binding of Danofloxacin
The protein binding ratio of danofloxacin alone or in combination with meloxicam in plasma samples of healthy lambs and lambs with respiratory infections is presented in Table 3. The protein binding ratio (mean ± SD) of danofloxacin for PBHD, PBHDM, PBID and PBIDM groups were 32.0 ± 4.8%, 22.7 ± 3.1%, 32.8 ± 3.9% and 21.6 ± 4.0%, respectively. No interaction of the health/infection condition, meloxicam and concentration of danofloxacin for the protein binding of danofloxacin was determined by the three-way ANOVA, but the meloxicam and concentration of danofloxacin acted independently. No significant effects on the plasma protein binding of danofloxacin were found for the healthy/infected conditions (p > 0.05). When danofloxacin was in combination with meloxicam (PBHDM and PBIDM groups), the plasma protein binding of danofloxacin decreased significantly (p < 0.05). In PBHD group, the protein binding ratio at the concentration of 5 µg/mL is statistically lower than that at the concentration of 0.05 µg/mL (p < 0.05). Preliminary studies found negligible binding (<1%) of danofloxacin to the ultrafiltration device. Table 3. Protein binding ratio (%) of danofloxacin alone or in combination with meloxicam in plasma samples of healthy lambs and lambs with respiratory infections. PBHD, healthy group who was spiked with danofloxacin alone; PBHDM, healthy group who was spiked with danofloxacin and meloxicam; PBID, infected group who was spiked danofloxacin alone; PBIDM, infected group who was spiked danofloxacin and meloxicam. Data are presented as the mean ± SD and were analyzed by three-way ANOVA with Tukey's test. * In PBHD group, the protein binding ratio at the concentration of 5 µg/mL is statistically lower than that at the concentration of 0.05 µg/mL (p < 0.05). a,b Different letters in the same row are statistically different (p < 0.05).

Pharmacodynamics
In the BAL fluid samples taken from the infected lambs, the E. coli (n = 6), Streptococcus spp. (n = 3), and M. haemolytica (n = 83) strains were isolated and identified. No growth was detected on the Mycoplasma cultures. The MIC values of danofloxacin were determined as 0.063-0.125 µg/mL for the E. coli strain and >16 µg/mL for the Streptococcus spp. strain. The breakpoints of danofloxacin for E. coli and Streptococcus spp. have not been defined. However, all isolates of E. coli isolated from the lambs could be susceptible to danofloxacin according to the susceptibility breakpoint (8 µg/mL) of danofloxacin reported for E. coli isolated from pigs [54]. The MIC 90 value could not be determined for the E. coli or Streptococcus spp. strains due to the insufficient number of isolates.
In this study, the MIC values of danofloxacin for the 83 M. haemolytica isolates isolated from the lambs ranged from 0.016 to >16 µg/mL. The percent MIC distribution of danofloxacin for the 83 isolates of M. haemolytica is presented in Figure 2

Pharmacokinetic/Pharmacodynamic Integration
The PK/PD surrogate indices determined using the pharmacokinetic parameters obtained from the free concentrations of danofloxacin and the MIC values determined for the bacterial strains isolated from the lambs with respiratory infections are presented in

Pharmacokinetic/Pharmacodynamic Integration
The PK/PD surrogate indices determined using the pharmacokinetic parameters obtained from the free concentrations of danofloxacin and the MIC values determined for the bacterial strains isolated from the lambs with respiratory infections are presented in Table 4. In the ID and IDM groups, the AUC 0-24 /MIC 90

Pharmacokinetics of Danofloxacin
Danofloxacin has been recommended for use at a dose of 6 mg/kg against M. haemolytica isolates with an MIC value of 0.25 µg/mL by PK/PD modeling in sheep [51,57]. In addition, the clinical efficacy of a 6 mg/kg dose of danofloxacin has been demonstrated in an experimental infection caused by M. haemolytica in lambs [17]. In this study, the injection route (SC) and dose (6 mg/kg) of danofloxacin administered to lambs were determined based on previous studies [17,50,56]. Meloxicam was administered at the approved dose (1 mg/kg) in sheep [20].

Danofloxacin Analysis
The plasma concentration of danofloxacin was analyzed using the high-pe liquid chromatography (HPLC)-UV system (Shimadzu, Tokyo, Japan) as per a p reported method [41][42][43][44]. First, 200 µL of acetonitrile was added to 200 µL of t sample. The mixture was vortexed for 30 s and centrifuged at 9167× g for 10 mi Then, 20 µL of supernatant was injected into the HPLC-UV system, comprisin (LC-20AT controlled CBM-20A), degasser (DGU-14A), autosampler (SIL-20A oven (CTO-10A), and ultraviolet detector (SPD-10A), which was set to 278 nm tographic separation was performed using a Gemini ™ C18 column (250 × 4.6 m nal diameter, 5 µ m; Phenomenex, Torrance, CA, USA). The column oven and au temperatures were kept at 30 and 22 °C, respectively. The mobile phase compri phosphoric acid of 0.4% in water, including 0.4% triethylamine and acetonitr v/v), pumped at a flow rate of 1 mL/min. HPLC system control and data ana performed with LC solution software (Shimadzu, Japan). To prepare the calibrat ards (0.04-10 µg/mL) and the quality control samples (0.05, 0.5, and 5 µg/mL plasma from animals that had received no treatment was spiked with working of danofloxacin (98%, analytical purity; Sigma-Aldrich, St. Louis, MO, USA) p water. The calibration standards and quality control samples were run with e The linearity of the standard curve created with the calibration standards was The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coefficient of v less than 20% and a bias of ±15%. To determine the recovery, precision, and a the assay, five replicates for quality control samples were tested for five consecu The recovery from the plasma was >91%. The percent coefficient of variation for and interday precision was <7.9%, while the percent bias for the intra-and inter racy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consu Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of acin for each lamb and to calculate the pharmacokinetic parameters by the non mental method using the formulae described by Gibaldi and Perrier [45]. After S istration, the elimination rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), a the curve (AUC), total body clearance (ClT/F), volume of distribution (Vdarea/F), residence time (MRT) were calculated. The volume of distribution at a steady sta after SC administration was calculated as MRT * ClT/F. The AUC was determin linear up/log down method. The ʎz was determined from the linear portion of nal phase by linear regression analysis. The peak concentration (Cmax) and th reach the Cmax (Tmax) were determined by direct observation from the individu concentration-time curves plotted for each lamb that received danofloxacin. The plasma concentration of danofloxacin was analyzed using the high-performance liquid chromatography (HPLC)-UV system (Shimadzu, Tokyo, Japan) as per a previously reported method [41][42][43][44]. First, 200 µL of acetonitrile was added to 200 µL of the plasma sample. The mixture was vortexed for 30 s and centrifuged at 9167× g for 10 min at 15 °C. Then, 20 µL of supernatant was injected into the HPLC-UV system, comprising a pump (LC-20AT controlled CBM-20A), degasser (DGU-14A), autosampler (SIL-20A), column oven (CTO-10A), and ultraviolet detector (SPD-10A), which was set to 278 nm. Chromatographic separation was performed using a Gemini ™ C18 column (250 × 4.6 mm; internal diameter, 5 µ m; Phenomenex, Torrance, CA, USA). The column oven and autosampler temperatures were kept at 30 and 22 °C, respectively. The mobile phase comprised orthophosphoric acid of 0.4% in water, including 0.4% triethylamine and acetonitrile (87:13, v/v), pumped at a flow rate of 1 mL/min. HPLC system control and data analysis were performed with LC solution software (Shimadzu, Japan). To prepare the calibration standards (0.04-10 µg/mL) and the quality control samples (0.05, 0.5, and 5 µg/mL), control plasma from animals that had received no treatment was spiked with working standards of danofloxacin (98%, analytical purity; Sigma-Aldrich, St. Louis, MO, USA) prepared in water. The calibration standards and quality control samples were run with each assay. The linearity of the standard curve created with the calibration standards was r 2 > 0.999. The lower limit of quantitation (LLOQ) was 0.04 µg/mL with a coefficient of variation of less than 20% and a bias of ±15%. To determine the recovery, precision, and accuracy of the assay, five replicates for quality control samples were tested for five consecutive days. The recovery from the plasma was >91%. The percent coefficient of variation for the intraand interday precision was <7.9%, while the percent bias for the intra-and interday accuracy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consulting Inc., Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of danofloxacin for each lamb and to calculate the pharmacokinetic parameters by the non-compartmental method using the formulae described by Gibaldi and Perrier [45]. After SC administration, the elimination rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), area under the curve (AUC), total body clearance (ClT/F), volume of distribution (Vdarea/F), and mean residence time (MRT) were calculated. The volume of distribution at a steady state (Vdss/F) after SC administration was calculated as MRT * ClT/F. The AUC was determined by the linear up/log down method. The ʎz was determined from the linear portion of the terminal phase by linear regression analysis. The peak concentration (Cmax) and the time to reach the Cmax (Tmax) were determined by direct observation from the individual plasma concentration-time curves plotted for each lamb that received danofloxacin. and interday precision was <7.9%, while the percent bias for the intra-and interday accuracy of the assay was ±5.3%.

Pharmacokinetic Analysis
WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consulting Inc., Sunnyvale, CA, USA) was used to plot the plasma concentration-time curve of danofloxacin for each lamb and to calculate the pharmacokinetic parameters by the non-compartmental method using the formulae described by Gibaldi and Perrier [45]. After SC administration, the elimination rate constant (ʎz), terminal elimination half-life (t1/2 ʎ z), area under the curve (AUC), total body clearance (ClT/F), volume of distribution (Vdarea/F), and mean residence time (MRT) were calculated. The volume of distribution at a steady state (Vdss/F) after SC administration was calculated as MRT * ClT/F. The AUC was determined by the linear up/log down method. The ʎz was determined from the linear portion of the terminal phase by linear regression analysis. The peak concentration (Cmax) and the time to reach the Cmax (Tmax) were determined by direct observation from the individual plasma concentration-time curves plotted for each lamb that received danofloxacin.

In Vitro Protein Binding Assay of Danofloxacin
In plasma samples obtained from healthy lambs and lambs with respiratory infections, the plasma protein binding ratio of danofloxacin alone or in combination with meloxicam was evaluated using ultrafiltration [46]. Studies were carried out at physiologic temperature and pH (7.4). Pooled plasma was separately collected from healthy lambs (n = 12, HD and HDM groups) and lambs (n = 14, ID and IDM groups) with respiratory infections that had received no drug treatment. Then, the obtained plasma samples were divided into 4 groups: PBHD and PBID groups who were spiked with danofloxacin only, and PBHDM and PBIDM groups who were spiked with danofloxacin and meloxicam simultaneously. Plasma samples were spiked with danofloxacin and meloxicam to make a 0.05, 0.5, and 5 µg/mL concentration. Three replicates of each concentration of danofloxacin were made. To achieve equilibrium between drugs and plasma proteins, the spiked plasma samples were incubated at 37 °C for 30 min prior to ultrafiltration. Samples z parameters in all groups. These results are also consistent with the results of studies in which the Cl T and Vd of enrofloxacin decreased, but its t 1/2 did not change [30,59].
In this study, the Cl T /F of danofloxacin significantly decreased with the administration (HDM and IDM groups) of meloxicam and the effect (ID and IDM groups) of infection. The lower Cl T /F of danofloxacin in the HDM (0.32 L/h·kg) and IDM (0.28 L/h·kg) groups compared to the HD (0.45 L/h·kg) and ID (0.38 L/h·kg) groups indicates that danofloxacin interacts with meloxicam. The elimination of danofloxacin and meloxicam has not been observed in sheep. The excretion of danofloxacin occurs approximately equally in urine and feces; while approximately 80% of the drug is removed unchanged, 20% of the drug is removed as a β-glucuronide conjugate of danofloxacin, desmethyldanofloxacin and danofloxacin-N-oxide, which are formed as a result of phase I and extensive phase II reactions [60]. Meloxicam is metabolized extensively in the liver through phase I reactions (conjugate derivative has not been determined) and less than <10% as unchanged meloxicam is excreted in the urine [61,62]. These data may indicate that danofloxacin and meloxicam interact primarily at the kidney level due to differences in their metabolism pathways. It is known that NSAIDs reduce the renal blood flow and glomerular filtration rate by inhibiting the synthesis of vasodilator prostaglandin (PG)E 2 and PGI 2 , which play a role in maintaining normal kidney function [63,64]. PGs are synthesized in the kidneys by both COX-1 and COX-2 enzymes. Selective COX-2 inhibitors have been shown to have similar effects on renal hemodynamics as non-selective NSAIDs [65,66]. The effect of meloxicam, a selective COX-2 inhibitor, on the Cl T /F of danofloxacin may be related to its decrease in renal blood flow and glomerular filtration rate.
Meloxicam is a substrate of BCRP, a member of the ATP-dependent efflux transporters [67,68]. Danofloxacin is the substrate of P-gp and MRP2, in addition to BCRP [16,43]. In sheep, ivermectin reduces the passage of danofloxacin entering the milk by a BCRPmediated interaction (40%) [43]. Ciprofloxacin, a substrate of BCRP but not of P-gp or MRP2, reduces the secretion and absorption of danofloxacin in a Caco-2 cell model [16]. In this study, the decrease in the Cl T /F of danofloxacin in the IDM and HDM groups may have resulted from the limitation of the excretion of danofloxacin due to a BCRP-mediated danofloxacin-meloxicam interaction in the excretion routes, such as the intestines, bile duct, or proximal tubule cells.
In this study, the lower Cl T /F of danofloxacin in the ID (0.38 L/h·kg) and IDM (0.28 L/h·kg) groups than that in the HD (0.45 L/h·kg) and HDM (0.32 L/h·kg) groups indicates the effect of infection. These findings are consistent with other studies that reported a reduced Cl T /F of danofloxacin, marbofloxacin, and tilmicosin in respiratory infections [69][70][71][72]. However, the Cl T /F of marbofloxacin increased in lambs naturally infected with M. haemolytica [73]. The Cl T of drugs is influenced by a variety of factors, particularly the blood flow to the excretion organs, the binding ratio of the drug to plasma proteins, and the activity of drug-metabolizing enzymes. These factors can be affected by sepsis and septic shock and may alter drug excretion and metabolism, leading to a reduced Cl T [74,75]. It is reported that inflammation and infection could lead to downregulation of drug-metabolizing enzymes and transporters (primarily of the ABC superfamily), resulting in higher plasma concentrations and altering some distribution processes. Many inflammatory mediators (interleukines, cytokines, transforming growth factor, tumor necrosis factor or interferons) reduced the gene expression of cytochrome P450 complex in liver. On the other hand, inflammation and infection also downregulate drug efflux transporters ATP-binding cassette (ABC) superfamily. These transporters are ubiquitous in the organism and play a major role in drug transport of antimicrobials, among other drugs. A specific ABC transporter, the BCRP, and ABCG2 family transporter, is present in the human mammary gland and is responsible to excrete fluoroquinolones to milk. BCRP was present in mammary glands of animals including sheep and goats, and it is reported that danofloxacin is a substrate of BCRP, and inflammation and infection could lead to a downregulation of this transporter, affecting the disposition of the drug in the organism [76][77][78][79][80].
In lambs infected with M. haemolytica, the release of pro-inflammatory cytokines such as interleukin-1β, interleukin-8, and tumor necrosis factor-alpha has been shown to increase [81]. Pro-inflammatory cytokines reduce the activities of various cytochrome P450 and phase II enzymes, as well as the expression and function of transporter proteins such as P-gp, BCRP, and MRP2, which play an important role in the pharmacokinetics of drugs [82][83][84][85]. These effects have been reported as the cause for the decreased Cl T of danofloxacin in infected animals [16,86]. In this study, the decreased Cl T /F of danofloxacin in the ID group may be related to changes in blood flow into the excretion organs caused by infection and the decrease in the functions of phase II enzymes and the P-gp, BCRP, and MRP2 transporters. The partial decrease in the Cl T /F of danofloxacin in the IDM group compared to the HDM group may be due to the therapeutic effect of meloxicam on the inflammation and fever caused by infection in the infected lambs.
In this study, the Vd area /F and Vd ss /F of danofloxacin were significantly decreased in the HDM and IDM groups compared to the HD group. These data show that the Vd area /F and Vd ss /F decreased with the administration of meloxicam (HDM and IDM). Similarly, the Vd of enrofloxacin and moxifloxacin has been shown to decrease with upon administration of some NSAIDs in healthy animals [30,31]. The protein binding ratio of danofloxacin in HD, HDM, ID and IDM groups were 32.0 ± 4.8%, 22.7 ± 3.1%, 32.8 ± 3.9% and 21.6 ± 4.0%, respectively. The binding ratio of meloxicam to plasma proteins is high (96% in dogs and 98% in calves) [60]. In this study, meloxicam decreased significantly the plasma protein binding of danofloxacin in HDM and IDM groups. A decrease in the binding ratio of danofloxacin to plasma proteins can be expected to cause an increased Vd. However, an increased concentration of danofloxacin in the plasma due to its reduced elimination by meloxicam can lead to a low calculation of Vd/F. Another reason for the decreased Vd/F in the HDM and IDM groups may be the limitation of the body disposition of danofloxacin, which is a substrate of P-gp, MRP2, and BCRP, by meloxicam [16,43,87]. Danofloxacin reaches 4 times higher concentrations in the lungs, bronchial mucosa, and bronchial secretions and 10 times higher in the milk than that in the plasma [12,57,88]. ABC transporters such as P-gp, MRP2, and especially BCRP are responsible for the high concentrations of danofloxacin in the lung tissues and milk [16,43]. The limitation of the excretion and passage of danofloxacin to highly accumulated tissues such as the lungs due to a BCRP-mediated danofloxacin-meloxicam interaction may be the reason for the decreased Vd/F in the HDM and IDM groups. In particular, given that the interest lies with the influence of meloxicam in the pharmacokinetics of danofloxacin, a more robust approach would be to also analyze the concentration-time profile of meloxicam to examine whether there is a potential concentration-dependent effect.
The rate of drug absorption from the site of administration is affected by conditions such as septicemia and septic shock, which affect the regional blood flow [74,89]. A systemic inflammatory response has been reported in pneumonia caused by M. haemolytica [81]. In this study, an increase (>40.5 • C) in body temperature, indicating the systemic effects of the infection, was determined in infected lambs. Therefore, the absorption rate of danofloxacin can be expected to change following SC administration in infected lambs. However, in this study, the C max /AUC and T max parameters used to evaluate the rate of drug absorption from the site of administration [90] were similar between the groups.
In this study, the C max of danofloxacin in the HD, HDM, ID, and IDM groups was 3.42, 3.76, 3.64, and 4.72 µg/mL, respectively. The administration of meloxicam (IDM) to infected lambs significantly increased the C max of danofloxacin compared to the HD group. The AUC of danofloxacin increased significantly in the HDM and IDM groups compared to in the HD and ID groups. The absorption extent, Cl, and Vd of a drug contribute to the formation of C max and AUC [91,92]. Danofloxacin, following SC administration, in sheep has been shown to achieve a high bioavailability of 94% [51,93]. In this study, the decreased Vd area /F and Cl T /F in the HDM and IDM groups may lead to the formation of a high C max and AUC. A high C max and AUC has been reported for enrofloxacin coadministered with flunixin meglumine and for danofloxacin in calves with respiratory tract infections [35,70]. Danofloxacin has been shown to demonstrate linear pharmacokinetics at doses of 1.25-10 mg/kg following SC administration and no toxic symptoms at doses of 10 and 20 mg/kg for six days or doses of 18 and 24 mg/kg for three days in calves [94]. In this study, a 6 mg/kg dose of danofloxacin in the IDM group created a high C max within the therapeutic range. This may have resulted in an increased therapeutic effect of danofloxacin in the IDM group.

Pharmacokinetic/Pharmacodynamic Integration
The PK/PD surrogate parameters showing the efficacy of fluoroquinolones are the ƒAUC 0-24 /MIC and ƒC max /MIC ratios [95,96]. In the treatment of infections caused by Gram-negative bacteria, an ƒAUC 0-24 /MIC ratio should be 100-125 and a ƒC max /MIC ratio should be 8-10 in order to achieve an ideal bactericidal effect and reduce the risk of resistance [97,98]. In in vitro studies, it has been shown that while fluoroquinolone dosages with a ƒC max /MIC ratio of >3 cause a 99% reduction in the number of bacteria, dosages with a ƒC max /MIC ratio of ≥8 are sufficient to prevent the emergence of resistant bacteria [99]. In the present study, the ƒAUC 0-24 /MIC and ƒC max /MIC ratios were calculated only for the ID and IDM groups. In the ID and IDM groups, the ƒAUC 0-24 /MIC ratio of danofloxacin for susceptible E. coli isolates with an MIC value of 0.125 µg/mL was 82.89 h and 118.43 h, respectively, while the ƒC max /MIC ratio was 19.39 and 28.80, respectively. Danofloxacin in the ID and IDM groups provided the ideal ƒC max /MIC ratios following SC administration at a dose of 6 mg/kg. However, the ideal ƒAUC 0-24 /MIC ratio was obtained for the IDM group only.
A susceptible MIC breakpoint of ≤0.25 µg/mL for danofloxacin against M. haemolytica has been reported [56,100]. In this study, the MIC 90 value of danofloxacin for M. haemolytica isolates isolated from lambs with respiratory infections was 0.25 µg/mL. In the ID and IDM groups, the ƒAUC 0-24 /MIC 90 ratio was 41.44 h and 59.21 h, respectively, while the ƒC max /MIC 90 ratio was 9.69 and 14.40. Danofloxacin at a dose of 6 mg/kg achieved the ideal ƒC max /MIC 90 value of 8-10 but did not reach the ƒAUC 0-24 /MIC 90 ratio of ≥125 h previously reported for fluoroquinolones [95,96]. However, studies have reported that the optimal ƒAUC 0-24 /MIC 90 value is not an absolute value, and the AUC 0-24 /MIC 90 value of danofloxacin, which can provide bacterial elimination of M haemolytica, is 2-4 times lower than 125 h [50][51][52]101]. The ideal ex vivo and in vivo AUC 0-24 /MIC values of danofloxacin for the elimination of M. haemolytica isolates from sheep have been reported to be 28.7 and 55.9 h, respectively [51]. When evaluated on the basis of these data, danofloxacin provided the ideal in vivo ƒAUC 0-24 /MIC 90 ratio (55.9 h) for M. haemolytica isolates with MIC values of ≤0.25 µg/mL in the ID and IDM groups, but not in the ID group. Furthermore, previous studies have shown that danofloxacin in the lung tissues and bronchial mucosa reaches approximately 5-and 3-times higher concentrations, respectively, than those obtained in the plasma [11]. Therefore, in the present study, the concentration of danofloxacin obtained in the lung tissues and bronchial mucosa of the lambs in the ID group may have significantly exceeded the MIC values required for the elimination of M. haemolytica, which is an extracellular localization [70,102].

Conclusions
The administration of meloxicam and the presence of infection in lambs reduced the Vd/F and Cl T /F of danofloxacin but did not affect the t 1/2λz . Meloxicam increased the plasma concentration of danofloxacin in both healthy lambs and lambs with respiratory infections. In lambs with respiratory infections, an increased plasma concentration of danofloxacin within the therapeutic range may result in a better therapeutic effect. However, the clear definition of an increase in the therapeutic effect requires the determination of the concentration of danofloxacin in the target tissues and the effects of the resulting concentration on bacterial eradication. In addition, repeated doses of danofloxacin are required for the treatment of infection. A danofloxacin-meloxicam interaction may not be predicted following administration at repeated doses. Therefore, it is necessary to deter-mine the pharmacokinetics of danofloxacin and meloxicam following the administration of repeated doses before simultaneous use in lambs with respiratory infections.
When co-administration with meloxicam, danofloxacin following SC administration at a dose of 6 mg/kg in lambs with respiratory infections provided optimum AUC 0-24 /MIC (>56 h) and C max /MIC (>8) values for susceptible M. haemolytica isolates with an MIC 90 value of 0.25 µg/mL and for susceptible E. coli isolates with an MIC value of ≤0.125 µg/mL. However, due to bacterial isolation in a small number of samples in this study, the determination of pharmacodynamic data on bacterial agents isolated from more lambs with respiratory infections will be more valuable for the rational use of danofloxacin.
Author Contributions: M.N.U. and K.U. contributed to conception, experimental design, and analysis, drafted and critically revised the manuscript, provided final approval, and agreed to be accountable for all aspects of the work, ensuring integrity and accuracy. All authors have read and agreed to the published version of the manuscript.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.